Keywords

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General Considerations

Neuroleptics have been defined as therapeutics effective against schizophrenia. One has to bear in mind that the effect of certain drugs has not been predicted by pharmacological tests but has been found in clinical trials by serendipity. The clinical discoveries were followed by pharmacological studies in many laboratories (Courvoisier 1956).

Various studies have demonstrated the blockade of postsynaptic catecholamine receptors, especially D2-receptors, to be the main mode of action of most neuroleptics. Several in vitro methods measure the receptor blockade by neuroleptics.

Pharmacological models in the development of antipsychotic drugs were reviewed by Costall et al. (1991).

References and Further Reading

  • Costall B, Domeney AM, Kelly ME, Naylor RJ (1991) Pharmacological models in the development of antipsychotic drugs – new strategies. In: Olivier B, Mos J, Slangen JL (eds) Animal models in psychopharmacology. Advances in pharmacological sciences. Birkhäuser, Basel, pp 253–263

  • Courvoisier S (1956) Pharmacodynamic basis for the use of chlorpromazine in psychiatry. J Clin Exp Psychopathol 17:25–37

In Vitro Methods

D1 Receptor Assay: [3H]-SCH 23390 Binding to Rat Striatal Homogenates

Purpose and Rationale

Dopamine receptors are the primary targets in the development of drugs for the treatment of schizophrenia, Parkinson’s disease, and Huntington’s chorea (Seeman and Van Tol 1994).

Reviews on dopamine receptors and their subtypes were given by Baldessarini and Tarazi (1996; Missale et al. 1998) and by the NC-IUPHAR subcommittee on dopamine receptors (Schwartz et al. 1998).

Multiple dopamine receptors are known. Two groups are most studied, designated as D1 and D2. In the group of D1-like dopamine receptors, the subtypes D1A and D5/D1B have been described. To D2-like dopamine receptors belong the D2S, the D2L, the D3, and the D4 receptor (Sokoloff et al. 1990; Civelli et al. 1991; Grandy et al. 1991; Van Tol et al. 1991; Lévesque et al. 1992; Baldessarini et al. 1993; Ginrich and Caron 1993; Todd and O’Malley 1993; Waddington and Deveney 1996).

D1 receptors are positively linked to adenylate cyclase, and the D2 receptor has been shown to be negatively linked to adenylate cyclase. For typical neuroleptic agents, like butyrophenones, a good correlation was found between D2 receptor binding and clinically effective doses. Atypical neuroleptics, like clozapine, were found to be potent inhibitors of D1 and D4 receptor binding, renewing interest in these receptor types. The compound SCH 23390 was found to be selective for the D1 receptor.

Procedure

Reagents

[N-Methyl-3H] Sch 23390 (Amersham Lab., specific activity 67–73 Ci/mmol). For IC 50 determinations, 3HSch 23390 is made up to a concentration of 10 nM and 50 μl is added to each tube. This yields a final concentration of 0.5 nM in the assay.

d-Butaclamol (Ayerst Laboratories). A 1 mM stock solution is made and diluted 1:20.

20 μl are added to three tubes for the determination of nonspecific binding.

For the test compounds α, 1 mM stock solution is made up in a suitable solvent and serially diluted, such that the final concentration in the assays ranges from 10−5 to 10−8 M.

Tissue Preparation

Male Wistar rats are decapitated, brains rapidly removed, striata dissected, and weighed. The striata are homogenized in 100 volumes of 0.05 M Tris buffer, pH 7.7, using a Tekmar homogenizer. The homogenate is centrifuged at 40,000 g for 20 min, and the final pellet is resuspended in the original volume of 0.05 Tris buffer, pH7.7, containing physiological ions (NaCl 120 mM, KCl 5 mM, MgCl2 1 mM, and CaCl2 2 mM).

Assay

50 μl

0.5 M Tris buffer, pH7.7, containing physiological ions

380 μl

H2O

20 μl

Vehicle or butaclamol or appropriate concentration of test compound

50 μl

3H-SCH 23390

500 μl

Tissue suspension

The tubes are incubated at 37 °C for 30 min. The assay is stopped by rapid filtration through Whatman GF/B filters using a Brandel cell harvester. The filter strips are then washed three times with ice-cold 0.05 M Tris buffer, pH7.7, and counted in 10 ml Liquiscint scintillation cocktail.

Evaluation

Specific binding is defined as the difference between total binding and binding in the presence of 1 μM butaclamol. IC 50 calculations are performed using log-probit analysis. The percent inhibition at each drug concentration is the average of duplicate determinations.

Modifications of the Method

Wamsley et al. (1992) recommended the radioactive form of a dopamine antagonist, [3H]SCH39166, as ligand for obtaining selective labeling of D1 receptors.

Sugamori et al. (1998) characterized the compound NNC 01–0012 as a selective and potent D1C receptor antagonist.

References and Further Reading

  • Anderson PH, Gronvald FC, Jansen JA (1985) A comparison between dopamine-stimulated adenylate cyclase and 3H-SCH 23390 binding in rat striatum. Life Sci 37:1971–1983

  • Anderson PH, Nielsen EB, Gronvald FC, Breastrup C (1986) Some atypical neuroleptics inhibit [3H]SCH 23390 binding in vivo. Eur J Pharmacol 120:143–144

  • Anderson PH, Gingrich JA, Bates MD, Dearry AD, Falardeau P, Senogles SE, Caron MG (1990) Dopamine receptor subtypes: beyond the D1/D2 classification. Trends Pharmacol Sci 11:213–236

  • Baldessarini RJ, Tarazi FI (1996) Brain dopamine receptors: a primer on their current status, basic and clinical. Harvard Rev Psychiatry 3:301–325

  • Baldessarini RJ, Kula NS, McGrath CR, Bakthavachalam V, Kebabian JW, Neumeyer JL (1993) Isomeric selectivity at dopamine D3 receptors. Eur J Pharmacol 239:269–270

  • Billard W, Ruperto V, Crosby G, Iorio LC, Barnett A (1984) Characterisation of the binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum. Life Sci 35:1885–1893

  • Chipkin RE, Iorio LC, Coffin VL, McQuade RD, Berger JG, Barnett A (1988) Pharmacological profile of SCH39166: a dopamine D1 selective benzonaphthazepine with potential antipsychotic activity. J Pharmacol Exp Ther 247:1093–1102

  • Civelli O, Bunzow JR, Grandy DK, Zhou QY, Van Tol HHM (1991) Molecular biology of the dopamine receptors. Eur H Pharmacol Mol Pharmacol Sect 207:277–286

  • Clement-Cormier YC, Kebabian JW, Petzold GR, Greengard P (1974) Dopamine-sensitive adenylate cyclase in mammalian brain. A possible site of action of anti-psychotic drugs. Proc Natl Acad Sci U S A 71:1113–1117

  • Creese I (1987) Biochemical properties of CNS dopamine receptors. In: Meltzer HY (ed) Psychopharmacology: the third generation of progress. Raven Press, New York, pp 257–264

  • Dawson TM, Gehlert DR, Yamamura HI, Barnett A, Wamsley JK (1985) D-1 dopamine receptors in the rat brain: autoradiographic localisation using [3H]SCH 23390. Eur J Pharmacol 108:323–325

  • Dearry A, Gingrich JA, Falardeau P, Fremeau RT, Bates MD, Caron MG (1990) Molecular cloning and expression of the gene for a human D1 dopamine receptor. Nature 347:72–76

  • DeNinno MP, Schoenleber R, MacKenzie R, Britton DR, Asin KE, Briggs C, Trugman JM, Ackerman M, Artman L, Bednarz L, Bhatt R, Curzon P, Gomez E, Kang CH, Stittsworth J, Kebabian JW (1991) A68930: a potent agonist selective for the dopamine D1 receptor. Eur J Pharmacol 199:209–219

  • Gerhardt S, Gerber R, Liebman JM (1985) SCH 23390 dissociated from conventional neuroleptics in apomorphine climbing and primate acute dyskinesia models. Life Sci 37:2355–2363

  • Ginrich JA, Caron MC (1993) Recent advances in the molecular biology of dopamine receptors. Annu Rev Neurosci 16:299–321

  • Grandy DK, Zhang Y, Bouvier C, Zhou QY, Johnson RA, Allen L, Buck K, Bunzow JR, Salon J, Civelli O (1991) Multiple human dopamine receptor genes: a functional D5 receptor and two pseudogenes. Proc Natl Acad Sci U S A 88:9175–9179

  • Hess E, Battaglia G, Norman AB, Iorio LC, Creese I (1986) Guanine nucleotide regulation of agonist Robinson T (ed) Interactions at [3H]SCH 23390-labelled D1 dopamine receptors in rat striatum. Eur J Pharmacol 121:31–38

  • Hyttel J (1983) SCH 23390 the first selective dopamine D-1 antagonist. Eur J Pharmacol 91:153–154

  • Iorio LC, Barnett A, Leitz FH, Houser VP, Korduba CA (1983) SCH 23390, a potential benzazepine antipsychotic with unique interactions on dopamine systems. J Pharmacol Exp Ther 226:462–468

  • Kebabian JW, Calne DB (1979) Multiple receptors for dopamine. Nature 277:93–96

  • Kebabian JW, Britton DR, DeNinno MP, Perner R, Smith L, Jenner P, Schoenleber R, Williams M (1992) A-77363: a potent and selective D1 receptor antagonist with antiparkinsonian activity in marmosets. Eur J Pharmacol 229:203–209

  • Kilpatrick GJ, Jenner P, Mardsen CD (1986) [3H]SCH 23390 identifies D-1 binding sites in rat striatum and other brain areas. J Pharm Pharmacol 38:907–912

  • Lévesque D, Diaz J, Pilon C, Martres MP, Giros B, Souil E, Schott D, Morgat JL, Schwartz JC (1992) Identification, characterization, and localization of the dopamine D3 receptor in rat brain using 7-[3H]hydroxy-N, N-di-n-propyl-2-aminotetralin. Proc Natl Acad Sci U S A 89:8155–8159

  • Missale C, Caron MG, Jaber M (1998) Dopamine receptors: from structure to function. Physiol Rev 78:189–225

  • Neumeyer JL, Kula NS, Baldessarini RJ, Baindur N (1992) Stereoisomeric probes for the D1 dopamine receptor: synthesis and characterization of R-(+) and S-(−) enantiomers of 3-allyl-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine and its 6-bromo analogue. J Med Chem 35:1466–1471

  • Niznik HB, Sunahara RK, van Tol HHM, Seeman P, Weiner DM, Stormann TM, Brann MR, O’Dowd BF (1992) The dopamine D1 receptors. In: Brann MR (ed) Molecular biology of G-protein coupled receptors. Birkhäuser, Boston/Basel/Berlin, pp 142–159

  • O’Boyle KM, Waddington JL (1992) Agonist and antagonist interaction with D1 dopamine receptors: agonist induced masking of D1 receptors depends on intrinsic activity. Neuropharmacol 31:177–183

  • Schwartz JC, Carlsson A, Caron M, Scatton B, Civelli O, Kebabian JW, Langer SZ, Sedvall G, Seeman P, Spano PF, Sokoloff P, Van Tol H (1998) Dopamine receptors. NCIUPHAR subcommittee for dopamine receptors. In: Gridlestone D (ed) The IUPHAR compendium of receptor characterization and classification. IUPHAR Media, London, pp 141–151

  • Seeman P (1977) Anti-schizophrenic drugs. Membrane receptor sites of action. Biochem Pharmacol 26:1741–1748

  • Seeman P, Van Tol HHM (1994) Dopamine receptor pharmacology. Trends Pharmacol Sci 15:264–270

  • Seeman P, Chau-Wong C, Tedesco J, Wong K (1975) Binding receptors for antipsychotic drugs and dopamine: direct binding assays. Proc Natl Acad Sci U S A 72:4376–4380

  • Snyder SH, Creese I, Burt DR (1975) The brain’s dopamine receptor: labeling with [3H]dopamine. Psychopharmacol Commun 1:663–673

  • Stoff JC, Kebabian JW (1982) Independent in vitro regulation by the D-2 dopamine receptor of dopamine-stimulated efflux of cyclic AMP and K+-stimulated release of acetylcholine from rat neostriatum. Brain Res 250:263–270

  • Sugamori KS, Hamdanizadeh SA, Scheideler MA, Hohlweg R, Vernier P, Niznik HB (1998) Functional differentiation of multiple dopamine D1-like receptors by NNC 01–0012. J Neurochem 71:1685–1693

  • Sunahara RK, Niznik HB, Weiner DM, Stormann TM, Brann MR, Kennedy JL, Gelernter JE, Rozmahel R, Yang Y, Israel Y, Seeman P, O’Dowd BF (1990) Human dopamine D1 receptor encoded by an intronless gene on chromosome 5. Nature 347:80–83

  • Todd RD, O’Malley KL (1993) Family ties: the dopamine D2-like receptor genes. Neurotransmiss 9(3):1–4

  • Trampus M, Ongini E, Borea PA (1991) The neutral endopeptidase-24.11 inhibitor SCH 34826 does not change opioid binding but reduces D1 dopamine receptors in rat brain. Eur J Pharmacol 194:17–23

  • Tricklebank MD, Bristow LJ, Hutson PH (1992) Alternative approaches to the discovery of novel antipsychotic agents. Progr Drug Res 38:299–336

  • Van Tol HHM, Bunzow JR, Guan HC, Sunahara RK, Seeman P, Niznik HB, Civelli O (1991) Cloning of the gene of a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 350:610–614

  • Waddington JL, Deveney AM (1996) Dopamine receptor multiplicity: ‘D1-like’-‘D2-like’ interactions and ‘D1-like’ receptors not linked to adenylate cyclase. Biochem Soc Transact 24:177–182

  • Wamsley JK, Alburges ME, McQuade RD, Hunt M (1992) CNS distribution of D1 receptors: use of a new specific D1 receptor antagonist, [3H]SCH39166. Neurochem Int 20(Suppl):123S–128S

  • Weinshank RL, Adham N, Macchi M, Olsen MA, Branchek TA, Hartig PR (1991) Molecular cloning and characterization of a high affinity dopamine receptor (D1 β) and its pseudogene. J Biol Chem 266:22427–22435

  • Zhou QY, Grandy DK, Thambi L, Kusher JA, Van Tol HHM, Cone R, Pribnow D, Salon J, Bunzow JR, Civelli O (1990) Cloning and expression of human and rat dopamine D1 receptors. Nature 347:76–80

D2 Receptor Assay: [3H]-Spiroperidol Binding

Purpose and Rationale

The neuroleptic compound haloperidol has been used as binding ligand to study the activity of other neuroleptics. The use of haloperidol has been superseded by spiroperidol. Dopamine receptor binding assays employing dopaminergic antagonists in mammalian striatal tissue, a dopamine-enriched area of the brain, have been shown to be predictive of in vivo dopamine receptor antagonism and antipsychotic activity. Significant correlations exist between neuroleptic binding affinities and their molar potencies in antagonism of apomorphine- or amphetamine-induced stereotypy, apomorphine-induced emesis in dogs, and antipsychotic activity in man. Spiroperidol is considered to be an antagonist specific for D2 receptors.

Procedure

Tissue Preparation

Male Wistar rats are decapitated, their corpora striata removed, weighed, and homogenized in 50 volumes of ice-cold 0.05 M Tris buffer, pH7.7. The homogenate is centrifuged at 40,000 g for 15 min. The pellet is rehomogenized in fresh buffer and recentrifuged at 40,000 g. The final pellet is then resuspended in Tris buffer containing physiological salts (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2) resulting in a concentration of 10 mg/ml.

Assay

The membrane preparations are incubated with 3H-spiroperidol (0.25 nM) and various concentrations of test drug at 37 °C for 20 min. in a K/Na phosphate buffer (50 mM, pH7.2), followed by cooling in an ice bath for 45 min. To determine nonspecific binding, samples containing 10 mM (+)-butaclamol are incubated under identical conditions without the test compound.

Bound ligand is separated by rapid filtration through Whatman GF/B glass fiber filters. The filters are washed three times with ice-cold buffer, dried, and shaken thoroughly with 3.5 ml scintillation fluid. Radioactivity is determined in a liquid scintillation counter. Specific binding is defined as the difference between total binding and the binding in the presence of 2.0 mM (+)-butaclamol.

Evaluation

The following parameters are determined:

  • Total binding of 3H-spiroperidol

  • Nonspecific binding: binding of samples containing 2 mM butaclamol

  • Specific binding: total binding nonspecific binding

  • Percent inhibition: 100-specific binding as percentage of the control value

IC 50 values are determined using at least 3–4 different concentrations of the test compound in triplicate. Results are presented as mean ± standard deviation.

Dissociation constants (K d) are determined, using 3H-spiroperidol concentrations ranging between 0.1 and 1.0 nM. Ki values (inhibitory constants) are calculated using the following equation:

$$ {K}_{\mathrm{i}}=\frac{I{C}_{50}}{1+c/{K}_{\mathrm{d}}} $$

c = 3H-spiroperidol concentrations used to determine IC 50.

Standard values: K i of haloperidol = 6.0 ± 1.2 nM.

Modifications of the Method

Two isoforms of the D2 receptor were found by alternative splicing: the long (D2L) and the short (D2S) isoform (Dal Toso et al. 1989; Giros et al. 1989; Monsma et al. 1989; Itokawa et al. 1996).

Niznik et al. (1985) recommended [3H]-YM-09151–2, a benzamide neuroleptic, as selective ligand for dopamine D2 receptors.

Hall et al. (1985) used [3H]-eticlopride, a substituted benzamide, selective for dopamine D2 receptors, for in vitro binding studies.

Radioactive ligands for the D2 and the D3 receptor were described by Seeman and Schaus (1991), Chumpradit et al. (1994), Booze and Wallace (1995), Gackenheimer et al. (1995), Seeman and van Tol (1995), and Van Vliet et al. (1996).

Vessotskie et al. (1997) characterized binding of [125I]S(−)5-OH-PIPAT to dopamine D2-like receptors.

Neve et al. (1992) used a special apparatus, the “cytosensor microphysiometer,” which measures the rate of proton excretion from cultured cells (McConnell et al. 1991, 1992; Owicki and Parce 1992). In C6 glioma cells and L fibroblasts expressing recombinant dopamine D2 receptors, the dopamine D2 receptor agonist, quinpirole, accelerated the rate of acidification of the medium dose-dependent up to 100 nM quinpirole. The response was inhibited by the D2 antagonist spiperone. The D2 receptor-stimulated acidification was due to transport of protons by a Na+/H+ antiporter which was verified by the inhibition with amiloride or methylisobutyl amiloride.

The isolated rabbit ear artery was recommended as a useful model to characterize dopamine D2 agonists and antagonists (Hieble et al. 1985).

Human Recombinant Dopamine D2A and D2B Receptors

Hayes et al. (1992) described functionally distinct human recombinant subtypes of the dopamine D2 receptor, D2A and D2B.

D2A Receptor Binding

In a radioligand binding assay, the binding of [3H]-spiperone to membranes prepared from COS cells transiently expressing a recombinant human dopamine D2A receptor is measured.

Twenty mg of membrane is incubated with [3H]-spiperone at a concentration of 2.0 nM for 2 h at 25 °C. Nonspecific binding is estimated in the presence of 10 mM haloperidol. Membranes are filtered and washed three times with binding buffer, and filters are counted to determine [3H]-spiperone bound.

D2B Receptor Binding

In a radioligand binding assay, the binding of [3H]-spiperone to membranes prepared from COS cells transiently expressing a recombinant human dopamine D2B receptor is measured.

Fifteen mg of membrane is incubated with [3H]-spiperone at a concentration of 0.7 nM for 2 h at 37 °C. Nonspecific binding is estimated in the presence of 10 mM haloperidol. Membranes are filtered and washed three times with binding buffer, and filters are counted to determine [3H]-spiperone bound.

References and Further Reading

  • Booze RM, Wallace DR (1995) Dopamine D2 and D3 receptors in the rat striatum and nucleus accumbens: use of 7-OHDPAT and [125I]iodosulpiride. Synapse 19:1–13

  • Bunzow JR, Van Tol HHM, Grandy DK, Albert P, Salon J, Christie MD, Machida CA, Neve KA, Civelli O (1988) Cloning and expression of rat D2 dopamine receptor cDNA. Nature 336:783–787

  • Chumpradit S, Kung MP, Vessotskie J, Foulon C, Mu M, Kung HF (1994) Iodinated 2-aminotetralins and 3-amino-1-benzopyrans: ligands for D2 and D3 receptors. J Med Chem 37:4245–4250

  • Civelli O, Bunzow J, Albert P, van Tol HHM, Grandy D (1992) The dopamine D2 receptor. In: Brann MR (ed) Molecular biology of G-protein coupled receptors. Birkhäuser, Boston/Basel/Berlin, pp 160–169

  • Dal Toso R, Sommer B, Ewert M, Pritchett DB, Bach A, Chivers BD, Seeberg P (1989) The dopamine D2 receptor: two molecular forms generated by alternative splicing. EMBO J 8:4025–4034

  • Fields JZ, Reisine TD, Yamamura HI (1977) Biochemical demonstration of dopaminergic receptors in rat and human brain using [3H]-spiroperidol. Brain Res 136:578–584

  • Gackenheimer SL, Schaus JM, Gehlert DE (1995) [3H]quinelorane binds to D2 and D3 dopamine receptors in the rat brain. J Pharmacol Exp Ther 274:1558–1565

  • Giros B, Sokoloff P, Matres MP, Riou JF, Emorine LJ, Schwartz JC (1989) Alternative splicing directs the expression of two D2 dopamine receptor isoforms. Nature 342:923–929

  • Grandy DK, Marchionni MA, Makam H, Stofko RE, Alfano M, Frothingham L, Fischer JB, Burke-Howie KJ, Bunzow JR, Server AC, Civelli O (1989) Cloning of the cDNA and gene for a human D2 dopamine receptor. Proc Natl Acad Sci U S A 86:9762–9766

  • Hall H, Köhler C, Gawell L (1985) Some in vitro receptor binding properties of [3H]eticlopride, a novel substituted benzamide, selective for dopamine-D2 receptors in the rat brain. Eur J Pharmacol 111:191–199

  • Hayes G, Biden TJ, Selbie LA, Shine J (1992) Structural subtypes of the dopamine D2 receptor are functionally distinct: expression of the D2A and D2B subtypes in a heterologous cell line. Mol Endocrinol 6:920–926

  • Hieble JP, Nelson SH, Steinsland OS (1985) Neuronal dopamine receptors of the rabbit ear artery: pharmacological characterization of the receptor. J Auton Pharmacol 5:115–124

  • Itokawa M, Toru M, Ito K, Tsuga H, Kameyama K, Haga T, Arinami T, Hamaguchi H (1996) Sequestration of the short and long isoforms of dopamine D2 receptors expressed in Chinese hamster ovary cells. Mol Pharmacol 49:560–566

  • Laduron PM, Janssen PFM, Leysen JE (1978) Spiperone: a ligand of choice for neuroleptic receptors. 2. Regional distribution and in vivo displacement of neuroleptic drugs. Biochem Pharmacol 27:317–328

  • Leysen JE, Gommeren W, Laduron PM (1978) Spiroperone: a ligand of choice for neuroleptic receptors. 1. Kinetics and characteristics of in vitro binding. Biochem Pharmacol 27:307–316

  • Locke KW, Dunn RW, Hubbard JW, Vanselous CL, Cornfeldt M, Fielding S, Strupczewski JT (1990) HP 818: a centrally acting analgesic with neuroleptic properties. Drug Dev Res 19:239–256

  • Martres MP, Bouthenet ML, Sales N, Sokoloff P, Schwartz JC (1985) Widespread distribution of brain dopamine receptors evidenced with [125I]iodosulpiride, a highly selective ligand. Science 228:752–755

  • McConnell HM, Rice P, Wada GH, Owicki JC, Parce JW (1991) The microphysiometer biosensor. Curr Opin Struct Biol 1:647–652

  • McConnell HM, Owicki JC, Parce JW, Miller DL, Baxter GT, Wada HG, Pitchford S (1992) The Cytosensor Microphysiometer: biological applications of silicon technology. Science 257:1906–1912

  • Monsma FJ, McVittie LD, Gerfen CR, Mahan LC, Sibley DR (1989) Multiple D2 dopamine receptors produced by alternative RNA splicing. Nature 342:926–929

  • Neve KA, Kozlowski MR, Rosser MP (1992) Dopamine D2 receptor stimulation of Na+/H+ exchange assessed by quantification of extracellular acidification. J Biol Chem 267:25748–25753

  • Niznik HB, Grigoriadis DE, Pri-Bar I, Buchman O, Seeman P (1985) Dopamine D2 receptors selectively labeled by a benzamide neuroleptic: [3H]-YM-09151–2. Naunyn Schmiedebergs Arch Pharmacol 329:333–343

  • Owicki JC, Parce JW (1992) Biosensors based on the energy metabolism of living cells: the physical chemistry and cell biology of extracellular acidification. Biosens Bioelectron 7:255–272

  • Seeman P (1981) Brain dopamine receptors. Pharmacol Rev 32:229–313

  • Seeman P, Schaus JM (1991) Dopamine receptors labelled by [3H]quinpirole. Eur J Pharmacol 203:105–109

  • Seeman P, van Tol HHM (1995) Deriving the therapeutic concentrations for clozapine and haloperidol: the apparent dissociation constant of a neuroleptic at the dopamine D2 and D4 receptors varies with the affinity of the competing radioligand. Eur J Pharmacol Mol Pharmacol Sect 291:59–66

  • Sibley DR, Monsma FJ Jr (1992) Molecular biology of dopamine receptors. Trends Pharmacol Sci 13:61–69

  • Strange PG (1992) Studies on the structure and function of D2 dopamine receptors. Biochem Soc Trans 20:126–130

  • Terai M, Hidaka K, Nakamura Y (1989) Comparison of [3H]YM-09151–2 with [3H]spiperone and [3H]raclopride for dopamine D-2 receptor binding to rat striatum. Eur J Pharmacol 173:177–182

  • Van Vliet LA, Tepper PG, Dijkstra D, Damstoa G, Wickstrom H, Pugsley DA, Akunne HC, Heffner TG, Glase SA, Wise LD (1996) Affinity for dopamine D2, D3, and D4 receptors of 2-aminotetralins. Relevance of agonist binding for determination of receptor subtype selectivity. J Med Chem 39:4233–4237

  • Vessotskie JM, Kung MP, Chumpradit S, Kung HF (1997) Characterization of S(−)5-OH-PIPAT binding to dopamine D2-like receptors expressed in cell lines. Neuropharmacol 36:999–1007

Dopamine D2 Receptor Autoradiography (3H-Spiperone Binding)

Purpose and Rationale

Autoradiography of 3H-spiperone binding sites using selective labeling conditions permits the visualization of the anatomical locations of D2-dopamine receptors (Palacios et al. 1981). Quantitative measurements of the binding to receptors can be obtained with computer-assisted video analysis of the autoradiograms with a greater anatomical resolution and sensitivity than in membrane homogenates (Altar et al. 1984; 1985). Using autoradiographic techniques, it has been demonstrated that striatal D2 receptors are present on intrinsic neurons (Trugman et al. 1986; Joyce and Marshall 1987) and that the distribution of D2 receptors within the striatum is not homogeneous (Joyce et al. 1985). Anatomically discrete interactions of drugs with D2 receptors can be examined in vitro with inhibition experiments and ex vivo following acute or chronic drug treatment of the whole animal.

Since 3H-spiperone labels serotonin-2 (5-HT2) sites in many brain regions, a masking concentration of a 5-HT2 receptor blocker, e.g., ketanserin, is included to selectively define binding to D2 receptors. This is necessary if the test compound inhibits 5-HT2 binding or if the brain region of interest has a low D2 receptor density.

The assay is used to determine potential antipsychotic activity of compounds via direct interaction with the D2 dopamine recognition site in discrete regions of the rat brain.

Procedure

Reagents
  1. 1a.

    0.5 M Tris + 1.54 M NaCl, pH7.4.

  2. 1b.

    0.05 M Tris + 0.154 M NaCl, pH7.4.

  3. 2.

    3H-spiperone (specific activity 70–90 Ci/mmol) is obtained from Amersham (TRK.818).

    For IC 50 determinations, 3H-spiperone is prepared at a concentration of 8 nM, and 0.55 ml is added to each slide mailer (yields a final concentration of 0.4 nM in the 11.0 ml assay volume).

    For saturation experiments, 3H-spiperone is prepared at a concentration of 20 nM. The final concentrations should range from 0.2 to 1.0 nM. Typically, six concentrations are used by adding 0.55 ml or less to each mailer (for smaller volumes, add water to bring total addition of 0.55 ml).

  4. 3.

    Sulpiride is obtained from sigma. A stock solution of 5 × 10−4 M is made by dissolving the sulpiride in 1.0 ml of 0.01 N acetic acid and bringing the final volume to 10 ml with distilled water. 0.22 ml of the stock solution is added to the nonspecific binding slide mailers (final concentration 10 μM). All other mailers receive 0.22 ml of vehicle (1 ml of 0.01 N acetic acid in a final volume of 10 ml with distilled water).

  5. 4.

    Ketanserin (free base or tartrate salt) is obtained from Janssen. A stock solution of 10−3 M is made by dissolving the ketanserin in 0.5 ml 1 N acetic acid and bringing the final volume to 10 ml with distilled water. The tartrate salt is water-soluble. This is further diluted to 5 × 10−6 M (50 μl q.s. to 10 ml). 0.22 ml is added to all mailers.

  6. 5.

    Test compounds (for IC 50 determinations). For most assays, a 5 × 10−3 M stock solution is made up in a suitable solvent and serially diluted, such that the final concentrations in the assay range from 10−5 to 10−8 M. Seven concentrations are used for each assay. Higher or lower concentrations may be used depending on the potency of the drug.

Tissue Preparation

Rat brain sections are collected from plates 9 (rostral nucleus accumbens) through plate 17 (caudal striatum) of The Rat Brain Atlas in Stereotaxic Coordinates by Paxinos and Watson.

  1. 1.

    For in vitro inhibition experiments, 3–5 sets of 10 slides are collected with 3–4 sections per slide.

  2. 2.

    For saturation experiments, 3–5 sets of 12 slides are collected with 3–4 sections per slide.

  3. 3.

    For ex vivo inhibition experiments, a set of 8 slides is used, 4 for total binding and 4 for nonspecific binding.

  4. 4.

    For experiments in which the tissue sections will be swabbed and counted with scintillation fluid, two sections per slide are collected.

Assay
  1. 1.

    Preparation of slide mailers (11.0 ml volume/slide mailer).

    Note: If slides with sections are to be wiped for scintillation counting, a final volume of 6.5 ml is sufficient to cover two sections. A proportional adjustment of the volumes to be pipetted is made.

    1. (a)

      In vitro inhibition experiments

      Separate mailers are prepared for total binding, nonspecific binding, and 7–8 concentrations of test compound. Ketanserin is included in all mailers to mask binding of [3H]-spiperone to 5-HT2 sites so that inhibition of binding is D2-selective.

      5.50 ml

      Buffer 1b

      0.55 ml

      Buffer 1a

      0.55 ml

      [3H]-spiperone, 0.4 nM final concentration

      3.96 ml

      Distilled water

      0.22 ml

      Ketanserin, 5 × 10−6 M, final concentration 100 nM or vehicle

      0.22 ml

      Test compound, final concentration 10−8 to 10−5 M or sulpiride 5 × 10−4, final conc. 10 μM or vehicle

    2. (b)

      Ex vivo inhibition experiments

      Separate mailers are prepared for total and nonspecific binding, as described above, including ketanserin to mask 5-HT2 receptor binding.

    3. (c)

      Saturation experiments

      Separate mailers are prepared for total and nonspecific binding at each radioligand concentration. Ketanserin is not included in the mailers, in saturation experiments, since specific binding is defined as sulpiride-displaceable.

      5.50 ml

      Buffer 1b

      0.55 ml

      Buffer 1a

      0.55 ml

      [3H]-spiperone, final concentration 0.2–1.0 nM

      4.18 ml

      Distilled water

      0.22 ml

      5 × 10–4 M sulpiride, final concentration 10 μM or vehicle

  2. 2.

    Slides are air-dried for 10–15 min at room temperature, preincubated in 0.05 M Tris + 0.154 M NaCl, pH7.4 for 5 min, and further incubated for 60 min with [3H]-spiperone. Slides are then rinsed with ice-cold solutions as follows: dipped in buffer 1b, rinsed in buffer 1b for 2 × 5 min, and dipped in distilled water.

    Slides used for wipes: both sections are wiped with one Whatman GF/B filter, and radioactivity is counted after addition of 10 ml of scintillation fluid. Slides used for autoradiography: slides are dried under a stream of air at room temperature and are stored in a desiccator under vacuum at room temperature (usually over night). Slides are then mounted onto boards, along with 3H-standards (Amersham RPA 506).

    In the dark room under safelight illumination (Kodak GBX-2 filter), slides are exposed to Amersham Hyperfilm or LKB Ultrofilm for 14–17 days.

References and Further Reading

  • Altar CA et al (1984) Computer-assisted video analysis of [3H]-spiroperidol binding autoradiographs. J Neurosci Methods 10:173–188

  • Altar CA et al (1985) Computer imaging and analysis of dopamine (D2) and serotonin (S2) binding sites in rat basal ganglia or neocortex labeled by [3H]-spiroperidol. J Pharmacol Exp Ther 233:527–538

  • Joyce JN, Marshall JF (1987) Quantitative autoradiography of dopamine D2 sites in rat caudate-putamen: localization to intrinsic neurons and not to neocortical afferents. Neuroscience 20:773–795

  • Joyce JN, Loeschen SK, Marshall JF (1985) Dopamine D2 receptors in rat caudate-putamen: the lateral to medial gradient does not correspond to dopaminergic innervation. Brain Res 378:209–218

  • Kobayashi Y, Ricci A, Rossodivita I, Amenta F (1994) Autoradiographic localization of dopamine D2-like receptors in the rabbit pulmonary vascular tree. Naunyn Schmiedebergs Arch Pharmacol 349:559–564

  • Palacios JM, Niehoff DL, Kuhar MJ (1981) [3H]-Spiperone binding sites in brain: autoradiographic localization of multiple receptors. Brain Res 213:277–289

  • Tarazi FI, Campbell A, Yeghiayan SK, Balldessarini RJ (1998) Localization of dopamine receptor subtypes in corpus striatum and nucleus accumbens septi of rat brain. Comparison of D1, D2 and D4-like receptors. Neuroscience 83:169–176

  • Trugman JM et al (1986) Localization of D2 dopamine receptors to intrinsic striatal neurons by quantitative autoradiography. Nature 323:267–269

Binding to the D3 Receptor

Purpose and Rationale

Sokoloff et al. (1990) reported molecular cloning and characterization of a dopamine receptor (D3) as a potential target for neuroleptics. The D3 receptor is localized in limbic areas of the brain which are associated with cognitive, emotional, and endocrine functions. Together with the D2S, the D2L, and the D4 receptor, the D3 receptor belongs to the group of D2-like dopamine receptors (Ginrich and Caron 1993). 7-[3H]hydroxy-N,N-di-n-propyi-2-aminotetralin (Lévesque et al. 1992), R(+)-7-OH-DPAT (Baldessarini et al. 1993), and [125I]trans-7-OHPIPAT-A (Kung et al. 1993) have been recommended as ligands for receptor binding studies.

Chio et al. (1993) compared the heterologously expressed D3 dopamine receptors with D2 receptors in Chinese hamster ovary cells.

Damsma et al. (1993) described R-(+)-7-OH-DPAT (R-(+)-7-hydroxy-2-(N,N-di-n-propylamino)tetralin) as a putative dopamine D3 receptor ligand.

Functional correlates of dopamine D3 receptor activation in the rat in vivo and their modulation by the selective agonist, (+)-S 14297, have been described by Millan et al. (1995).

Isoforms of the D3 receptor have been described (Pagliusi et al. 1993).

Akunne et al. (1995) described binding of the selective dopamine D3 receptor agonist ligand [3H]PD 128907 = 4aR,10bR-(+)-trans-3,4,4a,10b-tetrahydro-4-n-propyl-2H,5H-[1]benzopyrano[4,3-b]1,4-oxazin-9-ol.

Procedure

Human dopamine D3 receptor is expressed in Chinese hamster ovary cells. Cells are grown in Dulbecco’s modified Eagle’s medium containing 10 % fetal bovine serum. Cells are harvested by trypsin treatment (0.25 %) for 4–5 min and centrifugation at 2000 g for 5 min. They are homogenized with a Polytron in 10 mM Tris–HCl (pH7.5) containing 1 mM EDTA and are centrifuged at 35,000 g for 15 min. The pellet is then resuspended by sonication in a buffer containing 50 mM NaHepes, 1 mM EDTA, 50 μM 8-hydroxyquinoline, 0.005 % ascorbic acid, and 0.1 % bovine serum albumin (pH7.5) (incubation buffer). Membrane suspensions (15–25 μg protein) are added to polypropylene test tubes containing [3H]7-OH-DPAT (7-[3H]hydroxy-N,N-di-n-propyl-2-aminotetralin) for the D3 receptor assay. Competing drugs are dissolved in incubation buffer, the final volume being 1 ml. Tubes are incubated in triplicate for 1 h at room temperature. The incubations are stopped by rapid filtration under reduced pressure through Whatman GF/C glass filters coated with 0.1 % bovine serum albumin, followed by three rinses with 3–4 ml ice-cold buffer. Nonspecific binding is measured in the presence of 1 μM dopamine.

Evaluation

Saturation curves are analyzed by computer nonlinear regression using a one-site cooperative model to obtain equilibrium dissociation constants (K D) and maximal density of receptors (B max). Inhibition constants (K i) are estimated according to the equation

$$ {K}_{\mathrm{i}}=I{C}_{50}/1+L/{K}_{\mathrm{D}} $$

References and Further Reading

  • Akunne HC, Towers P, Ellis GJ, Dijkstra D, Wikstrom H, Heffner TG, Wise LD, Pugsley TA (1995) Characterization of binding of [3H]PD 128907, a selective dopamine D3 receptor agonist ligand to CHO-K1 cells. Life Sci 57:1401–1410

  • Baldessarini RJ, Kula NS, McGrath CR, Bakthavachalam V, Kebabian JW, Neumeyer JL (1993) Isomeric selectivity at dopamine D3 receptors. Eur J Pharmacol 239:269–270

  • Brucke T, Wenger S, Podreka I, Asenbaum S (1991) Dopamine receptor classification, neuroanatomical distribution and in vivo imaging. Wien Klin Wochenschr 103:639–646

  • Chio CL, Lajiness ME, Huff RM (1993) Activation of heterologously expressed D3 dopamine receptors: comparison with D2 dopamine receptors. Mol Pharmacol 45:51–60

  • Damsma G, Bottema T, Westerink BHC, Tepper PG, Dijkstra D, Pugsley TA, Mackenzie RG, Heffner TG, Wickstrom H (1993) Pharmacological aspects of R-(+)-7-OHDPAT, a putative dopamine D3 receptor ligand. J Pharmacol 249:R9–R10

  • Ginrich JA, Caron MC (1993) Recent advances in the molecular biology of dopamine receptors. Annu Rev Neurosci 16:299–321

  • Kung MP, Fung HF, Chumpradit S, Foulon C (1993) In vitro binding of a novel dopamine D3 receptor ligand: [125I]trans-7-OH-PIPAT-A. Eur J Pharmacol 235:165–166

  • Lévesque D, Diaz J, Pilon C, Martres MP, Giros B, Souil E, Schott D, Morgat JL, Schwartz JC (1992) Identification, characterization, and localization of the dopamine D3 receptor in rat brain using 7-[3H]hydroxy-N, N-di-n-propyl-2-aminotetralin. Proc Natl Acad Sci U S A 89:8155–8159

  • MacKenzie RG, Van Leeuwen D, Pugsley TA, Shih YH, Demattos S, Tang L, Todd RD, O’Malley KL (1994) Characterization of the human dopamine D3 receptor expressed in transfected cell lines. Eur J Pharmacol Mol Pharmacol Sect 266:79–85

  • Millan MJ, Peglion JL, Vian J, Rivet JM, Brocco M, Gobert A, Newman-Tancredi A, Dacquet C, Bervoets K, Girardon S, Jacques V, Chaput C, Audinot V (1995) Functional correlates of dopamine D3 receptor activation in the rat in vivo and their modulation by the selective agonist, (+)-S 14297: 1. Activation of postsynaptic D3 receptors mediates hypothermia, whereas blockade of D2 receptors elicits prolactin secretion and catalepsy. J Pharmacol Exp Ther 275:885–898

  • Pagliusi S, Chollet-Daemerius A, Losberger C, Mills A, Kawashima E (1993) Characterization of a novel exon within the D3 receptor gene giving rise to an mRNA isoform expressed in rat brain. Biochem Biophys Res Commun 194:465–471

  • Sibley DR (1991) Cloning of a ‘D3’ receptor subtype expands dopamine receptor family. Trends Pharmacol Sci 12:7–9

  • Sokoloff P, Giros B, Martres MP, Bouthenet ML, Schwartz JC (1990) Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347:146–151

  • Todd RD, O’Malley KL (1993) Family ties: the dopamine D2-like receptor genes. Neurotransmiss 9(3):1–4

Binding to D4 Receptors

Purpose and Rationale

Van Tol et al. (1991) reported cloning of the gene of a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Together with the D2S, the D2L, and the D3 receptor, the D4 receptor belongs to the group of D2-like dopamine receptors (Ginrich and Caron 1993). Recognition and characterization of this dopamine binding site may be useful in the design of new types of antipsychotic drugs.

Dopamine D4 receptors have been localized in GABAergic neurons of the primate brain (Mrzljak et al. 1996).

Procedure

A plasmid construct of a 3.9-kb gene-cDNA hybrid subcloned into the expression vector pCD-PS is introduced into COS-7 cells by calcium phosphate-mediated transfection. Cells are cultivated and homogenized (Teflon pestle) in 50 mM Tris–HCl (pH7.4 at 4 °C) buffer containing 5 mM EDTA, 1.5 mM CaCl2, 5 mM KCl, and 120 mM NaCl. Homogenates are centrifuged for 15 min at 39,000 g, and the resulting pellets resuspended in buffer at a concentration of 150–250 μg/ml. For saturation experiments, 0.25 ml of tissue homogenate are incubated in duplicate with increasing concentrations of [3H]-spiperone (70.3 Cl mmol−1; 10–3000 pM final concentration) for 120 min at 22 °C in a total volume of 1 ml. For competition binding experiments, assays are initiated by the addition of 0.25 ml membrane and incubated in duplicate with various concentrations of competing ligands (10−14–10−3 M) and [3H]spiperone (150–300 μM) either in the absence or the presence of 200 μM Gpp(NH)p for 120 min at 22 °C. Assays are terminated by rapid filtration through a Titertek cell harvester and filters then monitored for tritium. For all experiments, specific binding is defined as that inhibited by 10 μM (−)sulpiride.

Evaluation

Both saturation and competition binding data are analyzed by the nonlinear least-square curve-fitting program ligand run on a suitable PC.

Modifications of the Method

Human Recombinant Dopamine D4,2, D4,4, D4,7, and D5 Receptors

Van Tol et al. (1992) described multiple dopamine D4 receptor variants in the human population.

Sunahara et al. (1991) reported the cloning of the gene for a human D5 receptor.

Human Recombinant Dopamine D4,2 Receptor Binding

In a radioligand binding assay, the binding of [3H]-spiperone to membranes prepared from COS cells transiently expressing a recombinant human dopamine D4,2 receptor is measured.

Fifteen μg of membrane is incubated with [3H]-spiperone at a concentration of 0.7 nM for 2 h at 25 °C. Nonspecific binding is estimated in the presence of 10 μM haloperidol. Membranes are filtered and washed three times with binding buffer, and filters are counted to determine [3H]-spiperone bound.

Human Recombinant Dopamine D4,4 Receptor Binding

In a radioligand binding assay, the binding of [3H]-spiperone to membranes prepared from COS cells transiently expressing a recombinant human dopamine D4,4 receptor is measured.

Twenty-five μg of membrane are incubated with [3H]-spiperone at a concentration of 1.0 nM for 2 h at 25 °C. Nonspecific binding is estimated in the presence of 10 μM haloperidol. Membranes are filtered and washed three times with binding buffer, and filters are counted to determine [3H]-spiperone bound.

Human Recombinant Dopamine D4,7 Receptor Binding

In a radioligand binding assay, the binding of [3H]-spiperone to membranes prepared from COS cells transiently expressing a recombinant human dopamine D4,7 receptor is measured.

Fifteen μg of membrane is incubated with [3H]-spiperone at a concentration of 0.7 nM for 2 h at 25 °C. Nonspecific binding is estimated in the presence of 10 μM haloperidol. Membranes are filtered and washed three times with binding buffer, and filters are counted to determine [3H]-spiperone bound.

Human Recombinant Dopamine D5 Receptor

In a radioligand binding assay, the binding of [3H]SCH 23390 to membranes prepared from COS cells expressing a recombinant human dopamine D5 receptor is measured.

First, 40 μg of membrane is incubated with [3H]SCH 23390 at a concentration of 2 nM for 2 h at 25 °C. Nonspecific binding is estimated in the presence of 10 μM cis-flupentixol. Membranes are filtered and washed three times with binding buffer, and filters are counted to determine [3H]SCH 23390 bound.

Several selective dopamine D4 antagonists were described: Hidaka et al. (1996), Merchant et al. (1996), Rowley et al. (1996), and Birstow et al. (1997).

Some radioligands were proposed as being selective for dopamine D4 receptors: [3H]clozapine (Ricci et al. 1997a, b), [3H]NGD 94–1 (Thurkauf 1997; Primus et al. 1997), and RBI-257 (Kula et al. 1997).

References and Further Reading

  • Birstow LJ, Collinson N, Cook GP, Curtis N, Freedman SB, Kulagowski JJ, Leeson PD, Patel S, Ragan CI, Ridgill M, Saywell KL, Tricklebank MD (1997) L-745,870, a subtype selective dopamine D4 receptor antagonist, does not exhibit a neuroleptic-like profile in rodent behavior tests. J Pharmacol Exp Ther 283:1256–1263

  • Ginrich JA, Caron MC (1993) Recent advances in the molecular biology of dopamine receptors. Annu Rev Neurosci 16:299–321

  • Hidaka K, Tada S, Matsumoto M, Ohmori J, Maeno K, Yamaguchi T (1996) YM-50001: a novel, potent and selective dopamine D4 receptor antagonist. Neuroreport 7:2543–2546

  • Kula NS, Baldessarini RJ, Kebabian JW, Bakthavachalam V, Xu L (1997) RBI 257: a highly potent, dopamine D4 receptor-selective ligand. Eur J Pharmacol 331:333–336

  • Merchant KM, Gill KS, Harris DW, Huff RM, Eaton MJ, Lookingland K, Lutzke BS, McCall RB, Piercey MF, Schreur PJKD, Sethy VH, Smith MW, Svensson KA, Tang AH, von Voigtlander PF, Tenbrink RE (1996) Pharmacological characterization of U-101387, a dopamine D4 receptor selective antagonist. J Pharmacol Exp Ther 279:1392–1403

  • Mrzljak L, Bergson C, Pappy M, Huff R, Levenson R, Goldman-Rakic PS (1996) Localization of dopamine D4 receptors in GABAergic neurons of the primate brain. Nature 381:245–248

  • Primus J, Thurkauf A, Xu J, Yevich E, McInerney S, Shaw K, Tallman JF, Gallager DW (1997) Localization and characterization of dopamine D4 binding sites in rat and human brain by use of the novel D4 receptor-selective ligand [3H]NGD 94–1. J Pharmacol Exp Ther 282:1020–1027

  • Ricci A, Bronzetti E, Rossodivita I, Amenta F (1997a) Use of [3H]clozapine as a ligand of the dopamine D4 receptor subtype in peripheral tissues. J Auton Pharmacol 17:261–267

  • Ricci A, Bronzetti E, Felici L, Tayebati SK, Amenta F (1997b) Dopamine D4 receptor in human peripheral blood lymphocytes: a radioligand binding assay study. Neurosci Lett 229:130–134

  • Rowley M, Broughton HB, Collins I, Baker R, Emms F, Marwood R, Patel S, Ragan CI, Freedman SB, Leeson PD (1996) 5-(4-Chlorophenyl)-4-methyl-3-(1-(2-phenylethyl)piperidin-4-yl)isoxazole: a potent, selective antagonist at cloned dopamine D4 receptors. J Med Chem 39:1943–1945

  • Sunahara RK, Guan HC, O’Dowd BF, Seeman P, Laurier LG, Ng G, George SR, Torchia J, Van Tol HHM, Niznik HB (1991) Cloning of the gene for a human D5 receptor with higher affinity for dopamine than D1. Nature 350:614–619

  • Thurkauf A (1997) The synthesis of tritiated 2-phenyl-4-[4-(2-pyrimidyl)piperazinyl]methylimidazole ([3H]NGD 94–1), a ligand selective for the dopamine D4 receptor subtype. J Label Compd Radiopharm 39:123–128

  • Todd RD, O’Malley KL (1993) Family ties: the dopamine D2-like receptor genes. Neurotransmiss 9(3):1–4

  • Van Tol HHM, Bunzow JR, Guan HC, Sunahara RK, Seeman P, Niznik HB, Civelli O (1991) Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 350:610–614

  • Van Tol HHM, Wu CM, Guan HC, Ohara K, Bunzow JR, Civelli O, Kennedy J, Seeman P, Niznik HB, Jovanovic V (1992) Multiple dopamine D4 receptor variants in the human population. Nature 358:149–152

Determination of Dopamine Autoreceptor Activity

Purpose and Rationale

The method describes the procedure to determine if a compound possesses autoreceptor blocking activity without the interference from postsynaptic effects. Striatal DOPA (3,4-dihydroxyphenylalanine), DOPAC (3,4-dihydroxyphenylacetic acid), and DA (dopamine) are quantitated following in vivo treatment with drug, apomorphine, gamma butyrolactone, and NSD-1015. Antipsychotic compounds that block striatal dopaminergic presynaptic autoreceptors are believed to possess a greater liability for producing EPS.

Procedure

Reagents
  1. 1.

    0.1 M HCl

  2. 2.

    1 N NaOH

  3. 3.

    0.1 M perchloric acid (PCA) containing 4.3 mM EDTA

  4. 4.

    2 mM solutions of DOPAC, DA, and DOPA in 0.1 M HCl, with 0.5 ml aliquots stored at −60 °C until use

  5. 5.

    Preparation of 2° standard mixture

    10 μM solution of DOPAC, DA, and DOPA diluted from reagent 4 with 0.1 M PCA/EDTA

    The 2° standard solution is used for the preparation of standard curves.

  6. 6.

    Mobile phase/MeOH-buffer (4: 96, v/v) buffer: 0.012 mM sodium acetate, 0.036 M citric acid, and 152 μM sodium octane sulfonate (mobile phase); methanol/buffer (80 ml + 1920 ml) filtered through a 0.2 pm nylon 66 filter

  7. 7.

    Preparation of dosing solutions

    1. (a)

      Apomorphine (2 mg/kg) is prepared in saline containing 1 % Tween 80 + 0.1 % ascorbic acid to prevent oxidation.

    2. (b)

      GBL (750 mg/kg) is prepared as a solution in saline containing 1 % Tween 80.

    3. (c)

      NSD-1015 (100 mg/kg) is prepared as a solution in saline containing 1 % Tween 80.

HPLC-Instrumentation

Consists of the following:

  • Pump, model SP8810 (Spectra Physics)

  • Injector, WISP 710B (Waters Associates)

  • Detector, 5100A electrochemical with a 5011 analytical cell and 5020 guard cell (ESA)

  • Integrator, D-2000 (Hitachi), used as a backup for the data collection/integrator, CS 9000 (IBM) system

  • Analytical column: C18-ODS Hypersil, 3 pm, 100 × 4.6 mm (Shandon)

Tissue Preparation

Following treatment with test drug, rats are sacrificed by decapitation at the predetermined time. The brain is rapidly removed; the striatum is dissected on ice and frozen on dry ice. The tissue is analyzed by HPLC the same day.

Tissue is homogenized in 500 μl 0.1 M PCA/EDTA. The homogenate is centrifuged for 6 min using a microcentrifuge (model 5413, Eppendorf). The supernatant is transferred to 0.2 pm microfilterfuge™ tubes and centrifuged for 6–8 min as before. The filtrate is transferred to WISP vials. Standards are included every 12–15 samples.

Five μl of the striatum homogenate is injected into the HPLC column.

HPLC flow rate is 1.5 ml/min; run time is 20 min. Helium flow is constant in mobile phase.

For protein analysis, 1.0 ml 1 N NaOH is added to the tissue pellet. The next day, the protein analysis is performed as described by Bradford (1976) using the BioRad Assay Kit.

Evaluation

Peak area is used for quantitation. The mg of protein and pmoles of DOPAC, DA, and DOPA are calculated from linear regression analyses using the corresponding standard curve. Final data are reported as pmoles/mg protein.

References and Further Reading

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  • Magnusson O, Mohringe B, Fowler CJ (1987) Comparison of the effects of dopamine D1 and D2 receptor antagonists on rat striatal, limbic and nigral dopamine synthesis and utilization. J Neural Transm 69:163–177

  • Reinhard JF, Perry JA (1984) Fast analysis of tissue catechols using a short, high-efficiency (3 μM ) LC column and amperometric detection. J Liq Chromatogr 7:1211–1220

  • Wagner J et al (1979) Determination of DOPA, dopamine, DOPAC, epinephrine, norepinephrine, α-monofluoromethyldopa and α-difluoromethyldopa in various tissues of mice and rats using reversed-phase ion-pair liquid chromatography with electrochemical detection. J Chromatogr 164:41–54

  • Walters JR, Roth RH (1976) Dopaminergic neurons: an in vivo system for measuring drug interactions with presynaptic receptors. Naunyn Schmiedebergs Arch Pharmacol 296:5–14

Dopamine-Sensitive Adenylate Cyclase in Rat Striatum

Purpose and Rationale

Agonist stimulation of dopamine D1 receptors leads to increased cAMP formation mediated by a guanine nucleotide-binding regulatory protein. This effect is blocked by selective antagonists like SCH 23390.

Agonist stimulation of the dopamine D2 receptor leads to a decreased cAMP formation mediated by a guanine nucleotide-binding protein. Apomorphine is a potent agonist with full intrinsic activity at D2 receptors. Phenothiazines block both D1 and D2 receptors, whereas butyrophenones and related drugs are very potent antagonists at D2 receptors.

Studies on cAMP formation may be useful for differentiation of antipsychotic drugs.

Procedure

Tissue Preparation

Male Wistar rats are sacrificed by decapitation, the brains removed, and the striata dissected out, and weighed. Striatal tissue from two rats is homogenized in 25 volumes of ice-cold 0.08 M Tris-maleate buffer, pH7.4, containing 2 mM EGTA. Protein content of an aliquot is determined. A 50 μl aliquot is used in the cyclase enzyme assay.

Enzyme Assay

The following volumes are placed in conical centrifuge tubes kept in an ice-water bath:

200 μl

Incubation buffer (equal amounts of 0.8 mM Tris-maleate, pH 7.4; 60 mM MgSO4; 100 mM theophylline and 4 mM EGTA)

50 μl

1 mM dopamine HCl or water

25 μl

Test drug or water

125 μl

Distilled water

50 μl

Tissue homogenate

After incubation for 20 min at 0 °C, the enzyme reaction is started by addition of 50 μl of 15 mM ATP solution. The tube rack is placed in a shaking water bath preset at 30 °C for 2.5 min. The reaction is terminated by placing the tube rack in a boiling water bath for 4 min. Then, the tubes are centrifuged at 1000 g for 10 min.

A 25 μl aliquot of the supernatant in each tube is removed and the cAMP determined using a commercial RIA kit (Amersham).

Evaluation

Results are expressed as pmoles cAMP/mg protein of dopamine-stimulated versus nondopamine-stimulated level. Percentage inhibition of this dopamine-stimulated level by test drugs is calculated and IC 50 values determined by log-probit analysis.

References and Further Reading

  • Broaddus WC, Bennett JP Jr (1990) Postnatal development of striatal dopamine function. I. An examination of D1 and D2 receptors, adenylate cyclase regulation and presynaptic dopamine markers. Dev Brain Res 52:265–271

  • Clement-Cormier YC, Kebabian JW, Petzold GL, Greengard P (1974) Dopamine-sensitive adenylate cyclase in mammalian brain: a possible site of action of antipsychotic drugs. Proc Natl Acad Sci U S A 71:1113–1117

  • Clement-Cormier YC, Parrish RG, Petzold GL, Kebabian JW, Greengard P (1975) Characterisation of a dopamine-sensitive adenylate cyclase in the rat caudate nucleus. J Neurochem 25:143–149

  • Creese I (1987) Biochemical properties of CNS dopamine receptors. In: Meltzer HY (ed) Psychopharmacology: the third generation of progress. Raven Press, New York, pp 257–264

  • Gale K, Giudotti A, Costa E (1977) Dopamine-sensitive adenylate cyclase: location in substantia nigra. Science 195:503–505

  • Horn S, Cuello AC, Miller RJ (1974) Dopamine in the mesolimbic system of the rat brain: endogenous levels and the effect of drugs on the uptake mechanism and stimulation of adenylate cyclase activity. J Neurochem 22:265–270

  • Iversen LL (1975) Dopamine receptors in the brain. Science 188:1084–1089

  • Kebabian JW, Calne DB (1979) Multiple receptors for dopamine. Nature 277:93–96

  • Kebabian JW, Petzold GL, Greengard P (1972) Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain, and its similarity to the “dopamine receptor”. Proc Natl Acad Sci U S A 69:2145–2149

  • Magnusson O, Mohringe B, Fowler CJ (1987) Comparison of the effects of dopamine D1 and D2 receptor antagonists on rat striatal, limbic and nigral dopamine synthesis and utilisation. J Neural Transm 69:163–177

  • Setler PE, Rarau HM, Zirkle CL, Saunders HL (1978) The central effects of a novel dopamine agonist. Eur J Pharmacol 50:419–430

α 1-Adrenergic Receptor Binding in Brain

Purpose and Rationale

The use of neuroleptic and antidepressant drugs is sometimes limited by their side effects, such as orthostatic hypotension and sedation. These side effects are attributed to blockade of central and peripheral adrenergic α-receptors. For neuroleptics the ratio between their dopamine antagonistic and their receptor antagonistic potencies should be taken into account rather than their absolute α-blocking effect. WB-4101 is a specific and potent antagonist of the α 1-adrenoreceptor, characterized in vitro in rat brain, heart, vascular smooth muscle, and gastrointestinal smooth muscle.

The in vitro [3H]-WB 4101 receptor binding assay quantitates the α-adrenergic blocking properties of psychoactive agents and is used to assess a compound’s potential to cause orthostatic hypotension and sedation as well as primary blood pressure lowering effects through α 1-receptor blockade.

Procedure

Reagents

[Phenoxy-3-3H(N)]-WB 4101 = (2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane, New England Nuclear (specific activity 20–35 Ci/mmol).

For IC 50 determinations, [3H]-WB 4101 is made up to a concentration of 2 nM in Tris buffer and 500 μl is added to each tube (yields a final concentration of 0.5 nM in the 2 ml assay).

L-norepinephrine bitartrate (Sigma Chemical Company). A 800 μM solution is prepared in Tris buffer and 250 μl is added to each of three tubes to determine nonspecific binding. This yields a final concentration of 100 μM in the 2 ml assay.

Test compounds: A 80 μM stock solution is made up in a suitable solvent and serially diluted with Tris buffer, such that the final concentration in the assay ranges from 10−5 to 10−8 M. Usually, seven concentrations are studied for each assay.

Tissue Preparation

Male Wistar rats (100–150 g) are sacrificed by decapitation. The whole brain minus cerebellum is homogenized in 75 volumes of ice-cold 0.05 M Tris buffer, pH7.7. The homogenate is centrifuged at 40,000 g at 4 °C for 15 min. The supernatant is discarded and the pellet is rehomogenized in fresh Tris buffer and recentrifuged at 40,000 g at 4 °C for 15 min. The final pellet is resuspended in the original volume of ice-cold 0.05 M Tris buffer. The final tissue concentration in the assay is 10 mg/ml. Specific binding is approximately 80 % of total bound ligand.

Assay

1200 μl

Tissue suspension

500 μl

3H-WB 4101

250 μl

Vehicle (for total binding) or

800 μM

L-norepinephrine bitartrate (for nonspecific binding) or appropriate drug concentration

Sample tubes are kept in ice for additions, then vortexed, and incubated for 15 min at 25 °C. The binding is terminated by rapid vacuum filtration through Whatman GF/B filters, followed by three 5 ml washes with ice-cold 0.05 M Tris buffer. The filters are counted in 10 ml of Liquiscint scintillation cocktail.

Evaluation

Specific WB 4101 binding is defined as the difference between the total binding and that bound in the presence of 100 μM norepinephrine. IC 50 calculations are performed using computer-derived log-probit analysis.

References and Further Reading

  • Creese I (1978) Receptor binding as a primary drug screening device. In: Yamamura HI et al (eds) Neurotransmitter receptor binding. Raven Press, New York, pp 141–170

  • Creese I, Burt DR, Snyder SH (1976) Dopamine receptor binding predicts clinical and pharmacological potencies of antischizophrenic drugs. Science 192:481–483

  • Greenberg DA, U’Prichard DC, Snyder SH (1976) Alpha-noradrenergic receptor binding in mammalian brain: differential labelling of agonist and antagonist states. Life Sci 19:69–76

  • Huger FP, Smith CP, Chiang Y, Glamkowski EJ, Ellis DB (1987) Pharmacological evaluation of HP 370, a potential atypical antipsychotic agent. Drug Dev Res 11:169–175

  • Janowsky A, Sulser F (1987) Alpha and beta adrenoreceptors in brain. In: Meltzer HY (ed) Psychopharmacology: the third generation of progress. Raven Press, New York, pp 249–256

  • Mottram DR, Kapur H (1975) The α-adrenoceptor blocking effects of a new benzodioxane. J Pharm Pharmacol 27:295–296

  • Peroutka SJ, U’Prichard DC, Greenberg DA, Snyder SH (1977) Neuroleptic drug interactions with norepinephrine alpha receptor binding sites in rat brain. Neuropharmacology 16:549–566

  • U’Prichard DC, Snyder SH (1979) Distinct α-noradrenergic receptors differentiated by binding and physiological relationships. Life Sci 24:79–88

  • U’Prichard DC, Greenberg DA, Shehan PP, Snyder SH (1978) Tricyclic antidepressants: therapeutic properties and affinity for α-noradrenergic receptor binding sites in the brain. Science 199:197–198

  • Yamada S et al (1980) Characterisation of alpha-1 adrenergic receptors in the heart using [3H]-WB 4101: effect of 6-hydroxydopamine treatment. J Pharmacol Exp Ther 215:176–185

[3H]Spiroperidol Binding to 5-HT2 Receptors in Rat Cerebral Cortex

Purpose and Rationale

The purpose of this assay is to determine the anti-serotonin activity of neuroleptics, antidepressants, and antihypertensive compounds, by measuring the displacement of [3H]spiroperidol from serotonergic antagonist binding sites in cerebral cortical membranes. The regulation of 5-HT2 receptor density by chronic antidepressant treatment is discussed in a separate protocol (see chapter “Antidepressant Activity”).

The receptor binding of serotonergic sites in the CNS has been investigated using [3H]serotonin (5-HT) (Bennett and Snyder 1976), [3H]LSD (Peroutka and Snyder 1979), and [3H]spiroperidol (Peroutka and Snyder 1979; List and Seeman 1981; Leysen et al. 1978) as the radioligand. Receptor sites have been defined kinetically and classified as 5-HT1 sites (labeled by [3H]5-HT and displaced by agonists) and 5-HT2 sites (labeled by [3H]-spiroperidol and displaced by antagonists). [3H]LSD labels both 5-HT1 and 5-HT2 binding sites (Peroutka and Snyder 1979). Of the brain regions tested, the frontal cerebral cortex contained the greatest density of 5-HT2 binding sites. Lesioning studies indicate that 5-HT2 binding sites are postsynaptic and not linked to adenyiate cyciase (Peroutka et al. 1979).

The inhibition of 5-HT2 binding correlates with the inhibition of quipazine-induced head twitch, which may reflect decreased behavioral excitation. The physiological and pharmacological role of these receptors is not clear. Although numerous neuroleptics and antidepressants of varying chemical structures are potent inhibitors of 5-HT2 binding, there is no clear-cut relationship to the efficacy of these drugs. Methysergide and cyproheptadine are both potent inhibitors of 5-HT2 binding without having neuroleptic or antidepressant effects. However, potent interaction with 5-HT2 receptors may indicate a reduced potential for catalepsy, since methysergide blocks catalepsy induced by haloperidol (Rastogi et al. 1981). The interaction of serotonergic neurons with cholinergic neurons in the striatum (Samanin et al. 1978) may also be decreased by potent 5-HT2 antagonists. In addition, the ratio of activity at D2 and 5-HT2 receptors may be useful in the screening of atypical antipsychotic agents (Meltzer et al. 1989). Furthermore, it has been shown that ketanserin, a selective 5-HT2 antagonist, is an effective hypotensive agent which blocks peripheral vascular 5-HT receptors.

5-HT2 receptors have been subdivided into 5-HT2A, 5-HT2B, and 5-HT2C receptors. The new 5-HT receptor classification has been published by the VII. International Union of Pharmacology Classification of Receptors for 5-Hydroxytryptamine (Serotonin) (Hoyer et al. 1994). Further comments were given by Humphrey et al. (1993), Martin and Humphrey (1994), Saxena (1994), and Tricklebank (1996).

Several compounds with HT2A antagonistic activity are described, such as trazodone (Clements-Jewery et al. 1980; Hingtgen et al. 1984; Stryjer et al. 2003), MDL 100,907 (Kehne et al. 1996; Moser et al. 1996), and sarpogrelate (Hayashi et al. 2003).

McCullough et al. (2006) described the 5-HT2B antagonist and 5-HT4 agonist activities of tegaserod in the anesthetized rat.

Procedure

Reagents
  1. 1.

    0.5 M Tris buffer, pH 7.7

    1. (a)

      57.2 g Tris–HCl.

      16.2 g Tris base

      q.s. to 1 l (0.5 M Tris buffer, pH7.7)

    2. (b)

      Make a 1:10 dilution in distilled H2O (0.05 M Tris buffer, pH7.7).

  2. 2.

    Tris buffer containing physiological ions

    1. (a)

      Stock buffer

      NaCl

      7.014 g

      KCl

      0.372 g

      CaCl2

      0.222 g

      MgCl2

      0.204 g

      q.s. to 100 ml in 0.5 M Tris buffer.

    2. (b)

      Dilute 1:10 in distilled H2O.

    3. (c)

      This yields 0.05 M Tris–HCl, pH7.7; containing NaCl (120 mM), KCl (5 mM), CaCl2 (2 mM), and MgCl2 (1 mM).

  3. 3.

    [Benzene-3H] spiroperidol (20–35 Ci/mmol) is obtained from New England Nuclear. For IC 50 determinations, 3H-spiroperidol is made up to a concentration of 30 nM in 0.01 N HCl and 50 μl added to each tube (yields a final concentration of 1.5 nM in the 1 ml assay).

  4. 4.

    Methysergide maleate is obtained from Sandoz. Methysergide maleate stock solution is made up to 0.25 mM for determination of nonspecific binding. The final concentration in the assay is 5 μM, when 20 μl of the stock solution is added to the reaction tube.

  5. 5.

    Test compounds. For most assays, α 1 mM stock solution is made up in a suitable solvent and serially diluted, such that the final concentration in the assay ranges from 10−5 to 10−8 M. Seven concentrations are used for each assay, and higher or lower concentrations may be used, depending on the potency of the drug.

Tissue Preparation

Male Wistar rats are decapitated, and the cerebral cortical tissue is dissected, weighed, and homogenized in 50 volumes of 0.05 M Tris buffer, pH 7.7 (buffer 1b) with the Brinkman Polytron and then centrifuged at 40,000 g for 15 min. The supernatant is discarded and the pellet resuspended and recentrifuged as described above. This pellet is resuspended in 50 volumes of buffer 2b and stored in an ice bath. The final tissue concentration is 10 mg/ml. Specific binding is 7 % of the total added ligand and 50 % of total bound ligand.

Assay

50 μl

0.5 M Tris-physiological salts (buffer 2a)

380 μl

H2O

20 μl

Vehicle (for total binding) or 0.25 mM methysergide (for nonspecific binding) or appropriate drug concentration

50 μl

[3H] spiroperidol

500 μl

Tissue suspension

The samples are incubated for 10 min at 37 °C and then immediately filtered under reduced pressure using Whatman GF/B filters. The filters are washed with three 5 ml volumes of ice-cold 0.05 M Tris buffer, pH 7.7 mM methysergide.

Evaluation

IC 50 calculations are performed using log-probit analysis. The percent inhibition at each drug concentration is the mean of triplicate determinations.

Modification of the Method

The receptor binding properties of the 5-HT2 antagonist ritanserin were reported by Leysen et al. (1985).

Preclinical characterization of a putative antipsychotic as a potent 5-HT2A antagonist was reported by Kehne et al. (1996).

Using [125I]LSD and [3H]5-HT binding assays, Siegel et al. (1996) characterized a structural class of 5-HT2 receptor ligands.

[3H]Ketanserin has been described as a selective 3H-ligand for 5-HT2 receptor binding sites (Leysen et al. 1981).

[3H]RP 62203, a potent and selective 5-HT2 antagonist, was recommended for in vivo labeling of 5-HT2 receptors (Fajolles et al. 1992).

Other selective 5-HT2 receptor radioligands were recommended:

[125I]-EIL (radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide) (Lever et al. 1991); [3H]MDL100,907 (Lopez-Gimenez et al. 1998).

References and Further Reading

  • Altar CA, Wasley AM, Neale RF, Stone GA (1986) Typical and atypical antipsychotic occupancy of D2 and S2 receptors: an autoradiographic analysis in rat brain. Brain Res Bull 16:517–525

  • Bennett JP Jr, Snyder SH (1976) Serotonin and lysergic acid diethylamide binding in rat brain membranes: relationship to postsynaptic serotonin receptors. Mol Pharmacol 12:373–389

  • Clements-Jewery S, Robson PA, Chidley LJ (1980) Biochemical investigations into the mode of action of trazodone. Neuropharmacology 19:1165–1173

  • Costall B, Fortune DH, Naylor RJ, Marsden CD, Pycock C (1975) Serotonergic involvement with neuroleptic catalepsy. Neuropharmacology 14:859–868

  • Dugovic C, Leysen JE, Wauquier A (1991) Serotonin and sleep in the rat: the role of 5-HT2 receptors. In: Idzikowski C, Cowen PJ (eds) Serotonin, sleep and mental disorder. Wrightson Biomedical, Petersfield, pp 77–88

  • Fajolles C, Boireau A, Pochant M, Laduron PM (1992) [3H]RP 62203, a ligand of choice to label in vivo brain 5-HT2 receptors. Eur J Pharmacol 216:53–57

  • Gelders YG, Heylen SLE (1991) Serotonin 5-HT2 receptor antagonism in schizophrenia. In: Idzikowski C, Cowen PJ (eds) Serotonin, sleep and mental disorder. Wrightson Biomedical, Petersfield, pp 179–192

  • Hayashi T, Sumi D, Matsui-Hirai H, Fukatsu A, Rani JA, Kano H, Tsunekawa T, Iguchi A (2003) Sarpogrelate HCl, a selective 5-HT2A antagonist, retards the progression of atherosclerosis through a novel mechanism. Atherosclerosis 168:23–31

  • Hingtgen JN, Hendrie HC, Aprison MH (1984) Polysynaptic serotonergic blockade following chronic antidepressive treatment in an animal model of depression. Pharmacol Biochem Behav 20:425–428

  • Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphrey PP (1994) VII. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol Rev 46:157–203

  • Humphrey PPA, Hartig P, Hoyer D (1993) A proposed new nomenclature for 5-HT receptors. Trends Pharmacol Sci 14:233–236

  • Kehne JK, Baron BM, Carr AA, Chaney SF, Elands J, Feldman DJ, Frank RA, van Giersbergen PLM, McCloskey TC, Johnson MP, McCarty DR, Poirot M, Senyah Y, Siegel BW, Widmaier C (1996) Preclinical characterization of the potential of the putative atypical antipsychotic MDL 100,907 as a potent 5-HT2A antagonist with a favorable CNS safety profile. J Pharmacol Exp Ther 277:968–981

  • Lever JR, Scheffel UA, Musachio JL, Stathis M, Wagner HN Jr (1991) Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide: a ligand for in vitro and in vivo studies of serotonin receptors. Life Sci 48:PL-73–PL-78

  • Leysen JE, Niemegeers CJE, Tollenaere JP, Laduron PM (1978) Serotonergic component of neuroleptic receptors. Nature 272:168–171

  • Leysen JE, Niemegeers CJE, van Nueten JM, Laduron PM (1981) [3H]Ketanserin (R 41 468), a selective 3H-ligand for serotonin2 receptor binding sites. Binding properties, brain distribution, and functional role. Mol Pharmacol 21:301–314

  • Leysen JE, Niemegeers CJE, Van Nueten JM, Laduron PM (1982) [3H]Ketanserin (R41 468) a selective 3H-ligand for serotonin2 receptor binding sites. Mol Pharmacol 21:301–314

  • Leysen JE, de Chaffoy de Courcelles D, de Clerck F, Niemegeers CJE, van Nueten JM (1984) Serotonin-S2 receptor binding sites and functional correlates. Neuropharmacology 23:1493–1501

  • Leysen JE, Gommeren W, van Gompel P, Wynants J, Janssen PFM, Laduron PM (1985) Receptor-binding properties in vitro and in vivo by ritanserin. A very potent and long acting serotonin-S2 antagonist. Mol Pharmacol 27:600–611

  • List SJ, Seeman P (1981) Resolution of dopamine and serotonin receptor components of [3H]spiperone binding of rat brain regions. Proc Natl Acad Sci U S A 78:2620–2624

  • Lopez-Gimenez JF, Vilaro MT, Palacios JM, Mengod G (1998) [3H]-MDL100,907 labels serotonin 5-HT2A receptors selectively in primate brain. Neuropharmacology 37:1147–1158

  • Martin GR, Humphrey PPA (1994) Classification review. Receptors for 5-hydroxytryptamine: current perspectives on classification and nomenclature. Neuropharmacology 33:261–273

  • McCullough JL, Armstrong SR, Hegde SS, Beattie DT (2006) The 5-HT2B antagonist and 5-HT4 agonist activities of tegaserod in the anaesthetized rat. Pharmacol Res 53:353–358

  • Meert TF, Awouters F (1991) Serotonin 5-HT2 antagonists: a preclinical evaluation of possible therapeutic effects. In: Idzikowski C, Cowen PJ (eds) Serotonin, sleep and mental disorder. Wrightson Biomedical, Petersfield, pp 65–76

  • Meltzer HV, Matsubara S, Lee JC (1989) Classification of typical and atypical antipsychotic drugs on the basis of dopamine D1, D2 and serotonin2 pK i values. J Pharmacol Exp Ther 251:238–246

  • Morgan DG, Marcusson JO, Finch CE (1984) Contamination of serotonin-2 binding sites with an alpha-1 adrenergic component in assays with (3H)spiperone. Life Sci 34:2507–2514

  • Moser PC, Moran PM, Frank RA, Kehne JH (1996) Reversal of amphetamine-induced behaviours by MDL 100.907, a selective 5-HT2A antagonist. Behav Brain Res 73:163–167

  • Muramatsu M, Tamaki-Ohashi J, Usuki C, Araki H, Aihara H (1988) Serotonin-2 receptor mediated regulation of release of acetylcholine by minaprine in cholinergic nerve terminal of hippocampus of rat. Neuropharmacology 27:603–609

  • Palacios JM, Niehoff DL, Kuhar MJ (1981) [3H]Spiperone binding sites in brain: autoradiographic localization of multiple receptors. Brain Res 213:277–289

  • Pazos A, Cortés R, Palacios JM (1985) Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors. Brain Res 2346:231–249

  • Pedigo NW, Yamamura HI, Nelson DL (1981) Discrimination of multiple [3H]5-hydroxytryptamine binding sites by the neuroleptic spiperone in rat brain. J Neurochem 36:220–226

  • Peroutka SJ, Snyder SH (1979) Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [3H]-lysergic acid diethylamide and [3H]spiroperidol. Mol Pharmacol 16:687–699

  • Peroutka SJ, Lebovitz RM, Snyder SH (1979) Serotonin receptors binding sites affected differentially by guanine nucleotides. Mol Pharmacol 16:700–708

  • Rastogi RB, Singhal RL, Lapierre YD (1981) Effects of short- and long-term neuroleptic treatment on brain serotonin synthesis and turnover: focus on the serotonin hypothesis of schizophrenia. Life Sci 29:735–741

  • Samanin R, Quattrone A, Peri G, Ladinsky H, Consolo S (1978) Evidence of an interaction between serotonergic and cholinergic neurons in the corpus striatum and hippocampus of the rat brain. Brain Res 151:73–82

  • Saxena PR (1994) Modern 5-HT receptor classification and 5-HT based drugs. Expert Opin Investing Drugs 3:513–523

  • Siegel BW, Freedman J, Vaal MJ, Baron BM (1996) Activities of novel aryloxyalkylimidazolines on rat 5-HT2A and 5-HT2C receptors. Eur J Pharmacol 296:307–318

  • Stryjer R, Strous RD, Bar F, Poyurovsky M, Weizman A, Kotler M (2003) Treatment of neuroleptic-induced akathisia with the 5-HT2A antagonist trazodone. Clin Neuropharmacol 26:137–141

  • Tricklebank MD (1996) The antipsychotic potential of subtypeselective 5-HT-receptor ligands based on interactions with mesolimbic dopamine systems. Behav Brain Res 73:15–17

Serotonin 5-HT2 Receptor Autoradiography (3H-Spiperone Binding)

Purpose and Rationale

Autoradiography of 3H-spiperone binding sites with selective labeling conditions permits the visualization of the anatomical locations of 5-HT2 receptors (Palacios et al. 1981; Pazos et al. 1985; Altar et al. 1985). Quantitative measurements of the binding to receptors can be obtained with computer-assisted video analysis of the autoradiograms with a greater anatomical resolution and sensitivity than in membrane homogenates (Pazos et al. 1985; Altar et al. 1984). Using autoradiographic techniques, it has been demonstrated that there is a heterogeneous distribution of 5-HT2 receptors, with much higher levels in telencephalic areas such as the neocortex and the claustrum than in meso- or metencephalic areas. Within the cortex, 5-HT2 receptors are abundant in layers IV and V (Pazos et al. 1985). The high concentration of 5-HT2 receptors in the frontoparietal motor area and the claustrum which connects to the motor cortex and other motor areas suggests a physiological role for 5-HT2 receptors in some motor syndromes (Cadet et al. 1987; Costall et al. 1975; Kostowski et al. 1972). The high affinity of the atypical antipsychotic clozapine for 5-HT2 receptors (Fink et al. 1984; Altar et al. 1986) and the downregulation of 5-HT2 receptors following chronic administration of clozapine (Reynolds et al. 1983; Lee and Tang 1984; Wilmot and Szczepanik 1989) suggest that 5-HT2 receptor interaction may be a significant factor in the lack of extrapyramidal side effects and tardive dyskinesias with its clinical use.

Since3 H-spiperone labels a1-noradrenergic sites in the cerebral cortex, a masking concentration of the a1-blocker prazosin is included to selectively define binding to 5-HT2 receptors (Morgan et al. 1984). This is necessary if the test compound also inhibits a1receptors which may be present in the brain region of interest.

The assay is used to determine the direct interaction of potential antipsychotic compounds with the serotonin-5-HT2 recognition site in discrete regions of the rat brain either in vitro or after ex vivo treatment of the whole animal.

Procedure

Reagents
  1. 1a.

    0.5 M Tris + 1.54 M NaCl, pH7.4.

  2. 1b.

    0.05 M Tris + 0.154 M NaCl, pH7.4.

  3. 2.

    3H-spiperone (specific activity 70–90 Ci/mmol) is obtained from Amersham.

    • For IC 50 determinations, 3H-spiperone is made up to a concentration of 20 nM, and 0.55 ml is added to each slide mailer (yields a final concentration of 1.0 nM in the 11.0 ml assay volume).

    • For saturation experiments, 3H-spiperone is made up to a concentration of 20 nM. The final concentration should range from 0.5 to 2.5 nM. Typically, six concentrations are used by adding 0.55 ml or less to each mailer (for smaller volumes, add water to bring total addition of 0.55 ml).

  4. 3.

    Methysergide is used to determine nonspecific binding in brain sections of the frontal cortex. Methysergide maleate is obtained from Sandoz. A stock solution of 2.5 × 10−4 M is made by dissolving in distilled water. A volume of 0.22 ml of the stock solution is added to the nonspecific binding slide mailers (final concentration 5 μM). All other mailers receive 0.22 ml of vehicle (1 ml of 0.01 N acetic acid in a final volume of 10 ml with distilled water).

  5. 4.

    Ketanserin is used to determine nonspecific binding in those slide mailers containing sections with the nucleus accumbens and striatum. Ketanserin (free base or tartrate salt) is obtained from Janssen. A stock solution of 10−3 M is made by dissolving the ketanserin (free base) in 0.05 N acetic acid or the tartrate salt in distilled water. This is further diluted to 5 × 10−6 M (50 μM q.s 10 ml with distilled water). A volume of 0.22 ml is added to the slide mailers to give a final concentration of 100 nM.

  6. 5.

    Prazosin is used to mask α 1-receptors in cortical brain section.

    Prazosin HCl is obtained from Pfizer. A stock solution of 10−4 M is made by dissolving prazosin in 0.01 N acetic acid and bringing the final volume to 10 ml with distilled water. This is further diluted to 5 × 10−6 M (100 μM q.s 10 ml). A volume of 0.22 ml is added to those slide mailers to be used for cortical brain sections to give a final concentration of 100 nM.

  7. 6.

    Sulpiride is used to mask D2 receptor binding in brain sections from the nucleus accumbens and striatum.

    Sulpiride is obtained from sigma. A stock solution of 10−4 M is made by dissolving sulpiride in 1.0 ml of 0.01 N acetic acid and bringing the final volume to 10 ml with distilled water. A volume of 0.22 ml is added to the appropriate slide mailers to give a final concentration of 10 μM.

  8. 7.

    Test compounds (for in vitro IC 50 determinations). For most assays, a 5 × 10−3 M stock solution is made up in a suitable solvent and serially diluted, such that the final concentration in the assay ranges from 10−5 to 10−8 M. Seven concentrations are used for each assay. Higher or lower concentrations may be used depending on the potency of the drug.

Tissue Preparation

Frontal cortical brain sections are collected from plates 5 through 8, and nucleus accumbens/striatal sections are collected from plates 9 (rostral n. accumbens) through plate 17 (caudal striatum) of “The Rat Brain Atlas in Stereotaxic Coordinates” by Paxinos and Watson.

  1. 1.

    For in vitro inhibition experiments, 3–5 sets of 10 slides are collected with 4–5 sections per slide.

  2. 2.

    For saturation experiments, 3–5 sets of 12 slides are collected with 4–5 six sections per slide.

  3. 3.

    For ex vivo inhibition experiments, a set of 8 slides is used, 4 for total binding and 4 for nonspecific binding.

  4. 4.

    For experiments in which the tissue sections will be swabbed and counted with scintillation fluid, two sections per slide are collected.

Assay
  1. 1.

    Preparation of slide mailers (11.0 ml volume/slide mailer).

    Note: If slides with sections are to be wiped for scintillation counting, a final volume of 6.5 ml is sufficient to cover two sections. A proportional adjustment of the volumes to be pipetted is made.

    1. (a)

      In vitro inhibition experiments

      Separate mailers are prepared for total binding, nonspecific binding, and 7–8 concentrations of test compound.

      1. 1.

        For frontal cortical brain sections, prazosin is included in all mailers to mask the binding of [3H]-spiperone to α 1-receptors, and nonspecific binding is defined with 5 μM methysergide.

        5.50 ml

        Buffer 1b

        0.55 ml

        Buffer 1a

        0.55 ml

        [3H]-spiperone, 1.0 nM final concentration

        3.96 ml

        Distilled water

        0.22 ml

        Prazosin 5 × 10−6 M, final concentration 100 nM or vehicle

        0.22 ml

        Test compound, final concentration 10−8–10−5 M or methysergide 2.5 × 10−4 M, final concentration 5 μM or vehicle

      2. 2.

        For brain sections with the nucleus accumbens and striatum in which there is negligible binding of [3H]-spiperone to α 1-receptors, prazosin is not included. Since levels of 5-HT2 receptors in these brain areas are low, 10 μM sulpiride is included in all mailers to mask the binding of [3H]-spiperone to D2 receptors.

        Ketanserin, final concentration of 100 nM, is used to determine nonspecific binding since methysergide has a weak affinity for D2 receptors (IC 50 approximately 1–5 μM).

        5.50 ml

        Buffer 1b

        0.55 ml

        Buffer 1a

        0.55 ml

        [3H]-spiperone, 1.0 nM final concentration

        3.96 ml

        Distilled water

        0.22 ml

        Sulpiride 5 × 10−4 M, final concentration 10 μM or vehicle

        0.22 ml

        Test compound, final concentration 10−8 to 10−5 M or ketanserin 5 × 10−5 M, final concentration 100 nM or vehicle

    2. (b)

      Ex vivo inhibition experiments

      Separate mailers are prepared for total and nonspecific binding, as described above, including sulpiride to mask D2 receptor binding with brain sections through the nucleus accumbens and striatum and prazosin to mask α 1-receptors in cortical brain sections.

    3. (c)

      Saturation experiments

      Separate mailers are prepared for total and nonspecific binding at each radioligand concentration. Prazosin is not included in the mailers in saturation experiments, since specific binding is defined by methysergide which has negligible affinity for α 1-receptors.

      5.50 ml

      Buffer 1b

      0.55 ml

      Buffer 1a

      0.55 ml

      [3H]-spiperone, final concentrations 0.5–2.5 nM

      4.18 ml

      Distilled water

      0.22 ml

      2.5 × 10−4 M methysergide, final concentration 5 μM or vehicle

  2. 2.

    Slides are air-dried for 10–15 min at room temperature, preincubated in 0.05 M Tris + 0.154 M NaCl, pH7.4 for 5 min, and further incubated for 60 min with [3H]-spiperone. Slides are then rinsed with ice-cold solutions as follows: dipped in buffer 1b, 2 × 5 min rinsed in buffer 1b, dipped in distilled water.

    Slides used for wipes: both sections are wiped with one Whatman GF/B filter, and radioactivity is counted after addition of 10 ml of scintillation fluid. Slides used for autoradiography: slides are dried under a stream of air at room temperature and are stored in a desiccator under vacuum at room temperature (usually overnight). Slides are then mounted onto boards, along with 3H-standards (Amersham RPA 506).

    In the dark room under safelight illumination (Kodak GBX-2 filter), slides are exposed to Amersham Hyperfilm or LKB Ultrofilm for 14–17 days.

References and Further Reading

  • Altar CA et al (1984) Computer-assisted video analysis of [3H]spiroperidol binding autoradiographs. J Neurosci Methods 10:173–188

  • Altar CA et al (1985) Computer imaging and analysis of dopamine (D2) and serotonin (S2) binding sites in rat basal ganglia or neocortex labeled by [3H]-spiroperidol. J Pharmacol Exp Ther 233:527–538

  • Altar CA, Wasley AM, Neale RF, Stone GA (1986) Typical and atypical antipsychotic occupancy of D2 and S2 receptors: an autoradiographic analysis in rat brain. Brain Res Bull 16:517–525

  • Cadet JL, Kuyatt B, Fahn S, De Souza EB (1987) Differential changes in 125I-LSD-labeled 5-HT-2 serotonin receptors in discrete regions of brain in the rat model of persistent dyskinesias induced by iminodipropionitrile (IDPN): evidence from autoradiographic studies. Brain Res 437:383–386

  • Costall B, Fortune DH, Naylor RJ, Marsden CD, Pycock C (1975) Serotonergic involvement with neuroleptic catalepsy. Neuropharmacology 14:859–868

  • Fink H, Morgenstern R, Oelssner W (1984) Clozapine – a serotonin antagonist? Pharmacol Biochem Behav 20:513–517

  • Kostowski W, Gumulka W, Czlokowski A (1972) Reduced kataleptogenic effects of some neuroleptics in rats with lesioned midbrain raphe and treated with p-chlorophenylalanine. Brain Res 48:443–446

  • Lee T, Tang SW (1984) Loxapine and clozapine decrease serotonin (S2) but do not elevate dopamine (D2) receptor numbers in the rat brain. Psychiatry Res 12:277–285

  • Morgan DG, Marcusson JO, Finch CE (1984) Contamination of serotonin-2 binding sites with an alpha-1 adrenergic component in assays with (3H)spiperone. Life Sci 34:2507–2514

  • Palacios JM, Niehoff DL, Kuhar MJ (1981) [3H]Spiperone binding sites in brain: autoradiographic localization of multiple receptors. Brain Res 213:277–289

  • Pazos A, Cortés R, Palacios JM (1985) Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors. Brain Res 346:231–249

  • Reynolds CP, Garrett NJ, Rupniak N, Jenner P, Marsden CD (1983) Chronic clozapine treatment of rats down-regulates 5-HT2 receptors. Eur J Pharmacol 89:325–326

  • Wilmot CA, Szczepanik AM (1989) Effects of acute and chronic treatment with clozapine and haloperidol on serotonin (5HT2) and dopamine (D2) receptors in the rat brain. Brain Res 487:288–298

Binding to the Sigma Receptor

Purpose and Rationale

Sigma receptors, as a class of binding sites in the brain, were originally described as a subtype of the opiate receptors. Efforts to develop less addicting opiate analgesics led to the study of several benzomorphan derivatives which produce analgesia without causing the classical morphine-induced euphoria. Unfortunately, these compounds, like N-allylnormetazocine (SKF 10,047), produced a variety of psychotic symptoms. This psychotomimetic effect is thought to be mediated by sigma receptors. This binding site is sensitive to many neuroleptics, most notably the typical antipsychotic haloperidol, leading to the hypothesis that drug interactions with the sigma site may be a new approach for the discovery of novel antipsychotics which are not dopamine receptor antagonists. D2 receptor antagonism is thought to be linked with the occurrence of extrapyramidal symptoms in the form of hyperkinesia and Parkinson symptoms or tardive dyskinesia, limiting the therapeutic use of traditional antipsychotic medication. It is hoped that ligands to the sigma receptor do not produce these adverse reactions. The sigma site is believed to be distinct from the binding site for the psychotomimetic drug phencyclidine.

Procedure

Reagents

(+)-SKF 10,047 is prepared as a stock solution of 5 × 10−3 M with distilled water. 130 μl added to the 6.5 ml assay yields a final concentration of 10−4 M.

3H-(+)-SKF 10,047 (specific activity 40 Ci/mmol) is obtained from New England Nuclear. A 200 nM stock solution is made up with distilled water for IC 50 determinations. 325 μl added to each tube yields a final concentration of 10 nM in the 6.5 ml assay.

Test Compounds

A 5 mM stock solution is prepared in a suitable solvent and serially diluted, such that the final concentration in the assay ranges from 10−5 to 10−8 M.

Tissue Preparation

The assay utilizes slide-mounted cross sections of brain tissue from male Hartley guinea pigs. Whole brain sections of 10 pm thickness are obtained from the hippocampus, thaw-mounted onto gel-chrome alum subbed slides, freeze-dried, and stored at −70 °C until use. On the day of the assay, the sections are thawed briefly at room temperature until the slides are dry and then used in the assay at a final volume of 6.5 ml.

Assay

Incubation solutions are prepared in plastic slide mailer containers as follows:

3.250 ml

0.05 M Tris buffer, pH 7.7

2.470 ml

Distilled water

0.325 ml

0.5 M Tris buffer, pH 7.7

0.130 ml

(+)-SKF 10,047 or vehicle

0.325 ml

[3H](+)-SKF 10,047

Dried slides with tissue sections are added to the slide mailers and incubated at room temperature for 90 min. Non-bound radioligand is removed by rinsing the slides sequentially in two 5-min rinses in ice-cold 0.05 M Tris buffer and a dip in ice-cold distilled water. The sections are either swabbed with Whatman GF/B filters for scintillation counting of tissue-bound radioligand or exposed to tritium-sensitive film for autoradiography of the binding sites.

Evaluation

Specific binding is determined from the difference of binding in the absence or presence of 10−4 M (+)SKF 10,047 and is typically 60–70 % of total binding. IC 50 values for the competing drug are calculated by log-probit analysis of the data.

Modifications of the Method

[3H]-(+)-pentazocine has been recommended as a highly potent and selective radioligand for μ receptors (de Costa et al. 1989; DeHaven-Hudkins et al. 1992).

Classification of sigma binding sites into α 1 and α 2 receptors has been proposed (Walker et al. 1990; Quirion et al. 1992; Abou-Gharbia et al. 1993).

Hashimoto and London (1993) characterized [3H]ifenprodil binding to σ 2 receptors in rat brain.

Ganapathy et al. (1999) provided evidence for the expression of the type 1 sigma receptor in the Jurkat human T lymphocyte cell line.

References and Further Reading

  • Abou-Gharbia M, Ablordeppey SY, Glennon RA (1993) Sigma receptors and their ligands: the sigma enigma. Ann Rep Med Chem 28:1–10

  • Angulo JA, Cadet JL, McEwen BS (1990) σ receptor blockade by BMY 14802 affects enkephalinergic and tachykinin cells differentially in the striatum of the rat. Eur J Pharmacol 175:225–228

  • de Costa BR, Bowen WD, Hellewell SB, Walker JM, Thurkauf A, Jacobson AE, Rice KC (1989) Synthesis and evaluation of optically pure [3H]-(+)-pentazocine, a highly potent and selective radioligand for σ receptors. FEBS Lett 251:53–58

  • DeHaven-Hudkins DL, Fleissner LC, Ford-Rice FY (1992) Characterization of the binding of [3H](+)-pentazocine to σ recognition sites in guinea pig brain. Eur J Pharmacol 227:371–378

  • Deutsch SI, Weizman A, Goldman ME, Morihisa JM (1988) The sigma receptor: a novel site implicated in psychosis and anti-psychotic drug efficacy. Clin Neuropharmacol 11:105–119

  • Ferris RM, Tang FLM, Chang KJ, Russell A (1986) Evidence that the potential antipsychotic agent rimcazole (BW 234U) is a specific, competitive antagonist of sigma sites in brain. Life Sci 38:2329–2339

  • Ganapathy ME, Prasad PD, Huang W, Seth P, Leibach FH, Ganapathy V (1999) Molecular and ligand-binding characterization of the sigma receptor in Jurkat human T lymphocyte cell line. J Pharmacol Exp Ther 289:251–260

  • Goldman ME, Jacobson AE, Rice KC, Paul SM (1985) Differentiation of [3H]phencyclidine and (+)-[3H]SKF-10,047 binding sites in rat cerebral cortex. FEBS Lett 190:333–336

  • Hashimoto K, London ED (1993) Further characterization of [3H]ifenprodil binding to sigma receptors in rat brain. Eur J Pharmacol 236:159–163

  • Hoffman DW (1990) Neuroleptic drugs and the sigma receptor. Am J Psychiatry 147:1093–1094

  • Itzhak Y, Hiller JM, Simon EJ (1985) Characterisation of specific binding sites for [3H](d)-N-allylnormetazocine in rat brain membranes. Mol Pharmacol 27:46–52

  • Kaiser C, Pontecorvo MJ, Mewshaw RE (1991) Sigma receptor ligands: function and activity. Neurotransm 7:1–5

  • Khazan N, Young GA, El-Fakany EE, Hong O, Calligaro D (1984) Sigma receptors mediate the psychotomimetic effects of N-allylnormetazocine (SKF-0,047), but not its opioid agonistic-antagonistic properties. Neuropharmacology 23:983–987

  • Largent BL, Gundlach AL, Snyder SH (1986) Pharmacological and autoradiographic discrimination of sigma and phencyclidine receptor binding sites in brain with (+)-[3H]SKF 10,047, (+)-[3H]-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine and [3H]-1-[1-(2-thienyl)cyclohexyl]piperidine. J Pharmacol Exp Ther 238:739–748

  • Quirion R, Chicheportiche R, Contreras PC, Johnson KM, Lodge D, Tam SW, Woods JH, Zukin SR (1987) Classification and nomenclature of phencyclidine and sigma receptor sites. Trends Neurosci 10:444–446

  • Quirion R, Bowen WD, Itzhak Y, Junien JL, Musacchio JM, Rothman RB, Su TP, Tam SW, Taylor DP (1992) A proposal for the classification of sigma binding sites. Trends Pharmacol Sci 13:85–86

  • Sircar R, Nichtenhauser R, Ieni JR, Zukin SR (1986) Characterisation and autoradiographic visualisation of (+)-[3H]SKF 10,047 binding in rat and mouse brain: further evidence for phencyclidine I “sigma opiate” receptor commonalty. J Pharmacol Exp Ther 237:681–688

  • Su TP (1982) Evidence for sigma opioid receptor: binding of [3H]-SKF 10047 to etorphine-inaccessible sites in guinea pig brain. J Pharmacol Exp Ther 223:284–290

  • Tam SW, Cook L (1984) σ-opiates and certain antipsychotic drugs mutually inhibit (+)-[3H]-SKF 10,047 and [3H]haloperidol binding in guinea pig membranes. Proc Natl Acad Sci U S A 81:5618–5621

  • Taylor DP, Dekleva J (1987) Potential antipsychotic BMY 14802 selectively binds to sigma sites. Drug Dev Res 11:65–70

  • Vaupel DB (1983) Naltrexone fails to antagonize the σ effects of PCP and SKF 10.047 in the dog. Eur J Pharmacol 92:269–274

  • Walker JM, Bowen WD, Walker FO, Matsumoto RR, de Costa B, Rice KC (1990) Sigma receptors: biology and function. Pharmacol Rev 42:355–402

  • Weber E, Sonders M, Quarum M, McLean S, Pou S, Keana JFW (1986) 1,3-Di(2[5–3H]tolyl)guanidine: a selective ligand that labels σ-type receptors. Proc Natl Acad Sci 83:8784–8788

  • Zukin SR, Tempel A, Gardner EL, Zukin RS (1986) Interaction for psychotomimetic opiates and antipsychotic drugs. Proc Natl Acad Sci 83:8784–8788

Simultaneous Determination of Norepinephrine, Dopamine, DOPAC, HVA, HIAA, and 5-HT from Rat Brain Areas

Purpose and Rationale

To measure the effects of potential antipsychotic drugs on catecholamines and indols, a quantitative method for the determination of norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindolacetic acid (5HIAA), and 5-hydroxytryptamine (5-HT) from rat brain regions is used. These catecholamines and indols are measured in rat brain prefrontal cortex, nucleus accumbens, and striatum.

Procedure

Reagents
  1. 1.

    0.1 M HCl.

  2. 2.

    1 N NaOH.

  3. 3.

    2 mM solutions of DOPAC, DA, and DOPA in 0.1 M HCl;

    0.5 ml aliquots are stored at −60 °C until use.

  4. 4.

    Preparation of 2° standard mixture

    • 10 μM solution of NE, DOPAC, DA, HVA, 5HIAA, and 5-HT (diluted from reagent 3) in mobile phase (reagent 5).

    • The 2° standard solution is used for the preparation of standard curves.

  5. 5.

    Mobile phase/MeOH: buffer (7.5:92.5, v/v).

    • Buffer: 0.07 M sodium acetate, 0.04 M citric acid, 130 μM EDTA, and 230 μM sodium octane sulfonate

    • Mobile phase: methanol/buffer (150 ml + 1850 ml) is filtered through a 0.2 pm nylon 66 filter.

HΜLC-Instrumentation

  • Pump, model SP8810 (Spectra Physics).

  • Injector, WISP 710B (Waters Associates).

  • Detector, 5100A electrochemical with a 5011 analytical cell and 5020 guard cell (ESA).

  • Integrator, D-2000 (Hitachi), used as a backup for the data collection/integrator, CS 9000 (IBM) system.

  • Analytical column: C18-ODS Hypersil, 3 pm, 100 × 4.6 mm (Shandon).

Animal Treatment

Six rats per group (150–250 g) are dosed with 4–5 different concentrations of the putative antipsychotic drug; usual concentrations range from 0.03 to 30 mg/kg. At a predetermined time, usually 60 min, the rats are sacrificed.

Tissue Preparation

Following treatment with test drug, rats are sacrificed by decapitation. The brain is rapidly removed and placed on ice. The striatum, nucleus accumbens, and prefrontal cortex are dissected and placed in 1.5 ml microcentrifuge tubes. The tubes are capped and immediately placed in dry ice. The frozen brain sections are stored at −60 °C until HPLC analysis.

Tissue is homogenized in mobile phase (striatum, in 600 μl, nucleus accumbens and prefrontal cortex, in 300 μl). The homogenates are centrifuged for 6 min using a microcentrifuge (model 5413, Eppendorf). The supernatants are transferred to 0.2 pm microfilterfuge™ tubes and centrifuged for 6–8 min as before. The filtrate is transferred to WISP vials. Standards are included every 12–15 samples.

The following volumes are injected to the HPLC column:

  • Striatum, 5 μl; nucleus accumbens, 20 μl; prefrontal cortex, 50 μl.

  • HPLC flow rate is 1.0 mi/min; run time is 25 min.

    Helium flow is constant in mobile phase.

For protein analysis, 1 N NaOH is added to the tissue pellets as follows:

  • Striatum: 1.0 ml

  • Nucleus accumbens and prefrontal cortex: 0.5 ml

The next day, the protein analysis is run in duplicate with 5 μl of striatum and 20 μl of nucleus accumbens and prefrontal cortex as described by Bradford (1976) using the BioRad Assay Kit.

Evaluation

Peak area is used for quantitation. The mg of protein and pmoles of NE, DOPAC, DA, HVA, 5HIAA, and 5-HT are calculated from linear regression analysis using the corresponding standard curve. Final data are reported as pmoles/mg protein.

References and Further Reading

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  • Magnusson O, Nilsson LB, Westerlund D (1980) Simultaneous determination of dopamine, DOPAC and homovanillic acid. Direct injections of supernatants from brain tissue homogenates in a liquid chromatography-electrochemical detection system. J Chromatogr 221:237–247

  • Magnusson O, Fowler CJ, Köhler C, Ögren SO (1986) Dopamine D2 receptors and dopamine metabolism. Relationship between biochemical and behavioural effects of substituted benzamide drugs. Neuropharmacology 25:187–197

  • Raiteri M, Marchi M, Maura G (1984) Release of catecholamines, serotonin, and acetylcholine from isolated brain tissue. In: Lajtha A (ed) Handbook of neurochemistry, vol 6, 2nd edn. Plenum Press, New York/London, pp 431–462

  • Reinhard JF, Perry JA (1984) Fast analysis of tissue catechols using a short, high-efficiency (3 μM ) LC column and amperometric detection. J Liq Chromatogr 7:1211–1220

  • Shibuya T, Sato K, Salafsky B (1982) Simultaneous measurement of biogenic amines and related compounds by high performance liquid chromatography. Int J Clin Pharmacol Toxicol 20:297–301

  • Wagner J, Palfreyman M, Zraika M (1979) Determination of DOPA, dopamine, DOPAC, epinephrine, norepinephrine, α-monofluoromethyldopa and α-difluoromethyldopa in various tissues of mice and rats using reversed-phase ionpair liquid chromatography with electrochemical detection. J Chromatogr 164:41–54

  • Wagner J, Vitali P, Palfreyman MG, Zraika M, Hout S (1982) Simultaneous determination of 3,4-dihydroxyphenylalanine, 5-hydroxytryptophan, dopamine, 4-hydroxy-3-methoxyphenylalanine, norepinephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5hydroxyindolacetic acid in rat cerebrospinal fluid and brain by high-performance liquid chromatography with electrochemical detection. J Neurochem 38:1241–1254

Measurement of Neurotransmitters by Intracranial Microdialysis

Purpose and Rationale

Methods to measure neurotransmitters and their metabolites in specific areas of the brain by microdialysis were introduced by Ungerstedt and his group (Ungerstedt et al. 1982; Zetterström et al. 1982, 1983; Zetterström and Ungerstedt 1983; Ungerstedt 1984; Stähle et al. 1991; Lindefors et al. 1989; Amberg and Lindefors (1989) and by Imperato and di Chiara 1984, 1985). In brain dialysis, a fine capillary fiber is implanted in a selected brain area. Low molecular weight compounds diffuse down their concentration gradients from the brain extracellular fluid into a physiological salt solution that flows through the capillary fiber at a constant rate. The fluid is collected and analyzed.

Procedure

Several designs of dialysis probes have been used (Santiago and Westerink 1990; Kendrick 1991):

  1. 1.

    Horizontal Probe

    A straight tube (Vita Fiber, 3 × 50 Amicon) with an outer diameter of 0.34 mm and a molecular weight cutoff of 50,000 is used. The outer surface of the tube is porous and can easily be sealed by epoxy which is applied by passing the tube through a droplet of epoxy and then through a narrow hole corresponding to the outer diameter of the tube. The wall of the tube is sealed in this way except for the area where the dialysis is intended to take place. The length of this region can be varied from 2 to 8 mm depending upon which structure of the brain will be perfused. During the coating and all other handling of the tube, it is supported by a thin tungsten or steel wire inserted into its lumen. One end of the tube is glued into a steel cannula (6 mm long, outer diameter 0.64 mm).

    Male Sprague Dawley rats weighing 250–300 g are anesthetized with halothane and held in a stereotactic instrument. The animals are maintained under halothane anesthesia during the entire experiment.

    Holes are drilled bilaterally (5.7 mm below and 1.5 mm in front of bregma) in the temporal bones after the temporal muscles have been retracted from the bones and folded away.

    During the implantation, the cannula is held by the micromanipulator of the stereotactic instrument, and the dialysis tube is passed horizontally through the brain through the holes drilled on both sides of the skull. A polyethylene tubing carrying the perfusion fluid is connected to the steel cannula. The perfusate is collected at the other end.

  2. 2.

    Loop Probe

    The probe is made of a flexible cellulosic tubing (Dow 50, outer diameter 0.25 mm). Both ends of the tube are inserted into 0.64 mm diameter steel tubes, one of which is bent in an angle. A very thin microsuture (0.1 mm in diameter) is inserted into the tube and positioned half between the steel tubes. Before implantation, the tube is moistened and bent in such a way that the two steel tubes are held closely together in the micromanipulator of the stereotactic instrument. A tungsten wire is inserted into the straight steel tube and passed down the lumen of the dialysis tube in order to stretch it and make it rigid enough to be implanted into the brain. The tube is implanted vertically, and the steel cannulae are attached to the skull by dental cement. The tungsten wire is removed before starting the experiment. The cellulosic tube is flexible enough to withstand the bending at the lower end. The microsuture keeps the bend open.

    Loop-shaped or U-shaped microdialysis probes have been used by several authors, e.g., Ichikawa and Meltzer (1990), Jordan et al. (1994), Westerink and Tuinte (1985), and Auerbach et al. (1994).

  3. 3.

    Vertical Probe

    The probe is sealed at one end by epoxy. The other end is glued into a 0.64-mm-diameter steel tube. A thin inner cannula made of a steel tube or a glass capillary carries the fluid to the bottom of the dialysis tube where it leaves the inner capillary and flows upwards and leaves the probe by a lateral tube. This vertical probe can also be coated with epoxy. It is especially suited for reaching ventral parts of the brain and performing dialysis in small nuclei of the brain.

    A similar device has been described for continuous plasma sampling in freely moving rats by Chen and Steger (1993).

    Most of the commercially available microdialysis probes are based on this principle.

  4. 4.

    Commercially Available Microdialysis Probes

    The microdialysis probes CMA/10, manufactured by Carnegie Medicine, Stockholm, Sweden, consist of a tubular membrane (polycarbonate; length: 3 mm; outside diameter: 0.50 mm; and inside diameter: 0.44 mm) glued to a cannula (outside diameter, 0.60 mm) and sealed with a glue at the tip (Stähle et al. 1991). The perfusion medium is carried to the dialyzing part of the probe by a thin cannula inside the probe. The medium leaves the inner cannula through two holes, flows back between the membrane and the inner cannula, and is collected at the outlet of the probe. The perfusion medium is delivered by means of a high precision microsyringe pump.

    This probe was used by several authors, e.g., Wood et al. (1988), Benveniste et al. (1989), Rollema et al. (1989), Scheller and Kolb (1991), Wang et al. (1993), Kreiss and Lucki (1995), and Fink-Jensen et al. (1996).

    CMA/11 probes were used by Boschi et al. (1995), Romero et al. (1996), and Gobert et al. (1997).

    Dialysis fibers with a semipermeable membrane AN 69-HF, Hospal-Dasco, Bologna, Italy, were used by de Boer et al. (1994), Rayevsky et al. (1995), Arborelius et al. (1996), Gainetdinov et al. (1996), and Tanda et al. (1996).

Evaluation

Samples of the dialyzate are collected for different time intervals and analyzed for neurotransmitters. For the evaluation of neuroleptics, most authors measured dopamine, 3,4-dihydroxyphenyl acetic acid (DOPAC), and homovanillinic acid (HVA) by HPLC using appropriate detectors. See and Lynch (1996) analyzed dialysis samples for glutamate and GABA concentrations.

For the evaluation of antidepressants, the concentrations of 5-hydroxytryptamine (5-HT), 5-hydroxy indole acetic acid (5-HIAA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), or noradrenaline (NA) were measured in the effluent by HPLC. Wood et al. (1988) and Egan et al. (1996) used 3-methoxytyramine accumulation as an index of dopamine release.

Critical Assessment of the Method

The results obtained from brain dialysis depend on at least three variables: type of probe, post-implantation interval, and whether anesthetized or freely moving animals are used (Di Chiara 1990).

Several authors analyzed the diffusion processes underlying the microdialysis technique and described the limitations of the experiments (Jacobson et al. 1985; Amberg and Lindefors 1989; Benveniste et al. 1989; Scheller and Kolb 1991; Le Quellec et al. 1995).

As a matter of fact, brain microdialysis has been used for the evaluation of many drugs in various indications, such as:

  • For neuroleptics by Ichikawa and Meltzer (1990), Meil and See (1994), Hernandez and Hoebei (1994), See et al. (1995), Schmidt and Fadayel (1995), Semba et al. (1995), Rayevsky et al. (1995), Fink-Jensen et al. (1996), See and Lynch (1996), Gainetdinov et al. (1996), Egan et al. (1996), and Klitenick et al. (1996)

  • For antidepressants by de Boer et al. (1994), Jordan et al. (1994), Arborelius et al. (1996), Ascher et al. (1995), Auerbach et al. (1994), de Boer (1995, 1996), Casanovas and Artigas (1996), Gobert et al. (1997), Ichikawa and Meltzer (1995), Kreiss and Lucki (1995), Petty et al. (1996), Potter (1996), Romero et al. (1996), Sharp et al. (1996), and Tanda et al. (1996a, b)

  • For studies in Parkinson models by Rollema et al. (1989) and Parsons et al. (1991)

Modifications of the Method

Ferraro et al. (1990) continuously monitored ethanol levels in the brain by microdialysis.

Hernandez and Hoebei (1994) performed simultaneous cortical, accumbens, and striatal microdialysis in freely moving rats.

Hegarty and Vogel (1995) assayed dopamine, DOPAC, and HVA in the brain of rats after acute and chronic diazepam treatment and immobilization stress.

Casanovas and Artigas (1996) implanted microdialysis probes simultaneously in six different brain areas of rats (frontal cortex, dorsal striatum, ventral hippocampus, dorsal hippocampus, dorsal raphe nucleus, median raphe nucleus).

Beneviste et al. (1984) determined extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis.

Boschi et al. (1995) showed that microdialysis of small brain areas in mice is feasible using the smallest commercially available probes.

References and Further Reading

  • Amberg G, Lindefors N (1989) Intracerebral microdialysis: II. Mathematical studies of diffusion kinetics. J Pharmacol Methods 22:157–183

  • Arborelius L, Nomikus GG, Hertel P, Salmi P, Grillner P, Hök BB, Hacksell U, Svensson TH (1996) The 5-HT1A receptor antagonist (S)-UH-301 augments the increase in extracellular concentrations of 5-HT in the frontal cortex produced by both acute and chronic treatment with citalopram. Naunyn Schmiedebergs Arch Pharmacol 353:630–640

  • Ascher JA, Cole JO, Colin JN, Feighner JP, Ferris RM, Fibiger HC, Golden RN, Martin P, Zotter WZ, Richelson E, Sulser F (1995) Bupropion: a review of its mechanism of antidepressant activity. J Clin Psychiatry 56:395–401

  • Ashby CR, Wang RY (1996) Pharmacological actions of the atypical antipsychotic drug clozapine: a review. Synapse 24:349–394

  • Auerbach SB, Lundberg JF, Hjorth S (1994) Differential inhibition of serotonin release by 5-HT and NA reuptake blockers after systemic administration. Neuropharmacology 34:89–96

  • Beneviste H, Drejer J, Schousboe A, Diemer NH (1984) Elevation of extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem 43:1369–1374

  • Benveniste H, Hansen AJ, Ottosen NS (1989) Determination of brain interstitial concentrations by microdialysis. J Neurochem 52:1741–1750

  • Böschi G, Launay N, Rips R, Scherrmann JM (1995) Brain microdialysis in the mouse. J Pharmacol Toxicol Methods 33:29–33

  • Casanovas JM, Artigas F (1996) Differential effects of ipsapirone on 5-hydroxytryptamine release in the dorsal and median raphe neuronal pathways. J Neurochem 67:1945–1952

  • Chen Z, Steger RW (1993) Plasma microdialysis. A technique for continuous plasma sampling in freely moving rats. J Pharmacol Toxicol Methods 29:111–118

  • Chiara D (1990) In vivo brain dialysis of neurotransmitters. Trends Pharmacol Sci 11:116–121

  • De Boer T (1995) The effects of mirtazapine on central noradrenergic and serotonergic neurotransmission. Int Clin Psychopharmacol 10(Suppl 4):19–23

  • De Boer T (1996) The pharmacological profile of mirtazapine. J Clin Psychiatry 57(Suppl 4):19–25

  • De Boer T, Nefkens F, van Helvoirt A (1994) The α-2adrenenoceptor antagonist Org 3770 enhances serotonin transmission in vivo. Eur J Pharmacol 253:R5–R6

  • Egan MF, Chrapusta S, Karoum F, Lipska BK, Wyatt RJ (1996) Effects of chronic neuroleptic treatment on dopamine release: insights from studies using 3-methoxytyramine. J Neural Transm 103:777–805

  • Ferraro TN, Weyers P, Carrozza DP, Vogel WH (1990) Continuous monitoring of brain ethanol levels be intracerebral microdialysis. Alcohol 7:129–132

  • Fink-Jensen A, Hansen L, Hansen JB, Nielsen EB (1996) Regional differences in the effect of haloperidol and atypical neuroleptics on interstitial levels of DOPAC in the rat forebrain: an in vivo microdialysis study. J Psychopharmacol 10:119–125

  • Gainetdinov RR, Sotnikova TD, Grekhova TV, Rayevsky KS (1996) Simultaneous monitoring of dopamine, its metabolites and trans-isomer of atypical neuroleptic drug carbidine concentrations in striatal dialysates of conscious rats. Prog Neuropharmacol Biol Psychiatry 20:291–305

  • Gobert A, Rivet JM, Cistarelli L, Millan MJ (1997) Potentiation of fluoxetine-induced increase in dialysate levels of serotonin (5-HT) in the frontal cortex of freely moving rats by combined blockade of 5-HT1A and 5-HT1B receptors with WAY 100,635 and GR 127,935. J Neurochem 68:1159–1163

  • Hegarty AA, Vogel WH (1995) The effect of acute and chronic diazepam treatment on stress-induced changes in cortical dopamine in the rat. Pharmacol Biochem Behav 52:771–778

  • Hernandez L, Hoebei BG (1994) Chronic clozapine selectively decreases prefrontal cortex dopamine as shown by simultaneous cortical, accumbens, and striatal microdialysis in freely moving rats. Pharmacol Biochem Behav 52:581–589

  • Ichikawa J, Meltzer HY (1990) The effect of chronic clozapine and haloperidol on basal dopamine release and metabolism in rat striatum and nucleus accumbens studied by in vivo microdialysis. Eur J Pharmacol 176:371–374

  • Ichikawa J, Meltzer HY (1995) Effect of antidepressants on striatal and accumbens extracellular dopamine levels. Eur J Pharmacol 281:255–261

  • Imperato A, di Chiara G (1984) Trans-striatal dialysis coupled to reverse phase high performance liquid chromatography with electrochemical detection: a new method for the study of the in vivo release of endogenous dopamine and metabolites. J Neurosci 4:966–977

  • Imperato A, di Chiara G (1985) Dopamine release and metabolism in awake rats after systemic neuroleptics studied by trans-striatal dialysis. J Neurosci 5:297–306

  • Imperato A, Tanda G, Frau R, di Chiara G (1988) Pharmacological profile of dopamine receptor agonists studied by brain dialysis in behaving rats. J Pharmacol Exp Ther 245:257–264

  • Jacobson I, Sandberg M, Hamberger A (1985) Mass transfer in brain dialysis devices a new method for the estimation of extracellular amino acids concentration. J Neurosci Methods 15:263–268

  • Jordan S, Kramer GL, Zukas PK, Moeller M, Petty F (1994) In vivo biogenic amine efflux in medial prefrontal cortex with imipramine, fluoxetine, and fluvoxamine. Synapse 18:294–297

  • Kendrick KM (1991) In vivo measurement of amino acid, monoamine and neuropeptide release using microdialysis, Chapter 12. In: Greenstein B (ed) Neuroendocrine research methods, vol 1. Harwood Academic Publishers, Chur, pp 249–278

  • Klitenick MA, Taber MT, Fibiger HC (1996) Effects of chronic haloperidol on stress- and stimulation-induced increases in dopamine release: tests of the depolarization block hypothesis. Neuropsychopharmacology 15:424–428

  • Kreiss DS, Lucki I (1995) Effects of acute and repeated administration of antidepressant drugs on extracellular level of 5-hydroxytryptamine measured in vivo. J Pharmacol Exp Ther 274:866–876

  • Le Quellec A, Dupin S, Genissel P, Saivin S, Marchand B, Houin G (1995) Microdialysis probes calibration: gradient and tissue dependent changes in no net flux and reverse dialysis methods. J Pharmacol Toxicol Methods 33:11–16

  • Lindefors N, Amberg G, Ungerstedt U (1989) Intracerebral microdialysis: I. Experimental studies of diffusion kinetics. J Pharmacol Methods 22:141–156

  • Meil W, See RE (1994) Single pre-exposure to fluphenazine produces persisting behavioral sensitization accompanied by tolerance to fluphenazine-induced dopamine overflow in rats. Pharmacol Biochem Behav 48:605–612

  • Parsons LH, Smith AD, Justice JB Jr (1991) The in vivo microdialysis recovery of dopamine is altered independently of basal level by 6-hydroxydopamine lesions to the nucleus accumbens. J Neurosci Methods 40:139–147

  • Petty F, Davis LL, Kabel D, Kramer GL (1996) Serotonin dysfunction disorders: a behavioral neurochemistry prospective. J Clin Psychiatry 57(Suppl 8):11–16

  • Potter WZ (1996) Adrenoreceptor and serotonin receptor function: relevance to antidepressant mechanisms of action. J Clin Psychiatry 57(Suppl 4):4–8

  • Rayevsky KS, Gainetdinov RR, Grekhova TV, Sotnikova TD (1995) Regulation of dopamine release and metabolism in rat striatum in vivo: effects of dopamine receptor antagonists. Prog Neuro-Psychopharmacol Biol Psychiatry 19:1285–1303

  • Rollema H, Alexander GM, Grothusen JR, Matos FF, Castagnoli N Jr (1989) Comparison of the effects of intracerebrally administered MPP+ (1-methyl-4-phenylpyridinium) in three species: microdialysis of dopamine and metabolites in mouse, rat and monkey striatum. Neurosci Lett 106:275–281

  • Romero L, Hervás I, Artigas F (1996) The 5-HT1A antagonist WAY-100635 selectively potentiates the effects of serotonergic antidepressants in rat brain. Neurosci Lett 219:123–126

  • Sandberg M, Butcher S, Hagberg H (1986) Extracellular overflow of neuroactive amino acids during severe insulin-induced hypoglycemia: in vivo dialysis of the rat hippocampus. J Neurochem 47:178–184

  • Santiago M, Westerink BHC (1990) Characterization of the in vivo release of dopamine as recorded by different types of intracerebral microdialysis probes. Naunyn Schmiedebergs Arch Pharmacol 342:407

  • Scheller D, Kolb J (1991) The internal reference technique in microdialysis: a practical approach to monitoring dialysis efficiency and to calculating tissue concentrations from dialysate samples. J Neurosci Methods 40:31–38

  • Schmidt CJ, Fadayel GM (1995) The selective 5-HT2A receptor antagonist, MDL 100,907, increases dopamine efflux in the prefrontal cortex of the rat. Eur J Pharmacol 273:273–279

  • See RE, Lynch AM (1996) Duration-dependent increase in striatal glutamate following prolonged fluphenazine administration in rats. Eur J Pharmacol 308:279–282

  • See RE, Lynch AM, Aravagri M, Nemeroff CB, Owens MJ (1995) Chronic haloperidol-induced changes in regional dopamine release and metabolism and neurotensin content in rats. Brain Res 704:202–209

  • Semba J, Watanabe A, Kito S, Toru M (1995) Behavioural and neurochemical effects of OPC-14597, a novel antipsychotic drug, on dopamine mechanisms in rat brain. Neuropharmacology 34:785–791

  • Sharp T, Gartside SE, Umbers V (1996) Effects of co-administration of a monoamine oxidase inhibitor and a 5-HT1A receptor antagonist on 5-hydroxytryptamine cell firing and release. Eur J Pharmacol 320:15–19

  • Stähle L, Segersvärd S, Ungerstedt U (1991) A comparison between three methods for estimation of extracellular concentration of exogenous and endogenous compounds by microdialysis. J Pharmacol Methods 25:41–52

  • Tanda G, Bassareo V, di Chiara G (1996a) Mianserin markedly and selectively increases extracellular dopamine in the prefrontal cortex as compared to the nucleus accumbens in the rat. Psychopharmacology (Berl) 123:127–130

  • Tanda G, Frau R, di Chiara G (1996b) Chronic desipramine and fluoxetine differentially affect extracellular dopamine in the rat prefrontal cortex. Psychopharmacology (Berl) 127:83–87

  • Ungerstedt U (1984) Measurement of neurotransmitter release by intracranial dialysis. In: Marsden CA (ed) Measurement of neurotransmitter release in vivo. Wiley, New York, pp 81–105

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  • Westerink BHC, Tuinte MHJ (1985) Chronic use of intracerebral dialysis for the in vivo measurement of 3,4-dihydroxyphenylethylamine and its metabolite 3,4dihydroxyphenylacetic acid. J Neurochem 46:181–185

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  • Zetterström T, Vernet L, Ungerstedt U, Tossman U, Jonzon B, Fredholm BB (1982) Purine levels in the intact rat brain. Studies with an implanted perfused hollow fibre. Neurosci Lett 29:111–115

  • Zetterström T, Sharp T, Marsden CA, Ungerstedt U (1983) In vivo measurement of dopamine and its metabolites by intracerebral dialysis: changes after d-amphetamine. J Neurochem 41:1769–1773

Use of Push–Pull Cannulae to Determine the Release of Endogenous Neurotransmitters

Purpose and Rationale

Originally reported by Gaddum (1961), the push–pull cannula has become recognized and utilized as a powerful tool in conjunction with sufficiently sensitive assays to measure low levels of neuroregulator release in distinct brain areas in vivo (Philippu 1984).

This method has been used for various purposes, e.g.:

  • To perfuse the ventricles of the brain with drugs or to determine the release of labeled or endogenous compounds in the CSF (Bhattacharya and Feldberg 1958; Korf et al. 1976)

  • To perfuse distinct brain areas with drugs and to study their effects on functions of the central nervous system (Myers et al. 1976; Bhargava et al. 1978; Ruwe and Myers 1978)

  • To inject labeled monoamines or amino acids and to investigate the resting or induced release of radioactive compounds and their metabolites (Sulser et al. 1969; Strada and Sulser 1971; Kondo and Iwatsubo 1978)

  • To perfuse distinct brain areas with labeled transmitter precursors and to determine the patterns of release of the newly synthesized transmitters (Philippu et al. 1974; Chéramy et al. 1977; Nieoullon et al. 1977; Gauchy et al. 1980)

  • To perfuse distinct brain areas of anesthetized and conscious animals and to determine the release of endogenous neurotransmitters in the perfusate (Dluzen and Ramirez 1991)

Procedure

The superfusion of the hypothalamus of the conscious, freely moving rabbit has been described by Philippu et al. (1981) and Philippu (1984). Rabbits of both sexes are anesthetized with 40 mg/kg sodium pentobarbital i.p. Guide cannulae are mounted on a metal plate which is fixed on the skull with screws and dental cement. Some days after the operation, the guide cannulae are replaced with push–pull cannulae which are 4 mm longer than the guide cannulae, thus reaching the areas which are intended for superfusion. The push–pull cannulae are connected by tubing to two peristaltic pumps: one to push and another one to pull the fluid. The second pump is essential, because the superfusate is not directly collected from the side branch of the push–pull cannula but from tubing which is connected to the side branch. The superfusate is automatically collected every 10 s in fraction collectors.

Evaluation

The concentrations of neurotransmitters, e.g., epinephrine, norepinephrine, or dopamine, are determined with appropriate analytical methods (Wolfensberger 1984) before and after stimulation.

Modifications of the Method

Experiments in cats were described by Dietl et al. (1981) and in rats by Tuomisto et al. (1983).

The cortical cup technique for collection of neurotransmitters has been described by Moroni and Pepeu (1984).

References and Further Reading

  • Bhargava KP, Jain IP, Saxena AK, Sinha NJ, Tangri KK (1978) Central adrenoceptors and cholinoceptors in cardiovascular control. Br J Pharmacol 63:7–15

  • Bhattacharya BK, Feldberg W (1958) Perfusion of cerebral ventricles: effects of drugs on outflow from the cisterna and the aqueduct. Br J Pharmacol 13:156–162

  • Chéramy A, Nieoullon A, Glowinski J (1977) Effects of peripheral and local administration of picrotoxin on the release of newly synthesized 3H-dopamine in the caudate nucleus of the cat. Naunyn Schmiedebergs Arch Pharmacol 297:31–37

  • Dietl H, Sinha JN, Philippu A (1981) Presynaptic regulation of the release of catecholamines in the cat hypothalamus. Brain Res 208:143–147

  • Dluzen DE, Ramitez VD (1991) Push-pull cannula construction, application and considerations for use in neuroendocrinology, Chapter 8. In: Greenstein B (ed) Neuroendocrine research methods, vol 1. Harwood Academic Publishers, Chur, pp 163–186

  • Gauchy C, Kemel ML, Glowinski J, Besson JM (1980) In vivo release of endogenously synthesized (3H)GABA from the cat substantia nigra and the pallidoendopeduncular nuclei. Brain Res 193:129–141

  • Kondo A, Iwatsubo K (1978) Increased release of preloaded (3H)GABA from substantia nigra in vivo following stimulation of caudate nucleus and globus pallidus. Brain Res 154:305–400

  • Korf J, Boer PH, Fekkes D (1976) Release of cyclic AMP into push-pull perfusates in freely moving rats. Brain Res 113:551–561

  • Moroni F, Pepeu G (1984) The cortical cup technique. In: Marsden CA (ed) Measurements of neurotransmitter release in vivo. Wiley, Chichester/New York, pp 63–79

  • Myers RD, Simpson CW, Higgins D, Nattermann RA, Rice JC, Redgrave P, Metclaf G (1976) Hypothalamic Na+ and Ca2+ ions and temperature set-point: new mechanisms of action of a central or peripheral thermal challenge and intrahypothalamic 5-HT, NE, PGE1 and pyrogen. Brain Res Bull 1:301–327

  • Nieoullon A, Chéramy A, Glowinski J (1977) An adaptation of the push-pull cannula method to study the in vivo release of (3H)dopamine synthesized from (3H)tyrosine in the cat caudate nucleus: effects of various physical and pharmacological treatments. J Neurochem 28:819–828

  • Philippu A (1984) Use of push-pull cannulae to determine the release of endogenous transmitters in distinct brain areas of anesthetized and freely moving animals. In: Marsden CA (ed) Measurements of neurotransmitter release in vivo. Wiley, Chichester/New York, pp 3–37

  • Philippu A, Glowinski J, Besson JM (1974) In vivo release of newly synthesized catecholamines from the hypothalamus by amphetamine. Naunyn Schmiedebergs Arch Pharmacol 282:1–8

  • Philippu A, Dietl H, Eisert A (1981) Hypotension alters the release of catecholamines in the hypothalamus of the conscious rabbit. Eur J Pharmacol 69:519–523

  • Ruwe WD, Myers RD (1978) Dopamine in the hypothalamus of the cat: pharmacological characterization and push-pull perfusion analysis of sites mediating hypothermia. Pharmacol Biochem Behav 9:65–80

  • Strada SJ, Sulser F (1971) Comparative effects of p-chloroamphetamine and amphetamine on metabolism and in vivo release of 3H-norepinephrine in the hypothalamus of the rat in vivo. Eur J Pharmacol 15:45–51

  • Sulser F, Owens ML, Strada SJ, Dingell NJ (1969) Modification by desipramine (DMI) of the availability of epinephrine released by reserpine in the hypothalamus of the rat in vivo. J Pharmacol Exp Ther 168:272–282

  • Tuomisto L, Yamatodani A, Dietl H, Waldmann U, Philippu A (1983) In vivo release of endogenous catecholamines, histamine and GABA in the hypothalamus of Wistar Kyoto and spontaneously hypertensive rats. Naunyn Schmiedebergs Arch Pharm 323:183

  • Wolfensberger M (1984) Gaschromatographic and mass-fragmentographic measurement of amino acids released into brain perfusates collected in vivo by push–pull cannula techniques. In: Marsden CA (ed) Measurements of neurotransmitter release in vivo. Wiley, Chichester/New York, pp 39–61

Fos Protein Expression in Brain

Purpose and Rationale

The proto-oncogene c-fos encodes a 55,000 mol wt, 380 amino acid phosphoprotein (FOS), which after translation in the cytoplasm reenters the nucleus and binds to DNA (Morgan and Curran 1989). C-fos induction can occur as a consequence of synaptic activation. An increase in fos immunoreactivity is associated with an increased metabolic demand on a neuron, i.e., a marker for neurons that is metabolically activated. Intermediate early genes such as c-fos have been tentatively classified or linked to third messengers, whose function is to produce a long-term effect on the recipient neuron.

Acute administration of antipsychotics induces c-fos expression in several areas of the rat forebrain as was shown with immunocytochemical methods (Dragunow et al. 1990; Nguyen et al. 1992; Robertson and Fibiger 1992; MacGibbon et al. 1994). Fos protein is believed to act as an initiator of long-term cellular changes (neural plasticity) in response to a variety of extracellular stimuli, including drugs (Graybiel et al. 1990; Rogue and Vicendon 1992). Typical (e.g., haloperidol) and atypical (e.g., clozapine) neuroleptic drugs have different antipsychotic effects and side effects. A differential FOS-protein induction in rat forebrain regions after haloperidol and clozapine treatment was found (Deutch et al. 1992; Fibiger 1994; Fink-Jensen and Kristensen 1994; Merchant et al. 1994; Sebens et al. 1995). The induction pattern of Fos-like immunoreactivity in the forebrain could serve as predictor of atypical antipsychotic drug activity (Robertson et al. 1994).

Procedure

Groups of 4–6 male Wistar rats weighing 350–450 g are injected subcutaneously with saline (control) or with various doses of the standard drugs or compounds with putative antipsychotic activity. After 2 h, the animals are deeply anesthetized by intraperitoneal injection of 100 mg/kg pentobarbital and perfused with 200 ml saline followed by 200 ml of 4 % paraformaldehyde in phosphate buffer solution (PBS). Each brain is removed immediately after perfusion and placed in fresh fixative for at least 12 h.

After the post-fixative period, 30-pm sections are cut from each brain using a vibratome. Several antisera to detect Fos can be used, such as a sheep polyclonal antibody directed against residues 2–16 of the N-terminal region of the Fos molecule or a polyclonal antiserum raised in rabbits against Fos peptide (4–17 amino acids of human Fos).

Sections are washed three times with 0.02 mM PBS and then incubated in PBS containing 0.3 % hydrogen peroxide for 10 min to block endogenous peroxidase activity. Sections are then washed three times in PBS and incubated in PBS containing 0.3 % Triton X-100, 0.02 % azide, and Fos primary antisera (diluted 1:200) for 48 h. The sections are then washed three times with PBS and incubated with a biotinylated rabbit antisheep secondary antibody (diluted 1:200) for 1 h. The sections are washed three times with PBS and incubated for 1 h with PBS containing 0.3 % Triton X-100 and 0.5 % avidin-biotinylated horseradish peroxidase complex. After three washes in PBS, the sections are rinsed in 0.1 M acetate buffer, pH6.0. Fos immunoreactivity is revealed by placing the sections in a solution containing 0.05 % 3,3′-diaminobenzidine, 0.2 % ammonium nickel sulfate, and 0.01 % H2O2. The reaction is terminated with a washing in acetate buffer. The sections are mounted on chrome-alum-coated slides, dehydrated, and prepared for microscopic observation.

Drug-induced changes in Fos-like immunoreactivity are quantified by counting the number of immunoreactive nuclei in the medial prefrontal cortex, nucleus accumbens, medial and dorsolateral striatum, and the lateral septal nucleus. The number of Fos-positive nuclei is counted with a 550 × 550 pm grid placed over each of these regions with a 100 × magnification.

Typical and atypical antipsychotics can be classified on the basis of difference between Fos-like immunoreactivity in the nucleus accumbens and lateral striatum. For this purpose, the data are corrected for the effects which are produced by the injection procedure itself. The injection-corrected value for the dorsolateral striatum is subtracted from the corresponding accumbal value for each drug dose.

This manipulation yields a value termed the atypical index, i.e., number of Fos-positive neurons in the nucleus accumbens minus the number in the lateral striatum = atypical index. A negative index indicates the probability of side effects, like extrapyramidal syndrome, exerted by the typical neuroleptics, a positive value to be devoid of it.

Evaluation

A one-way analysis of variance is performed on the cell count data for each dose and the corresponding vehicle control. If the analysis of variance is significant, multiple comparisons are performed by using the Newman–Keuls test.

Modifications of the Method

Graybiel et al. (1990) reported a drug-specific activation of c-fos gene in striosome-matrix compartments and limbic subdivisions of the striatum by amphetamine and cocaine.

Deutch et al. (1991) found that stress selectively increases Fos protein in dopamine neurons innervating the prefrontal cortex.

Gogusev et al. (1993) described modulation of c-fos and other proto-oncogene expression by phorbol diester in a human histiocytosis DEL cell.

Deutch et al. (1995) studied the induction of Fos protein in the thalamic paraventricular nucleus as locus of antipsychotic drug action.

References and Further Reading

  • Ashby CR, Wang RY (1996) Pharmacological actions of the atypical antipsychotic drug clozapine: a review. Synapse 24:349–394

  • Deutch AY (1994) Identification of the neural systems subserving the actions of clozapine: clues from immediate early gene expression. J Clin Psychiatry 55(Suppl):37–42

  • Deutch AY, Lee MC, Gillham MH, Cameron DA, Goldstein M, Iadarola MJ (1991) Stress selectively increases Fos protein in dopamine neurons innervating the prefrontal cortex. Cereb Cortex 1:273–292

  • Deutch AY, Lee M, Iadarola MJ (1992) Regionally specific effects of atypical antipsychotic drugs on striatal fos expression: the nucleus accumbens shell as a locus of antipsychotic action. Mol Cell Neurosci 3:332–341

  • Deutch AY, Öngur D, Duman RS (1995) Antipsychotic drugs induce Fos protein in the thalamic paraventricular nucleus: a novel locus of antipsychotic drug action. Neuroscience 66:337–346

  • Dragunow M, Robertson GS, Faull RLM, Robertson HA, Jansen K (1990) D2 Dopamine receptor antagonists induce FOS and related proteins in rat striatal neurons. Neuroscience 37:287–294

  • Fibiger HC (1994) Neuroanatomical targets of neuroleptic drugs as revealed by Fos immunochemistry. J Clin Psychiatry 55(Suppl B):33–36

  • Fink-Jensen A, Kristensen P (1994) Effects of typical and atypical neuroleptics on Fos protein expression in the rat forebrain. Neurosci Lett 182:115–118

  • Gogusev J, Barbey S, Nezelof C (1993) Modulation of C-myc, C-myb, C-fos, C-sis and C-fms proto-oncogene expression and of CSF-1 transcripts and protein by phorbol diester in human histiocytosis DEL cell line with 5q 35 break point. Anticancer Res 13:1043–1048

  • Graybiel AM, Moratalla R, Robertson HA (1990) Amphetamine and cocaine induce drug-specific activation of the c-fos gene in striosome-matrix compartments and limbic subdivisions of the striatum. Proc Natl Acad Sci U S A 87:6912–6916

  • MacGibbon GA, Lawlor PA, Bravo R, Dragunow M (1994) Clozapine and haloperidol produce a different pattern of immediate early gene expression in rat caudate-putamen, nucleus accumbens, lateral septum and islets of Calleja. Mol Brain Res 23:21–32

  • Merchant KM, Drosa DM (1993) Differential induction of neurotensin and c-fos gene expression by typical versus atypical antipsychotic drugs. Proc Natl Acad Sci U S A 90:3447–3451

  • Merchant KM, Dobie DJ, Filloux FM, Totzke M, Aravagiri M, Dorsa DM (1994) Effects of chronic haloperidol and clozapine treatment on neurotensin and c-fos mRNA in rat neostriatal subregions. J Pharmacol Exp Ther 271:460–471

  • Morgan JI, Curran T (1989) Stimulus-transcription coupling in neurons: role of cellular immediate early genes. Trends Neurosci 12:459–462

  • Morgan JI, Curran T (1991) Stimulus-transcription coupling in the nervous system: involvement of the inducible protooncogens fos and jun. Annu Rev Neurosci 14:421–451

  • Nguyen TV, Kosofsky BE, Birnbaum R, Cohen BM, Heyman SE (1992) Differential expression of c-Fos and Zif628 in rat striatum after haloperidol, clozapine and amphetamine. Proc Natl Acad Sci U S A 89:4720–4724

  • Robertson GS, Fibiger HC (1992) Neuroleptics increase c-fos expression in the forebrain. Contrasting effects of haloperidol and clozapine. Neuroscience 46:315–328

  • Robertson GS, Matsumara H, Fibiger HC (1994) Induction pattern of fos-like immunoreactivity in the forebrain as predictors of atypical antipsychotic activity. J Pharmacol Exp Ther 271:1058–1066

  • Rogue P, Vincendon G (1992) Dopamine D2 receptor antagonists induce immediate early genes in the rat striatum. Brain Res Bull 29:469–472

  • Sebens JB, Koch T, Ter Horst GJ, Korf J (1995) Differential Fosprotein induction in rat forebrain regions after acute and long-term haloperidol and clozapine treatment. Eur J Pharmacol 273:175–182

Neurotensin

General Considerations on Neurotensin and Neurotensin Receptors

Neurotensin is a 13-amino acid peptide originally isolated from calf hypothalamus (Carraway and Leeman 1973). It is secreted by peripheral and neuronal tissues and produces numerous pharmacological effects in animals, suggesting analgesic (Coguerel et al. 1988; Clineschmidt and McGuffin 1977; Smith et al. 1997), wound healing (Brun et al. 2005), cardiovascular (Carraway and Leeman 1973; Schaeffer et al. 1998; Seagard et al. 2000), endocrine (Rostene and Alexander 1997), hypothermic (Bissette et al. 1976; Benmoussa et al. 1996; Tyler-McMahon et al. 2000), and antipsychotic (Nemeroff 1986; Sarhan et al. 1997; Feifel et al. 1999; Kinkead et al. 1999; Cusack et al. 2000) actions. Neurotensin is even considered to be an endogenous neuroleptic (Ervin and Nemeroff 1988; Gully et al. 1995). Radke et al. (1998) studied synthesis and efflux of neurotensin in different brain areas after acute and chronic administration of typical and atypical antipsychotic drugs.

Neurotensin affects gastrointestinal functions , such as stimulating the growth of various gastrointestinal tissues (Feurle et al. 1987), modulating pre- and postprandial intestinal motility (Pellissier et al. 1996), inhibiting gastric acid secretion (Zhang et al. 1989a), stimulating responses in rat stomach strips (Quirion et al. 1980), inducing contractile responses in intestinal smooth muscle (Unno et al. 1999), and maintaining gastric mucosal blood flow during cold water restraint (Zhang et al. 1989b; Xing et al. 1998).

Neurotensin acts as a growth factor on a variety of normal and cancer cells (Wang et al. 2000).

Like other neuropeptides, neurotensin is synthesized as part of a larger precursor which also contains neuromedin N, a six amino acid neurotensin-like peptide belonging to the gastrin-releasing peptide/bombesin family (see J.3.1.8).

Several peptidic and non-peptidic neurotensin agonists and antagonists have been synthesized and analyzed in pharmacological tests as potential drugs mainly in psychopharmacology (Gully et al. 1995, 1996, 1997; Azzi et al. 1996; Castagliuolo et al. 1996; Chapman and See 1996; Mule et al. 1996; Hong et al. 1997; Johnson et al. 1997; Sarhan et al. 1997; Betancur et al. 1998; Gudasheva et al. 1998; Schaeffer et al 1998; Kitabgi 2002). Furthermore, inhibitors of neurotensin-degrading enzymes were described (Bourdel et al. 1996). Binder et al. (2001) reviewed neurotensin and dopamine interactions.

References and Further Reading

  • Azzi M, Boudin H, Mahmudi N, Pelaprat D, Rostene W, Berod A (1996) In vivo regulation of neurotensin receptors following long-term pharmacological blockade with a specific receptor antagonist. Mol Brain Res 42:213–221

  • Benmoussa M, Chait A, Loric G, de Beaurepaire R (1996) Low doses of neurotensin in the preoptic area produce hypothermia. Comparison with other brain sites and with neurotensin-induced analgesia. Brain Res Bull 39:275–279

  • Betancur C, Canton M, Burgos A, Labeeuw B, Gully D, Rostene W, Pelaprat D (1998) Characterization of binding sites of a new neurotensin receptor antagonist, 3H-SR 142948A, in the rat brain. Eur J Pharmacol 343:67–77

  • Binder EB, Kinkead B, Owens MJ, Nemeroff CB (2001) Neurotensin and dopamine interactions. Pharmacol Rev 53:453–486

  • Bissette G, Nemeroff CB, Loosen PT, Prange AJ Jr, Lipton MA (1976) Hypothermia and cold intolerance induced by the intracisternal administration of the hypothalamic peptide neurotensin. Nature 262:607–609

  • Bourdel E, Doulut S, Jarretou G, Labbé-Juilié C, Fehrentz JA, Doumbia O, Kitabgi P, Martinez J (1996) New hydroxamate inhibitors of neurotensin-degrading enzymes: synthesis and enzyme active-site recognition. Int J Pept Protein Res 48:148–155

  • Brun P, Mastrotto C, Beggiao E, Stefani A, Barzon L, Sturniolo GC, Palù G, Castagliuolo I (2005) Neuropeptide neurotensin stimulates intestinal wound healing following chronic intestinal inflammation. Am J Physiol 288:G621–G629

  • Carraway R, Leeman SE (1973) The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J Biol Chem 248:6854–6861

  • Castagliuolo I, Leeman SE, Bartolak-Suki E, Nikulasson S, Quiu B, Carraway RE (1996) A neurotensin antagonist, SR 48692, inhibits colonic responses to immobilization stress in rats. Proc Natl Acad Sci U S A 93:12611–12615

  • Chapman MA, See RE (1996) The neurotensin receptor antagonist SR 48692 decreases extracellular striatal GABA in rats. Brain Res 729:124

  • Clineschmidt R, McGuffin JC (1977) Neurotensin administered intracisternally inhibits responsiveness of mice to noxious stimuli. Eur J Pharmacol 49:395–396

  • Coguerel A, Dubuc I, Kitabgi P, Costentin J (1988) Potentiation by thiorphan and bestatin of the naloxon-insensitive analgesic effects of neurotensin and neuromedin N. Neurochem Int 12:361–366

  • Cusack B, Boules M, Tyler BM, Fauq A, McCormick DJ, Richelson E (2000) Effects of a novel neurotensin peptide analog given extracranially on CNS behaviors mediated by apomorphine and haloperidol. Brain Res 856:48–54

  • Ervin GN, Nemeroff CB (1988) Interactions of neurotensin with dopamine-containing neurons in the central nervous system. Prog Neuropsychopharmacol Biol Psychiatry 12:S53–S69

  • Feifel D, Reza TL, Wustrow DJ, Davis MD (1999) Novel antipsychotic-like effects on prepulse inhibition of startle produced by a neurotensin agonist. J Pharmacol Exp Ther 288:710–713

  • Feurle GE, Muller B, Rix E (1987) Neurotensin induces hyperplasia of the pancreas and growth of the gastric antrum in rats. Gut 28(Suppl 1):19–23

  • Gudasheva TA, Voronina TA, Ostrovskaya RU, Zaitseva NI, Bondarenko NA, Briling VK (1998) Design of Nacylprolyltyrosine ‘tripeptoid’ analogues of neurotensin as potential atypical antipsychotic agents. J Med Chem 41:284–290

  • Gully D, Jeanjean F, Poncelet M, Steinberg R, Soubrié P, Le Fur G, Maffrand JP (1995) Neuropharmacological profile of non-peptide neurotensin antagonists. Fundam Clin Pharmacol 9:513–521

  • Gully D, Lespy L, Canton M, Rostene W, Kitabgi P, le Fur G, Maffrand JP (1996) Effect of the neurotensin receptor antagonist SR 48692 on rat blood pressure modulation by neurotensin. Life Sci 58:665–674

  • Gully G, Labeeuw B, Boigegrain R, Oury-Donat F, Bachy A, Poncelet M, Steinberg R, Suaud-Chagny MF, Santucci V, Vita N, Pecceu F, Labbé-Jullié C, Kitabgi B, Soubriè P (1997) Biochemical and pharmacological activities of SR 142948A, a new potent neurotensin receptor antagonist. J Pharmacol Exp Ther 280:802–812

  • Hong F, Cusack B, Fauq A, Richelson E (1997) Peptidic and non-peptidic neurotensin analogs. Curr Med Chem 4:421–434

  • Johnson SJ, Akunne HC, Heffener TG, Kesten SR, Pugsley TA, Wise LD, Wustrow DJ (1997) Novel small molecule neurotensin antagonists: 3-(1,5-diaryl-1,5-dioxopentan-3-yl) benzoic acids. Bioorg Med Chem Lett 7:561–566

  • Kinkead B, Binder EB, Nemeroff CB (1999) Does neurotensin mediate the effects of antipsychotic drugs? Biol Psychiatry 46:340–351

  • Kitabgi P (2002) Targeting neurotensin receptors with agonists and antagonists for therapeutic purposes. Curr Opin Drug Discov Devel 5:764–776

  • Mule F, Serio R, Postorino A, Vetri T, Bonvissuto F (1996) Antagonism by SR 48692 on mechanical responses to neurotensin in rat intestine. Br J Pharmacol 117:488–492

  • Nemeroff CB (1986) The interaction of neurotensin with dopaminergic pathways in the central nervous system: basic neurobiology and implications for the pathogenesis and treatment of schizophrenia. Psychoneuroendocrinology 11:15–37

  • Pellissier S, Eribon O, Chabert J, Gully D, Roche M (1996) Peripheral neurotensin participates in the modulation of pre and postprandial intestinal motility in rats. Neuropeptides 30:412–419

  • Quirion R, Regoli D, Rioux F, St-Pierre S (1980) The stimulatory effect of neurotensin and related peptides in rat stomach strips and guinea pig atria. Br J Pharmacol 68:83–91

  • Radke JM, Owens MJ, Ritchie JC, Nemeroff CB (1998) Atypical antipsychotic drugs selectively increase neurotensin efflux in dopamine terminal regions. Proc Natl Acad Sci U S A 95:11462–11464

  • Rostene W, Alexander MJ (1997) Neurotensin and neuroendocrine regulation. Front Neuroendocrinol 18:115–173

  • Saegard JL, Dean C, Hopp FA (2000) Neurochemical transmission of the baroreceptor input in the nucleus tractus solitarius. Brain Res Bull 51:111–118

  • Sarhan S, Hitchcock JM, Grauffel CA, Wettstein JG (1997) Comparative antipsychotic profiles of neurotensin and a related systematically active peptide agonist. Peptides 18:1223–1227

  • Schaeffer P, Laplace MC, Bernat A, Prabonaud V, Gully D, Lespy L, Herbert JM (1998) SR142948A is a potent antagonist of the cardiovascular effects of neurotensin. J Cardiovasc Pharmacol 31:545–550

  • Smith DJ, Hawranko AA, Monroe PJ, Gully D, Urban MO, Craig CR, Smith JP, Smith DI (1997) Dose-dependent pain facilitatory and -inhibitory actions of neurotensin are revealed by SR 48692, a nonpeptide neurotensin antagonist: influence on the antinociceptive effect of morphine. J Pharmacol Exp Ther 282:899–908

  • Tyler-McMahon BM, Steward JA, Farinas F, McCormick DJ, Richelson E (2000) Highly potent neurotensin analog that causes hypothermia and antinociception. Eur J Pharmacol 390:107–111

  • Unno T, Komori S, Ohashi H (1999) Characterization of neurotensin receptors in intestinal smooth muscle using a nonpeptide antagonist. Eur J Pharmacol 369:73–80

  • Vincent JP, Mazella J, Kitagbi P (1999) Neurotensin and neurotensin receptors

  • Wang L, Friess H, Zhu Z, Graber H, Zimmermann A, Korc M, Reubi JC, Buchler MW (2000) Neurotensin receptor-1 mRNA analysis in normal pancreas and pancreatic disease. Clin Cancer Res 6:566–571

  • Xing L, Karinch AM, Kauffman GL Jr (1998) Mesolimbic expression of neurotensin and neurotensin receptor during stress-induced gastric mucosal injury. Am J Physiol 274:R38–R45, Regul Integr Comp Physiol

  • Zhang L, Xing L, Demers L, Washington J, Kauffman GL Jr (1989a) Central neurotensin inhibits gastric acid secretion: an adrenergic mechanism in rats. Gastroenterology 97:1130–1134

  • Zhang L, Colony PC, Washington JH, Seaton JF, Kauffman GL Jr (1989b) Central neurotensin affects rat gastric integrity, prostaglandin E2, and blood flow. Am J Physiol 256:G226–G232, Gastrointest Liver Physiol 19

Neurotensin Receptor Binding

Purpose and Rationale

Neurotensin interacts with two cloned receptors that were originally differentiated on the basis of their affinity to the antihistaminic drug levocabastine (Schotte et al. 1986). The high sensitive, levocabastine-insensitive rat neurotensin receptor (NTR1) was cloned first (Tanaka et al. 1990) and shown to mediate a number of peripheral and central neurotensin responses, including the neuroleptic-like effects of the peptide (Labbé-Jullié et al. 1994). The human NTR1 has been cloned from the colonic adenocarcinoma cell line HT29 (Vita et al. 1993) and shown to consist of a 416 amino acid protein that shares 84 % homology with rat NTR1. A second human NTR1 receptor differing only in one amino acid has been cloned from substantia nigra by Watson et al. (1993).

The lower-affinity, levocabastine-sensitive neurotensin receptor (NTR2) was cloned by Chalon et al. (1996) and Mazella et al. (1996) and characterized by Yamada et al. (1998). Studies by Dubuc et al. (1999) indicate that NTR2 mediates neurotensin-induced analgesia.

A third neurotensin receptor (NTR3) was cloned from a human brain cDNA library (Mazella et al. 1998; Vincent et al. 1999; Mazella 2001; Mazella and Vincent 2006). It is identical with sortilin, a receptor-like protein, cloned from human brain (Petersen et al. 1997, 1999). The NT3/gp95/sortilin protein is a transmembrane neuropeptide receptor which does not belong to the superfamily of G-protein-coupled receptors.

Gully et al. (1997) described a binding assay for the neurotensin1 receptor.

Procedure
Cell Culture

CHO cells transfected with cDNA of the human neurotensin receptor cloned from HT 29 cells (h-NTR1-CHO cells) are cultured at 37 °C in modified Eagle’s medium without nucleosides, containing 10 % fetal calf serum, 4 mM glutamine, and 300 μg /ml geneticin (G418), in a humidified incubator under 5 % CO2 in O2. The colonic adenocarcinoma HT 29 cell line (American Type Culture Collection, Rockville, MD) is cultured under similar conditions in Dulbecco’s modified Eagle’s medium/F-12 medium supplemented with 10 % fetal calf serum, 4 mM glutamine, 200 IU/ml penicillin, and 200 mg/ml streptomycin. One week after seeding, confluent monolayer cultures are washed three times with 3 ml PBS and harvested by enzymatic dissociation with trypsin. After dilution with PBS, cells are resuspended in the same culture medium at a density of 5 × 104 cells/ml and are plated into 35-mm diameter, fibronectin-coated Petri culture dishes.

Membrane Homogenate Preparation and Binding Assay

Whole brains of male Sprague Dawley rats albino guinea pigs or cell pellets are homogenized in 10 volumes of ice-cold 50 mM Tris–HCl buffer (pH7.4) for 30 s, using a polytron homogenizer (setting 5). After 20 min. centrifugation at 30,000 g, the pellet is washed; centrifuged again under the same conditions; resuspended in a storage buffer containing 50 mM Tris–HCl (pH7.4), 1 mM EDTA, 0.1 % BSA, 40 mg/l bacitracin, 1 mM 1,10-orthophenanthroline, and 5 mM dithiothreitol; and stored as aliquots in liquid nitrogen until used.

Aliquots of membranes (10, 50, 300, and 500 μg of protein for h-NTR1-CHO cells, HT 29 cells, rat brain, and guinea pig brain, respectively) are incubated for 20 min at 20 °C in the incubation buffer (0.5 ml final volume) containing appropriate concentrations of [125I-Tyr3]neurotensin (25–100 pM) and unlabeled drugs. After incubation, the assay medium is diluted with 4 ml of ice-cold 50 mM Tris–HCl buffer (pH 7.4) supplemented with 0.1 % BSA and 1 mM EDTA, and the mixture is rapidly filtered under reduced vacuum through Whatman GF/B glass fiber filters that have been pretreated with 0.1 % polyethyleneimine. The filters are washed under the same conditions three times and radioactivity is measured. Nonspecific binding is determined in the presence of 1 μM unlabeled neurotensin. All experiments are performed in triplicate, and data are expressed as the mean ± SEM of at least three separate determinations.

Evaluation

The IC 50 is the value of ligand that inhibits 50 % of the specific binding and is determined using an iterative nonlinear regression program (Munson and Rodbard 1980).

Modifications of the Method

Cusack et al. (1995) studied species selectivity of neurotensin analogs at the rat and two human NTR1 receptors.

Lugrin et al. (1991) produced a series of pseudopeptide analogs of neurotensin by systematically replacing peptide bonds in neurotensin with CH2NH bonds. The compounds were screened in vitro for agonist or antagonist activity and for metabolic stability.

Le et al. (1997) cloned the human neurotensin receptor gene and determined the structure.

Labbé-Jullié et al. (1998) attempted to identify residues in the rat NTR1 that are involved in binding of a nonpeptide neurotensin antagonist.

Souazé et al. (1997) and Najimi et al. (1998) studied the effects of a neurotensin agonist and showed in human colonic adenocarcinoma HT 29 cells after short incubation an increase, after prolonged exposure a decrease of mRNA levels, and in the human neuroblastoma cell line CHP 212 a high-affinity neurotensin receptor gene activation.

Ovigne et al. (1998) described a monoclonal antibody specific for the human NTR1.

Nouel et al. (1999) found that both NT2 and NT3 neurotensin receptor subtypes were expressed by cortical glial cells in culture.

Cusack et al. (2000) developed a neurotensin analog, NT34, which can distinguish between rat and human neurotensin receptors and exhibits more than a 100-fold difference in binding affinities.

Neuromedin N , a peptide belonging to the gastrin-releasing peptide/bombesin family (see chapter “Pharmacological Effects on Gastric Function”), shows a high affinity to brain neurotensin receptors and is rapidly inactivated by brain synaptic peptidases (Checler et al. 1990).

References and Further Reading

  • Chalon P, Vita N, Kaghad M, Guillemot M, Bonnin J, Delpech P, Le Fur G, Ferrara P, Caput D (1996) Molecular cloning of a levocabastine-sensitive binding site. FEBS Lett 400:211–214

  • Checler F, Vincent JP, Kitabgi P (1986) Neuromedin N: high affinity interaction with brain neurotensin receptors and rapid inactivation by brain synaptic peptidases. Eur J Pharmacol 126:239–244

  • Cusack B, McCormick DJ, Pang Y-P, Souder T, Garcia R, Fauq A, Richelson E (1995) Pharmacological and biochemical profiles of unique neurotensin 8–13 analogs exhibiting species selectivity, stereoselectivity, and superagonism. J Biol Chem 270:18359–18366

  • Cusack B, Chou T, Jansen K, McCormick DJ, Richelson E (2000) Analysis of binding sites and efficacy of a species-specific peptide at rat and human neurotensin receptors. J Pept Res 55:72–80

  • Dubuc I, Sarret P, Labbé-Jullié C, Botto JM, Honoré E, Bourdel E, Martinez J, Costentin J, Vincent JP, Kitabgi P, Mazella J (1999) Identification of the receptor subtype involved in the analgesic effect of neurotensin. J Neurosci 19:503–510

  • Gully D, Labeeuw B, Boigegrain R, Oury-Donat F, Bachy B, Poncelet M, Steinberg R, Suaud-Chagny MF, Santucci V, Vita N, Pecceu F, Labbé-Jullié C, Kitabgi P, Soubrié P, Le Fur G, Maffrand JP (1997) Biochemical and pharmacological activities of SR 142948A, a new potent neurotensin receptor antagonist. J Pharmacol Exp Ther 280:802–812

  • Labbé-Jullié C, Dubuc I, Brouard A, Doulut S, Bourdel E, Pelaprat D, Mazella J, Martinez J, Rostène W, Costentin J, Kitabgi P (1994) In vivo and in vitro structure-activity studies with peptide and pseudopeptide neurotensin analogs suggest the existence of distinct central neurotensin receptor subtypes. J Pharmacol Exp Ther 268:328–336

  • Labbé-Jullié C, Barroso S, Nicolas-Etève D, Reversat JL, Botto JM, Mazella J, Barnassau JM, Kitabgi P (1998) Mutagenesis and modeling of the neurotensin receptor NTR1. Identification of residues that are critical for binding of SR 48692, a nonpeptide neurotensin. J Biol Chem 273:16351–16357

  • Le F, Groshan K, Zeng X-P, Richelson E (1997) Characterization of the genomic structure, promotor region, and a tetranucleotide repeat polymorphism of the human neurotensin receptor gene. J Biol Chem 272:1315–1322

  • Lugrin D, Vecchini F, Doulut S, Rodriguez M, Marinez J, Kitabgi P (1991) Reduced peptide bond pseudopeptide analogues of neurotensin: binding and biological activities, and in vitro metabolic stability. Eur J Pharmacol 205:191–198

  • Mazella J (2001) Sortilin/neurotensin receptor-3: a new tool to investigate neurotensin signaling and cellular trafficking? Cell Signal 13:1–6

  • Mazella J, Vincent JP (2006) Functional roles of the NTS2 and NTS3 receptors. Peptides 27:2469–2475

  • Mazella J, Botto JM, Guillemare E, Coppola T, Sarret P, Vincent JP (1996) Structure, functional expression, and cerebral localization of the levocabastine-sensitive neurotensin/neuromedin N receptor from mouse brain. J Neurosci 16:5613–5620

  • Mazella J, Zsürger N, Navarro V, Chabry J, Kaghad M, Caput D, Ferrara P, Vita N, Gully D, Maffrand JP, Vincent JP (1998) The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein coupled receptor. J Biol Chem 273:26273–26276

  • Munson PJ, Rodbard D (1980) LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220–239

  • Najimi M, Souzé F, Méndez M, Hermans E, Berbar T, Rostène W, Forgez P (1998) Activation of receptor gene transcription is required to maintain cell sensitization after agonist exposure. Studies on neurotensin receptor. J Biol Chem 273:21634–21641

  • Nouel D, Sarret P, Vincent JP, Mazella J, Beaudet A (1999) Pharmacological, molecular and functional characterization of glial neurotensin receptors. Neuroscience 94:1189–1197

  • Ovigne JM, Vermot-Desroches C, Lecron JC, Portier M, Lupker J, Pecceu F, Wijdenes J (1998) An antagonistic monoclonal antibody (B-N6) specific for the human neurotensin receptor-1. Neuropeptides 32:247–256

  • Petersen CM, Nielson MS, Nykjar A, Jacobsen L, Tommerup N, Rasmussen HH, Roigaard H, Gliemann J, Madsen P, Moestrup SK (1997) Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography. J Biol Chem 272:3599–3605

  • Petersen CM, Nielson MS, Jacobsen C, Tauris J, Jacobsen L, Gliemann J, Moestrup SK, Madsen P (1999) Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding. EMBO J 18:595–604

  • Schotte A, Leysen JE, Laduron PM (1986) Evidence for a displaceable non-specific 3H-neurotensin binding site in rat brain. Naunyn Schmiedebergs Arch Pharmacol 333:400–405

  • Souazé F, Rostène W, Forgez P (1997) Neurotensin agonist induces differential regulation of neurotensin receptor mRNA. Identification of distinct transcriptional and post-transcriptional mechanisms. J Biol Chem 272:10087–10094

  • Tanaka K, Masu M, Nakanishi S (1990) Structure and functional expression of the cloned rat neurotensin receptor. Neuron 4:847–854

  • Vincent JP, Mazella J, Kitagbi P (1999) Neurotensin and neurotensin receptors

  • Vita N, Laurent P, Lefort S, Chalon P, Dumont X, Kaghad M, Gully D, Le Fur G, Ferrara P, Caput D (1993) Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor. FEBS Lett 317:139–142

  • Watson M, Isackson PJ, Makker M, Yamada MS, Yamada M, Cusack B, Richelson E (1993) Identification of a polymorphism in the human neurotensin receptor gene. Mayo Clin Proc 68:1043–1048

  • Yamada M, Lombet A, Forgez P, Rostène W (1998) Distinct functional characteristics of levocabastine-sensitive rat neurotensin NT2 receptor expressed in Chinese hamster ovary cells. Life Sci 62:PL375–PL379

Genetically Altered Monoamine Transporters

Monoamine transporters, such as the dopamine transporter, 5-hydroxytryptamine transporter, and noradrenaline transporter, in the plasma membrane provide effective control over the intensity of monoamine-mediated signaling by recapturing neurotransmitters released by presynaptic neurons (Gainetdinov et al. 2002). These transporters act also as molecular gateways for neurotoxins (Uhl and Kiayama 1993; Miller et al. 1999; Vincent et al. 1999).

Takahashi et al. (1997) found that heterozygote animals of VMAT2 knockout mice display reduced amphetamine-conditioned reward, enhanced amphetamine locomotion, and enhanced MPTP toxicity.

References and Further Reading

  • Gainetdinov RR, Sotnikova TD, Caron MG (2002) Monoamine transporter pharmacology and mutant mice. Trends Pharmacol Sci 23:367–373

  • Miller GW, Gainetdinov RR, Levey AI, Caron MG (1999) Dopamine transporters and neuronal injury. Trends Pharmacol Sci 20:424–429

  • Takahashi N, Miner LL, Sora I, Ujike H, Revay RS, Kostic V, Jackson-Lewis V, Przedborski S, Uhl GR (1997) VMAT2 knockout mice: heterozygotes display reduced amphetamine-conditioned reward, enhanced amphetamine locomotion, and enhanced MPTP toxicity. Proc Natl Acad Sci U S A 94:9938–9943

  • Uhl GR, Kiayama S (1993) A cloned dopamine transporter. Potential insights into Parkinson’s disease pathogenesis. Acta Neurol 60:321–324

  • Vincent JP, Mazella J, Kitabgi P (1999) Neurotensin and neurotensin receptors. Trends Pharmacol Sci 20:302–309

Dopamine Transporter Knockout Mice

Many drugs exert their psychotropic action via dopamine transporters (Amara and Kuhar 1993; Giros and Caron 1993).

Dopamine transporter knockout mice, which are generated by disruption of the gene encoding the dopamine transporter by homologous recombination (Giros et al. 1996; Sora et al. 1998), have a distinct biochemical and behavioral phenotype. At the neurochemical level, the homoeostasis of dopamine-containing neurons is altered markedly, including disrupted clearance of dopamine, an elevated extracellular concentration of dopamine, and dramatically decreased intraneuronal storage of dopamine (Jones et al. 1998; Gainetdinov et al. 1998; Benoit-Marand et al. 2000).

In response to the elevated dopamine-mediated tone, both presynaptic and postsynaptic dopamine receptors are downregulated (Giros et al. 1996), but although autoreceptor functions are lost (Jones et al. 1999), some postsynaptic responses appear to be enhanced (Gainetdinov et al. 1999a; Fauchey et al. 2000).

Dopamine transporter knockout mice are hyperactive (Gainetdinov et al. 1999b; Spielewoy et al. 2000) and have a much reduced body size (Bossé et al. 1997). These animals have cognitive deficits (Gainetdinov et al. 1999a, b), disrupted sensorimotor gating (Ralph et al. 2001), and sleep dysregulation (Wisor et al. 2001). Dopamine transporter knockout mice appear to provide a model of some aspects of manic behavior (Ralph-Williams et al. 2003).

Abnormalities in skeletal structure (Bliziotes et al. 2000) and altered regulation of gastrointestinal tract motility (Walker et al. 2000) are also observed.

References and Further Reading

  • Amara SG, Kuhar MJ (1993) Neurotransmitter transporters: recent progress. Annu Rev Neurosci 16:73–93

  • Benoit-Marand M, Jaber M, Gonon F (2000) Release and elimination of dopamine in vivo in mice lacking the dopamine transporter: functional consequences. Eur J Neurosci 12:2985–2992

  • Bliziotes M, McLoughlin S, Gunness M, Fumagalli F, Jones SR, Caron MG (2000) Bone histomorphometric and biochemical abnormalities in mice homozygous for deletion of the dopamine transporter gene. Bone 26:15–19

  • Bossé R, Fumagalli F, Jaber M, Giros B, Gainetdinov RR, Wetsel WC, Missale C, Caron MG (1997) Anterior pituitary hypoplasia and dwarfism in mice lacking the dopamine transporter. Neuron 19:127–138

  • Fauchey V, Jaber M, Caron MG, Bloch B, Le Moine C (2000) Differential regulation of the dopamine D1, D2 and D3 receptor gene expression and changes in the phenotype of the striatal neurons in mice lacking the dopamine receptor. Eur J Neurosci 12:19–26

  • Gainetdinov RR, Jones SR, Fumagalli F, Wightman RM, Caron MG (1998) Re-evaluation of the role of the dopamine transporter in dopamine homeostasis. Brain Res Brain Res Rev 26:148–153

  • Gainetdinov RR, Jones SR, Caron MG (1999a) Functional hyperdopaminergia in dopamine transporter knockout mice. Biol Psychiatry 46:303–311

  • Gainetdinov RR, Wetsel WC, Jones SR, Levin ED, Jaber M, Caron MG (1999b) Role of serotonin in the paradoxical calming effect of psychostimulants on hyperactivity. Science 283:397–401

  • Giros B, Caron MG (1993) Molecular characteristics of the dopamine transporter. Trends Pharmacol Sci 14:43–49

  • Giros B, Jaber M, Jones SR, Wightman RM, Caron MG (1996) Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379:606–612

  • Jones SR, Gainetdinov RR, Jaber M, Giros B, Wightman RM, Caron MG (1998) Profound neuronal plasticity in response to inactivation of the dopamine transporter. Proc Natl Acad Sci U S A 95:4029–4034

  • Jones SR, Gainetdinov RR, Hu XT, Cooper DC, Wightman RM, White FJ, Caron MG (1999) Loss of autoreceptor functions in mice lacking the dopamine transporter. Nat Neurosci 2:649–655

  • Ralph RJ, Paulus MP, Fumagalli F, Caron MG, Geyer MA (2001) Prepulse inhibition deficits and perseverative motor patterns in dopamine transporter knock-out mice: differential effects of D1 and D2 receptor antagonists. J Neurosci 21:305–313

  • Ralph-Williams RJ, Pauluis MP, Zhuang X, Hen R, Geyer MA (2003) Valproate attenuates hyperactive and perseverative behaviors in mutant mice with a dysregulated dopamine system. Biol Psychiatry 53:352–359

  • Sora I, Wichems C, Takahashi M, Li XF, Zeng Z, Revay R, Lesch KP, Murphy DL, Uhl GR (1998) Cocaine reward models: conditioned place preference can be established in dopamine- and serotonin-transporter knockout mice. Proc Natl Acad Sci U S A 95:7699–7704

  • Spielewoy C, Roubert C, Hamon M, Nosten-Bertrand M, Betancur C, Giros B (2000) Behavioural disturbances associated with hyperdopaminergia in dopamine-transporter knockout mice. Behav Pharmacol 11:279–290

  • Walker JK, Gainetdinov RR, Mangel AW, Caron MG, Shetzline MA (2000) Mice lacking the dopamine transporter display altered regulation of distal colon motility. Am J Physiol 279:G311–G318

  • Wisor JP, Nishino S, Sora I, Uhl GH, Mignot R, Edgar M (2001) Dopaminergic role in stimulant-induced wakefulness. J Neurosci 21:1787–1794

Serotonin Transporter Knockout Mice

The serotonin transporter has a key role in regulating the intensity of 5-HT-mediated transmission and is the primary target for several antidepressants and psychostimulants (Amara and Kuhar 1993; Bengel et al. 1996).

Disruption of 5-HT uptake in serotonin transporter knockout mice increases the extracellular concentration of 5-HT sixfold and reduces intracellular concentration by 60 % – 80 % (Fabre et al. 2000).

Holmes et al. (2003) found that mice lacking the serotonin transporter exhibit 5-HT1A receptor-mediated abnormalities in tests for anxiety-like behavior.

Lira et al. (2003) reported altered depression-related behaviors and functional changes in the dorsal raphe nucleus of serotonin-transporter-deficient mice.

Marked desensitization of both presynaptic and postsynaptic 5-HT1A receptors is observed in electrophysiological studies (Gobbi et al. 2001).

There is a significant decrease in 5-HT1A receptor binding sites, mRNA, and protein in some, but not all, 5-HT-containing brain areas. Altered hypothermic and neuroendocrine responses to 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) are also reported (Li et al. 1999).

Thermal hyperalgesia in mice after chronic constrictive sciatic nerve injury was absent in serotonin transporter-deficient mice (Vogel et al. 2003).

Decreases in 5-HT1A and 5-HT1B receptor coupling are observed, accompanied by disruption of the neurochemical responses to the 5-HT1A receptor agonist ipsapirone and the 5-HT1A/5-HT1D receptor agonist GR127935 (Fabre et al. 2000).

The hyperlocomotor effect of MDMA, but not that of high doses of d-amphetamine, is disrupted in serotonin receptor knockout mice (Bengel et al. 1996).

In double knockout mice that lack the dopamine transporter and have no or one copy of the gene that encodes the serotonin transporter, no place preference for cocaine was observed (Sora et al. 2001).

References and Further Reading

  • Amara SG, Kuhar MJ (1993) Neurotransmitter transporters: recent progress. Annu Rev Neurosci 16:73–93

  • Bengel D, Murphy DL, Andrews AM, Wichems CH, Feltner D, Heils A, Mössner R, Westphal H, Lesch KP (1996) Altered brain homeostasis and locomotor insensitivity to 3,4-methylenedioxy methamphetamine (“Ecstasy”) in serotonin transporter-deficient mice. Mol Pharmacol 53:649–655

  • Fabre V, Beaufour C, Evrad A, Rioux A, Hanoun N, Lesch KP, Murphy DL, Lanfumey L, Hamon M, Martres MP (2000) Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter. Eur J Neurosci 12:2299–2310

  • Gobbi G, Murphy DL, Lesch K, Blier P (2001) Modifications of the serotonergic system in mice lacking serotonin transporters: an in vivo electrophysiological study. J Pharmacol Exp Ther 296:987–995

  • Holmes A, Yang RJ, Lesch KP, Crawley JN, Murphy DL (2003) Mice lacking the serotonin transporter exhibit 5-HT1A receptor-mediated abnormalities in tests for anxiety-like behavior. Neuropsychopharmacology 28:2077–2088

  • Li Q, Wichems C, Heils A, Van De Kar LD, Lasch KP, Murphy DL (1999) Reduction of 5-hydroxytryptamine (5-HT1A)-mediated temperature and neuroendocrine responses and 5-HT1A binding sites in 5-HT transporter knockout mice. J Pharmacol Exp Ther 291:999–1007

  • Lira A, Zhou M, Castanon N, Ansorge MS, Gordon JA, Francis JH, Bradley-Moore M, Lira J, Underwood MD, Arango V, Kung HF, Hofer MA, Hen R, Gingrich JA (2003) Altered depression-related behaviors and functional changes in the dorsal raphe nucleus of serotonin transporter deficient mice. Biol Psychiatry 54:960–971

  • Sora I, Hall FS, Andrews AM, Itokawa M, Li XF, Wei HB, Wichems C, Lesch KP, Murphy DL, Uhl GR (2001) Molecular mechanisms of cocaine award: combined dopamine and serotonin transporter knockouts eliminate cocaine place preference. Proc Natl Acad Sci U S A 98:5300–5305

  • Vogel C, Mössner R, Gerlach H, Heinemann T, Murphy DL, Riederer P, Lesch KP, Sommer C (2003) Absence of thermal hyperalgesia in serotonin transporter-deficient mice. J Neurosci 23:708–715

Noradrenaline Transporter Knockout Mice

The noradrenaline transporter has a role similar to that of the dopamine transporter and the serotonin transporter with respect to noradrenaline-mediated transmission (Blakely et al. 1994).

Noradrenaline-transporter knockout mice have been generated using homologous recombination (Xu et al. 2000).

Wang et al. (1999) reviewed genetic approaches to studying norepinephrine function using knockout of the mouse norepinephrine transporter gene.

The prolonged synaptic lifetime of noradrenaline in noradrenaline transporter knockout mice results in elevation of the extracellular concentration of noradrenaline and depletion of the intraneuronal stores. In addition, in noradrenaline transporter knockout mice, the α 1-adrenoceptor decreased in the hippocampus (Xu et al. 2000), although α 2A-adrenoceptor density did not change in the spinal cord (Bohn et al. 2000).

Noradrenaline-transporter knockout mice have a lower body weight and reduced locomotor responses to novelty. In the tail-suspension test used for screening antidepressant drugs, noradrenaline transporter knockout mice behaved like antidepressant-treated, wild-type animals, and no additional effects of the antidepressants desipramine, paroxetine, and bupropion were observed in mutant mice in this test (Xu et al. 2000).

In the tail-flick assay, morphine induced greater analgesia in noradrenaline transporter knockout mice compared with wild-type mice (Bohn et al. 2000).

In synaptosomes from the frontal cortex of noradrenaline transporter knockout mice, cocaine and nisoxetine had no inhibitory effect on the uptake of dopamine, whereas in the nucleus accumbens, the effectiveness of cocaine was somewhat reduced. Uptake of dopamine in brain regions that have low levels of dopamine transporter may depend primarily on the noradrenaline transporter (Morón et al. 2002).

Locomotor responses to cocaine and amphetamine are elevated in noradrenaline knockout mice, and chronic administration of cocaine did not induce further sensitization. The enhanced responses to psychostimulants in noradrenaline transporter knockout mice correlate with the suppression of presynaptic dopamine function and supersensitivity to postsynaptic D2 and D3 receptors (Xu et al. 2000).

Haller et al. (2002) studied behavioral responses to social stress in noradrenaline transporter knockout mice.

References and Further Reading

  • Blakely RD, De Felice LJ, Hartzell HC (1994) Molecular physiology of norepinephrine and serotonin transporters. J Exp Biol 196:263–281

  • Bohn LM, Xu F, Gainetdinov RR, Caron MG (2000) Potentiated opioid analgesia in norepinephrine transporter knockout mice. J Neurosci 20:9040–9045

  • Haller J, Bakos N, Rodriguiz RM, Carin MG, Wetsel WC, Liposits Z (2002) Behavioral responses to social stress in noradrenaline transporter knockout mice: effects on social behavior and depression. Brain Res Bull 58:279–284

  • Morón JA, Brockington A, Wise RA, Rocha BA, Hope BT (2002) Dopamine uptake through the norepinephrine transporter in brain regions with low levels of the dopamine transporter: evidence from knock-out mice lines. J Neurosci 22:389–395

  • Wang YM, Xu F, Gainetdinov RR, Caron MG (1999) Genetic approaches to studying norepinephrine function: knockout of the mouse norepinephrine transporter gene. Biol Psychiatry 46:1124–1130

  • Xu F, Gainetdinov RR, Wetsel WC, Jones SR, Bohn LM, Miller GW, Wang YM, Caron MG (2000) Mice lacking the norepinephrine transporter are supersensitive to psychostimulants. Nat Neurosci 3:465–471

In Vivo Tests

Golden Hamster Test

Purpose and Rationale

“Innate behavior” of many species including man has been described by Lorenz (1943, 1966). The “golden hamster test” (Ther et al. 1959) uses the innate behavior of this species (Mesocricetus auratus) for differentiation between neuroleptic and sedative – hypnotic activity. The aggressive behavior of male golden hamsters is suppressed by neuroleptics in doses which do not impair motor function.

Procedure

Ten to 20 male golden hamsters with an average weight of 60 g are crowded together in Makrolon(R) cages for at least 2 weeks. During this time, the animals develop a characteristic fighting behavior. For the test, single animals are placed into glass jars of 2 l. In this situation, the hamsters assume a squatting and resting position during the day. If the animals are touched with a stick or a forceps, they wake up from their daytime sleep and arouse immediately from the resting position. If one tries to hold the hamster with a blunted forceps, a characteristic behavior is elicited: The hamster throws himself onto his back, tries to bite and to push the forceps away with his legs, and utters angry shrieks. Touching the animals is repeated up to six times followed by punching with the forceps. Only animals responding to the stimulus with all three defense reactions (turning, vocalizing, and biting) are included into the test.

The test compounds are applied either subcutaneously, intraperitoneally, or orally. Six animals are used for each dose.

Evaluation

The stimuli are applied every 20 min for 3 h. The number of stimuli until response is recorded. Furthermore, the suppression of the defense reactions (turning, biting, and vocalizing) is evaluated. An animal is regarded to be completely “tamed” if all defense reactions are suppressed even after punching with the forceps at least once during the test period.

After each stimulation, the “tamed” animal is placed on an inclined board with 20° inclination. Normal hamsters and hamsters tamed by neuroleptics are able to support themselves or to climb on the board. Impaired motor function causes sliding down. This experiment is repeated three times after each testing of the defense reactions. An animal’s coordination is considered to be disturbed if it falls three times during two tests of the experiments.

For each dose, the number of tamed hamsters and the number of animals with impaired motor function are recorded. Using different doses, ED 50 values can be calculated for the taming effect and for impairment of motor function.

The ED 50 values of taming were 1.5 mg/kg for chlorpromazine s.c. and 0.2 mg/kg for reserpine s.c. Much higher doses (ten times of chlorpromazine and five times of reserpine) did not elicit motor disturbances. On the contrary, while ED 50 values of 10 mg/kg phenobarbital s.c. and 180 mg/kg meprobamate p.o. for the taming effect were found, these doses already caused severe motor disturbances. The taming dose of diazepam was 10 mg/kg p.o. which already showed some muscle-relaxing activity. The term “neuroleptic width” indicates the ratio between the ED 50 for taming and the ED 50 for motor disturbances. Only for neuroleptic drugs are ratios found between 1:5 and 1:30.

Critical Assessment of the Method

The method has the advantage that neuroleptics can easily be differentiated from sedative and hypnotic drugs. Anxiolytics with pronounced muscle-relaxing activity also show no significant differences between taming and impaired motor function. Moreover, the method has the advantage that no training of the animals and no expensive apparatus are needed.

References and Further Reading

  • Kreiskott H, Vater W (1959) Verhaltensstudien am Goldhamster unter dem Einfluß zentralwirksamer Substanzen. Naunyn Schmiedebergs Arch Exp Pathol Pharmakol 236:100–105

  • Lorenz K (1943) Die angeborenen Formen möglicher Erfahrung. Z Tierpsychol 5:235–409

  • Lorenz K (1965) Evolution and modification of behavior. University of Chicago Press, Chicago

  • Lorenz K (1966) Evolution and modification of behavior. Methuen, London

  • Ther L, Vogel G, Werner P (1959) Zur pharmakologischen Differenzierung und Bewertung von Neuroleptica. Arzneim Forsch/Drug Res 9:351–354

Influence on Behavior of the Cotton Rat

Purpose and Rationale

The “cotton-rat test” is another attempt to use the innate behavior as described for several animal species by Lorenz (1943, 1966) for the differentiation of psychotropic drugs (Vogel and Ther 1960). The cotton rat (Sigmodon hispidus) is a very shy animal which conceals himself at any time. This innate flight reflex is suppressed by centrally active drugs. Simultaneous evaluation of motor function allows the differentiation between neuroleptic and sedative drugs.

Procedure

Cotton rats are bred in cages equipped with a clay cylinder of 20 cm length and 10 cm diameter. This cylinder is used by the animals for hiding, sleeping, and breeding. Moreover, the animals which bite easily can be transported from one cage to another just by closing the cylinder on both ends. For the test, young animals with a body weight of 40 g are used. Young animals are as shy as the old ones but less vicious. Nevertheless, leather gloves have to be used for handling of cotton rats. Normal cages (25 × 30 × 20 cm) with a wire lid are used. A tunnel of sheet metal (half of a cylinder) 20 cm long and 7 cm high is placed into the cage. The cotton rats hide immediately in this tunnel. If the tunnel is lifted and placed on another site of the cage, the cotton rats immediately hide again.

Three rats are placed in one cage and tested for their behavior. Selective shaving of the fur enables the observer to recognize each animal. If the rats behave as described, they are then treated with the test compound subcutaneously or orally. At least six animals divided in two cages are used for each dose of test compound or standard. Fifteen min after application of the drug, the test period of three h is started. The tunnel is lifted and placed to another site. If the animals do not show the immediate flight reflex, an airstream of short duration is blown through the wire lid. If the animal still does not respond with the flight reflex, it is considered to be positively influenced. Afterwards, the animal is placed on an inclined board with 35° of inclination and tested for disturbance of motor coordination. A normal animal is able to climb upwards. If coordination is disturbed, the rat slides down.

Evaluation

The test procedure is repeated every 15 min over a period of 3 h. The animals which show at least one suppression of the flight reflex during the test period are counted as well as those who slide down on the inclined board. Using different doses, ED 50 values are calculated for both parameters. The ratio between these two ED 50 values is regarded as “neuroleptic width” which is 1:20 for chlorpromazine and 1:30 for reserpine, whereas ratios of 1:2 for phenobarbital and 1:1.5 for meprobamate indicate the absence of neuroleptic activity.

Critical Assessment of the Method

The method allows the differentiation of drugs with neuroleptic activity against other centrally active drugs. No training of the animals and no expensive equipment are necessary.

References and Further Reading

  • Lorenz K (1943) Die angeborenen Formen möglicher Erfahrung. Z Tierpsychol 5:235–409

  • Lorenz K (1966) Evolution and modification of behavior. Methuen, London

  • Vogel G, Ther L (1960) Das Verhalten der Baumwollratte zur Beurteilung der neuroleptischen Breite zentral-depressiver Stoffe. Arzneim Forsch/Drug Res 10:806–808

Artificial Hibernation in Rats

Purpose and Rationale

Giaja (1938, 1940, 1953, 1954) studied the effects of reduced oxygen tension and cold environment on rats. The animals were placed in hermetically closed glass vessels which were submerged in ice water. Due to the respiratory activity, the oxygen tension diminishes and the carbon dioxide content increases. Under the influence of cooling and of hypoxic hypercapnia, the rectal temperature falls to 15 °C, and the animal is completely anesthetized and immobilized. The rat can survive in this poikilothermic state for more than 20 h. Complete recovery occurs after warming up. This kind of artificial hibernation was augmented by chlorpromazine (Courvoisier et al. 1953; Giaja and Markovic-Giaja 1954). Vogel (1959) and Ther et al. (1959, 1963) used these observations for evaluation of neuroleptics and opioid analgesics.

Procedure

Male Wistar rats weighing 100–150 g are deprived of food with free access to tap water overnight. The test compounds are injected subcutaneously 15 min prior to the start of the experiment. First, the rats are placed in ice-cold water to which surfactant is added in order to remove the air from the fur for 2 min. Then, the animals are placed into hermetically closed glass vessels of 750 ml volume which are placed into a refrigerator at 2 °C temperature. During the following hour, the vessels are opened every 10 min for exactly 10 s, allowing some exchange of air and reducing the carbon dioxide accumulation. At each time, animals are removed from the glass vessel and observed for signs of artificial hibernation which are not shown by control animals under these conditions. Treated animals, lying on the side, are placed on the back and further examined. An animal is considered positive, when it remains on the back, even if the extremities are stretched out. In this state, cardiac and respiration frequency are reduced, and the rectal temperature has fallen to 12–15 °C. The rigor of the musculature allows only slow movements of the extremities. The animals recover completely within a few hours if they are brought to their home cages at room temperature. Artificial hibernation is induced dose-dependent by neuroleptics of the phenothiazine type and by some opioid analgesics like meperidine and methadone. In contrast, morphine shows only slight activity.

Evaluation

Various doses are applied to groups of ten animals. Percentage of positive animals is calculated for each group, and ED 50 values with confidence limits are estimated according to Litchfield and Wilcoxon.

References and Further Reading

  • Courvoisier S, Fournel J, Ducrot R, Kolsky M, Koeschet P (1953) Propriétés pharmacodynamiques du chlorhydrate de chloro-3-(diméthylamino-3′-propyl)-10-phenothiazine (4.560 R.P.). Arch Int Pharmacodyn 92:305–361

  • Giaja J (1938) Sur l’analyse de la fonction de calorification de l’homéotherme par la dépression barométrique. C R Soc Biol 127:1355–1359

  • Giaja J (1940) Léthargie obtenue che le Rat par la dépression barométrique. C R Acad Sci 210:80–84

  • Giaja J (1953) Sur la physiologie de l’organisme refroidi. Presse Med 61:128–129

  • Giaja J, Markovic-Giaja L (1954) L’hyperthermie produite par la chlorpromazine et la résistance a l’asphyxie. Bull Soc Chim Biol 36:1503–1506

  • Litchfield J, Wilcoxon F (1949) A simplified method of evaluating dose effect experiments. J Pharmacol Exp Ther 96:99–113

  • Ther L, Vogel G, Werner P (1959) Zur pharmakologischen Differenzierung und Bewertung der Neuroleptica. Arzneim Forsch/Drug Res 9:351–354

  • Ther L, Lindner E, Vogel G (1963) Zur pharmakologischen Wirkung der optischen Isomeren des Methadons. Dtsch Apoth Ztg 103:514–520

  • Vogel G (1959) Über die Wirkung von Dolantin und Polamidon im Vergleich zu anderen stark wirksamen Analgetica an der unterkühiten Ratte nach Giaja. Naunyn Schmiedebergs Arch Exp Pathol Pharmakol 236:214–215

Catalepsy in Rodents

Purpose and Rationale

Catalepsy in rats is defined as a failure to correct an externally imposed, unusual posture over a prolonged period of time. Neuroleptics which have an inhibitory action on the nigrostriatal dopamine system induce catalepsy (Costall and Naylor 1974; Chermat and Simon 1975; Sandberg et al. 1986), while neuroleptics with little or no nigrostriatal blockade produce relatively little or no cataleptic behavior (Honma and Fukushima 1976). Furthermore, cataleptic symptoms in rodents have been compared to the Parkinson-like extrapyramidal side effects seen clinically with administration of antipsychotic drugs (Duvoisin 1976).

Procedure

Groups of six male Sprague Dawley or Wistar rats with a body weight between 120 and 250 g are used. They are dosed intraperitoneally with the test drug or the standard. Then, they are placed individually into translucent plastic boxes with a wooden dowel mounted horizontally 10 cm from the floor and 4 cm from one end of the box. The floor of the box is covered with approximately 2 cm of bedding material. White noise is presented during the test. The animals are allowed to adapt to the box for 2 min. Then, each animal is grasped gently around the shoulders and under the forepaws and placed carefully on the dowel. The amount of time spent with at least one forepaw on the bar is determined. When the animal removes its paws, the time is recorded, and the rat is repositioned on the bar. Three trials are conducted for each animal at 30, 60, 120, and 360 min.

Evaluation

An animal is considered to be cataleptic if it remains on the bar for 60 s. Percentage of cataleptic animals is calculated. For dose–response curves, the test is repeated with various doses and more animals. ED 50 values can be calculated. A dose of 1 mg/kg i.p. of haloperidol was found to be effective.

Critical Assessment of the Method

The phenomenon of catalepsy can be used for measuring the efficacy and the potential side effects of neuroleptics.

Modifications of the Method

Catalepsy induced by neuroleptic drugs can also be measured by the PAW test , which measures increase in forelimb and hindlimb retraction time in rats (Ellenbroek et al. 1987, 2001; Ellenbroek and Cools 1988, 2000; Prinssen et al. 1994, 1995).

The test is performed 30 min after intraperitoneal injection of test drug. Male Wistar rats weighing 220–300 g are placed on a Perspex platform (30 × 30 cm with a height of 20 cm) containing two holes for the forelimbs (40 mm) and two for the hindlimbs (50 mm), and a slit for the tail. The distance between the right and left forelimb holes is 15 mm, and the distance between forelimb and hindlimb holes is 55 mm. The rat is held behind the forelimbs, and the hindlimbs are gently placed in the holes. The forelimb retraction time and the hindlimb retraction time are defined as the time the animal needs to withdraw one forelimb and one hindlimb, respectively. The average forelimb retraction time and hindlimb retraction time (the mean of three measurements) is calculated for each rat.

Extrapyramidal syndromes after treatment with typical and atypical neuroleptics were measured in nonhuman primates (Cebus monkeys) by Casey (1989, 1991, 1993) and Gerlach and Casey (1990).

References and Further Reading

  • Casey DE (1989) Serotonergic aspects of acute extrapyramidal syndromes in nonhuman primates. Psychopharmacol Bull 25:457–459

  • Casey DE (1991) Extrapyramidal syndromes in nonhuman primates: typical and atypical neuroleptics. Psychopharmacol Bull 27:47–50

  • Casey DE (1993) Serotonergic and dopaminergic aspects of neuroleptic-induced extrapyramidal syndromes in nonhuman primates. Psychopharmacology (Berl) 112:S55–S59

  • Chermat R, Simon P (1975) Appréciation de la catalepsie chez le rat. J Pharmacol 6:493–496

  • Costall B, Naylor RJ (1973) Is there a relationship between the involvement of extrapyramidal and mesolimbic brain areas with the cataleptic action of neuroleptic agents and their clinical antipsychotic effects? Psychopharmacology (Berl) 32:161–170

  • Costall B, Naylor RJ (1974) On catalepsy and catatonia and the predictability of the catalepsy test for neuroleptic activity. Psychopharmacology (Berl) 34:233–241

  • Duvoisin R (1976) Parkinsonism: animal analogues of the human disorder. In: Yahr M (ed) The basai ganglia. Raven Press, New York, pp 293–303

  • Ellenbroek B, Cools AR (1988) The PAW test: an animal model for neuroleptic drugs which fulfils the criteria for pharmacological isomorphism. Life Sci 42:1205–1213

  • Ellenbroek B, Cools AR (2000) Animal models for the negative symptoms of schizophrenia. Behav Pharmacol 11:223–233

  • Ellenbroek BA, Peeters BWE, Honig WM, Cools AR (1987) The paw test: a behavioural paradigm for differentiating between classical and atypical neuroleptic drugs. Psychopharmacology (Berl) 93:343–345

  • Ellenbroek B, Liégeois JF, Bruhwyler J, Cools AR (2001) Effects of JL 13, a pyridobenzoxazepine with potential atypical antipsychotic activity, in animal models of schizophrenia. J Pharmacol Exp Ther 298:386–391

  • Gerlach J, Casey DE (1990) Remoxipride, a new selective D2 antagonist, and haloperidol in Cebus monkeys. Prog Neuropsychopharmacol Biol Psychiatry 14:103–112

  • Honma T, Fukushima H (1976) Correlation between catalepsy and dopamine decrease in the rat striatum induced by neuroleptics. Neuropharmacology 15:601–607

  • Locke KW, Dunn RW, Hubbard JW, Vanselous CL, Cornfeldt M, Fielding S, Strupczewski JT (1990) HP 818: a centrally analgesic with neuroleptic properties. Drug Dev Res 19:239–256

  • Moore NA, Tye NC, Axton MS, Risius FC (1992) The behavioral pharmacology of olanzapine, a novel “atypical” antipsychotic agent. J Pharmacol Exp Ther 262:545–551

  • Prinssen EP, Ellenbroek BA, Cools AR (1994) Peripheral and central adrenoceptor modulation of the behavioural effects of clozapine in the paw test. Br J Pharmacol 112:769–774

  • Prinssen EPM, Ellenbroek BA, Stamatovic B, Cools AR (1995) Role of striatal dopamine D2 receptors in the paw test, an animal model for the therapeutic efficacy and extrapyramidal side effects of neuroleptic drugs. Brain Res 673:283–289

  • Szewczak MR, Cornfeldt ML, Dunn RW, Wilker JC, Geyer HM, Glamkowski EJ, Chiang Y, Fielding S (1987) Pharmacological evaluation of HP 370, a potential atypical antipsychotic agent. 1. In vivo profile. Drug Dev Res 11:157–168

Pole Climb Avoidance in Rats

Purpose and Rationale

The pole-climb avoidance paradigm is an avoidance escape procedure used to separate neuroleptics from sedatives and anxiolytics. Whereas sedative compounds suppress both avoidance and escape responding at approximately the same doses, neuroleptic drugs reduce avoidance responding at lower doses than those affecting escape responding (Cook and Catania 1964).

Procedure

Male rats of the Long-Evans strain with a starting body weight of 250 g are used. The training and testing of the rats is conducted in a 25 × 25 × 40 cm chamber that is enclosed in a dimly lit, sound-attenuating box. Scrambled shock is delivered to the grid floor of the chamber. A 2.8-kHz speaker and a 28-V light are situated on top of the chamber. A smooth stainless-steel pole, 2.5 cm in diameter, is suspended by a counterbalance weight through a hole in the upper center of the chamber. A microswitch is activated when the pole is pulled down 3 mm by a weight greater than 200 g. A response is recorded when a rat jumps on the pole and activates the microswitch. The rat cannot hold the pole down while standing on the grid floor because of the counterbalance tension and cannot remain on the pole any length of time because of its smooth surface. The activation of the light and the speaker together is used as the conditioning stimulus. The conditioning stimulus is presented alone for 4 s and then is coincident with the unconditioned stimulus, a scrambled shock delivered to the grid floor, for 26 s. The shock current is maintained at 1.5 mA. A pole climb response during the conditioned stimulus period terminates the conditioned stimulus and the subsequent conditioned and unconditioned stimuli. This is considered an avoidance response. A response during the time when both the conditioned and unconditioned stimuli are present terminates both stimuli and is considered an escape response. Test sessions consist of 25 trials or 60 min, whichever comes first. There is a minimum intertrial interval of 90 s. Any time remaining in the 30 s allotted to make the pole climb is added to the 90 s intertrial interval. Responses during this time have no scheduled consequences; however, rats having greater than ten intertrial interval responses should not be used in the experiment. Before testing experimental compounds, rats are required to make at least 80 % avoidance responses without any escape failures.

Evaluation

Data are expressed in terms of the number of avoidance and escape failures relative to the respective vehicle control data. ED 50 values can be calculated using different doses.

References and Further Reading

  • Cook L, Catania AC (1964) Effects of drugs on avoidance and escape behavior. Fed Proc 23:818–835

  • Cook L, Weidley E (1957) Behavioral effects of some psychopharmacological agents. Ann N Y Acad Sci 66:740–752

  • Dunn RW, Carlezon WA, Corbett R (1991) Preclinical anxiolytic versus antipsychotic profiles of the 5-HT3-antagonists Ondansedron, Zacopride, 3α-tropanyl-1Hindole-3-carboxylic ester, and 1αH, 3α, 5αH-tropan-3-yl-3,5-dichlorobenzoate. Drug Dev Res 23:289–300

  • Locke KW, Dunn RW, Hubbard JW, Vanselous CL, Cornfeldt M, Fielding S, Strupczewski JT (1990) HP 818: a centrally acting analgesic with neuroleptic properties. Drug Dev Res 19:239–256

  • Szewczak MR, Cornfeldt ML, Dunn RW, Wilker JC, Geyer HM, Glamkowski EJ, Chiang Y, Fielding S (1987) Pharmacological evaluation of HP 370, a potential atypical antipsychotic agent. 1. In vivo profile. Drug Dev Res 11:157–168

Footshock-Induced Aggression

Purpose and Rationale

The test as described by Tedeschi et al. (1959) using mice which fight after footshock-induced stimulation is useful to detect neuroleptics but also shows positive effects with anxiolytics and other centrally effective drugs. The method has been used by several authors to test drugs with neuroleptic activity. The test is described in chapter “Tests for Anxiolytic Activity”.

References and Further Reading

  • Tedeschi RE, Tedeschi DH, Mucha A, Cook L, Mattis PA, Fellows EJ (1959) Effects of various centrally acting drugs on fighting behavior of mice. J Pharmacol Exp Ther 125:28–34

Brain Self-Stimulation

Purpose and Rationale

In several species, electrical stimulation of selected brain loci produces effects which are positively reinforcing and pleasurable (Olds and Milner 1954; Olds 1961, 1972). Most of the data available have been obtained from experiments using rats with electrodes chronically implanted in the median forebrain bundle at the level of hypothalamus. Minute electrical pulses sustain a variety of operant behaviors such as lever pressing. Neuroleptics have been shown to be potent blockers of self-stimulation (Broekkamp and Van Rossum 1975; Koob et al. 1978; Gallistel and Freyd 1987). Conversely, compounds that facilitate catecholaminergic transmission such as d-amphetamine and methylphenidate will increase responding for such stimulation.

Procedure

Male Wistar rats (350–400 g) are anesthetized with 50 mg/kg pentobarbital i.p. and their heads placed on a level plane in a Kopf stereotactic instrument. A midline incision is made in the scalp and the skin held out of the way by muscle retractors. A small hole is drilled in the scull with a dental burr at the point indicated by the stereotactic instrument for the structure it is desired to stimulate. Using bregma as a reference point, the electrode (Plastic Products MS303/1) is aimed at the medium forebrain bundle according to the atlas of Paxinos and Watson (1986), using the coordinates of AP = −0.8 mm, Lat = +2.8 mm, and DV = −7.2 mm below dura. The assembly is then permanently affixed to the scull using stainless-steel screws and bone cement.

After a minimum of 10 days for recovery, the animals are trained to bar press for electrical stimulation on a continuous reinforcement schedule in a standard operant box outfitted with a single lever. The reward stimulus is a train of biphasic square-wave pulses generated by a Haer stimulator (Pulsar 4i). The parameters are set at a pulse duration of 0.5 ms with 2.5 ms between each pulse pair. The train of pulses may vary between 16 and 30/s, and the intensity of the pulses that are delivered range from 0.1 to 0.5 mA using the lowest setting that will sustain maximal responding. After consistent baseline responding is obtained for five consecutive 30-min session, the animals are ready for testing with standard agents. Compounds are administered 60 min. prior to testing. All data are collected on both cumulative recorders and counters.

Evaluation

The number of drug responses is compared to the number of responses made during each animal’s 30-min control session on the preceding day, which is considered to be equal to 100 %. Testing various doses, ED 50 values with 95 % confidence limits can be calculated.

Critical Assessment of the Method

Since there is sufficient evidence that self-stimulation behavior is maintained by catecholamines, the method gives indirectly insight into the catecholaminergic facilitating or blocking properties of a compound. Active neuroleptic drugs inhibit the self-stimulation behavior in very small doses. The relative potency observed in this test of clinically efficacious drugs parallels their potency in the treatment of schizophrenia.

Modifications of the Method

Reinforcing brain stimulation by electrodes placed in the medial forebrain bundle of rats is decreased after lesion of the internal capsule in the region of the diencephalic–telencephalic border. This decrement in rewarding processing can be reversed by antidepressant drugs (Cornfeldt et al. 1982).

Depoortere et al. (1996) used electrical self-stimulation of the ventral tegmental area to study the behavioral effects of a putative dopamine D3 agonist in the rat.

Anderson et al. (1995) examined the interaction of aversive and rewarding stimuli in self-stimulating rats in terms of duration and direction. The rats were implanted with two moveable electrodes, one in a region supporting self-stimulation (the ventral tegmental area) and another in a region supporting escape (the nucleus reticularis gigantocellularis).

Kokkinidis et al. (1986) used amphetamine withdrawal for a behavioral evaluation. Mice implanted with stimulating electrodes in the lateral hypothalamus demonstrated stable and reliable rates of self-stimulation responding. After exposure to a chronic schedule of amphetamine treatment, response rates were severely depressed.

Post-amphetamine depression of self-stimulation from the substantia nigra can be reversed by cyclic antidepressants (Kokkinidis et al. 1980).

Moreau et al. (1992) reported that antidepressant treatment prevents chronic unpredictable mild stress-induced anhedonia as assessed by ventral tegmentum self-stimulation in rats.

References and Further Reading

  • Anderson R, Diotte M, Miliaressis E (1995) The bidirectional interaction between ventral tegmental rewarding and hindbrain aversive stimulation effects in rats. Brain Res 688:15–20

  • Brodie DA, Moreno OM, Malis JE, Boren JJ (1960) Rewarding properties of intracranial stimulation. Science 131:920–930

  • Broekkamp CLE, Van Rossum JM (1975) The effect of microinjections of morphine and haloperidol into the neostriatum and the nucleus accumbens on self-stimulation behavior. Arch Int Pharmacodyn 217:110–117

  • Corbett D, Laferriere A, Milner P (1982) Plasticity of the medial prefrontal cortex: facilitated acquisition of intracranial self-stimulation by pretraining stimulation. Physiol Behav 28:531–543

  • Cornfeldt M, Fisher B, Fielding S (1982) Rat internal capsule lesion: a new test for detecting antidepressants. Fed Proc 41:1066

  • Depoortere R, Perrault G, Sanger DJ (1996) Behavioral effects in the rat of the putative dopamine D3 receptor agonist 7-OH-DPAT: comparison with quinpirole and apomorphine. Psychopharmacology (Berl) 124:231–240

  • Dunn RW, Carlezon WA, Corbett R (1991) Preclinical anxiolytic versus antipsychotic profiles of the 5-HT3 antagonists ondansetron, zacopride, 3α-tropanyl-1H-indole-3-carboxylic acid ester, and 1αH, 3α, 5αH-tropan-3-yl-3,5-dichlorobenzoate. Drug Dev Res 23:289–300

  • Fielding S, Lal H (1978) Behavioral actions of neuroleptics. In: Iversen LL, Iversen SD, Snyder SH (eds) Neuroleptics and Schizophrenia, vol 10. Plenum Press, New York, pp 91–128

  • Gallistel CR, Freyd G (1987) Quantitative determination of the effects of catecholaminergic agonists and antagonists on the rewarding efficacy of brain stimulation. Pharmacol Biochem Behav 26:731–741

  • Goldstein JM, Malick JB (1983) An automated descending rate intensity self-stimulation paradigm: usefulness for distinguishing antidepressants from neuroleptics. Drug Dev Res 3:29–35

  • Kokkinidis L, Zacharko RM, Predy PA (1980) Post-amphetamine depression of self-stimulation from the substantia nigra: reversal by cyclic antidepressants. Pharmacol Biochem Behav 13:379–383

  • Kokkinidis L, Zacharko RM, Anisman H (1986) Amphetamine withdrawal: a behavioral evaluation. Life Sci 38:1617–1623

  • Koob GF, Fray PJ, Iversen SD (1978) Self-stimulation at the lateral hypothalamus and locus coeruleus after specific unilateral lesions of the dopamine system. Brain Res 146:123–140

  • Mekarski JE (1989) Main effects of current and pimozide on prepared and learned self-stimulation behaviors are on performance not reward. Pharmacol Biochem Behav 31:845–853

  • Mora F, Vives F, Alba F (1980) Evidence for an involvement of acetylcholine in self-stimulation of the prefrontal cortex in the rat. Experientia 36:1180–1181

  • Moreau JL, Jenck F, Martin JR, Mortas P, Haefely WE (1992) Antidepressant treatment prevents chronic unpredictable mild stress-induced anhedonia as assessed by ventral tegmentum self stimulation in rats. Eur Neuropsychopharmacol 2:43–49

  • Olds J (1961) Differential effects of drives and drugs on self-stimulation at different brain sites. In: Sheer DE (ed) Electrical stimulation of the brain. University of Texas Press, Austin, pp 350–366

  • Olds ME (1972) Alterations by centrally acting drugs of the suppression of self-stimulation behavior in the rat by tetrabenazine, physostigmine, chlorpromazine and pentobarbital. Psychopharmacology (Berl) 25:299–314

  • Olds J, Milner P (1954) Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain. J Comp Physiol Psychol 47:419–427

  • Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates, 2nd edn. Academic, New York

  • Roberts DCS, Zito KA (1987) Interpretation of lesion effects on stimulant self-administration. In: Bozarth MA (ed) Methods for assessing the reinforcing properties of abused drugs. Springer, New York/Berlin/Heidelberg, pp 87–103

  • Szewczak MR, Cornfeldt ML, Dunn RW, Wilker JC, Geyer HM, Glamkowski EJ, Chiang Y, Fielding S (1987) Pharmacological evaluation of HP 370, a potential atypical antipsychotic agent. 1. In vivo profile. Drug Dev Res 11:157–168

Prepulse Inhibition of Startle Response

Purpose and Rationale

Prepulse inhibition is a model of sensorimotor gating which can be assessed in both animals and humans using the startle reflex response. When a fixed startle-eliciting stimulus (i.e., the pulse) is preceded by 30–500 ms by a weak, non-startle-eliciting stimulus (i.e., the prepulse), the magnitude of the startle response is significantly reduced to the pulse alone. Schizophrenic patients have decreased prepulse inhibition relative to normal control subjects, and this is thought to reflect an impairment in their ability to filter irrelevant sensory stimuli (Braff and Geyer 1990; Geyer 1998). Similar reductions in prepulse inhibition are produced in rats by administration of psychotomimetic drugs such as the dopamine agonists amphetamine and apomorphine or the noncompetitive NMDA antagonists phencyclidine and dizocilpine (MK801) (Mansbach and Geyer 1989; Swerdlow et al. 1998; Geyer et al. 2001; Rowley et al. 2001; Weiss and Feldon 2001; Pouzet et al. 2002). Most antipsychotics tested are able to antagonize prepulse inhibition disruption produced by dopamine antagonists, whereas prepulse inhibition disruption by NMDA antagonists may be selectively sensitive to antipsychotics with atypical features (Bakshi and Geyer 1995; Bubenikova et al. 2005; Fox et al. 2005). Haloperidol failed to block the effects of phencyclidine and dizocilpine prepulse inhibition of startle (Keith et al. 1991).

Feifel et al. (Feifel and Reza 1999; Feifel et al. 1999a, b) tested the effects of a neurotensin agonist on prepulse inhibition of startle in rats.

Procedure

Male Sprague Dawley rats were treated with various doses of test compound or saline s.c. Immediately afterwards, rats receive a second s.c. injection consisting of 2 mg/kg d-amphetamine, or 0.5 mg/kg apomorphine, or 0.1 mg/kg dizocilpine or saline. Then, 10 min later, animals were placed in special startle chambers (SR-LAB, San Diego Instruments, San Diego, Calif., USA). Startle chambers consist of a Plexiglas cylinder 8.2 cm in diameter, resting on a 12.5 × 25.5 cm Plexiglas frame within a ventilated enclosure housed in a sound-attenuated room exposed to 70-dB background noise. After a 5-min acclimation period, acoustic stimuli were presented via a speaker mounted 24 cm above the animal. Acoustic stimuli consisted of a 120-dB pulse by itself (pulse alone) or a 120-dB pulse preceded by 100 ms by prepulses 3, 5, and 10 dB above background noise. There was an average of 15 s between stimuli. A piezoelectric accelerometer mounted below the Plexiglas frame detected and transduced the motion within the cylinder. Startle amplitude was defined as the degree of motion detected by this accelerometer. Each rat was tested on four separate occasions separated by 7 non-test days. On each test day, the dose of test compound was kept constant, but the specific psychotomimetic agent was alternated across test days in a counterbalanced fashion.

Evaluation

Prepulse inhibition was calculated as the percentage of the pulse-alone startle amplitude using the following formula: [1 (startle amplitude after prepulse–pulse pair/startle amplitude after pulse only)] × 100. Analysis of data was then carried out using a three-factor repeated-measures analysis of variance (ANOVA). Significant factor results from the ANOVA were followed up with separate one-way ANOVAs for each psychotomimetic agent and then, when indicated, with individual group mean comparisons using post hoc t-tests for multiple comparisons using the Bonferroni method.

Modifications of the Method

Sipes and Geyer (1995) studied the disruption of prepulse inhibition of the startle response in the rat by DOI [(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride], which is mediated by 5-HT2A receptors. The authors suggested that studies of the serotonergic substrates of prepulse inhibition may provide a model of the possible serotonergic role in the sensorimotor gating abnormalities in patients with schizophrenia and with obsessive-compulsive disorder.

Ellenbroeck et al. (1998) described the effects of an early stressful life event on sensorimotor gating in adult rats.

Andersen and Pouzet (2001) compared the effects of acute versus chronic treatment with typical or atypical antipsychotics on d-amphetamine-induced sensorimotor gating deficits in rats.

Heidbreder et al. (2000) used the prepulse inhibition of acoustic startle for behavioral, neurochemical, and endocrinological characterization of the early social isolation syndrome.

Krebs-Thomson et al. (2001) reported that postweanling handling attenuates isolation-rearing disruption of prepulse inhibition in rats.

Weiss et al. (2001) studied the dissociation between the effects of preweaning and/or postweaning social isolation on prepulse inhibition and latent inhibition in adult Sprague Dawley rats.

Dirks et al. (2003) reported reversal of startle gating deficits in transgenic mice overexpressing corticotropin-releasing factor by antipsychotic drugs.

Andreasen et al. (2006) studied the effect of nicotinic agents on prepulse inhibition (PPI) in mice using a startle response/PPI system from TSE Systems, Bad Homburg, Germany.

Lind et al. (2004) described prepulse inhibition of the acoustic startle reflex in pigs and its disruption by d-amphetamine.

References and Further Reading

  • Andersen PM, Pouzet B (2001) Effects of acute versus chronic treatment with typical or atypical antipsychotics on d-amphetamine-induced sensorimotor gating deficits in rats. Psychopharmacology (Berl) 156:291–304

  • Andreasen JT, Andersen KK, Nielsen EØ, Mathiasen L, Mirza NR (2006) Nicotine and clozapine selectively reverse a PCP-induced deficit of PPI in BALB/cByJ but not in NMRI mice: comparison with risperidone. Behav Brain Res 167:118–127

  • Bakshi VP, Geyer MA (1995) Antagonism of phencyclidine-induced deficits in prepulse inhibition by the putative “atypical” antipsychotic olanzapine. Psychopharmacology (Berl) 122:198–201

  • Braff DL, Geyer MA (1990) Sensorimotor gating and schizophrenia: human and animal models. Arch Gen Psychiatry 47:181–188

  • Bubenikova V, Votava M, Horacek J, Palenicek T, Dockery C (2005) The effect of zotepine, risperidone, clozapine and olanzapine on MK-801-disrupted sensorimotor gating. Pharmacol Biochem Behav 80:591–596

  • Dirks A, Groenink L, Westphal KGC, Olivier JDA, Verdouw PM, van der Gugten J, Ma G, Olivier B (2003) Reversal of startle gating deficits in transgenic mice overexpressing corticotropin-releasing factor by antipsychotic drugs. Neuropsychopharmacology 28:1790–1798

  • Ellenbroeck BA, van den Kroonenberg PTJM, Cools AR (1998) The effects of an early stressful life event on sensorimotor gating in adult rats. Schizophr Res 30:251–260

  • Feifel D, Reza TL (1999) Effects of neurotensin administered into the tegmental area on prepulse inhibition of startle. Behav Brain Res 106:189–193

  • Feifel D, Reza TL, Wustro DJK, Davis D (1999a) Novel antipsychotic-like effects on prepulse inhibition of startle produced by a neurotensin agonist. J Pharmacol Exp Ther 288:710–713

  • Feifel D, Reza T, Robeck S (1999b) Antipsychotic potential of CCK-based treatments: an assessment using the prepulse inhibition model of psychosis. Neuropsychopharmacology 20:141–149

  • Fox GB, Esbenshade TA, Pan JB, Radek RJ, Krueger KM, Yao BB, Browman KE, Buckley MJ, Ballard ME, Komater VA, Miner H, Zhang M, Faghih R, Rueter LE, Bitner RS, Drescher KU, Wetter J, Marsh K, Lemaire M, Porsolt RD, Bennani YL, Sullivan JP, Cowart MD, Decker MW, Hancock AA (2005) Pharmacological properties of ABT-239 [4-(2-2-[(2R)-2-methylpyrrolidinyl]ethyl-benzofuran-5-yl)bezonitrile]: II. Neuropharmacological characterization and broad preclinical efficacy in cognition and schizophrenia of a potent and selective histamine H3 receptor antagonist. J Pharmacol Exp Ther 313:176–190

  • Geyer MA (1998) Behavioural studies of hallucinogenic drugs in animals: implications for schizophrenia research. Pharmacopsychiatry 31:73–79

  • Geyer MA, Krebs-Thomsen K, Braff DL, Swerdlow NR (2001) Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacology (Berl) 156:117–154

  • Heidbreder CA, Weiss IC, Domeney AM, Pryce C, Homberg J, Hedou G, Feldon J, Moran MC, Nelson P (2000) Behavioral, neurochemical and endocrinological characterization of the early social isolation syndrome. Neuroscience 100:749–768

  • Keith VA, Mansbach RS, Geyer MA (1991) Failure of haloperidol to block the effects of phencyclidine and dizocilpine prepulse inhibition of startle. Biol Psychiatry 30:557–566

  • Krebs-Thomson K, Giracello D, Solis A, Geyer MA (2001) Post-weanling handling attenuates isolation-rearing disruption of prepulse inhibition in rats. Behav Brain Res 120:221–224

  • Lind NM, Arnfred SM, Hemmingsen RP, Hansen AK (2004) Prepulse inhibition of the acoustic startle reflex in pigs and its disruption by D-amphetamine. Behav Brain Res 155:217–222

  • Mansbach RS, Geyer MA (1989) Effects of phencyclidine and phencyclidine biologs on sensorimotor gating in the rat. Neuropsychopharmacology 2:299–306

  • Pouzet B, Didriksen M, Arnt J (2002) Effects of the 5-HT6 receptor antagonist, SB-271046, in animal models of schizophrenia. Pharmacol Biochem Behav 71:635–643

  • Rowley M, Bristow LJ, Hutson PH (2001) Current and novel approaches to the drug treatment of schizophrenia. J Med Chem 44:477–501

  • Sipes TE, Geyer MA (1995) DOI disruption of prepulse inhibition of startle in the rat is mediated by 5-HT2A and not by 5-HT2C receptors. Behav Pharmacol 6:839–842

  • Swerdlow NR, Taaid N, Oostwegel JL, Randolph E, Geyer MA (1998) Towards a cross-species pharmacology of sensorimotor gating: effects of amantadine, bromocriptine, pergolide and ropinirole on prepulse inhibition of acoustic startle in rats. Behav Pharmacol 9:389–396

  • Weiss IC, Feldon J (2001) Environmental animal models for sensorimotor gating deficiencies in schizophrenia: a review. Psychopharmacology (Berl) 156:305–326

  • Weiss IC, Domeney AM, Moreau JL, Russig H, Feldon J (2001) Dissociation between the effects of pre-weaning and/or post-weaning social isolation on prepulse inhibition and latent inhibition in adult Sprague–Dawley rats. Behav Brain Res 121:207–218

N40 Sensory Gating

Purpose and Rationale

The N40 auditory-evoked potential has been used to develop an animal model for the study of sensory gating mechanisms (Boutros et al. 1997a; Boutros and Kwan 1998). The method has been applied to evaluation of psychotropic compounds (Adler et al. 1986; Boutros et al. 1994, 1997b). Bickford-Wimer et al. (1990) localized one possible source of the N40 waveform to the CA3 region of the hippocampus.

Fox et al. (2005) used the N40 sensory gating model in mice for evaluation of a potential antipsychotic drug.

Procedure

Male DBA/2 mice were stereotaxically implanted with tripolar stainless-steel wire head stages for EEG recordings in the CA3 region of the hippocampus. The mice were first anesthetized with a solution of 2.8 % ketamine, 0.28 % xylazine, and 0.05 % acepromazine. Three access holes for the electrodes were made at AP −1.8 mm from the bregma, and in a plane perpendicular to the suture, ML 0.6 (cortical electrode), 1.6 (reference electrode), and 2.6 mm electrode directed at the hippocampus). The depth of the hippocampal electrode tip was DV 1.65–1.70 mm below the surface of the cortex. The depths of the cortical and reference electrodes were DV 0.5 mm from the surface of the skull, resulting in contact, but not penetration, of the cortical tissue. The tripolar electrode was lowered into position with a stereotaxic electrode holder and affixed using cyanoacrylic gel and dental acrylic and two anchor screws. Mice were allowed to recover for 3 days before commencement of the experiments. Awake mice were recorded in acoustically isolated chambers. Flexible tethers and electrical swivels were used to convey EEG signals to differential AC EEG amplifiers and allowed the mice free movement within the chambers. The EEG was amplified 1000 × with a 50- to 60-Hz notch filter engaged, and high- and low-pass filters were set at 1 and 100 Hz, respectively. Hippocampal auditory-evoked potentials were generated by presentation of 60 sets of 3 kHz-paired tone bursts from a speaker within the recording chamber at a distance of 15–20 cm to the mouse. The first tone of the pair is referred to as the conditioning stimulus, and the second is referred to as the test stimulus. The duration of both the condition and test stimuli was 5 ms, with 0.5 s between the stimuli and 20 s between pairs. Data acquisition software recorded EEG signals 100 ms before and for 899 ms after the initial conditioning stimulus. The software averaged the 60-paired responses into one composite-evoked response. Various doses of test drug were administered i.p. 20–30 min before mice were placed into the recording chambers and initiation of auditory-evoked potential recording. Recording of paired auditory potentials continued for two 20-min sessions, each comprised of 60 paired stimuli. Each mouse was administered every treatment dose and a control vehicle treatment in a balanced order on separate days with at least 48 h between treatments. This within-subject design allowed each mouse to serve as its own control. The hippocampal response to auditory stimuli was identified as the highest positive peak deflection in the ongoing EEG at a latency of 10–20 ms after the stimulus (P20), followed by the lowest negative peak deflection in the ongoing EEG at 20–45 ms after the stimulus (N40). The difference in amplitude between P20 and N40 was defined as the N40 amplitude in microvolts.

Evaluation

N40 amplitudes were determined for both the averaged conditioning and test-evoked potentials, and a ratio was derived between the two responses by dividing the test amplitude by the conditioning amplitude (T/C ratio).

Modifications of the Method

Flack et al. (1996) studied sensory gating in a computer model of the CA3 neural network of the hippocampus.

Stevens et al. (1998) investigated changes in auditory information processing after kainic acid lesions in adult rats used as a model of schizophrenia.

References and Further Reading

  • Adler LE, Rose G, Freedman R (1986) Neurophysiological studies of sensory gating in rats: effects of amphetamine, phencyclidine, and haloperidol. Biol Psychiatry 21:787–798

  • Bickford-Wimer PC, Nagamoto H, Johnson R, Adler LE, Egan M, Rose GM, Freedman R (1990) Auditory sensory gating in hippocampal neurons: a model system in the rat. Biol Psychiatry 27:183–192

  • Boutros NN, Kwan SW (1998) Test-retest reliability of the rat N40 auditory evoked response: preliminary data. Psychiatry Res 81:269–276

  • Boutros NN, Uretsky N, Berntson G, Bornstein R (1994) Effects of cocaine on sensory inhibition in rats: preliminary data. Biol Psychiatry 36:242–248

  • Boutros NN, Bonnet KA, Millana R, Liu J (1997a) A parametric study of the N40 auditory evoked response in rats. Biol Psychiatry 42:1051–1059

  • Boutros NN, Uretsky NJ, Lui JJ, Millana RB (1997b) Effects of repeated cocaine administration on sensory inhibition in rats: preliminary data. Biol Psychiatry 41:461–466

  • Flack KA, Adler LE, Gerhardt GA, Miller C, Bickford P, Mac-Gregor RJ (1996) Sensory gating in a computer model of the CA3 neural network of the hippocampus. Biol Psychiatry 40:1230–1245

  • Fox GB, Esbenshade TA, Pan JB, Radek RJ, Krueger KM, Yao BB, Browman KE, Buckley MJ, Ballard ME, Komater VA, Miner H, Zhang M, Faghih R, Rueter LE, Bitner RS, Drescher KU, Wetter J, Marsh K, Lemaire M, Porsolt RD, Bennani YL, Sullivan JP, Cowart MD, Decker MW, Hancock AA (2005) Pharmacological properties of ABT-239 [4-(2-2-[(2R)-2-methylpyrrolidinyl]ethyl-benzofuran-5-yl)bezonitrile]: II. Neuropharmacological characterization and broad preclinical efficacy in cognition and schizophrenia of a potent and selective histamine H3 receptor antagonist. J Pharmacol Exp Ther 313:176–190

  • Stevens KE, Nagamoto H, Johnson RG, Adams CE, Rose GM (1998) Kainic acid lesions in adult rats as a model of schizophrenia: changes in auditory information processing. Neuroscience 82:701–708

Latent Inhibition

Purpose and Rationale

Latent inhibition has been recommended as an animal model of schizophrenia (Feldon and Weiner 1992; Swerdlow et al. 1996; Vaitl and Lipp 1997; Moser et al. 2000; Bender et al. 2001). Latent inhibition refers to the retarded acquisition of a conditioned response that occurs if the subject being tested is first preexposed to the to-be-conditioned stimulus without the paired unconditioned stimulus. Because the “irrelevance” of the to-be-conditioned stimulus is established during non-contingent preexposure, the slowed acquisition of the conditioned stimulus-unconditioned stimulus association is thought to reflect the process of overcoming this learned irrelevance. Latent inhibition has been reported to be diminished in acutely hospitalized schizophrenia patients. Several authors used the latent inhibition model in rats to test psychotropic compounds (Solomon et al. 1981; Feldon and Weiner 1991; Moran and Moser 1992; De la Casa et al. 1993; Lacroix et al. 2000; Alves et al. 2002). Trimble et al. (2002) tested the effects of selective D1 antagonists on latent inhibition in the rat.

Procedure

Animals

Male Sprague Dawley rats weighing 300–400 g were housed two to a cage under a 12-h reversed cycle lighting with food and water ad libitum. All experimental manipulations were carried out in the dark phase of the dark/light cycle.

Apparatus

Modified metal Skinner boxes (24.5 × 24.5 × 21 cm measured from a raised grid floor) were located in darkened, sound-insulated, ventilated outer boxes. A removable water bottle was located on one side of each Skinner box through a hole of 1.0 cm diameter, positioned 2 cm above the grid floor. When water was not required, the water bottle was removed. Licks at the spout of each water bottle were recorded using a lickometer (model 453, Campden Instruments, London, UK). The preexposed stimulus was a flashing light (10 s duration with three light flashes per second) situated in the middle of the roof of each Skinner box. The grid floor consisted of steel bars (0.5 cm in diameter) spaced 1 cm apart. Shock generators with scramblers were calibrated to produce 0.5-mA shocks via the grid floor.

Procedure

Rats were randomly assigned to experimental groups and were allocated to a particular Skinner box. They had experience of only that box for the duration of the experiment. After adaptation to the housing conditions for 1 week, rats were placed immediately on a 23-h water deprivation schedule that continued until the end of the experiments. Food remained freely available.

Baseline Days (Days 15–19)

After 7 days on the water deprivation period, 5 days of pretraining commenced. Each rat was placed in a Skinner box for 15 min. The water bottle was present and each rat could drink freely. After the baseline session was over, each rat was returned to its home cage and allowed access to water for 45 min.

Preexposure (Day 20)

With the water bottle removed, each rat was placed in a Skinner box. Rats received ten stimulus (flashing house-light) presentations of 10 s duration (three light flashes per second) with a fixed stimulus interval of 50 s. Afterwards the rats were returned to their home cages and allowed access to water for 1 h.

Conditioning (Day 21)

With the water bottle removed, each rat was placed in a Skinner box. Then, 5 min later, each rat received the first of two light footshock pairings. House-light parameters were identical to those of the preexposure period. The house-light was immediately followed by the footshock (0.5 mA, 1 s). The second light-shock pairing was given 5 min later. After the conditioning period had terminated, animals were returned to their home cages and allowed access to water for 1 h.

Re-baseline Day (Day 22)

With the water bottle present, each rat was placed in a Skinner box and allowed to drink as in the baseline sessions.

Test Day (Day 23)

With the water bottle present, each rat was placed in a Skinner box and allowed to drink. When each rat completed 75 licks, the flashing house-light was presented and continued until 5 min had elapsed from stimulus onset. Time bins of 30-s duration commenced from the time of stimulus presentation, and the number of licks made by each rat within every time bin was recorded. This measure allowed the pattern of drinking over the course of stimulus presentation to be shown. The amount of suppression of licking for each rat was assessed using a suppression ratio calculated from the time (in seconds) to complete licks 51–75 (pre-stimulus) divided by the time (in seconds) to complete licks 51–100 (pre-stimulus + stimulus on). A suppression ratio of 0.01 indicates total suppression of licking (no latent inhibition), while a ratio of 0.5 indicates no change in licking rate from the pre-stimulus period to the stimulus-on period (latent inhibition).

DRUG Treatment

Test drugs or vehicle was administered by subcutaneous injection in various doses 30 min prior to preexposure and conditioning.

Evaluation

Times to complete licks and the suppression ratios were analyzed independently using a 2 × 6 ANOVA with main factors of preexposure and drugs.

Modifications of the Method

Lehmann et al. (1998) studied the long-term effects of repeated maternal separation on three different latent inhibition paradigms.

Pouzet et al. (2004) reported that latent inhibition is spared by NMDA-induced ventral hippocampal lesions, but is attenuated following local activation of the ventral hippocampus by intracerebral NMDA infusion.

Bethus et al. (2005) examined the effects of prenatal stress and gender in latent inhibition.

References and Further Reading

  • Alves CRR, Delucia R, Silva MTA (2002) Effects of fencamfamine on latent inhibition. Prog Neuropsychopharmacol Biol Psychiatry 26:1089–1093

  • Bender S, Müller B, Oades RD, Sartory G (2001) Conditioned blocking and schizophrenia: a replication and study of the role of symptoms, age, onset, onset-age of psychosis and illness-duration. Schizophr Res 49:157–170

  • Bethus I, Lemaire V, Lhomme M, Goodall G (2005) Does prenatal stress affect latent inhibition? It depends on the gender. Behav Brain Res 158:331–338

  • De la Casa LG, Ruiz G, Lubow RE (1993) Amphetamine-produced attenuation of latent inhibition is modulated by stimulus preexposure duration: implications for schizophrenia. Biol Psychiatry 15:707–711

  • Feldon J, Weiner I (1991) The latent inhibition model of schizophrenic attention disorder: haloperidol and sulpiride enhance rats’ ability to ignore irrelevant stimuli. Biol Psychiatry 29:635–646

  • Feldon J, Weiner I (1992) From an animal model of an attentional deficit towards new insights into pathophysiology of schizophrenia. J Psychiatr Res 26:345–366

  • Lacroix L, Broersen LM, Feldon J, Weiner I (2000) Effect of local infusions of dopaminergic drugs into the medial prefrontal cortex of rats on latent inhibition, prepulse inhibition and amphetamine induced activity. Behav Brain Res 107:111–121

  • Lehmann J, Stöhr T, Schuller J, Domeney A, Heidbreder C, Feldon J (1998) Long-term effects of repeated maternal separation on three different latent inhibition paradigms. Pharmacol Biochem Behav 59:873–882

  • Moran PM, Moser PC (1992) MDL 73,147EF, a 5-HT3 antagonist, facilitates latent inhibition in the rat. Pharmacol Biochem Behav 42:519–522

  • Moser PC, Hitchcock JM, Lister S, Moran PM (2000) The pharmacology of latent inhibition as an animal model of schizophrenia. Brain Res Rev 33:275–307

  • Pouzet B, Zhang WN, Weiner I, Feldon J, Yee BK (2004) Latent inhibition is spared by N-methyl-D-aspartate (NMDA)induced ventral hippocampal lesions, but is attenuated following local activation of the ventral hippocampus by intracerebral NMDA infusion. Neuroscience 124:183–194

  • Solomon PR, Crider A, Winkelman JW, Turi A, Kamer RM, Kaplan LJ (1981) Disrupted latent inhibition in the rat with chronic amphetamine or haloperidol-induced hypersensitivity: relationship to schizophrenic attention disorder. Biol Psychiatry 16:519–537

  • Swerdlow NR, Braff DL, Hartston H, Perry W, Geyer MA (1996) Latent inhibition in schizophrenia. Schizophr Res 20:91–103

  • Trimble KM, Bell R, King DJ (2002) Effects of the selective D1 antagonists NNC 01–0112 and SCH 39166 on latent inhibition in the rat. Physiol Behav 77:115–123

  • Vaitl D, Lipp OV (1997) Latent inhibition and autonomic response: a psychophysiological approach. Behav Brain Res 88:85–93

Tests Based on the Mechanism of Action

Amphetamine Group Toxicity

Purpose and Rationale

It is well known that aggregation of mice in small cages greatly enhances the toxicity of amphetamine. The death rate can be reduced by pretreatment with neuroleptics. This phenomenon is generally accepted as an indicator of neuroleptic activity. The increased toxicity results from increased behavioral activation due to aggregation inducing an increase of circulating catecholamines. The mechanism can be understood by the fact that amphetamine is an indirectly acting sympathomimetic amine that exerts its effects primarily by releasing norepinephrine from storage sites in the sympathetic nerves. After administration of high doses of amphetamine, mice exhibit an elevated motor activity which is highly increased by aggregation. This increased behavioral activation is followed by death within 24 h in 80–100 % of control animals. Neuroleptics reduce this death rate. In contrast, non-neuroleptic sympatholytics and psychosedative agents like the barbiturates do not produce a dose-related protection. Moreover, anxiolytic agents like benzodiazepines are also found to be ineffective in the prevention of amphetamine group toxicity.

Procedure

Ten male mice of the NMRI-strain are used for each group. They are dosed with the test compound or the standard either orally or intraperitoneally and all placed in glass jars of 18 cm diameter. Untreated animals serve as controls. The test has to be performed at room temperature of 24 °C. Thirty min after i.p. or 1 h after oral administration, the mice receive 20 mg/kg d-amphetamine subcutaneously. The mortality is assessed 1, 4, and 24 h after dosing.

Evaluation

The mortality of amphetamine-only treated animals is at least 80 %. If less than 80 % die due to low ambient temperature, the test has to be repeated. The estimation of ED 50 values for protection and their confidence limits are calculated by probit analysis of the data using the number of dosed versus the number of surviving animals. Doses of 10 mg/kg chlorpromazine p.o. and 1 mg/kg haloperidol have been found to be effective.

Critical Assessment of the Method

The amphetamine group toxicity test has been used by many investigators and has been found to be a reliable method for detecting neuroleptic activity.

References and Further Reading

  • Chance MRA (1946) Aggregation as a factor influencing the toxicity of sympathomimetic amines in mice. J Pharmacol 87:214–217

  • Derlet RW, Albertson TE, Rice P (1990) Protection against d-amphetamine toxicity. Am J Emerg Med 8:105–108

  • Locke KW, Dunn RW, Hubbard JW, Vanselous CL, Cornfeldt M, Fielding S, Strupczewski JT (1990) HP 818: a centrally acting analgesic with neuroleptic properties. Drug Dev Res 19:239–256

Inhibition of Amphetamine Stereotypy in Rats

Purpose and Rationale

Amphetamine is an indirect acting sympathomimetic agent which releases catecholamines from its neuronal storage pools. In rats the drug induces a characteristic stereotypic behavior. This behavior can be prevented by neuroleptic agents.

Procedure

Groups of six Wistar rats with a body weight between 120 and 200 g are used. They are injected simultaneously with d-amphetamine (10 mg/kg s.c.) and the test compound intraperitoneally and then placed individually in stainless-steel cages (40 × 20 × 18 cm). The control groups receive d-amphetamine and vehicle. Stereotypic behavior is characterized by continuous sniffing, licking or chewing and compulsive gnawing. The animals are observed 60 min after drug administration. An animal is considered to be protected, if the stereotypic behavior is reduced or abolished.

Evaluation

The percent effectiveness of a drug is determined by the number of animals protected in each group. A dose–response is obtained by using ten animals per group at various doses. ED 50 values can be calculated. The standard neuroleptic drugs have the following ED 50 values: chlorpromazine 1.75 mg/kg i.p. and haloperidol 0.2 mg/kg i.p.

Critical Assessment of the Method

Inhibition of amphetamine-induced stereotypies in rats can be regarded as a simple method to detect neuroleptic activity. However, this may reflect the effects in the corpus striatum which are thought to be responsible for the Parkinsonism-like side effects of neuroleptics.

Modifications of the Method

Ljungberg and Ungerstedt (1985) described a rapid and simple behavioral screening method for simultaneous assessment of limbic and striatal blocking effects of neuroleptic drugs. A low dose of 2 mg/kg d-amphetamine i.p. induces both increased locomotion, thought to reflect an increased dopamine transmission in the nucleus accumbens, and weak stereotypies, thought to reflect an increased dopamine transmission in the neostriatum. The behavior is measured in a combined open-field apparatus with holes on the bottom to measure nose-pocking and registration of time spent in the corners. Neuroleptics with less propensity to induce unwanted extrapyramidal side effects can be differentiated from classical drugs with more extrapyramidal adverse reactions.

Segal and Kuczenski (1997) described an escalating dose “binge” model of amphetamine psychosis. Rats were exposed to escalating doses of amphetamine (1.0–8.0 mg/kg) before multiple daily injections of relatively high doses of the drug (8 mg/kg every 2 h × 4 injections).

Atkins et al. (2001) described stereotypic behaviors in mice selectively bred for high and low methamphetamine-induced stereotypic chewing.

Machiyama (1992) recommended chronic methylamphetamine intoxication in Japanese monkeys (Macaca fuscata) as a model of schizophrenia in animals.

Ellenbroek (1991) described the ethological analysis of Java monkeys (Macaca fascicularis) in a social setting as an animal model for schizophrenia.

Sams-Dodd and Newman (1997) described the effects of the administration regime on the psychotomimetic properties of d-amphetamine in the Squirrel monkey (Saimiri sciureus).

References and Further Reading

  • Atkins AL, Helms ML, O’Toole LA, Belknap JK (2001) Stereotypic behaviors in mice selectively bred for high and low methamphetamine-induced stereotypic chewing. Psychopharmacology (Berl) 157:96–104

  • Ellenbroek BA (1991) The ethological analysis of monkeys in a social setting as an animal model for schizophrenia. In: Olivier B, Mos J, Slangen JL (eds) Animal models in psychopharmacology. Advances in pharmacological sciences. Birkhäuser, Basel, pp 265–284

  • Ljungberg T, Ungerstedt U (1985) A rapid and simple behavioral screening method for simultaneous assessment of limbic and striatal blocking effects of neuroleptic drugs. Pharmacol Biochem Behav 23:479–485

  • Locke KW, Dunn RW, Hubbard JW, Vanselous CL, Cornfeldt M, Fielding S, Strupczewski JT (1990) HP 818: a centrally acting analgesic with neuroleptic properties. Drug Dev Res 19:239–259

  • Machiyama Y (1992) Chronic methylamphetamine intoxication model of schizophrenia in animals. Schizophr Bull 18:107–113

  • Sams-Dodd F, Newman JD (1997) Effects of administration regime on the psychotomimetic properties of d-amphetamine in the Squirrel monkey (Saimiri sciureus). Pharmacol Biochem Behav 56:471–480

  • Segal DS, Kuczenski R (1997) An escalating dose ‘binge’ model of amphetamine psychosis: behavioral and neurochemical characteristics. J Neurosci 17:2551–2566

  • Simon P, Chermat R (1972) Recherche d’une interaction avec les stéréotypies provoquées par l’amphétamine chez le rat. J Pharmacol 3:235–238

Inhibition of Apomorphine Climbing in Mice

Purpose and Rationale

Administration of apomorphine to mice results in a peculiar climbing behavior characterized initially by rearing and then full-climbing activity, predominantly mediated by the mesolimbic dopamine system (Costall et al. 1978). The ability of a drug to antagonize apomorphine-induced climbing behavior in the mouse has been correlated with neuroleptic potential (Protais et al. 1976; Costall et al. 1978).

Procedure

Groups of ten male mice (20–22 g) are treated i.p. or orally with the test substance or the vehicle and placed individually in wire-mesh stick cages. Thirty min afterwards, they are injected s.c. with 3 mg/kg apomorphine. Ten, 20, and 30 min after apomorphine administration, they are observed for climbing behavior and scored as follows:

  • 0 = four paws on the floor

  • 1 = four feet holding the vertical bars

  • 2 = four feet holding the bars

Evaluation

The average values of the drug-treated animals are compared with those of the controls, and the decrease is expressed as percent. The ED 50-values and confidence limits are calculated by probit analysis. Three dose levels are used for each compound and the standard with a minimum of ten animals per dose level.

Critical Assessment of the Test

Similar to the enhancement of compulsive gnawing of mice after apomorphine by antidepressant drugs, the suppression of climbing behavior of mice after apomorphine can be used for testing neuroleptic drugs. The test has been modified by various authors.

In contrast to other strains of mice, apomorphine climbing is not induced in DBA2 mice unless subchronic manipulations of brain dopamine transmission are performed (Duterte-Boucher and Costentin 1989).

References and Further Reading

  • Bischoff S, Christen P, Vassout A (1988) Blockade of hippocampal dopamine (DA) receptors: a tool for antipsychotics with low extrapyramidal side effects. Prog Neuropsychopharmacol Biol Psychiatry 12:455–467

  • Brown F, Campell W, Clark MSG, Graves DS, Hadley MS, Hatcher J, Mitchell P, Needham P, Riley G, Semple J (1988) The selective dopamine antagonist properties of BRL 34779: a novel substituted benzamide. Psychopharmacology (Berl) 94:350–358

  • Cabib S, Puglisi-Allegra S (1988) A classical genetic analysis of two apomorphine-induced behaviors in the mouse. Pharmacol Biochem Behav 30:143–147

  • Corral C, Lissavetzky J, Valdeolmillos A, Bravo L, Darias V, Sänchez Mateo C (1992) Neuroleptic activity of 10-(4-methyl-1-piperazinyl)-thieno(3,2-b)(1,5)benzothiazepine derivatives. Arzneim Forsch/Drug Res 42:896–900

  • Costall B, Naylor RJ, Nohria V (1978) Climbing behavior induced by apomorphine in mice: a potent model for the detection of neuroleptic activity. Eur J Pharmacol 50:39–50

  • Duterte-Boucher D, Costentin J (1989) Appearance of a stereotyped apomorphine-induced climbing in unresponsive DBA2 mice after chronic manipulation of brain dopamine transmission. Psychopharmacology (Berl) 98:56–60

  • Horváth K, Andrási P, Berzsenyi P, Pátfalusy M, Patthy M, Szabó G, Sebestyén L, Bagdy E, Körösi J, Botka P, Hamaori T, Láng T (1989) A new psychoactive 5H-2,3-benzodiazepine with an unique spectrum of activity. Arzneim Forsch/Drug Res 39:894–899

  • Moore NA, Axton MS (1988) Production of climbing behaviour in mice requires both D1 and D2 receptor activation. Psychopharmacology (Berl) 94:263–266

  • Moore NA, Tye NC, Axton MS, Risius FC (1992) The behavioral pharmacology of olanzapine, a novel “atypical” antipsychotic agent. J Pharmacol Exp Ther 262:545–551

  • Protais P, Costentin J, Schwartz JC (1976) Climbing behavior induced by apomorphine in mice: a simple test for the study of dopamine receptors in the striatum. Psychopharmacology (Berl) 50:1–6

  • Szewczak MR, Cornfeldt ML, Dunn RW, Wilker JC, Geyer HM, Glamkowski EJ, Chiang Y, Fielding S (1987) Pharmacological evaluation of HP 370, a potential atypical antipsychotic agent. 1. In vivo profile. Drug Dev Res 11:157–168

  • Vasse M, Chagraoui A, Protais P (1988) Climbing and stereotyped behaviors in mice require the stimulation of D-1 dopamine receptors. Eur J Pharmacol 148:221–229

Inhibition of Apomorphine Stereotypy in Rats

Purpose and Rationale

Apomorphine induces a stereotyped behavior in rats, characterized by licking, sniffing, and gnawing in a repetitive, compulsive manner, which is an indication of striatal dopaminergic stimulation (Anden et al. 1967; Ernst 1967; Costall and Naylor 1973). Compounds which prevent apomorphine-induced stereotypy antagonize dopamine receptors in the nigrostriatal system (Ljungberg and Ungerstedt 1978; Tarsy and Baldessarini 1974). Furthermore, antagonism of this behavior is predictive of propensity for the development of extrapyramidal side effects and tardive dyskinesias (Klawans and Rubovits 1972; Tarsy and Baldessarini 1974; Christensen et al. 1976; Clow et al. 1980).

Procedure

For screening, groups of six male Wistar rats with a body weight between 120 and 200 g are used. The test drug or the standard is administered i.p. 60 min. prior to apomorphine dosage. Apomorphine HCl is injected s.c. at a dose of 1.5 mg/kg. The animals are placed in individual plastic cages. A 10 s observation period is used to measure the presence of stereotypic activity such as sniffing, licking, and chewing 10 min after apomorphine administration. An animal is considered protected if this behavior is reduced or abolished.

Evaluation

The percent effectiveness of a drug is determined by the number of animals protected in each group. With a group size of ten animals, dose–response curves are obtained and ED 50 values calculated. ED 50 values were found to be 0.2 mg/kg s.c. for haloperidol and 5.0 mg/kg for chlorpromazine, whereas clozapine was ineffective even at high doses.

Modifications of the Methods

Puech et al. (1978) studied the effects of several neuroleptic drugs on hyperactivity induced by a low dose of apomorphine in mice.

Apomorphine induces stereotypic behavior in a variety of species including pigeons. The symptoms in pigeons are manifested as pecking against the wall of the cage or on the floor. Akbas et al. (1984) described a method registering the pecking after apomorphine by a microphone, amplification through a pulse preamplifier, and registration with a polygraph. The effect of apomorphine was dose-dependent decreased by yohimbine and neuroleptics.

Stereotyped behavior in guinea pigs induced by apomorphine or amphetamine consisting in continuous gnawing and sniffing of the cage floor was described by Klawans and Rubovits (1972) and used as an experimental model of tardive dyskinesia.

References and Further Reading

  • Akbas O, Verimer T, Onur R, Kayaalp SO (1984) The effects of yohimbine and neuroleptics on apomorphine-induced pecking behavior in the pigeon. Neuropharmacology 23:1261–1264

  • Andén NE, Rubenson A, Fuxe K, Hoekfelt T (1967) Evidence for dopamine receptor stimulation by apomorphine. J Pharm Pharmacol 19:627–629

  • Christensen A, Fjalland B, Møller Nielsen I (1976) On the supersensitivity of dopamine receptors, induced by neuroleptics. Psychopharmacology (Berl) 48:1–6

  • Clow A, Theodorou A, Jenner P, Marsden CD (1980) A comparison of striatal and mesolimbic dopamine function in the rat during a 6-month trifluoperazine administration. Psychopharmacology (Berl) 69:227–233

  • Costall B, Naylor RJ (1973) On the mode of action of apomorphine. Eur J Pharmacol 21:350–361

  • Dall’Olio R, Gandolfi O (1993) The NMDA positive modulator D-cycloserine potentiates the neuroleptic activity of D1 and D2 dopamine receptor blockers in the rat. Psychopharmacology (Berl) 110:165–168

  • Ernst AM (1967) Mode of action of apomorphine and dexamphetamine on gnawing compulsion in rats. Psychopharmacologia (Berlin) 10:316–323

  • Janssen PAJ, Niemegeers CJC, Jageneau AHM (1960) Apomorphine-antagonism in rats. Arzneim Forsch 10:1003–1005

  • Jolicoeur FB, Gagne MA, Rivist R, Drumheller A, St Pierre S (1991) Neurotensin selectively antagonizes apomorphine-induced stereotypic climbing. Pharmacol Biochem Behav 38:463–465

  • Klawans HL, Rubovits R (1972) An experimental model of tardive dyskinesia. J Neural Transm 33:235–246

  • Kostowski W, Krzascik P (1989) Research for evaluating the role of dopaminergic mechanisms in the action of valproate. Biog Amin 6:169–176

  • Ljungberg T, Ungerstedt U (1978) Classification of neuroleptic drugs according to their ability to inhibit apomorphine-induced locomotion and gnawing: evidence for two different mechanisms of action. Psychopharmacology (Berl) 56:239–247

  • Locke KW, Dunn RW, Hubbard JW, Vanselous CL, Cornfeldt M, Fielding F, Strupczewski JT (1990) HP 818: a centrally acting analgesic with neuroleptic properties. Drug Dev Res 19:239–256

  • Puech AJ, Simon P, Boissier JR (1978) Benzamides and classical neuroleptics: comparison of their action using 6 apomorphine-induced effects. Eur J Pharmacol 50:291–300

  • Szewczak MR, Cornfeldt ML, Dunn RW, Wilker JC, Geyer HM, Glamkowski EJ, Chiang Y, Fielding S (1987) Pharmacological evaluation of HP 370, a potential atypical antipsychotic agent. 1. In vivo profile. Drug Dev Res 11:157–168

  • Tarsy D, Baldessarini RJ (1974) Behavioral supersensitivity to apomorphine following chronic treatment with drugs which interfere with the synaptic function of catecholamines. Neuropharmacology 13:927–940

Yawning/Penile Erection Syndrome in Rats

Purpose and Rationale

Yawning is a phylogenetically old, stereotyped event that occurs alone or associated with stretching and/or penile erection in humans and in animals from reptiles to birds and mammals under different conditions (Argiolas and Melis 1998). The yawning–penile erection syndrome can be induced in rats by apomorphine and other dopamine autoreceptor stimulants (Ståhle and Ungerstedt 1983; Gower et al. 1984) and can be antagonized by haloperidol and other dopamine antagonists. Antagonism against this syndrome can be regarded as indication of antipsychotic activity (Furukawa 1996).

Besides the dopaminergic system in this behavior (Mogilnicka and Klimek 1977; Baraldi et al. 1979; Benassi-Benelli et al. 1979; Nickolson and Berendsen 1980; Gower et al. 1984, 1986; Dourish et al. 1985; Doherty and Wisler 1994; Kurashima et al. 1995; Bristow et al. 1996; Fujikawa et al. 1996a; Asencio et al. 1999) also the serotonergic (Baraldi et al. 1977; Berendsen and Broekkamp 1987; Berendsen et al. 1990; Protais et al. 1995; Millan et al. 1997), the cholinergic (Yamada and Furukawa 1980; Fujikawa et al. 1996b), the GABAergic (Zarrindast et al. 1995), the NO system (Melis et al. 1995, 1996, 1997a, b), and steroid as well as peptide hormones (Bertolini and Baraldi 1975; Bertolini et al. 1978; Holmgren et al. 1980; Berendsen and Nickolson 1981; Berendsen and Gower 1986; Gully et al. 1995) are involved (Argiolas and Melis 1998).

Procedure

Naive male Wistar rats, weighing 220–280 g, are housed under controlled 12 h light–dark cycle with free access to standard food pellets and tap water. Rats are pretreated with subcutaneous injection of the antagonist 30 min prior to injections of the agonist, such as apomorphine (0.02 to 0.25 mg/kg s.c.) or physostigmine (0.02 to 0.3 mg/kg s.c. or i.p.). After administration of the agonist, rats are placed in individual transparent Perspex cages. A mirror is placed behind the row of observation cages to facilitate observation of the animals for penile erections and yawns. Yawning is a fixed innate motor pattern characterized by a slow, wide opening of the mouth. A penile erection is considered to occur when the following behaviors are present: repeated pelvic thrusts immediately followed by an upright position and an emerging, engorged penis which the rats proceed to lick while eating the ejaculate. The number of penile erections and yawns is counted for 30 min following the last injection.

Evaluation

The results are expressed as the mean number of yawns and of penile erections per group ± SEM. The statistical significance is determined by comparing the results of each group with the results of the relevant control group using a nonparametric rank sum test.

Critical Assessment of the Method

Ferrari et al. (1993) published some evidence that yawning and penile erection in rats underlie different neurochemical mechanisms. Nevertheless, the procedure can be regarded as a useful behavioral tool to study putative antipsychotic activity of new compounds.

Modifications of the Method

Two sublines of Sprague Dawley rats were bred for high- and low-yawning frequency in males (Eguibar and Moyaho 1997).

Apomorphine produced more yawning in Sprague Dawley rats than in F344 rats (Tang and Himes 1995).

Sato-Suzuki et al. (1998) evoked yawning by electrical or chemical stimulation in the paraventricular nucleus of anesthetized rats.

The yawning–penile erection syndrome in rats can be elicited by injections of 50 ng NMDA or AMPA (Melis et al. 1994, 1997b) into the paraventricular nucleus of the hypothalamus or intracerebroventricular injection of 50 ng oxytocin (Melis et al. 1997a) or ACTH (Genedani et al. 1994; Poggioli et al. 1998) or α-MSH (Vergoni et al. 1998).

Champion et al. (1997) and Bivalacqua et al. (1998) studied the effect of intracavernosal injections of adrenomedullin and other peptide hormones on penile erections in cats.

Dopaminergic influences on male sexual behavior of rhesus monkeys were studied by Pomerantz (1990, 1992).

References and Further Reading

  • Argiolas A, Melis MR (1998) The neuropharmacology of yawning. Eur J Pharmacol 343:1–16

  • Asencio M, Delaquerriere B, Cassels BK, Speisky H, Comoy E, Protais P (1999) Biochemical and behavioral effects of boldine and glaucine on dopaminergic systems. Pharmacol Biochem Behav 62:7–13

  • Baraldi M, Benassi-Benelli A, Lolli M (1977) Penile erections in rats after fenfluramine administration. Riv Farmacol Ter 8:375–379

  • Baraldi M, Benassi-Benelli A, Bernabei MT, Cameroni R, Ferrari F, Ferrari P (1979) Apocodeine-induced stereotypies and penile erection in rats. Neuropharmacology 18:165–169

  • Benassi-Benelli A, Ferrari F, Pellegrini-Quarantotti B (1979) Penile erection induced by apomorphine and N-npropylnorapomorphine in rats. Arch Int Pharmacodyn 242:241–247

  • Berendsen HHG, Broekkamp CLE (1987) Drug-induced penile erections in rats: indication of serotonin1B receptor mediation. Eur J Pharmacol 135:279–287

  • Berendsen HHG, Gower AJ (1986) Opiate-androgen interaction in drug-induced yawning and penile erections in the rat. Neuroendocrinology 42:185–190

  • Berendsen HHG, Jenk F, Broekkamp CLE (1990) Involvement of 5-HT1C-receptors in drug-induced penile erections in rats. Psychopharmacology (Berl) 101:57–61

  • Bertolini A, Baraldi M (1975) Anabolic steroids: permissive agents of ACTH-induced penile erections in rats. Life Sci 17:263–266

  • Bertolini A, Genedani S, Castelli M (1978) Behavioural effects of naloxone in rats. Experientia 34:771–772

  • Bivalacqua TJ, Rajasekaran M, Champion HC, Wang R, Sikka SC, Kadowitz PJ, Hellstrom WJG (1998) The influence of castration of pharmacologically induced penile erection in the cat. J Androl 19:551–557

  • Bristow LJ, Cook GP, Gay JC, Kulagowski J, Landon L, Murray F, Saywell KL, Young L, Hutson PH (1996) The behavioral and neurochemical profile of the putative dopamine D3 agonist, (+)-PD 128907, in the rat. Neuropharmacology 35:285–294

  • Champion HC, Wang R, Shenassa BB, Murphy WA, Coy DH, Hellstrom WJG, Kadowitz PJ (1997) Adrenomedullin induces penile erection in the cat. Eur J Pharmacol 319:71–75

  • Doherty PC, Wisler PA (1994) Stimulatory effects of quinelorane on yawning and penile erection in the rat. Life Sci 54:507–514

  • Dourish CT, Cooper SJ, Philips SR (1985) Yawning elicited by systemic and intrastriatal injection of piribedil and apomorphine in the rat. Psychopharmacology (Berl) 86:175–181

  • Eguibar JR, Moyaho A (1997) Inhibition of grooming by pilocarpine differs in high- and low-yawning sublines of Sprague Dawley rats. Pharmacol Biochem Behav 58:317–322

  • Ferrari F, Pelloni F, Giuliani D (1993) Behavioural evidence that different neurochemical mechanisms underlie stretching-yawning and penile erection induced in male rats by SND 919, a new selective D2 dopamine receptor agonist. Psychopharmacology (Berl) 113:172–276

  • Fujikawa M, Nagashima M, Inuoe T, Yamada K, Furukawa T (1996a) Potential agonistic effects of OPC-14597, a potential antipsychotic agent, on yawning behavior in rats. Pharmacol Biochem Behav 53:903–909

  • Fujikawa M, Yamada K, Nagashima M, Domae M, Furukawa T (1996b) The new muscarinic M1-receptor agonist YM796 evokes yawning and increases oxytocin secretion from the posterior pituitary in rats. Pharmacol Biochem Behav 55:55–60

  • Furukawa T (1996) Yawning behavior for preclinical drug evaluation. Methods Find Exp Clin Pharmacol 18:141–155

  • Genedani S, Bernardi M, Bertolini A (1994) Influence of ifenprodil on the ACTH-induced behavioral syndrome in rats. Eur J Pharmacol 252:77–80

  • Gower AJ, Berendsen HHG, Princen MM, Broekkamp CLE (1984) The yawning-penile erection syndrome as a model for putative dopamine autoreceptor activity. Eur J Pharmacol 103:81–89

  • Gower AJ, Berendsen HHG, Broekkamp CLE (1986) Antagonism of drug-induced yawning and penile erections in rats. Eur J Pharmacol 122:239–244

  • Gully D, Jeanjean F, Poncelet M, Steinberg R, Soubriè P, La Fur G, Maffrand JP (1995) Neuropharmacologic profile of nonpeptide neurotensin antagonists. Fundam Clin Pharmacol 9:513–521

  • Holmgren B, Urbá-Holmgren R, Aguiar M, Rodriguez R (1980) Sex hormone influences on yawning behavior. Acta Neurobiol Exp 40:515–519

  • Kurashima M, Katsushi Y, Nagashima M, Shirakawa K, Furukawa T (1995) Effects of putative D3 receptor agonists, 7-OH.DPAT, and quinpirole, on yawning, stereotypy, and body temperature in rats. Pharmacol Biochem Behav 52:503–508

  • Melis MR, Stancampiano R, Argiolas A (1994) Penile erection and yawning induced by paraventricular NMDA injection in male rats are mediated by oxytocin. Pharmacol Biochem Behav 48:203–207

  • Melis MR, Stancampiano R, Argiolas A (1995) Role of nitric oxide in penile erection and yawning induced by 5-HT1C receptor agonists in male rats. Naunyn Schmiedebergs Arch Pharmacol 351:439–445

  • Melis MR, Succu S, Argiolas A (1996) Dopamine agonists increase nitric oxide production in the paraventricular nucleus of the hypothalamus: correlation with penile erection and yawning. Eur J Neurosci 8:2056–2063

  • Melis MR, Succu S, Iannucci U, Argiolas A (1997a) Oxytocin increases nitric oxide production in the paraventricular nucleus of the hypothalamus of male rats: correlation with penile erection and yawning. Regul Pept 69:105–111

  • Melis MR, Succu S, Iannucci U, Argiolas A (1997b) N-Methyl-D-aspartic acid-induced penile erection and yawning: role of hypothalamic paraventricular nitric oxide. Eur J Pharmacol 328:115–123

  • Millan MJ, Peglion JL, Lavielle G, Perrin-Monneyron S (1997) 5-HT2C receptors mediate penile erections in rats: actions of novel and selective agonists and antagonists. Eur J Pharmacol 325:9–12

  • Mogilnicka E, Klimek V (1977) Drugs affecting dopamine neurons and yawning behavior. Pharmacol Biochem Behav 7:303–305

  • Nickolson VJ, Berendsen HHG (1980) Effects of the potential neuroleptic peptide des-tyrosine1 -γ-endorphin and haloperidol on apomorphine-induced behavioural syndromes in rats and mice. Life Sci 27:1377–1385

  • Poggioli R, Arletti R, Benelli A, Cavazzuti E, Bertolini A (1998) Diabetic rats are unresponsive to the penile erection-inducing effect of intracerebroventricularly injected adrenocorticotropin. Neuropeptides 32:151–155

  • Pomerantz SM (1990) Apomorphine facilitates male sexual behavior of rhesus monkeys. Pharmacol Biochem Behav 35:659–664

  • Pomerantz SM (1992) Dopaminergic influences on male sexual behavior of rhesus monkeys: effects of dopamine agonists. Pharmacol Biochem Behav 41:511–517

  • Protais P, Windsor M, Mocaër E, Comoy E (1995) Post-synaptic 5-HT1A receptor involvement in yawning and penile erections induced by apomorphine, physostigmine and mCCP in rats. Psychopharmacology (Berl) 120:376–383

  • Sato-Suzuki I, Kita I, Oguri M, Arita H (1998) Stereotyped yawning responses induced by electrical and chemical stimulation of paraventricular nucleus of the rat. J Neurophysiol 80:2765–2775

  • Ståhle L, Ungerstedt U (1983) Assessment of dopamine autoreceptor properties of apomorphine, (+)-3-PPP and (−)-3-PPP by recording of yawning behaviour in rats. Eur J Pharmacol 98:307–310

  • Tang AH, Himes CS (1995) Apomorphine produced more yawning in Sprague Dawley rats than in F344 rats: a pharmacological study. Eur J Pharmacol 284:13–18

  • Vergoni AV, Bertoline A, Mutulis F, Wikberg JES, Schioth HB (1998) Differential influence of a selective melanocortin MC4 receptor antagonist (HS014) on melanocortin-induced behavioral effects in rats. Eur J Pharmacol 362:95–101

  • Yamada K, Furukawa T (1980) Direct evidence for involvement of dopaminergic inhibition and cholinergic activation in yawning. Psychopharmacology (Berl) 67:39–43

  • Zarrindast MR, Toloui V, Hashemi B (1995) Effects of GABAergic drugs on physostigmine-induced yawning in rats. Psychopharmacology (Berl) 122:297–300

Inhibition of Mouse Jumping

Purpose and Rationale

Lal et al. (1975) described a jumping response in mice after administration of L-dopa in amphetamine pretreated animals where the number of jumps can be objectively counted. The mouse jumping is due to dopaminergic overstimulation similar to that seen in rats when stereotypy is induced by higher doses of amphetamine. The phenomenon can be blocked by neuroleptics.

Procedure

Male CD-1 mice weighing 22–25 g are injected with 4 mg/kg d-amphetamine sulfate, followed 15 min later by an i.p. injection of 400 mg/kg L-dopa. The mice spontaneously begin to jump at a high rate. A median of 175 jumps can be observed in these mice during 60 min. Since mice do not show any jumping after saline administration, the responses after drug administration are specific and can be measured automatically through a pressure-sensitive switch closure or properly positioned photoelectric beam disruptions. Test compounds are administered 60 min prior to L-dopa injection.

Evaluation

Jumps of mice treated with test drugs or standard are counted and expressed as percentage of jumps in amphetamine-/L-dopa-treated animals. Using various doses, ED 50 values with 95 % confidence limits are calculated.

Critical Assessment of the Method

The method has been found to be sensitive and rather specific for neuroleptic drugs.

References and Further Reading

  • Fielding S, Lal H (1978) Behavioral actions of neuroleptics. In: Iversen LL, Iversen SD, Snyder SH (eds) Neuroleptics and Schizophrenia, vol 10. Plenum Press, New York, pp 91–128

  • Fielding S, Marky M, Lal H (1975) Elicitation of mouse jumping by combined treatment with amphetamine and L-dopa: blockade by known neuroleptics. Pharmacologist 17:210

  • Lal H, Colpaert F, Laduron P (1975) Narcotic withdrawal-like mouse jumping produced by amphetamine and L-dopa. Eur J Pharmacol 30:113–116

  • Lal H, Marky M, Fielding S (1976) Effect of neuroleptic drugs on mouse jumping induced by L-dopa in amphetamine treated mice. Neuropharmacology 15:669–671

Antagonism Against MK-801-Induced Behavior

Purpose and Rationale

Dizocilpine (MK-801), a noncompetitive NMDA antagonist, induces a characteristic behavior in rat and mice, which is regarded as a model of psychosis (Andiné et al. 1999). In mice MK-801 induces a characteristic stereotypy marked by locomotion and falling behavior through both dopamine-dependent and dopamine-independent mechanisms (Carlson and Carlson 1989; Verma and Kulkarni 1992). Antipsychotic agents dose-dependently antagonize this MK-801-induced behavior.

Procedure

Male CD-1 mice (20–30 g) are individually placed in activity boxes lined with wire-mesh flooring and allowed to acclimate for 60 min. The animals are then dosed with compounds 30 min prior to subcutaneous administration of MK-801 at 0.2 mg/kg. The mice are observed for locomotion and the presence of falling behavior 15 min following MK-801 administration.

Evaluation

ED 50 values and 95 % confidence limits are calculated by the Litchfield and Wilcoxon method.

Modifications of the Method

Deutsch and Hitri (1993), Rosse et al. (1995), Deutsch et al. (2002, 2003), and Mastropaolo et al. (2004) described methods to measure the MK-801-induced explosive behavior in mice, called “popping.”

Farber et al. (1996) showed that neuroleptic drugs can prevent neuronal vacuolization and necrosis induced by MK-801 (Fix et al. 1993).

References and Further Reading

  • Andiné P, Widermark N, Axelsson R, Nyberg G, Olafsson U, Marrtensson E, Sandberg M (1999) Characterization of MK-801-induced behavior as a putative rat model of psychosis. J Pharmacol Exp Ther 290:1393–1408

  • Carlson M, Carlson A (1989) The NMDA antagonist MK-801 causes marked locomotor stimulation in monoamine-depleted mice. J Neural Transm 75:221–226

  • Deutsch SI, Hitri A (1993) Measurement of an explosive behavior in the mouse, induced by MK-801, a PCP analogue. Clin Neuropharmacol 16:251–257

  • Deutsch SI, Rosse RB, Billingslea EN, Bellack AS, Mastropaolo J (2002) Topiramate antagonizes MK-801 in an animal model of schizophrenia. Eur J Pharmacol 449:121–125

  • Deutsch SI, Rosse RB, Billingslea EN, Bellack AS, Mastropaolo J (2003) Modulation of MK-801-elicited mouse popping behavior by galantamine is complex and dose-dependent. Life Sci 73:2355–2361

  • Farber NB, Foster J, Duhan NL, Olney JW (1996) Olanzapine and fluperlapine mimic clozapine in preventing MK-801 neurotoxicity. Schizophr Res 21:33–37

  • Fix AS, Horn JW, Wigtman KA, Johnson CA, Long GG, Storts RW, Farber N, Wozniak DF, Olney JW (1993) Neuronal vacuolization and necrosis induced by the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK(+)801 (dizocilpine maleate): a light and electron microscopic evaluation of the rat retrosplenial cortex. Exp Neurol 123:204–215

  • Litchfield J, Wilcoxon F (1949) A simplified method of evaluating dose effect experiments. J Pharmacol Exp Ther 96:99–113

  • Mastropaolo J, Rosse RB, Deutsch SI (2004) Anabasine, a selective nicotinic acetylcholine receptor agonist, antagonizes MK801-elicited mouse popping behavior, an animal model of schizophrenia. Behav Brain Res 153:419–422

  • Rosse RB, Mastropaolo J, Sussman DM, Koetzner L, Morn CB, Deutsch SI (1995) Computerized measurement of MK-801-elicited popping and hyperactivity in mice. Clin Neuropharmacol 18:448–457

  • Verma A, Kulkarni SK (1992) Modulation of MK-801 response by dopaminergic agents in mice. Psychopharmacology 107:431–436

Phencyclidine Model of Psychosis

Purpose and Rationale

Phencyclidine (PCP)-induced symptoms in rats are considered as a model of psychosis (Ogawa et al. 1994; Halberstadt 1995; Steinpreis 1996; Abi-Saab et al. 1998; Sams-Dodd 1998a; Jentsch and Roth 1999; Phillips et al. 2000; Farber 2003; Morris et al. 2005).

PCP-induced symptoms can be antagonized by neuroleptic drugs (Witkin et al. 1997; Sams-Dodd 1998b; Javitt et al. 2004).

Cartmell et al. (1999) found that metabotropic glutamate receptor agonists selectively attenuate phencyclidine versus d-amphetamine motor behaviors in rats.

Procedure

Behavior of male Sprague Dawley rats weighing 250–300 g was monitored while in transparent, plastic shoebox cages of the dimensions 45 × 25 × 20 cm, with 1 cm depth of wood clips as bedding, and a metal grill on the top of the cage. Motor monitors consisted of a rectangular rack of 12 photobeams arranged in an 8 × 4 formation. Shoe boxes were placed inside these racks, enabling the activity of the rat to be monitored in a home-cage environment. The lower rack was positioned at a height of 5 cm, which allowed the detection of PCP-induced head movements in addition to movements of the body of the rat. Rearing activity was detected by a second rack placed 10 cm above the first. Rats were placed in the cage for an acclimation period of 30 min, and then removed, administered the test compounds s.c. or sterile water, and then returned to the same cages. After 30 min, the rats were given an s.c. injection of PCP or amphetamine or sterile water and once again returned to the cages. Motor activity was monitored for the following 60 min resulting in the measurement of three different parameters: ambulations (pattern of beam breaks indicating that the animal had relocated its entire body), fine movements (nonambulatory beam breaks), and time at rest. An indication of rearing activity was detected in the upper rack of photobeams.

Evaluation

Data were analyzed by a one-way ANOVA, and then post hoc comparisons for each dose group versus control or PCP alone or PCP and test compound were made using Newman–Keuls multiple comparison test.

Modifications of the Method

Furuya et al. (1998) investigated the effects of a strychnine-insensitive glycine site antagonist on the hyperactivity and the disruption of prepulse inhibition induced by phencyclidine (PCP) in rats.

Redmond et al. (1999) tested the effects of acute and chronic antidepressant administration on PCP-induced locomotor hyperactivity.

Boulay et al. (2004) tested a putative atypical antipsychotic for improvement of social interaction deficits induced by PCP in rats.

References and Further Reading

  • Abi-Saab WM, D’Souza DC, Moghaddam B, Krystal JH (1998) The NMDA antagonist model for schizophrenia: promise and pitfalls. Pharmacopsychiatry 31(Suppl 2):104–109

  • Boulay D, Depoortère R, Louis C, Perrault G, Griebel G, Soubrié P (2004) SSR181507, a putative atypical antipsychotic with dopamine D2 agonist and 5-HT1A agonist activities: improvement of social interaction deficits induced by phencyclidine in rats. Neuropharmacology 46:1121–1129

  • Cartmell J, Monn JA, Schopp DD (1999) The metabotropic glutamate 2/3 receptor agonists LY354740 and LY379268 selectively attenuate phencyclidine versus d-amphetamine motor behaviors in rats. J Pharmacol Exp Ther 291:161–170

  • Farber NB (2003) The NMDA receptor hypofunction model of psychosis. Ann N Y Acad Sci 2003:119–130

  • Furuya Y, Kagaya T, Nishizawa Y, Ogura H (1998) Differential effects of the strychnine-insensitive glycine site antagonist (+)-HA-966 on the hyperactivity and the disruption of prepulse inhibition induced by phencyclidine in rats. Brain Res 781:227–235

  • Halberstadt AL (1995) The phencyclidine-glutamate model of schizophrenia. Clin Neuropharmacol 18:237–249

  • Javitt DC, Balla A, Burch S, Suckow R, Xie S, Sershen H (2004) Reversal of phencyclidine-induced dopaminergic dysregulation by N-methyl-D-aspartate receptor/glycine-site agonists. Neuropsychopharmacology 29:300–307

  • Jentsch JD, Roth RH (1999) The neuropsychopharmacology of phencyclidine: from NMDA receptor hypofunction to the dopamine hypothesis of schizophrenia. Neuropsychopharmacology 20:201–225

  • Morris BJ, Cochran SM, Pratt JA (2005) PCP: from pharmacology to modeling schizophrenia. Curr Opin Pharmacol 5:101–106

  • Ogawa S, Okuyama S, Araki H, Nakazato A, Otomo S (1994) A rat model of phencyclidine psychosis. Life Sci 55:1605–1610

  • Phillips M, Wang C, Johnson KM (2000) Pharmacological characterization of locomotor sensitization induced by chronic phencyclidine administration. J Pharmacol Exp Ther 296:905–913

  • Redmond AM, Harkin A, Kelly JP, Leonard BE (1999) Effects of acute and chronic antidepressant administration on phencyclidine (PCP) induced locomotor hyperactivity. Eur Neuropsychopharmacol 9:165–170

  • Sams-Dodd F (1998a) Effects of continuous D-amphetamine and phencyclidine administration on social behaviour, stereotyped behaviour, and locomotor activity in rats. Neuropsychopharmacology 19:18–25

  • Sams-Dodd F (1998b) Effects of diazepam, citalopram, methadone and naloxone on PCP-induced stereotyped behavior and social isolation in the rat social interaction test. Neurosci Behav Rev 23:287–293

  • Steinpreis RE (1996) The behavioral and neurochemical effects of phencyclidine in humans and animals: some applications for modeling psychosis. Behav Brain Res 74:45–55

  • Witkin JM, Steele TD, Sharpe LG (1997) Effects of strychnine-insensitive glycine receptor ligands in rats discriminating dizocilpine or phencyclidine from saline. J Pharmacol Exp Ther 280:46–52

Inhibition of Apomorphine-Induced Emesis in the Dog

Purpose and Rationale

The blockade of centrally acting dopaminergic mechanisms is considered to play a major role in suppression of psychotic reactions in schizophrenia. Apomorphine, regarded as a direct dopaminergic agonist, produces a pronounced emetic effect in dogs, and the blockade of apomorphine emesis is used as an indication of dopaminergic blockade. However, although both antiemetic activity and antipsychotic activity are thought to be due to dopaminergic blockade, the sites of action are in different brain areas, and there is a lack of complete correlation of these activities.

Procedure

Adult beagle dogs of either sex are used in treatment groups of three to nine dogs/dose. The dogs are given the test compounds in a gelatin capsule; they are then dosed with 0.15 mg/kg apomorphine s.c. at various intervals after administration of the test compound. The dogs are first observed for overt behavioral effects, e.g., pupillary response to light, changes in salivation, sedation, tremors, etc.; then, after the administration of apomorphine, the dogs are observed for stereotypic sniffing, gnawing, and the emetic response. Emesis is defined as retching movements followed by an opening of the mouth and either attempted or successful ejection of stomach content.

Evaluation

If the experimental compound is antiemetic in the primary screen, the dose is progressively lowered to obtain a minimal effective dose or an ED 50 value. The ED 50 values for haloperidol and chlorpromazine were found to be 0.06 mg/kg p.o. and 2.0 mg/kg p.o., respectively. Clozapine was not effective at doses between 2 and 10 mg/kg. p.o.

Critical Assessment of the Method

The method has been extensively used by several laboratories. However, since nonclassical neuroleptics like clozapine did not show pronounced activity, the test has been abandoned. Moreover, tests in higher animals like dogs are limited due to regional regulations.

References and Further Reading

  • Chipkin RE, Iorio LC, Coffin VL, McQuade RD, Berger JG, Barnett A (1988) Pharmacological profile of SCH39166: a dopamine D1 selective benzonaphthazepine with potential antipsychotic activity. J Pharmacol Exp Ther 247:1093–1102

  • Janssen PAJ, Niemegeers CJE (1959) Chemistry and pharmacology of compounds related to 4-(4-hydroxy-4-phenylpiperidino)-butyrophenone. Part II – Inhibition of apomorphine vomiting in dogs. Arzneim Forsch 9:765–767

  • Janssen PA, Niemegeers CJE, Shellekens HL (1965) Is it possible to predict the clinical effects of neuroleptic drugs (major tranquilizers) from animal data? Arzneim Forsch 15:1196–1206

  • Rotrosen J, Wallach MB, Angrist B, Gershon S (1972) Antagonism of apomorphine-induced stereotypy and emesis in dogs by thioridazine, haloperidol and pimozide. Psychopharmacology (Berl) 26:185–195

Purposeless Chewing in Rats

Purpose and Rationale

Purposeless chewing can be induced in rats by directly acting cholinergic drugs or cholinesterase inhibitors (Rupniak et al. 1983), which can be blocked by antimuscarinic agents. The chewing behavior has been proposed to be mediated through central M2 receptors rather than via central M1 sites (Stewart et al. 1989). Chewing can also be induced by chronic administration of neuroleptics in rats (Clow et al. 1979; Iversen et al. 1980). Purposeless chewing is mediated by dopaminergic and nicotinic mechanisms.

Procedure

Male albino rats are housed 10 per cage at room temperature and kept on a 12 h light–dark cycle. For the experiments, rats are placed individually in a large glass cylinder (height 30 cm, diameter 20 cm) at 21 ± 1°C and allowed to habituate for 15 min before injection of drugs. The antagonists, e.g., sulpiride or mecamylamine as standards, are given at different doses 30 min before treatment either with 0.01 mg/kg nicotine or 1 mg/kg pilocarpine i.p. Number of chewings are counted by direct observation immediately after drug administration. The results are presented as number of chews in a 30-min period.

Evaluation

Analysis of variance (ANOVA) , followed by Newman–Keuls tests, are used to evaluate the significance of the results obtained. P < 0.05 is considered as significant.

Modifications of the Method

Tremulous jaw movements induced by tacrine (Cousins et al. 1999) can be antagonized by antipsychotic drugs (Betz et al. 2005; Ishiwarii et al. 2005).

References and Further Reading

  • Betz A, Ishiwari K, Wisniecki A, Huyn N, Salamone JD (2005) Quetiapine (Seroquel) shows a pattern of behavioral effects similar to the atypical antipsychotics clozapine and olanzapine: studies with tremulous jaw movements in rats. Psychopharmacology (Berl) 179:383–392

  • Clow A, Jenner P, Marsden CD (1979) Changes in dopamine-mediated behaviour during one year neuroleptic treatment. Eur J Pharmacol 57:365–375

  • Collins P, Broekkamp CLE, Jenner P, Marsden CD (1991) Drugs acting at D-1 and D-2 dopamine receptors induce identical purposeless chewing in rats which can be differentiated by cholinergic manipulation. Psychopharmacology (Berl) 103:503–512

  • Cousins MS, Finn M, Trevitt J, Carriero DL, Conlan A, Salamone JD (1999) The role of ventrolateral striatal acetylcholine in the production of tacrine-induced jaw movements. Pharmacol Biochem Behav 62:439–447

  • Ishiwarii K, Betz A, Weber S, Felsted J, Salamone JD (2005) Validation of the tremulous jaw movement model for assessment of motor effects of typical and atypical antipsychotics: effects of pimozide (Orap) in rats. Pharmacol Biochem Behav 80:351–362

  • Iversen SD, Howells RB, Hughes RP (1980) Behavioural consequences of long-term treatment with neuroleptic drugs. Adv Biochem Psychopharmacol 24:305–313

  • Rupniak NMJ, Jenner P, Marsden CD (1983) Cholinergic manipulation of perioral behaviour induced by chronic neuroleptic administration to rats. Psychopharmacology (Berl) 79:226–230

  • Samini M, Yekta FS, Zarrindast MR (1995) Nicotine-induced purposeless chewing in rats: possible dopamine receptor mediation. J Psychopharmacol 9:16–19

  • Stewart BR, Jenner P, Marsden CD (1989) Assessment of the muscarinic receptor subtype involved in the mediation of pilocarpine-induced purposeless chewing behaviour. Psychopharmacology (Berl) 97:228–234

  • Zarrindast MR, Moini-Zanjani T, Manaheji H, Fathi F (1992) Influence of dopamine receptors on chewing behaviour in rats. Gen Pharmacol 23:915–919

Models of Tardive Dyskinesia

Purpose and Rationale

Tardive dyskinesia is a severe side effect of traditional neuroleptics affecting a considerable number of patients probably based on a genetic disposition, being characterized by involuntary movements of the oral region. Various authors used rats as animal model for tardive dyskinesia, either after treatment with reserpine (Waddington 1990; Neisewander et al. 1994; Bergamo et al. 1997; Queiroz and Frussa-Filho 1999; Andreassen and Jorgensen 2000; Casey 2000; Van Kampen and Stoessl 2000; Calvente et al. 2002; Abílio et al. 2003; Peixoto et al. 2003) or haloperidol (Takeuchi et al. 1998; Harvey and Nel 2003; Naidu et al. 2003; Burger et al. 2005). Several authors compared the effects of different neuroleptics (See and Ellison 1990; Tamminga et al. 1994) or studied potential antagonistic effects (Takeuchi et al. 1998; Queiroz and Frussa-Filho 1999; Abílio et al. 2003; Naidu et al. 2003; Peixoto et al. 2003).

Burger et al. (2005) found that ebselen attenuates haloperidol-induced orofacial dyskinesia and oxidative stress in rat brain.

Procedure

Male Wistar rats weighing 270–320 g were injected s.c. once a week with 12 mg/kg haloperidol decanoate for 4 weeks. Another group was pretreated with 30 mg/kg ebselen and received in addition to haloperidol every other day an i.p. injection of 30 mg/kg ebselen.

The animals were observed for the quantification of orofacial dyskinesia just before haloperidol administration and on the 7th, 14th, 21st, and 28th day after the first administration of haloperidol.

Rats were placed individually in cages (20 × 20 × 19 cm) containing mirrors under the floor and behind the back wall of the cage to allow behavioral quantification when the animal was faced away from the observer. To quantify the occurrence of oral dyskinesia, the incidence of tongue protrusions, vacuous chewing movements frequency, and the duration of facial twitching were recorded for 15 min. Observers were blind to drug treatment.

Evaluation

Data were analyzed by a three-way ANOVA, followed, when appropriate, by univariate analysis and Duncan’s post hoc test.

Modifications of the Method

Several authors used monkeys (Cebus apella or Macaca speciosa) to evaluate the effect of neuroleptics to induce tardive dyskinesia-like symptoms (Gunne and Barany 1979; Domino 1985; Werge et al. 2003).

References and Further Reading

  • Abílio VC, Araujo CCS, Bergamo M, Calvente PRV, D’Almeida V, Ribeiro RA, Frussa-Filho R (2003) Vitamin E attenuates reserpine-induced oral dyskinesia and striatal oxidized glutathione/reduced glutathione ratio (GSSSG/GSH) enhancement in rats. Prog Neuropsychopharmacol Biol Psychiatry 27:109–114

  • Andreassen OA, Jorgensen HA (2000) Neurotoxicity associated with neuroleptic-induced oral dyskinesias in rats, implications for tardive dyskinesia. Prog Neurobiol 61:525–531

  • Bergamo M, Abílio VC, Queiroz MT, Barbösa-Júnior HN, Prussa-Filho R (1997) The effects of age on a new animal model of tardive dyskinesia. Neurobiol Aging 18:623–629

  • Burger ME, Fachinetto R, Zeni G, Rocha JBT (2005) Ebselen attenuates haloperidol-induced orofacial dyskinesia and oxidative stress in rat brain. Pharmacol Biochem Behav 81:608–615

  • Calvente PRV, Araujo CCS, Bergamo M, Abílio VC, D’Almeida V, Riberiro RA, Frussa-Filho R (2002) The mitochondrial toxin 3-nitropropionic acid aggravates reserpine-induced oral dyskinesias in rats. Prog Neuropsychopharmacol Biol Psychiatry 26:401–405

  • Casey DE (2000) Tardive dyskinesia: pathophysiology and animal models. J Clin Psychiatry 61:5–9

  • Domino EF (1985) Induction of tardive dyskinesia in Cebus apella and Macaca speciosa monkeys. A review. Psychopharmacology Suppl 2:217–223

  • Gunne LM, Barany S (1979) A monitoring test for the liability of neuroleptic drugs to induce tardive dyskinesia. Psychopharmacology (Berl) 63:195–198

  • Harvey BH, Nel A (2003) Role of aging and striatal nitric oxide synthase activity in an animal model of tardive dyskinesia. Brain Res Bull 61:407–416

  • Naidu PS, Singh A, Kaur P, Sandhir R, Kulkarni SK (2003) Possible mechanism of action in melatonin attenuation of haloperidol-induced orofacial dyskinesia. Pharmacol Biochem Behav 74:641–648

  • Neisewander JL, Castańeda E, Davis DA (1994) Dose-dependent differences in the development of reserpine-induced dyskinesia in rats: support for a model of tardive dyskinesia. Psychopharmacology (Berl) 116:79–84

  • Peixoto MF, Abílio VC, Silva RH, Frussa-Filho R (2003) Effects of valproic acid on an animal model of tardive kinesia. Behav Brain Res 142:229–233

  • Queiroz CMT, Frussa-Filho R (1999) Effects of buspirone on a model of tardive dyskinesia. Prog Neuropsychopharmacol Biol Psychiatry 22:1405–1418

  • See RE, Ellison G (1990) Comparison of chronic administration of haloperidol and the atypical neuroleptics, clozapine and raclopride, in an animal model of tardive dyskinesia. Eur J Pharmacol 181:175–186

  • Takeuchi H, Ishigooka J, Kobayashi K, Watanabe S, Miura S (1998) Study on the suitability of a rat model for tardive dyskinesia and the preventive effects of various drugs. Prog Neuropsychopharmacol Biol Psychiatry 22:679–691

  • Tamminga CA, Thaker GK, Moran M, Kakigi T, Gao XM (1994) Clozapine in tardive dyskinesia: observations from human and animal studies. J Clin Psychiatry 55(Suppl B):102–106

  • Van Kampen JM, Stoessl AJ (2000) Dopamine D1A receptor function in a rodent model of tardive dyskinesia. Neuroscience 101:629–635

  • Waddington JL (1990) Spontaneous orofacial movements induced in rodents by very long-term neuroleptic drug administration. Phenomenology, pathophysiology and putative relationship to tardive dyskinesia. Psychopharmacology (Berl) 101:431–447

  • Werge T, Elbaek Z, Andersen MB, Lundbaek JA, Rasmussen HB (2003) Cebus apella, a nonhuman primate highly susceptible to neuroleptic side effects, carries the GLY9 dopamine reporter associated with tardive dyskinesia in humans. Pharmacogenomics J 3:97–100

Single-Unit Recording of A9 and A10 Midbrain Dopaminergic Neurons

Purpose and Rationale

Interactions with central nervous system dopamine pathways are crucial for the expression of antipsychotic effects seen with clinically effective neuroleptics. These interactions also have a role in the expression of several of the neurological side effects seen with these agents. Extracellular single-unit recording techniques of rat A9 (substantia nigra) and A10 (ventral tegmental area) dopamine neurons show that after acute treatment with neuroleptics, the number of spontaneously firing cells is increased in both areas. After repeated treatment (21 days), a decrease was found with all neuroleptics in the A10 neurons, whereas in the A9 cell, only compounds with clinically evident extrapyramidal side effects induced a decrease. Clozapine which is believed not to produce extrapyramidal side effects resulted in the depolarization inactivation of A10 neurons but not A9 cells. The method provides a prediction of a compound’s antipsychotic potential as well as potential neurological side effects (Chiodo and Bunney 1983).

Procedure

Male Wistar rats weighing 280–360 g are anesthetized with chloral hydrate intraperitoneally. The animal is mounted in a stereotaxic apparatus (Kopf, model 900). The cranium is exposed, cleaned of connective tissue, and dried. The skull overlying both the substantia nigra (A9: anterior (A) 3000–3400 μm, lateral (L) 1800–2400 μm from lambda) and the ventral tegmental area (A10: A 3000–3400 μm, L 400–1000 μm from lambda) (Paxinos and Watson 1986) is removed. Using the dura as point of reference, a micropipette driven by a hydraulic microdrive is lowered through the opening of the skull at vertical 6000–8500 μm. Spontaneously firing dopamine neurons within both the substantia nigra and the ventral tegmental area are counted by lowering the electrode into twelve separate tracks (each track separated from the other by 200 μm) in each region. The sequence of these tracks is kept constant, forming a block of tissue which can be reproducibly located from animal to animal.

Extracellular neuronal signals are sampled using a single barrel micropipette approximately one μm at its tip and filled with 2 M NaCl saturated with 1 % pontamine sky blue dye (in vitro impedance between 5 and 10 MΩ). Electrical potentials are passed through a high-impedance preamplifier, and the signal is sent to a window discriminator which converts potentials above background noise levels to discrete pulses of fixed amplitude and duration. Only cell whose electrophysiological characteristics match those previously established for midbrain dopamine neurons are counted. In an anesthetized rat, a neuron is considered to be dopaminergic if it displays a triphasic positive–negative–positive spike profile of 0.4 to 1.5 mV amplitude and 2.5 ms duration, firing in an irregular pattern of 3 to 9 Hz with occasional bursts characterized by progressively decreasing spike amplitude and increasing spike duration.

At the end of each experiment, the location of the last recorded track tip is marked by passing 25 microampere cathodal current through the recording micropipette barrel for 15 min in order to deposit a spot of dye. The rat is sacrificed, and the brain is then removed, dissected, and frozen on a bed of dry ice. Frozen serial sections (20 μm in width) are cut, mounted and stained with cresyl violet, and examined using a light microscope.

Animals pretreated with vehicle prior to neuronal sampling serve as controls. For animals that are used in an acute single-unit dopamine neuron sampling assay, test compounds are administered intraperitoneally 1 h prior to the beginning of dopamine neuron sampling. For animals used in a chronic single-unit dopamine sampling assay, the compounds are administered once a day for 21 days, and dopamine neuron sampling is started 2 h after the last dose on the 21st day.

Evaluation

Drug treatment groups are compared to vehicle groups with a one-way ANOVA with a post hoc Newman–Keuls analysis for significance.

Modifications of the Method

Nyback et al. (1975) tested the influence of tricyclic antidepressants on the spontaneous activity of norepinephrine-containing cells of the locus coeruleus in anesthetized rats.

Scuvée-Moreau and Dreese (1979) studied the effect of various antidepressant drugs on the firing rate of locus coeruleus and dorsal raphe neurons of the anesthetized rat with extracellular microelectrodes.

Using the method of single-unit recording of spontaneous firing of locus coeruleus neurons in rats, Cedarbaum and Aghajanian (1977) studied the inhibition by microiontophoretic application of catecholaminergic agonists.

Marwaha and Aghajanian (1982) examined in single-unit studies the actions of adrenoceptor antagonists at alpha-1 adrenoceptors of the dorsal raphe nucleus and the dorsal lateral geniculate nucleus and alpha-2 adrenoceptors of the nucleus locus coeruleus.

Mooney et al. (1990) studied the organization and actions of the noradrenergic input to the superior colliculus of the hamster using microiontophoretic techniques together with extracellular single-unit recording.

Bernardini et al. (1991) studied in vitro with brain slices of mice the amphetamine-induced and spontaneous release of dopamine from A9 and A10 cell dendrites.

Santucci et al. (1997) investigated the effects of synthetic neurotensin receptor antagonists on spontaneously active A9 and A10 neurons in rats.

References and Further Reading

  • Bernardini GL, Gu X, Viscard E, German DC (1991) Amphetamine-induced and spontaneous release of dopamine from A9 and A10 cell dendrites: an in vitro electrophysiological study in the mouse. J Neural Transm 84:183–193

  • Bowery B, Rothwell LA, Seabrock GR (1994) Comparison between the pharmacology of dopamine receptors mediating the inhibition of cell firing in rat brain slices through the substantia nigra pars compacta and ventral tegmental area. Br J Pharmacol 112:873–880

  • Bunney BS, Grace AA (1978) Acute and chronic haloperidol treatment: comparison of effects on nigral dopaminergic cell activity. Life Sci 23:1715–1728

  • Cedarbaum JM, Aghajanian GK (1977) Catecholamine receptors on locus coeruleus neurons: pharmacological characterization. Eur J Pharmacol 44:375–385

  • Chiodo LA, Bunney BS (1983) Typical and atypical neuroleptics: differential effect of chronic administration on the activity of A9 and A10 midbrain dopaminergic neurons. J Neurosci 3:1607–1619

  • Marwaha J, Aghajanian GK (1982) Relative potencies of alpha-1 and alpha-2 antagonists in the locus coeruleus, dorsal raphe and dorsal lateral geniculate nuclei: an electrophysiological study. J Pharmacol Exp Ther 222:287–293

  • Mooney RD, Bennett-Clarke C, Chiaia NL, Sahibzada N, Rhoades RW (1990) Organization and actions of the noradrenergic input to the hamster’s superior colliculus. J Comp Neurol 292:214–230

  • Nyback HV, Walters JR, Aghajanian GK, Roth RH (1975) Tricyclic antidepressants: effects on the firing rate of brain noradrenergic neurons. Eur J Pharmacol 32:302–312

  • Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates, 2nd edn. Academic, Sydney

  • Santucci V, Gueudet C, Steinberg R, Le Fur G, Soubrie P (1997) Involvement of cortical neurotensin in the regulation of rat mesocortico-limbic dopamine neurons: evidence from changes in the number of spontaneously active A10 cells after neurotensin receptor blockade. Synapse 26:370–380

  • Schmidt CJ, Black CK, Taylor VL, Fadayel GM, Humphreys TM, Nieduzak TR, Sorensen SM (1992) The 5-HT2 receptor antagonist, MDL 28,133A, disrupts the serotonergic-dopaminergic interaction mediating the neurochemical effects of 3,4-methylenedioxymethylamphetamine. Eur J Pharmacol 220:151–159

  • Scuvée-Moreau JJ, Dreese AE (1979) Effect of various antidepressant drugs on the spontaneous firing rate of locus coeruleus and dorsal raphe neurons of the rat. Eur J Pharmacol 57:219–225

  • Todorova A, Dimpfel W (1994) Multiunit activity from the A9 and A10 areas in rats following chronic treatment with different neuroleptic drugs. Eur Neuropsychopharmacol 4:491–501

  • White FJ, Wang RY (1983a) Comparison of the effects of chronic haloperidol treatment on A9 and A10 dopamine neurons in the rat. Life Sci 32:983–993

  • White FJ, Wang RY (1983b) Differential effects of classical and atypical antipsychotic drugs on A9 and A10 dopamine neurons. Science 221:1054–1057

In Vivo Voltammetry

Purpose and Rationale

Various groups (Lane et al. 1979, 1987, 1988; Blaha and Lane 1983, 1984, 1987; Crespi et al. 1984; Marsden et al. 1984; Maidment and Marsden 1987a, b; Armstrong-James and Millar 1979, 1984; Kawagoe et al. 1993) described in vivo voltammetry as an electrochemical technique that uses carbon fiber microelectrodes stereotactically implanted in brain areas to monitor monoamine metabolism and release. De Simoni et al. (1990) reported on a miniaturized optoelectronic system for telemetry of in vivo voltammetric signals in freely moving animals.

Procedure

Carbon fiber working electrodes are made from pyrolytic carbon fibers supported in a pulled glass capillary (Armstrong-James and Millar 1979; Sharp et al. 1984) and electrically pretreated for simultaneous recording of ascorbic acid DOPAC and 5-HIAA (Crespi et al. 1984).

Male Sprague Dawley rats weighing 270–340 g are anesthetized with a 2–3 % halothane O2/NO2 mixture (1:1) and held in a stereotactic frame. Reference and auxiliary electrodes are positioned on the surface of the dura through 1 mm holes drilled in the cranium and held in place with dental cement. Holes, approx. 2 mm in diameter, are drilled in the cranium above the left or right nucleus accumbens and contralateral anterior striatum, and the underlying dura is broken with a hypodermic needle. A working electrode is lowered in one of the above regions and cemented in place. A second electrode is then implanted in the remaining structure. The coordinates, measured from the bregma, are as follows: nucleus accumbens–rostrocaudal +3.4 mm, mediolateral ± 1.4 mm, dorsoventral −7 mm, striatum–rostrocaudal +2.8 mm, mediolateral ± 2.6 mm, and dorsoventral −5.5 mm.

Drugs are injected subcutaneously. Voltammograms are recorded using a Princeton Applied Research 174A polarographic analyzer alternatively from each region every 5 min and after a 1 h stabilization period.

Evaluation

Voltammetric data are expressed as percentage changes from preinjection control values using the mean of the last six peak heights before administration of drug as the 100 % value. However, statistical analysis of the data is carried out on the absolute peak heights using a paired Student’s t-test to compare six preinjection control peak heights with those after administration of drug at selected time points.

Modifications of the Method

Swiergiel et al. (1997) constructed voltammetric probes from stainless steel and fused silica tubing sheathing carbon fibers and compared them with commercially available glass-sealed IVEV-5 electrodes. This type of electrodes can be easily manufactured and does not require any special equipment.

Parada et al. (1994, 1995) described a triple-channel swivel suitable for intracranial fluid delivery and microdialysis experiments which can be equipped with three electrical channels for in vivo voltammetry and measurement of intracranial temperature with a thermocouple.

Frazer and Daws (1998) used electrodes coated with a perfluorinated ion exchange resin (Nafion) to assess serotonin transporter function in vivo by chronoamperometry whereby voltage is applied to the electrode in a pulsed manner and the current obtained measured as a function of time.

References and Further Reading

  • Armstrong-James M, Millar J (1979) Carbon fibre microelectrodes. J Neurosci Methods 1:279–287

  • Armstrong-James M, Millar J (1984) High-speed cyclic voltammetry and unit recording with carbon fibre microelectrodes. In: Marsden CA (ed) Measurement of neurotransmitter release in vivo. Wiley, Chichester/New York, pp 209–224

  • Blaha CD, Lane RF (1983) Chemically modified electrode for in vivo monitoring of brain catecholamines. Brain Res Bull 10:861–864

  • Blaha CD, Lane RF (1984) Direct in vivo electrochemical monitoring of dopamine release in response to neuroleptic drugs. Eur J Pharmacol 98:113–117

  • Blaha CD, Lane RF (1987) Chronic treatment with classical and atypical antipsychotic drugs differentially decreases dopamine release in striatum and nucleus accumbens in vivo. Neurosci Lett 78:199–204

  • Buda M, Gonon FG (1987) Study of brain noradrenergic neurons by use of in vivo voltammetry. In: Justice JB Jr (ed) Voltammetry in the neurosciences: principles, methods and applications. Humana Press, Clifton, pp 239–272

  • Cespuglio R, Faradji H, Hahn Z, Jouvet M (1984) Voltammetric detection of brain 5-hydroxyindolamines by means of electrochemically treated carbon fibre electrodes: chronic recordings for up to one month with movable cerebral electrodes in the sleeping or waking rat. In: Marsden CA (ed) Measurement of neurotransmitter release in vivo. Wiley, Chichester/New York, pp 173–191

  • Crespi F, Sharp T, Maidment NT, Marsden CA (1984) Differential pulse voltammetry: simultaneous in vivo measurement of ascorbic acid, catechols and 5-hydroxyindoles in the rat striatum. Brain Res 322:135–138

  • de Simoni MG, de Luigi A, Imeri L, Algerin S (1990) Miniaturized optoelectronic system for telemetry of in vivo voltammetric signals. J Neurosci Methods 33:233–240

  • Frazer A, Daws LC (1998) Serotonin transporter function in vivo: assessment by chronoamperometry. In: Martin GR, Eglen RM, Hoyer D, Hamblin MW, Yocca F (eds) Advances in serotonin research. Molecular biology, signal transduction, and therapeutics, vol 861, Annals of the New York Academy Sciences. New York Academy of Sciences, New York, pp 217–229

  • Gonon FG (1987) In vivo electrochemical monitoring of dopamine release. In: Justice JB Jr (ed) Voltammetry in the neurosciences: principles, methods and applications. Humana Press, Clifton, pp 163–183

  • Gonon F, Buda M, Oujol JF (1984) Treated carbon fibre electrodes for measuring catechols and ascorbic acid. In: Marsden CA (ed) Measurement of neurotransmitter release in vivo. Wiley, Chichester/New York, pp 153–171

  • Justice JB Jr (1987) Introduction to in vivo voltammetry. In: Justice JB Jr (ed) Voltammetry in the Neurosciences: principles, methods and applications. Humana Press, Clifton, pp 3–102

  • Justice JB Jr, Michael AC (1987) Monitoring extracellular DOPAC following stimulated release of dopamine. In: Justice JB Jr (ed) Voltammetry in the neurosciences: principles, methods and applications. Humana Press, Clifton, pp 185–208

  • Kawagoe KT, Zimmerman JB, Wightman RM (1993) Principles of voltammetry and microelectrode surface states. J Neurosci Methods 48:225–240

  • Lane RF, Blaha CD (1986) Electrochemistry in vivo: application to CNS pharmacology. Ann N Y Acad Sci 473:50–69

  • Lane RF, Hubbard AT, Blaha CD (1979) Application of semidifferential electroanalysis to studies of neurotransmitters in the central nervous system. J Electroanal Chem 95:117–122

  • Lane RF, Blaha CD, Hari SP (1987) Electrochemistry in vivo: monitoring dopamine release in the brain of the conscious, freely moving rat. Brain Res Bull 19:19–27

  • Lane RF, Blaha CD, Rivet JM (1988) Selective inhibition of mesolimbic dopamine release following chronic administration of clozapine: involvement of α 1-noradrenegic receptors demonstrated by in vivo voltammetry. Brain Res 460:389–401

  • Maidment NT, Marsden CA (1985) In vivo voltammetric and behavioral evidence for somatodendritic autoreceptor control of mesolimbic dopamine neurons. Brain Res 338:317–325

  • Maidment NT, Marsden CA (1987a) Acute administration of clozapine, thioridazine, and metoclopramide increases extracellular DOPAC and decreases extracellular 5-HIAA, measured in rat nucleus accumbens and striatum of the rat using in vivo voltammetry. Neuropharmacology 26:187–193

  • Maidment NT, Marsden CA (1987b) Repeated atypical neuroleptic administration: effects on central dopamine metabolism monitored by in vivo voltammetry. Eur J Pharmacol 136:141–149

  • Marsden CA, Brazell MP, Maidment NT (1984) An introduction to in vivo electrochemistry. In: Marsden CA (ed) Measurement of neurotransmitter release in vivo. Wiley/New York, Chichester, pp 127–151

  • Marsden CA, Martin KF, Brazell MP, Maidment NT (1987) In vivo voltammetry: application to the identification of dopamine and 5-hydroxytryptamine receptors. In: Justice JB Jr (ed) Voltammetry in the neurosciences: principles, methods and applications. Humana Press, Clifton, pp 209–237

  • Nagatsu T, Ikeda M, Fujita K, Shinzato M, Takahashi H, Adachi T (1987) Application of in vivo voltammetry to behavioral pharmacology. In: Justice JB Jr (ed) Voltammetry in the neurosciences: principles, methods and applications. Humana Press, Clifton, pp 313–324

  • Parada MA, Puig de Parada M, Hoebel BG (1994) A new triple-channel swivel for fluid delivery in the range of intracranial (10 nl) and intravenous (100 μl) self-administration volumes and also suitable for microdialysis. J Neurosci Methods 54:1–8

  • Parada MA, Puig de Parada M, Hernandez L, Hoebel BG (1995) Triple electrical channels on a triple fluid swivel and its use to monitor intracranial temperature with a thermocouple. J Neurosci Methods 60:133–139

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Genetic Models of Psychosis

The Heterozygous Reeler Mouse

Purpose and Rationale

Reelin is an extracellular matrix protein secreted by GABAergic interneurons that, acting through pyramidal neuron integrin receptors, provides a signal for dendritic spine plasticity. The gene responsible for a mouse mutant strain is called reeler (D’Arcangelo and Curran 1998; Lombroso and Goldowitz 1998; Fatemi 2001; Pappas et al. 2003). Heterozygous reeler mice that exhibit a 50 % downregulation of reelin expression replicate the dendritic spine and GABAergig defects described in human schizophrenia (Larson et al. 2003). The heterozygote reeler mouse was recommended as a model for the development of a new generation of antipsychotics (Tueting et al. 1999; Rowley et al. 2001; Costa et al. 2002). This view has been challenged by Podhorna and Didriksen (2004).

Tomasiewicz et al. (1993) and Wood et al. (1998) proposed NCAM-180 knockout mice with a deletion of the neural cell adhesion molecule variant (NCAM-180) displaying increased lateral ventricle size and a reduced prepulse inhibition of startle response as model for schizophrenia.

Dirks et al. (2003) reported reversal of startle gating deficits in transgenic mice overexpressing corticotropin-releasing factor by antipsychotic drugs.

Van den Buuse (2003) showed deficient prepulse inhibition of acoustic startle in Hooded-Wistar rats compared with Sprague Dawley rats, suggesting that the Hooded-Wister line could be a useful genetic animal model to study the interaction of glutaminergic and dopaminergic mechanisms in anxiety and schizophrenia.

References and Further Reading

  • Costa E, Davis J, Pesold C, Tueting P, Guidotti A (2002) The heterozygote reeler mouse as a model for the development of a new generation of antipsychotics. Curr Opin Pharmacol 2:56–62

  • D’Arcangelo G, Curran T (1998) Reeler: new tales on an old mutant mouse. Bioassays 20:235–244

  • Dirks A, Groenink L, Westphal KGC, Olivier JDA, Verdouw PM, van der Gugten J, Geyer MA, Olivier B (2003) Reversal of startle gating deficits in transgenic mice overexpressing corticotropin-releasing factor by antipsychotic drugs. Neuropsychopharmacology 28:1790–1798

  • Fatemi SH (2001) Reelin mutations in mouse and man: from reeler mouse to schizophrenia, mood disorders, autism and lissencephaly. Mol Psychiatry 6:129–133

  • Larson J, Hoffman JS, Guidotti A, Costa E (2003) Olfactory discrimination learning deficit in heterozygous reeler mice. Brain Res 971:40–46

  • Lombroso PJ, Goldowitz D (1998) Brain development, VIII: the reeler mouse. Am J Psychiatry 155:1660

  • Pappas GD, Kriho V, Liu WS, Tremolizzo L, Lugli G, Larson J (2003) Immunocytochemical localization of reelin in the olfactory bulb of the heterozygous reeler mouse. An animal model for schizophrenia. Neurol Res 25:819–830

  • Podhorna J, Didriksen M (2004) The heterozygous reeler mouse: behavioural phenotype. Behav Brain Res 153:43–54

  • Rowley M, Bristow LJ, Hutson PH (2001) Current and novel approaches to the drug treatment of schizophrenia. J Med Chem 44:477–501

  • Tomasiewicz H, Ono K, Yee D, Thompson C, Goridis C, Rutishauser U, Magnuson T (1993) Genetic deletion of a neural cell adhesion molecule variant (N-CAM-180) produces defects in the central nervous system. Neuron 11:1163–1174

  • Tueting P, Costa E, Dwivedi Y, Guidotti A, Impagnatiello F, Manev R, Pesold C (1999) The phenotypic characterization of heterozygous reeler mouse. Neuroreport 10:13291334

  • van den Buuse M (2003) Deficient prepulse inhibition of acoustic startle in Hooded-Wistar rats compared with Sprague–Dawley rats. Clin Exp Pharmacol Physiol 30:254–261

  • Wood GK, Tomasiewicz H, Rutishauser U, Magnuson T, Quirion R, Rochford J, Srivasta LK (1998) NCAM-180 knockout mice display increased lateral ventricle size and reduced prepulse inhibition of startle. Neuroreport 16:461–466

The Hooded-Wistar Rat

Purpose and Rationale

Van den Buuse (2003), Lodge et al. (2003), and Martin et al. (2004) suggested that the Hooded-Wistar line (fawn-hooded rats) could be a useful genetic animal model to study the interaction of glutamatergic and dopaminergic mechanisms in anxiety and schizophrenia.

Broderick (2002) compared hippocampal serotonin and norepinephrine release during open-field behavior in Sprague Dawley animals with the Fawn-Hooded animals model of depression.

References and Further Reading

  • Broderick PA (2002) Interleukin 1α alters hippocampal serotonin and norepinephrine release during open-field behavior in Sprague–Dawley animals: difference from the Fawn-Hooded animals model of depression. Prog Neuropsychopharmacol Biol Psychiatry 26:1355–1372

  • Lodge DJ, Roques BP, Lawrence AJ (2003) Atypical behavioural responses to CCK-B receptor ligands in Fawn-Hooded rats. Life Sci 74:1–12

  • Martin S, Lawrence AJ, van den Buuse M (2004) Prepulse inhibition in fawn-hooded rats: increased sensitivity to 5-HT1A receptor stimulation. Eur Neuropsychopharmacol 14:373–379

  • van den Buuse M (2003) Deficient prepulse inhibition of acoustic startle in Hooded-Wistar rats compared with Sprague–Dawley rats. Clin Exp Pharmacol Physiol 30:254–261