Elevated voltage-gated Ca2+ channel immunoreactivities in the hippocampus of seizure-prone gerbil

Brain Research 1029 (2004) 168 – 178 www.elsevier.com/locate/brainres

Research report

Elevated voltage-gated Ca2+ channel immunoreactivities in the hippocampus of seizure-prone gerbil Tae-Cheon Kanga,*, Duk-Soo Kima, Ki-Yeon Yooa, In-Koo Hwanga, Sung-Eun Kwaka, Ji-Eun Kima, Ju-Young Junga, Moo Ho Wona, Jun-Gyo Suhb, Yang-Seok Ohb b

a Department of Anatomy, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, South Korea Department of Medical Genetics and Experimental Animal Center, College of Medicine, Hallym University, Chunchon, Kangwon-Do 200-702, South Korea

Accepted 27 September 2004 Available online 2 November 2004

Abstract In present study, we investigated voltage-gated Ca2+ channel (VGCC) expressions in the hippocampus of the Mongolian gerbil and its association with different sequelae of spontaneous seizures, in an effort to identify the epileptogenesis in this animal. In the hippocampus of pre-seizure seizure sensitive (SS) gerbils, VGCC subunit expressions were significantly elevated, as compared with seizure-resistant (SR) gerbils. In 3 h postictal group, the alteration of VGCC expressions showed regional- and neuronal-specific manners; VGCC immunoreactivities in principal neurons were markedly decreased; however, their immunoreactivities in interneurons were significantly elevated. These results are the first comprehensive description of the distribution of VGCC immunoreactivities in the normal and epileptic hippocampus of gerbils, and suggest that these alterations in the hippocampus of the SS gerbil may be related with tissue excitability and have a role in modulating recurrent excitation following seizures. D 2004 Elsevier B.V All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: human studies and animal models Keywords: Voltage-gated Ca2+ channel; Epilepsy; Hippocampus; Seizure; Gerbil

1. Introduction Rapid Ca2+ influx into neurons is largely mediated through glutamate receptors. Glutamatergic impulses from the entorhinal cortex constitute the major excitatory input to the hippocampus and a shift in glutamate-mediated excitability may be involved in the pathogenesis of epileptic discharges. Moreover, N-methyl-d-aspartate (NMDA) receptor may be responsible for the seizure-induced selective excitotoxic cell death of certain hippocampal neuronal populations, since NMDA receptor antagonists provide protection against such damage [4,5]. However, in

* Corresponding author. Fax: +82 33 256 1614. E-mail address: [email protected] (T.-C. Kang). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.09.040

our previous study [35], no differences were observed in the NMDA receptor systems of seizure-sensitive (SS) gerbils and seizure-resistant (SR) gerbils. For this reason, NMDA receptor expression may not initiate seizure activity in SS gerbils, although it could be that other glutamate receptor (e.g., kainate-, AMPA-, or metabotropic glutamate receptor) might be involved in seizure activity in gerbils. On the other hand, the voltage-gated Ca2+ channel (VGCC) family also provides Ca2+ translocation pathways to the cytosol. Based on their electrophysiological and pharmacological properties, VGCC family members have been classified into three groups [37,39]. Among them, P/Q-type (a1A) VGCC and N-type VGCC (a1B) are predominantly localized in cell bodies and presynaptic nerve terminals, and regulate presynaptic activity including neurotransmitter release and modulate Ca2+

T.-C. Kang et al. / Brain Research 1029 (2004) 168–178

influx into the cell body [26,33,42]. In contrast to these VGCC subtypes, L-type (a1C and D) VGCCs are localized in clusters on cell bodies and dendrites, and are implicated in specialized somatic functions, but not in neurotransmitter release [13]. Therefore, VGCC regulates neurotransmitter release and thus is essential for controlling cell-to-cell communication and plasticity in the hippocampus. In addition, VGCC is implicated in hippocampal pathophysiological processes including epilepsy [3,11,30]. Indeed, the influx of Ca2+ ions through VGCC contributes to neuronal excitability, and is an important feature of epileptogenesis. The Mongolian gerbil exhibits spontaneous seizures in response to a variety of stimuli without the neuronal degeneration that would be induced by neurotoxins such as kainate. Moreover, this model allows epileptic and nonepileptic animals to be directly compared, which allows the identification of differences in brain anatomy and electrophysiology that correlate with seizure behavior. In particular, these animals also provide an opportunity to investigate self-recovery mechanisms after seizure onset [23,24,22,20,21,18,19,27,32]. However, to the best of our knowledge, differences in the patterns of VGCC expression in the epileptic gerbil hippocampus remain to be clarified, for example, as to whether the altered expressions of VGCC correlate with seizure activity in this animal model. Therefore, in the present study, we provide the first comprehensive description of the distribution of VGCC in the normal and epileptic hippocampus of gerbils and association between VGCC and the different sequelae of spontaneous seizure. This finding suggest that that altered VGCC expressions in the hippocampus may be closely related with seizure activity in this animal model.

169

seizures were classified as grade 1, a brief pause in normal activity accompanied by vibrissae twitches and retraction of the pinnae; grade 2, motor arrest with twitching of the vibrissae and retraction of the pinnae; grade 3, motor arrest with myoclonic jerks; grade 4, clonic–tonic seizures; grade 5, clonic–tonic seizures with body rollover. Only animals with a consistent stage 4 or 5 seizure score were included in the present study as seizure-sensitive (SS) gerbils. Seizure-resistant (SR) gerbils never demonstrated the seizure activity, thus they were assigned seizure severity score of 0 [23–25]. Sixty five SS and 13 SR gerbils (about 8 months old) were used in the present experiment. To examine the temporal changes of CRF and CRF-BP expressions following seizure, the SS gerbils were divided into five groups; pre-seizure group (n= 13), and post-seizure group I, II, III, and IV (n=13, respectively) that recovered normally at 3, 6, 12, or 24 h after the onset of tonic– clonic generalized seizure, respectively. Pre-seizure SS gerbils showed no seizure activity at least 36 h prior to the perfusion [18,19]. 2.2. Tissue processing The gerbils were anesthetized with ketamine, and perfused via the ascending aorta with 200 ml of 4% paraformaldehyde in phosphate buffer (PB). The brains were removed, postfixed in the same fixative for 4 h, and rinsed in PB containing 30% sucrose at 4 8C for 2 days. Thereafter, the tissues were frozen and sectioned with a cryostat at 30 Am and consecutive sections were collected in six-well plates containing phosphate-buffered saline (PBS). 2.3. Immunohistochemistry

2. Materials and methods 2.1. Experimental animals These studies utilized the progeny of Male Mongolian gerbil (Meriones unguiculatus) weighing 80–88 g obtained from the Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were housed at constant temperature (23 8C) and relative humidity (60%) with a fixed 12-h light/dark cycle and free access to food and water. The procedures involving animals and their care were conducted in conformity with the institutional guidelines and in compliance with international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 8523, 1985). Each animal was stimulated by vigorous stroking on the back with a pencil as described by Paul et al. [32] and tested a minimum of three times. According to the seizure severity rating scale of Loskota et al. [27],

Free-floating sections were first incubated with 3% bovine serum albumin in PBS for 30 min at room temperature. Sections were then incubated in rabbit antiP/Q-type (a1A), N-type (a1B), L-type (a1C or a1D) VGCC antisera (diluted 1:200, Chemicon, USA) in PBS containing 0.3% Triton X-100 and 2% normal goat serum overnight at room temperature. After washing three times for 10 min with PBS, sections were incubated sequentially, in goat anti-rabbit IgG and ABC complex (Vector, USA), diluted 1:200 in the same solution as the primary antiserum. Between incubations, the tissues were washed three times for 10 min with PBS. The sections were visualized with 3,3V-diaminobenzidine (DAB) in 0.1 M Tris buffer and mounted on gelatin-coated slides. Immunoreactions were observed under the Axioscope microscope (Carl Zeiss, Germany) [30]. In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of primary antibody, or a

170

T.-C. Kang et al. / Brain Research 1029 (2004) 168–178

pre-absorption test for VGCC antibodies was performed using control antigen peptides (20 mg/ml, Chemicon). The immunohistochemical controls showed the absence of immunoreactivity in all structures. All experiment procedures in the present study were performed in parallel [30]. 2.4. Western blot Three animals in each group were used in the immunoblot study. For tissue preparation, animals were anaesthetized under ketamine, decapitated, hippocampus was removed, and then each tissue was homogenized in 10 mM PB containing 0.1 mM EDTA, 1 mM 2mercaptoethanol, and 1 mM PMSF. After centrifugation, the protein concentrations in the supernatants were determined by using a Micro BCA protein assay kit with bovine serum albumin as the standard (Pierce Chemical, USA). Aliquots containing 30 Ag of total protein were boiled with an equal volume of 2 SDS sample buffer and boiled for 3 min, and then, each mixture was loaded onto a 10% polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Schleicher and Schuell, USA). To reduce background staining, the filters were incubated with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 45 min, sequentially incubated with primary antisera (1:10,000), peroxidase-conjugated goat anti-guinea pig IgG (Sigma, USA), and then with ECL kit (Amersham, USA).

spatial-specific alterations in VGCC expression patterns in the pre- and postictal stages. 3.1. P/Q-type (a1A) VGCC In SR gerbils, P/Q-type VGCC immunoreactivity was observed in all regions of the hippocampus (Fig. 1A). Briefly, P/Q-type VGCC immunoreactivity was mainly detected in the pyramidal cells in CA1–3 regions (Fig. 1B– C) and in the granule cells of the dentate gyrus (Fig. 1D) in the hippocampus of SR gerbils. Although the localization of P/Q-type VGCC immunoreactivity in the hippocampus was similar in SR and pre-seizure SS gerbils (Fig. 1E), the immunodensity of P/Q-type VGCC in the hippocampus was significantly higher in pre-seizure SS gerbils (CA1 region, Pb0.01; CA2–3 regions and dentate gyrus, Pb0.05, respectively) than in SR gerbils (Fig. 1F–H and M–O). Hilar neurons and granule cells in the dentate gyrus also showed strong P/Q-type VGCC immunoreactivity, as compared with SR gerbils (Fig. 1H). In the 3 h postictal group, P/Q-type VGCC immunoreactivity in the hippocampus was apparently lower than that in the preseizure group. However, P/Q-type VGCC immunoreactivity in interneurons and granule cells was unaltered, as compared with pre-seizure animals (Fig. 1I–L). This distribution pattern of P/Q-type VGCC immunoreactivity was maintained up to 6 h after seizure onset, and recovered to the pre-seizure level at 12 h following seizure onset (Fig. 1M–O). Immunoblot data were consistent with the immunohistochemical results (Fig. 5).

2.5. Quantitation of data and statistical analysis 3.2. N-type (a1B) VGCC Sections (15 sections per one animal) were viewed through a microscope connected via CCD camera to a PC monitor. At a magnification of 25–50, the region was outlined on the monitor. Images of VGCC immunoreactivity in CA1, CA2–CA3 regions of each section on the monitor at a magnification of 100 were captured by using an Applescanner. All measurements were carried out under the same optical and light conditions. The brightness and contrast of each image file were uniformly calibrated using Adobe Photoshop version 2.4.1, and then analyzed using NIH Image 1.59 software. Intensity measurements were represented as the mean number of a 256 gray scale. All data obtained from the quantitative measurements were analyzed using one-way ANOVA test to determine statistical significance. Bonferroni’s test was used for post hoc comparisons. P value below 0.01 or 0.05 was considered statistically significant [18–22].

3. Results In the present study, VGCC expression levels in the hippocampus of pre- and postictal groups differed from those of SR gerbils. Thus, we describe temporal- and

In SR gerbils, N-type VGCC immunoreactivity was detected in the strata oriens, radiatum, and lacunosummoleculare of the hippocampus proper, particularly in the CA1 region (Fig. 2A–D). However, N-type VGCC immunoreactivity was rare in the strata pyramidale of Ammon’s horn, thus this immunoreactivity was mainly detected within neuropil, not within the perikarya in the hippocampus proper (Fig. 2B–C). The N-type VGCC immunodensity in the hippocampus of the pre-seizure SS gerbils was significantly greater than that of SR gerbils (Fig. 2E); thus, its immunoreactivity was also detected in pyramidal cells of the CA1–3 region ( Pb0.05) and in the granule cells of the dentate gyrus ( Pb0.01; Fig. 2F–H and M–O). In 3 h postictal group, elevated N-type VGCC immunoreactivity in pyramidal cells was observed in CA1 region, while its immunoreactivity in neuropil was reduced in this region (Fig. 2I). In contrast to CA1 region, decreased N-type VGCC immunoreactivity was detected in CA2–3 regions and the dentate gyrus (Fig. 2K–L). The N-type VGCC immunoreactivity recovered to the preseizure level at 12 h postictal (Fig. 2M–O). Immunoblot data were consistent with the immunohistochemical results (Fig. 5).

T.-C. Kang et al. / Brain Research 1029 (2004) 168–178

171

Fig. 1. P/Q-type VGCC immunoreactivity in gerbil hippocampus. In SR gerbils (A–D), P/Q-type VGCC immunoreactivity is detected in all regions of the hippocampus. Compared with SR gerbils, P/Q-type VGCC immunoreactivity is significantly higher in pre-seizure SS gerbils (E–H). In the 3-h postictal group (I–L), decreased P/Q-type VGCC immunoreactivity is observed, although P/Q-type VGCC immunoreactivity in hilar neurons was unaltered. Arrows in A, E, and I indicate stratum lacunosum-moleculare. Bar=250 Am (A, E, I) and 35 Am (B–D, F–H, J–L). Abbreviations: SLM, stratum lacunosum-moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; ML, molecular layer; GL, granule cell layer; H, Hilar region. (M–O) The relative densitometric analysis of the immunoreactivity for P/Q-type VGCC in the gerbil hippocampus. Significant differences from the SR gerbil (basal level, bar 100 %) *Pb0.05, **Pb0.01.

3.3. L-type (a1C) VGCC The distribution pattern of L-type (a1C) VGCC immunoreactivity in pre-seizure SS gerbils (Fig. 3E–H)

was similar to that in SR gerbils (Fig. 3A–D). The density of L-type (a1C) VGCC immunoreactivity in the CA1 regions (Fig. 3F) and the dentate gyrus (Fig. 3H) was similar to SR gerbils. In pre-seizure SS gerbils, however,

172

T.-C. Kang et al. / Brain Research 1029 (2004) 168–178

Fig. 2. Alteration in N-type (a1B) VGCC immunoreactivity in the gerbil hippocampus. In SR gerbils (A–D), N-type VGCC immunoreactivity is observed within neuropil in the strata oriens, radiatum, and lacunosum-moleculare of the hippocampus proper, particularly in the CA1 region. The N-type VGCC immunoreactivity in the hippocampus of the pre-seizure SS gerbils (E–H) is significantly greater than that of SR gerbils; thus, this immunoreactivity was also detected in pyramidal cells of the CA1–3 region (F, G). In 3 h postictal group (I–L), N-type VGCC immunoreactivity in CA1 pyramidal cells is elevated (J), while its immunoreactivity is reduced in CA2–3 regions (K). Decreased N-type VGCC immunoreactivity is also detected in the dentate gyrus (L). Arrows in A, E, and I indicate stratum lacunosum-moleculare. Bar=250 Am (A, E, I) and 35 Am (B–D, F–H, J–L). Abbreviations shown Fig. 1. (M–O) The relative densitometric analysis of the immunoreactivity for N-type VGCC in the gerbil hippocampus. Significant differences from the SR gerbil (basal level, bar 100%) *Pb0.05, **Pb0.01.

T.-C. Kang et al. / Brain Research 1029 (2004) 168–178

173

Fig. 3. Changed L-type (a1C) VGCC immunoreactivity in the gerbil hippocampus. The distribution pattern of L-type (a1C) VGCC immunoreactivity in SR gerbils (A–D) is similar to that in pre-seizure SS gerbils (E–H). In CA2–3 regions of pre-seizure group (G), however, L-type (a1C) VGCC immunoreactivity is higher, as compared with SR gerbils (C). In the 3 h postictal group (I–L), L-type (a1C) VGCC immunoreactivity in CA1 region is increased in GABAergic neurons (arrowheads), although its immunoreactivity in pyramidal cells was unaltered (J). However, in the CA2–3 region, L-type (a1C) VGCC immunoreactivity in GABAergic neurons is elevated, while its immunoreactivity in pyramidal cells is reduced (K). L-type (a1C) VGCC immunoreactivity in the dentate gyrus is unaltered, as compared with pre-seizure group (L). Arrows in A, E, and I indicate stratum lacunosum-moleculare. Bar=250 Am (A, E, I) and 35 Am (B–D, F–H, J– L). Abbreviations shown Fig. 1. (M–O) The relative densitometric analysis of the immunoreactivity for L-type (a1C) VGCC in the gerbil hippocampus. Significant differences from the SR gerbil (basal level, bar 100%) *Pb0.05, **Pb0.01.

174

T.-C. Kang et al. / Brain Research 1029 (2004) 168–178

elevated L-type (a1C) VGCC immunoreactivity was detected in CA2–3 pyramidal cells ( Pb0.01), as compared with SR gerbils (Fig. 3G,N). In the 3 h postictal group, L-

type (a1C) VGCC immunoreactivity in CA1 region was increased in GABAergic neurons, although its immunoreactivity in pyramidal cells was unaltered (Fig. 3I–J) as

Fig. 4. L-type (a1D) VGCC immunoreactivity in the gerbil hippocampus. The L-type (a1C) VGCC immunoreactivity in SR gerbils (A–D) is similar to that in pre-seizure SS gerbils (E–H). However, L-type (a1D) VGCC immunoreactivity in CA2–3 pyramidal cells of pre-seizure SS gerbils (G) is higher than that in SR gerbils (C). In 3 h postical group (I–L), L-type (a1D) VGCC immunoreactivity is elevated in GABAergic neurons of CA2–3 regions (arrowheads), despite of reduction of its immunoreactivity in pyramidal cells (K), as compared with pre-seizure animals. Elevated L-type (a1D) VGCC immunoreactivity is also detected in hilar neurons of the dentate gyrus (arrowheads, L). Arrows in A, E, and I indicate stratum lacunosum-moleculare. Bar = 250 Am (A, E, I) and 35 Am (B–D, F–H, J–L). Abbreviations shown Fig. 1. (M–O) The relative densitometric analysis of the immunoreactivity for L-type (a1D) VGCC in the gerbil hippocampus. Significant differences from the SR gerbil (basal level, bar 100%) *Pb0.05, **Pb0.01.

T.-C. Kang et al. / Brain Research 1029 (2004) 168–178

Fig. 5. Western blot analysis of gerbil hippocampal aliquots containing 30 Ag total protein.

compared with pre-seizure SS gerbils. Unlike the CA1 region, L-type (a1C) VGCC immunoreactivity in GABAergic neurons was elevated in the CA2–3 regions, while its immunoreactivity in pyramidal cells was reduced as compared with pre-seizure SS gerbils (Fig. 3K). L-type (a1C) VGCC immunoreactivity in the dentate gyrus was unchanged in 3 h postictal groups (Fig. 3L). L-type (a1C) VGCC immunoreactivity recovered to the pre-seizure levels at 12 h postictal (Fig. 3M–O). Immunoblot data were consistent with the immunohistochemical results (Fig. 5). 3.4. L-type (a1D) VGCC Similar to L-type (a1C) VGCC immunoreactivity, L-type (a1D) VGCC immunoreactivity in CA2–3 pyramidal cells of pre-seizure SS gerbils (Fig. 4G,N) was higher ( Pb0.01) than that in SR gerbils (Fig. 4C,N), although in other regions it was similar (Fig. 4A–B, D, E–F, H). In 3 h postical group (Fig. 4I–L), L-type (a1D) VGCC immunoreactivity was elevated in GABAergic neurons, despite of reduction of its immunoreactivity in pyramidal cells of CA1–3 regions (Fig. 4G–K), as compared with pre-seizure animals. Elevated L-type (a1D) VGCC immunoreactivity was also detected in hilar neurons of the dentate gyrus (Fig. 4L). In the 12 h postictal group, L-type (a1D) VGCC immunoreactivity was recovered to pre-seizure level (Fig. 4M–O). Immunoblot data were consistent with the immunohistochemical results (Fig. 5).

4. Discussion 4.1. Regional specific up-regulation of VGCC expression in the hippocampus of SS gerbils In the present study, the localization of VGCC immunoreactivities in the gerbil hippocampus was consistent with that reported in our previous report [30]. In addition, the immunoblot data demonstrated single immunoreactive band

175

for each VGCC. Briefly, P/Q-type VGCC was M.W. ~195 kDa, N-type VGCC was ~160 kDa, L-type (a1C) was ~135 kDa, and L-type (a1D) was ~250 kDa. These results were consistent with previous studies [42–44] demonstrating the VGCC immunoreactive band in the rat brain. Regarding these immunoblot results, the antibodies used in the present study may have cross reactivity between gerbils and rats. VGCC expressions in the hippocampus of SS gerbils, however, were up-regulated vs. SR gerbils in spatialspecific manner. These findings are consistent with previous studies that demonstrated that the VGCC is functionally enhanced in different regions of the hippocampus in the kindling epilepsy model [10,39,41]. In terms of the hippocampal circuits and the functional properties of VGCC [13,26,33,42], the present findings indicate that altered VGCC expression in the pre-seizure group of SS gerbils may be closely related to the excitatory circuitry pathway of the hippocampus. Enhanced N-type VGCC immunoreactivity in the stratum lacunosum-moleculare, which contains the perforant pathway, implies an elevation of the excitatory input into the dentate gyrus. Furthermore, the enhancements of both P/ Q-type and N-type VGCCs immunoreactivities in the dentate gyrus accompanied by elevated L-type (a1C and D) VGCC immunoreactivities in the CA2–3 pyramidal cells may represent an enhancement of Ca2+-dependent excitability in the CA2–3 regions. The higher P/Q-type and N-type VGCC immunoreactivities in the strata radiatum and pyramidale of CA1 also indicate that axonal activity associated to Schaffer’s collaterals may be elevated. Therefore, these findings provide definitive evidence that the elevated expressions of VGCC subtypes may increase Ca2+ dependent excitatory transmission in the hippocampus of the SS gerbil. 4.2. Temporal alterations in VGCC immunoreactivity following spontaneous seizure In the present study, VGCC expressions in principal neurons in the hippocampus of SS gerbil was significantly reduced at 3 h after seizure onset, and recovered to the pre-seizure level at 12 h postictal. It is postulated that this may play an important role in the modulation of hyperexcitability by diminishing VGCC functions. The fact is that Ca2+ entry blockers can prevent or reduce convulsion in kindling—or in chemical-induced epileptiform seizures [2,7]. In addition, our previous study has reported that a decline in NMDA receptor expression in the gerbil hippocampus following seizure onset [35]. Following seizure onset, on the other hand, L-type (a1C and D) VGCC immunoreactivities were elevated in GABAergic neurons [22,30], despite reductions of their immunoreactivities in pyramidal neurons. These elevations of L-type (a1C and D) VGCC immunoreactivities may represent an enhancement of GABA-mediated inhibition, resulting in an alleviation of seizure activity. This is

176

T.-C. Kang et al. / Brain Research 1029 (2004) 168–178

because in the hippocampus of SS gerbils, vesicular glutamate transporter (VGAT) and glutamic acid decarboxylase (GAD) immunoreactivities were significantly elevated following seizure onset [25,20]. Furthermore, Ltype (a1C and D) VGCCs have been linked to somal action potentials [34] and gene expression [28,29]. Therefore, temporal alterations in VGCC immunoreactivities following seizure onset may be a compensatory reaction to regulate seizure activity. Unexpectedly, N-type VGCC was elevated in CA1 pyramidal neurons following seizure onset. Hippocampal output fibers from CA1, strongly activate deep layers of the entorhinal cortex, which can then drive synchronous discharges to the superficial layers (II and III). Layer II, which sends the major excitatory input to the granular cell layer in the hippocampus, is under strong local inhibitory control and layer III projects into the CA1 neurons where it evokes predominant inhibitory effects by presumably stimulating GABA interneurons [9,17]. With respect to this hippocampal–entorhinal feedback circuit [9,17], elevated Ntype VGCC in the CA1 region may play an important role in regulating neuronal activity into and out of the gerbil hippocampal complex during the postictal stage. 4.3. Comparison of alterations in VGCC expressions with other epilepsy models The relationships between changes in VGCC receptor expression and seizure activity remain controversial, particularly in the kainate (KA) model. Vigues et al. [39] reported an up-regulation of VGCC expression in the hippocampus. In contrast, Westenbroek et al. [44] reported no change in the expression of any of the VGCCs in neurons after KA injection. These discrepancies may have been caused by properties of KA models, which induces severe neuronal degeneration in the hippocampus [12,31]. Therefore, in KA models, it is uneasy to determine whether altered VGCC immunoreactivity may be linked with the seizure activity. In fact, we have reported corroborative evidence concerning elevations of neuronal VGCC in the neurodegenerative process induced by ischemia [30]. In addition, the enhancement of VGCC mRNA in the hippocampus was not found in KA-treated rats with nonlesioned hippocampus [39]. Thus, in KA models, changes in VGCC could be linked both to neuronal damage and to epileptogenesis. In the electrical kindling model, a significant enhancement of VGCC was observed in the hippocampus [10,41]. Hendriksen et al. [14] reported that during the initial stages of epileptogenesis in the kindling model, the a1A and a1D expression levels were significantly increased in the hippocampus, despite decreased a1B and unaltered a1C expressions. In addition, at the fully kindled stage, only the a1B expression level was up-regulated, and no change in VGCC expression was found in animals investigated long-term after the establishment of the fully kindled state. Thus, it was suggested that VGCC

expression may be altered in a subclass-specific manner during the early stages of kindling, and that it may play a role in the establishment of a kindled focus, possibly caused by altering the population of VGCC subunits involved in neurotransmitter release. Similarly, in the present study, the enhancement of VGCC expressions without neuronal degeneration was found in the SS gerbil hippocampus. Therefore, we postulate that the altered VGCC immunoreactivities may systematically affect neuronal transmission within the gerbil hippocampus. Furthermore, it is conceivable that enhanced VGCC immunoreactivities, accompanied by reduced inhibitory transmission in the hippocampus of SS gerbils, may be related to the effects of abnormal hyperdischarge, and the generation of seizure activity. 4.4. Speculative mechanism for up-regulation of VGCC expression in the hippocampus of SS gerbils Epilepsy in the Mongolian gerbil has been linked to abnormalities in the inhibitory systems (e.g., GABA, neuropeptide Y and somatostatin) [23–25,20,21,18,19]. Recently, we reported that somatostatin receptor (SST) expression in the hippocampus of SS gerbils was lower than that of SR gerbils [24]. SST was found to be downregulated in the dentate gyrus, in the CA2–3 region and in the stratum radiatum (Schaffer’s collateral) of SS gerbils, where VGCC expression was elevated in the present study. Thus, spatial alterations in SST expression are similar to those of VGCC. The elevation of VGCC immunoreactivity in the SS gerbil hippocampus could be the result of reduced SST-mediated inhibition, since the activation of SST inhibits the pre- and postsynaptic VGCC functions [6,8,15,36,38,40,45]. Indeed, somatostatin was found to modulate VGCC in hippocampal CA1 pyramidal neurons [16], and lost SST function could result in abnormal synaptic potentiation through N-type VGCC in the mouse dentate gyrus [1], which contributes to epileptogenesis. Therefore, it is presumable that the elevation of VGCC expression in the hippocampus of SS gerbils may be a consequence of reduced SST function. To explain the precise roles of SST in the elevated VGCC expressions in the gerbil hippocampus, further studies are needed. In conclusion, our findings provide the first comprehensive description of the distribution of VGCC immunoreactivities in the normal and epileptic gerbil hippocampus, and suggest that enhanced VCGG expressions in the hippocampus may be closely related with seizure activity in this animal.

Acknowledgements This study was supported by Korea Research Foundation Grant (KRF-2003-041-E20004).

T.-C. Kang et al. / Brain Research 1029 (2004) 168–178

References [1] M.V. Baratta, T. Lamp, M.K. Tallent, Somatostatin depresses long-term potentiation and Ca2+ signaling in mouse dentate gyrus, J. Neurophysiol. 88 (2002) 3078 – 3086. [2] D.E. Braun, W.J. Freed, Effect of nifedipine and anticonvulsants on kainic acid-induced seizures in mice, Brain Res. 533 (1990) 157 – 160. [3] D.L. Burgess, J.M. Jones, M.H. Meisler, J.L. Noebels, Mutation of the Ca2+ channel beta subunit gene Cchb4 is associated with ataxia and seizures in the lethargic (lh) mouse, Cell 88 (1997) 385 – 392. [4] D.W. Choi, Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage, Trends Neurosci. 11 (1988) 465 – 469. [5] D.W. Choi, Excitotoxic cell death, J. Neurobiol. 23 (1992) 1261 – 1276. [6] C.B. Cooper, M.I. Arnot, Z.P. Feng, S.E. Jarvis, J. Hamid, G.W. Zamponi, Cross-talk between G-protein and protein kinase C modulation of N-type calcium channels is dependent on the G-protein beta subunit isoform, J. Biol. Chem. 275 (2000) 40777 – 40781. [7] G. De Sarro, A. De Sarro, F. Federico, B.S. Meldrum, Anticonvulsant properties of some calcium antagonists on soundinduced seizures in genetically epilepsy prone rats, Gen. Pharmacol. 21 (1990) 769 – 778. [8] V.E. Degtiar, B. Wittig, G. Schultz, F. Kalkbrenner, A specific G(o) heterotrimer couples somatostatin receptors to voltage-gated calcium channels in RINm5F cells, FEBS Lett. 380 (1996) 137 – 141. [9] R. Empson, U. Heinemann, The perforant path projection to hippocampal area CA1 in the rat hippocampal–entorhinal cortex combined slice, J. Physiol. 4843 (1995) 707 – 720. [10] G.C. Faas, M. Vreugdenhil, W.J. Wadman, Calcium currents in pyramidal CA1 neurons in vitro after kindling epileptogenesis in the hippocampus of the rat, Neuroscience 75 (1996) 57 – 67. [11] C.F. Fletcher, C.M. Lutz, T.N. O’Sullivan, J.D. Shaughnessy Jr., R. Hawkes, W.N. Frankel, N.G. Copeland, N.A. Jenkins, Absence epilepsy in tottering mutant mice is associated with calcium channel defects, Cell 87 (1996) 607 – 617. [12] L.K. Friedman, Selective reduction of GluR2 protein in adult hippocampal CA3 neurons following status epilepticus but prior to cell loss, Hippocampus 8 (1998) 511 – 525. [13] J.W. Hell, R.E. Westenbroek, C. Warner, M.K. Ahlijanian, W. Prystay, M.M. Gilbert, T.P. Snutch, W.A. Catterall, Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits, J. Cell Biol. 123 (1993) 949 – 962. [14] H. Hendriksen, W. Kamphuis, F.H. Lopes da Silva, Changes in voltage-dependent calcium channel alpha1-subunit mRNA levels in the kindling model of epileptogenesis, Brain Res. Mol. Brain Res. 50 (1997) 257 – 266. [15] M. Inoue, M. Yoshii, Modulation of ion channels by somatostatin and acetylcholine, Prog. Neurobiol. 38 (1992) 203 – 230. [16] H. Ishibashi, N. Akaike, Somatostatin modulates high-voltageactivated Ca2+ channels in freshly dissociated rat hippocampal neurons, J. Neurophysiol. 74 (1995) 1028 – 1036. [17] R.S.G. Jones, J.D.C. Lambert, Entorhinal–hippocampal connections: a speculative view of their function, Trends Neurosci. 16 (1990) 58 – 64. [18] T.C. Kang, S.H. Park, S.K. Park, J.C. Lee, S.M. Jo, S.G. Do, J.G. Suh, Y.S. Oh, J.Y. Lee, M.H. Won, The temporal and spatial expressions of neuropeptide Y induced by seizure in the hippocampal complex of gerbil, Brain Res. 870 (2000) 179 – 184. [19] T.C. Kang, S.K. Park, S.G. Do, J.G. Suh, S.M. Jo, Y.S. Oh, Y.G. Jeong, M.H. Won, The over-expression of somatostatin in the gerbil entorhinal cortex induced by seizure, Brain Res. 882 (2000) 55 – 61. [20] T.C. Kang, H.S. Kim, M.O. Seo, S.Y Choi, O.S. Kwon, N.I. Baek, H.Y. Lee, M.H. Won, The temporal alteration of GAD67/GAD65 ratio in the gerbil hippocampal complex following seizure, Brain Res. 920 (2001) 159 – 169. [21] T.C. Kang, S.K. Park, J.H. Bahn, S.G. Jeon, S.M. Jo, S.W. Cho, S.Y Choi, M.H. Won, The alteration of gamma-aminobutyric acid-trans-

[22]

[23]

[24]

[25]

[26] [27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

177

aminase expression in the gerbil hippocampus induced by seizure, Neurochem. Int. 38 (2001) 609 – 614. T.C. Kang, S.K. Park, I.K. Hwang, S.J. An, J.H. Bahn, D.W. Kim, S.Y Choi, O.S. Kwon, N.I. Baek, H.Y. Lee, M.H. Won, Changes in pyridoxal kinase immunoreactivity in the gerbil hippocampus following spontaneous seizure, Brain Res. 957 (2002) 242 – 250. T.C. Kang, S.J. An, S.K. Park, I.K. Hwang, M.O. Seo, H.S. Kim, J.H. Kang, O.S. Kwon, M.H. Won, The somatostatin receptors in the normal and epileptic hippocampus of the gerbil: subtype-specific localization and its alteration, Brain Res. 986 (2003) 91 – 102. T.C. Kang, S.K. Park, I.K. Hwang, S.J. An, S.Y. Choi, O.S. Kwon, N.I. Baek, H.Y. Lee, M.H. Won, The altered expression of GABA shunt enzymes in the gerbil hippocampus before and after seizure generation, Neurochem. Int. 42 (2003) 239 – 249. T.C. Kang, S.J. An, S.K. Park, I.K. Hwang, J.C. Bae, M.H. Won, Changed vesicular GABA transporter immunoreactivity in the gerbil hippocampus following spontaneous seizure and vigabatrin administration, Neurosci. Lett. 335 (2003) 207 – 211. M.J. Lin, S.Y. Lin-Shiau, Multiple types of Ca2+ channels in mouse motor nerve terminals, Eur. J. Neurosci. 9 (1997) 817 – 823. W.J. Loskota, P. Lomax, S.T. Rich, The gerbil as a model for the study of the epilepsies. Seizure patterns and ontogenesis, Epilepsia 15 (1974) 109 – 119. R.P. Misra, A. Bonni, C.K. Miranti, V.M. Rivera, M. Sheng, M.E. Greenberg, L-type voltage-sensitive calcium channel activation stimulates gene expression by a serum response factor-dependent pathway, J. Biol. Chem. 269 (1994) 25483 – 25493. T.H. Murphy, P.F. Worley, J.M. Baraban, L-type voltage-sensitive calcium channels mediate synaptic activation of immediate early genes, Neuron 7 (1991) 625 – 635. S.K. Park, S.J. An, I.K. Hwang, J.G. Suh, Y.S. Oh, M.H. Won, T.C. Kang, Temporal alterations in voltage gated Ca2+ channel immunoreactivities in the gerbil hippocampus following ischemic insults, Brain Res. 970 (2003) 87 – 96. A. Patel, K. Bulloch, Type II glucocorticoid receptor immunoreactivity in the mossy cells of the rat and the mouse hippocampus, Hippocampus 13 (2003) 59 – 66. L.A. Paul, I. Fried, K. Watanabe, A.B. Forsythe, A.B. Scheibel, Structural correlates of seizure behavior in the Mongolian gerbil, Science 213 (1981) 924 – 926. T. Sakurai, R.E. Westenbroek, J. Rettig, J. Hell, W.A. Catterall, Biochemical properties and subcellular distribution of the BI and rbA isoforms of alpha 1A subunits of brain calcium channels, J. Cell Biol. 134 (1996) 511 – 528. N.C. Spitzer, Development of voltage-dependent and ligand-gated channels in excitable membranes, Prog. Brain Res. 102 (1994) 169 – 179. J.G. Suh, Z.W. Ryoo, M.H. Won, Y.S. Oh, T.C. Kang, Differential alteration of NMDA receptor subunits in the gerbil dentate gyrus and subiculum following seizure, Brain Res. 904 (2001) 104 – 111. Q.Q. Sun, J.R. Huguenard, D.A. Prince, Somatostatin inhibits thalamic network oscillations in vitro: actions on the GABAergic neurons of the reticular nucleus, J. Neurosci. 22 (2002) 5374 – 5386. R.W. Tsien, D. Lipscombe, D.V. Madison, K.R. Bley, A.P. Fox, Multiple types of neuronal calcium channels and their selective modulation, Trends Neurosci. 11 (1988) 431 – 438. F. Viana, B. Hille, Modulation of high voltage-activated calcium channels by somatostatin in acutely isolated rat amygdaloid neurons, J. Neurosci. 16 (1996) 6000 – 60011. S. Vigues, M. Gastaldi, C. Chabret, A. Massacrier, P. Cau, J. Valmier, Regulation of calcium channel alpha(1A) subunit splice variant mRNAs in kainate-induced temporal lobe epilepsy, Neurobiol. Dis. 6 (1999) 288 – 301. C. Vilchis, J. Bargas, T. Perez-Rosello, H. Salgado, E. Galarraga, Somatostatin modulates Ca2+ currents in neostriatal neurons, Neuroscience 109 (2002) 555 – 567.

178

T.-C. Kang et al. / Brain Research 1029 (2004) 168–178

[41] M. Vreugdenhil, W.J. Wadman, Enhancement of calcium currents in rat hippocampal CA1 neurons induced by kindling epileptogenesis, Neuroscience 49 (1992) 373 – 381. [42] R.E. Westenbroek, J.W. Hell, C. Warner, S.J. Dubel, T.P. Snutch, W.A. Catterall, Biochemical properties and subcellular distribution of an Ntype calcium channel alpha 1 subunit, Neuron 9 (1992) 1099 – 1115. [43] R.E. Westenbroek, T. Sakurai, E.M. Elliott, J.W. Hell, T.V. Starr, T.P. Snutch, W.A. Catterall, Immunochemical identification and subcellular distribution of the 1A subunits of brain Ca2+ channels, J. Neurosci. 15 (1995) 6403 – 6418.

[44] R.E. Westenbroek, S.B. Bausch, R.C. Lin, J.E. Franck, J.L. Noebels, W.A. Catterall, Upregulation of L-type Ca2+ channels in reactive astrocytes after brain injury, hypomyelination, and ischemia, J. Neurosci. 18 (1998) 2321 – 2334. [45] Y. Zhu, J.L. Yakel, Calcineurin modulates G protein-mediated inhibition of N-type calcium channels in rat sympathetic neurons, J. Neurophysiol. 78 (1997) 1161 – 1165.