Increased GABAA-Receptor α1-Subunit Expression in Hippocampal Dentate Gyrus after Early-life Status Epilepticus

Authors


  • The first two authors contributed equally to this work.

Address correspondence and reprint requests to Dr. A. Brooks-Kayal at Division of Neurology, Abramson Pediatric Research Center, Rm 502, 3615 Civic Center Blvd., Philadelphia, PA 19104, U.S.A. E-mail: kayal@email.chop.edu

Abstract

Summary: Purpose: Previous studies in neonatal (postnatal day 10) and adult rats suggest that status epilepticus (SE) induces changes in the α1 subunit of the GABAA receptor (GABRA1) in dentate granule neurons (DGNs) that are age dependent and vary inversely with the likelihood of epilepsy development. In the present study, we examined GABRA1 expression after SE at postnatal day 20 (P20), an intermediate age when only a subset of SE-exposed animals develop epilepsy.

Methods: SE was induced with lithium-pilocarpine or kainate at P20. Animals were video-EEG monitored after SE to determine the presence or absence of spontaneous seizures. GABRA1 mRNA and protein levels were determined 7 days or 3 months later in SE-exposed and control animals by using a combination of aRNA amplification, Western blotting, and immunohistochemistry techniques.

Results: GABRA1 mRNA levels in DGNs of SE-exposed rats that did not become epileptic were higher than those in control rats, but were not different from DGNs in epileptic SE-exposed rats. GABRA1 protein levels in dentate gyrus were significantly increased in both epileptic and nonepileptic SE-exposed rats compared with controls. GABRA1 mRNA changes were region specific and did not occur in CA1 or CA3 areas of hippocampus. GABRA1 alterations were present by 1 week after P20 SE and were similar whether pilocarpine or kainate was used to induced SE.

Conclusions: P20 SE results in persistent increases in GABRA1 levels selectively in dentate gyrus. These changes preceded the onset of epilepsy, were not model specific, and occurred in both epileptic and nonepileptic animals.

Status-epilepticus (SE) is characterized by prolonged seizures lasting ≥30 min, during which patients do not regain consciousness (ILAE, 1981). Occurrence of SE is more common in children than in adults (Shinnar et al., 1997), and retrospective studies suggest that a strong link exists between SE in early childhood and development of epilepsy later in life (Falconer et al., 1964; Aicardi and Chevrie, 1983; Sagar and Oxbury, 1987). However, the exact mechanism that leads to development of epilepsy after prolonged seizures remains unclear. Several laboratories have demonstrated age-dependent changes in inhibitory neurotransmission after prolonged SE (Buhl et al., 1996; Gibbs et al, 1997; Schwarzer et al., 1997; Brooks-Kayal et al., 1998; Fritschy et al., 1999; Zhang et al., 2004a); however, the functional importance of these changes for the process of epileptogenesis remains unclear.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, and its fast inhibitory action is mediated via the GABAA receptor (GABAR). GABARs are heteromeric proteins composed of multiple subunits [α(1-6), β(1-4), γ(1-3), δ, ɛ, φ, π] (Macdonald and Olsen, 1994). Earlier studies using various models of SE have found that changes in GABAR subunit expression vary with the age at SE induction (Schwarzer et al., 1997; Brooks-Kayal et al., 1998; Fritschy et al., 1999; Zhang et al., 2004a; Laurén et al., 2005). In our studies in the pilocarpine-induced SE model, we found that dentate granule neurons (DGNs) of rats that experience SE in adulthood have reduced levels of α1-subunit expression (Brooks-Kayal et al., 1998). Further, in this model, all rats that experience SE in adulthood go on to develop spontaneous seizures (epilepsy) after a latency of 5–15 days, and changes in α1-subunit expression precede epilepsy onset (Brooks-Kayal et al., 1998). In contrast, we found that DGNs of adult rats that experience SE at postnatal day 10 (P10) have increased expression of the α1 subunit (Zhang et al., 2004a), and in none does epilepsy develop later in life (Cavalheiro et al., 1987; Priel et al., 1996, Dubé et al., 2000; Zhang et al., 2004a). These findings suggest that decreased α1-subunit levels in dentate may be associated with an increased risk of developing spontaneous seizures. To investigate this possible association further, in the current study, we examined the effect of SE on α1-subunit expression at an intermediate age (P20) when epilepsy develops in only a fraction of animals after a long latent period (Sankar et al., 1998; Dubé et al., 2001; Roch et al., 2002; Raol et al., 2003). Rats were video-EEG monitored after SE at P20, and then levels of the α1 subunit in dentate were compared between epileptic and nonepileptic SE-exposed rats as well as controls in adulthood. Levels of α1-subunit expression were also examined in the latent period before development of epilepsy, in different regions of hippocampus and in a different chemoconvulsant model to determine the time course of GABAR expression and whether changes were area or model specific.

METHODS

Lithium-pilocarpine and kainate injections

Sprague-Dawley rats obtained from Charles River (Kingston, PA, U.S.A.) were used for the study. The Joseph Stokes Research Institute Animal Care and Use Committee approved all the protocols used in the study. At postnatal day 19 (P19), rat pups were briefly separated from the mother and injected intraperitoneally (i.p) with 3 mEq/kg body weight lithium chloride (Sigma, St. Louis, MO, U.S.A.). Fourteen to 18 h after the lithium injection, pups were again separated from their mother and received i.p injection of either 60 mg/kg pilocarpine (Sigma) or 0.9% saline (control group). To induce SE by using kainate, P20 rat pups were injected i.p with 8 mg/kg kainate (Silveira et al., 2002). Control rat pups received 0.9% saline instead of kainate. All rats were monitored for the appearance of behavioral seizures. The pilocarpine- and kainite-treated pups uniformly developed prolonged status epilepticus (SE) within 15–30 min of injection, and seizures occurred intermittently over a period of 5 to 6 h. Rats that did not exhibit stage III seizures (according to the Racine classification) were not included in the study. All pups were returned to the mother after 6 h and then were weaned at P21. Because our earlier studies found effects of handling and maternal separation at P10 on GABAR-subunit mRNA expression (Hsu et al., 2003), we compared the GABAR-subunit mRNA expression between P20 naïve control and lithium-injected rats that were separated from the mother on P19 and P20. We did not find any significant difference in GABAR-subunit mRNA expression between lithium-injected rats and naïve control rats, and therefore in the present studies, only lithium-injected rats were used for the control group.

Behavioral and electroencephalogram monitoring for spontaneous seizures

Lithium-pilocarpine–injected rat pups were implanted with electrodes beginning 5–40 days after SE induction and were video-EEG monitored, as previously described in detail (Raol et al., 2003) until two spontaneous seizures were observed [animals then classified as with spontaneous seizures (SS)] or until ≥4 months of age (100 days after SE) if seizures were never observed [animals then classified as without spontaneous seizures (NSS)]. Rats were regularly checked for handling-induced seizures throughout the monitoring period until they were killed for the experiment. Based on the video-EEG monitoring results, the lithium-pilocarpine injected rats (n = 30) were divided into two groups: those with spontaneous seizures (SS; n = 18) and those without spontaneous seizures (NSS; n = 12). Some of the rats from all the groups have been used for more than one biochemical analysis to minimize the number of animals required to complete the study.

Isolation of dentate gyrus, CA1, and CA3

Animals were killed and brains were dissected in chilled oxygenated HEPES medium. Longitudinal hippocampal slices 500 μm in thickness were cut, and dentate gyrus (DG), CA1, and CA3 were identified and dissected under an anatomic microscope. For the analysis in microdissected regions, DG, CA1, and CA3 were put in separate tubes and stored at −80°C until further use. To isolate dentate granule neurons (DGN) from DG, tissue cubes were enzymatically digested at 30°C for 30–45 min under oxygen-infused PIPES-buffered saline, as described in detail in previous publications (Hsu et al., 2003; Zhang et al., 2004a). After digestion, tissue cubes were rinsed thoroughly and incubated in enzyme-free medium for 1 h before use. Neurons were isolated by trituration in 1 ml of oxygenated HEPES buffer by using a series of descending diameter of fire-polished glass pipettes. The cell suspension was then plated onto a Petri dish, and the cells were allowed to settle for 10–15 min before the buffer was replaced with extracellular solution. DGN were identified based on their size (∼10 μm) and characteristic morphology (oval cell body with single neuron process).

Single-cell aRNA amplification

Amplification

Relative expression of 16 GABAR subunit [α(1-6), β(1-3), γ(1-3), δ, ɛ, θ, π] mRNAs in individual DGN was measured by using single-cell antisense mRNA (aRNA) amplification, as described previously (Brooks-Kayal et al., 1998). DGN were triturated, plated, and 10-μm round cells with a single dendrite were aspirated. Nineteen DGN from six control rats, 19 cells from six NSS rats, and 21 DGNs from seven SS rats were harvested for the study. Amplification of cellular mRNA was performed by using the single-cell aRNA amplification method of Eberwine (1992), modified as we previously described in detail (Brooks-Kayal et al., 1998; Zhang et al., 2004a). After two rounds of amplification, a final aRNA probe was synthesized by using 25 pmol of [α-32P]CTP (Perkin–Elmer, Wellesley, MA, U.S.A.).

Slot-blot preparation and expression profiles

Each blot was prehybridized for 12 h, as previously described in the literature (Brooks-Kayal et al., 1998; Zhang et al., 2004a), and then hybridized with the radiolabeled aRNA probe from an individual cell for 60 h (42°C). After washing, the blots were directly exposed for 4 h to a Molecular Dynamics Phosphor-Image screen (Amersham Biosciences, Piscataway, NJ, U.S.A.) with a linear dynamic range over five orders of magnitude. All hybridization signals fell well within this dynamic range.

Tissue RNA amplification

RNA isolation and cDNA synthesis

Dentate gyrus, CA1, and CA3 were microdissected from control rats (five lithium- and four saline-injected controls) and rats that had experienced SE at P20 (six pilocarpine- and five kainite-induced SE). For the studies in microdissected regions, RNA was isolated by using an RNeasy kit (Qiagen, Valencia, CA, U.S.A.) according to the manufacturer's protocol. Syntheses of cDNA and cRNA were carried out as described later. Then 500 ng of total RNA and 1 μl of 20 μM T7-(dT)24 oligonucleotide primer were added to a final volume of 5.5 μl reaction mixture. The mixture was incubated for 10 min at 70°C and immediately put on ice for 3 min. After brief centrifugation, 2 μl of 5× first-strand buffer, 0.5 μl of 10 mM dNTPs mixture, 1 μl of 100 mM DTT, 0.5 μl RNAse, and 0.5 μl SuperScript II RNase H-reverse transcriptase (200 U/μl) were added to make a final volume of 10 μl. After the incubation at 42°C for 90 min, 15 μl of 5× second-strand buffer, 1.5 μl of 10 mM dNTP mixture, 0.5 μl Escherichia coli DNA ligase (10 U/μl), 0.5 μl RNase (2 U/μl), and 2 μl DNA polymerase (10 U/μl) were added for second-strand cDNA synthesis. The mixture was incubated for 2 h at 16°C; 1 μl T4 DNA polymerase was then added, and the mixture was again incubated 16°C for 15 min. The cDNA was extracted with phenol-chloroform and washed twice with 75% ethanol, and the pellet was dried and resuspended in 15 μl DEPC water.

T7 RNA polymerase amplification (cRNA synthesis)

The AmpliScribe T7 High Yield Transcription Kit (Epicentre Technologies, Madison, WI, U.S.A.) was used for cRNA synthesis. Then 8 μl double-strand cDNA was added to a mixture of 2 μl of 10 × AmpliScribe T7 buffer, 1.5 μl each of 100 mM ATP, CTP, GTP, and UTP, 100 mM DTT (2 μl), and 2 μl of T7 RNA polymerase, and incubated at 42°C for 4 h. The cRNA was purified by using the RNeasy MinElute Cleanup kit protocol (Qiagen) according to the manufacturer's protocol.

RNA target labeling and hybridization

The 6 μl cRNA (500 ng) and 2 μl random hexamers (1 μg/ul) were incubated for 10 min at 70°C and then immediately put on ice for 3 min. After brief centrifugation to collect sample, 4 μl of 5× first-strand buffer, 1 μl of 20 mM dNTPs (A,G,T) mix, 1 μl of 0.5 mM dCTP, 1 μl of 100 mM DTT, 4 μl [32P]dCTP (10 mCi/ml-800 Ci/mmol), and 1 μl SuperScript II RNase H-reverse transcriptase (200 U/μl) were added to make a final volume of 20 μl. The reaction was incubated at 37°C for 90 min. After incubation, the labeled cDNA/cRNA was washed and purified with 500 μl DEPC water in a Microcon-50 column. The radiolabeled cDNA probe was hybridized for 18 h against a slot-blot containing GABAR cDNAs.

Slot-blot preparation and expression profiles

Each blot was prehybridized for 2 h at 42°C in 5 ml of prehybridization solution (MicroHyb Buffer, Invitrogen) with salmon sperm (1 μg/ml), hybridized with the radiolabeled cDNA probes in hybridization solution (MicroHyb Buffer, Invitrogen, Carlsbad, CA, U.S.A.) with Poly dA (1 μg/ml) and Cot-1 DNA (1 μg/ml) for 18 h at 42°C. The blots were washed in a final concentration of 2× SSC, 1% SDS at 50°C for 20 min, 0.5× SSC, 1% SDS at 55°C for 25 min, 0.2× SSC, 1% SDS at 55°C for 20 min, and then directly exposed for 4 h to Molecular Dynamics Phosphor-Image screen.

Western blot analysis

The DG was dissected from control (n = 5), NSS (n = 4), and SS (n = 6) groups of rats for the chronic time point. To determine the levels of α1-subunit levels after 1–2 weeks of SE at P20, DG was isolated from seven control and seven pilocarpine-induced SE rats. Tissue was sonicated in 50 mM Tris/HCl, 140 mM NaCl, 10 mM EDTA, 2% SDS, 0.5 mM, 10 mM NaF, 30 mM sodium pyrophosphate, 50 mMβ-glycerophosphate, and the following protease inhibitors: 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 mM sodium orthovandate, and a 1:1000 dilution of protease inhibitor cocktail (Sigma, St. Louis, MO, U.S.A.). Protein concentrations of samples were analyzed by using the Bio-Rad RC/DC reagent kit (Bio-Rad Laboratories, Hercules, CA, U.S.A.). Identical quantities of protein homogenate from each animal were loaded onto 10% SDS/polyacrylamide gels and run for 1 h at 100 V and then transferred to nitrocellulose by electroblotting (100 V, 1 h). Protein, 12.5 μg, was loaded on the gels. Nitrocellulose membranes were blocked in 5% nonfat dry milk made with 0.05% Tween 20 in Tris-buffered saline (pH 7.4) for 2 h at room temperature. The blots were then incubated with 1:400 anti α1-subunit primary antibody (Upstate Biotechnology, Lake Placid, NY, U.S.A.) diluted in 5% nonfat dry milk overnight at 4°C followed by incubation with anti-rabbit immunoglobulin G (IgG) secondary antibody conjugated with horseradish peroxidase (HRP; 1:5000 dilution; Amersham, Buckinghamshire, U.K.) for 2 h at room temperature. Protein bands were visualized by using Super Signal West Pico chemiluminescent substrate kit (Pierce, Rockford, IL, U.S.A.). Bands of the appropriate size were quantified by using NIH IMAGE software (National Institutes of Health, Bethesda, MD, U.S.A.). To control for potential variability in loading, all the blots were normalized to β-actin, for which the blots were stripped and reprobed with anti-β-actin polyclonal antibody, 1:10,000 dilution (Sigma) and anti-rabbit IgG secondary antibody conjugated with HRP. The results are presented as percentage of control values.

Immunocytochemistry

Rats were anaesthetized with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg; Sigma) and perfused intracardially with cold phosphate-buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde. Brains remained in 4% paraformaldehyde overnight before they were put into 10–30% sucrose solution. Once the brains were dehydrated, they were put in Tissue-tek (Sakura Finetek, Torrance, CA, U.S.A.) and frozen on dry ice. Brains were then cut into 10- to 12-μm-thick sections by using a cryostat (Leica Microsystems, Exton, PA, U.S.A.). For the immunostaining, sections were washed 2–3 times in PBS containing 1% Triton and then with PBS before incubating with 10% goat serum for 20 min at room temperature. The sections were then incubated at 4°C for 48 h with anti-α1-subunit antibody diluted at 1:100 (Upstate Biotechnology), as was previously described (Jeon et al., 2002; McDonald et al., 2004). After washing with PBS, sections were then incubated with anti-rabbit IgG secondary antibody (1:25 dilution; Jackson ImmunoResearch laboratories, West Grove, PA, U.S.A.) for 5 h at room temperature. After washing with PBS, sections were covered with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, U.S.A.). Intensity of immunofluorescence was quantified by using confocal microscopy (Leica Microscopy). At 10x magnification, part of the upper blade of the dentate gyrus containing the dentate granule cell layer and molecular layer was selected. All the sections (four sections from each animal in all three groups) selected from control (n = 6), NSS (n = 4), and SS (n = 11) groups were at a similar plane. The intensity of the fluorescence was expressed as “mean amplitude.”

Statistical analysis

Statistical comparison between the three groups (control, NSS, and SS groups) for the aRNA analysis involving whole tissue, immunocytochemistry, and Western blot analysis was done by using one-way analysis of variance (ANOVA) followed by post hoc Tukey–Kramer multiple comparison test when the ANOVA showed a significant effect of group. Unpaired Student's t test was used to analyze the mRNA and protein levels for the α1-subunits that were measured 1–2 weeks after SE that involved only two groups. For single-cell data, the effect of group on the various outcomes, as measured repeatedly across different cells, was analyzed based on a longitudinal mixed-effects approach, the SAS Proc Mixed models (SAS, 1999). The SAS Proc Mixed procedure fits a variety of mixed linear models to data, and the data are permitted to exhibit correlation and nonconstant variability, allowing modeling of the means of the data as well as their variances and covariances. One-way ANOVA and t test analysis was performed by using GraphPad Instat 3.0a version for Macintosh (San Diego, CA, U.S.A.). SAS Proc Mixed Model analysis was performed by using SAS software, version 8. A p value of ≤0.05 was considered statistically significant.

RESULTS

Early-life seizures increase the expression of α1-subunit mRNA

We examined the effect of prolonged seizures induced by pilocarpine at P20 on expression of GABAR-subunit mRNA [α (1-6), β (1-3), γ (1-3), δ, ɛ, θ, π] in DGN isolated from the hippocampus of animals when they reached adulthood (Fig. 1A). Overall, the relative expression of α1-subunit mRNA (compared with β-actin in the same cells) was higher in DGNs of rats that had experienced SE at P20 compared with control rats (Fig. 1B; F5, 38, p < 0.05 for the effect of group). Pairwise group comparison indicates that the overall differences in α1-subunit levels result mainly from differences between the nonepileptic SE-experienced rats (NSS) and the controls (p < 0.01). No statistically significant differences were found in α1-subunit mRNA levels between SSs and controls or between NSS and SS groups (p = 0.13 and 0.097, respectively). No other statistically significant difference in mRNA levels for any of the other GABAR subunits were found between the three groups by using aRNA profiling.

Figure 1.

GABAR subunit mRNA levels in dentate granule neurons (DGN) isolated from hippocampus of adult rats that were treated at P20 with lithium injection only (control) or lithium-pilocarpine–induced status epilepticus (SE) without subsequent epilepsy (no spontaneous seizures; NSS) and with subsequent epilepsy (spontaneous seizures; SS). A: aRNA expression profiles from single DGNs acutely isolated from control, NSS, and SS groups of rats. Slot-blot demonstrates hybridization intensities of radiolabeled aRNA probe to slot-blot containing GABAR subunit cDNA a1-3 (α1-3), b1-2 (α4-5), b3 (β1), c1-2 (β2-3), c3 (γ1), d1 (γ2), d2 (δ3), d3 (ɛ), e1 (π), e2 (β-actin). Only α1-subunit mRNA levels in adult rats that did not develop epilepsy (NSSs) after SE at P20 increased significantly compared with controls, whereas expression of other GABAR subunits was not different between the groups. B: Bar graph illustrating the mean (±SEM) relative expression of α1-subunit mRNA (compared with β-actin) in the same three groups. Note the significant increase in α1-subunit mRNA levels in adult rats that did not develop epilepsy (NSS) after SE at P20 compared with controls (n = 19 cells from six rats for control, 19 cells from six rats for NSS, and 21 cells from seven rats for the SS group; **p < 0.01).

α1-Subunit protein levels increase after prolonged seizure at P20

The α1-subunit protein levels were higher in dentate gyrus (DG) of both NSS and SS groups of rats that were killed 2–4 months after SE at P20 as determined by western blot of microdissected DG (Fig. 2A and B; F= 6.48; p = 0.01 for the effect of group; pairwise comparison, p < 0.05 for NSS and SS). Immunocytochemistry using an α1 subunit–specific antibody showed higher intensity of staining in the inner molecular layer of dentate gyrus of both NSS and SS groups of rats compared with controls (Fig. 2C). Quantification of the intensity of fluorescence by using confocal microscopy confirmed significant differences between the three groups (F= 11.34; p < 0.01, for the effect of group; pairwise comparison with control, p < 0.05 for NSS and p < 0.01 for SS).

Figure 2.

P20 status epilepticus (SE) caused long-term alterations in GABAR α1-subunit protein levels. A: Representative Western blots demonstrating intensities of α1-subunit and β-actin protein levels in homogenates of dentate gyrus from control, NSS, and SS groups of rats. B: Bar graph illustrating the mean (±SEM) protein levels for each group measured by using Western blot technique. Note the significant increase in α1-subunit protein levels in both NSS (n = 4) and SS (n = 6) adult rats that experienced SE at P20 compared with control rats (n = 5). C: Photographs taken at 5x (upper panel) and 10x (lower panel) show the dentate gyrus stained with antibody directed against α1-subunit protein. Note increased fluorescence intensity in the inner molecular layer (IML) of DG of NSS and SS rats. IML, inner molecular layer; GCL, granule cell layer; HIL, hilus. *p < 0.05.

Changes in α1-subunit levels begin by 1 week after SE

To determine the time course of α1-subunit changes, we examined α1-subunit expression in microdissected dentate gyrus 24 h and 1–2 weeks after P20 SE, before the onset of spontaneous seizures in any animals. The levels of α1-subunit mRNA (Fig. 3A; p < 0.01) and protein (Fig. 3C and D; p < 0.01) were higher in the microdissected DG of rats that were killed 7–14 days after P20 SE when compared with control animals. No change was seen in α1-subunit mRNA expression levels in rats killed 24 h after P20 SE.

Figure 3.

Levels of α1 subunit increased by 1–2 weeks after status epilepticus (SE), selectively in dentate gyrus. Note that animals were killed during the latent period before onset of spontaneous seizures in any animals. A: Histogram shows the mean (±SEM) levels of α1-subunit mRNA relative to β-actin in microdissected dentate gyrus (DG), CA1, and CA3 regions of hippocampus in rats killed 1–2 weeks after lithium-pilocarpine–induced SE. Note that the levels of α1-subunit mRNA increased significantly in DG but not in CA1 or CA3 regions (n = 6 for pilocarpine group, and n = 5 for control group). **p < 0.01. B: The levels of α1-subunit mRNA, presented as mean (±SEM) values relative to β-actin, were higher in DG but not in CA1 or CA3 areas of rats that were killed 1–2 weeks after kainate-induced SE at P20 (n = 5) compared with control rats (n = 4). **p < 0.01. C: Representative Western blots demonstrating intensities of α1-subunit and β-actin protein levels in homogenates of dentate gyrus from control and pilocarpine (Pilo)-treated rats at 1–2 weeks after SE. D: Bar graph illustrating the mean (±SEM) protein levels measured by using Western blot technique. The levels of α1-subunit protein were significantly higher in dentate gyrus of pilocarpine-treated rats that were killed at 1–2 weeks after SE at P20 (n = 7) as compared with control rats (n = 7). **p < 0.01.

Changes in α1-subunit levels are region specific but not model specific

The levels of α1-subunit mRNA were also analyzed in microdissected CA1 and CA3 regions of hippocampus after SE at P20 to determine whether changes observed in α1-subunit levels were region specific. No significant change was noted in α1-subunit levels in CA1 or CA3 areas 1–2 weeks after P20 SE induced by pilocarpine (Fig. 3A). To assure that higher levels of α1 subunit observed in the DG of the rats that had experienced P20 SE induced by pilocarpine were due to the effect of prolonged seizures and not solely related to the effects of pilocarpine, we determined the α1-subunit levels in DG of rats that had SE induced by kainic acid (KA) at P20. Similar to our findings after pilocarpine-induced SE, the levels of α1-subunit mRNA were higher in microdissected DG of rats killed 1–2 weeks after P20 KA-induced SE compared with saline-injected controls (Fig. 3B; p < 0.01). No significant change was found in α1-subunit levels in CA1 or CA3 areas after kainate-induced P20 SE.

DISCUSSION

The results of the current study demonstrate that lithium-pilocarpine–induced SE at P20 causes long-term increase in expression of GABAR α1 subunit in dentate gyrus beginning within 1–2 weeks after SE. At the single-cell level, a statistically significant increase occurs in α1-subunit mRNA levels in DGN of rats that did not develop epilepsy (NSS) compared with controls; however, trend was observed toward increased α1-subunit mRNA levels in the epileptic group (SS) as well, but the difference fell short of statistical significance. At a protein level, the α1-subunit levels were higher in microdissected DG in all the rats that had experienced SE at P20, both epileptic and nonepileptic. The current study also found that the change in α1 subunit preceded the development of epilepsy, beginning 1 week after P20 SE. These findings suggest that the change observed in α1 subunit is not resulting from spontaneous seizures. The changes observed in α1-subunit levels after pilocarpine-induced SE were also not model specific, because similar changes were also observed in DG of the rats that experienced kainate-induced SE at P20. The changes in α1-subunit expression, however, did appear to be region specific, being limited only to DG in both the models, with no significant change in α1-subunit being observed in CA1 or CA3 area of hippocampus.

The higher levels of α1 subunit observed in DGN after P20 SE are similar to those observed in our previous studies of P10 rats subjected to SE (Zhang et al., 2004a) but opposite the decreased α1-subunit expression we found in DGN of rats that had SE in adulthood (Brooks-Kayal et al., 1998, but also see Schwarzer et al., 1997; Fritschy et al., 1999; Pirker et al., 2003; Laurén et al., 2005). We and others also found that rats that experience pilocarpine-induced SE at P10 do not become epileptic as adults (Cavalheiro et al., 1987; Priel et al., 1996; Dubé et al., 2000; Zhang et al., 2004a). In contrast, adult rats subjected to pilocarpine-induced SE uniformly become epileptic in our model, with a latency of 5–15 days and a seizure frequency of 22–50 seizures/week (unpublished observation; Shumate et al., 1998; Arida et al., 1999; Wallace et al., 2003). After P20 SE, only two thirds of rats subjected to SE develop spontaneous seizures (Roch et al., 2002; Raol et al., 2003), and a longer latency occurs (45–74 days; Roch et al., 2002; Raol et al., 2003) and lower seizure frequency (two seizures/week; Raol et al., 2003) compared with rats exposed to SE as adults. Taken together with our current results, these findings suggest that the higher levels of α1 subunit in DG after early-life SE may play a role in decreasing the frequency and/or severity of spontaneous seizures (compared with adult) but is not sufficient to prevent development of epilepsy in all animals. The increased GABAR α1-subunit levels in DGN after early-life SE could augment inhibition in the DG, which has been postulated to serve as an inhibitory “gate” that filters excessive excitation and reduces the frequency and severity of seizure activity propagating through the hippocampus (Heinemann et al., 1992). This hypothesis is supported by studies of fast- and slow-kindling strains of rats showing that slow-kindling rats had 70% higher α1-subunit levels compared with control rats (Poulter et al., 1999). To test this hypothesis directly, however, additional studies examining the effects of enhancing α1-subunit expression in the DGN on subsequent development of epilepsy after SE are needed.

In summary, our data suggest that increased levels of α1 subunit may act as an adaptive mechanism to reduce the occurrence or severity of epilepsy after early-life SE. Our findings do not answer the question of what mechanism(s) promotes the development of epilepsy in two thirds of the animals after SE at P20. We have previously demonstrated that of P20 SE rats that became epileptic, only 33% had evidence of mossy fiber sprouting, and only 22% had any hippocampal cell loss (Raol et al., 2003), indicating that morphologic changes in hippocampus were not necessary prerequisites for the development of epilepsy in this model. A number of studies from other laboratories have also reported a paucity of chronic morphologic changes after early-life seizures (Albala et al., 1984; Sperber et al., 1991; Stafstrom et al., 1992; Toth et al., 1998; Baram et al., 2002; Riviello et al., 2002; Dubé et al., 2006). In contrast to developmental seizure models, rats that experience SE in adulthood have a number of obvious morphologic changes, such as mossy fiber sprouting and cell loss in hippocampus (for review, see Baram et al., 2002; Holmes, 2002). These studies highlight the marked differences in the neuropathologic consequences of SE in the immature and adult brain. In adult rats, the extent of lesions is large and triggers major circuit reorganization. However, after P20 SE, the extent of morphologic damage is more subtle and less uniform between animals. Several recent studies using noninvasive approaches such as magnetic resonance imaging (Roch et al., 2002; Dubé et al., 2004; Nairismägi et al., 2004, 2006), however, have shown changes in a variety of brain regions including hippocampus, amygdala, and piriform, perirhinal, and entorhinal cortex after prolonged seizures in immature rats, suggesting that nonstructural plastic changes may play a potentially critical role in the development of epilepsy after early-life seizures. In support of this concept, multiple laboratories have reported a variety of cellular and molecular changes after early-life seizures, including alterations in dendritic structure (Jiang et al., 1998), as well as expression of AMPA- and NMDA-receptor subunits, glutamic acid decarboxylase (GAD65), and HCN channels (Sanchez et al., 2001; Brewster et al., 2002; Bender et al., 2003; Zhang et al., 2004b; Valotta et al., 2005) that may be critical determinants of the later development of epilepsy. Clearly, additional studies are needed to elucidate which changes are critical for the process of epileptogenesis after early-life seizures.

Acknowledgments

Acknowledgment:  This study was supported by funding from the National Institutes of Health (RO1NS-38595 to A.R.B-K. I.V. Lund was supported by training grant F31NS051943–01.

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