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.

Benzodiazepines remain widely used for the treatment of anxiety disorders despite prominent, often limiting side effects including sedation, muscle relaxation, and ataxia. A compound producing a robust anxiolytic action comparable to benzodiazepines, but lacking these limiting side effects at therapeutic doses (an anxioselective agent), would represent an important advance in the treatment of generalized anxiety disorder, and perhaps other anxiety disorders. Here we report that the pyrazolo[1,5-a]-pyrimidine, ocinaplon, exhibits an anxioselective profile in both preclinical procedures and in patients with generalized anxiety disorder, the most common of the anxiety disorders. In rats, ocinaplon produces significant muscle relaxation, ataxia, and sedation only at doses >25-fold higher than the minimum effective dose (3.1 mg͞kg) in the Vogel ''conflict'' test. This anticonflict effect is blocked by flumazenil (Ro 15-1788), indicating that like benzodiazepines, ocinaplon produces an anxiolytic action through allosteric modulation of GABA A receptors. Nonetheless, in eight recombinant GABAA receptor isoforms expressed in Xenopus oocytes, the potency and efficacy of ocinaplon to potentiate GABA responses varied with subunit composition not only in an absolute sense, but also relative to the prototypical benzodiazepine, diazepam. In a double blind, placebo controlled clinical trial, a 2-week regimen of ocinaplon (total daily dose of 180 -240 mg) produced statistically significant reductions in the Hamilton rating scale for anxiety scores. In this study, the incidence of benzodiazepine-like side effects (e.g., sedation, dizziness) in ocinaplon-treated patients did not differ from placebo. These findings indicate that ocinaplon represents a unique approach both for the treatment and understanding of anxiety disorders.generalized anxiety disorder ͉ benzodiazepines

The regulated expression of type A ␥-aminobutyric acid receptor (GABAAR) subunit genes is postulated to play a role in neuronal maturation, synaptogenesis, and predisposition to neurological disease. Increases in GABA levels and changes in GABA AR subunit gene expression, including decreased ␤1 mRNA levels, have been observed in animal models of epilepsy. Persistent exposure to GABA down-regulates GABAAR number in primary cultures of neocortical neurons, but the regulatory mechanisms remain unknown. Here, we report the identification of a TATA-less minimal promoter of 296 bp for the human GABAAR ␤1 subunit gene that is neuron specific and autologously down-regulated by GABA. ␤1 promoter activity, mRNA levels, and subunit protein are decreased by persistent GABAAR activation. The core promoter, 270 bp, contains an initiator element (Inr) at the major transcriptional start site. Three concatenated copies of the 10-bp Inr and its immediate 3 flanking sequence produce full neural specific activity that is down-regulated by GABA in transiently transfected neocortical neurons. Taking these results together with those of DNase I footprinting, electrophoretic mobility shift analysis, and 2-bp mutagenesis, we conclude that GABA-induced down-regulation of ␤1 subunit mRNAs involves the differential binding of a sequencespecific basal transcription factor(s) to the Inr. The results support a transcriptional mechanism for the down-regulation of ␤1 subunit GABA AR gene expression and raises the possibility that altered levels of sequence-specific basal transcription factors may contribute to neurological disorders such as epilepsy.transcription ͉ neural specific ͉ epilepsy G ABA (␥-aminobutyric acid) is the major transmitter at inhibitory chemical synapses; yet little is known about the mechanisms underlying the genetic regulation of type A GABA receptor (GABA A R) number and composition. GABA A R number and subunit mRNA levels are down-regulated in primary brain cultures as a response to persistent activation of the GABA A R (1-3). Moreover, down-regulation of subunit mRNAs precedes down-regulation of receptor number, indicating that GABA-induced GABA A R down-regulation is a consequence of decreased mRNA levels (4).Whereas the effects of GABA are inhibitory in the adult brain, they are excitatory during embryogenesis and early postnatal life (5). A variety of observations indicate that GABA A R subunit gene expression is regulated by receptor activation (6, 7) regardless of age. GABA A Rs may play a critical role in the development of the central nervous system and so determine its future susceptibility to seizures (8). Extracellular GABA is increased during paroxysmal hippocampal activity in epileptic patients (9), and GABA uptake is inhibited in genetic absence epilepsy rats (10). Both of these changes may lead to increases in the activity of cell surface GABA A Rs and subsequent changes in GABA A R function through down-regulation of subunit specific gene expression. A decrease in the levels of ␣1 and ␤1 GABA A R subunit...

Expression of metabotropic GABA B receptors is essential for slow inhibitory synaptic transmission in the CNS, and disruption of GABA B receptor-mediated responses has been associated with several disorders, including neuropathic pain and epilepsy. The location of GABA B receptors in neurons determines their specific role in synaptic transmission, and it is believed that sorting of subunit isoforms, GABA B R1a and GABA B R1b, to presynaptic or postsynaptic membranes helps to determine this role. GABA B R1a and GABA B R1b are thought to arise by alternative splicing of heteronuclear RNA. We now demonstrate that alternative promoters, rather than alternative splicing, produce GABA B R1a and GABA B R1b isoforms. Our data further show that subunit gene expression in hippocampal neurons is mediated by the cAMP response element-binding protein (CREB) by binding to unique cAMP response elements in the alternative promoter regions. Double-stranded oligonucleotide decoys selectively alter levels of endogenous GABA B R1a and GABA B R1b in primary hippocampal neurons, and CREB knock-out mice show changes in levels of GABA B R1a and GABA B R1b transcripts, consistent with decoy competition experiments. These results demonstrate a critical role of CREB in transcriptional mechanisms that control GABA B R1 subunit levels in vivo. In addition, the CREB-related factor activating transcription factor-4 (ATF4) has been shown to interact directly with GABA B R1 in neurons, and we show that ATF4 differentially regulates GABA B R1a and GABA B R1b promoter activity. These results, together with our finding that the depolarization-sensitive upstream stimulatory factor (USF) binds to a composite CREB/ATF4/USF regulatory element only in the absence of CREB binding, indicate that selective control of alternative GABA B R1 promoters by CREB, ATF4, and USF may dynamically regulate expression of their gene products in the nervous system.

Tonic inhibition mediated by extrasynaptic GABA A receptors (GABARs) sensing ambient levels of GABA can profoundly alter the membrane input resistance to affect cellular excitability. Therefore, regulation of tonic inhibition is an attractive mechanism to control the levels of cortical firing. In cortical pyramidal cells, tonic inhibition is regulated by age and several neurotransmitters and is affected by stroke and epilepsy. However, the possible role of sensory experience has not been examined. Here, we report that a brief 2-day exposure to dark reduces by 1/3 the inhibitory tonic conductance recorded in layer II/III pyramidal cells of the mouse juvenile (postnatal day 12-27) visual cortex. In these cells, tonic inhibition is carried primarily by GABARs containing the ␦ subunit. Consistently, the dark exposure reduction in conductance was associated with a reduction in ␦ subunit levels, which were not affected in control frontal cortex. We propose that a deprivation-induced reduction in tonic inhibition might serve a homeostatic function by increasing the firing levels of cells in deprived cortical circuits.

Abstract:The ability of nerve cells to regulate the expression of specific neurotransmitter receptors is of central importance to nervous system function, but little is known about the DNA elements that mediate neuron specific gene expression. The type A ␥-aminobutyric acid (GABA A ) receptor ␣6-subunit gene, which is expressed exclusively in cerebellar granule cells, presents a unique opportunity to study the cis elements involved in restricting gene expression to a distinct neuronal population. In an effort to identify the regulatory elements that govern cerebellar granule cell-specific gene expression, the proximal 5Ј flanking regions for the human, rat, and mouse ␣6 genes were cloned and sequenced, and a major transcriptional initiation site was identified in the rodent genes. Functional analysis of rat ␣6 gene -reporter constructs in primary neuronal cultures reveals that a 155-bp TATA-less promoter region (Ϫ130 to ϩ25 bp) constitutes a minimal promoter that can drive cerebellar granule cell-specific expression. Internal deletion and decoy competition studies demonstrate that the minimal promoter contains a 60-bp region (Ϫ130 to Ϫ70 bp) that is critical for enhanced promoter activity in cerebellar granule cells. Activity of the compromised promoter containing the deletion cannot be rescued by placing the 60-bp region downstream of the reporter gene, demonstrating that it is not a classical enhancer but rather a positionally dependent regulator. An additional cerebellar-specific activating sequence is located between Ϫ324 and Ϫ130 bp, and a downstream negative regulatory region (ϩ158 to ϩ294) has been shown to be active in fibroblasts but inactive in cerebellar granule cells. Taken together, the results suggest a possible mechanism for the control of cerebellar granule cell-specific expression of the GABA A receptor ␣6 subunit gene.

We demonstrate an accurate, quantitative, and label-free optical technology for high-throughput studies of receptor-ligand interactions, and apply it to TATA binding protein (TBP) interactions with oligonucleotides. We present a simple method to prepare single-stranded and double-stranded DNA microarrays with comparable surface density, ensuring an accurate comparison of TBP activity with both types of DNA. In particular, we find that TBP binds tightly to single-stranded DNA, especially to stretches of polythymine (poly-T), as well as to the traditional TATA box. We further investigate the correlation of TBP activity with various lengths of DNA and find that the number of TBPs bound to DNA increases >7-fold as the oligomer length increases from 9 to 40. Finally, we perform a full human genome analysis and discover that 35.5% of human promoters have poly-T stretches. In summary, we report, for the first time to our knowledge, the activity of TBP with poly-T stretches by presenting an elegant stepwise analysis of multiple techniques: discovery by a novel quantitative detection of microarrays, confirmation by a traditional gel electrophoresis, and a full genome prediction with computational analyses.