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W Wisden

Publications and source records attributed to W Wisden.

At least 37 records · Page 2Linked to original sources
PubMed1999

Alterations in the expression of GABAA receptor subunits in cerebellar granule cells after the disruption of the alpha6 subunit gene.

Any given subunit of the heteromultimeric type-A gamma-aminobutyric acid (GABA) GABAA receptor may be present in several receptor subtypes expressed by individual neurons. Changes in the expression of a subunit may result in differential changes in the expression of other subunits depending on the subunit composition of the receptor subtype, leading to alterations in neuronal responsiveness to GABA. We used the targeted disruption of the alpha6 subunit gene to test for changes in the expression of other GABAA receptor subunits. Immunoprecipitation and ligand binding experiments indicated that GABAA receptors were reduced by approximately 50% in the cerebellum of alpha6 -/- mice. Western blot experiments indicated that the alpha6 subunit protein completely disappeared from the cerebellum of alpha6 -/- mice, which resulted in the disappearance of the delta subunit from the plasma membrane of granule cells. The amount of beta2, beta3 and gamma2 subunits was reduced by approximately 50%, 20% and 40%, respectively, in the cerebella of alpha6 -/- mice. A comparison of the reduction in the level of alpha1, beta2, beta3, gamma2, or delta-subunit-containing receptors in alpha6 -/- cerebellum with those observed after removal of alpha6-subunit-containing receptors from the cerebella of alpha6 +/+ mice by immuno-affinity chromatography demonstrated the presence of a significantly higher than expected proportion of receptors containing beta3 subunits in alpha6 -/- mice. The receptors containing alpha1, beta2, beta3 and gamma2 subunits were present in the plasma membrane of granule cells of alpha6 -/- mice at both synaptic and extrasynaptic sites, as shown by electron microscopic immunocytochemistry. Despite the changes, the alpha1 subunit content of Golgi-cell-to-granule-cell synapses in alpha6 -/- animals remained unaltered, as did the frequency of alpha1 immunopositive synapses in the glomeruli. Furthermore, no change was apparent in the expression of the alpha1, beta2 and gamma2 subunits in Purkinje cells and interneurons of the molecular layer. These results demonstrate that in alpha6 -/- mice, the cerebellum expresses only half of the number of GABAA receptors present in wild-type animals. Since these animals have no gross motor deficits, synaptic integration in granule cells is apparently maintained by alpha1-subunit-containing receptors with an altered overall subunit composition, and/or by changes in the expression of other ligand and voltage gated channels.

Animals↗
PubMed1999

The intrinsic specification of gamma-aminobutyric acid type A receptor alpha6 subunit gene expression in cerebellar granule cells.

The patterns of gamma-aminobutyric acid type A (GABAA) receptor subunit gene expression in the brain are complex. For example, mouse hippocampal dentate granule cells express many subunit genes, whereas adult cerebellar granule cells, which may share differentiation mechanisms, have a smaller compliment and uniquely express the alpha6 subunit gene. To see how the alpha6 expression component arises, i.e. if intrinsically or environmentally specified, we used a mouse line (Deltaalpha6lacZ) with a beta-galactosidase reporter inserted into the alpha6 gene. Precursor cells from postnatal day 1 Deltaalpha6lacZ cerebellum were transplanted to the adult hippocampus and cerebellum of wild-type mice; 4 weeks after transplantation, Deltaalpha6lacZ cells expressed alpha6-lacZ in the hippocampus, amygdala and cerebellum. Thus, different adult environments support both the development and maintenance of alpha6 gene expression from cerebellar granule cell precursors. Establishing alpha6 gene expression is not likely to require specific patterns of neurotransmitter innervation or other factors present only in the developing brain; instead, alpha6 expression can be timed and maintained autonomously.

Amygdala↗
PubMed1998

Interleukin (IL)-4-independent induction of immunoglobulin (Ig)E, and perturbation of T cell development in transgenic mice expressing IL-13.

Recent studies using interleukin (IL)-4-deficient animals have highlighted the existence of IL-4-independent immunoglobulin (Ig)E induction. We have established transgenic mice expressing IL-13 from a transgene comprising a genomic fragment containing the IL-13 gene and the human CD2 locus control region. The transgenes were expressed in lymphoid tissues and induced by T cell activators, suggesting regulation by elements of the IL-13 promoter. IL-13 transgenic lines expressed 10-100-fold higher levels of serum IgE than their littermate controls, but had normal levels of other serum Ig isotypes. Elevated IgE levels were also detected in sera from IL-4-deficient mice carrying IL-13 transgenes, indicating that IL-4 is not required for IL-13-induced IgE expression in the mouse. Expression of IL-13 also perturbed the development of thymocytes. Although thymocyte development was normal up to 4 wk of age, thymocyte number decreased dramatically thereafter, reaching 10% of normal by 10 wk, and despite normal size and appearance, histological examination demonstrated that transgenic thymi contained only small foci of thymocytes. The reduction in thymocyte number was due mainly to a depletion of CD4(+)CD8(+) thymocytes, and did not affect significantly the composition of peripheral T cell populations. These data indicate that expression of IL-13 transgenes in vivo can regulate IgE production in the mouse, and that IL-13 may also influence thymocyte development.

Animals↗
PubMed1998

Mouse cerebellar granule cell differentiation: electrical activity regulates the GABAA receptor alpha 6 subunit gene.

GABAA receptor alpha6 subunit gene expression marks cerebellar granule cell maturation. To study this process, we used the Deltaalpha6lacZ mouse line, which has a lacZ reporter inserted into the alpha6 gene. At early stages of postnatal cerebellar development, alpha6-lacZ expression is mosaic; expression starts at postnatal day 5 in lobules 9 and 10, and alpha6-lacZ is switched on inside-out, appearing first in the deepest postmigratory granule cells. We looked for factors regulating this expression in cell culture. Membrane depolarization correlates inversely with alpha6-lacZ expression: granule cells grown in 25 mM [K+]o for 11-15 d do not express the alpha6 gene, whereas cultures grown for the same period in 5 mM [K+]o do. This is influenced by a critical early period: culturing for >/=3 d in 25 mM [K+]o curtails the ability to induce the alpha6 gene on transfer to 5 mM [K+]o. If the cells start in 5 mM [K+]o, however, they still express the alpha6-lacZ gene in 25 mM [K+]o. In contrast to granule cells grown in 5 mM [K+]o, cells cultured in 25 mM [K+]o exhibit no action potentials, mEPSCs, or mIPSCs. In chronic 5 mM [K+]o, factors may therefore be released that induce alpha6. Blockade of ionotropic and metabotropic GABA and glutamate receptors or L-, N-, and P/Q-type Ca2+ channels did not prevent alpha6-lacZ expression, but inhibition of action potentials with tetrodotoxin blocked expression in a subpopulation of cells.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

DNA regions supporting hippocalcin gene expression in cell lines.

The rat hippocalcin gene -3.2 to +0.6 kb region activates reporter gene expression in the NG108-15 and PC12 neuronal cell lines, but not in NIH3T3 or HEK-293 cells. Three fragments (-3.2 to -2.6, -2.6 to -2.3 and -2.3 to -1.8 kb) weakly activate transcription, and "-1.8 to -1.5" kb is a strong activator. Thus cell type-specific expression of the rat hippocalcin gene is regulated by distributed elements in the -3.2 to -1.5 kb region.

3T3 Cells↗
PubMed1997

[Better cure for anxiety, sleep disorders, epilepsy?].

Gamma-aminobutyric acid (GABA), the most important inhibitory neurotransmitter, is characterised by rapid effects that are mediated via GABAA, receptors. These receptors are also targets for many drugs including benzodiazepines, barbiturates and general anaesthetics. Recognition of the heterogeneity of GABAA receptors has opened up new possibilities for the development of more selective therapeutic agents. In particular, subtype-specific receptor ligands varying in intrinsic activity are likely to give rise to fewer side effects than do currently available drugs.

Anti-Anxiety Agents↗
PubMed1997

Directing gene expression to cerebellar granule cells using gamma-aminobutyric acid type A receptor alpha6 subunit transgenes.

Expression of the gamma-aminobutyric acid type A receptor alpha6 subunit gene is restricted to differentiated granule cells of the cerebellum and cochlear nucleus. The mechanisms underlying this limited expression are unknown. Here we have characterized the expression of a series of alpha6-based transgenes in adult mouse brain. A DNA fragment containing a 1-kb portion upstream of the start site(s), together with exons 1-8, can direct high-level cerebellar granule cell-specific reporter gene expression. Thus powerful granule cell-specific determinants reside within the 5' half of the alpha6 subunit gene body. This intron-containing transgene appears to lack the cochlear nucleus regulatory elements. It therefore provides a cassette to deliver gene products solely to adult cerebellar granule cells.

Animals↗
PubMed1997

Ligand-gated ion channel subunit partnerships: GABAA receptor alpha6 subunit gene inactivation inhibits delta subunit expression.

Cerebellar granule cells express six GABAA receptor subunits abundantly (alpha1, alpha6, beta2, beta3, gamma2, and delta) and assemble various pentameric receptor subtypes with unknown subunit compositions; however, the rules guiding receptor subunit assembly are unclear. Here, removal of intact alpha6 protein from cerebellar granule cells allowed perturbations in other subunit levels to be studied. Exon 8 of the mouse alpha6 subunit gene was disrupted by homologous recombination. In alpha6 -/- granule cells, the delta subunit was selectively degraded as seen by immunoprecipitation, immunocytochemistry, and immunoblot analysis with delta subunit-specific antibodies. The delta subunit mRNA was present at wild-type levels in the mutant granule cells, indicating a post-translational loss of the delta subunit. These results provide genetic evidence for a specific association between the alpha6 and delta subunits. Because in alpha6 -/- neurons the remaining alpha1, beta2/3, and gamma2 subunits cannot rescue the delta subunit, certain potential subunit combinations may not be found in wild-type cells.

Animals↗
PubMed1997

Prominent dendritic localization in forebrain neurons of a novel mRNA and its product, dendrin.

A recently cloned rat brain cDNA derives from a novel gene, termed dendrin (DEN), expressed exclusively in forebrain structures, particularly in neocortex, olfactory bulb, hippocampus, caudate-putamen, and limbic system. In these structures, the cognate mRNA is present in neuronal cell bodies and their dendrites, whereas near exclusive dendritic localization is observed for the polypeptide product. In the hippocamus, DEN mRNA is highly expressed in the cell laminae and dendritic layers of the dentate gyrus and CA1 field, but expression is markedly reduced in the CA3 and CA4 areas. The predicted primary structure of the hydrophilic, highly basic 653-amino-acid polypeptide does not suggest a function. The restricted expression and dendritic location are compatible with a role for DEN in synaptic plasticity of central neocortical forebrain neurons.

Amino Acid Sequence↗
PubMed1997

Cerebellar gamma-aminobutyric acid type A receptors: pharmacological subtypes revealed by mutant mouse lines.

The vast molecular heterogeneity of brain gamma-aminobutyric acid type A (GABAA) receptors forms the basis for receptor subtyping. Using autoradiographic techniques, we established the characteristics of cerebellar granule cell GABAA receptors by comparing wild-type mice with those with a targeted disruption of the alpha6 subunit gene. Cerebellar granule cells of alpha6(-/-) animals have severe deficits in high affinity [3H]muscimol and [3H]SR 95531 binding to GABA sites, in agonist-insensitive [3H]Ro 15-4513 binding to benzodiazepine sites, and in furosemide-induced increases in tert-[35S]butylbicyclophosphorothionate binding to picrotoxin-sensitive convulsant sites. These observations agree with the known specific properties of these sites on recombinant alpha6beta2/3gamma2 receptors. In the presence of GABA concentrations that fail to activate alpha1 subunit-containing receptors, methyl-6,7-dimethoxy-4-ethyl-beta-carboline (30 microM), allopregnanolone (100 nM), and Zn2+ (10 microM) are less efficacious in altering tert-[35S]butylbicyclophosphorothionate binding in the granule cell layer of the alpha6(-/-) than alpha6(+/+) animals. These data concur with the deficiency of the cerebellar alpha6 and delta subunit-containing receptors in the alpha6(-/-) animals and could also account for the decreased affinity of [3H]muscimol binding to alpha6(-/-) cerebellar membranes. Predicted additional alterations in the cerebellar receptors of the mutant mice may explain a surplus of methyl-6,7-dimethoxy-4-ethyl-beta-carboline-insensitive receptors in the alpha6(-/-) granule cell layer and an increased diazepam-sensitivity in the molecular layer. These changes may be adaptive consequences of altered GABAA receptor subunit expression patterns in response to the loss of two subunits (alpha and delta) from granule cells.

Animals↗
PubMed1997

GABA(A)-receptor subtypes: clinical efficacy and selectivity of benzodiazepine site ligands.

The main inhibitory neurotransmitter receptor of the brain, the gamma-aminobutyric acid type A receptor (GABA[A]), mediates the actions of several classes of clinically important drugs, such as benzodiazepines, barbiturates and general anaesthetics. This review summarizes the current knowledge on how classical benzodiazepines and novel nonbenzodiazepine compounds act on the benzodiazepine site of GABA(A) receptors and on their clinical pharmacology related to anxiolytic, sedative, hypnotic and cognitive effects or side-effects. Partial agonism, receptor subtype selectivity and novel binding sites are discussed as possible strategies to develop new drugs with fewer adverse effects than are seen in the clinical use of benzodiazepines.

Benzodiazepines↗
PubMed1996

Characterization of the rat hippocalcin gene: the 5' flanking region directs expression to the hippocampus.

Hippocalcin is an EF-hand [Persechini A. et al. (1989) Trends Neurosci. 12, 462-467] Ca2+ binding protein encoded by a neuron-specific gene. A detailed atlas of hippocalcin messenger RNA expression in the adult rat brain was complied using in situ hybridization. Highest levels of messenger RNA are found in the hippocampus, where messenger RNA is localized in proximal dendrites of CA pyramidal cells. Expression is also seen in other brain regions, including the neocortex, caudate-putamen, taenia tecti, claustrum, olfactory tubercle, anterior olfactory nucleus, and granule cell and glomerular layers of the olfactory bulb. The rat hippocalcin gene spans approximately 9 kb and consists of three exons, separated by introns of 6.7 and 0.25 kb. Sequence analysis of the putative proximal promoter region identified two clusters of multiple E-box sites which may regulate the cell-specific expression. Two lacZ fusion constructs carrying 0.9 and 3.4 kb of rat hippocalcin gene upstream region were used to create transgenic mice. With the 3.4 kb construct, transgene expression varied between founder mice, but was always found in the dentate gyrus and CA1-CA4 regions of the hippocampus, thus partly mimicking the expression of the endogenous gene. For the 0.9 kb construct, the levels of lacZ expression were weaker and more variable. Neither construct showed expression in any peripheral tissues examined. To establish an in vitro model of transcriptional regulation, the 3.4 and 0.9 kb 5' upstream regions were fused to a promoterless reporter gene encoding chloramphenicol acetyltransferase and transiently transfected into the hippocalcin-positive NG-108 cells. The 3.4 kb construct was strongly expressed, whilst the 0.9 kb construct was not expressed. In this paper, we describe the detailed expression pattern of the rat hippocalcin gene, the gene structure and its neuron-specific promoter.

Animals↗
PubMed1996

Blunted furosemide action on cerebellar GABAA receptors in ANT rats selectively bred for high alcohol sensitivity.

Furosemide specifically reverses the inhibition by gamma-aminobutyric acid (GABA) of t-[35S]-butylbicyclophosphorothionate ([35S]TBPS) binding and increases the basal [35S]TBPS binding to the cerebellar granule cell layer GABAA receptors. For the selectivity of furosemide, an interplay between GABAA receptor alpha 6 and beta 2 or beta 3 subunits is needed. We have now investigated the furosemide sensitivity of cerebellar [35S]TBPS binding in the alcohol-sensitive (ANT) rat line that harbors a pharmacologically critical point mutation in the alpha 6 subunit [alpha 6 (Q1000)], increasing benzodiazepine affinity of the normally insensitive alpha 6-containing receptors. ANT receptors were less efficiently affected by furosemide, while a normal GABAA receptor antagonism was observed with a specific GABAA receptor antagonist SR 95531. Reduced [3H]muscimol binding in ANT samples and small alterations in situ hybridization signals for alpha 1, alpha 6, beta 2, beta 3, gamma 2 and delta subunit mRNAs failed to correlate with impaired cerebellar furosemide efficacy in individual animals. The alpha 6 (q100) ANT mutation was not responsible for the reduced efficacy of furosemide in the ANT rat line, as judged from the potent furosemide antagonism in recombinant ANT-type alpha 6 (Q100)beta 3 gamma 2 receptors. This data suggest that presence of a novel abnormality in the structure and/or expression of alpha 6 subunit-containing GABAA receptors in the ANT rat line.

Animals↗
PubMed1996

The cerebellum: a model system for studying GABAA receptor diversity.

The basic unsolved questions concerning GABAA receptors are: "How many receptor subtypes exist?", "What subtypes are used by which types of neuron and where are they located on the cell?", and "What are the functions of the different subtypes?" As described in this Review, the cerebellum is an ideal vertebrate brain region for investigating these issues.

Animals↗
PubMed1996

Conservation of gamma-aminobutyric acid type A receptor alpha 6 subunit gene expression in cerebellar granule cells.

The gamma-aminobutyric acid type A receptor cDNAs encoding the alpha 6 subunit homologues from chicken and goldfish have been cloned and sequenced. These proteins exhibit 83 and 75% identity, respectively, to the rat alpha 6 polypeptide. In situ hybridization has demonstrated that, as in mammals, the avian and teleost fish alpha 6 subunit genes are predominantly expressed in cerebellar granule cells. Correspondingly, flunitrazepam-non-displaceable binding of [3H]Ro 15-4513 (a benzodiazepine partial inverse agonist), which is a major characteristic of gamma-aminobutyric acid type-A receptors that contain the alpha 6 polypeptide, is also mainly found for cerebellar granule cells of fish and chick. The conservation of this expression pattern suggests that gamma-aminobutyric acid type A receptors possessing the alpha 6 subunit are of fundamental importance for cerebellar function and that the corresponding gene regulatory elements, e.g., granule cell-specific enhancers, have also been conserved.

Amino Acid Sequence↗
PubMed1996

Characterization of a cerebellar granule cell-specific gene encoding the gamma-aminobutyric acid type A receptor alpha 6 subunit.

The alpha 6 subunit of gamma-aminobutyric type A receptors is a marker for cerebellar granule cells and is an attractive candidate to study cell-specific gene expression in the brain. The mouse alpha 6 subunit gene has nine exons and spans approximately 14 kb. The largest intron (intron 8) is approximately 7 kb. For a minority of mRNAs, a missplice of the first exon was identified that disrupts the signal peptide and most likely results in the production of nonfunctional protein. The gene is transcribed from a TATA-less promoter that uses multiple start sites. Using transgenic mice, it was found that the proximal 0.5 kb of the rat alpha 6 gene upstream region confers expression on a beta-galactosidase reporter gene. One founder gave rise to a line with cerebellar granule cell-specific expression, although expression varied with lobule region. Other founders had ectopic but neuron-specific expression, with beta-galactosidase found in cerebellar Purkinje cells, neocortex, thalamus, hippocampus, caudate-putamen, and inferior colliculi. Thus, we have defined a region containing the basal promoter of the alpha 6 subunit gene and that confers neuron-specific expression.

Alternative Splicing↗
PubMed1995

Flip and Flop variants of AMPA receptors in the rat lumbar spinal cord.

The expression of eight messenger RNA splice forms encoding the Flip and Flop variants of AMPA receptor subunits GluR-A to -D in the rat lumbar spinal cord was examined by in situ hybridization using specific oligonucleotides. In the dorsal horn (laminae I, II and III) the predominant mRNA was GluR-B Flip. Much lower levels of GluR-A Flip were found in lamina I and in superficial parts of lamina II outer. In the ventral horn, motor neurons expressed mainly GluR-B Flip, GluR-C Flip and Flop, and GluR-D Flip. Serial sectioning through large motor neurons indicated that a given cell contained, for example, both GluR-C Flip and Flop splice types.

Animals↗