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In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the γ2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase–induced swapping of the γ2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits. [ABSTRACT FROM AUTHOR]

Abstract: In rodent models, γ-aminobutyric acid A (GABAA) receptors with the α6 and δ subunits, expressed in the cerebellar and cochlear nucleus granule cells, have been linked to ethanol sensitivity and voluntary ethanol drinking. Here, we review the findings. When considering both in vivo contributions and data on cloned receptors, the evidence for direct participation of the α6-containing receptors to increased ethanol sensitivity is poor. The α6 subunit-knockout mouse lines do not have any changed sensitivity to ethanol, although these mice do display increased benzodiazepine sensitivity. However, in general the compensations occurring in knockout mice (regardless of which particular gene is knocked out) tend to fog interpretations of drug actions at the systems level. For example, the α6 knockout mice have increased TASK-1 channel expression in their cerebellar granule cells, which could influence sensitivity to ethanol in the opposite direction to that obtained with the α6 knockouts. Indeed, TASK-1 knockout mice are more impaired than wild types in motor skills when given ethanol; this might explain why GABAA receptor α6 knockout mice have unchanged ethanol sensitivities. As an alternative to studying knockout mice, we examined the claimed δ subunit-dependent/γ2 subunit-independent ethanol/[3H]Ro 15-4513 binding sites on GABAA receptors. We looked at [3H]Ro 15-4513 binding in HEK 293 cell membrane homogenates containing rat recombinant α6/4β3δ receptors and in mouse brain sections. Specific high-affinity [3H]Ro 15-4513 binding could not be detected under any conditions to the recombinant receptors or to the cerebellar sections of γ2(F77I) knockin mice, nor was this binding to brain sections of wild-type C57BL/6 inhibited by 1–100mM ethanol. Since ethanol may act on many receptor and channel protein targets in neuronal membranes, we consider the α6 (and α4) subunit-containing GABAA receptors unlikely to be directly responsible for any major part of ethanol''s actions. Therefore, we finish the review by discussing more generally alcohol and GABAA receptors and by suggesting potential future directions for this research. [Copyright &y& Elsevier]

Abstract: The main inhibitory neurotransmitter system in the brain, the γ-aminobutyric acid (GABA) system, is the target for many clinically used drugs to treat, for example, anxiety disorders and epilepsy and to induce sedation and anesthesia. These drugs facilitate the function of pentameric A-type GABA (GABAA) receptors that are extremely widespread in the brain and composed from the repertoire of 19 subunit variants. Modern genetic studies have found associations of various subunit gene polymorphisms with neuropsychiatric disorders, including alcoholism, schizophrenia, anxiety, and bipolar affective disorder, but these studies are still at their early phase because they still have failed to lead to validated drug development targets. Recent neurobiological studies on new animal models and receptor subunit mutations have revealed novel aspects of the GABAA receptors, which might allow selective targeting of the drug action in receptor subtype-selective fashion, either on the synaptic or extrasynaptic receptor populations. More precisely, the greatest advances have occurred in the clarification of the molecular and behavioral mechanisms of action of the GABAA receptor agonists already in the clinical use, such as benzodiazepines and anesthetics, rather than in the introduction of novel compounds to clinical practice. It is likely that these new developments will help to overcome the present problems of the chronic treatment with nonselective GABAA agonists, that is, the development of tolerance and dependence, and to focus the drug action on the neurobiologically and neuropathologically relevant substrates. [Copyright &y& Elsevier]

Background: γ-Aminobutyric acid type A (GABAA) receptors provide the main inhibitory control in the brain. Their heterogeneity may make it possible to precisely target drug effects to selected neuronal populations. In situ hybridization using rat brain sections has revealed a unique expression of GABAA receptor ϵ and θ subunit transcripts in the locus coeruleus, where they are accompanied at least by α3, α2, β1 and β3 subunits. Here, we studied the pharmacology of the human α3β1, α3β1ϵ, α3β1θ and α3β1ϵθ receptor subtypes expressed in Xenopus oocytes and compared them with the 㬲 subunit-containing receptors. Results: The GABA sensitivites and effects of several positive modulators of GABAA receptors were studied in the absence and the presence of EC25 GABA using the two-electrode voltage-clamp method. We found 100-fold differences in GABA sensitivity between the receptors, α3β1ϵ subtype being the most sensitive and α3β1㬲 the least sensitive. Also gaboxadol dose-response curves followed the same sensitivity rank order, with EC50 values being 72 and 411 μM for α3β1ϵ and α3β1㬲 subtypes, respectively. In the presence of EC25 GABA, introduction of the ϵ subunit to the receptor complex resulted in diminished modulatory effects by etomidate, propofol, pregnanolone and flurazepam, but not by pentobarbital. Furthermore, the α3β1ϵ subtype displayed picrotoxinsensitive spontaneous activity. The θ subunit-containing receptors were efficiently potentiated by the anesthetic etomidate, suggesting that θ subunit could bring the properties of β2 or β3 subunits to the receptor complex. Conclusion: The ϵ and θ subunits bring additional features to α3β1 GABAA receptors. These receptor subtypes may constitute as novel drug targets in selected brain regions, e.g., in the brainstem locus coeruleus nuclei. [ABSTRACT FROM AUTHOR]

Two-pore domain potassium (K2P ) channel expression is believed to underlie the developmental emergence of a potassium leak conductance [IK(SO)] in cerebellar granule neurons (CGNs), suggesting that K2P function is an important determinant of the input conductance and resting membrane potential. To investigate the role that different K2P channels may play in the regulation of CGN excitability, we generated a mouse lacking TASK-1, a K2P channel known to have high expression levels in CGNs. In situ hybridization and real-time PCR studies in wild-type and TASK-1 knock-outs (KOs) demonstrated that the expression of other K2P channels was unaltered in CGNs. TASK-1 knock-out mice were healthy and bred normally but exhibited compromised motor performance consistent with altered cerebellar function. Whole-cell recordings from adult cerebellar slice preparations revealed that the resting excitability of mature CGNs was no different in TASK-1 KO and littermate controls. However, the modulation of IK(SO) by extracellular Zn2+, ruthenium red, and H+ was altered. The IK(SO)) recorded from TASK-1 knock-out CGNs was no longer sensitive to alkalization and was blocked by Zn2+ and ruthenium red. These results suggest that a TASK-1-containing channel population has been replaced by a homodimeric TASK-3 population in the TASK-1 knock-out. These data directly demonstrate that TASK-1 channels contribute to the properties of IK(SO) in adult CGNs. However, TASK channel subunit composition does not alter the resting excitability of CGNs but does influence sensitivity to endogenous modulators such as Zn2+ and H+. [ABSTRACT FROM AUTHOR]

Abstract: The preparation of hippocampal slices results in loss of input neurons to dentate granule cells, which leads to the reorganization of their axons, the mossy fibers, and alters their functional properties in long-term cultures, but its temporal aspects in the immature hippocampus are not known. In this study, we have focused on the early phase of this plastic reorganization process by analyzing granule cell function with field potential and whole cell recordings during the in vitro maturation of hippocampal slices (from 1 to 17 days in vitro, prepared from 6 to 7-day-old rats), and their morphology using extracellular biocytin labelling technique. Acute slices from postnatal 14–22-day-old rats were analyzed to detect any differences in the functional properties of granule cells in these two preparations. In field potential recordings, small synaptically-evoked responses were detected at 2 days in vitro, and their amplitude increased during the culture time. Whole cell voltage clamp recordings revealed intensive spontaneous excitatory postsynaptic currents, and the susceptibility to stimulus-evoked bursting increased with culture time. In acutely prepared slices, neither synaptically-evoked responses in field potential recordings nor any bursting in whole cell recordings were detected. The excitatory activity was under the inhibitory control of γ-aminobutyric acid type A receptor. Extracellularily applied biocytin labelled dentate granule cells, and revealed sprouting and aberrant targeting of mossy fibers in cultured slices. Our results suggest that reorganization of granule cell axons takes place during the early in vitro maturation of hippocampal slices, and contributes to their increased excitatory activity resembling that in the epileptic hippocampus. Cultured immature hippocampal slices could thus serve as an additional in vitro model to elucidate mechanisms of synaptic plasticity and cellular reactivity in response to external damage in the developing hippocampus. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

GABAA receptors mediate fast phasic inhibitory postsynaptic potentials and participate in slower tonic extrasynaptic inhibition. Thy1α6 mice with ectopic forebrain expression of GABAA receptorα6 subunits exhibit increased extrasynaptic GABAA receptor-mediated background conductance and reduced synaptic GABAA receptor currents in hippocampal CA1 neurons[W. Wisdenet al. (2002)Neuropharmacology43, 530–549]. Here we demonstrate that isolated CA1 neurons of these mice showed furosemide-sensitivity of GABA-evoked currents, confirming the functional expression ofα6 subunit. In addition, receptor autoradiography of the CA1 region of Thy1α6 brain sections revealed pharmacological features that are unique forα6βγ2 andα6β receptors. The existence of atypicalα6β receptors was confirmed after completely eliminating GABAA receptors containingγ1,γ2,γ3 orδ subunits using serial immunoaffinity chromatography on subunit-specific GABAA receptor antibodies. Behaviourally, the Thy1α6 mice showed normal features with slightly enhanced startle reflex and struggle-escape behaviours. However, they were more sensitive to GABAA antagonists DMCM (shorter latency to writhing clonus) and picrotoxinin (shorter latency to generalized convulsions). Tiagabine, an antiepileptic GABA-uptake inhibitor that increases brain GABA levels, delayed picrotoxinin-induced convulsions at a low dose of 3.2 mg/kg in Thy1α6 mice, but not in control mice; however, the overall effect of higher tiagabine doses on the convulsion latency remained smaller in the Thy1α6 mice. Altered balance between extrasynaptic and synaptic receptors thus affects seizure sensitivity to GABAergic convulsants. Importantly, the increased extrasynaptic inhibition, even when facilitated in the presence of tiagabine, was not able fully to counteract enhanced seizure induction by GABAA antagonists. [ABSTRACT FROM AUTHOR]

Compares the development of tolerance to the motor-impairing effects of repeated moderate diazepam doses between mice deficient in the cerebellar granule cell-restricted GABA receptor of alpha6 subunit and their wild-type controls. Complete tolerance in both mouse lines produced by chronic treatment; Tolerance to motor-impairing actions of diazepam.

The possible effect of atipamezole, a potent and specific α2-adrenoceptor antagonist, on the development and expression of d-amphetamine-induced behavioural sensitization was evaluated in mice. Male (C57Bl/6J) mice were given daily doses of d-amphetamine (2 mg/kg). In addition, groups of mice received injections of atipamezole (0.3 or 1 mg/kg) 20 min before d-amphetamine or vehicle administration. Idazoxan (1 mg/kg) was used in some experiments to extend the results to other α2-adrenoceptor antagonists. Challenge doses of d-amphetamine were administered to the mice on days 7–9 to evaluate the effects of α2-adrenoceptor antagonists on the d-amphetamine sensitization, evidenced by increased locomotor activation. Mice treated repeatedly with d-amphetamine developed strong locomotor sensitization that was reduced by pretreatment with α2-adrenoceptor antagonists. Acute atipamezole at both doses attenuated the expression of d-amphetamine-induced sensitization. Atipamezole at 1 mg/kg alone had no effect on locomotor activity, but the lower dose (0.3 mg/kg) increased locomotor activity after repeated administration. These results indicate that α2-adrenoceptor antagonists modulate the actions of d-amphetamine in a manner not explicable by their enhancing actions on noradrenaline and dopamine release, and may thus provide a novel approach to the treatment of motor complications caused by dopaminergic agents, such as dyskinesias, and perhaps also drug dependence. [ABSTRACT FROM AUTHOR]

Neurotransmitter receptor systems have been the focus of intensive pharmacological research for more than 20 years for basic and applied scientific reasons, but only recently has there been a better understanding of their key features. One of these systems includes the type A receptor for the γ-aminobutyric acid (GABA), which forms an integral anion channel from a pentameric subunit assembly and mediates most of the fast inhibitory neurotransmission in the adult vertebrate central nervous system. Up to now, depending on the definition, 16–19 mammalian subunits have been cloned and localized on different genes. Their assembly into proteins in a poorly defined stoichiometry forms the basis of functional and pharmacological GABAA receptor diversity, i.e. the receptor subtypes. The latter has been well documented in autoradiographic studies using ligands that label some of the receptors’ various binding sites, corroborated by recombinant expression studies using the same tools. Significantly less heterogeneity has been found at the physiological level in native receptors, where the subunit combinations have been difficult to dissect.This review focuses on the characteristics, use and usefulness of various ligands and their binding sites to probe GABAA receptor properties and to gain insight into the biological function from fish to man and into evolutionary conserved GABAA receptor heterogeneity. We also summarize the properties of the novel mouse models created for the study of various brain functions and review the state-of-the-art imaging of brain GABAA receptors in various human neuropsychiatric conditions.The data indicate that the present ligands are only partly satisfactory tools and further ligands with subtype-selective properties are needed for imaging purposes and for confirming the behavioral and functional results of the studies presently carried out in gene-targeted mice with other species, including man. [Copyright &y& Elsevier]

Chronic alcohol administration increases the expression of cerebellum-specific GABA[sub A] receptor alpha 6 subunit mRNA, protein and selective autoradiographical finger print on rat and mouse brain sections. We have tested whether the alpha 6 gene is activated by chronic alcohol administration (daily p.o. injection of 2 g/kg during the first 3 days and 2.5 g/kg during the next 17 days) that produced tolerance in the rotarod test to motor impairment by acute challenge of ethanol (2 g/kg, i.p.). We utilized a mouse line engineered to express E. coli beta-galactosidase enzyme and an unfunctional truncated alpha 6 subunit under the control of the alpha 6 gene promoter. Chronic ethanol treatment failed to alter the cerebellar beta-galactosidase activity when compared with no treatment and isocaloric sucrose treatment in groups of alpha 6 subunit-deficient mice. The results suggest that tolerance to motor-impairing effects of ethanol can be achieved in the absence of alpha 6 subunit-containing GABA[sub A] receptors, but that the reported upregulation of alpha 6 gene transcription by ethanol treatment requires functional alpha 6 subunits. [ABSTRACT FROM AUTHOR]

Abstract Benzodiazepine- and alcohol-induced ataxias in rodents have been proposed to be affected by the γ-aminobutyric acid type A (GABAA) receptor α6 subunit, which contributes to receptors specifically expressed in cerebellar granule cells. We have studied an α6 –/– mouse line for motor performance and drug sensitivity. These mice, as a result of a specific genetic lesion, carry a precise impairment at their Golgi-granule cell synapses. On motor performance tests (rotarod, horizontal wire, pole descending, staircase and swimming tests) there were no robust baseline differences in motor function or motor learning between α6 –/– and α6 +/+ mice. On the rotarod test, however, the mutant mice were significantly more impaired by diazepam (5–20 mg/kg, i.p.), when compared with α6 +/+ control and background C57BL/6J and 129/SvJ mouse lines. Ethanol (2.0–2.5 g/kg, i.p.) produced similar impairment in the α6 –/– and α6 +/+ mice. Diazepam-induced ataxia in α6 –/– mice could be reversed by the benzodiazepine site antagonist flumazenil, indicating the involvement of the remaining α1β2/3γ2 GABAA receptors of the granule cells. The level of activity in this synapse is crucial in regulating the execution of motor tasks. We conclude that GABAA receptor α6 subunit-dependent actions in the cerebellar cortex can be compensated by other receptor subtypes; but if not for the α6 subunit, patients on benzodiazepine medication would suffer considerably from ataxic side-effects. [ABSTRACT FROM AUTHOR]

To determine the roles of the alternatively spliced short and long forms of the γ2 subunit in the effect of ethanol on the GABAA receptor function, picrotoxin-sensitive [35S] t-butylbicyclophosphorothionate ([35S]TBPS) binding was studied in recombinant rat α1β2γ2 and α6β2γ2 receptors expressed in human embryonic kidney 293 cells. Ethanol (10-500 mM) in the absence of added GABA had only minor effects on [35S]TBPS binding irrespective of the γ2 splice variant, its effects being greater in α6β2γ2 than in α1β2γ2 receptors. Ethanol (100 mM) decreased the binding in all four subunit combinations at various concentrations of GABA, again an effect independent of the γ2 variant. The two γ2 variants had different effects on GABA modulation of the binding, with the long γ2 variant decreasing the efficiency of GABA inhibition in α6β2γ2 receptors and enhancing the biphasic GABA stimulation and inhibition in α1β2γ2 receptors. The findings confirm the importance of the α subunits in the allosteric interactions between the convulsant binding site and other effector sites, which can be modified only to a minor extent by the type of the γ2 splice variant. [ABSTRACT FROM AUTHOR]

While interest in psychedelic drugs in the fields of psychiatry and neuroscience has re-emerged in recent last decades, the general understanding of the effects of these drugs remains deficient. In particular, there are gaps in knowledge on executive functions and goal-directed behaviors both in humans and in commonly used animal models. The effects of acute doses of psychedelic lysergic acid diethylamide (LSD) on reward-driven decision making were explored using the mouse version of the Iowa Gambling Task. A total of 15 mice were trained to perform in a touch-screen adaptation of the rodent version of the Iowa Gambling Task, after which single acute doses of LSD (0.025, 0.1, 0.2, 0.4 mg/kg), serotonin 2A receptor-selective agonist 25CN-NBOH (1.5 mg/kg), d-amphetamine (2.0 mg/kg), and saline were administered before the trial. 25CN-NBOH and the three lowest doses of LSD showed no statistically significant changes in option selection or in general functioning during the gambling task trials. The highest dose of LSD (0.4 mg/kg) significantly decreased premature responding and increased the omission rate, but had no effect on option selection in comparison with the saline control. Amphetamine significantly decreased the correct responses and premature responding while increasing the omission rate. In conclusion, mice can perform previously learned, reward-driven decision-making tasks while under the acute influence of LSD at a commonly used dose range. [ABSTRACT FROM AUTHOR]