Introduction

Status epilepticus (SE) is a medical emergency with significant mortality (DeLorenzo et al. 1995). Status epilepticus has been defined as a continuous seizure activity involving severe and prolonged hypoxia, enough to induce a sustained encephalopathy (Rossetti et al. 2007). Therefore, SE causes neuronal cell death (Fujikawa 1995; Rice and DeLorenzo 1998), epileptogenesis (Rice and DeLorenzo 1998), and learning impairment (Stewart and Persinger 2001). Pilocarpine (PILO) acts on muscarinic receptors; both M1 and M2 receptors appear to be involved. M2 receptor activation results in the inhibition of adenylate cyclase, decreasing the release of acetylcholine and neuronal excitation (Smolders et al. 1997). However, M1 receptor activation induces synthesis of phospholipase C producing diacylglycerol (DAG), and inositol-1,4,5 triphosphate (IP3), in turn, increases intracellular Ca2+ release (ICR) from endoplasmic reticulum via IP3 receptor and ryanodine receptors (Kim and Kang 2011).

Transient receptor potential canonical channel (TRPC) is a nonselective cation channel permeable to Ca2+, which is expressed in many cell types, including neurons (Wes et al. 1995; Zhu et al. 1995; Montell et al. 2002; Harteneck et al. 2000). The TRPC subfamily is divided into seven subgroups based on their sequence similarity (Clapham 2003; Vazquez et al. 2004). The TRPC subfamily has different roles, including growth cone guidance (Li et al. 2005), neurite outgrowth (Greka et al. 2003), and neuronal survival (Jia et al. 2007). In particular, the TRPC6 protein in neurons was specifically down-regulated by the N-Methyl-D-aspartic acid (NMDA) receptor-dependent calpain proteolysis in ischemic insults. Blocking its degradation protected neurons and brains against cerebral ischemia (Du et al. 2010). Thus, TRPC6 plays a critical role in promoting neuronal survival against NMDA receptor-mediated neuronal death (Du et al. 2010). Interestingly, the TRPC is organized into supramolecular signaling complexes, including intracellular receptors, such as IP3, ryanodine receptors, and adaptor proteins in native tissues (Kiselyov et al. 1999; Lepage et al. 2006; Redondo et al. 2004; Rosado et al. 2005; Lee et al. 2006). Among them, TRPC3 and TRPC6 are directly activated by DAG analogs (Beech et al. 2009), indicating that PILO might activate TRPC. However, the alterations in TRPC expression in response to M1 receptor-mediated SE have not been explored. Therefore, the present study was designated to elucidate the roles of TRPC3 and TRPC6 in neuronal death following SE induced by PILO.

Experimental Procedures

Experimental Animals and Chemicals

This study utilized the progeny of male Sprague–Dawley (SD) rats (7 weeks old) obtained from Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity, and lighting conditions (22 ± 2 °C, 55 ± 5 %, and a 12:12 light/dark cycle with lights). Procedures involving animals and their care were conducted in accordance with our institutional guidelines that comply with NIH Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, 1996). In addition, all possible efforts were taken to avoid animals’ suffering and to minimize the number of animals used at each stage of the experiment. All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), except as noted.

SE Induction

In order to examine the effect of SE on TRPC3 and TRPC6 expression in the hippocampus, animals were given LiCl (3 mEq/kg i.p) 24 h before the PILO treatment. Some animals were intraperitoneally (i.p) treated with pilocarpine (30 mg/kg, i.p., Sigma, St. Louis, MO) 20 min after atropine methylbromide (5 mg/kg i.p.) and were placed in individual observation chambers where seizure activity was scored according to the system of Racine 1972). Animals that entered SE typically did so within 20 to 30 min of the administration of pilocarpine and exhibited continuous seizure activity between 2 and 5 on the Racine scale (including akinesia, facial automatisms, limbic seizures consisting of forelimb clonus with rearing, salivation, masticatory jaw movements, and falling). Diazepam (Valium, Hoffman Ia Roche, Neuilly sur-Seine, France; 10 mg/kg i.p.) was administered 2 h after the onset of SE and repeated, as needed. Age-matched animals (n = 5) were used as non-SE experienced controls. Non-SE animals received saline in place of PILO.

Intracerebroventricular Drug Infusion

In order to investigate the direct roles of TRPC3 and TRPC6 in SE-induced neuronal death, we performed intracerebroventricular infusion of pyrazole-3 (Pyr3, a TRPC3 receptor inhibitor) and hyperforin (TRPC6 receptor activator) prior to SE induction. The other rats were divided into three groups (n = 6, respectively): vehicle (0.001 % DMSO/saline, v/v), Pyr3 (0.1 mg/kg /day), and hyperforin (6 μM) treated animals. The dosage of each compound was determined as the highest dose that did not affect seizure threshold in the preliminary study. Animals were anesthetized (Zolretil, 50 mg/kg, i.m.; Virbac Laboratories) and placed in a stereotaxic frame. For the osmotic pump implantation, holes were drilled through the skull for introducing a brain infusion kit 1 (Alzet, Cupertino, CA, USA) into the right lateral ventricle (1 mm posterior; 1.5 mm lateral; −3.5 mm depth; flat skull position with bregma as reference), according to the atlas of the rat brain (Paxinos and Watson 1997). The infusion kit was sealed with dental cement and connected to an osmotic pump (1007D, Alzet, Cupertino, CA, USA). The pump was placed in a subcutaneous pocket in the dorsal region. Animals received 0.5 μl/hr of vehicle or compound for 1 week (Siuciak et al. 1996; Pencea et al. 2001; Kim et al. 2010). Three days after surgery, rats were induced with SE using the same methods described above. Rest of the animals (n = 3 in each group) were used as non-SE experienced controls. Since neuronal damage was first detectable at 3 days after SE (Kang et al. 2006), we determined 3 days after SE as the best time point to evaluate the effects of Pyr3 and hyperforin infusion on SE-induced neuronal damages. Therefore, these animals were stained with fluoro-Jade B (FJB) and double immunofluorescence study was conducted 3 days after SE using the methods described below.

Tissue Processing

At the designated time points (Non-SE, 1 day, 3 days, and 1 week after SE, n = 5, respectively), animals were perfused transcardially with phosphate-buffered saline (PBS, pH 7.4) followed by 4 % paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) under urethane anesthesia (1.5 g/kg i.p.). Drug-infused animals were perfused using the same methods, 3 days after SE. The brains were removed, postfixed in the same fixative for 4 h, and rinsed in PB containing 30 % sucrose at 4 °C for 2 days. Thereafter, the tissues were frozen and sectioned with a cryostat at 30 μm.

FJB Staining

Fluoro-Jade B (FJB) staining was used to identify degenerating neurons in tissues obtained from non-SE and 3-days-post-SE animals in drug-infused groups. In brief, the brain sections were rinsed in distilled water, mounted onto gelatin-coated slides, and then dried on a slide warmer. The slides were immersed in 100 % ethanol for 3 min, followed by 70 % ethanol for 2 min and distilled water for 2 min. The slides were then transferred to 0.06 % potassium permanganate for 15 min and gently agitated. After rinsing in distilled water for 2 min, the slides were incubated for 30 min in 0.001 % FJB (Histo-Chem Inc. Jefferson, AR, USA), freshly prepared by adding 20 ml of a 0.01 % stock FJB solution to 180 ml of 0.1 % acetic acid, with gentle shaking in the dark. After rinsing for 1 min in each of three changes of distilled water, the slides were dried, dehydrated in xylene, and coverslipped with DPX.

Immunohistochemistry

In each animal, the free-floating sections were first incubated with 10 % normal chicken serum (Vector, Burlingame, CA, USA) for 30 min at room temperature. Some sections were incubated in either of the following antibodies : rabbit anti-TRPC3 (diluted 1:200, Alomone labs, Jerusalem, Israel) or TRPC6 antibody (diluted 1:200, Millipore Corporation, Billerica, MA, USA) in PBS containing 0.3 % Triton X-100 and 2 % normal chicken serum (Vector, Burlingame, CA, USA) overnight at room temperature. After washing three times for 10 min with PBS, the sections were incubated sequentially in donkey anti-rabbit IgG (Vector, Burlingame, CA, USA) and ABC complex (Vector, Burlingame, CA, USA), diluted 1:200 in the same solution as the primary antiserum. Between the incubations, the tissues were washed with PBS three times for 10 mins each. The sections were visualized with 3,3′-diaminobenzidine in 0.1 M Tris buffer and mounted on the gelatin-coated slides. The immunoreactions were observed under the Axioscope microscope (Carl Zeiss, Munchen-Hallbergmoos, Germany).

For double immunofluorescence study, some sections were incubated in either mixture of antibodies: rabbit anti-TRPC3 (diluted 1:100) or TRPC6 antibody (diluted 1:100)/mouse anti-neuronal nuclear antigen (NeuN) antibody (a neuronal marker, Millipore Corporation, Billerica, MA, USA, diluted 1:1,000) in PBS containing 0.3 % Triton X-100 overnight at room temperature. After washing three times for 10 min with PBS, the sections were also incubated in a mixture of fluorescein isothio-cyanate-conjugated secondary antiserum and Cy3-conjugated secondary antiserum (diluted 1:200, Amersham, San Francisco, CA, USA) for 1 h at room temperature. The sections were washed three times for 10 min with PBS and mounted on gelatin-coated slides. For nuclei counterstaining, we used Vectashield mounting medium with DAPI (Vector, Burlingame, CA, USA). For negative control, the rat hippocampal tissues were incubated with 1 μg of the antibody that was preincubated with 1 μg of purified peptide for 1 h at room temperature (for TRPC3 and TRPC6), or incubated with pre-immune serum instead of primary antibody (for NeuN). The negative control resulted in the absence of immunoreactivity in any of the structures. All images were captured using an Axiocam HRc camera and Axio Vision 3.1 software.

Stereology

The hippocampal volumes (V) were estimated according to the formula based on the modified Cavalieri method: \( V = \Upsigma a \times t_{nom} \times 1/ssf \), where a is area of the region of the delineated subfield measured by AxioVision Rel. 4.8 software, t nom is the nominal section thickness (of 30 μm in this study), and ssf is the fraction of the sections sampled or section sampling fraction (of 1/6 in this study). The subfield areas were delineated with a 2.5 × objective lens. The volumes are reported as mm3 (Bedi et al. 1991; Madeira et al. 1995). The optical fractionator was used to estimate the cell numbers. The optical fractionator (combination of performing counting with the optical dissector, with fractionator sampling) is a stereological method based on a properly designed systematic random sampling method that, by definition, yields unbiased estimates of population number. The sampling procedure is accomplished by focusing through the depth of the tissue (the optical dissector height, h, of 15 μm in all cases for this study). The number of each cell type (C) in each of the subregions is estimated as: \( C = \Upsigma Q^{ - } \times t/h \times 1/asf \times 1/ssf \), where Q − is the number of cells actually counted in the dissectors that fell within the sectional profiles of the subregion seen on the sampled sections, and asf is the area sampling fraction calculated by the area of the counting frame of the dissector, a(frame) (of 50 × 50 μm2 in this study) and the area associated with each x, y movement, grid (x, y step) (of 250 × 250 μm 2 in this study) \( \{ {\text{asf}} = [a({\text{frame}})/a(x,y\,{\text{step}})]\} \). Fluoro-Jade B (FJB) positive cells were counted with a 40× objective lens. All FJB positive cells were counted regardless of the intensity of labeling. Cell counts were performed by two different investigators who were blind to the classification of tissues. The SE-induced hippocampal atrophy is evident (Niessen et al. 2005; Roch et al. 2002), so changes in cell number may be caused by an alteration in the volume of the hippocampus. Therefore, the total number of cells was corrected by multiplying with appropriate correction factors representing the degree of shrinkage (or swelling) compared with the non-SE animals (Kim et al. 2011a).

Quantification of Data and Statistical Analysis

For quantification of immunohistochemical data, images were captured using an AxioImage M2 microscope and AxioVision Rel. 4.8 software (15 sections per animal). Figures were mounted with Adobe PhotoShop v 8.0. Images were converted into gray and white images. The range of intensity values was obtained from the selected images using Adobe PhotoShop v. 8.0. Based on the mean range of intensity values, each image was normalized by adjusting the black and white range of the image using Adobe PhotoShop v. 8.0. Manipulation of the images was restricted to threshold and brightness adjustments to the whole image. After regions were outlined, 10 areas/rat (500 μm2/area) were selected from the hippocampus and the gray values were measured. Intensity measurements were represented as the mean number of a 256 gray scale (NIH Image 1.59 software and AxioVision Rel. 4.8 software). Values for background staining were obtained from the corpus callosum. Optical density values were then corrected by subtracting the average values of background noise obtained from 15 image inputs. All data obtained from the quantitative measurements were analyzed using one-way analysis of variance to determine statistical significance. Bonferroni’s test was used for post hoc comparisons. A p value below 0.05 was considered statistically significant (Kim et al. 2009a– c).

Results

Up-regulation of Neuronal TRPC3 Expression Following SE

In non-SE animals (Figs. 1a, 2a, b), TRPC3 immunoreactivity was abundantly detected in the dendrites of pyramidal cells and the cell bodies of dentate granule cells. One day after SE (Fig. 1b, e), elevated TRPC3 immunoreactivity was observed only in NeuN positive neurons. Transient receptor potential canonical channel 3 (TRPC3) immunoreactivity was obviously detected in the dendrites of pyramidal cells and dentate granule cells. Transient receptor potential canonical channel 3 (TRPC3) expression in CA1-, CA3- pyramidal cells, and dentate granule cells was increased to 2.4, 2.8, and 3.1-fold of the non-SE level, respectively (p < 0.05 vs. non-SE, n = 5, respectively). Three days after SE (Fig. 1c, e), elevated TRPC3 expression was maintained in CA1-, CA3- and dentate granule cells. One week after SE (Fig. 1d, e), TRPC3 expression in CA1- and CA3- pyramidal cells was reduced to 0.5 and 0.7-fold of the non-SE level, respectively (p < 0.05 vs. non-SE, n = 5, respectively), due to massive neuronal death. However, TRPC3 expression in dentate granule cells was similar to that observed in non-SE animals. Pyrazole-3 pretreatment effectively prevented up-regulation of neuronal TRPC3 expression induced by SE (p < 0.05 vs. vehicle, n = 3, respectively, Fig. 2).

Fig. 1
figure 1

TRPC3 expression in the hippocampus following SE. a Non-SE animal. Bar = 400 (panel 1) and 50 μm (panel 2–4). b One day-post SE animals. Bar = 400 (panel 1) and 50 μm (panel 2–4). c Three days-post SE animals. Bar = 400 (panel 1) and 50 μm (panel 2–4). d One week-post SE animals. Bar = 400 (panel 1) and 50 μm (panel 2–4). e Quantitative analysis of TRPC3 expression in the hippocampus following SE (mean ± SEM). Significant differences from vehicle-treated groups (*p < 0.05 vs. non-SE animals). DG dentate granule cell layer; CA1 pyramidal cell layer; CA3 pyramidal cell layer

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Fig. 2
figure 2

Effect of TRPC3 inhibitor (Pyr3) on neuronal death in the hippocampus following SE. a, b Non-SE animals; c, d three days-post SE animals treated with vehicle; e, f three days-post SE animals treated with Pyr-3. b, d, f High magnification of CA1 pyramidal cells. Panels 1, 2, 3, and 4 are TRPC3, NeuN, DAPI counterstaining, and merge images, respectively. Bar = 400 (a, c, e) and 12.5 μm (b, d, f). g Quantitative analysis of TRPC3 expression in the hippocampus following SE (mean ± SEM). Significant differences from non-SE animals and vehicle-treated animals (* and # p < 0.05 vs. non-SE animals and vehicle-treated animals, respectively)

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Down-regulation of Neuronal TRPC6 Expression Following SE

In non-SE animals (Figs. 3a, 4a, b), TRPC6 immunoreactivity was mainly detected in the dendrites of pyramidal cells and the cell bodies of dentate granule cells. In addition, TRPC6 immunoreactivity was also strongly observed in neuropils within the molecular layer of the dentate gyrus. One day after SE (Fig. 3b, e), TRPC6 expression in CA1-, CA3- pyramidal cells and dentate granule cells was reduced to 0.51, 0.48, and 0.52-fold of the non-SE level, respectively (p < 0.05 vs. non-SE, n = 5, respectively). In addition, reduced TRPC6 immunoreactivity was detected in the inner molecular layer of the dentate gyrus. Three days after SE (Figs. 3c, e, 4c, d), TRPC6 expression was unaltered in the hippocampus except the molecular layer of the dentate gyrus where TRPC6 expression was reduced to 0.3-fold of the non-SE level (p < 0.05 vs. non-SE), as compared to 1 day-post-SE animals (n = 5, respectively). Following SE, TRPC6 expression was also detected in astrocytes in the CA1 subfield of the hippocampus proper following SE (data not shown). One week after SE (Fig. 3d, e), TRPC6 expression in CA1-, CA3- pyramidal cells, and dentate granule cells was reduced to 0.21-, 0.31-, and 0.18-fold of the non-SE level, respectively (p < 0.05 vs. non-SE, n = 5, respectively). Hyperforin pretreatment effectively prevented down-regulation of neuronal TRPC6 expression induced by SE (p < 0.05 vs. vehicle, n = 3, respectively, Fig. 4).

Fig. 3
figure 3

TRPC6 expression in the hippocampus following SE. a Non-SE animal. Bar = 400 (panel 1) and 50 μm (panel 2–4). b One day-post SE animals. Bar = 400 (panel 1) and 50 μm (panel 2–4). c Three days-post SE animals. Bar = 400 (panel 1) and 50 μm (panel 2–4). d One week-post SE animals. Bar = 400 (panel 1) and 50 μm (panel 2–4). e Quantitative analysis of TRPC6 expression in the hippocampus following SE (mean ± SEM). Significant differences from vehicle-treated groups (*p < 0.05 vs. non-SE animals). DG dentate granule cell layer; CA1 pyramidal cell layer; CA3 pyramidal cell layer

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Fig. 4
figure 4

Effect of TRPC6 activator (hyperforin) on neuronal death in the hippocampus following SE. a, b Non-SE animals; c, d three days-post SE animals treated with vehicle; e, f three days-post SE animals treated with hyperforin. b, d, f High magnification of CA1 pyramidal cells. Panels 1, 2, 3, and 4 are TRPC3, NeuN, DAPI counterstaining, and merge images, respectively. Bar = 400 (a, c, e) and 12.5 μm (b, d, f). g Quantitative analysis of TRPC6 expression in the hippocampus following SE (mean ± SEM). Significant differences from non-SE animals and vehicle-treated animals (* and # p < 0.05 vs. non-SE animals and vehicle-treated animals, respectively)

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Neuronal Damages

In our previous (Ryu et al. 2011) and preliminary data, neuronal damage was first detectable 3 days after SE. Therefore, we applied FJB stains in 3 days-post SE animals that were pretreated with vehicle, Pyr3, and hyperforin. Few FJB positive neurons were detected in the hippocampus of non-SE induced animals of all groups (data not shown). In vehicle-treated animals, FJB positive neurons were detected in CA1-3 pyramidal cells and dentate hilus neurons (Fig. 5a). The number of FJB positive neurons in the dentate hilus, the CA1, and the CA3 region was 16,102 ± 4,325, 262,038 ± 48,174, and 298,681 ± 15,269, respectively (Fig. 5d, f). As compared to vehicle-treated animals, Pyr3- or hyperforin-treated animals showed significant reductions in neuronal damages induced by SE within the hippocampus (p < 0.05 vs. vehicle, n = 3, respectively): In Pyr3-pretreated animals, the number of FJB positive neurons in the dentate hilus, the CA1, and the CA3 region was significantly reduced to 4,601 ± 1,272, 72,788 ± 8,352, and 123,141 ± 13,983, respectively (Fig. 5b, d–f). In hyperforin-pretreated animals, the number of FJB positive neurons in the dentate hilus, the CA1, and the CA3 region was significantly reduced to 5,367 ± 1,364, 87,312 ± 6,312, and 103,108 ± 12,038, respectively (Fig. 5c–f). These findings indicate reverse roles of TRPC3 and TRPC6 in SE-induced neuronal death.

Fig. 5
figure 5

Effects of Pyr3 and hyperforin on neuronal damages in the hippocampus following SE. a Vehicle-treated animals; b pyr3-treated animals; c hyperforin-treated animals. Bar = 20 μm. (d–f) Quantitative analysis of FJB positive neurons in the hippocampus following SE (mean ± SEM). Significant differences from vehicle-treated animals (*p < 0.05 vs. non-SE animals). DH dentate hilar cell; CA1 pyramidal cell; CA3 pyramidal cell

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Discussion

In epileptic hippocampus, severe neurodegeneration occurs both in principal neurons and in interneurons (Mathern et al. 1995; Wittner et al. 2001). Neurodegeneration in the hippocampus is not uniform, but heterogeneous among the principal hippocampal cell layers. For example, dentate granule cells are remarkably resistant to neuronal damage caused by SE. Conversely, dentate hilar neurons, CA1-, and CA3- pyramidal neurons are extremely vulnerable to SE (Kim et al. 2011b; Ryu et al. 2011). Malfunction of muscarinic acetylcholine receptor transmission is one of the candidates for the prolonged depolarization underlying seizure-related neuronal death (Kim et al. 2011b; Kim and Kang 2011). Recently, TRPC functions are activated by the formation of DAG and IP3, followed by activation of IP3 receptors in intracellular Ca2+ stores (Clapham 2003; Montell et al. 2002). Therefore, it is likely that TRPC may participate in the regulation of muscarinic receptor-mediated neuronal death. Indeed, the present study showed that TRPC6 expression was reduced in CA1-3 neurons as well as dentate granule cells. Furthermore, hyperforin pretreatment effectively protected neuronal death from SE insults. These findings are consistent that TRPC6 function is suppressed in ischemia and the maintenance of TRPC6 expression is neuroprotective via the cAMP response element-binding-dependent mechanism (Du et al. 2010). Thus, our findings suggest that activation of TRPC6 function or inhibition of TRPC6 degradation may protect neuronal damages from SE, like ischemic brain damage.

In the present study, TRPC3 expression was significantly elevated in the hippocampus following SE. Since TRPC3 mRNA expression is increased in TRPC6 knockout mice (Tiruppathi et al. 2002), it is likely that increase in TRPC3 expression induced by SE may be a compensatory mechanism for reduction in TRPC6 function. However, TRPC3 and TRPC6 are not functionally interchangeable (Carrillo et al. 2012; Urban et al. 2012; Cheung et al. 2011). Furthermore, the present study showed that up-regulated TRPC3 expression may not be correlated with altered TRPC6 expression and that the inhibition of TRPC3 function by Pyr3 effectively prevented SE-induced neuronal death. On the other hand, TRPC3 is needed to activate a nonselective cationic current by brain-derived neurotrophic factor (BDNF) (Amaral and Pozzo-Miller 2007), which can facilitate and potentially initiate seizure activity (Scharfman et al. 2002). Blockade of BDNF function effectively decreases SE-induced neuronal death, indicating an injury-facilitating role for BDNF in vivo (Unsain et al. 2009). Based on these previous studies, it is likely that up-regulated TRPC3 expression may be involved in the BDNF-mediated neuronal death following SE. Further studies are needed to elucidate the role of TRPC3 in neuronal death.

In conclusion, the present study yields novel information regarding the role of TRPC3 and 6 in epileptogenic insults, and suggest that TRPC 3 and 6 may be involved in neurodegeneration following SE.