Investigators' WorkshopPoster Session11:30 a.m.-1:30 p.m.

Epilepsia 48(s6):238-248 (2007)

IW.36IN VIVO IMAGING OF ACUTE DENDRITIC SPINE LOSS AND ACTIN DEPOLYMERIZATION WITH KAINATE SEIZURES Lin Xu1, L. Zeng1, N. R. Rensing1, P. Sinatra1, S. M. Rothman1 and M. Wong1 ( 1Neurology, Washington University School of Medicine, Saint Louis, MO) Rationale: Seizures may cause brain injury via a variety of mechanisms, potentially contributing to cognitive deficits in epilepsy patients. Although seizures induce neuronal death in some situations, they may also have "non-lethal" pathophysiological effects on neuronal structure and function, such as modifying dendritic morphology. Previous studies involving conventional fixed tissue analysis have demonstrated a chronic loss of dendritic spines following seizures in animal models and human tissue. More recently, in vivo time-lapse imaging methods have been used to monitor acute changes in spines directly during seizures, but documented spine loss only under severe conditions. Here, we examined effects of secondary generalized seizures induced by kainate, on dendritic structure of neocortical neurons utilizing multiphoton imaging in live mice in vivo and investigated molecular mechanisms mediating these structural changes. Methods: Multiphoton imaging was performed through a craniotomy window to visualize dendrites and associated spines of neocortical neurons in GFP-expressing transgenic mice before and after kainate-induced seizures of varying severity. The same dendrities were followed sequentially by time-lapse imaging, monitoring for overt morphological changes (beading) and changes in spine number over a several hour period. Phalloidin-rhodamine labeling and Western blotting were used to analyze changes in actin polymerization and actin-regulatory factors. Results: Higher stage kainate-induced seizures caused dramatic dendritic beading and loss of spines within minutes, in the absence of neuronal death or changes in systemic oxygenation. Although the dendritic beading improved rapidly following the seizures, the spine loss recovered only partially over a several hour period. Kainate seizures also resulted in activation of the actin-depolymerizing factor, cofilin, and a corresponding decrease in filamentous actin, indicating that depolymerization of actin may mediate the morphological dendritic changes. Finally, an inhibitor of the calcium-dependent phosphatase, calcineurin, antagonized the effects of seizures on cofilin activation and spine morphology. Conclusions: These dramatic in vivo findings demonstrate that seizures produce acute dendritic injury in neocortical neurons via calcineurin-dependent regulation of the actin cytoskeleton and suggest novel therapeutic targets for preventing seizure-induced brain injury. IW.37AUTOSOMAL DOMINANT JUVENILE MYOCLONIC EPILEPSY RESULTS FROM GABA-A RECEPTOR ALPHA 1 SUBUNIT MISFOLDING AND DEGRADATION Martin J. Gallagher1, E. J. Botzolakis1 and L. Ding1 ( 1Neurology, Vanderbilt University School of Medicine, Nashville, TN) Rationale: Although uncommon, monogenic forms of idiopathic generalized epilepsy (IGE) syndromes provide invaluable models for the elucidation of the pathogenic mechanisms of the corresponding polygenic and/or sporadic IGE syndrome. A single missense mutation (A322D) in the GABAA receptor 03B11 subunit causes an autosomal dominant form of juvenile myoclonic epilepsy. We demonstrated previously that the A322D mutation reduced 03B11 subunit expression and that residual 03B11 subunit localized to the endoplasmic reticulum. Here we determined the mechanism by which a single missense mutation substantially reduced 03B11 expression. Methods: We transfected heterologous mammalian cells with wild type or mutant 03B11 subunits and performed [35S]methionine pulse-chase experiments to determine the rates of 03B11 subunit degradation and biosynthesis. We elucidated the role of the ubiquitin-proteasome system in 03B11 degradation by performing pulse-chase experiments in the presence or absence of proteasome inhibitors and also by directly identifying polyubiquitinated 03B11 subunits via immunoblot. We determined the effect of the A322D mutation as well as the effects of a series of other 03B11 mutations on 03B11 subunit folding by performing glycosylation topology assays. Finally, to determine the proportion of surface GABAA receptors in heterozygous cells that contained the 03B11(A322D) subunit, we transfected cells with wild type and mutant subunits that contained different epitope tags and quantified the surface wild type and mutant subunits by flow cytometry. Results: The A322D mutation caused rapid 03B11 subunit degradation (t1/2= 23 min) through the ubiquitin proteasome system. Because of their altered hydrophobicity, mutant 03B11 subunits misfold by adopting an aberrant transmembrane topology. Because of this rapid degradation, in heterozygous expression, only 6 ± 1% of surface 03B11 subunits contained the A322D mutation. Conclusions: The A322D mutation decreases the hydrophobicity of an 03B11 subunit transmembrane domain causing misfolding and rapid 03B11 subunit degradation through the ubiquitin proteasome system resulting in haploinsufficiency in heterozygous expression. Thus, the A322D mutation provides the first example of a protein folding disease that produces an IGE syndrome. The A322D mutation decreased the hydrophobicity of the M3 transmembrane segment which inhibited its insertion into the membrane. We identified misfolded subunits by detecting glycosylation of N365. IW.38REGULATION OF THALAMIC EXCITABILITY BY CHLORIDE CHANNELS Mark P. Beenhakker1, T. A. Lew1, M. Maduke2 and J. R. Huguenard1 ( 1Neurology & Neurol. Science, Stanford University, Stanford, CA and 2Molecular & Cellular Physiology, Stanford University, Stanford, CA) Rationale: Channelopathies underlie several neurological disorders, particularly those associated with excessive neural excitability such as epilepsy. Recent work has demonstrated that mutations in the gene encoding the chloride channel subtype CLCN2 are associated with certain human generalized epilepsies, including absence epilepsy (1). While it remains unknown how chloride channel dysfunction leads to such epilepsies, altered neuronal excitability within substructures of the thalamus, particularly the reticular thalamic nucleus (RT), can promote neural activity patterns characteristic of absence epilepsy. As previous studies have shown that CLCN2 expression in RT is high (2), we tested the hypothesis that CLCN2 dysfunction in RT causes hyperexcitability and promotes epileptiform activity. Methods: We examined chloride channel function using both genetic and pharmacological manipulations. In a first series of experiments, we compared the activity of RT neurons recorded in wild type mice to that of mice deficient in CLCN2 (ie CLCN2 knock mice, generous gift of James Melvin, U. Rochester). Individual RT neurons in horizontal brain slices of mice (postnatal day 11201314) were recorded using whole-cell patch-clamp electrodes. Spontaneous excitatory (sEPSCs) and inhibitory postsynaptic currents (sIPSCs) were isolated and recorded. The instantaneous frequency and amplitude of individual currents were measured. In a second set of experiments, chloride channel regulation of RT neuron excitability was evaluated by blocking CLCN2 channels in wild type brain slices. We compared sEPSCs and sIPSCs generated in control conditions (ACSF only) to that observed during bath application of the CLCN2 antagonist NPPB (100 uM). Results: Preliminary EEG studies indicate that CLCN2 knockout mice express the electrophysiological signature (ie spike and wave activity) of absence seizures. Our slice experiments indicated RT neuron hyperexcitability is a likely candidate mechanism for the generation of these seizures. Specifically, spontaneous synaptic activity of RT neurons was altered by inactivation of CLCN2. The frequency but not amplitude of sEPSCs recorded in CLCN2 knockout mice was higher than in wild type mice. In contrast, sIPSCs were indistinguishable in recordings of RT neurons from wild type versus knockout mice. Pharmacological blockade of CLCN2 yielded results consistent with the knockout mouse data. Specifically, the frequency (but not amplitude) of sEPSCs was increased during NPPB application. Also, sIPSCs were unaffected by NPPB. Conclusions: Our results reveal a potential link between chloride channel dysfunction and generalized seizure activity. Specifically, these data show that CLCN2 deficiency increases synaptic excitation in RT, and suggest that altered excitability of RT may play a mechanistic role in generation of the absence seizures. Haug, K. et al. (2003). Nat Genet 33, 5272013532. Smith, R.L. et al. (1995). J Neurosci 15, 405720134067. IW.39REVERSAL OF SEIZURE-INDUCED INCREASES IN KINASE ACTIVITY AND GLUR1 PHOSPHORYLATION BY POST TREATMENT WITH AMPAR ANTAGONISTS IN A RAT MODEL OF HYPOXIA-INDUCED SEIZURES Sanjay Rakhade1, C. Zhou1, P. K. Aujla1, N. J. Sucher1 and F. E. Jensen1,2 ( 1Children's Hospital and Harvard Medical School, Boston, MA and 2Program in Neuroscience, Harvard Medical School, Boston, MA) Rationale: Hypoxic encephalopathy is the most common cause of neonatal seizures. We have established a rodent model that mimics the acute seizures and chronic pro-epileptogenic effects of neonatal hypoxia. We have previously shown enhanced LTP and kindling in hippocampal slices following hypoxic seizures, as well as enhanced suscpetibilty to later life "second-hit" seizures. The long term effects of hypoxia-induced early life seizures can be attenuated by early post-insult treatment(1201348 h) with the AMPA receptor (AMPAR) antagonists NBQX and topiramate. We recently showed that there is a significant increase in the phosphorylation of AMPA receptors GluR1-S831, GluR1-S845 and GluR2-S880 and a concomitant increase in PKA, PKC and CamK II activity 12201348 hours following a hypoxic seizure at P10 (Rakhade et al, Epilepsia, Oct 2006, 47 (s4), 27.) We hypothesize that AMPAR antagonists may interrupt the acute and sub-acute effects of hypoxia-induced seizures kinase activity as well as AMPAR phosphorylation, thereby mediating the long-term protective effects observed in the rodent model of early-life seizures. Methods: Rat pups were subjected to hypoxia (15 minutes at 4 7% O2) on postnatal day (P) 10. NBQX (20 mg/kg) and TPM (30 mg/kg) were dissolved in PBS and administered intraperitoneally (i.p.) to the pups, within 30 minutes of global hypoxia-induced seizures, vehicle-treated pups (0.1 ml PBS) served as controls. Protein extracts were prepared from hippocampal tissue obtained at 1, 6, 12, 24 and 48 hours post hypoxia. PKA, PKC and CamK II kinase activity was assayed using ELISA assays. Immunoblot analysis was performed using phospho-specific antibodies against serine 831 and 845 epitope of AMPAR subunit GluR1. Results: We observe treatment with the AMPAR antagonists NBQX and topiramate significantly decreased the activity of CamK II (128% and 167% n = 4, p < 0.05 compared to 331% in vehicle-treated subjects), PKA (178% and 199%, n = 4, p < 0.05; compared to 317% in vehicle-treated subjects) and PKC (155% and 181%, n = 4, p < 0.05; compared to 191% in vehicle-treated subjects). Furthermore, we also observe a reversal in the increased phosphorylation of GluR1-S831 (133% and 87%, n = 5, p < 0.05) and GluR1-S845 (110% and 113%, n = 5, p < 0.05) following post-hypoxia administration of AMPAR antagonists NBQX and topiramate respectively. Conclusions: Here we show that early post-hypoxia treatment with the AMPAR antagonists NBQX and topiramate appear to reverse the rapid post-translational modification of the GluR1 AMPAR subunits. Importantly, we have previously shown that identical post-treatment paradigm protect against the long term increases in seizure susceptibility after hypoxia-induced early life seizures (Koh et al Epilepsia, 2004; 45(6):569201375.) and neuronal injury can be attenuated by early post-insult treatment with the AMPAR antagonists NBQX and topiramate. Taken together, these data imply that the early post-translational changes are dependent upon activation of AMPARs, and may contribute to long term epileptogenesis in this model. IW.40ANALYSIS OF GLUTAMATE RECYCLING IN MAINTENANCE OF EPILEPTIFORM ACTIVITY THE ACUTE SLICE Richard J. Reimer1, H. Tani1, C. G. Dulla1 and J. R. Huguenard1 ( 1Neurology, Stanford University, Stanford, CA) Rationale: The epilepsies are characterized by an increase in network excitability. This suggests that dynamic regulation of the synthesis and release of the excitatory neurotransmitter glutamate will likely influence the abnormal activity that underlies these disorders. Recent molecular and pharmacological advances have refined our understanding of how glutamine, the immediate precursor of synaptically released glutamate, is metabolized by neurons. Using a combined pharmacological and physiological approach we investigated the role of glutamate recycling through glutamine in maintenance of epileptiform activity in acute brain slice preparations treated with GABA A and GABA B receptor inhibitors. Methods: Brains were removed from anesthetized rats and transferred into ice cold low NaCl slicing solution bubbled with 95%O2 /5% CO2 and 400 micron coronal slices were made using a vibratome. Slices were incubated in aCSF at 32 degrees for 1 hour prior to recording. The solution was continuously bubbled with 95% O2/5% CO2. Slices were placed in an interface recording chamber partially submerged in and superfused continuously (223C2.0 ml/min) with ACSF equilibrated with 95% O2/5% CO2. Extracellular field potentials were recorded from Layer V of the neocortex by using glass micropipettes (223C1 M ohm) filled with ACSF. A bipolar stimulating electrode was placed to stimulate the layer VI/white matter boundary. Slices were stimulated with varying intensities and at varying intervals in the absence and presence of glutamine or glutamate as well as inhibitors of the components of the glutamine-glutamate shuttle and metabolic pathways for de novo synthesis of glutamate. Results: MeAIB, a specific inhibitor of neuronal glutamine uptake through the system A transporters SNAT1 and SNAT2, partially inhibited epileptiform discharges in the disinhibited slice2013responses were weakened but not abolished. Histidine, a general inhibitor of glutamine transport, however, completely blocked prolonged epileptiform field depolarizations. Inhibition of de novo synthesis of glutamate through anaplerosis in astrocytes by blocking amino acid transferase with AOAA also completely blocked the epileptiform discharges. This latter effect could be overcome by the addition of exogenous glutamine or glutamate. Conclusions: Maintenance of epileptiform discharges in the disinhibited slice requires a continuous source of neuronal glutamate. Exogneously applied glutamate or its immediate metabolic precursor glutamine can suffice, but in their absence, synaptically released glutamate is derived through de novo synthesis via anaplerosis in astrocytes. Transfer of the glutamate carbon backbone to neurons occurs via a glutamine intermediate that is taken up by neurons through system A dependent and system A independent transport processes. These findings indicate that glutamate metabolism, and in particular, the de novo synthesis of glutamate and/or transfer of glutamine between astrocytes and neurons should be considered for rational therapeutic intervention in the epilepsies. IW.41A COMPUTER MODEL OF CA3 EPILEPTIFORM ACTIVITY EXACERBATED BY LOSS OF FUNCTION IN EXCITATORY CONNECTIONS Waldemar Swiercz1,2, H. R. Sabolek1,2 and K. J. Staley1,2 ( 1MGH, Charlestown, MA and 2Harvard University, Cambridge, MA) Rationale: Our primary goal is to understand the mechanisms leading to temporal lobe epileptic seizures and to develop more effective treatments. We are studying the influence of synaptic connectivity on neural network activity, with emphasis on the spatial patterns of propagation related to epileptic seizures. Our computer model of the CA3 region of hippocampus allows us to observe network activity patterns and to analyze their relationship to neural and network parameters. We tested the hypothesis that under conditions supporting periodic synchronous discharge of area CA3, reductions in excitatory synaptic transmission could result in repeated patterns of network activity. These repetitive patterns could underlie the local rhythmic EEG activity that precedes focal seizures. This activity would be analogous to the reentrant activity observed in the heart as a consequence of loss of function mutations of cardiac sodium channels. Methods: We tested this hypothesis with our computer model of a neural network (Swiercz et al. J Neuroph 2007;24(2):165201374). This model includes 10,000 pyramidal and 225 interneurons. Since we are modeling CA3 structure, the majority of connections are excitatory recurrent collateral synapses that demonstrate use-dependent depression. We implemented synaptic loss of function by reducing the rate of glutamate release and increasing the time of glutamate replenishment. Results: As predicted from experimental data, use-dependent depression terminated the synchronous population activity in the control network. However, the networks with reduced rates of glutamate release exhibited prolonged network activation. These results are consistent with the in vitro results obtained by Jones et al. (J Neuroph 2007;97(5):381220138) in which the rate of glutamate release was reduced by replacement of extracellular Ca2 + with Sr2 + . The spatial patterns of activity differed dramatically between normal networks and those with reduced rates of glutamate release. In control networks, we observed circular patterns of excitation while spiral-like patterns of activity were observed in networks with reduced glutamate release. Conclusions: The reentrant activity we observed computationally can be attributed to reductions in the degree of activity-dependent synaptic depression. Under these conditions, when circular waves of excitation return to their origin, the origin has already recovered from a activity-dependent depression, so that repeated activation of the same pathway can take place, and spiral-like activity may occur. The following conditions increase the probability of repeated pathway activation: 1) the conduction time along the circular pathway closely matches the time required to recover from synaptic depression 2) glutamate release rates must be sufficiently high to support synaptic transmission, but low enough to reduce the degree of synaptic depression. Importantly, even under these conditions spiral-like waves often terminate upon collision with other spontaneous waves of activity, providing a possible explanation for the relative rarity of seizures vs. interictal spikes. IW.42EARLY INCREASE OF CALBINDIN-D28K IMMUNOREACTIVITY IN THE DENTATE GYRUS IN KAINIC ACID-INDUCED SEIZURES IN NEWBORN RATS Ignacio Valencia1, C. D. Katsetos1,2, O. P. Mishra3, M. Delivoria-Papadopoulos3 and A. Legido1 ( 1Section of Neurology, Department of Pediatrics, St. Christopher's Hospital for Children, Drexel University College of Medicine, Philadelphia, PA; 2Department of Pathology and Laboratory Medicine, Drexel University College of Medicine, St. Christopher's Hospital for Children, Philadelphia, PA and 3Section of Neonatology, Department of Pediatrics, St. Christopher's Hospital for Children, Drexel University College of Medicine, Philadelphia, PA) Rationale: Calbindin-D28k (CB) is an EF-hand calcium binding protein (CaBP) that plays an important role in neuronal excitability and neuroprotection/excitoprotection through its calcium buffering and anti-apoptotic properties. In the hippocampus, CB is widely expressed in granule neurons of the dentate gyrus and a subpopulation of CA1 pyramidal neurons, two regions that are known for their resistance to excitotoxic neuronal damage during epilepsy, but it is absent in CA3. The aim of this study is to examine whether seizures induced by kainic acid (KA) in newborn rats alter CB-immunoreactivity (IR) in granule cells of the dentate gyrus, and whether KA-induced CB changes are mediated by nitric oxide (NO). Methods: Ten-day-old Long Evans rats were divided into 3 groups of 6 animals each: I. Seizure-free control group2013II. Seizure group treated with KA (2 mg/kg, intraperitoneally) and III. Group with KA-induced seizures pretreated with 7-nitroindazole (7-NINA, 1 mg/kg, intraperitoneally), a relatively selective neuronal NO synthase inhibitor. All animals had implantation of brain electrodes 24 hours before the experiments. Digital EEG monitoring was continuously recorded. Animals were euthanized 2 hours after KA injection. The cellular distribution of CB-IR was evaluated in the dentate gyrus by immunohistochemistry, using an affinity purified rabbit polyclonal antibody to calbindin-D28k (gift of Dr. Sylvia Christakos, Biochemistry/Mol Biol UMDNJ, Newark). The labeling index (LI) for each case was expressed as the percentage of immunolabeled CB neurons out of the total number of neurons counted in 10 non-overlapping high power/40x fields. Group means were compared using one-way analysis of variance (ANOVA, SPSS). Results: CB-IR was expressed in the dentate gyrus and CA1 sector of the hippocampus. Cellular distribution in all groups was neuronal and exhibited a dual cytoplasmic and nuclear compartmentalization. Increased somato-dendritic and nuclear CB staining was detected in granule cells of the dentate gyrus of KA-treated animals with seizures versus non-seizure controls (Table 1). No morphological evidence of neuronal necrosis (eosinophilic neurons with karyopyknosis/karyorrhexis) was seen. Conclusions: Our study shows a marked increase of CB-IR in the dentate granule cells following acute KA induced seizures, which is -in part- mediated by NO. These findings in neonatal seizures mirror the results previously reported in KA induced seizures in adult rats insofar as there is enhanced CB expression in the dentate gyrus. This early response supports the role of CB in excitoprotection/neuroprotection through nuclear and cytosolic calcium buffering and anti-apoptotic mechanisms. Further studies to elucidate the role of CB and its interaction with NO in epileptogenesis at different maturational stages are warranted. Table 1. Mean labeling indices of calbindin-D28k immunoreactivity in the dentate gyrus in different groups. Note: LI: Labeling index, SD: standard deviation, KA: kainic acid, SZ: seizures, 7-NINA: 7-nitroindazole. *p: 0.01 compared to Group I. IW.43MOLECULAR MECHANISMS OF DENTATE GYRUS GRANULE CELL RESISTANCE TO SEIZURE-INDUCED DAMAGE Synphen H. Wu1, J. C. Arevalo1, G. H. Malthankar-Phatak2, T. M. Hintz2, D. P. McCloskey2, L. Tessarollo3, M. V. Chao1 and H. E. Scharfman2 ( 1Physiology and Neuroscience, New York University School of Medicine, New York, NY; 2Helen Hayes Hospital, West Haverstraw, NY and 3National Cancer Institute, Frederick, MD) Rationale: Granule cells of the dentate gyrus are relatively resistant to seizure-induced neuronal damage compared to adjacent hippocampal neurons, but the reasons for this resistance are unclear. Because granule cells express a higher concentration of the neurotrophin brain-derived neurotrophic factor (BDNF) than other hippocampal neurons, and BDNF has been shown to be neuroprotective, we explored BDNF-mediated mechanisms that might explain granule cell resistance. BDNF binds and signals through the TrkB receptor, and one signaling substrate of TrkB that may play a role in granule cell resistance is ARMS (ankyrin repeat-rich membrane spanning protein, also called KIDINS-220). Therefore, we compared the damage following experimental status epilepticus in mice with a heterozygous mutation in ARMS (±; Het) to wild type (WT) mice. Methods: Status epilepticus was induced at 12201313 week of age using the chemoconvulsant pilocarpine hydrochloride (300 mg/kg, s.c.) 30 minutes after an injection of 5 mg/kg (s.c.) atropine methylbromide. One hour after the onset of status, determined using the Racine scale, the anticonvulsant diazepam was administered (5 mg/kg, i.p.). Animals were perfusion-fixed 3, 7, 14 days or 2 months after status and 50 03BCm sections were cut with a vibratome. Immunocytochemistry was conducted to examine neuronal loss using an antibody to NeuN, a neuronal marker (mouse monoclonal; Chemicon). Fluorojade B was used to detect neurodegeneration. Neuropeptide Y (NPY; rabbit polyclonal; Chemicon) was used to identify mossy fibers. Degenerating hilar neurons were quantified using an optical fractionator (StereoInvestigator; Microbrightfield, Inc.). Results: Granule cell and hippocampal pyramidal cell morphology and density appeared identical in WT and Het mice that had received pilocarpine but did not have status (n = 5/group). Two months after status, there was greater loss of granule cells in the Het mice compared to WT mice (n = 5/group). However, latency to status, incidence of status, and the latency to the first seizure were not statistically different, and differences in the behavioral signs of status could not be discriminated, suggesting that the results could not be explained by more severe status in Het mice. Moreover, stereological estimates of hilar neuronal loss were not statistically different in WT and Het mice (n = 5/group), and fluorojade labeling 3 (n = 5/group), 7 (n = 2/group) and 14 days (n = 2/group) after status was comparable in pyramidal cell layers of both WT and Het mice. NPY labeling showed more mossy fiber sprouting in Het mice when they were examined 2 months after status despite the loss of granule cells (n = 6/group). Conclusions: The results suggest a role for ARMS in the resistance of dentate gyrus granule cells to seizure-induced neuronal damage. The high expression of BDNF, its receptor TrkB, and downstream molecular components of neurotrophin signaling, such as ARMS, could be a powerful combination that ensures survival of the granule cell population even after insults that damage the hippocampal pyramidal cells and hilar neurons. IW.44TWO CLASSES OF RODENT GLIAL CELLS IN THE HIPPOCAMPUS IN THE KAINIC ACID-INDUCED STATUS EPILEPTICUS MODEL OF TEMPORAL LOBE EPILEPSY Daniel K. Takahashi1 and K. S. Wilcox1,2 ( 1Neuroscience, University of Utah, Salt Lake City, UT and 2Pharmacology and Toxicology, University of Utah, Salt Lake City, UT) Rationale: Profound astrogliosis occurs throughout the hippocampus in animal models and in human temporal lobe epilepsy (TLE). While the membrane properties of astrocytes have been evaluated in the hippocampus from normal rodents and in resected hippocampus from patients with TLE, to our knowledge, very few studies have examined, in brain slices, the electrophysiological properties of astrocytes in the kainic acid (KA)-induced status epilepticus (SE) model of TLE. The present study uses patch clamp and immunohistochemical techniques to characterize the membrane properties of glial fibrillary acidic protein (GFAP)-positive and GFAP-negative glial cells in the CA1 region of the hippocampus in animals subjected to KA-induced SE. Methods: Brain slices were prepared from male Sprague-Dawley rats 7201314 days after systemic KA treatment (Smith et al., 2007). Following decapitation, horizontal brain slices (300 03BCm) were cut on a vibroslicer and placed in oxygenated standard artificial cerebrospinal fluid (ACSF) at 37 C for one hour. Slices were then incubated at room temperature until transferred to the recording chamber. In some experiments, 1003BCM glycine was added and MgCl2 was omitted from the recording solution. Glial cells in the stratum radiatum of the hippocampus were visualized by IR-DIC microscopy and recorded in the whole cell configuration with biocytin-containing electrodes (520139 M03A9). Results: Voltage clamp experiments revealed heterogeneity in the resting membrane potentials, input resistances, and voltage dependent membrane currents of hippocampal glial cells. In a subset of cells, post-hoc staining for biocytin and GFAP revealed two classes of glia, GFAP-positive and GFAP-negative, which were correlated with differences observed in membrane properties. Interestingly, cells with linear current-voltage profiles and low input resistance (GFAP-positive) were found to have recurrent spontaneous inward currents in the high glycine, low MgCl2 ACSF (n = 5) at a frequency of 0.17 ± 0.07 Hz (mean ± st.dev.). In contrast, spontaneous recurrent inward currents were never observed in the other type of glial cell (with high input resistance, non-linear current-voltage relationships, and GFAP negative). Conclusions: Astrocytes with a linear current profile from the hippocampus of KA-treated rats were found to be GFAP-positive and dye-coupled with other glial cells. These cells were also found to exhibit recurrent, spontaneous inward currents under permissive conditions for neuronal bursting. In contrast, glial cells with a non-linear current-voltage relationship were often GFAP-negative, were not dye-coupled, and did not exhibit recurrent spontaneous inward currents. Ongoing experiments will identify the cellular mechanism underlying the spontaneous inward currents observed in the GFAP-positive cells. It is anticipated that an increased understanding of the role of astrocytes in TLE will provide innovative molecular targets for the treatment of this frequently therapy-resistant seizure disorder. (Sources of funding: KSW: R01 NS44210, DKT: EFA pre-doctoral fellowship) IW.45THE INTEGRATION OF ADULT-GENERATED HIPPOCAMPAL GRANULE CELLS IS DRAMATICALLY AND SELECTIVELY DISRUPTED DURING EPILEPTOGENESIS Steve Danzer1,2, C. Walter1 and B. Murphy4,1 ( 1Anesthesia, Cincinnati Children's Hospital, Cincinnati, OH; 2Pediatrics, University of Cincinnati, Cincinnati, OH; 3Anesthesia, University of Cincinnati, Cincinnati, OH and 4Program in Neuroscience, University of Cincinnati, Cincinnati, OH) Rationale: Aberrantly-interconnected granule cells are characteristic of temporal lobe epilepsy. By reducing network stability, these abnormal neurons may contribute directly to disease development. Only subsets of granule cells, however, exhibit abnormalities. Why this is the case is not known. Ongoing neurogenesis in the adult hippocampus may provide an explanation. Newly-generated granule cells may be uniquely vulnerable to environmental disruptions relative to their mature neighbors. Here, we determine whether there is a critical period following neuronal birth, during which neuronal integration can be disrupted by an epileptogenic insult. We predict that immature and newborn granule cells 2013 but not mature ones 2013 will develop aberrant connections during epileptogenesis. Methods: To establish the age and reveal the morphology of mature, immature and newborn granule cells, BrdU was given to Thy1-GFP-expressing transgenic mice eight week before (mature), one week before (immature) or three week after (newborn) pilocarpine-induced epileptogenesis. The neuronal morphology of the subset of dentate granule cells labeled with both BrdU and GFP was examined both four and eight week after pilocarpine treatment (when all cells were mature) using confocal microscopy. Dendritic orientation, branching pattern, spine density and neuronal position were assessed. Results: Almost 50% of immature granule cells exposed to pilocarpine-induced epileptogenesis exhibited aberrant hilar basal dendrites. In contrast, only 9% of mature granule cells exposed to the identical insult possessed basal dendrites. In addition, newborn cells were even more severely impacted than immature cells, with 40% exhibiting basal dendrites and an additional 20% exhibiting migration defects. By comparison, less than 5% of neurons from normal animals exhibited either abnormality, regardless of age. Conclusions: Together, these data demonstrate the existence of a critical period following the birth of adult-generated neurons during which they are vulnerable to being recruited into epileptogenic neuronal circuits. Pathological brain states, therefore, may pose a significant hurdle for the appropriate integration of newly-born endogenous (and exogenously implanted) neurons. Moreover, thousands of new neurons are added to the brain in the week after epileptogenesis, and, disturbingly, our findings indicate that the majority of these cells integrate abnormally. Abnormal integration of such large numbers of neurons may contribute to the development of epilepsy, and/or to co-morbid conditions associated with epilepsy, such as cognitive impairment and depression. IW.46RNA EDITING OF GABAA RECEPTORS: A POTENTIAL MECHANISM FOR SHAPING DEVELOPING SYNAPSES Andre H. Lagrange1, E. Y. Rula2, R. B. Emeson2 and R. L. Macdonald1 ( 1Neurology, Vanderbilt University, Nashville, TN and 2Pharmacology, Vanderbilt University, Nashville, TN) Rationale: The 03B13 subunit is a predominant GABAA receptor isoform expressed during brain development, a period when GABA evokes depolarizing currents that may play an important role in synaptogenesis. Recent studies by Ohlson et al (2007) reported that editing of the 03B13 subunit mRNA occurs at about the same time. This process involves a site-selective deamination of a single adenosine in transmembrane domain 3, thereby converting a genomically encoded isoleucine (I) to a methionine (M) codon. Expanding on this previous work, we found that RNA transcripts encoding the adult mouse 03B13 subunit, but not the 03B11, 2, 4, 5 or 6 subunits, are edited to 90% completion in most brain regions, although 30% of the unedited form persists in the adult hippocampus. By contrast, the extent of editing is very low at embryonic day 15 (E15) and increases during development, reaching maximal levels by postnatal day 7 (P7). Methods: To explore the functional effects of editing, cDNAs encoding either non-edited 03B13(I) or edited 03B13(M) human subunits were cotransfected with human 03B23 and 03B32L subunit cDNAs into HEK 293T cells. Whole cell voltage clamp of lifted cells was used to record the currents elicited by GABA applied with a rapid drug delivery system. Results: Compared to 03B1103B2203B32 receptors (the predominant combination in the mature brain), 03B13(I/M)03B2303B32L receptors were insensitive to low levels of GABA and activated very slowly but also desensitized and deactivated slowly. After excluding the possibility that RNAs encoding the non-edited 03B13(I) subunit were modified in HEK cells, we found that currents from receptors containing the non-edited 03B13 subunit [03B13(I)03B2303B32L] activated more rapidly, responded to slightly lower GABA concentrations and deactivated much more slowly than currents from edited 03B13(M)03B2303B32L receptors. Since the non-edited 03B13(I) subunit is prevalent at a developmental period during which GABA-evoked currents are depolarizing, ramp current-voltage (IV) plots were generated before and during exposure to a low concentration of GABA. The edited form of the receptor conducted current in a nearly linear manner, whereas the non-edited form had strong outward rectification, consistent with enhanced influx of Cl- anions. Conclusions: Taken together, these results suggest that regulation of GABAA receptor 03B13 subunits allows a developmental period during which a predominant receptor isoform is particularly responsive to GABA. Since this occurs when GABA evokes depolarizing currents, the non-edited 03B13(I) form expressed in developing brain would allow for robust excitatory responses that may trigger sodium- and/or calcium-dependent action potentials. However, once the membrane potential exceeds the chloride reversal potential, GABAA receptors would conduct a strong influx of Cl-, thereby shunting their depolarizing response. Therefore, RNA editing of 03B13 GABAA proteins may provide a developmental window in which GABA is stimulatory, while at the same time maintaining inhibition during excessive excitation. IW.47USING CALCIUM IMAGING TO INVESTIGATE "CEREBRAL FIBRILLATION" AS A MECHANISM OF EPILEPTIFORM ACTIVITY FOLLOWING LOSS OF FUNCTION IN EXCITATORY PATHWAYS Helen Sabolek1,2, W. B. Swiercz1,2, V. I. Dzhala1,2 and K. J. Staley1,2 ( 1Neurology, Massachusetts General Hospital, Charlestown, MA and 2Harvard University, Cambridge, MA) Rationale: Seizures are generally thought to arise from a shift from balanced excitation and inhibition to increased excitation. However, recent genetic studies of human epilepsy demonstrate loss of function mutations in voltage-gated Na2 + channels that presumably disrupt excitatory mechanisms. Similar loss-of-function mutations occur in cardiac Na2 + channels, and reduce the conduction velocity of excitatory transmission. When the rate is sufficiently low, propagating circular waves of excitation return to their origin following recovery from a refractory state, and pathological reentrant activity occurs. Repeated activation of excitatory pathways may occur during seizures in patients with Na2 + channel mutations, and this may account for the local rhythmic EEG activity that precedes focal seizures and the activity-dependent degradation in inhibition that leads to the spread of seizure activity Methods: To test the feasibility of this "cerebral fibrillation" hypothesis of epileptogenesis, we combined electrophysiological recordings with calcium imaging of hippocampal CA3 organotypic slice cultures. We used the roller-tube technique to obtain the thinnest possible cultures, which allows entire networks of CA3 neurons to be imaged simultaneously. Calcium transients associated with epileptiform-related paroxysmal depolarizing shifts were imaged with AM dyes (eg Fluo-4), slow glial calcium signals were removed, and neuronal signals were mapped onto a Cartesian coordinate system. Propagation of epileptiform activity was quantified using 3-D bubble plots: bubble diameter represent the number of active neurons; x,y coordinates represent median x,y positions of all neurons within the network; and the z axis represents time. Propagation pathways were compared under control conditions (100 uM picrotoxin/1 uM CGP 55845), and after loss of function modeled by 1) reducing synchrony of glutamate release by substituting extracellular calcium with strontium and 2) reducing sodium conductance (nanomolar concentrations of TTX). Results: Organotypic CA3 slices disinhibited by picrotoxin/CGP displayed epileptiform activity that was recorded extracellularly. Altering excitatory transmission changed the pattern of burst propagation visible with calcium imaging. Strontium increased the duration of CA3 epileptiform discharges. Conclusions: Reduced excitatory mechanisms (eg synchronous glutamate release) can prolong epileptiform discharges, likely due to 1) a reduced rate of onset of the synaptic depression that typically terminates CA3 bursting and 2) reduced conduction velocity allowing waves of excitation to return to the area of origin after recovery from a refractory state 3) reduced fidelity of axonal conduction (ie more failures in TTX). Our modeling studies have demonstrated spiral waves of excitation consistent with reentrant patterns of neuronal excitation, and here we report calcium-imaging studies testing whether corresponding patterns of activation are observed in synaptically coupled neurons. IW.48SUBUNIT SPECIFIC, ACTIVITY DEPENDENT TRAFFICKING OF GABAA RECEPTORS DURING STATUS EPILEPTICUS Howard Goodkin1, S. Joshi1, J. Brar1 and J. Kapur1 ( 1Dept of Neurology, University of Virginia Medical Center, Charlottesville, VA) Rationale: The reduction in GABA-mediated synaptic inhibition that occurs during the prolonged seizures of status epilepticus (SE) is, in part, the result of a decrease in the surface expression and an increase in the intracellular accumulation of GABAA receptors (GABARs). It is not known whether this rapid modification in the GABAR pool results from the selective or non-selective internalization of GABARs. Methods: In this study, biochemical, electrophysiological, and immunohistochemical methods were used to test for subunit-dependent trafficking of GABARs during SE. Results: The surface expression of GABARs in hippocampal slices acutely obtained from male Sprague Dawley rats in SE (SE-treated) and age matched controls was studied by means of a biotinylation pull down assay. The SE-treated slices were obtained at a point when SE is known to be resistant to treatment with benzodiazepines (BDZ). The surface expression of the 03B22/3 and 03B32 GABAR subunits, which are expressed in synaptic GABARs, was diminished in SE-treated slices. In contrast, the surface expression of the 03B4 subunit, which is expressed in extrasynaptic GABARs, was similar in SE-treated and control slices. Complementary electrophysiological findings were observed: GABAergic synaptic currents (mIPSCs) recorded from SE-treated dentate granule cells (DGCs) were smaller in amplitude and less frequent than control mIPSCs. In contrast, extrasynaptic tonic currents in SE-treated DGCs, measured in response to the competitive GABAR antagonists bicuculline and SR 95531 and to the open channel blocker penicillin, was similar to that recorded from control DGCs. Reduced surface expression of GABARs could result from excessive extracellular GABA or increased excitation/neuronal activity. After hippocampal neuronal cultures (> 2 week in culture) were incubated in 100 03BCM GABA for 30 minutes, the surface immunoreactivity of the 03B32 subunit remain unchanged. In contrast, increased neuronal activity (induced via a high external concentration of KCl) reduced the surface expression of the 03B32 subunit but not that of the 03B4 subunit. Conclusions: These studies demonstrate that there is differential modulation of the surface expression of synaptic and extrasynaptic receptors during SE that occurs in response to the increase in neuronal activity and not ligand binding. As the synaptic receptors are BDZ-sensitive whereas the extrasynaptic receptors are not, subunit-specific, activity-dependent trafficking of GABARs is a potential mechanism to explain the development of BDZ pharmacoresistance during SE. IW.49POST-SEIZURE TREATMENT WITH TOPIRAMATE REVERSES CA1 PYRAMIDAL NEURONAL HYPEREXCITABILITY IN A RAT NEONATAL SEIZURE MODEL Chengwen Zhou1, P. K. Aujla1, S. N. Rakhade1, N. J. Sucher1 and F. E. Jensen1 ( 1Neurology, Children's Hospital, Harvard Medical School, Boston, MA) Rationale: Hypoxic encephalopathy is the most common cause of neonatal seizures, and is associated with long term sequela such as epilepsy. In our rat model of hypoxia-induced neonatal seizures we have observed long-term increase in hippocampal excitability and susceptibility to seizure induced neuronal injury(Jensen et al., J Neurophysiol. 1998;79:73201381; Koh and Jensen, Ann Neurol. 2001;50:366201372). Moreover, we have demonstrated rapid increases in hippocampal network excitability and enhanced spontaneous AMPAR EPSCs in hippocampal CA1 neurons, and that these are in part associated with post-translational modification of AMPAR GluR1 subunits (Sanchez et al., J Neurosci. 2005;25:3442201351; Rakhade et al., AES abstract 2006). We have shown that post-insult treatment with the AMPAR antagonists topiramate and NBQX attenuate the long term increases in seizure susceptibility (Koh et al., Epilepsia. 2004;45:569201375). Here we hypothesized that AMPAR activation may mediate these early functional alterations of the AMPAR currents. Methods: Rat pups were subjected to hypoxia (15 minutes at 420137% O2) on postnatal day (P)10. Immediately following hypoxia, the rats received topiramate (30 mg/kg i.p) or vehicle (PBS) and compared to normoxic rats. After 1 hour, hippocampal slices were prepared from the rats. The whole-cell patch clamp technique was used to examine spontaneous(s) and miniature(m) EPSCs in CA1 pyramidal neurons. The pipette solution contained 110 mM Cs, 10 mM TEA and 5 mM QX-314 to block voltage-sensitive K + and Na + currents. ACSF contained picrotoxin (30 03BCM) and DL-AP5 (100 03BCM) to block GABA and NMDA receptor currents, respectively. TTX (1 03BCM) was used to block sodium channels when miniature EPSCs were studied. Results: Similar to our previous reports, following hypoxic seizures (vehicle treatment), AMPAR-mediated sEPSCs were increased in their amplitude (158 ± 15.9%, n = 15) and frequency (261.6 ± 83.1%, n = 15), compared with those from normoxic rats (100%, n = 10). Consistently, AMPAR-mediated mEPSCs also showed similar increase in amplitude (188 ± 22.3%, n = 5) and frequency (226 ± 47%, n = 5). In contrast, with in vivo injection of NBQX or topiramate following hypoxia exposure, sEPSCs were decreased in both amplitude (topiramate 116.3 ± 10.9%, n = 5) and frequency (topiramate 48.0 ± 7.4%, n = 5), compared to slices from the vehicle-treated hypoxic animals (p < 0.001), and were not significantly different from those in slices from the naïve normoxic rats. Conclusions: Our data suggest that hypoxia-induced seizures induce early increases in AMPAR-mediated sEPSCs. Topiramate administration in vivo blocks the development of these changes. These data suggest that ongoing AMPAR activation is required to effect these rapid alterations in function. In a related study (Rakhade et al., AES abstract 2007), NBQX and topiramate block post-translational changes in the GluR1 subunit, providing a potential mechanism for the functional change. AMPAR antagonist treatment following seizures may prevent early epileptogenic changes in neuronal glutamate receptors in the developing brain. IW.50EFFECT OF METABOTROPIC GLUTAMATE RECEPTOR ACTIVATION ON EVOKED FIELD POTENTIALS IN MALFORMED CORTEX Kimberle Jacobs1, P. Wolfgang1 and A. L. George1 ( 1Anatomy & Neurobiology, Virginia Commonwealth University, Richmond, VA) Rationale: Low-threshold spiking (LTS) but not fast-spiking neocortical interneurons are excited by Group I metabotropic glutamate receptor (mGluR) agonists. Rhythmic spiking in LTS cells during mGluR application synchronizes the activity of surrounding pyramidal neurons. We hypothesize that LTS cells are altered in number or function in the freeze-lesion model of microgyria, creating a hyper-synchronous cortical state in the epileptogenic paramicrogyral area (PMG). Our preliminary whole cell patch clamp data show that application of the mGluR type I agonist, (S)-3,5-Dihydroxyphenylglycine hydrate (DHPG) produces a significantly larger increase in sIPSC frequency in PMG compared to control layer V pyramidal neurons (George & Jacobs 2007 SFN abstract). This suggests that PMG interneurons are either more sensitive to DHPG or are increased in number. Here we have examined the effects of mGluR agents on the cortical network using field potential recordings. Methods: Transcranial freeze lesions over somatosensory cortex were made in rats on postnatal day (P) 1. Coronal slices were taken on P13201319. Field potential recordings were made simultaneously in superficial (SF) and deep layers (DF) in the cortical column above the stimulating electrode within layer VI, 223C1 mm from the sulcus, or in homologous control cortex from unlesioned rats. Fields were evoked at a mid-range intensity, 1/30 sec, and averaged over 2.5 min epochs. Drugs were applied in the aCSF at the following concentrations: 10 03BCM DHPG; 30 03BCM 1-Amino-2,3-dihydro-1H-indene-1,5-dicarboxylic acid (AIDA); 10 03BCM 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Measures of the field potential after 30201340 minutes of application were compared within each slice to predrug conditions and are reported as% change ± SEM. Results: In 6 control slices, DHPG decreased the peak, and increased the area and halfwidth of the SF (221214 ± 8%; 68 ± 49%; 15 ± 12%, t-test, p < 0.05) but decreased the peak and area of the DF (221228 ± 11%; 221227 ± 14%, t-test, p < 0.05). In 6 PMG slices, the SF showed no change in peak or area, and a small increase in halfwidth (11 ± 11%, N.S.; 7 ± 6%, p < 0.05). In striking contrast to control cortex, the PMG DF showed no change in peak but a 112 ± 58% increase in area and a 34 ± 29% increase in halfwidth (p < 0.05). Application of the mGluR5 selective antagonist, MPEP did not affect any of the fields in either PMG or control cortex (12 slices each). The mGluR1 selective antagonist, AIDA produced effects opposite to DHPG in 14 control slices: (SF peak 9 ± 5%, DF peak 6 ± 3%, DF area 33 ± 13%, t-tests, p < 0.05), and 18 PMG slices (DF area 221214 ± 7, t-test, p < 0.05). Conclusions: The deep layers of PMG cortex respond to mGluR agonists similar to the superficial layers of control cortex and significantly different from the control deep layers (t-test, p < 0.05). Thus, there is an increased sensitivity to mGluR type I agonists in deep PMG cortex, and perhaps an decreased sensitivity in superficial PMG cortex. This may arise from an abnormal distribution of interneurons containing mGluR receptors within malformed epileptogenic cortex. (Supported by the Epilepsy Foundation) IW.51GABAA RECEPTOR-MEDIATED EXCITATION IN HUMAN HYPOTHALAMIC HAMARTOMAS: A POTENTIAL EPILEPTOGENIC MECHANISM Jie Wu1, J. DeChon1, F. Xue2, G. Li1, Q. Li1, K. Ellsworth1, D. Kim1, J. Rho1, J. Kerrigan1 and Y. Chang2 ( 1Neurology, Barrow Neurological Institute, Phoenix, AZ and 2Neurobiology, Barrow Neurological Institute, Phoenix, AZ) Rationale: Human hypothalamic hamartomas (HH) are rare, intrinsically epileptogenic, developmental malformations. Recently, we reported that HH lesions contain two predominant types of neurons, small- and large-sized, and that the majority of small HH neurons are GABAergic and show spontaneous, intrinsic pacemaker-like firing activity.1,2 However, the mechanisms involved in epileptogenesis remain unknown. Methods: Here we tested the hypothesis that GABAA receptor-mediated excitation occurs in some HH neurons, contributing to inherent epileptogenesis. Gramicidin-perforated and cell-attached recordings in acutely dissociated HH neurons were applied, and cell and molecular biological approaches were combined. Results: Gramicidin-perforated and cell-attached recordings demonstrated that the GABAA receptor agonist, muscimol, excited (depolarized) large HH neurons, but inhibited (hyperpolarized) small HH neurons in most cases. Based on current-voltage relationships for GABA responses, the estimated intracellular Cl- concentrations were 39.1 ± 2.8 and 19.2 ± 3.7 mM in large and small neurons, respectively (p < 0.01). Single-cell RT-PCR and immunostaining studies revealed lower expression of the chloride-extruding, K + -Cl- cotransporter (KCC2) in large HH neurons. Microtransplantation of HH or control (normal human hypothalamic) tissue membrane fractions into Xenopus oocytes produced cells showing GABA-mediated whole-cell current responses with Cl- equilibrium potentials of 221213.2 ± 1.3 or 221223.9 ± 0.8 mV (p < 0.001), respectively, further associating HH tissues with elevated internal Cl- concentrations. Conclusions: Collectively, these results suggest that GABAA receptor signaling on most large acutely-dissociated HH neurons, and perhaps in HH tissue, is excitatory rather than inhibitory due to low expression of KCC2 and abnormally elevated internal Cl- levels. The immature profile for Cl- distribution, KCC2 expression, and GABAA receptor excitatory activity may play important roles in HH epileptogenicity. References This work was supported by an NIH grant (NS-056104, J.W., Y.C.) and grants from the Neuroscience Foundation and Women's Board of the Barrow Neurological Institute (J.W., J.F.K.). IW.52RAPAMYCIN PREVENTS THE DEVELOPMENT OF EPILEPSY IN A MOUSE MODEL OF TUBEROUS SCLEROSIS COMPLEX Linghui Zeng1, L. Xu1, D. H. Gutmann1 and M. Wong1 ( 1Neurology, Washington University School of Medicine, St. Louis, MO) Rationale: Patients with Tuberous Sclerosis Complex (TSC) often have severe, intractable epilepsy. Mutation of the TSC1 or TSC2 genes in TSC has recently been shown to cause hyperactivation of the mammalian target of rapamycin (mTOR) pathway, leading to a number of downstream cellular and molecular events related to glioneuronal growth and proliferation. mTOR inhibitors, such as rapamycin, have been proposed as novel therapeutic agents to prevent tumor growth in TSC, but the efficacy of such drugs in treating epilepsy has not been tested. In this study, we have evaluated the effect of rapamycin on seizures and other brain abnormalities in a mouse model of TSC with conditional inactivation of the Tsc1 gene primarily in glia (Tsc1GFAPCKO mice). Methods:Tsc1GFAPCKO mice and littermate controls were treated with rapamycin (3 mg/kg/d, i.p., 5 days/week) or vehicle starting at P14. Mice were monitored for seizures by video-EEG and survival from 4 week of age until 11 week or death. In other mice, brains were examined by immunohistochemical staining with GFAP-specific antibody and Nissl staining for glia and neuronal density in hippocampus and cortex. Phospho-S6 expression was also assayed by Western blotting to determine inhibition of the mTOR pathway by rapamycin. Results: Seizures developed in vehicle-treated Tsc1GFAPCKO mice between 420136 week of age and became progressively more frequent through 10 week of age, with most mice dying by 10 week. In contrast, rapamycin-treated mice exhibited no seizures during this time period. Furthermore, rapamycin caused a dramatic increase in survival, with only 1 of 10 mice dying prior to 5 months of age. Correspondingly, rapamycin prevented abnormal hyperactivation of phospho-S6, glial proliferation, neuronal disorganization, and increased brain size in Tsc1GFAPCKO mice. Conclusions: Rapamycin has strong efficacy for preventing seizures and prolonging survival in Tsc1GFAPCKO mice. IW.53MRI CHARACTERISTICS OF STATUS EPILEPTICUS-INDUCED EARLY HIPPOCAMPAL INJURY AND THE ASSOCIATION WITH SUBSEQUENT BRAIN INJURY IN THE LITHIUM-PILOCARPINE RAT MODEL ManKin Choy1, M. F. Lythgoe1, D. L. Thomas2, D. G. Gadian1 and R. C. Scott1 ( 1Radiology and Physics, UCL Institute of Child Health, London, United Kingdom and 2Department of Medical Physics and Bioengineering, University College London, London, United Kingdom) Rationale: Status epilepticus (SE) in humans may be associated with acute hippocampal injury that may progress onto mesial temporal sclerosis (MTS) and temporal lobe epilepsy. However, the mechanisms that underlie these events remain unclear. Clinical and experimental MRI studies have demonstrated that acute and transient MRI changes occur in the hippocampus after SE, but the relationships between these early changes and MTS are not known. Therefore we used MRI to characterise these early hippocampal changes after SE with quantitative T2, apparent diffusion coefficient (ADC) and cerebral blood flow (CBF) in the rat lithium-pilocarpine model. We measured these parameters over a period of 21 days and investigated the relationships between these early changes and brain injury. Methods: Sixteen adult Sprague-Dawley rats were given lithium chloride (3 mEq/kg) intraperitoneally (i.p.) 18 to 20 h prior to either pilocarpine (30 mg/kg) (n = 9) or saline (n = 7). Methylscopolamine i.p. (1 mg/kg) was given to reduce mortality. Diazepam (10 mg/kg) was administered i.p. 90 min after SE onset. Imaging was performed on a 2.35T MRI scanner. Quantitative T2, CBF and ADC were measured before injections and on days 0, 1, 2, 3, 7, 14, 21 after SE. High-resolution anatomical spin-echo images were acquired on day 21 for assessing whole brain injury and these were scored from 1 (no injury) to 5 (severe injury) by 3 independent assessors. The sum of the scores was used to give a final brain injury score. Results: In the pilocarpine animals, hippocampal T2 increases were first detected on day 0, and reached a peak on day 2, before returning to pre-SE levels on day 7. CBF followed a similar pattern. In contrast, ADC decreased on days 2 and 3 only, in a region localised to the CA1 subfield. Furthermore, our analysis indicated that there was a strong relationship between T2 on day 2 and brain injury on day 21 (R2= 0.70, p = 0.005). No significant MR changes were observed in the saline-injected animals. Conclusions: In the SE-injured hippocampus, we observed T2, ADC and CBF changes that occur within the first few days after SE, which is similar to the pattern seen in humans. The T2 and ADC changes are thought to reflect oedema formation and energy failure respectively, and these are consistent with previous reports that have demonstrated that cell death and mitochondrial dysfunction occur during this period. In addition, inflammation has also been reported at this time and may be driving the observed increase in CBF. All of these changes peaked on day 2. Furthermore, the T2 measurements made on day 2 were clearly associated with the degree of damage on day 21. Further studies are necessary to elucidate the mechanisms that underlie this period and also the role that this period may play in epileptogenesis. In conclusion, we have used MRI to characterise early SE-induced hippocampal injury in the lithium-pilocarpine rat model and we have identified an early biomarker of subsequent injury. This project was funded by the Epilepsy Research Foundation UK. IW.54ARE MRI CHANGES PREDICTIVE OF EPILEPTOGENESIS AFTER EXPERIMENTAL PROLONGED FEBRILE SEIZURES? Celine M. Dube1, M. Hamamura2, M. Williams1, A. Ring1, N. Abbott1, O. Nalcioglu2 and T. Z. Baram1 ( 1Anatomy & Neurobiology and Pediatrics, University of California at Irvine, Irvine, CA and 2Tu & Yuen Center for Functional Onco-Imaging, University of California at Irvine, Irvine, CA) Rationale: Whereas most febrile seizures carry a benign outcome, a subpopulation of individuals with prolonged febrile seizures (FS) are at risk for later temporal lobe epilepsy. Signal changes on MRI may provide markers for changes associated with epileptogenesis in such individuals. A prospective multicenter study of children with prolonged FS (S. Shinnar, PI) found MRI changes in 13% of children with prolonged FS (>90 minutes), and 2 of these children developed epilepsy. We previously demonstrated that: an experimental FS in immature rats led to subtle, limbic, 'temporal lobe' epilepsy in 35% of the rats. In a separate study, we found abnormal MRI T2 signal in hippocampus and other limbic structures of a subset of FS-experiencing rats. Therefore we tested the hypothesis that MRI changes were predictive of the development of limbic epilepsy after experimental prolonged FS, i.e. they would distinguish animals that would become epileptic from those who would not. Methods: Immature rats (n = 11) experienced experimental prolonged FS lasting 223C66 minutes on postnatal (P) day 10. They were imaged one month later using a 2D multi-echo spin echo sequence in a 7 Tesla, 15 cm bore magnet (Oxford). Absolute T2 signal intensity maps were generated for control rats (n = 5). These served as comparison for T2 signal intensities on T2 maps of the 'FS' group. Following the MRI, rats were implanted bilaterally with hippocampal bipolar and cortical electrodes, and video EEG monitoring was initiated on P90, in comparison with controls. Seizures were defined as events with both EEG and behavioral criteria: On EEG ictal activity included polyspikes or sharp-waves longer than 6 seconds with amplitude > 200% of background. Interictal events were defined as: 1) polyspikes or sharp-waves discharges lasting less than five seconds; 2) isolated spikes and/or sharp waves; 3) spikes trains not associated with a change of behavior. Results: Rats experiencing experimental FS segregated into two groups: those with minimal changes of MRI T2 values compared to controls (n = 3), and those with significantly abnormal T2 values in hippocampus and often in other limbic regions (n = 8). EEGs were normal in all control rats (over 200 hours) and none of these rats developed spontaneous seizures. Early-life prolonged FS evoked interictal events (spikes trains) in 8 out of the 11 rats (73% of the total, over 2100 hours) with a mean duration of 135 ± 10.2 seconds, n = 73. Preliminary data suggest that 3 out these 8 animals, all with abnormal T2 signal on MRI, now have spontaneous seizures lasting 41 ± 8.4 seconds. Ictal behaviors consist of forelimb clonus and rearing in one rat and of facial automatism and body jerks in the 2 others. Conclusions: These preliminary data suggest that MR T2 signal changes after experimental prolonged FS may provide a biomarker for epileptogenesis. Supported by NS 35439 (TZB) and NS 49618 (TZB/GLH). IW.55LATENT STEM CELLS IN THE HIPPOCAMPUS ARE ACTIVATED BY PROLONGED SEIZURES Robyn H. Wallace1, T. Walker1 and B. Bartlett1 ( 1Queensland Brain Institute, University of Queensland, Brisbane, QLD, Australia) Rationale: Neurogenesis in the adult hippocampus has been shown to be influenced by prolonged seizures. Such hippocampal neurogenesis would be expected to be dependent on a resident or nearby stem cell population. However, studies of the hippocampus have only identified precursors resembling progenitor cells, in that they have limited self-renewal capacity. This raises the questions of whether there is a quiescent latent population of stem cells in the hippocampus and, more importantly, how this population is regulated. Methods: We used 20 mM KCl to depolarize cultures of hippocampal cells, freshly isolated from an adult mouse, and then assessed the number of neurospheres generated 10 days later using a previously described in vitro assay. To investigate neurogenesis following prolonged seizures, we used the pilocarpine mouse model. Results: We found there was a 3-fold increase in the total number of neurospheres generated following high extracellular KCl concentrations. Application of high extracellular KCl led to the emergence of a subpopulation (223C5%) of very large neurospheres, 2502014550 03BCm in diameter. Approximately 36% of the large neurospheres could be expanded for at least 10 passages. After differentiation, small hippocampal neurospheres exclusively gave rise to astrocytes, while those derived from large neurospheres generated a large number of neurons. Thus, neural excitation caused by depolarization activated a latent population with the characteristics of stem cells: self-renewal and multipotentiality. We next investigated whether the latent hippocampal population identified in vitro could also be activated by synaptic excitation in vivo. The increased prolonged synaptic activity found in status epilepticus (SE) results in increased neurogenesis in the dentate gyrus. We found that mice which experienced SE for at least 2 hours and were studied after 2 days had a four fold increase in the number of hippocampal neurospheres compared to control mice, similar to the effect observed in the KCl-stimulated cultures. In contrast, mice that suffered only sporadic seizures showed no such increase, suggesting that only SE stimulates the latent KCl-activatable precursor population. The latent precursor population in mice subjected to SE was almost completely activated, and subsequent KCl induced-depolarization in vitro resulted in no further activation. Prolonged excitation was also a requirement for activation in vitro; at least 24 hours exposure to high levels of KCl was required to produce a significant effect. Thus, it seems that the in vitro and in vivo mechanisms of activation are similar and that both are linked to prolonged neural excitation. Conclusions: We conlcude that the adult hippocampus contains a large number of latent precursors, including a self-renewing stem cell population, which only become activated following hyperpolarization in vitro or after prolonged seizures in vivo. The discovery of this latent population provides the first demonstration of an activatable stem cell in the adult hippocampus. IW.56LOCALIZED INCREASES IN GAMMA FREQUENCY AND FAST OSCILLATIONS RECORDED IN VITRO DURING THE LATENT PERIOD IN THE PILOCARPINE MODEL OF TEMPORAL LOBE EPILEPSY Daniel P. McCloskey1, C. A. Schevon2, S. K. Ng2, J. Cappell2, F. G. Gilliam2, R. G. Emerson2 and H. E. Scharfman1,3 ( 1CNRRR, Helen Hayes Hospital, West Haverstraw, NY; 2Neurology, Columbia Univ. College of Physicians and Surgeons, New York, NY and 3Neurology & Pharmacology, Columbia Univ. College of Physicians and Surgeons, New York, NY) Rationale: Epilepsy may be driven by an increase in the magnitude of gamma activity (20201380 Hz) and fast oscillations (802013500 Hz), which has been reported in the hippocampus and entorhinal cortecx (EC) of patients with temporal lobe epilepsy and animal models of this disorder. However, determining differences in baseline frequency in vivo can be influenced by behavioral and peripheral factors in awake subjects, and pharmacological factors in anesthetized subjects. Therefore, we addressed whether changes in neuronal activity that have been identified during epileptogenesis in vivo can also be detected in the hippocampus and entorhinal cortex in vitro under stable recording conditions. Methods: Male Sprague Dawley rats (38201344 days) were injected with atropine methylnitrate (2.5 mg/kg s.c.) followed by pilocarpine HCl (380 mg/kg s.c.). Diazepam (5 mg/kg i.p.) was given 1 hr after the onset of status epilepticus (status), or 3 hrs after saline or pilocarpine injection if status did not occur (control). Horizontal hippocampal-EC slices (400 03BCm thick) were made using standard procedures, 120133 week after status (n = 21 slices in 7 rats), or 220136 week after saline (n = 7 slices in 3 rats). A 96 tip multielectrode array (Cyberkinetics Neurotechnology Inc.; 1 mm electrode depth; 400 03BCm interelectrode distance) was lowered 223C100 03BCm into slices, maintained in an interface recording chamber. Forty consecutive 50 msec windows were sampled from a 2 kHz sample rate recording, which was made >10 min after the electrode array was lowered, and was absent of any detectable spontaneous field potential. The magnitude of the FFT was calculated for each channel in three different frequency bands (20201380 Hz, 802013200 Hz, 2002013500 Hz), and this value was averaged across 3 neighboring channels selected from regions of interest (CA3, CA1, subiculum, dentate gyrus, EC). A t-test was used with a criterion value of 0.05 to determine differences between slices from control rats and slices from rats that had status. Results: When slices from control animals were compared to slices from rats that had status, there was a significant increase in gamma oscillations in CA3, CA1, the subiculum, and the EC, but not the dentate gyrus. When the frequency band 802013200 Hz was examined, there were significant increases in the FFT magnitude in area CA1 and the dentate gyrus, but not in other regions. There were no significant changes in FFT magnitude in any region in the 2002013500 Hz frequency band. Conclusions: Pilocarpine-induced status epilepticus leads to an increase in gamma oscillations in all of the regions examined except for the dentate gyrus, and an increase in fast oscillations (802013200 Hz) in area CA1 and the dentate gyrus. These changes were detected in the hippocampus and EC at time points that precede the onset of seizures and in vitro epileptiform activity. These results confirm previous reports that changes in network activity begin early in epileptogenesis, and suggest the in vitro model as a useful system to examine the physiological markers of epileptogenesis. IW.57TUBERIN REGULATES CONTRACTILITY IN MIGRATING NEURONS Tristan T. Sands1 and A. R. Kriegstein2 ( 1Pathology, Columbia University, NYC, NY and 2Neurology, UCSF, San Francisco, CA) Rationale: Tuberous Sclerosis is a genetic cause of epilepsy characterized by cortical dysplasia thought to result, in part, from defective neuronal migration during development. However, the pathogenesis of cortical tubers is poorly understood and the role of the TSC gene products, tuberin and hamartin, in neuronal migration remains unknown. Methods: In utero injection and electroporation of short hairpin RNAs into the developing cortices of embryonic rats on the 16th day of gestation; time-lapse microscopy of cultured cortical sections Results: To determine the effects of acute tuberin knockdown on migrating neurons, we used in utero injection and electroporation to introduce RNA interference (RNAi) targeting the TSC2 transcript into embryonic rat cortices. Whereas labeled neurons in the control conditions were observed to enter the developing cortex in large numbers by the fourth day following electroporation, tuberin deficient neurons appeared unable to penetrate the cortical plate. Moreover, neurons in the TSC2 RNAi condition ultimately failed to achieve their proper laminar fate in the adult brain, often coming to reside in the white matter. Thus loss of tuberin function led to a neuronal migration defect and replicated aspects of cortical tubers. To learn why tuberin is required for neuronal migration, we examined the morphology of tuberin-deficient cells en route to the cortex three days following electroporation. Bipolar neurons in the TSC2 RNAi condition were found to have longer leading processes and more spherical cell somas than controls. Additionally, the point at which the leading process joins the cell soma was constricted in these cells, while the proximal leading process was swollen relative to control neurons. Most of these attributes are exaggerations of features manifested by migrating neurons just prior to movement of their cell soma into the leading process, suggesting that loss of tuberin specifically disrupts this aspect of migration, known as "somal translocation." We used time-lapse imaging to examine dynamic features of migration and to confirm that tuberin is required for somal translocation. Acute sections of electroporated brains were cultured and imaged hourly for nine to twelve hours on the third day. While control cells were observed to undergo periodic saltatory displacements of their somas, tuberin deficient cells displayed disrupted somal translocation. Whereas contractions in the control condition served to propel the cell soma forward, contractions in the TSC2 RNAi condition often resulted in reduced or even negative displacement of the cell soma. In addition, the membranes of TSC2 RNAi neurons were frequently stretched by isolated contractions, occasionally resulting in cytoplasmic amputations. Conclusions: We propose that tuberin, likely in conjunction with hamartin, regulates the position and/or timing of contractile events required for somal translocation of migrating cortical neurons. IW.58HEPATOCYTE GROWTH FACTOR (HGF) REDUCES SEIZURES AND BEHAVIORAL DEFICITS IN A MOUSE MODEL OF FRONTAL LOBE EPILEPSY Mihyun Bae1, G. B. Bissonette1,2, T. Suresh4, T. M. Franz1, D. A. Depireux1,2 and E. M. Powell1,3 ( 1Anatomy & Neurobiology, University of Maryland School of Medicine, Baltimore, MD; 2Program in Neuroscience, Graduate Program in Life Sciences, University of Maryland, Baltimore, Baltimore, MD; 3Psychiatry, University of Maryland School of Medicine, Baltimore, MD and 4University of Maryland Baltimore County, Baltimore, MD) Rationale: Imbalances between excitatory and inhibitory signals due to altered GABA transmission causes neurological disorders, including epilepsy, autism, mental retardation. The urokinase plasminogen activator receptor (uPAR), as an activator of hepatocyte growth factor/scatter factor (HGF/SF), mediates multiple biological functions. The uPAR null mouse lacks the GABAergic interneurons in frontal cortical regions suggesting altered frontal lobe function. The uPAR2212/2212 mouse has intact learning and memory function, but displays abnormal emotional and social behavior, indicating the uPAR2212/2212 mouse is rodent model of frontal lobe epilepsy. Loss of uPAR decreases HGF/SF levels, and we hypothesized that genetic supplementation of HGF/SF will recover the deficits observed in the uPAR2212/2212 mouse. Methods: The uPAR2212/2212:Gfap-HGF mouse, generated by crossing a mouse that expresses HGF/SF under the control of the glial fibrillary acidic protein promoter (Gfap) with the uPAR2212/2212 mouse, showed anatomical recovery of cortical GABAergic interneurons, as compared to the uPAR2212/2212 mouse. Previously the uPAR2212/2212 mouse demonstrated dramatically increased susceptibility to pentylenetetrazole (PTZ) seizure induction, spontaneous seizures, and abnormal EEG activity Results: The seizure behavior was reversed in the uPAR2212/2212:Gfap-HGF mice that had increased levels of active HGF/SF in the postnatal brain. We tested mice with inhibitory GABAergic defects on behavior tests such as the resident-intruder assay, elevated plus maze, social interaction paradigm, and the mouse reversal/set-shifting test. The uPAR2212/2212 mouse has a difficulty on these tests, suggesting that loss of uPAR alters social behavior, anxiety, and cognition. We are investigating whether overexpressing HGF/SF recovers the behavioral defects. We are also performing cortical EEG recordings in the awake mouse to probe whether exogenous HGF/SF changes the abnormal cortical neural circuitry in uPAR2212/2212 mouse. Conclusions: This study demonstrates a successful genetic approach to remediate the adverse effects of frontal lobe epilepsy in a rodent model. IW.59KNOCKOUT MOUSE DATA SUPPORT BRD2 AS A GENE FOR JUVENILE MYOCLONIC EPILEPSY David A. Greenberg1,5, E. Shang1, J. Luo6, C. Beseler5, I. Tsai2, D. A. Talmage4, L. W. Role6,3, X. Wang7 and D. J. Wolgemuth2,7 ( 1Division of Statistical Genetics, Columbia University Medical Center, New York, NY; 2Genetics and Devlopment, Columbia University Medical Center, New York, NY; 3Psychiatry, Columbia University Medical Center, New York, NY; 4Dept. of Pediatrics; Institute of Human Nutrition, Columbia University Medical Center, New York, NY; 5Genetic Epidemiology, NY State Psychiatric Institute, New York, NY; 6Center for Neurobiology and Behavior, Columbia University Medical Center, New York, NY and 7OB/GYN, Columbia University Medical Center, New York, NY) Rationale: The BRD2 gene has been identified as the EJM1 gene for JME, located at chromosome 6p21. The locus was originally located and replicated by linkage analysis [120134], and identified and replicated by association studies [520137], in some European populations. While the statistical genetic evidence for BRD2's involvement is strong, we now present biological evidence that reveal its profound effect on brain development and structure. Mutational evidence in the mouse model supports the population human genetic results showing polymorphisms of BRD2 that influence seizure susceptibility. Methods: We examined brain development, anatomy, brain structure, and seizure susceptibility in a Brd2-null (Brd22212/2212) mouse and the null/wild-type heterozygote (Brd2 ±). Results: The knock out of the mouse Brd2 gene is an embryonic lethal: At embryonic day 9.5, the homozygous Brd2 2212/2212 embryos are noticeably smaller and exhibit profound neural tube abnormalities (fig. 1). In situ hybridization shows the highest expression levels of Brd2 are in the developing nervous system. Although heterozygous Brd2 ± mice appear grossly normal, closer inspection reveals selective anatomical and behaviorial abnormalities. Brd2 ± females appear to more susceptible to pentylenetetrazole-induced (50 mg/kg; IP) seizures than same age wild-type mice. Anatomically, the Brd2 ± mice have a notably lower density of parvalbuimin-positive GABAergic interneurons in prefrontal cortical regions (anterior cingulate, prelimbic and infralimbic cortex) (fig. 2) without alteration in the density of calbindin-positive GABAergic interneurons in the same brain regions. Conclusions: The data from the null mutation show that mouse Brd2 has a profound influence on brain structure, supporting a role for human BRD2's influence on brain structure. The observations from the heterozygous Brd2 ± mice further suggest that a decrease in BRD2 protein levels may be the mechanism underlying the dominant inheritance of JME, indicated by the linkage data. That is, small decreases in BRD2 protein levels during development could lead to subtle changes in frontal lobe structure that predispose to JME and brain hyperexcitability when other seizure-related genes are present. Greenberg, DA et al. Am J Med Genet 1988; 31:1852013192. Weissbecker, KA et al. Am J Med Genet 1991; 38:32201336. Durner, M et al. Ann Neurol 2001; 49:3282013335. Greenberg, DA et al. American Journal of Human Genetics 2000; 66:5082013516. Pal, DK, et al. Am J Hum Genet 2003; 73:2612013270 Epub 2003 Jun 2025. Lorenz, S. Neurosci Lett 2006; 400:1352013139. Cavalleri, GL et al. Epilepsia 2007; 48:7062013712. Fig 1.  Sections of 9.5 day old embryos comparing wild type (left) with Brd22212/2212 knockout mice. Note severely abnormal neural tube development in knockout. Fig 2.  Density comparison of parvalbumin-stained neurons in Brd2 ± mice vs wild type in four regions of frontal lobe. IW.60EARLY DEVELOPMENTAL EXPRESSION OF A FAMILIAL GABAA EPILEPSY GENE INCREASES ADULT SEIZURE SUSCEPTIBILITY Christopher A. Reid1, C. Chiu1, H. O. Tan1, P. Davies1, s. Tan1, M. Jones2 and s. Petrou1 ( 1Ion channels and disease group, Howard Florey Institute, Melbourne, VIC, Australia and 2Dept of Physiology, University of Wisconsin, Madison, WI) Rationale: A GABAA 03B32 gene mutation (R43Q) has been linked to inherited forms of epilepsy in man. Like patients with the mutation, gene targeted knock-in mice display increased seizure susceptibility. Epilepsy develops in this model presumably as a consequence of GABAA receptor dysfunction. GABAA receptors can exert their action "directly" by altering the moment-to-moment excitability of the brain. Alternatively, GABAA receptors play an integral role in the developing brain, dysfunction of which may lead to long-term changes in neuronal network stability. Discerning between direct and development effects is vital to our understanding of epileptogenesis. Methods: We developed a conditional knock-in mouse model harbouring the R43Q mutation to address this question. Comparison of allelic expression of mice containing a loxP flanked Neomycin cassette with a Cre-recombinase excised line revealed a relative reduction in expression of the 03B32(Q) neo allele by 90%, forming the basis of our switch. The spatio-temporal resolution of the unsilencing excision event was explored using the Z/EG Cre reporter strain crossed with a forebrain-specific tTA regulated Cre-recombinase line. Results: In the absence of doxycycline, gene activation was confirmed in the hippocampus and cortex within useful timeframes. Increased sensitivity to the proconvulsant, pentylenetetrazol (PTZ) is an established phenotype of mice harbouring the R43Q mutation. Using the conditional approach we 'silenced' the mutant allele from inception to post natal day 21. Interestingly, PTZ susceptibility was significantly reduced in these mice. Conclusions: Expression of mutant 03B32 GABAA receptors in early development increase seizure susceptibility in adulthood, presumably as a consequence of altered brain development. (This study was in part supported by a Program grant from the Australian National Health and Medical Research Council (NH&MRC) IW.61ASSOCIATION BETWEEN VARIATION IN THE GABRA4 GENE AND HUMAN TEMPORAL LOBE EPILEPSY Russell J. Buono1,2, M. R. Sperling2, D. J. Dlugos3, M. D. Privitera4, J. A. French5, W. Lo6, S. C. Schachter7, P. Cossette8, H. Zhao9, J. Y. Lee9, N. J. Collins4, T. Scattergood5, F. W. Lohoff10, W. H. Berrettini10 and T. N. Ferraro10 ( 1Research Service, Department of Veteran Affairs, Coatesville, PA; 2Neurology, Thomas Jefferson University, Philadelphia, PA; 3Neurology, The Children's Hospital of Philadelphia, Philadelphia, PA; 4Neurology, The University of Cincinnati, Cincinnati, OH; 5Neurology, The University of Pennsylvania, Philadelphia, PA; 6Neurology, Columbus Children's Research Institute, Columbus, OH; 7Neurology, Beth Israel Deconess Medical Center, Boston, MA; 8Neurology, CHU Hospital Notre Dame, Montreal, QC, Canada; 9School of Public Health, Yale University, New Haven, CT and 10Psychiatry, The University of Pennsylvania, Philadelphia, PA) Rationale: Quantitative trait loci mapping (QTL) in DBA/2 (relatively seizure sensitive) and C57BL6 (relatively seizure resistant) strains of mice identified several chromosomal regions linked to seizure phenotypes. One of these is on chromosome 5, a region which is homologous to a segment of human chromosome 4p12, and that contains a cluster of four GABA receptor subunit genes (GABRG1, GABRA2, GABRA4, and GABRB1). We have begun to study variation in these genes systematically using DNA samples from a cohort of human epilepsy patients and controls collected in the US and Canada. We report preliminary findings on the GABRA4 gene as prior literature suggests a role for this receptor in Temporal Lobe Epilepsy (TLE). Methods: Single nucleotide polymorphisms (SNPs) rs1512132 and rs2229940 (http://www.ncbi.nlm.nih.gov/SNP/) were genotyped using TaqMan assays in patients with common forms of epilepsy (n = 572), including both idiopathic generalized (IGE, n = 309) and focal (TLE, n = 263) and in unrelated healthy control individuals (n = 203). All subjects were of European ancestry. Hardy-Weinberg equilibrium (HWE) was tested and alleles were studied for disease associations using chi square analysis. Linkage disequilibrium (LD) between SNPs was tested using likelihood ratio analysis. Haplotype estimation and association analysis were performed using HAPSTAT and Plink software. Results: Markers were in HWE for all controls and IGE patients; however, the TLE group did not exhibit HWE: rs1512132 p = 0.007, rs2229940 p = 0.01. The two markers do not exhibit strong LD in IGE patients or controls with r2 values of 0.39 and 0.54, respectively. In TLE patients, however, the LD is strong (r2 = 0.85). In addition, minor allele frequencies (MAF) for controls and IGE patients are in agreement with data published by Applied Biosystems and the International HAPMAP project, but MAF for the TLE group is significantly different for rs1512132 (0.36 compared to 0.47) leading to a positive association with single marker analysis p = 0.0007. Haplotype analysis shows a less significant result (p = 0.02, dominant model) as the markers are separated by 40KB in the locus. Conclusions: Results suggest that variation at SNP rs1512132 may be associated with susceptibility to TLE. Lack of HWE in the TLE group could have occured because of genotyping errors, non random mating, genetic drift or other geographical considerations. However, this result can also indicate the presence of a skewed allelic distribution consistent with a mutation or susceptibility allele. Preliminary examination of data files does not indicate the presence of genotyping errors and it is improbable that non random mating occured in our cohort. Since the MAF for controls and IGE patients are in agreement with published figures, but the MAF for rs1512132 is significantly different in the TLE group, we hypothesize that variation in this SNP is in fact associated with susceptibility to TLE. Further studies at this locus are required to confirm this association. Support: R01NS40396 to RJB; UPenn Ctr for Neuro and Behav; Dept Veteran Affairs; Neurosci Inst, U Cincinnati.