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Previous work from this laboratory indicates that the KDI (Lys-Asp-Ile) domain of gamma 1-laminin promotes functional regeneration of adult rat spinal cord injuries and protects adult rat hippocampal neurons against massive neuronal death induced by intracerebral injection of the glutamate analogue kainic acid. In the present study, we used patch clamp recordings on cultured human embryonic neocortical neurons and HEK 293 cells expressing recombinant glutamate receptor subunits to study a putative interaction of the KDI with the glutamate system. We show that the KDI domain of gamma 1-laminin is a universal and potent inhibitor of AMPA, kainate, and NMDA subclasses of glutamate receptors, with a noncompetitive action on the AMPA receptor channel activity. Glutamate neurotoxicity plays a key role in both CNS trauma and neurodegenerative disorders, so this unexpected, novel function of the gamma 1-laminin-derived tripeptide may prove clinically valuable in treatment of CNS trauma and/or disease., ((c) 2005 Wiley-Liss, Inc.)

Inhaled anesthetics are believed to produce anesthesia by their actions on ion channels. Because inhaled anesthetics robustly enhance GABA A receptor (GABA(A)-R) responses to GABA, these receptors are considered prime targets of anesthetic action. However, the importance of GABA(A)-Rs and individual GABA(A)-R subunits to specific anesthetic-induced behavioral effects in the intact animal is unknown. We hypothesized that inhaled anesthetics produce amnesia, as assessed by loss of fear conditioning, by acting on the forebrain GABA(A)-Rs that harbor the alpha1 subunit. To test this, we used global knockout mice that completely lack the alpha1 subunit and forebrain-specific, conditional knockout mice that lack the alpha1 subunit only in the hippocampus, cortex, and amygdala. Both knockout mice were 75 to 145% less sensitive to the amnestic effects of the inhaled anesthetic isoflurane. These results indicate that alpha1-containing GABA(A)-Rs in the hippocampus, amygdala, and/or cortex influence the amnestic effects of inhaled anesthetics and may be an important molecular target of the drug isoflurane.

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 gamma2 subunit is highly expressed throughout the brain. Global gamma2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous gamma2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the gamma2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated gamma2 expression, i.e., gamma2 knockdown mice., Results: Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the gamma2 gene. Knockdown mice, on average, showed a 65% reduction of gamma2 subunit mRNA compared to controls; however gamma2 gene expression was highly variable in these mice, ranging from 10-95% of normal. Immunohistochemical studies demonstrated that gamma2 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 gamma2 knockdown mouse line can be used to create gamma2 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 gamma2 gene variably reduced the amount of gamma2, and that 2) attenuated expression of gamma2 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.

GABAA receptors mediate fast phasic inhibitory postsynaptic potentials and participate in slower tonic extrasynaptic inhibition. Thy1alpha6 mice with ectopic forebrain expression of GABAA receptor alpha6 subunits exhibit increased extrasynaptic GABAA receptor-mediated background conductance and reduced synaptic GABAA receptor currents in hippocampal CA1 neurons [W. Wisden et al. (2002) Neuropharmacology 43, 530-549]. Here we demonstrate that isolated CA1 neurons of these mice showed furosemide-sensitivity of GABA-evoked currents, confirming the functional expression of alpha6 subunit. In addition, receptor autoradiography of the CA1 region of Thy1alpha6 brain sections revealed pharmacological features that are unique for alpha6betagamma2 and alpha6beta receptors. The existence of atypical alpha6beta receptors was confirmed after completely eliminating GABAA receptors containing gamma1, gamma2, gamma3 or delta subunits using serial immunoaffinity chromatography on subunit-specific GABAA receptor antibodies. Behaviourally, the Thy1alpha6 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 Thy1alpha6 mice, but not in control mice; however, the overall effect of higher tiagabine doses on the convulsion latency remained smaller in the Thy1alpha6 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.

To image the possible alterations in brain regional GABAA receptor subtype properties in a genetic animal model of human anxiety, mice heterozygous for the deletion of GABAA receptor gamma2 subunit (gamma2+/-) were studied using ligand autoradiographic assays on brain cryostat sections. The [35S]TBPS binding assay was designed to reveal impaired GABA and channel site coupling shown to be more prominent in recombinant alpha1/6beta3 than in alpha1/2beta3gamma2 or beta2 subunit-containing GABAA receptors expressed in HEK 293 cells. Increased GABA-insensitive [35 S]TBPS binding in the gamma2+/- mouse brains was evident in the cerebral cortex and in subcortical regions, the alterations being regionally similar to the loss of gamma2 subnunit-dependent benzodiazepine (BZ) sites as revealed by [3H]Ro 15-4513 autoradiography. As the gamma2 subunit protein is needed for synaptic clustering of GABAA receptors, these results indicate that the extrasynaptic alphabeta3 receptors can be visualized in vitro as atypical GABA-insensitive [35S]TBPS binding sites. The results suggest that GABAAergic synaptic inhibition is widely decreased in the brains of anxiety-prone gamma2+/- mice, while extrasynaptic GABAA receptors are increased. These autoradiographic imaging findings further demonstrate the need to develop GABAA receptor subtype-selective in vivo ligands to aid in assessing the contributions of various subcellular receptor populations in anxious and other patient groups.

We have utilized quantitative autoradiography with the GABA(A) receptor chloride channel blocker [35S]t-butylbicyclophosphorothionate ([35S]TBPS) in rodent brain sections to investigate if differential proton modulation of various GABA(A) receptor subtypes expressed in various brain regions are differentially sensitive to pH alternations. Acidic and basic pHs decreased the binding, the mean values at pH 5.4 and pH 9.4 being 17% and 76% of the binding at pH 7.4, respectively. The regional profiles of the pH effects could be divided into two types. In regions with high basal binding at pH 7.4. the pH profile was usually 'bell-shaped,' with maximal binding at pH 7.4 (type 1). In regions with low basal binding at pH 7.4, the pH profile (type 2) revealed very low binding at pH 5.4, lower sensitivity to high pH, and usually maximal binding at pH 8.4. In brain regions with type 1 pH modulation alpha1 and beta2 subunits are abundantly expressed, whereas alpha2 and beta3 subunits are abundant in type 2 regions. Therefore the alpha1beta2gamma2 and alpha2beta3gamma2 receptor subtypes are suggested to be preferentially responsible for brain regional heterogeneity of the pH modulation of [35S]TBPS binding.

Ethanol actions on alpha-amino-3-hydyroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors were studied using voltage-clamp recordings from mouse cortical and hippocampal neurons. During whole-cell recordings ethanol (EtOH) inhibited AMPA receptor-mediated currents in a dose-dependent manner at concentrations from 10 to 500 mM. The steady-state component of AMPA-activated current was more sensitive to EtOH than the peak component. To examine the effect of EtOH on a well resolved peak current component, patches were excised from cultured cortical neurons, to which AMPA and EtOH were applied using a piezoelectric solution application system. Under this condition, the peak current was not inhibited significantly by EtOH. To further study possible mechanisms of EtOH inhibition, kainate and AMPA were used to evoke currents in the absence and presence of cyclothiazide. Ethanol inhibition was stronger when receptors were activated by low than high kainate concentrations. Cyclothiazide reduced inhibition by EtOH regardless of the agonist used to activate the receptor. Finally, EtOH inhibition was reduced in a point mutated (L497Y) GluRAi receptor that lacks desensitization. These findings suggest that EtOH inhibits AMPA receptors by stabilizing the desensitized state. Our results can explain some of the variation observed in EtOH inhibition in previous studies, and support the idea that physiologically relevant concentrations of EtOH can have a strong effect on AMPA receptor function.

We studied the effect of a novel delta-opioid receptor antagonist N,N(CH(3))(2)Dmt-Tic-OH (Me(2)-Dmt-Tic-OH) on voluntary ethanol intake in an alcohol-preferring AA (Alko, Alcohol) rat line using a 4-hour limited access paradigm. Acute injections of Me(2)-Dmt-Tic-OH (10 and 30 mg/kg, i.p.) did not reduce 1-hour or 4-hour ethanol intake. Subtype non-selective opioid receptor antagonist naltrexone [0.1 and 0.3 mg/kg, subcutaneously (s.c.)] significantly reduced 1-hour ethanol drinking but had no effect on 4-hour ethanol consumption. Locomotor stimulation induced by the delta-opioid receptor agonist Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE; 15 microg, intracerebroventricularly) was significantly attenuated by Me(2)-Dmt-Tic-OH (10 and 30 mg/kg, i.p.), which confirmed its efficacy as a delta-opioid receptor antagonist in rat brain. Our results support the idea that delta-opioid receptors do not mediate alcohol reward in AA rats.

Development of tolerance to motor-impairing effects of repeated administration of moderate diazepam doses (5.0-7.5 mg/kg; three times daily PO 3 weeks) was compared between mice deficient in the cerebellar granule cell-restricted GABAA receptor alpha6 subunit and their wild-type controls. The alpha6 -/- mice were more impaired by the initial challenge doses of diazepam (5 or 10 mg/kg) than their controls, but acquired partial tolerance by the second tests with the same doses 4-7 days later. Chronic treatment produced complete tolerance in both mouse lines. Ligand autoradiography revealed a significant reduction in baseline benzodiazepine and chloride channel site-bindings in various regions of the alpha6 -/- brains, but the chronic diazepam treatment did not consistently alter baseline or benzodiazepine site agonist and inverse agonist-modulated binding in the alpha6 -/- and wildtype mice. The results indicate that tolerance to motor-impairing actions of diazepam is independent of the diazepam-insensitive alpha6 subunit-containing receptors, which rules out the possibility that tolerance emerges as an increase in structurally benzodiazepine-insensitive receptor population.

The possible effect of atipamezole, a potent and specific alpha(2)-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 alpha(2)-adrenoceptor antagonists. Challenge doses of d-amphetamine were administered to the mice on days 7-9 to evaluate the effects of alpha(2)-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 alpha(2)-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 alpha(2)-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.