The γ-aminobutyric acid (GABA) type A receptor (GABA A R) is the major inhibitory neurotransmitter receptor in the brain. Its multiple subunits show regional, developmental, and disease-related plasticity of expression; however, the regulatory networks controlling GABA A R subunit expression remain poorly understood. We report that the seizure-induced decrease in GABA A R α1 subunit expression associated with epilepsy is mediated by the Janus kinase (JAK)/ signal transducer and activator of transcription (STAT) pathway regulated by brain-derived neurotrophic factor (BDNF). BDNF-and seizure-dependent phosphorylation of STAT3 cause the adenosine 3′,5′-monophosphate (cAMP) response element-binding protein (CREB) family member ICER (inducible cAMP early repressor) to bind with phosphorylated CREB at the Gabra1:CRE site. JAK/STAT pathway inhibition prevents the seizure-induced decrease in GABA A R α1 abundance in vivo and, given that BDNF is known to increase the abundance of GABA A R α4 in a JAK/STATindependent manner, indicates that BDNF acts through at least two distinct pathways to influence GABA A R-dependent synaptic inhibition.

Differential expression of GABA A receptor (GABR) subunits has been demonstrated in hippocampus from patients and animals with temporal lobe epilepsy (TLE), but whether these changes are important for epileptogenesis remains unknown. Previous studies in the adult rat pilocarpine model of TLE found reduced expression of GABR ␣1 subunits and increased expression of ␣4 subunits in dentate gyrus (DG) of epileptic rats compared with controls. To investigate whether this altered subunit expression is a critical determinant of spontaneous seizure development, we used adeno-associated virus type 2 containing the ␣4 subunit gene (GABRA4) promoter to drive transgene expression in DG after status epilepticus (SE). This novel use of a condition-dependent promoter upregulated after SE successfully increased expression of GABR ␣1 subunit mRNA and protein in DG at 1-2 weeks after SE. Enhanced ␣1 expression in DG resulted in a threefold increase in mean seizure-free time after SE and a 60% decrease in the number of rats developing epilepsy (recurrent spontaneous seizures) in the first 4 weeks after SE. These findings provide the first direct evidence that altering GABR subunit expression can affect the development of epilepsy and suggest that ␣1 subunit levels are important determinants of inhibitory function in hippocampus.

Fig. 1. Protein-protein interactions between gp32 and gp59 on fDNA. (A) The fluorescence from individual molecules of fDNA with the proteins bound in the order as indicated at the side of each row. The gp32 protein is labeled with A488 (gp32 D ) and the gp59 protein is labeled with A555 (gp59 A ). The filter sets are described in Experimental Methods: F1 is for A488 emission, F2 for FRET between A488 and A555, and F3 for A555 emission. (B) Ensemble FRET studies of Oregon-green-488-maleimide-labeled gp59 titrated into a solution of 400 nM CPM-labeled gp32 and 100 nM fDNA. The fluorescence spectra of 400 nM CPM-gp32 alone (black line), the endpoint of the titration at 1 M Oregon-green-488-maleimide-gp59 (dark gray line), and several intermediate spectra (light gray lines) are shown. (C) Analysis of the donor quenching and acceptor sensitization plotted against the gp59 concentration determines the stoichiometry among gp32, gp59, and fDNA to be 1:1:1 with a calculated binding constant of Ϸ40 nM.

Long-term GABA A receptor alterations occur in hippocampal dentate granule neurons of rats that develop epilepsy after status epilepticus in adulthood. Hippocampal GABA A receptor expression undergoes marked reorganization during the postnatal period, however, and the effects of neonatal status epilepticus on subsequent GABA A receptor development are unknown. In the current study, we utilize single cell electrophysiology and antisense mRNA amplification to determine the effect of status-epilepticus induced by lithium-pilocarpine in postnatal day 10 rat pups on GABA A receptor subunit expression and function in hippocampal dentate granule neurons. We find that rats subjected to lithium-pilocarpine-induced status epilepticus at postnatal day 10 show long-term GABA A receptor changes including a two-fold increase in α1 subunit expression (compared with lithiuminjected controls) and enhanced type I benzodiazepine augmentation that are opposite of those seen after status epilepticus in adulthood and may serve to enhance dentate gyrus inhibition. Further, unlike adult rats, postnatal day 10 rats subjected to status epilepticus do not become epileptic. These findings suggest age-dependent differences in the effects of status epilepticus on hippocampal GABA A receptors that could contribute to the selective resistance of the immature brain to epileptogenesis. Keywordsepilepsy; seizure; developing brain; dentate gyrus; benzodiazepine; mRNA amplification GABA A receptors (GABARs) are heteromeric protein complexes that mediate most fast synaptic inhibition in fore-brain. Many distinct subunit subtypes exist, and their distribution varies in a regional and cell type-specific manner Sperk et al., 1997;Barnard et al., 1998). GABARs can be assembled in different subunit combinations to produce a variety of different receptor compositions, pharmacological profiles, and intrinsic receptor characteristics (Pritchett et al., 1989;Sieghart, 1995;Barnard et al., 1998 refractory temporal lobe epilepsy demonstrate marked alterations in GABAR properties in hippocampal dentate granule neurons (DGNs; Buhl et al., 1996;Gibbs et al., 1997; BrooksKayal et al., 1998 BrooksKayal et al., , 1999. GABAR subunit expression in DGNs vary during early postnatal development Fritschy et al., 1994;Brooks-Kayal et al., 2001), however, and the effects of neonatal SE on GABARs is not known. Here we examine the effect of SE in postnatal day 10 (P10) rat pups on subsequent GABAR development in DGN and find increased α1 subunit expression and type I benzodiazepine augmentation when the rats reach adulthood. These findings are opposite of those seen after SE in adulthood and could contribute to the selective resistance of the immature brain to epileptogenesis. EXPERIMENTAL PROCEDURES Lithium-pilocarpine injectionsAll studies were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with the approval of The Children's Hospital of Philadelphia Animal Care and Use Committee. Maximum care ...

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 GABA A 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.

Objective-Neonatal seizures occur frequently, are often refractory to anticonvulsants, and are associated with considerable morbidity and mortality. Genetic and electrophysiological evidence indicates that KCNQ voltage-gated potassium channels are critical regulators of neonatal brain excitability. This study tests the hypothesis that selective openers of KCNQ channels may be effective for treatment of neonatal seizures.Methods-We induced seizures in postnatal day 10 rats with either kainic acid or flurothyl. We measured seizure activity using quantified behavioral rating and electrocorticography. We compared the efficacy of flupirtine, a selective KCNQ channel opener, with phenobarbital and diazepam, two drugs in current use for neonatal seizures.Results-Unlike phenobarbital or diazepam, flupirtine prevented animals from developing status epilepticus (SE) when administered prior to kainate. In the flurothyl model, phenobarbital and diazepam increased latency to seizure onset, but flupirtine completely prevented seizures throughout the experiment. Flupirtine was also effective in arresting electrographic and behavioral seizures when administered after animals had developed continuous kainate-induced SE. Flupirtine caused doserelated sedation and suppressed EEG activity, but did not result in respiratory suppression or result in any mortality.Interpretation-Flupirtine appears more effective than either of two commonly used anti-epileptic drugs, phenobarbital and diazepam, in preventing and suppressing seizures in both the kainic acid and flurothyl models of symptomatic neonatal seizures. KCNQ channel openers merit further study as potential treatments for seizures in infants and children.Epileptic seizures occur commonly in the first days after birth. 1 Such neonatal seizures are usually symptomatic, arising as a result of developmental abnormalities, in utero injuries, perinatal hypoxia-ischemia, infection, and other causes. Patients experiencing neonatal seizures are at substantial risk of mortality and long-term morbidity, including static encephalopathy, cerebral palsy, and chronic epilepsy. 2, 3 The first-line drugs given to neonates, phenobarbital and phenytoin, are effective in less than 50% of cases 4 . Moreover, phenobarbital, benzodiazepines and phenytoin have been shown to cause widespread neuronal apoptosis when given to young rodents, raising concerns about administration of these drugs in human infants. Genetic, physiological, and pharmacological evidence calls attention to KCNQ potassium channels (also termed Kv7 and M-channels) as potential molecular targets for treatment of neonatal seizures. 9 Loss-of-function mutations in the KCNQ2 and KCNQ3 genes cause benign familial neonatal seizures (BFNS), an uncommon but highly penetrant, dominantly-inherited syndrome characterized by seizures that recur frequently over the first weeks of life. 10-12 Experimental inhibition of KCNQ channels in rodents, by pharmacological and genetic methods, dramatically promotes neuronal and network hyperexcitabi...

Altered excitatory amino acid (EAA) neurotransmission, mediated primarily by glutamate, is a major cause of the imbalance of excitation and inhibition which characterizes both early development and epileptogenesis. Glutamate's actions are mediated by three classes of receptors: NMDA, non-NMDA (AMPA and kainate), and metabotropic. Several features of normal EAA development contribute to hyperexcitability in the immature brain, making it more prone to development of seizures. These features include increased density of NMDA receptors, differences in NMDA receptor subunit composition and activation kinetics, which result in reduced voltage-dependent Mg(2+) blockade and longer receptor openings in early development. Also, the unique subunit composition of AMPA receptors present at synapses during early development results in increased Ca(2+) influx. These and other differences in EAA signaling, in combination with developmental alterations in inhibitory neurotransmission, contribute to the increased seizure susceptibility seen in young animals and children. In turn, seizures themselves may alter EAA neurotransmission in an age-dependent manner. Age related changes in excitatory neurotransmission may, therefore, lead to differences in basic mechanisms of epileptogenesis between the immature and mature brain, and may also alter the activity and efficacy of antiepileptic drugs in the pediatric age group.

Together, these data suggest that ES may influence immediate secretion of 3alpha,5alpha-THP and corticosterone and have pervasive effects in adulthood on the biosynthesis and/or metabolism of progestins in the hippocampus.