Deletion of the gabra2 gene results in hypersensitivity to the acute effects of ethanol but does not alter ethanol self administration

Human genetic studies have suggested that polymorphisms of the GABRA2 gene encoding the GABA(A) α2-subunit are associated with ethanol dependence. Variations in this gene also convey sensitivity to the subjective effects of ethanol, indicating a role in mediating ethanol-related behaviours. We therefore investigated the consequences of deleting the α2-subunit on the ataxic and rewarding properties of ethanol in mice. Ataxic and sedative effects of ethanol were explored in GABA(A) α2-subunit wildtype (WT) and knockout (KO) mice using a Rotarod apparatus, wire hang and the duration of loss of righting reflex. Following training, KO mice showed shorter latencies to fall than WT littermates under ethanol (2 g/kg i.p.) in both Rotarod and wire hang tests. After administration of ethanol (3.5 g/kg i.p.), KO mice took longer to regain the righting reflex than WT mice. To ensure the acute effects are not due to the gabra2 deletion affecting pharmacokinetics, blood ethanol concentrations were measured at 20 minute intervals after acute administration (2 g/kg i.p.), and did not differ between genotypes. To investigate ethanol's rewarding properties, WT and KO mice were trained to lever press to receive increasing concentrations of ethanol on an FR4 schedule of reinforcement. Both WT and KO mice self-administered ethanol at similar rates, with no differences in the numbers of reinforcers earned. These data indicate a protective role for α2-subunits, against the acute sedative and ataxic effects of ethanol. However, no change was observed in ethanol self administration, suggesting the rewarding effects of ethanol remain unchange

GABA Receptors and the Pharmacology of Sleep

Current GABAergic sleep-promoting medications were developed pragmatically, without making use of the immense diversity of GABAA receptors. Pharmacogenetic experiments are leading to an understanding of the circuit mechanisms in the hypothalamus by which zolpidem and similar compounds induce sleep at α2βγ2-type GABAA receptors. Drugs acting at more selective receptor types, for example, at receptors containing the α2 and/or α3 subunits expressed in hypothalamic and brain stem areas, could in principle be useful as hypnotics/anxiolytics. A highly promising sleep-promoting drug, gaboxadol, which activates αβδ-type receptors failed in clinical trials. Thus, for the time being, drugs such as zolpidem, which work as positive allosteric modulators at GABAA receptors, continue to be some of the most effective compounds to treat primary insomnia

Phase-amplitude coupled persistent theta and gamma oscillations in rat primary motor cortex in vitro

In vivo, theta (4-7 Hz) and gamma (30-80 Hz) neuronal network oscillations are known to coexist and display phase-amplitude coupling (PAC). However, in vitro, these oscillations have for many years been studied in isolation. Using an improved brain slice preparation technique we have, using co-application of carbachol (10 μM) and kainic acid (150 nM), elicited simultaneous theta (6.6 ± 0.1 Hz) and gamma (36.6 ± 0.4 Hz) oscillations in rodent primary motor cortex (M1). Each oscillation showed greatest power in layer V. Using a variety of time series analyses we detected significant cross-frequency coupling 74% of slice preparations. Differences were observed in the pharmacological profile of each oscillation. Thus, gamma oscillations were reduced by the GABAA receptor antagonists, gabazine (250 nM and 2 μM), and picrotoxin (50 μM) and augmented by AMPA receptor antagonism with SYM2206 (20 μM). In contrast, theta oscillatory power was increased by gabazine, picrotoxin and SYM2206. GABAB receptor blockade with CGP55845 (5 μM) increased both theta and gamma power, and similar effects were seen with diazepam, zolpidem, MK801 and a series of metabotropic glutamate receptor antagonists. Oscillatory activity at both frequencies was reduced by the gap junction blocker carbenoxolone (200 μM) and by atropine (5 μM). These data show theta and gamma oscillations in layer V of rat M1 in vitro are cross-frequency coupled, and are mechanistically distinct. The development of an in vitro model of phase-amplitude coupled oscillations will facilitate further mechanistic investigation of the generation and modulation of coupled activity in mammalian cortex

GABA receptors on the cell-body membrane of an identified insect motor neuron

The pharmacology of a gamma-aminobutyric acid (GABA) receptor on the cell body of an identified motor neuron of the cockroach (Periplaneta americana) was investigated by current-clamp and voltage-clamp methods. Iontophoretic application of GABA increased membrane conductance to chloride ions, and prolonged application resulted in desensitization. Hill coefficients, determined from dose-response data, indicated that binding of at least two GABA molecules was required to activate the chloride channel. Differences between vertebrate GABAA receptors and insect neuronal GABA receptors were detected. For the GABA receptor of motor neuron Df, the following rank order of potency was observed: isoguvacine greater than muscimol greater than or equal to GABA greater than 3-aminopropanesulphonic acid. The GABAB receptor agonist baclofen was inactive. Of the potent vertebrate GABA receptor antagonists (bicuculline, pitrazepin, RU5135 and picrotoxin), only picrotoxin (10(-7) M) produced a potent, reversible block of the response to GABA of motor neuron Df. Both picrotoxinin and picrotin also blocked GABA-induced currents. Bicuculline hydrochloride (10(-4) M) and bicuculline methiodide (10(-4) M) were both ineffective when applied at resting membrane potential (-65 mV), although at hyperpolarized levels partial block of GABA-induced current was sometimes observed. Pitrazepin (10(-4) M) caused a partial, voltage-independent block of GABA-induced current. The steroid derivative RU5135 was inactive at 10(-5) M. In contrast to the potent competitive blockade of vertebrate GABAA receptors by bicuculline, pitrazepin and RU5135, none of the weak antagonism caused by these drugs on the insect GABA receptor was competitive. Flunitrazepam (10(-6) M) potentiated GABA responses, providing evidence for a benzodiazepine site on an insect GABA-receptor-chloride-channel complex.</p

A study of subunit selectivity, mechanism and site of action of the delta selective compound 2 (DS2) at human recombinant and rodent native GABAA receptors

BACKGROUND AND PURPOSE: Most GABA(A) receptor subtypes comprise 2α, 2β and 1γ subunit, although for some isoforms, a δ replaces a γ-subunit. Extrasynaptic δ-GABA(A) receptors are important therapeutic targets, but there are few suitable pharmacological tools. We profiled DS2, the purported positive allosteric modulator (PAM) of δ-GABA(A) receptors to better understand subtype selectivity, mechanism/site of action and activity at native δ-GABA(A) receptors. EXPERIMENTAL APPROACH: Subunit specificity of DS2 was determined using electrophysiological recordings of Xenopus laevis oocytes expressing human recombinant GABA(A) receptor isoforms. Effects of DS2 on GABA concentration–response curves were assessed to define mechanisms of action. Radioligand binding and electrophysiology utilising mutant receptors and pharmacology were used to define site of action. Using brain-slice electrophysiology, we assessed the influence of DS2 on thalamic inhibition in wild-type and δ(0/0) mice. KEY RESULTS: Actions of DS2 were primarily determined by the δ-subunit but were additionally influenced by the α, but not the β, subunit (α4/6βxδ > α1βxδ >> γ2-GABA(A) receptors > α4β3). For δ-GABA(A) receptors, DS2 enhanced maximum responses to GABA, with minimal influence on GABA potency. (iii) DS2 did not act via the orthosteric, or known modulatory sites on GABA(A) receptors. (iv) DS2 enhanced tonic currents of thalamocortical neurones from wild-type but not δ(0/0) mice. CONCLUSIONS AND IMPLICATIONS: DS2 is the first PAM selective for α4/6βxδ receptors, providing a novel tool to investigate extrasynaptic δ-GABA(A) receptors. The effects of DS2 are mediated by an unknown site leading to GABA(A) receptor isoform selectivity