The gamma-aminobutyric acid type A (GABAA) receptor represents an elementary switching mechanism integral to the functioning of the central nervous system and a locus for the action of many mood- and emotion-altering agents such as benzodiazepines, barbiturates, steroids, and alcohol. Anxiety, sleep disorders, and convulsive disorders have been effectively treated with therapeutic agents that enhance the action of GABA at the GABAA receptor or increase the concentration of GABA in nervous tissue. The GABAA receptor is a multimeric membrane-spanning ligand-gated ion channel that admits chloride upon binding of the neurotransmitter GABA and is modulated by many endogenous and therapeutically important agents. Since GABA is the major inhibitory neurotransmitter in the CNS, modulation of its response has profound implications for brain functioning. The GABAA receptor is virtually the only site of action for the centrally acting benzodiazepines, the most widely prescribed of the anti-anxiety medications. Increasing evidence points to an important role for GABA in epilepsy and various neuropsychiatric disorders. Recent advances in molecular biology and complementary information derived from pharmacology, biochemistry, electrophysiology, anatomy and cell biology, and behavior have led to a phenomenal growth in our understanding of the structure, function, regulation, and evolution of the GABAA receptor. Benzodiazepines, barbiturates, steroids, polyvalent cations, and ethanol act as positive or negative modulators of receptor function. The description of a receptor gene superfamily comprising the subunits of the GABAA, nicotinic acetylcholine, and glycine receptors has led to a new way of thinking about gene expression and receptor assembly in the nervous system. Seventeen genetically distinct subunit subtypes (alpha 1-alpha 6, beta 1-beta 4, gamma 1-gamma 4, delta, p1-p2) and alternatively spliced variants contribute to the molecular architecture of the GABAA receptor. Mysteriously, certain preferred combinations of subunits, most notably the alpha 1 beta 2 gamma 2 arrangement, are widely codistributed, while the expression of other subunits, such as beta 1 or alpha 6, is severely restricted to specific neurons in the hippocampal formation or cerebellar cortex. Nervous tissue has the capacity to exert control over receptor number, allosteric uncoupling, subunit mRNA levels, and posttranslational modifications through cellular signal transduction mechanisms under active investigation. The genomic organization of the GABAA receptor genes suggests that the present abundance of subtypes arose during evolution through the duplication and translocations of a primordial alpha-beta-gamma gene cluster. This review describes these varied aspects of GABAA receptor research with special emphasis on contemporary cellular and molecular discoveries.

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1 The neurosteroid pregnenolone sulphate (PS) potentiates N-methyl-D-aspartate (NMDA) receptor mediated responses in various neuronal preparations. The NR1 subunit can combine with NR2A, NR2B, NR2C, or NR2D subunits to form functional receptors. Dierential NR2 subunit expression in brain and during development raises the question of how the NR2 subunit in¯uences NMDA receptor modulation by neuroactive steroids. 2 We examined the eects of PS on the four diheteromeric NMDA receptor subtypes generated by co-expressing the NR1 100 subunit with each of the four NR2 subunits in Xenopus oocytes. Whereas PS potentiated NMDA-, glutamate-, and glycine-induced currents of NR1/NR2A and NR1/NR2B receptors, it was inhibitory at NR1/NR2C and NR1/NR2D receptors. 3 In contrast, pregnanolone sulphate (3a5bS), a negative modulator of the NMDA receptor that acts at a distinct site from PS, inhibited all four subtypes, but was approximately 4 fold more potent at NR1/NR2C and NR1/NR2D than at NR1/NR2A and NR1/NR2B receptors. 4 These ®ndings demonstrate that residues on the NR2 subunit are key determinants of modulation by PS and 3a5bS. The modulatory eects of PS, but not 3a5bS, on dose-response curves for NMDA, glutamate, and glycine are consistent with a two-state model in which PS either stabilizes or destabilizes the active state of the receptor, depending upon which NR2 subunit is present. 5 The selectivity of sulphated steroid modulators for NMDA receptors of speci®c subunit composition is consistent with a neuromodulatory role for endogenous sulphated steroids. The results indicate that it may be possible to develop therapeutic agents that target steroid modulatory sites of speci®c NMDA receptor subtypes. British Journal of Pharmacology (2002) 135, 901 ± 909

Steroid sulfation occurs in nervous tissue and endogenous sulfated steroids can act as positive or negative modulators of N-methyl-D-aspartate (NMDA) receptor function. In the current study, structure-activity relationships for sulfated steroids were examined in voltage-clamped chick spinal cord and rat hippocampal neurons in culture and in Xenopus laevis oocytes expressing NR1(100) and NR2A subunits. The ability of pregnenolone sulfate (a positive modulator) and epipregnanolone sulfate (a negative modulator) to compete with each another, as well as with other known classes of NMDA receptor modulators, was examined. The results show that steroid positive and negative modulators act at specific, extracellularly directed sites that are distinct from one another and from the spermine, redox, glycine, Mg2+, MK-801, and arachidonic acid sites. Sulfated steroids are effective as modulators of ongoing glutamate-mediated synaptic transmission, which is consistent with their possible role as endogenous neuromodulators in the CNS.

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N-methyl-D-aspartate (NMDA) receptor function is modulated by several endogenous molecules, including zinc, polyamines, protons, and sulfated neurosteroids. Zinc, polyamines, and phenylethanolamines exert their respective modulatory effects by exacerbating or relieving tonic proton inhibition. Here, we report that pregnenolone sulfate (PS) uses a unique mechanism for enhancement of NMDA receptor function that is independent of the proton sensor. We identify a steroid modulatory domain, SMD1, on the NMDA receptor NR2B subunit that is critical for both PS enhancement and proton sensitivity. This domain includes the J͞K helices in the S2 region of the glutamate recognition site and the fourth membrane transmembrane region (M4). A molecular model based on ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor structure suggests that steroid modulatory domain 1 contributes residues to a hydrophobic pocket that is capable of accommodating PS. The results demonstrate that the J͞K helices and the fourth membrane transmembrane region participate in transducing allosteric interactions induced by steroid and proton binding to their respective sites. N-methyl-D-aspartate (NMDA) receptors mediate fast glutamatergic synaptic transmission, a core element of nervous system function, and are key loci for control of synaptic plasticity, learning and memory, and neuronal development. In particular, memory consolidation involves NMDA receptor-dependent synaptic reinforcement (1) and augmentation of N-methyl-Daspartate receptor subtype 2B (NR2B) subunit expression leads to enhancement of learning and memory in mice (2). Abnormal activation of NMDA receptors may, however, be associated with certain acute and chronic neurological disorders, including neuropathic pain, stroke, and neurodegenerative diseases.NMDA receptor function is known to be regulated pharmacologically, and in some cases physiologically, by endogenous molecules such as zinc, polyamines, protons, arachidonic acid, and sulfated neurosteroids, but the mechanism(s) by which modulators function to control transmitter-induced gating of ionotropic glutamate receptors (iGluRs) is unresolved. Moreover, the development of small molecule modulators provides a basis for the use of the NMDA receptor as a key target for future drug discovery (3, 4).Pregnenolone sulfate (PS) is one of the most abundant neurosteroids synthesized de novo in the nervous system (5-7). In vivo administration of PS promotes the release of acetylcholine in the cerebral cortex and hippocampus (8, 9), and dopamine in the nucleus accumbens (10), whereas exogenous and endogenous alterations of PS correlates with changes in spatial recognition and cognitive functions (11)(12)(13)(14)(15)(16). At the molecular level, PS enhances NMDA receptor function while inhibiting several other ligand gated ion channels, including ␥-aminobutyric acid type A and ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (17)(18)(19)(20).The proton sensor of the NMDA receptor has been demonstrated to...

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

Learning and memory deficits associated with age-related mild cognitive impairment have long been attributed to impaired processing within the hippocampus. Hyperactivity within the hippocampal CA3 region that is associated with aging is mediated in part by a loss of inhibitory interneurons and thought to underlie impaired performance in spatial memory tasks, including the abnormal tendency in aged animals to pattern complete spatial representations. Here, we asked whether the spatial firing patterns of simultaneously recorded CA3 and CA1 neurons in young and aged rats could be manipulated pharmacologically to selectively reduce CA3 hyperactivity and thus, according to hypothesis, the associated abnormality in spatial representations. We used chronically implanted high-density tetrodes to record the spatial firing properties of CA3 and CA1 units during animal exploration for food in familiar and novel environments. Aged CA3 place cells have higher firing rates, larger place fields, less spatial information content, and respond less to a change from a familiar to a novel environment than young CA3 cells. We also find that the combination of levetiracetam (LEV) + valproic acid (VPA), previously shown to act as a cognitive enhancer in tests of spatial memory, attenuate CA3 place cell firing rates, reduce place field area, and increase spatial information content in aged but not young adult rats. This is consistent with drug enhancing the specificity of neuronal firing with respect to spatial location. Contrary to expectation, however, LEV + VPA reduces place cell discrimination between novel and familiar environments, i.e., spatial correlations increase, independent of age even though drug enhances performance in cognitive tasks. The results demonstrate that spatial information content, or the number of bits of information encoded per action potential, may be the key correlate for enhancement of spatial memory by LEV + VPA.