PubMed Health⌕ Search

Biomedical subjects

W Wisden

Publications and source records attributed to W Wisden.

At least 19 recordsLinked to original sources
PubMed2005

Agonistic effects of the beta-carboline DMCM revealed in GABA(A) receptor gamma 2 subunit F77I point-mutated mice.

Affinity of the inverse agonist methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) to the benzodiazepine binding site of the GABA(A) receptor is abolished by a phenylalanine (F) to isoleucine (I) substitution at position 77 of the gamma2 subunit. We tested the effects of DMCM in gene knockin gamma2I77 mice carrying this mutation. Unlike in wild-type mice, DMCM was not able to reverse the GABA-induced reduction of the picrotoxin-sensitive t-butylbicyclophosphoro-[35S]thionate ([35S]TBPS) binding to GABA(A) receptor channels in the forebrain sections of gamma2I77 mice. Accordingly, DMCM was not convulsant in the mutant mice even at doses 20-fold higher (60mg/kg, i.p.) than those producing convulsions in wild-type littermate controls (3 mg/kg, i.p.). Neither did DMCM raise the c-Fos levels in gamma2I77 mouse brain. DMCM additionally exhibits a less well described agonistic effect on GABA(A) receptors that is normally masked by its strong inverse agonist effect. DMCM agonistically enhanced the GABA-induced reduction in [35S]TBPS binding to the cerebellar granule cell layer in control and mutant mice. In vivo DMCM (20-60 mg/kg i.p.) produced modest anxiolytic-like effects in gamma2I77 mice as assessed by elevated plus maze and staircase tests, but no motor impairment was found in the rotarod test. The results suggest only minor agonistic efficacy for the beta-carboline DMCM.

Animals↗
PubMed2005

Loss of zolpidem efficacy in the hippocampus of mice with the GABAA receptor gamma2 F77I point mutation.

Zolpidem is a hypnotic benzodiazepine site agonist with some gamma-aminobutyric acid (GABA)(A) receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of gamma2 subunit (gamma2F77I) point mutant mice. Analysis of forebrain GABA(A) receptor expression with immunocytochemistry, quantitative [(3)H]muscimol and [(35)S] t-butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with [(3)H]flunitrazepam and [(3)H]muscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous gamma2I77/I77 and gamma2F77/F77 mice. However, quantitative immunoblot analysis of gamma2I77/I77 hippocampi showed some increased levels of gamma2, alpha1, alpha4 and delta subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 microm) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL/6J, gamma2F77/F77) mice by approximately 60%, and peak amplitude by approximately 20% at 33-34 degrees C in vitro. The actions of zolpidem (100 nm or 1 microm) were substantially reduced in gamma2I77/I77 mice, although residual effects included a 9% increase in decay and 5% decrease in peak amplitude. Similar results were observed in CA1 stratum oriens/alveus interneurons. At network level, the effect of zolpidem (10 microm) on carbachol-induced oscillations in the CA3 area of gamma2I77/I77 mice was significantly different compared with controls. Thus, the gamma2F77I point mutation virtually abolished the actions of zolpidem on GABA(A) receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the gamma2 subunit.

Action Potentials↗
PubMed2004

Abolition of zolpidem sensitivity in mice with a point mutation in the GABAA receptor gamma2 subunit.

Agonists of the allosteric benzodiazepine site of GABAA receptors bind at the interface of the alpha and gamma subunits. Here, we tested the in vivo contribution of the gamma2 subunit to the actions of zolpidem, an alpha1 subunit selective benzodiazepine agonist, by generating mice with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the gamma2 subunit. The gamma2F77I mutation has no major effect on the expression of GABAA receptor subunits in the cerebellum. The potency of zolpidem, but not that of flurazepam, for the inhibition of [3H]flunitrazepam binding to cerebellar membranes is greatly reduced in gamma2I77/I77 mice. Zolpidem (1 microM) increased both the amplitude and decay of miniature inhibitory postsynaptic currents (mIPSCs) in Purkinje cells of control C57BL/6 (34% and 92%, respectively) and gamma2F77/F77 (20% and 84%) mice, but not in those of gamma2F77I mice. Zolpidem tartrate had no effect on exploratory activity (staircase test) or motor performance (rotarod test) in gamma2I77/I77 mice at doses up to 30 mg/kg (i.p.) that strongly sedated or impaired the control mice. Flurazepam was equally effective in enhancing mIPSCs and disrupting performance in the rotarod test in control and gamma2I77/I77 mice. These results show that the effect of zolpidem, but not flurazepam, is selectively eliminated in the brain by the gamma2F77I point mutation.

Amino Acid Substitution↗
PubMed2003

Cerebellar granule cell Cre recombinase expression.

The cerebellum maintains balance and orientation, refines motor action, stores motor memories, and contributes to the timing aspects of cognition. We generated two mouse lines for making Cre recombinase-mediated gene disruptions largely confined to adult cerebellar granule cells. For this purpose we chose the GABA(A) receptor alpha6 subunit gene, whose expression marks this cell type. Here we describe mouse lines expressing Cre recombinase generated by 1) Cre knocked into the native alpha6 subunit gene by homologous recombination in embryonic stem cells; and 2) Cre recombined into an alpha6 subunit gene carried on a bacterial artificial chromosome (BAC) genomic clone. The fidelity of Cre expression was tested by crossing the mouse lines with the ROSA26 reporter mice. The particular alpha6BAC clone we identified will be valuable for delivering other gene products to cerebellar granule cells.

Animals↗
PubMed2002

Ectopic expression of the GABA(A) receptor alpha6 subunit in hippocampal pyramidal neurons produces extrasynaptic receptors and an increased tonic inhibition.

We generated transgenic (Thy1alpha6) mice in which the GABA(A) receptor alpha6 subunit, whose expression is usually confined to granule cells of cerebellum and cochlear nuclei, is ectopically expressed under the control of the pan-neuronal Thy-1.2 promoter. Strong Thy1alpha6 subunit expression occurs, for example, in deep cerebellar nuclei, layer V iscocortical and hippocampal pyramidal cells and dentate granule cells. Ligand binding and protein biochemistry show that most forebrain alpha6 subunits assemble as alpha6betagamma2-type receptors, and some as alpha1alpha6betagamma2 and alpha3alpha6betagamma2 receptors. Electron microscopic immunogold labeling shows that most Thy1-derived alpha6 immunoreactivity is in the extrasynaptic plasma membrane of dendrites and spines in both layer V isocortical and CA1pyramidal cells. Synaptic immunolabeling is rare. Consistent with the alpha6 subunits' extrasynaptic localization, Thy1alpha6 CA1 pyramidal neurons have a five-fold increased tonic GABA(A) receptor-mediated current compared with wild-type cells; however, the spontaneous IPSC frequency and the mIPSC amplitude in Thy1alpha6 mice decrease 37 and 30%, respectively compared with wild-type. Our results strengthen the idea that GABA(A) receptors containing alpha6 subunits can function as extrasynaptic receptors responsible for tonic inhibition and further suggest that a homeostatic mechanism might operate, whereby increased tonic inhibition causes a compensatory decrease in synaptic GABA(A) receptor responses.

Action Potentials↗
PubMed2001

Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance.

Many neurons receive a continuous, or 'tonic', synaptic input, which increases their membrane conductance, and so modifies the spatial and temporal integration of excitatory signals. In cerebellar granule cells, although the frequency of inhibitory synaptic currents is relatively low, the spillover of synaptically released GABA (gamma-aminobutyric acid) gives rise to a persistent conductance mediated by the GABA A receptor that also modifies the excitability of granule cells. Here we show that this tonic conductance is absent in granule cells that lack the alpha6 and delta-subunits of the GABAA receptor. The response of these granule cells to excitatory synaptic input remains unaltered, owing to an increase in a 'leak' conductance, which is present at rest, with properties characteristic of the two-pore-domain K+ channel TASK-1 (refs 9,10,11,12). Our results highlight the importance of tonic inhibition mediated by GABAA receptors, loss of which triggers a form of homeostatic plasticity leading to a change in the magnitude of a voltage-independent K + conductance that maintains normal neuronal behaviour.

Adaptation, Physiological↗
PubMed2001

Expression of GABA(A) receptor subunits in rat brainstem auditory pathways: cochlear nuclei, superior olivary complex and nucleus of the lateral lemniscus.

Inhibition by GABA is important for auditory processing, but any adaptations of the ionotropic type A receptors are unknown. Here we describe, using in situ hybridization, the subunit expression patterns of GABA(A) receptors in the rat cochlear nucleus, superior olivary complex, and dorsal and ventral nuclei of the lateral lemniscus. All neurons express the beta3 and gamma2L subunit messenger RNAs, but use different alpha subunits. In the dorsal cochlear nucleus, fusiform (pyramidal) and giant cells express alpha1, alpha3, beta3 and gamma2L. Dorsal cochlear nucleus interneurons, particularly vertical or tuberculoventral cells and cartwheel cells, express alpha3, beta3 and gamma2L. In the ventral cochlear nucleus, octopus cells express alpha1, beta3, gamma2L and delta. Spherical cells express alpha1, alpha3, alpha5, beta3 and gamma2L. In the superior olivary complex, the expression profile is alpha3, alpha5, beta3 and gamma2L. Both dorsal and ventral cochlear nucleus granule cells express alpha1, alpha6, beta3 and gamma2L; unlike their cerebellar granule cell counterparts, they do not express beta2, gamma2S or the delta subunit genes. The delta subunit's absence from cochlear nucleus granule cells may mean that tonic inhibition mediated by extrasynaptic GABA(A) receptors is less important for this cell type. In both the dorsal and ventral nuclei of the lateral lemniscus, alpha1, beta3 and gamma2L are the main subunit messenger RNAs; the ventral nucleus also expresses the delta subunit. We have mapped, using in situ hybridization, the subunit expression patterns of the GABA(A) receptor in the auditory brainstem nuclei. In contrast to many brain regions, the beta2 subunit gene and gamma2S splice forms are not highly expressed in auditory brainstem nuclei. GABA(A) receptors containing beta3 and gamma2L may be particularly well suited to auditory processing, possibly because of the unique phosphorylation profile of this subunit combination.

Animals↗
PubMed2001

GABA(A) receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1.

Controlling the number of functional gamma-aminobutyric acid A (GABA(A)) receptors in neuronal membranes is a crucial factor for the efficacy of inhibitory neurotransmission. Here we describe the direct interaction of GABA(A) receptors with the ubiquitin-like protein Plic-1. Furthermore, Plic-1 is enriched at inhibitory synapses and is associated with subsynaptic membranes. Functionally, Plic-1 facilitates GABA(A) receptor cell surface expression without affecting the rate of receptor internalization. Plic-1 also enhances the stability of intracellular GABA(A) receptor subunits, increasing the number of receptors available for insertion into the plasma membrane. Our study identifies a previously unknown role for Plic-1, a modulation of GABA(A) receptor cell surface number, which suggests that Plic-1 facilitates accumulation of these receptors in dendritic membranes.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden, W., 1997. Ligand-gated ion channel partnerships: GABA(A) receptor alpha(6) subunit inactivation inhibits delta subunit expression. Journal of Neuroscience 17, 1350-1362).

Animals↗
PubMed2000

Long-range interactions in neuronal gene expression: evidence from gene targeting in the GABA(A) receptor beta2-alpha6-alpha1-gamma2 subunit gene cluster.

Clustering of GABA(A) receptor alpha1, alpha6, beta2, and gamma2 subunit genes on mouse chromosome 11/human chromosome 5 may have functional significance for coordinating expression patterns, but until now there has been no evidence for cross-talk between the genes. However, altering the structure of the alpha6 gene, specifically expressed in the cerebellum, with neomycin gene insertions in two different experiments unexpectedly reduced the expression of the widespread alpha1 and beta2 genes in the forebrain. There were corresponding reductions in the levels of alpha1 and beta2 subunit proteins and in autoradiographic ligand binding densities to GABA(A) receptors in the forebrain of alpha6-/- mice. The gamma2 mRNA level was not changed, nor were beta3 and delta mRNAs. The data suggest that elements in the neo gene may have an influence over long distances in the GABA(A) subunit gene complex on as yet undefined structures coordinating the expression of the alpha1 and beta2 genes.

Animals↗
PubMed2000

Expression of the neuronal calcium sensor protein family in the rat brain.

The neuronal calcium sensor proteins are members of the calcium-binding protein superfamily. They control localized calcium signalling on membranes and may make G-protein cascades sensitive to cytosolic calcium. The family members are recoverin (visinin, S-modulin), neuronal calcium sensor-1 (frequenin), hippocalcin, neuronal visinin-like protein-1 (visinin-like protein, neurocalcin-alpha), neuronal visinin-like protein-2 and neuronal visinin-like protein-3. Recoverin is expressed only in the retina and pineal gland. Using in situ hybridization, we mapped the expression of the other neuronal calcium sensor protein genes in the adult rat brain. Neuronal visinin-like protein-1 messenger RNA has a widespread distribution and is abundant in all brain areas except the caudate-putamen. Neuronal calcium sensor-1 gene expression is pan-neuronal. Neuronal calcium sensor-1 messenger RNA is present in the dendrites of hippocampal pyramidal and granule cells, suggesting a specific role in dendritic function. Hippocalcin and neuronal visinin-like protein-2 are mainly expressed in the forebrain and have similar expression patterns (neocortex, hippocampus and caudate-putamen). Neuronal visinin-like protein-3 has the most restricted expression; its highest expression level is in the cerebellum (Purkinje and granule cells). However, the neuronal visinin-like protein-3 gene is also expressed in many ventral nuclei throughout the fore- and midbrain, in the medial habenulae, and in the superior and inferior colliculi. The neuronal calcium sensor proteins are a relatively unexplored family of Ca(2+)-binding proteins. They are likely to be involved in many diverse areas of neuronal signalling. In this paper, we describe their expression in the rat brain as determined by in situ hybridization. As all five neuronal calcium sensor protein genes have distinctive expression patterns, they probably perform specific functions.

Animals↗
PubMed1999

Loreclezole and La3+ differentiate cerebellar granule cell GABA(A) receptor subtypes.

The effects of loreclezole and La3+ on native cerebellar GABA(A) receptors were compared between GABA(A) receptor alpha6 subunit-deficient (alpha6-/-) and wildtype mouse lines, produced through homologous recombination, using t-[35S]butylbicyclophosphorothionate ([35S]TBPS) autoradiography in brain sections. In the alpha6 subunit-deficient mice, the GABA receptor antagonistic ability of La3+ was abolished in the cerebellar granule cell layer, consistent with its opposite actions on alpha6- and of alpha1 subunit-containing receptors. La3+ significantly potentiated the action of GABA in the molecular layer of the alpha6-/- mice, but not in that of the wildtype mice. The potentiation of agonistic GABA inhibition of [35S]TBPS binding by loreclezole in alpha6-/- granule cells was reduced, suggesting an emergence of low-affinity GABA(A) receptors. The present results thus identified two ligands that may be useful in studying functional roles of cerebellar alpha1 and alpha6 subunit-containing GABA(A) receptor subtypes.

Animals↗
PubMed1999

Cerebellar granule-cell-specific GABAA receptors attenuate benzodiazepine-induced ataxia: evidence from alpha 6-subunit-deficient mice.

Benzodiazepine- and alcohol-induced ataxias in rodents have been proposed to be affected by the gamma-aminobutyric acid type A (GABAA) receptor alpha 6 subunit, which contributes to receptors specifically expressed in cerebellar granule cells. We have studied an alpha 6 -/- 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 alpha 6 -/- and alpha 6 +/+ mice. On the rotarod test, however, the mutant mice were significantly more impaired by diazepam (5-20 mg/kg, i.p.), when compared with alpha 6 +/+ control and background C57BL/6J and 129/SvJ mouse lines. Ethanol (2.0-2.5 g/kg, i.p.) produced similar impairment in the alpha 6 -/- and alpha +/+ mice. Diazepam-induced ataxia in alpha 6 -/- mice could be reversed by the benzodiazepine site antagonist flumazenil, indicating the involvement of the remaining alpha 1 beta 2/3 gamma 2 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 alpha 6 subunit-dependent actions in the cerebellar cortex can be compensated by other receptor subtypes; but if not for the alpha 6 subunit, patients on benzodiazepine medication would suffer considerably from ataxic side-effects.

Animals↗