JN Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Neurophysiol 95: 2446-2455, 2006. First published January 11, 2006; doi:10.1152/jn.01173.2005
0022-3077/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
95/4/2446    most recent
01173.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kumar, S. S.
Right arrow Articles by Buckmaster, P. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kumar, S. S.
Right arrow Articles by Buckmaster, P. S.

GABAA Receptor–Mediated IPSCs and {alpha}1 Subunit Expression Are Not Reduced in the Substantia Nigra Pars Reticulata of Gerbils With Inherited Epilepsy

Sanjay S. Kumar1, Xiling Wen1, Yufeng Yang3 and Paul S. Buckmaster1,2

1Department of Comparative Medicine, 2Department of Neurology and Neurological Sciences, and 3Graduate Program in Neuroscience, Stanford University, Stanford, California

Submitted 7 November 2005; accepted in final form 9 January 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Domestic Mongolian gerbils, a model of inherited epilepsy, begin having spontaneous seizures at ~1.5 mo of age, making it possible to evaluate them during epileptic and pre-epileptic stages. Previous studies have shown that GABA binding is reduced in the substantia nigra pars reticulata (SNr) of both epileptic and pre-epileptic gerbils compared with controls, suggesting that reduced expression of GABAA receptors in SNr might be epileptogenic in this model. To test this hypothesis, we measured the expression of the GABAA receptor {alpha}1 subunit, the dominant {alpha} subunit expressed in the SNr, and evaluated GABAA receptor–mediated postsynaptic currents in SNr neurons. GABAA {alpha}1 subunit mRNA levels in substantia nigra–rich tissue from pre-epileptic animals were similar to controls, and immunocytochemistry for the {alpha}1 subunit showed similar strong expression in the SNr in both groups. Western analysis confirmed that expression of the {alpha}1 subunit protein was similar in substantia nigra–rich tissue from pre-epileptic and control gerbils. The frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) and the frequency of miniature (m)IPSCs in SNr neurons of pre-epileptic gerbil were similar to those of controls. The amplitude of mIPSCs in the pre-epileptics was significantly larger than controls. Zolpidem, an {alpha}1 subunit–specific modulator of the GABAA receptor, was equally efficacious in prolonging the decay time of mIPSCs in both groups. Hence, contrary to the predictions of the hypothesis, mRNA and protein expression levels of the major GABAA receptor {alpha} subunit were normal, and neurons of the SNr in pre-epileptic gerbils displayed normal or enhanced IPSC frequencies and amplitudes. Therefore reduced expression of GABAA receptors in SNr is not likely to be an epileptogenic mechanism in this model.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Genetic factors play an important etiological role in many forms of epilepsy. At least 13 genes have been identified in which mutations are directly associated with human epilepsy (Scheffer and Berkovic 2003Go). Animal models provide opportunities to discover new genes and mechanisms of inherited epilepsy. Mongolian gerbils (Meriones unguiculatus) are a model of inherited epilepsy that displays spontaneous, recurrent, generalized tonic-clonic seizures beginning at ~1.5 mo of age (Buckmaster and Wong 2002Go; Buckmaster et al. 1996Go; Loskota et al. 1974Go; Seto-Ohshima et al. 1992Go). The cause of seizures is unknown, but their predictable development allows investigators to evaluate pre-epileptic animals that are about to become epileptic, which is advantageous because seizures themselves have many side effects. For example, seizure activity changes the expression of GABAA receptors (Brooks-Kayal et al. 1998Go; Kapur and Macdonald 1997Go; Naylor et al. 2005Go).

Pre-epileptic and epileptic gerbils display abnormalities that involve GABAergic synaptic transmission (Buckmaster et al. 1996Go; Kato et al. 2000Go; Löscher 1987Go; Peterson and Ribak 1987Go; Seto-Ohshima et al. 2001Go), which is consistent with the unusually high anticonvulsant potency of GABAmimetic drugs in this model (Löscher and Frey 1984Go; Löscher et al. 1983Go). Mutations in the {alpha}1, {gamma}2, and {delta} subunits of the GABAA receptor have been linked to human familial epilepsy (Macdonald et al. 2004Go). Radioligand binding to membrane homogenates revealed fewer GABAA receptor binding sites in the midbrain of epileptic gerbils compared with controls, and receptor autoradiography localized the reduction to the periaqueductal gray and substantia nigra, with the largest deficits in the substantia nigra pars reticulata (SNr) (Olsen et al. 1985Go). The deficiency was attributed to the number of receptors and not to differences in the affinity of the ligand for the receptor. Importantly, 1-mo-old pre-epileptic gerbils showed similar reductions in the SNr, suggesting that the difference was not just a side effect of seizures but instead might be a genetically determined epileptogenic mechanism.

The SNr strongly influences seizure activity (reviewed in Moshé et al. 1995Go). It contains GABAergic neurons that project to and inhibit portions of the thalamus and brain stem (the superior colliculus and pedunculopontine nucleus) and provides local inhibition to neurons within the SNr itself. Lesioning (Garant and Gale 1983Go; Hayashi 1952Go) or inhibiting (Iadarola and Gale 1982Go; McNamara et al. 1984Go; Thompson and Suchomelova 2004Go) the SNr suppresses seizure activity. As reviewed in Depaulis et al. (1994)Go, bilateral inhibition of the SNr clearly suppresses seizures in many different animal models of epilepsy, including maximal electroshock seizures, systemically or intracerebrally administered chemoconvulsants, flurothyl inhalation, kindling, and genetic or chemically induced absence seizures. Conversely, disinhibition of the SNr may be proepileptic. Fewer GABAA receptors in SNr suggests that a deficit in GABA-mediated inhibition could be present, and this might contribute to seizure susceptibility in gerbils with inherited epilepsy (Olsen et al. 1984Go, 1985Go, 1986Go).

This study tested the hypothesis that reduced expression of GABAA receptors contributes to epileptogenesis in gerbils with inherited epilepsy. We compared 1-mo-old pre-epileptic gerbils from a domestic, epileptic strain with age-matched nondomestic, control gerbils. We measured mRNA and protein expression levels of the dominant {alpha} subunit of the GABAA receptor expressed in the SNr and evaluated inhibitory postsynaptic currents of SNr neurons.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals

The Mongolian gerbils (M. unguiculatus) used in this study were 1 mo old and of both sexes. Pre-epileptic gerbils were the offspring of epileptic parents from a line of epileptic gerbils originally derived from a seizure-sensitive strain (WJL/UC) (Loskota et al. 1974Go) and subsequently maintained and selectively bred in our laboratory. The parents of pre-epileptic gerbils had exhibited at least three seizures during four weekly novel environment exposures that began when they were ≥2 mo old, as describe previously (Buckmaster et al. 1996Go). Virtually all of the gerbils in this line exhibit seizures beginning at ~1.5 mo of age. Therefore it is highly likely that all of the pre-epileptic gerbils used in this study would have developed epilepsy. Nondomestic gerbils had been obtained during an expedition to Mongolia in 1995 (Neumann et al. 2001Go), and descendents of the wild-caught gerbils were kindly provided by Drs. R. Weinandy and R. Gatterman (Martin Luther University, Halle, Germany). The control gerbils used in this study were the offspring of nondomestic parents. The nondomestic parents never displayed seizures when they were tested with novel environment exposure at least four times at weekly intervals beginning when they were ≥2 mo old. None of the control gerbils used in this study or their parents were ever observed to have a seizure. All experiments were performed in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Stanford University Institutional Animal Care and Use Committee.

Molecular biology

TISSUE PREPARATION. Real-time RT-PCR and Western analysis were done using substantia nigra–rich tissue. Gerbils were killed with an overdose of urethane (2 g/kg, ip) and decapitated. The brain was quickly removed, placed in a chilled gerbil brain matrix (ASI Instruments, Warren, MI), and blocked coronally to isolate a 3-mm-thick anterior-posterior segment that contained the substantia nigra. On a chilled platform, substantia nigra–rich tissue was isolated along dissection lines shown in Fig. 1A. Immediately after dissection, the tissue was frozen in liquid nitrogen and preserved at –70°C for subsequent isolation of RNA or protein.


Figure 1
View larger version (123K):
[in this window]
[in a new window]
  		FIG. 1. Tyrosine hydroxylase immunoreactivity (TH), Nissl staining, and GABAA {alpha}1 subunit immunoreactivity ({alpha}1) of the substantia nigra in control and pre-epileptic gerbils. A1 and A2: immunoreactivity for TH identifies dopaminergic neurons and fibers that are more concentrated in the substantia nigra pars compacta (SNc) than in the pars reticulata (SNr). Broken lines in A1 indicate where coronal slices were dissected to isolate substantia nigra–rich tissue for measuring mRNA and protein levels. Boxes in A1 are shown at higher magnification in A2. B: Nissl stain reveals no major differences in cell distribution or density of the SNr. C: immunocytochemistry for the {alpha}1 subunit of the GABAA receptor shows strong staining of neuropil and somata in the SNr of a control and pre-epileptic gerbil.

 
REAL-TIME RT-PCR. The investigators processing the tissue and analyzing the data were blind to the experimental groups. Total RNA was isolated from substantia nigra–rich tissue in each animal (6 control and 6 pre-epileptic gerbils) using RNAstat 60 (Tel-Test, Friendswood, TX). Total RNA concentration was adjusted to 0.8 ng/µl according to ultraviolet absorbance at 260 nm for reverse transcription and PCR. The purity of the isolated RNA was assessed by measuring the ratio of absorption at 260 and 280 nm and was found to be >1.8 for all samples. Real-time PCR was performed using ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). We sequenced the gerbil GABAA receptor {alpha}1 subunit and used the sequence to design the primer/probe set using PrimerExpress software: probe, 5'-ATGCCTAACAAACTCCTGCGCATCACAG-3'; forward primer, 5'-TCTGTGGCCCACAACATGAC-3'; and reverse primer, 5'-GTGTAGAGCAGCGTGCCATC-3'. Rodent GAPDH control reagent VIC probe (Applied Biosystems) was used as an internal control. The {alpha}1 subunit and GAPDH genes were amplified in separate tubes, and each sample was analyzed in duplicate. CT values were analyzed using the comparative CT ({Delta}{Delta}CT) method as described by the manufacturer. Relative efficiencies of target and reference amplification were approximately equal as confirmed by separate experiments. Control experiments revealed no evidence of genomic DNA contamination.

WESTERN ANALYSIS. Isolation of protein from brain cell lysates was carried out using substantia nigra–rich tissue. Because of the small volume of tissue from each gerbil, samples from eight to nine gerbils were pooled. Three sets of eight to nine animals were analyzed in triplicate for both the control and pre-epileptic groups. Tissue samples were homogenized in 50 mM Tris (pH 7.5), with 5 mM EDTA, 1% Triton X-100, and Complete, Mini (Roche, Indianapolis, IN) on ice. Homogenates were centrifuged at 150,000g at 4°C for 1 h. Protein concentrations were determined using a BCA assay kit (Pierce, Rockford, IL), and the aliquots were stored at –70°C until further use. To prevent proteolysis, all isolation procedures were carried out on ice, and all buffers contained a cocktail of protease inhibitors. Similarly, isolation of protein from membrane fractions was carried out using substantia nigra–rich tissue from three pools of eight to nine animals in each group. Tissue was homogenized in 320 mM sucrose, and homogenates were centrifuged at 2000g for 10 min at 4°C. The supernatant was recentrifuged at 100,000g for 1 h at 4°C, and the resulting pellets were resuspended in 320 mM sucrose. Storage of the supernatant and methods for determining protein concentration were similar to those described for the cell lysate fractions.

Expression levels of the GABAA receptor {alpha}1 subunit protein were measured by Western blotting. Isolated proteins were loaded on 10% SDS-polyacrylamide gels and electrophoresed at 30 mA for 1.5 h before being transferred onto nitrocellulose at 200 mA for 2 h. The blotted nitrocellulose was blocked with freshly prepared PBS containing 5% nonfat milk and 0.05% Tween-20 (PBST-milk). The nitrocellulose was incubated in rabbit anti-GABAA receptor {alpha}1 subunit serum (0.5 µg/ml; Upstate Biotechnology, Charlottesville, VA) in PBST-milk overnight with agitation at 4°C. After a wash, the nitrocellulose was incubated in peroxidase-conjugated AffiniPure goat anti-rabbit secondary IgG (1:15,000; Jackson Laboratories, West Grove, PA) in 5% nonfat milk for 1.5 h at room temperature with agitation. The nitrocellulose was subsequently washed with PBS-0.1% Tween 20. ECL Western blotting detection reagents and autoradiography film (Hyperfilm ECL, Amersham Biosciences, Piscataway, NJ) were used for band detection. After completing the analysis of GABAA {alpha}1 receptor subunit bands, the blotted nitrocellulose was washed with Restore Western blot stripping buffer (Pierce) and incubated with anti-actin serum (Sigma, St. Louis, MI), using protocols similar to those described above. GABAA {alpha}1 receptor subunit and actin levels were quantified by densitometry using National Institutes of Health image software. Ratiometric data for each group, consisting of triplicate sample tubes, were averaged together.

Anatomy

Gerbils were killed by urethane overdose (2 g/kg, ip) and perfused through the ascending aorta at 15 ml/min with 0.9% NaCl for 2 min and 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 30 min. Brains were postfixed at 4°C overnight, equilibrated in 30% sucrose in 0.1 M PB, and sectioned coronally with a sliding microtome set at 30 µm. Adjacent sections (1-in-6 series) were processed for Nissl stain (0.25% thionin), tyrosine hydroxylase, or GABAA receptor {alpha}1 subunit immunoreactivity. Immunostaining was performed as described previously (Buckmaster et al. 1996Go). Briefly, endogenous peroxidases were quenched in 1% H2O2 for 1 h. Free-floating sections were exposed to blocking solution consisting of 3% goat serum (Vector Laboratories, Burlingame, CA), 2% bovine serum albumin, and 0.3% Triton X-100 in 0.05 M Tris-buffered saline (TBS) for 1 h. Sections incubated in rabbit anti-tyrosine hydroxylase serum (1:1,200; Chemicon, Temecula, CA) or rabbit anti-GABAA receptor {alpha}1 subunit (0.5 µg/ml; Upstate Biotechnology) in 1% goat serum, 0.2% bovine serum albumin, and 0.3% Triton X-100 in 0.05 M TBS overnight at 4°C. Sections were exposed to biotinylated goat anti-rabbit serum (1:500; Vector Laboratories) in secondary diluent (2% bovine serum albumin and 0.3% Triton X-100 in 0.05 M TBS) for 2 h and avidin-biotin-horseradish peroxidase complex (1:500; Vector Laboratories) in secondary diluent for 2 h. Color was developed with 0.02% diaminobenzidine, 0.04% NH4Cl, 0.015% glucose oxidase, and 0.1% beta-D-glucose in 0.1 M tris buffer for 12 min. Sections from both experimental groups (6 pre-epileptic and 6 control gerbils) were processed together.

Electrophysiology

Coronal slices, 350 µm thick, were cut from brains of gerbils that were deeply anesthetized with urethane (1.5 g/kg, ip). Slices were prepared with a microslicer (VT1000S, Leica) in a chilled (4°C) low-Ca2+, low-Na+ "cutting solution" containing (in mM) 230 sucrose, 10 D-glucose, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, and 0.5 CaCl2, equilibrated with a 95%-5% mixture of O2 and CO2. Slices were stored in oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 26 NaHCO3, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, and 10 D-glucose (pH 7.4), first at 32°C for 1 h and subsequently at room temperature before being transferred to a recording chamber.

Recordings were obtained at 32 ± 1°C from neurons in the SNr under Nomarski optics (Nikon, Tokyo, Japan) using a visualized infrared setup (Hamamatsu Photonics, Hamamatsu, Japan). Recording electrodes (1.2–2.0 µm tip diameter, 3–6 M{Omega}) contained (in mM) 100 potassium gluconate, 40 HEPES, 10 EGTA, 5 MgCl2, 2 disodium-ATP, 0.3 sodium-GTP, and 20 biocytin for current-clamp recordings and 120 cesium methanesulfonate, 10 HEPES, 8 NaCl, 2 magnesium-ATP, 0.3 sodium-GTP, 5 QX-314, 0.1 BAPTA, and 20 biocytin for voltage-clamp recordings. Internal solutions were adjusted to a pH of 7.3 with KOH or CsOH and to an osmolarity of 300 mOsm. The presence of QX-314 and cesium in the pipette solution precluded the recording of GABAB receptor–mediated inhibitory postsynaptic currents (IPSCs). Slices were maintained in oxygenated (95% O2-5% CO2) ACSF, and drugs were applied through the perfusate (2 ml/min). The following compounds were bath-applied as required for specific protocols: D(–)-2-amino-5-phosphonopentanoic acid (D-APV), 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX, diluted in dimethylsulfoxide, <0.1% final concentration), TTX, and picrotoxin (all from Sigma). Zolpidem was a gift from Huguenard Laboratory (Stanford University).

Spontaneous and miniature IPSC data, obtained from 2-min-long continuous recordings, were analyzed using Mini Analysis (Synaptosoft, Decatur, GA). The threshold for event detection was set at three times root mean square noise level. Average noise level of recordings obtained from control and pre-epileptic gerbils was not significantly different. Postsynaptic currents and potentials were recorded with an Axopatch-1D amplifier and pClamp software (Axon Instruments, Foster City, CA), filtered at 1–2 kHz (10 kHz for current clamp), digitized at 10–20 kHz, and stored digitally. Series resistance was monitored continuously, and those cells in which this parameter exceeded 15 M{Omega} or changed by >20% were rejected. Series resistance compensation was not used. The frequency and amplitude of postsynaptic currents were averaged from all detected events. Miniature IPSCs (mIPSCs) whose rising and decay phases did not overlap other events were used to compute decay kinetics and half widths. Several hundred events of this type were aligned and averaged and the peak-to-baseline decay phase of the resulting trace was fitted by the following double exponential function

Formula
where Afast and Aslow are the fast and slow amplitude components, and {tau}fast and {tau}slow are the fast and slow decay time constants, respectively. The weighted decay time constant ({tau}d,w) was calculated using the following equation

Formula
To visualize biocytin-labeled neurons after recording, slices were fixed at least overnight in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 4°C. After fixation, slices were stored in 30% ethylene glycol and 25% glycerol in 50 mM PB at –20°C, before being processed with counterstaining by NeuN-immunocytochemistry. Slices were rinsed in 0.5% Triton X-100 and 0.1 M glycine in 0.1 M PB and placed in a blocking solution containing 0.5% Triton X-100, 2% goat serum (Vector Laboratories), and 2% bovine serum albumin in 0.1 M PB for 4 h. Slices were incubated in mouse anti-NeuN serum (1:1,000; MAB377, Chemicon) in blocking solution overnight. After a rinsing step, slices were incubated with the fluorophores Alexa 594 streptavidin (5 µg/ml) and Alexa 488 goat anti-mouse (10 µg/ml; Molecular Probes, Eugene, OR) in blocking solution overnight. Slices were rinsed, mounted on slides, and coverslipped with Vectashield (Vector Laboratories) before being examined with a confocal microscope (LSM 5 Pascal, Zeiss, Oberkochen, Germany).

Data are expressed as mean ± SE. Differences between groups were determined using the unpaired Student's t-test, unless otherwise indicated, with P < 0.05 considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
GABAA receptor {alpha}1 subunit expression

Real-time RT-PCR was used to quantify GABAA receptor {alpha}1 mRNA expression in substantia nigra–rich tissue of 1-mo-old pre-epileptic and control gerbils. GABAA {alpha}1 subunit mRNA levels in pre-epileptic animals were 96% relative to controls (P > 0.9, t-test; n = 6 animals in each group; range for pre-epileptics: 0.6–1.8; controls: 0.6–1.7; Fig. 2).


Figure 2
View larger version (29K):
[in this window]
[in a new window]
  		FIG. 2. Real-time RT-PCR of GABAA {alpha}1 subunit mRNA in control and pre-epileptic gerbils. A: expression of {alpha}1 mRNA relative to internal control in SNr-rich tissue from control and pre-epileptic gerbils. Box plots specify lower (25th percentile) and upper quartiles (75th percentile), 5–95% range (whiskers), median (lines within boxes), and mean (circles within boxes). B: log scale amplification plots for the GABAA {alpha}1 subunit (B1) and GAPDH, an internal control (B2), showing overlapping expression curves for multiple samples of tissue taken from control (solid lines) and pre-epileptic animals (dashed lines). Amplification threshold (T) is indicated by horizontal solid line on semi-log plots of the change in fluorescence ({Delta}Rn) vs. cycle number. ns = not statistically significant, t-test.

 
Immunocytochemistry was used to evaluate the level of expression and location of GABAA receptor {alpha}1 subunit protein. Tyrosine hydroxylase staining revealed that the substantia nigra pars compacta (SNc) was qualitatively similar in both groups and helped localize the SNr (Fig. 1A). Adjacent Nissl-stained sections revealed qualitatively similar neuron densities in the SNr (Fig. 1B). Immunocytochemistry for the GABAA receptor {alpha}1 subunit revealed intense labeling of the neuropil and cell bodies in the SNr in control and pre-epileptic gerbils (Fig. 1C). Immunostaining levels suggested that the expression of the {alpha}1 subunit was not reduced in pre-epileptic gerbils and might be slightly increased.

Western analysis was used to quantify the levels of GABAA receptor {alpha}1 subunit protein expressed in pre-epileptic and control animals. Cell lysates were analyzed to assess the expression of the 51-kDa GABAA receptor {alpha}1 subunit protein relative to actin. In pre-epileptic gerbils, the average expression of the {alpha}1 subunit protein relative to the internal control was 0.24 ± 0.05 compared with 0.27 ± 0.02 in controls (P > 0.8; Fig. 3). Because protein expression in the cytoplasm can differ from expression in the plasma membrane, membrane fractions were analyzed for GABAA receptor {alpha}1 protein content. The averaged expression of {alpha}1 protein in the membrane fractions of the pre-epileptic gerbils was similar to that of controls (0.70 ± 0.01 vs. 0.72 ± 0.03; P > 0.7; Fig. 4).


Figure 3
View larger version (35K):
[in this window]
[in a new window]
  		FIG. 3. Western analysis of GABAA receptor {alpha}1 subunit protein from whole cell lysates. A: results from substantia nigra–rich tissue from control and pre-epileptic gerbils showing a 51-kDa band (top) corresponding to the GABAA receptor {alpha}1 subunit and a 42-kDa band (bottom) corresponding to actin (internal control). B: bar plot indicates averaged ratios of densitometry measurements of bands corresponding to the {alpha}1 subunit and actin in control and pre-epileptic gerbils. Error bars indicate SE. ns = not statistically significant, t-test.

 

Figure 4
View larger version (16K):
[in this window]
[in a new window]
  		FIG. 4. Western analysis of GABAA receptor {alpha}1 subunit protein from membrane fractions. A: results from substantia nigra–rich tissue from control and pre-epileptic gerbils showing the 51- (top) and 42-kDa (bottom) bands corresponding to the {alpha}1 subunit and actin (internal control), respectively. B: bar plot shows similar averaged densitometry ratios for control and pre-epileptic gerbils, suggesting similar levels of protein expression. Error bars indicate SE. ns = not statistically significant, t-test.

 
Electrophysiological and anatomical characterization of neurons in the SNr

Whole cell recordings were obtained from visualized neurons in the SNr (Fig. 5A). Under infrared optics, the SNr was distinguishable from the denser and more opaque SNc. In contrast with the densely packed SNc, neurons in the SNr were sparse and distributed diffusely in groups throughout the nucleus.


Figure 5
View larger version (55K):
[in this window]
[in a new window]
  		FIG. 5. Electrophysiological and anatomical characterization of SNr neurons. A1 and A2: brain slice visualized with infrared differential interference contrast optics showing the SNr (*). Inset in A1: typical SNr neuron under high magnification. A2: outline of a section highlighting region visualized through the microscope in A1 (dashed boxed). III indicates the third ventricle. B: current-clamp recordings from 2 types of electrophysiologically defined neurons in the SNr: a nonadapting (top) and adapting neuron (bottom) in response to current injections. Note prominent sag current (open arrowhead) and rebound burst firing (filled arrowhead) in adapting neuron. C1–C3: typical biocytin-labeled neurons in SNr showing differences in soma size and dendritic arborization (C1). These neurons discharged action potentials differently in response to equivalent amounts of current injection, as shown in C2 and C3. C2: neuron with the small, round cell body and multipolar dendrites with extensive branching close to the soma displayed sustained firing in response to current injection, whereas the neuron with the large, fusiform cell body and dendrites that tended not to branch until farther from the soma displayed spike-firing adaptation. C3: individual action potentials of the small, round, multipolar neuron were narrower and displayed a large amplitude afterhyperpolarization, whereas the larger, fusiform neuron displayed longer duration action potentials with smaller amplitude spike afterhyperpolarizations.

 
Within the SNr, the vast majority of neurons are GABAergic (Ribak et al. 1976Go), but there also is a small subpopulation of dopaminergic neurons (Grofova et al. 1982Go; Juraska et al. 1977Go; Richards et al. 1997Go). We used electrophysiology and biocytin-labeling to distinguish between these cell types. Current-clamp recording revealed two types of neurons based on intrinsic electrophysiological properties. The majority of cells sampled (6/8) fired rapidly in response to current injection, displayed little or no spike frequency adaptation, and had brief action potentials (<1.5 ms) with large amplitude afterhyperpolarizations (Fig. 5, B and C). In contrast, two of the eight cells fired fewer action potentials in response to equivalent amounts of current injection, showed prominent sag currents, rebound burst firing, spike frequency adaptation, and had longer action potentials (>2 ms) with smaller amplitude afterhyperpolarizations. Previous studies have reported two types of electrophysiologically defined neurons in the SNr: one that displays tonic-firing with brief action potentials and large amplitude spike afterhyperpolarizations and another that shows phasic-firing with sag current and longer action potentials (Nakanishi et al. 1987Go). Previous biocytin-labeling studies revealed that the tonic-firing cells were GABAergic, had a small somata and multipolar dendritic projections, and their dendrites branched closer to the cell body; in contrast, phasic-firing cells were dopaminergic, had a larger somata, and their dendrites tended not to branch until they were further from the cell body (Richards et al. 1997Go). Our findings suggest that the same two types of electrophysiologically and neurochemically defined SNr neurons in rats also are present in gerbils.

To optimize voltage-clamp recordings of GABAA receptor–mediated currents in this study, the pipette solution contained cesium and QX-314, which precluded analysis of action potentials. To ensure that voltage-clamp data were obtained from GABAergic SNr neurons, we selected cells with a small somata and only included data obtained from biocytin-labeled neurons with multipolar dendritic projections and dendrites that branched close to the cell body.

Inhibitory synaptic drive of SNr neurons

Whole cell voltage-clamp recordings of sIPSCs (outward events at a holding potential of 0 mV; Fig. 6A) were obtained from SNr neurons. The frequency and amplitude of sIPSCs in SNr neurons from pre-epileptic gerbils were comparable with those from controls (Table 1). Averaged cumulative amplitude distributions of sIPSCs recorded in SNr neurons from control and pre-epileptic gerbils were overlapping (Fig. 6A3). Miniature IPSCs were isolated in the presence of the glutamatergic receptor antagonists (10 µM NBQX and 50 µM D-AP5) and TTX (1 µM). The average frequency of mIPSCs in neurons from pre-epileptic gerbils was comparable with that of controls (Table 1; Fig. 6B), but averaged mIPSC amplitude in the pre-epileptic gerbils was significantly larger (144% of controls). The averaged cumulative amplitude distribution of mIPSCs recorded in SNr neurons from pre-epileptic gerbils was right-shifted compared with controls (Fig. 6B3). Reversal potentials for mIPSCs obtained by extrapolating linear fits of averaged current-voltage relationships (holding potential ≥ 0 mV) were –31 ± 6 mV in controls and –37 ± 2 mV in the pre-epileptics (P > 0.3). Chord conductances associated with GABAA-mediated mIPSCs in control and pre-epileptic gerbils, deduced from a linear fit of the averaged current-voltage relationships, were similar: 551 ± 31 and 523 ± 83 pS, respectively.


Figure 6
View larger version (34K):
[in this window]
[in a new window]
  		FIG. 6. Spontaneous inhibitory postsynaptic currents (IPSCs; outward events at 0-mV holding potential) from SNr neurons in control and pre-epileptic gerbils. A1: bottom traces show time-expanded views of regions indicated by bars in top traces. A2: bar plots show mean frequency and amplitude of events for indicated number of neurons in each group. Error bars indicate SE. A3: cumulative amplitude distributions of spontaneous IPSCs recorded in SNr neurons from control and pre-epileptic gerbils averaged over indicated number of neurons in each group (bin size = 5 pA). Distributions are similar (P > 0.9, Kolmogorov-Smirnov test). B1: miniature IPSCs in SNr neurons of control (left) and pre-epileptic gerbils (right). B2: bar plots represent averages from indicated number of cells and show no significant difference in frequency but significantly larger amplitude in the pre-epileptic animals. **P < 0.01 (t-test). B3: averaged cumulative amplitude distribution of miniature IPSCs recorded in SNr neurons from pre-epileptic gerbils is right-shifted compared with controls (P < 0.003, Kolmogorov-Smirnov test).

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. Frequency and amplitude of spontaneous and miniature inhibitory postsynaptic currents in substantia nigra pars reticulata neurons of control and pre-epileptic gerbils

 
GABAA receptor {alpha}1 subunit–specific pharmacology

To evaluate GABAA receptor {alpha}1 subunit–specific changes in SNr neurons, we examined the effects of zolpidem, an {alpha}1 subunit-specific modulator of the GABAA receptor (Korpi et al. 2002Go; Mohler et al. 2002Go). Bath application of 200 nM zolpidem caused a broadening of mIPSCs without significantly affecting their amplitude or frequency. This effect was observed in both control and pre-epileptic animals (Fig. 7, A and B). The weighted decay time constant ({tau}dw) in controls increased from 4.7 ± 0.6 to 7.3 ± 0.9 ms in zolpidem (P < 0.01, paired t-test, n = 6 cells in each group; Fig. 7C), whereas in the pre-epileptic gerbils, {tau}dw increased from 5.9 ± 0.5 to 7.7 ± 0.5 ms (P < 0.02, paired t-test, n = 6). Comparing the mean percent change in both groups revealed that zolpidem was equally efficacious in altering mIPSC duration (% change in {tau}dw in zolpidem for pre-epileptics: 32% vs. controls: 35%, P > 0.8, t-test, n = 6; Fig. 7D).


Figure 7
View larger version (30K):
[in this window]
[in a new window]
  		FIG. 7. Effects of zolpidem, a GABAA receptor {alpha}1 subunit–specific modulator, on miniature IPSCs in control and pre-epileptic gerbils. A: recordings of mIPSCs in a SNr neuron from a control gerbil before (left) and after (right) application of zolpidem (200 nM). Below each trace is the corresponding averaged response of all nonoverlapping events detected during a 2-min-long continuous recording. Effects of zolpidem are apparent in the overlay of averaged events shown on right, which shows average mIPSC before (black) and after (gray) zolpidem application. B: recordings of mIPSCs in an SNr neuron from a pre-epileptic gerbil obtained before (left) and after (right) application of zolpidem (200 nM). Average mIPSCs are shown below. Overlay shows average mIPSC before (black) and after (gray) zolpidem application. C: bar plots of averaged weighted decay time constant ({tau}d,w) of mIPSCs from indicated number of cells in control and pre-epileptic gerbils before (–) and after (+) zolpidem application. D: comparison of averaged percent change in {tau}d,w in animals from the 2 groups. *P < 0.02; **P < 0.01 (paired t-test); ns = not statistically significant, t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The principal findings of this study are that control and pre-epileptic gerbils display similar expression levels of the GABAA receptor {alpha}1 subunit in substantia nigra–rich tissue, and the frequency and amplitude of IPSCs are not reduced in SNr neurons of pre-epileptic gerbils. These findings suggest that, contrary to a previous hypothesis based on reduced GABA binding (Olsen et al. 1985Go), reduced expression of GABAA receptors in the SNr is an unlikely mechanism of inherited epilepsy in this model.

Normal expression of the GABAA receptor {alpha}1 subunit

Principal neurons in the SNr of rats express the GABAA receptor subunits {alpha}1, beta(2–3), and {gamma}1 (Schwarzer et al. 2001Go). The {alpha} subunit is important for GABA binding (Smith and Olsen 1995Go). Our immunocytochemistry findings confirm that the {alpha}1 subunit is strongly expressed in SNr neurons of gerbils. If reduced GABA binding were attributable to reduced expression of GABAA receptors, one might expect that the level of {alpha}1 subunit mRNA or protein would be lower in pre-epileptic gerbils compared with controls. That was not the case. Quantitative PCR and Western analysis of cell lysates and membrane fractions of substantia nigra–rich tissue revealed no differences between groups. The tissue used in these experiments, however, was not purely SNr, and differences in that specific region could have been diluted or obscured by neighboring structures. If so, one might expect to observe reduced immunostaining for the {alpha}1 subunit in the SNr of pre-epileptic gerbils. Again, that was not the case.

The quantitative PCR, Western, and immunocytochemical data suggest that GABAA receptor {alpha}1 subunit expression is not reduced in pre-epileptic gerbils. These findings, however, do not exclude the possibility that the subunit may be expressed at normal levels but fail to incorporate into functional receptors in the cell membrane, which could result in reduced GABA binding. To test that possibility, we evaluated the effect of zolpidem on mIPSCs in SNr neurons. Zolpidem binds with the GABAA receptor {alpha}1 subunit and prolongs the decay time of IPSCs (Korpi et al. 2002Go; Mohler et al. 2002Go). Zolpidem had similar effects on mIPSCs in SNr neurons of pre-epileptic and control gerbils, suggesting that the {alpha}1 subunit was present in functional GABAA receptors at appropriate synaptic sites in pre-epileptic gerbils.

IPSCs are not reduced in SNr neurons of pre-epileptic gerbils

We focused on the GABAA {alpha}1 subunit because of its high level of expression in neurons of the SNr (Schwarzer et al. 2001Go). It is possible, however, that an epileptogenic defect reduces the expression of functional GABAA receptors in SNr but does not involve the {alpha}1 subunit. If there were fewer GABAergic synapses, one might expect to find reduced frequency of IPSCs in pre-epileptic gerbils. In fact, the trend was the opposite, suggesting that the number of GABAA synapses and their probability of release are not reduced in pre-epileptic gerbils. However, a normal frequency of mIPSCs does not exclude the possibility that fewer receptors were expressed per synapse, which would reduce GABA binding. If there were fewer receptors per synapse, one might expect to find lower amplitude mIPSCs in pre-epileptic gerbils. On the contrary, the amplitude of mIPSCs was significantly larger in SNr neurons of pre-epileptic gerbils compared with controls.

Increased amplitude of mIPSCs in pre-epileptic gerbils could be attributable to increased electrochemical driving force on chloride ions, increased single channel conductance, or more channels/synapse. Using chord conductances of mIPSCs, assuming mean single-channel conductance for a synaptic GABAA receptor to be 27 pS (Angelotti and Macdonald 1993Go; Brickley et al. 1999Go), and not taking into account the stochastic nature of channel opening, we estimate a similar number of GABAA receptors/synapse (19–20) in control and pre-epileptic gerbils. Under our recording conditions (intracellular cesium), the extrapolated reversal potential tended to be more hyperpolarized in pre-epileptic gerbils compared with controls, but further studies are needed to measure more physiological chloride reversal potentials.

If the density of GABAA binding sites is reduced (Olsen et al. 1985Go), it is unclear why IPSC frequency or amplitude of SNr neurons are not reduced in pre-epileptic gerbils. One possibility is that the number of synapses and receptors expressed by each cell is normal, but there are fewer cells in the SNr. This seems unlikely, because Nissl staining and GABAA {alpha}1 subunit-immunocytochemistry in this study did not reveal an obvious reduction in SNr cell densities, and the number and size of glutamic acid decarboxylase (GAD)-immunoreactive neurons in SNr are similar in epileptic and control gerbils (Peterson and Ribak 1987Go).

What causes epilepsy in Mongolian gerbils?

Many different mechanisms have been proposed to account for inherited epilepsy in gerbils (reviewed in Buckmaster 2005Go). The "disinhibition" hypothesis of epilepsy in gerbils contends that seizures begin in the hippocampal dentate gyrus because an overabundance of GABAergic interneurons selectively inhibits basket cells and thereby disinhibits granule cells (Farias et al. 1992Go; Peterson and Ribak 1987Go; Peterson et al. 1985Go). However, subsequent studies found similar numbers of GABAergic interneurons in control and epileptic gerbils (Buckmaster et al. 1996Go; Scotti et al. 1997aGo,bGo) and no evidence of reduced inhibition in the dentate gyrus at seizure onset (Buckmaster et al. 2000Go). Another hypothesis of gerbil epilepsy holds that a defect in neocortical inhibition underlies epilepsy in gerbils (Kato et al. 2000Go; Seto-Ohshima et al. 2001Go). GAD activity is reduced in parts of neocortex in epileptic gerbils (Löscher 1987Go). This hypothesis requires further testing.

This study tested the hypothesis that reduced expression of GABAA receptors in the SNr is an epileptogenic mechanism in this model (Olsen et al. 1984Go, 1985Go, 1986Go). Our findings are not consistent with that hypothesis. However, they do not exclude the possibility of an epileptogenic defect in GABAergic synaptic transmission. In fact, the amplitude of mIPSCs was significantly higher in pre-epileptic gerbils. These findings suggest that SNr neurons may be more inhibited in pre-epileptic gerbils. Enhanced inhibition also has been reported in the hippocampus of juvenile epileptic gerbils. Gerbils that are 2 mo old and have just begun to display seizures exhibit enhanced paired-pulse depression of field potential responses in the dentate gyrus in response to perforant path stimulation at short interstimulus intervals (Buckmaster et al. 1996Go). These findings support the idea that abnormalities in GABAergic synaptic transmission of multiple brain regions may precede and potentially contribute to the development of epilepsy in this model.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by the National Institute of Neurological Disorders and Stroke.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank C. Neuwohner for assistance with anatomy, Drs. R. Weinandy and R. Gatterman (Martin Luther University, Halle, Germany) for providing nondomestic control gerbils, Dr. David Lyons (Stanford University) for sharing equipment, and Dr. John Huguenard (Stanford University) and members of his laboratory for zolpidem and advice.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. Buckmaster, Dept. of Comparative Medicine, Stanford Univ., 300 Pasteur Dr., R321 Edwards Bldg., Stanford, CA 94305-5342 (E-mail: psb{at}stanford.edu)


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Angelotti TP and Macdonald RL. Assembly of GABAA receptor subunits: alpha 1 beta 1 and alpha 1 beta 1 gamma 2S subunits produce unique ion channels with dissimilar single-channel properties. J Neurosci 13: 1429–1440, 1993.[Abstract]

Brickley SG, Cull-Candy SG, and Farrant M. Single-channel properties of synaptic and extrasynaptic GABAA receptors suggest differential targeting of receptor subtypes. J Neurosci 19: 2960–2973, 1999.[Abstract/Free Full Text]

Brooks-Kayal AR, Shumate MD, Jin H, Rikhter TY, and Coulter DA. Selective changes in single cell GABAA receptor subunit expression and function in temporal lobe epilepsy. Nat Med 4: 1166–1172, 1998.[CrossRef][ISI][Medline]

Buckmaster PS. Inherited epilepsy in Mongolian gerbils. In: Models of Seizures and Epilepsy, edited by Pitkänen A, Schwartzkroin PA, and Moshé SL. San Diego, CA: Elsevier, 2005, chapt. 21, p. 273–294.

Buckmaster PS, Jongen-Rêlo AL, Davari SB, and Wong EH. Testing the disinhibition hypothesis of epileptogenesis in vivo and during spontaneous seizures. J Neurosci 20: 6232–6240, 2000.[Abstract/Free Full Text]

Buckmaster PS, Tam E, and Schwartzkroin PA. Electrophysiological correlates of seizure sensitivity in the dentate gyrus of epileptic juvenile and adult gerbils. J Neurophysiol 76: 2169–2180, 1996.[Abstract/Free Full Text]

Buckmaster PS and Wong EH. Evoked responses of the dentate gyrus during seizures in developing gerbils with inherited epilepsy. J Neurophysiol 88: 783–793, 2002.[Abstract/Free Full Text]

Depaulis A, Vergnes M, and Marescaux C. Endogenous control of epilepsy: the nigral inhibitory system. Prog Neurobiol 42: 33–52, 1994.[CrossRef][ISI][Medline]

Farias PA, Low SQ, Peterson GM, and Ribak CE. Morphological evidence for altered synaptic organization and structure in the hippocampal formation of seizure-sensitive gerbils. Hippocampus 2: 229–246, 1992.[CrossRef][ISI][Medline]

Garant DS and Gale K. Lesions of substantia nigra protect against experimentally induced seizures. Brain Res 273: 156–161, 1983.[CrossRef][ISI][Medline]

Grofova I, Deniau JM, and Kitai ST. Morphology of the substantia nigra pars reticulata projection neurons intracellularly labeled with HRP. J Comp Neurol 208: 352–368, 1982.[CrossRef][ISI][Medline]

Hayashi T. A physiological study of epileptic seizures following cortical stimulation in animals and its application to human clinics. Jpn J Physiol 3: 46–64, 1952.[Medline]

Iadarola MJ and Gale K. Substantia nigra: site of anticonvulsant activity mediated by gamma-aminobutyric acid. Science 218: 1237–1240, 1982.[Abstract/Free Full Text]

Juraska JM, Wilson CJ, and Groves PM. The substantia nigra of the rat: a Golgi study. J Comp Neurol 172: 585–600, 1977.[CrossRef][ISI][Medline]

Kapur J and Macdonald RL. Rapid seizure-induced reduction of benzodiazepine and Zn2+ sensitivity of hippocampal dentate granule cell GABAA receptors. J Neurosci 17: 7532–7540, 1997.[Abstract/Free Full Text]

Kato M, Ito M, and Seto-Ohshima AS. Cortical somatosensory evoked potentials of seizure-sensitive and seizure-resistant gerbils. Epilepsy Res 40: 129–139, 2000.[CrossRef][ISI][Medline]

Korpi ER, Grunder G, and Luddens H. Drug interactions at GABA(A) receptors. Prog Neurobiol 67: 113–159, 2002.[CrossRef][ISI][Medline]

Löscher W. Hippocampal glutamate decarboxylase activity is not altered in gerbils with high seizure susceptibility. Biochem Pharmacol 36: 979–982, 1987.[CrossRef][ISI][Medline]

Löscher W and Frey HH. Evaluation of anticonvulsant drugs in gerbil with reflex epilepsy. Drug Res 34: 1484–1488, 1984.[Medline]

Löscher W, Frey HH, Reiche R, and Schultz D. High anticonvulsant potency of {gamma}-aminobutyric acid (GABA)mimetic drugs in gerbils with genetically determined epilepsy. J Pharmacol Exp Ther 226: 839–844, 1983.[Abstract/Free Full Text]

Loskota WJ, Lomax P, and Rich ST. The gerbil as a model for the study of the epilepsies. Epilepsia 15: 109–119, 1974.[ISI][Medline]

Macdonald RL, Gallagher MJ, Feng HJ, and Kang J. GABA(A) receptor epilepsy mutations. Biochem Pharmacol 68: 1497–1506, 2004.[CrossRef][ISI][Medline]

McNamara JO, Galloway MT, Rigsbee LC, and Shin C. Evidence implicating substantia nigra in regulation of kindled seizure threshold. J Neurosci 4: 2410–2417, 1984.[Abstract]

Mohler H, Fritschy JM, and Rudolph U. A new benzodiazepine pharmacology. J Pharmacol Exp Ther 300: 2–8, 2002.[Abstract/Free Full Text]

Moshé SL, Garant DS, Sperber EF, Veliskova J, Kubova H, and Brown LL. Ontogeny and topography of seizure regulation by the substantia nigra. Brain Dev 17: 61–72, 1995.

Nakanishi H, Kita H, and Kitai ST. Intracellular study of rat substantia nigra pars reticulata neurons in an in vitro slice preparation: electrical membrane properties and response characteristics to subthalamic stimulation. Brain Res 437: 45–55, 1987.[CrossRef][ISI][Medline]

Naylor DE, Liu H, and Wasterlain CG. Trafficking of GABAA receptors, loss of inhibition, and a mechanism for pharmacoresistance in status epilepticus. J Neurosci 25: 7724–7733, 2005.[Abstract/Free Full Text]

Neumann K, Maak S, Stuermer IW, von Lengerken G, and Gatterman R. Low microsatellite variation in laboratory gerbils. J Hered 92: 71–74, 2001.[Abstract/Free Full Text]

Olsen RW, Snowman AM, Lee R, Lomax P, and Wamsley JK. Role of the gamma-aminobutyric acid receptor-ionophore complex in seizure disorders. Ann Neurol 16: S90–S97, 1984.

Olsen RW, Wamsley JK, Lee RJ, and Lomax P. Benzodiazepine/barbiturate/GABA receptor-chloride ionophore complex in a genetic model for generalized epilepsy. Adv Neurol 44: 365–378, 1986.[Medline]

Olsen RW, Wamsley JK, McCabe RT, Lee RJ, and Lomax P. Benzodiazepine/gamma-aminobutyric acid receptor deficit in the midbrain of the seizure-susceptible gerbil. Proc Natl Acad Sci USA 82: 6701–6705, 1985.[Abstract/Free Full Text]

Peterson GM and Ribak CE. Hippocampus of the seizure-sensitive gerbil is a specific site for anatomical changes in the GABAergic system. J Comp Neurol 261: 405–422, 1987.[CrossRef][ISI][Medline]

Peterson GM, Ribak CE, and Oertel WH. A regional increase in the number of hippocampal GABAergic neurons and terminals in the seizure-sensitive gerbil. Brain Res 340: 384–389, 1985.[CrossRef][ISI][Medline]

Ribak CE, Vaughn JE, Saito K, Barber R, and Roberts E. Immunocytochemical localization of glutamate decarboxylase in rat substantia nigra. Brain Res 116: 287–298, 1976.[CrossRef][ISI][Medline]

Richards CD, Shiroyama T, and Kitai ST. Electrophysiological and immunocytochemical characterization of GABA and dopamine neurons in the substantia nigra of the rat. Neuroscience 80: 545–557, 1997.[CrossRef][ISI][Medline]

Scheffer IE and Berkovic SF. The genetics of human epilepsy. Trends Pharmacol Sci 24: 428–433, 2003.[CrossRef][Medline]

Schwarzer C, Berresheim U, Pirker S, Wieselthaler A, Fuchs K, Sieghart W, and Sperk G. Distribution of the major gamma-aminobutyric acid(A) receptor subunits in the basal ganglia and associated limbic brain areas of the adult rat. J Comp Neurol 433: 526–549, 2001.[CrossRef][ISI][Medline]

Scotti A, Bollag O, Kalt G, and Nitsch C. Loss of perikaryal parvalbumin immunoreactivity from surviving GABAergic neurons in the CA1 field of epileptic gerbils. Hippocampus 7: 524–535, 1997a.[CrossRef][ISI][Medline]

Scotti A, Kalt G, Bollag O, and Nitsch C. Parvalbumin disappears from GABAergic CA1 neurons of the gerbil hippocampus with seizure onset while its presence persists in the perforant path. Brain Res 760: 109–117, 1997b.[CrossRef][ISI][Medline]

Seto-Ohshima A, Ito M, Katoh M, Kitajima S, and Kishikawa M. Manipulation of the somatosensory cortex modulates stimulus-induced repetitive ear movements in a seizure-sensitive strain of gerbil. Zoo Sci 18: 1217–1223, 2001.[CrossRef]

Seto-Ohshima A, Ito M, Kudo T, and Mizutani A. Intrinsic and drug-induced seizures of adult and developing gerbils. Acta Neurol Scand 85: 311–317, 1992.[ISI][Medline]

Smith GB and Olsen RW. Functional domains of GABAA receptors. Trends Pharmacol Sci 16: 162–168, 1995.[CrossRef][Medline]

Thompson KW and Suchomelova LM. Transplants of cells engineered to produce GABA suppress spontaneous seizures. Epilepsia 45: 4–12, 2004.[ISI][Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
95/4/2446    most recent
01173.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kumar, S. S.
Right arrow Articles by Buckmaster, P. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kumar, S. S.
Right arrow Articles by Buckmaster, P. S.

HOME HELP FEEDBACK