Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

The alpha5 subunit of the GABA(A) receptor is localized mainly to the hippocampus of the mammalian brain. The significance of this rather distinct localization and the function of alpha5-containing GABA(A) receptors has been explored by targeted disruption of the alpha5 gene in mice. The alpha5 -/- mice showed a significantly improved performance in a water maze model of spatial learning, whereas the performance in non-hippocampal-dependent learning and in anxiety tasks were unaltered in comparison with wild-type controls. In the CA1 region of hippocampal brain slices from alpha5 -/- mice, the amplitude of the IPSCs was decreased, and paired-pulse facilitation of field EPSP (fEPSP) amplitudes was enhanced. These data suggest that alpha5-containing GABA(A) receptors play a key role in cognitive processes by controlling a component of synaptic transmission in the CA1 region of the hippocampus.

Both relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/ INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(B⌬23-27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly 23 to Arg, a truncation at the C terminus (Gly 24 -Trp 27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(B⌬23-27)R/I5 binds to human GPCR135 (IC 50 ‫؍‬ 0.67 nM) and GPCR142 (IC 50 ‫؍‬ 2.29 nM) with high affinity and is a potent functional GPCR135 antagonist (pA2 ‫؍‬ 9.15) but is not a human LGR7 ligand. Furthermore, R3(B⌬23-27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC 50 ‫؍‬ 0.25 nM, pA2 ‫؍‬ 9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(B⌬23-27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(B⌬23-27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.Relaxin-3 (R3) 2 (1) is the most recently identified member of the insulin-relaxin peptide family. Both relaxin-3 and its receptor, GPCR135 (2), are predominantly expressed in the brain (2, 3). GPCR135, an inhibitory receptor, is expressed in many regions of the rodent brain such as the superior colliculus, sensory cortex, olfactory bulb, amygdale, and paraventricular nucleus (4 -6), suggesting potential physiological involvement in neuroendocrine and sensory processing. Recent in vivo studies have further shown that relaxin-3 and GPCR135 are involved in the stress response and in regulation of feeding. More specifically, water restraint stress or intracerebroventricular corticotrophin-releasing factor (CRF) infusion induces relaxin-3 expression in cells of the nucleus incertus, a region where CRF receptor-1 is also expressed (7), and central administration of relaxin-3 induces feeding in rat (8, 9). These findings suggest that GPCR135 and relaxin-3 may be involved in multiple physiological processes, some of which might be as yet unknown.In vitro relaxin-3 activates GPCR135 (2), GPCR142 (10), and LGR7 (11) receptors. The predominant brain expression of both relaxin-3 and GPCR135, coupled with their high affinity interaction, strongly suggests that relaxin-3 is the endogenous ligand for GPCR135 (2). Phar...

The specific mechanisms underlying general anesthesia are primarily unknown. The intravenous general anesthetic etomidate acts by potentiating GABA(A) receptors, with selectivity for beta2 and beta3 subunit-containing receptors determined by a single asparagine residue. We generated a genetically modified mouse containing an etomidate-insensitive beta2 subunit (beta2 N265S) to determine the role of beta2 and beta3 subunits in etomidate-induced anesthesia. Loss of pedal withdrawal reflex and burst suppression in the electroencephalogram were still observed in the mutant mouse, indicating that loss of consciousness can be mediated purely through beta3-containing receptors. The sedation produced by subanesthetic doses of etomidate and during recovery from anesthesia was present only in wild-type mice, indicating that the beta2 subunit mediates the sedative properties of anesthetics. These findings show that anesthesia and sedation are mediated by distinct GABA(A) receptor subtypes.

ABSTRACT␣5IA is a compound that binds with equivalent subnanomolar affinity to the benzodiazepine (BZ) site of GABA A receptors containing an ␣1, ␣2, ␣3, or ␣5 subunit but has inverse agonist efficacy selective for the ␣5 subtype. As a consequence, the in vitro and in vivo effects of this compound are mediated primarily via GABA A receptors containing an ␣5 subunit. In a mouse hippocampal slice model, ␣5IA significantly enhanced the burst-induced long-term potentiation of the excitatory postsynaptic potential in the CA1 region but did not cause an increase in the paroxysmal burst discharges that are characteristic of convulsant and proconvulsant drugs. These in vitro data suggesting that ␣5IA may enhance cognition without being proconvulsant were confirmed in in vivo rodent models. Hence, ␣5IA significantly enhanced performance in a rat hippocampaldependent test of learning and memory, the delayed-matchingto-position version of the Morris water maze, with a minimum effective oral dose of 0.3 mg/kg, which corresponded to a BZ site occupancy of 25%. However, in mice ␣5IA was not convulsant in its own right nor did it potentiate the effects of pentylenetetrazole acutely or produce kindling upon chronic dosing even at doses producing greater than 90% occupancy. Finally, ␣5IA was not anxiogenic-like in the rat elevated plus maze nor did it impair performance in the mouse rotarod assay. Together, these data suggest that the GABA A ␣5-subtype provides a novel target for the development of selective inverse agonists with utility in the treatment of disorders associated with a cognitive deficit.Agonists at the benzodiazepine (BZ) binding site of the GABA A receptor, such as diazepam, enhance the inhibitory effects of GABA and have been used as anxiolytics and hypnotics for more than 40 years (Argyropoulos and Nutt, 1999). In addition, they have therapeutic utility in inducing not only sedation and muscle relaxation but also amnesia before surgical procedures (Williams and Bowie, 1999). Although the amnesic effects of BZ agonists in animals and humans have been known for some time (Ghoneim and Mewaldt, 1990), the precise nature of the processes underlying these effects are still uncertain. Since the anterograde rather than retrograde amnesia (McNaughton and Morris, 1987) produced by BZ agonists is similar to deficits induced by hippocampal lesions in animals and humans, it has been suggested that these drugs may exert their amnesic effects by modulating hippocampal function.At present, 19 GABA A receptor subunits have been identified (␣1-␣6, ␤1-␤3, ␥1-␥3, ␦, ⑀, , 1-3, and ; Simon et al.,

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The alpha1beta2gamma2 is the most abundant subtype of the GABA(A) receptor and is localized in many regions of the brain. To gain more insight into the role of this receptor subtype in the modulation of inhibitory neurotransmission, we generated mice lacking either the alpha1 or beta2 subunit. In agreement with the reported abundance of this subtype, >50% of total GABA(A) receptors are lost in both alpha1-/- and beta2-/- mice. Surprisingly, homozygotes of both mouse lines are viable, fertile, and show no spontaneous seizures. Initially half of the alpha1-/- mice died prenatally or perinatally, but they exhibited a lower mortality rate in subsequent generations, suggesting some phenotypic drift and adaptive changes. Both adult alpha1-/- and beta2-/- mice demonstrate normal performances on the rotarod, but beta2-/- mice displayed increased locomotor activity. Purkinje cells of the cerebellum primarily express alpha1beta2gamma2 receptors, and in electrophysiological recordings from alpha1-/- mice GABA currents in these neurons are dramatically reduced, and residual currents have a benzodiazepine pharmacology characteristic of alpha2- or alpha3-containing receptors. In contrast, the cerebellar Purkinje neurons from beta2-/- mice have only a relatively small reduction of GABA currents. In beta2-/- mice expression levels of all six alpha subunits are reduced by approximately 50%, suggesting that the beta2 subunit can coassemble with alpha subunits other than just alpha1. Our data confirm that alpha1beta2gamma2 is the major GABA(A) receptor subtype in the murine brain and demonstrate that, surprisingly, the loss of this receptor subtype is not lethal.

Benzodiazepines exert their effects by binding to multiple subtypes of the GABAA receptor, the predominant subtypes in the brain being those that contain ␣1-, ␣2-, ␣3-, and ␣5-subunits. To understand the potentially different roles of these subtypes in the therapeutic and side effects of benzodiazepines, we evaluated GABAA receptor subtype-preferring compounds in nonhuman primate models predictive of anxiolytic, sedative, motor, subjective, and reinforcing effects of benzodiazepine-type drugs. These compounds included zolpidem, which shows preferential binding to GABAA receptors containing ␣1-subunits (␣1GABAA receptors); L-838,417, which shows functional selectivity for ␣2GABAA, ␣3GABAA, and ␣5GABAA receptors; and nonselective conventional benzodiazepines. The results provide evidence in nonhuman primates that ␣1GABAA receptors do not play a key role in the anxiolytic and muscle-relaxant properties of benzodiazepine-type drugs; instead, these effects involve ␣2GABAA, ␣3GABAA, and͞or ␣5GABAA subtypes. Our results also suggest that the ␣1GABAA receptor subtype might be critically involved in the subjective, sedative, and motor effects of benzodiazepine-type drugs. In contrast, stimulation of ␣1GABAA receptors is sufficient, but not necessary, for mediation of the abuse potential of these drugs.addiction ͉ anxiety R eceptors for the neurotransmitter GABA, in particular the type A subtype (GABA A receptor), have received considerable attention as the site of action for drugs acting as anxiolytics, sedatives, anticonvulsants, and muscle relaxants. These clinically beneficial effects are exhibited by the benzodiazepines, which act by allosterically binding to GABA A receptors and enhancing the ability of GABA to increase chloride conductance. The therapeutic use of benzodiazepines is constrained, however, by other characteristic effects of these drugs, such as daytime drowsiness and impairment of motor coordination. Benzodiazepines additionally have subjective and reinforcing effects that might contribute to their widespread abuse (1). Recent studies have revealed the existence of multiple subtypes of the GABA A receptor (2, 3), and research with transgenic mice has postulated that the diverse behavioral effects of benzodiazepine-like drugs may reflect action at different subtypes of GABA A receptors (3-5). Although provocative, the extent to which these findings in transgenic mice are applicable to other species, especially primates, is not known. Moreover, virtually no information is available regarding the role of specific GABA A receptor subtypes in the addictive properties of benzodiazepines in any species.The GABA A receptors in the central nervous system are pentamers constituted from structurally distinct proteins, with each protein family consisting of different subunits (for review, see ref.3). The majority of GABA A receptors consist of ␣-, ␤-, and ␥-subunit families, and benzodiazepine action appears to be determined by the presence of particular ␣-subunits. Benzodiazepine-like drugs bind predominantly to...