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[Gamma]-Aminobutyric acid type A receptors ([GABA.sub.A]-Rs) mediate the bulk of rapid inhibitory synaptic transmission in the central nervous system. The [Beta]3 subunit is an essential component of the [GABA.sub.A]-R in many brain regions, especially during development, and is implicated in several pathophysiologic processes. We examined mice harboring a [Beta]3 gene inactivated by gene targeting. [GABA.sub.A]-R density is approximately halved in brain of [Beta]3-deficient mice, and [GABA.sub.A]-R function is severely impaired. Most [Beta]3-deficient mice die as neonates; some neonatal mortality, but not all, is accompanied by cleft palate. [Beta]3-deficient mice that survive are runted until weaning but achieve normal body size by adulthood, although with reduced life span. These mice are fertile but mothers fail to nurture offspring. Brain morphology is grossly normal, but a number of behaviors are abnormal, consistent with the widespread location of the [Beta]3 subunit. The mice are very hyperactive and hyperresponsive to human contact and other sensory stimuli, and often run continuously in tight circles. When held by the tail, they hold all paws in like a ball, which is frequently a sign of neurological impairment. They have difficulty swimming, walking on grids, and fall off platforms and rotarods, although they do not have a jerky gait. [Beta]3-deficient mice display frequent myoclonus and occasional epileptic seizures, documented by electroencephalographic recording. Hyperactivity, lack of coordination, and seizures are consistent with reduced presynaptic inhibition in spinal cord and impaired inhibition in higher cortical centers and/or pleiotropic developmental defects.

Alcohol-non-tolerant (ANT) rats carry a gene that expresses the alpha-six subunit of the granule-cell-specific gamma-aminobutyric acid (GABA-A). A point mutation of this gene identified through polymerase chain reaction causes the ANT rats to be susceptible to abnormal motor function due to diazepam and other benzodiazepine agonists. The potentiation of GABA-A may cause this sensitivity to both benzodiazepine-related compounds and to alcohol.

In the brain, extrasynaptically expressed ionotropic, δ subunit-containing γ-aminobutyric acid A-type receptors (δ-GABAARs) have been implicated in drug effects at both neuronal and behavioral levels. These alterations are supposed to be caused via drug-induced modulation of receptor ionophores affecting chloride ion-mediated inhibitory tonic currents. Often, a transgenic mouse model genetically lacking the δ-GABAARs (δ-KO) has been used to study the roles of δ-GABAARs in brain functions, because a specific antagonist of the δ-GABAARs is still lacking. We have previously observed with these δ-KO mice that activation of δ-GABAARs is needed for morphine-induced conditioning of place preference, and others have suggested that δ-GABAARs act as targets selectively for low doses of ethanol. Furthermore, activation of these receptors via drug-mediated agonism induces a robust increase in the slow-wave frequency bands of electroencephalography (EEG). Here, we tested δ-KO mice (compared to littermate wild-type controls) for the pharmaco-EEG responses of a broad spectrum of pharmacologically different drug classes, including alcohol, opioids, stimulants, and psychedelics. Gaboxadol (THIP), a known superagonist of δ-GABAARs, was included as the positive control, and as expected, δ-KO mice produced a blunted pharmaco-EEG response to 6 mg/kg THIP. Pharmaco-EEGs showed notable differences between treatments but also differences between δ-KO mice and their wild-type littermates. Interestingly mephedrone (4-MMC, 5 mg/kg), an amphetamine-like stimulant, had reduced effects in the δ-KO mice. The responses to ethanol (1 g/kg), LSD (0.2 mg/kg), and morphine (20 mg/kg) were similar in δ-KO and wild-type mice. Since stimulants are not known to act on δ-GABAARs, our findings on pharmaco-EEG effects of 4-MMC suggest that δ-GABAARs are involved in the secondary indirect regulation of the brain rhythms after 4-MMC. [ABSTRACT FROM AUTHOR]

Abstract Background Gamma-aminobutyric acid type A receptors (GABAA-Rs) are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice. Results Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10–95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line. Conclusion We conclude that: 1) insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably reduced the amount of γ2, and that 2) attenuated expression of γ2 increased anxiety-like behaviors but did not lead to differences in the hypnotic response to benzodiazepine site ligands. This suggests that reduced synaptic inhibition can lead to a phenotype of increased anxiety-like behavior. In contrast, normal drug effects can be maintained despite a dramatic reduction in GABAA-R targets.

Histamine/gamma-aminobutyric acid (GABA) neurons of posterior hypothalamus send wide projections to many brain areas and participate in stabilizing the wake state. Recent research has suggested that GABA released from the histamine/GABA neurons acts on extrasynaptic GABAA receptors and balances the excitatory effect of histamine. In the current study, we show the presence of vesicular GABA transporter mRNA in a majority of quantified hypothalamic histaminergic neurons, which suggest vesicular release of GABA. As histamine/GABA neurons form conventional synapses infrequently, it is possible that GABA released from these neurons diffuses to target areas by volume transmission and acts on extrasynaptic GABA receptors. To investigate this hypothesis, mice lacking extrasynaptic GABAA receptor δ subunit (Gabrd KO) were used. A pharmacological approach was employed to activate histamine/GABA neurons and induce histamine and presumably, GABA, release. Control and Gabrd KO mice were treated with histamine receptor 3 (Hrh3) inverse agonists ciproxifan and pitolisant, which block Hrh3 autoreceptors on histamine/GABA neurons and histamine-dependently promote wakefulness. Low doses of ciproxifan (1 mg/kg) and pitolisant (5 mg/kg) reduced locomotion in Gabrd KO, but not in WT mice. EEG recording showed that Gabrd KO mice were also more sensitive to the wake-promoting effect of ciproxifan (3 mg/kg) than control mice. Low frequency delta waves, associated with NREM sleep, were significantly suppressed in Gabrd KO mice compared with the WT group. Ciproxifan-induced wakefulness was blocked by histamine synthesis inhibitor α-fluoromethylhistidine (αFMH). The findings indicate that both histamine and GABA, released from histamine/GABA neurons, are involved in regulation of brain arousal states and δ-containing subunit GABAA receptors are involved in mediating GABA response. [ABSTRACT FROM AUTHOR]

THIP (gaboxadol), a superagonist of the δ subunit-containing extrasynaptic GABAA receptors, produces persistent neuroplasticity in dopamine (DA) neurons of the ventral tegmental area (VTA), similarly to rewarding drugs of abuse. However, unlike them THIP lacks abuse potential and induces conditioned place aversion in mice. The mechanism underlying the aversive effects of THIP remains elusive. Here, we show that mild aversive effects of THIP were detected 2 h after administration likely reflecting an anxiety-like state with increased corticosterone release and with central recruitment of corticotropin-releasing factor corticotropin-releasing factor receptor 1 (CRF1) receptors. A detailed immunohistochemical c-Fos expression mapping for THIP-activated brain areas revealed a correlation between the activation of CRF-expressing neurons in the oval nucleus of the bed nuclei of stria terminalis and THIP-induced aversive effects. In addition, the neuroplasticity of mesolimbic DA system (24 h after administration) and conditioned place aversion by THIP after four daily acute sessions were dependent on extrasynaptic GABAA receptors (abolished in δ-GABAA receptor knockout mice) and activation of the CRF1 receptors (abolished in wildtype mice by a CRF1 receptor antagonist). A selective THIP-induced activation of CRF-expressing neurons in the oval part of the bed nucleus of stria terminalis may constitute a novel mechanism for inducing plasticity in a population of VTA DA neurons and aversive behavioral states. [ABSTRACT FROM AUTHOR]

[Gamma]-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the ~ subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the [Delta] subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids.

Endogenous neurosteroids and neuroactive steroids have potent and widespread actions on the brain via inhibitory GABAA receptors. In recombinant receptors and genetic mouse models their actions depend on the α, β, and δ subunits of the receptor, especially on those that form extrasynaptic GABAA receptors responsible for non-synaptic (tonic) inhibition, but they also act on synaptically enriched γ2 subunitcontaining receptors and even on ab binary receptors. Here we tested whether behavioral sensitivity to the neuroactive steroid agonist 5β-pregnan-3α-ol-20-one is altered in genetically engineered mouse models that have deficient GABAA receptormediated synaptic inhibition in selected neuronal populations. Mouse lines with the GABAA receptor γ2 subunit gene selectively deleted either in parvalbumin-containing cells (including cerebellar Purkinje cells), cerebellar granule cells, or just in cerebellar Purkinje cells were trained on the accelerated rotating rod and then tested for motor impairment after cumulative intraperitoneal dosing of 5β-pregnan-3α-ol-20-one. Motorimpairing effects of 5β-pregnan-3α-ol-20-one were strongly increased in all three mouse models in which γ2 subunit-dependent synaptic GABAA responses in cerebellar neurons were genetically abolished. Furthermore, rescue of postsynaptic GABAA receptors in Purkinje cells normalized the effect of the steroid. Anxiolytic/explorative effects of the steroid in elevated plus maze and light:dark exploration tests in mice with Purkinje cell γ2 subunit inactivation were similar to those in control mice. The results suggest that, when the deletion of γ2 subunit has removed synaptic GABAA receptors from the specific cerebellar neuronal populations, the effects of neuroactive steroids solely on extrasynaptic ab or abd receptors lead to enhanced changes in the cerebellum-generated behavior. [ABSTRACT FROM AUTHOR]