INTRODUCTION

Aggressive behavior is an important evolutionary adaptation that serves as a tool for competition over resources, territory, and mating partners. Various hormones and neurotransmitters participate in the regulation of aggression, but a special role in the mechanisms underlying this behavior is attributed to serotonin (5-hydroxytryptamine, 5-HT). The latter is one of the most important neurotransmitters in the brain. Serotonergic neurons, whose cell bodies are located in raphe nuclei of the midbrain, interact with almost all areas of the central nervous system. Serotonin influences many physiological systems of the body, mood, and various types of behavior [1].

Key enzymes of the serotonergic system are tryptophan hydroxylase 2 (TPH2; EC 1.14.16.4, tryptophan 5-monooxygenase, which catalyzes the rate-limiting step of 5-HT biosynthesis: the addition of an OH group at position 5 of tryptophan) and monoamine oxidase A (MAOA; EC 1.4.3.4, which oxidizes serotonin to 5-hydroxyindoleacetaldehyde. The connection of these enzymes with aggressive behavior is well known. Numerous articles have revealed a positive correlation between intermale aggression and the expression and activity of TPH2 [2-6]. In a study on mice of 10 genotypes, it was shown that animals with reduced TPH2 activity in the brain exhibit less profound intermale aggression [3]. Investigation of a polymorphism affecting TPH2 activity has revealed its relation to the intensity of intermale aggression [4]. TPH2 inhibitor para-chlorophenylalanine reduces intermale aggression in C57BL/6 mice [2]. Studies on humans have uncovered a similar trend: decreased aggression levels in individuals with low-activity alleles of the TPH2 gene [7, 8]. Nonetheless, some researchers have demonstrated a reverse correlation between TPH2 and aggression. For instance, mice with a knockout of this gene (Tph2−/−) show greater aggression as compared to wild-type animals in the resident–intruder test [9]. Furthermore, rats with a knockout of Tph2 exhibit more aggressive behavior during social interactions [10].

A deficiency in MAOA leads to increased intermale aggression in mice [11, 12]. Animals with a genetic knockout of the Maoa gene are more prone to retaliatory aggression, engage in fights more often, and have shorter first-attack latency [12, 13]. Pharmacological inhibition of this enzyme during prenatal development in mice and rats enhances aggressive tendencies in adulthood [14, 15]. In humans, a MAOA deficiency also leads to increased manifestation of aggressive behavior [16], and diminished MAOA enzymatic activity is associated with the expression of hostility, anger, and aggressive traits [17, 18].

Experimental models based on genetically selected animals are widely used to study mechanisms behind specific types of behavior. At the Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences (ICG SB Russian Academy of Sciences; Novosibirsk, Russia) gray rats are selectively bred to investigate fear-induced aggression and domestication. The selection is conducted in two directions: for a high level of aggression toward humans (aggressive rats) and for its complete absence (tame rats). The obtained strains, among other differences, demonstrate variations in the serotonergic system: in the overall level of this neurotransmitter and its metabolite in the brain [19, 20], in the expression and activity of serotonin receptors [20-25], and in the expression of the serotonin transporter gene [26]. In aggressive rats compared to tame ones, in the 24th generation of the selection, a decrease in TPH2 activity in the midbrain was observed [20]. On the other hand, MAOA activity did not differ between the two strains in generations 35-36 [27].

It was recently found that benzopentathiepin TC-2153 reduces aggression in the aggressive rats [28]. Furthermore, this substance exerts a number of other beneficial effects on behavior, particularly anxiolytic [28, 29], anticataleptic [30], and antidepressant actions [31]. Moreover, it is known that TC-2153 affects the serotonergic system: overall levels of both serotonin and its metabolite [32] and the expression and functional activity of serotonin receptors [22, 31, 33].

On the other hand, the influence of TC-2153 on the expression of TPH2 and MAOA in aggressive and tame rats has not been researched before. Furthermore, levels of these proteins and the expression of genes encoding them have not been investigated in these animals. Thus, the aim of this study was to examine the impact of single administration of TC-2153 on the activity and expression of TPH2 and MAOA in the brain of aggressive and tame rats.

MATERIALS AND METHODS

Animals. The experiments were performed on adult outbred male gray rats (Rattus norvegicus) 4-5 months old and weighing 300-350 g, selectively bred for over 85 generations at the ICG SB Russian Academy of Sciences for a high level of aggression toward humans (aggressive, 24 animals) or its complete absence (tame, 24 animals) [34, 35].

The animals were housed in groups of four in metal cages measuring 50×33×20 cm under standard laboratory conditions on a 12-h light/dark cycle with ad libitum access to feed and water. The study was conducted at the Center for Genetic Resources of Laboratory Animals of the federal research center ICG SB Russian Academy of Sciences (RFMEFI62119X0023).

Pharmacological intervention. The study involved the substance TC-2153 [8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride], which was synthesized at N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB Russian Academy of Sciences (Novosibirsk, Russia). The compound was diluted with a solution (vehicle) consisting of 0.05% of Tween 20 (v/v), 0.05% of DMSO (v/v), and 0.9% of NaCl (w/v) [31] and was administered intraperitoneally (i.p.) at 100 µl per 100 g of an animal’s body weight at a dose of 10 or 20 mg/kg.

Experimental design. Twenty-four aggressive rats (and 24 tame rats) were subdivided into three groups of eight animals each. Control animals received the vehicle solution. The other two groups received TC-2153 i.p. at a dose of 10 or 20 mg/kg. Five hours after the injection of the vehicle or the drug, the animals were euthanized with decapitation. The doses and timing were chosen based on previously obtained data about effects of TC-2153 on behavior and on the serotonergic system [29-33, 36]. The dissected brain structures (midbrain, hippocampus, and hypothalamus) were frozen in liquid nitrogen and stored at –80°C until molecular analyses were performed.

Each brain structure was homogenized in 300 µl of Tris-HCl buffer (50 mM, pH 7.6) at 4°C using a mechanical homogenizer (Z359971, Sigma-Aldrich, USA), and aliquots of the homogenate were used for chromatography and total-RNA and total-protein extraction.

Gene expression quantification. Total RNA was extracted from 60 µl of a homogenate by means of the TRIzol Reagent (Life Technologies, USA) according to the manufacturer’s instructions and a previously described protocol [28]. Complementary DNA (cDNA) was synthesized from the obtained total RNA, and gene expression was measured via detection of the fluorescence of intercalating dye SYBR Green I (R-402 Master mix, Syntol, Russia). The primers used are presented in table. Genomic DNA isolated from hepatocytes of Wistar male rats was used as an external standard at concentrations of 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, and 64 ng/µl [37-39]. Gene expression was evaluated as the number of cDNA copies of a target gene per 100 copies of housekeeping gene DNA-dependent RNA polymerase 2 (Polr2a), serving as an internal standard.

The sequences of utilized primers, their annealing temperatures, and amplicon lengths

Gene

Primer sequence

Annealing temperature, °C

Product length, bp

Polr2a

F: 5′-TTGTCGGGCAGCAGAACGTG-3′

R: 5′-CAATGAGACCTTCTCGTCCTCCC-3′

63

186

Maoa

F: 5′-GAAATTACCCACACCTTCTTAGAG-3′

R: 5′-CACTTTCTTTCACATGCGATG-3′

60

178

Tph2

F: 5′-CCCAGGCAGATGACCATTCAG-3′

R: 5′-GGAATTGTGTGAGGAATGTTGGC-3′

64

146

Protein quantification with western blot analysis. The levels of proteins TPH2 and MAOA were determined by western blotting via a previously described method [28]. Components of total protein were separated by SDS polyacrylamide gel electrophoresis (20 µg of protein per lane) using a 10% separating gel. A polyclonal rabbit antibody to the TPH2 protein (1 : 1000 dilution, cat. # ab184505, Abcam, UK) and a monoclonal rabbit antibody to the MAOA protein (1 : 500, ab126751, Abcam) were used for target protein detection. A polyclonal rabbit antibody to GAPDH (1 : 2000, ab9485, Abcam) were employed as an internal control. Protein levels were expressed in relative units normalized to the GAPDH protein level. The TPH2 protein was detected at the 56 kDa level, MAOA at 60 kDa, and GAPDH at 37 kDa.

Monoamine oxidase activity assay. MAO activity was determined by a previously described procedure [40] with a modular chromatographic analysis system (Shimadzu Corporation, USA) equipped with a Luna C18(2) column (5 μm particle size, L × I.D. 100 × 4.6 mm, Phenomenex, USA), gradient pump (LC-20AD) with a vacuum degasser (DGU-20A5R), an autosampler with a 100 µl loop (SIL-20A), and an electrochemical detector (750 mV, DECADE II, Antec, Netherlands). The concentration of the substrate (5-HT) in the reaction mixture was 0.15 mM. This method measures the total activity of MAOA and MAOB. MAO activity was calculated as the amount of synthesized 5-hydroxyindoleacetaldehyde (nmol) per minute with normalization to the amount of total protein in the sample, as measured by the Bradford method (assay kit from Bio-Rad, USA) on a Multiscan spectrophotometer (Thermo Fisher Scientific Inc., USA).

Tryptophan hydroxylase 2 activity assay. TPH2 activity was determined using a previously described method [41] with the modular chromatographic analysis system described above. The concentration of the substrate (L-tryptophan) in the reaction mixture was 0.4 mM. TPH2 activity was calculated as the amount of synthesized 5-hydroxytryptophan (pmol) per minute with normalization to the amount of total protein in the sample.

Statistical analysis. Data from measurements of genes’ expression, protein levels, and enzymatic activities were tested for normality of distribution and equality of variances by Lilliefors and Bartlett tests. Data are presented as mean ± standard error and were subjected to two-way analysis of variance (ANOVA) with independent factors “genotype” (strain) and “treatment”. If a significant effect was detected, then the difference between groups was evaluated by a post hoc analysis (Fischer’s least significant difference test for pairwise comparisons). The threshold for statistical significance was set to p < 0.05. Outliers were excluded by Dixon’s test.

RESULTS

Effects of single TC-2153 administration on TPH2 activity in vitro in the brain of aggressive and tame rats. Two-way ANOVA did not detect differences between aggressive and tame rats in the activity of TPH2 in vitro in the midbrain (F1,41 < 1), hippocampus (F1,40 < 1), or hypothalamus (F1,38 < 1) (Fig. 1). We found an effect of the single TC-2153 treatment on this parameter in the hypothalamus (F2,38 = 5.38, p < 0.01) but not in the midbrain (F2,41 = 2.05, p > 0.05) and hippocampus (F2,41 = 2.1, p > 0.05). Therefore, TC-2153 reduced TPH2 activity in the hypothalamus in both aggressive and tame rats (p < 0.05). There was no significant genotype–drug interaction in any of the assayed brain regions (midbrain: F2,41 < 1; hippocampus: F2,40 < 1; hypothalamus: F2,38 < 1).

Fig. 1.
figure 1

The influence of single administration of vehicle (control) or TC-2153 at 10 or 20 mg/kg on TPH2 activity in vitro (pmol/min per 1 mg of protein) in the midbrain (a), hippocampus (b), and hypothalamus (c) of aggressive and tame rats. # p < 0.05 in comparison with the control group (7-8 animals per group). 1) Control aggressive rats, 2) aggressive rats TC-2153 10 mg/kg, 3) aggressive rats TC-2153 20 mg/kg, 4) control tame rats, 5) tame rats TC-2153 10 mg/kg, 6) tame rats TC-2153 20 mg/kg.

Full size image

Effects of the single TC-2153 treatment on Tph2 gene expression in the midbrain of aggressive and tame rats. The assay of Tph2 gene expression in the midbrain by real-time PCR showed an elevated level of mRNA of this gene in aggressive rats compared to tame ones (genotype effect: F1,34 = 13.31, p < 0.001). At the same time, there was no effect of the single TC-2153 treatment (F2,34 < 1) or of genotype–drug interaction (F2,34 < 1) on this parameter (Fig. 2).

Fig. 2.
figure 2

The influence of single administration of vehicle (control) or TC-2153 at 10 or 20 mg/kg on Tph2 mRNA levels in the midbrain of aggressive and tame rats. The expression levels were evaluated as the number of transcript copies per 100 copies of rPol2a mRNA. *** p < 0.001 for the genotype effect (6-7 animals per group). 1) Control aggressive rats, 2) aggressive rats TC-2153 10 mg/kg, 3) aggressive rats TC-2153 20 mg/kg, 4) control tame rats, 5) tame rats TC-2153 10 mg/kg, 6) tame rats TC-2153 20 mg/kg.

Full size image

Effects of the single TC-2153 treatment on TPH2 protein levels in the brain of aggressive and tame rats. A significant effect of the genotype on the TPH2 protein level was observed in the hippocampus (F1,41 = 16.45, p < 0.001) (Fig. 3b) and the hypothalamus (F1,37 = 7.01, p < 0.05) (Fig. 3c) but not in the midbrain (F1,35 = 1.55, p > 0.05) (Fig. 3a). The expression level of this protein proved to be elevated in the hippocampus and hypothalamus of aggressive rats compared to tame ones. There was no impact of single TC-2153 administration in any of the analyzed brain structures (midbrain: F2,35 = 1.26, p > 0.05; hippocampus: F2,41 < 1; hypothalamus: F2,37 < 1), and no genotype–drug interaction was detectable (midbrain: F2,35 < 1; hippocampus: F2,41 = 1.68, p > 0.05; hypothalamus: F2,37 = 1.26, p > 0.05).

Fig. 3.
figure 3

The influence of single administration of vehicle (control) or TC-2153 at 10 or 20 mg/kg on the TPH2 protein levels in the midbrain (a), hippocampus (b), and hypothalamus (c) of aggressive and tame rats. The protein levels are presented in relative units normalized to the corresponding level of the GAPDH protein. * p < 0.05, *** p < 0.001 for the genotype effect (5-8 animals per group). 1) Control aggressive rats, 2) aggressive rats TC-2153 10 mg/kg, 3) aggressive rats TC-2153 20 mg/kg, 4) control tame rats, 5) tame rats TC-2153 10 mg/kg, 6) tame rats TC-2153 20 mg/kg. Some immunoblot data from the membrane were used for TPH2 protein quantitation. I) Control aggressive rats, II) aggressive rats TC-2153 10 mg/kg, III) aggressive rats TC-2153 20 mg/kg, IV) control tame rats, V) tame rats TC-2153 10 mg/kg, VI) tame rats TC-2153 20 mg/kg.

Full size image

Effects of the single TC-2153 treatment on MAO activity in vitro in the brain of aggressive and tame rats. We observed a significantly increased MAO activity in the midbrain (F1,41 = 11.73, p < 0.01) (Fig. 4a) and the hippocampus (F1,41 = 9.61, p < 0.01) (Fig. 4b) of aggressive rats compared to tame ones. No interstrain differences were detectable in the hypothalamus (F1,42 = 3.15, p > 0.05) (Fig. 4c). Furthermore, TC-2153 influenced this parameter in the midbrain of rats (F2,41 = 4.19, p < 0.05) by significantly reducing MAO activity in tame animals (p < 0.05) while the decrease in aggressive rats was not statistically significant (p = 0.07). In the hippocampus (F2,41 < 1) and hypothalamus (F2,42 = 1.24, p > 0.05), the ANOVA did not show an effect of the treatment. There was also no significant effect of interaction between the factors “genotype” and “drug” (midbrain: F2,41 = 2.22, p > 0.05; hippocampus: F2,41 < 1; hypothalamus: F2,42 = 1.97, p > 0.05).

Fig. 4.
figure 4

The influence of single administration of vehicle (control) or TC-2153 at 10 or 20 mg/kg on MAO activity in vitro (nmol/min per 1 mg of protein) in the midbrain (a), hippocampus (b), and hypothalamus (c) of aggressive and tame rats. ** p < 0.01 for the genotype effect, # p < 0.05, %p = 0.07 in comparison with the control group (7-8 animals per group). 1) Control aggressive rats, 2) aggressive rats TC-2153 10 mg/kg, 3) aggressive rats TC-2153 20 mg/kg, 4) control tame rats, 5) tame rats TC-2153 10 mg/kg, 6) tame rats TC-2153 20 mg/kg.

Full size image

Effects of the single TC-2153 treatment on Maoa mRNA expression in the brain of aggressive and tame rats. The expression of the Maoa gene did not differ between aggressive and tame rats in any of the assayed brain regions (midbrain: F1,35 = 1.17, p > 0.05; hippocampus: F1,31 < 1; hypothalamus: F1,34 < 1) (Fig. 5). No effect of single TC-2153 administration was noticeable in the midbrain (F2,35 = 1.72, p > 0.05), hippocampus (F2,31 = 1.54, p > 0.05), or hypothalamus (F2,34 < 1). An effect of genotype–drug interaction was not statistically significant either (midbrain: F2,35 < 1; hippocampus: F2,31 < 1; hypothalamus: F2,34 < 1).

Fig. 5.
figure 5

The influence of single administration of vehicle (control) or TC-2153 at 10 or 20 mg/kg on Maoa mRNA levels in the midbrain (a), hippocampus (b), and hypothalamus (c) of aggressive and tame rats. The expression levels were evaluated as the number of transcript copies per 100 copies of rPol2a mRNA (5-7 animals per group). 1) Control aggressive rats, 2) aggressive rats TC-2153 10 mg/kg, 3) aggressive rats TC-2153 20 mg/kg, 4) control tame rats, 5) tame rats TC-2153 10 mg/kg, 6) tame rats TC-2153 20 mg/kg.

Full size image

Effects of the single TC-2153 treatment on MAOA protein levels in the brain of aggressive and tame rats. The level of the MAOA protein was significantly higher in the hypothalamus of aggressive rats compared to tame ones (F1,32 = 16.88, p < 0.001) (Fig. 6c), while in the midbrain (F1,32 = 3.21, p > 0.05) (Fig. 6a) and hippocampus (F1,39 = 2.44, p > 0.05) (Fig. 6b), the effect of the genotype was not statistically significant. Single administration of TC-2153 did not affect the expression of this protein (midbrain: F2,32 < 1; hippocampus: F2,39 < 1; hypothalamus: F2,32 < 1), and there was no genotype–drug interaction in the midbrain (F2,32 < 1) and hippocampus (F2,39 < 1). Meanwhile, a significant effect of interaction of these factors was noted in the hypothalamus of aggressive and tame rats (F2,32 = 5.71, p < 0.01): TC-2153 reduced the level of MAOA protein in this structure in tame animals (p < 0.05), while in aggressive animals, there was a tendency toward an increase (p = 0.056).

Fig. 6.
figure 6

The influence of single administration of vehicle (control) or TC-2153 at 10 or 20 mg/kg on MAOA protein levels in the midbrain (a), hippocampus (b), and hypothalamus (c) of aggressive and tame rats. The protein levels are presented in relative units normalized to the corresponding level of the GAPDH protein. *** p < 0.001 for the genotype effect, # p < 0.05, % p = 0.056 in comparison with the control group (4-8 animals per group). 1) Control aggressive rats, 2) aggressive rats TC-2153 10 mg/kg, 3) aggressive rats TC-2153 20 mg/kg, 4) control tame rats, 5) tame rats TC-2153 10 mg/kg, 6) tame rats TC-2153 20 mg/kg. Some immunoblot data from the membrane were used for MAOA protein quantitation. I) Control aggressive rats, II) aggressive rats TC-2153 10 mg/kg, III) aggressive rats TC-2153 20 mg/kg, IV) control tame rats, V) tame rats TC-2153 10 mg/kg, VI) tame rats TC-2153 20 mg/kg.

Full size image

DISCUSSION

Currently, there are conflicting data regarding the role of TPH2 in the regulation of aggressive behavior. Some researchers have reported a positive correlation of the expression and activity of TPH2 with this type of behavior [2-6]. Nonetheless, a knockout of the Tph2 gene results in higher aggression in mice and rats [9, 10]. It is worth noting that consequences of a gene knockout likely reflect compensatory changes caused by the absence of the gene product rather than by effects of the gene itself. In our study, we demonstrated that the expression of the Tph2 gene in the midbrain of aggressive rats is higher as compared to tame rats. At the same time, the level of the TPH2 protein was found to be increased in aggressive animals in the hippocampus and hypothalamus but not in the midbrain (Fig. 7). These results are consistent with the majority of the data on the positive correlation between TPH2 expression and aggressive behavior. It is known that cell bodies of serotonergic neurons are located in midbrain raphe nuclei, from where they project to other parts of the central nervous system. The enzyme TPH2 is produced in the cell bodies of serotonergic neurons in the midbrain and is transported along their axons to other brain structures, where it ensures the synthesis of serotonin at synaptic terminals. Accordingly, in our study, the observed increase in mRNA levels in the midbrain mainly can be attributed to serotonergic neurons projecting to the hippocampus and hypothalamus, where upregulation of protein levels was observed. The enzymatic activity of TPH2 did not differ between aggressive and tame rats. These results contradict earlier findings in rats of selection generations 24-27, where an increase in the activity of this enzyme was observed in the midbrain of tame rats compared to aggressive ones [20]. The observed discrepancy may be explained by ongoing changes in the serotonergic system in these strains during the past 30 years of selection.

Fig. 7.
figure 7

Interstrain differences in the activity and expression of key enzymes of the serotonergic system – tryptophan hydroxylase 2 (TPH2) and monoamine oxidase A (MAOA) – in the midbrain, hippocampus, and hypothalamus of aggressive and tame rats.

Full size image

It is known that underexpression of the main serotonin-degrading enzyme MAOA enhances aggression [11-13, 16]. In the current study, however, we found an elevated basal level of this protein in the hypothalamus of aggressive rats compared to tame ones, along with increased levels of MAOA activity in the midbrain and hippocampus (Fig. 7). This observed upregulation of the protein level and enzymatic activity may be associated with this specific type of aggression. For instance, previous studies have shown diminished MAOA activity in the brainstem of tame foxes compared to wild ones [42], whereas the MAOA activity does not differ between wild gray rats and aggressive and tame ones of the 35-36th generation of selection [27]. Of note, we failed to detect genotype-related differences in mRNA levels of the Maoa gene or changes in its protein levels. Moreover, the observed differences in protein levels and enzymatic activity did not coincide in localization, implying different regulatory pathways for MAOA depending on a brain region.

Previously, we have found lower expression of the 5-HT1A receptor in the midbrain of aggressive rats [22], where it is mainly expressed presynaptically, and its activation leads to hyperpolarization of serotoninergic neurons and inhibition of serotonin signaling (for review see [43]). Conversely, the expression of 5-HT2A receptors, known activators of the serotonergic system (for review see [44]), proved to be elevated in the midbrain, hippocampus, and hypothalamus of aggressive rats [22]. Thus, on the basis of the observed expression patterns of these receptor types and earlier data on increased serotonin levels in the cingulate cortex, nucleus accumbens, and putamen of aggressive rats [19], we hypothesized that this strain is characterized by a more active serotonergic system as compared to tame rats. This hypothesis is supported by our present data on greater activity and expression of TPH2 and MAOA (the key enzymes of serotonin synthesis and catabolism) in aggressive rats.

In previous studies, we have revealed that aggressive rats have higher levels of striatal-enriched protein tyrosine phosphatase (STEP) relative to tame rats [28]. STEP plays a major role in the pathogenesis of neurodegenerative diseases. Furthermore, in mice, inhibition of TPH2 activity using para-chlorophenylalanine lowers mRNA levels of the gene encoding the STEP protein in the striatum [45]. In the current study, we demonstrated an increase in TPH2 expression in aggressive animals. These results may indicate a connection between these two enzymes. It is possible that this interaction is mediated by brain-derived neurotrophic factor (BDNF) because the absence of TPH2 in knockout mice leads to BDNF overexpression [46, 47], which negatively correlates with STEP.

The compound TC-2153 is an inhibitor of the STEP protein [48] and affects the brain serotonergic system [31-33]. Additionally, we have reported that TC-2153 reduces aggression and the STEP protein level in aggressive rats and has an anxiolytic effect on both aggressive and tame animals [28].

In the present work, we found that single administration of TC-2153 had no impact on mRNA levels of the Tph2 gene or the amount of TPH2 protein. Earlier, chronic administration of TC-2153 to mice has not affected Tph2 gene expression in the midbrain either [33]. By contrast, TC-2153 did affect TPH2 activity, by reducing this parameter in the hypothalamus of both aggressive and tame rats (Fig. 8). Preliminary experiments in vitro indicated that TC-2153 at concentrations 0.01 mM or less – presumed concentration in the rat brain at doses used in this work – does not affect TPH2 activity directly and therefore has an indirect effect. TPH2 activity is regulated by calcium/calmodulin-dependent protein kinase II [49], whose activity depends on calcium concentration. We have previously shown that TC-2153 reduces the activity of serotonin 5-HT2A receptors [31], whose activation is accompanied by an increase in intracellular calcium concentration and that 5-HT2A receptor antagonists block this calcium concentration elevation [50-52]. Consequently, by reducing the functional activity of 5-HT2A receptors, TC-2153 may affect calcium concentration, yielding a decrease in TPH2 activity.

Fig. 8.
figure 8

The effect of single administration of TC-2153 on the activity and expression of tryptophan hydroxylase 2 (TPH2) and monoamine oxidase A (MAOA) in the midbrain, hippocampus, and hypothalamus of aggressive and tame rats.

Full size image

The reduction in TPH2 activity under the influence of TC-2153 is consistent with its antiaggressive and anxiolytic effects [28]. Given that TPH2 activity and expression positively correlate with aggressive behavior, it is fair to assume that a decline of TPH2 activity will be accompanied by less intense aggression. Nonetheless, the effect of TC-2153 on TPH2 activity was observed here in both aggressive and tame rats, although the antiaggressive effect was only evident in the aggressive strain. Tame rats are characterized by almost highest scores of friendly behavior in the glove test, and perhaps the reduction in TPH2 activity no longer exerts visible behavioral effects in this direction. On the other hand, the anxiolytic effect of TC-2153 is observed in both rat strains. This phenomenon may also be mediated by reduced TPH2 activity because numerous articles have also shown a positive correlation between TPH2 and anxiety (for review see [53]).

TC-2153 also affected the enzymatic activity of MAO in the present work (Fig. 8). Administration of TC-2153 reduced the activity of this enzyme in the midbrain of aggressive rats (with marginal significance) just as in tame rats. It is known that MAO inhibitors exert anxiolytic effects [54-56]. Earlier, we have demonstrated that single administration of TC-2153 reduces anxiety in both aggressive and tame rats in the elevated plus maze test [28]. It is possible that the observed behavioral changes are also related to the action of this substance on MAO activity in the midbrain. Preliminary experiments indicated that TC-2153 does not bind directly to this enzyme either. The influence of TC-2153 on MAO activity may be mediated by mitogen-activated protein kinase p38, which phosphorylates serine at position 209 and deactivates MAOA [57]. The activity of p38 is also regulated by phosphorylation, and this protein is one of targets of phosphatase STEP [58]. Thus, by inhibiting STEP, TC-2153 presumably extends its effects onto MAO activity via p38.

Besides, TC-2153 altered the level of the MAOA protein, by reducing it in the hypothalamus of tame rats, while an insignificant increase was registered in the hypothalamus of aggressive rats. Nevertheless, no effect on Maoa gene expression was detectable. It is possible that at this time point (5 h after injection), mRNA levels returned to normal, and we observed only changes in protein levels. The hypothalamus is a key component of the hypothalamic–pituitary–adrenal axis and regulates the stress response. Aggressive and tame rats differ in the baseline state of this axis and in their response to stress [34, 35, 59]. Meanwhile, pathways regulating MAOA expression are influenced by stress and are interconnected with the hypothalamic–pituitary–adrenal axis (for review see [60]). Thus, interstrain differences (between aggressive and tame rats) in this axis may account for the divergent responses within the hypothalamus to single administration of TC-2153. It is known that transcription factor FoxO1 acts as a repressor of the Maoa gene [61]. The activity of FoxO1 in turn is regulated by kinases p38 and ERK1/2 [62]: substrates of phosphatase STEP [58], which is inhibited by TC-2153. These two kinases have opposite effects on FoxO1: p38 activates this transcription factor, whereas ERK1/2 deactivates it. The divergent actions of the STEP inhibitor benzopentathiepin TC-2153 on the level of MAOA protein in aggressive and tame rats may indicate different states of these signaling pathways in the two strains.

In conclusion, this study revealed significant differences in the activity and expression of key serotonin synthesis and catabolism enzymes – TPH2 and MAOA – in rats selectively bred for >85 generations for pronounced aggressive behavior toward humans or its absence. Furthermore, we demonstrated that in aggressive and tame rats, the STEP inhibitor TC-2153 reduces the activity of TPH2 and MAOA in the hypothalamus and midbrain, respectively, and differently (between the strains) affects the level of MAOA protein in the hypothalamus. The data obtained in this work provide deeper insights into the contribution of key enzymes of the serotonergic system to processes underlying fear-induced aggressive behavior as well as into mechanisms of action of compound TC-2153 on the brain serotonergic system.