GABAB receptors make it to the top--as dimers.
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Animals↗
Biomedical subjects
H Möhler
Publications and source records attributed to H Möhler.
[Pharmacotherapy with benzodiazepines: basic principles and new developments].
Pharmacotherapy of various neurologic and psychiatric disorders is based on amplification of the effects of the inhibitory neurotransmitter GABA in the CNS. Of particular importance is the modulation of GABAA receptors by benzodiazepines. Their effects are activity-dependent and self limiting. With the development of new ligands for the benzodiazepine receptorsite selective activity-profiles with minimal side-effects are sought. Progress is to be expected from partial agonists and in particular from ligands with selectivity for receptor subtypes.
Anti-Anxiety Agents↗
Immunohistochemical evidence for dysregulation of the GABAergic system ipsilateral to photochemically induced cortical infarcts in rats.
Deficits of GABAergic transmission have been reported to occur in tissue surrounding ischemic cortical lesions between a few days and several weeks after the insult. In the present experiments, we used immunohistochemistry with antibodies against parvalbumin and two major subunits of the GABA(A) receptor (alpha1, alpha2) to characterize the events that underlie these changes at different levels of circuit organization. Neocortical infarcts (2 mm diameter) consistently affecting medial parts of the primary somatosensory cortex were induced photochemically in adult male Wistar rats; animals were allowed to recover for one week before perfusion-fixation. When compared to controls the pattern of immunoreactivity had changed for the al subunit of the GABA(A) receptor seven days after the insult. Ipsilateral to the ischemic lesions, we found a decrease in staining intensity reaching up to 4 mm laterally, resulting in a partial or complete absence of the normal laminar staining pattern. No consistent changes were observed for the alpha2 subunit. Parvalbumin staining revealed pathological alterations in a rim of tissue surrounding the infarct, measuring up to 1 mm from the border of the infarcts. Parvalbumin-positive interneurons in this region showed signs of degeneration; both a reduction of the number of dendrites and, to a lesser extent and only immediately adjacent to the ischemic lesions, a reduction of the number of parvalbumin-positive neurons was readily apparent. The results provide evidence for both a differential regulation of two GABA(A) receptor subunits and degenerative changes of parvalbumin-containing interneurons ipsilateral to cortical infarcts. The relevance of these findings for mechanisms underlying long-term recovery, transient functional deficits and postinfarct seizures warrants further investigation.
Animals↗
Preclinical and clinical studies of a collagen membrane (Bio-Gide).
Membranes are used to guide the repopulation of defects by preferred cells and to achieve a specific healing effect. The collagen membrane studied, Bio-Gide, was developed particularly for periodontal, peri-implant applications or to improve the ossification of bone defects of any origin. Bio-Gide is a bilayer membrane; one compact and smooth layer is covered by a particularly dense film, designed to prevent the invasion of soft tissue in a membrane-protected bone defect. The other, rough side of Bio-Gide must be placed towards the bone defect in order to make bone ingrowth possible. As a prerequisite for its therapeutic use in humans, the collagen matrix must be devoid of major immunogenicity. The immunological response to the membrane material was analysed in rabbits. Later, a clinical prospective study provided information about the bone regeneration effect under the Bio-Gide membrane inserted in six patients selected at random. The immune response to the collagen membrane Bio-Gide and the bone healing was tested in these patients when undergoing oral surgery.
Animals↗
Characterization of agonist-induced down-regulation of NMDA receptors in cerebellar granule cell cultures.
Exposure of cerebellar granule cells to NMDA in culture at 5 days in vitro, when cells are not yet vulnerable to NMDA, evoked a pronounced reduction in NMDA receptor activity, measured by NMDA-induced 45Ca2+ influx, and counteracted the normal developmental increase in NMDA receptors. The effect was concentration and time dependent, the half-maximal effect being reached at about 45 microM and by 4-5 h. The decrease in NMDA receptor function was accompanied by a significant reduction in the protein level of the obligatory NMDA receptor subunit (NR) NR1. Both parameters remained at a low level as long as the agonist was present. However, receptor down-regulation was reversible, as receptor protein levels and NMDA responses were restored to control values upon NMDA removal, this process requiring protein synthesis. NMDA treatment also elicited a decrease in NR1, NR2A, and NR2B subunit messenger RNA (mRNA) levels. However, in comparison with NMDA receptor proteins, the decrease was faster, and NMDA receptor mRNA content recovered to control levels within 24 h in spite of the presence of NMDA. Concerning the mechanisms of agonist-induced regulation of NMDA receptor expression, it seems that protein kinase C-mediated protein phosphorylation is not involved, whereas inhibition of Ca2+/calmodulin-dependent kinase II/IV by KN-62 does depress NMDA receptor expression even in the absence of NMDA.
Animals↗
Agonist-induced down-regulation of NMDA receptors in cerebellar granule cells in culture.
In contrast to the acute toxic effect of NMDA on mature cerebellar granule cells, chronic treatment with NMDA (140 microM from 1 to 9 days in vitro) did not compromise cell survival. Such treatment markedly suppressed NMDA receptor activity: at 8 days in vitro NMDA-induced 45Ca2+ influx was reduced by approximately 60% and acute exposure to NMDA (highest concentration tested, 1 mM) at 9 days in vitro did not cause detectable toxicity. The reduction in NMDA receptor activity was accompanied by a significant decrease (approximately 80% at 9 days in vitro) in the level of the NR1 and the NR2A NMDA receptor subunit protein, detected using the selective photoaffinity ligand [125I]CGP55802A. It seems, therefore, that the agonist-induced decrease in NMDA receptor activity is due to receptor down-regulation. In contrast to the marked influence of chronic NMDA exposure on the cellular content of the NMDA receptor subunit proteins, mRNA levels of the different subunits (NR1, NR2A, NR2B and NR2C) were not significantly affected. It seems, therefore, that agonist-induced down-regulation of the NMDA receptor involves critically mRNA translation and/or post-translational regulation.
Animals↗
Colocalization of gephyrin and GABAA-receptor subunits in the rat retina.
Gephyrin is a protein that copurifies with the glycine receptor (GlyR) and is required for the clustering of GlyRs at postsynaptic sites. Previously, it was thought that antibody mAb 7a, directed against gephyrin, was a specific marker for GlyR. However, there is evidence that gephyrin can also be found at nonglycinergic synapses. Here, immunocytochemistry was applied to show this directly for the rat retina. Both gephyrin and different subunits of the gamma-aminobutyric acid (GABA)A receptor were localized to discrete puncta in the inner plexiform layer, and these puncta were shown by electron microscopy to represent synaptic sites. Double immunocytochemistry revealed that GABAA receptors and GlyRs are not colocalized. However, gephyrin and different subunits of GABAA receptors were found to occur at the same synapses. The amount of colocalization varied with the GABAA receptor subunit composition and was most extensive for the alpha 2 subunit, less for the alpha 3 subunit, and minimal for the alpha 1 subunit. The gephyrin present at GABAergic synapses of the retina might also be involved with clustering of receptors at the postsynaptic sites. Hence, localization of gephyrin can no longer be considered as a unique marker of glycinergic synapses.
Animals↗
GABAA receptor subunits have differential distributions in the rat retina: in situ hybridization and immunohistochemistry.
The distributions of nine different subunits of the gamma-aminobutyric acidA (GABAA) receptor (alpha 1, alpha 2, alpha 3, alpha 5; beta 1, beta 2, beta 3; gamma 2; delta) were investigated in the rat retina using immunocytochemistry and in situ hybridization. With the exception of the alpha 5 subunit, all subunits could be localized. Each subunit was expressed in characteristic strata within the inner plexiform layer (IPL). Some subunits (e.g., gamma 2) showed a ubiquitous distribution, while others (e.g., delta) were restricted to narrow sublayers. Double labeling experiments using different combinations of the subunit-specific antibodies revealed colocalizations of subunits within individual neurons. Additionally, GABAA receptor subunits were mapped to distinct populations of retinal neurons by coapplication of defined immunocytochemical markers and subunit-specific antibodies. Cholinergic amacrine cells were found to express the alpha 2, beta 1, beta 2/3 and delta subunits, while dopaminergic amacrine cells express the alpha 2, alpha 3 and gamma 2 subunits. Dissociated rod bipolar cells express the alpha 1 and gamma 2 subunits. In summary, this study provides evidence for the existence of multiple GABAA receptor subtypes in the retina. The distinct stratification pattern of the subunits in the IPL suggests that different functional circuits involve specific subtypes of GABAA receptors.
Animals↗
Differential distribution of GABAA receptor subunits in soma and processes of cerebellar granule cells: effects of maturation and a GABA agonist.
Quantitative analysis of the density of alpha 1 and beta 2/3 GABAA receptor subunits was performed at the electron microscope level after indirect pre-embedding immunogold labeling with subunit-specific antibodies of rat cerebellar granule cell cultures grown for 4 or 8 days and in the presence or absence of the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridin-3-ol (THIP). THIP (150 microM) induced a 2-fold increase in the number of alpha 1 and beta 2/3 subunits in both cell bodies and processes in 4-day-old cultures. Extending the culture period to 8 days led to a polarization of the receptor expression, since the increase in the number of subunits selectively was observed in the processes. Moreover, a general subcellular differentiation of the receptor population was observed in all culture conditions, since the ratio between the two subunits (beta 2/3; alpha 1) was four times higher in cell bodies compared to processes. A detailed analysis of the less mature (4-day-old) cultures revealed the existence of two populations of neurons exhibiting differences in the average number of receptors. During maturation neurons with few receptors developed into cells with a higher density of receptors resulting in a single population of the latter neurons, a process enhanced by exposure to THIP. This may indicate that receptor development is a discontinuous process with individual neurons following different temporal patterns. In double-labeling experiments, a spatially close association of the alpha 1 and beta 2/3 subunits could be seen, but the subunits were more frequently found separated from each other. In spite of the fact that exposure of the neurons to THIP increased the total number of receptor subunits, its presence apparently prevented formation of receptors with this subunit composition. Interestingly, receptor subunit clusters, consisting of alpha 1 alone, were more frequently observed than composite (alpha 1; beta 2/3) clusters. This substantiates the view that receptors not having alpha 1 and beta 2/3 subunits in the same complex may exist.
Animals↗
Immunocytochemical localization of GABAA receptor beta 2/beta 3-subunits in the brain of Atlantic salmon (Salmo salar L).
In order to obtain a basis for future investigations concerning the possible interactions between melatonin, GABA and benzodiazepines in the central nervous system of a teleost fish, the Atlantic salmon, we have studied the expression of immunoreactivity with a monoclonal antibody against the GABAA-receptor beta 2/beta 3-subunits (bd-17) in the salmon brain. Immunoreactivity was found in all parts of the brain, mostly as a diffuse labelling of discrete neuropil areas but in some instances as a granular perikaryal labelling. Strong neuropil labelling is located in the telencephalon, dorsal thalamus/pretectum, optic tectum, torus semicircularis, and ventrolateral tegmentum. Perikaryal labelling was observed in the stratum periventriculare of the optic tectum, torus longitudinalis, torus semicircularis, ventrolateral tegmentum, and in the granular layer of the cerebellum. The general pattern of distribution is similar to that observed in mammals, in which high receptor densities are found in the telencephalon (cerebral cortex), superior and inferior colliculi, and cerebellum. There is a good correlation with the distribution of melatonin binding sites, observed in a previous study, in areas receiving visual input such as the optic tectum, pretectum, and torus semicircularis. Moreover, a correlation was found in the inferior lobes and regions connected with them. Regions containing both bd-17-immunoreactivity and melatonin binding sites may constitute areas of functional interaction between melatonin, GABA and benzodiazepines in the central nervous system.
Animals↗
Regulation of the expression of NMDA receptor subunits in rat cerebellar granule cells: effect of chronic K(+)-induced depolarization and NMDA exposure.
The influence of K(+)-induced membrane depolarization and NMDA treatment on the regulation of NMDA receptor subunit (NR) expression was investigated during the development of granule cells in culture, as a follow-up of previous work on NMDA receptor activity. In spite of the increase in NMDA receptor activity elicited by these treatments (K25 or K10 + NMDA cultures), the main developmental changes in receptor mRNA levels were similar to those in untreated cells (K10) (a threefold increase in total NMDA receptor mRNA, quantitative dominance of NR1 mRNA, late expression of NR2C, and virtual absence of NR2D. However, high K+ and NMDA treatment resulted in a greater increase of NR2A mRNA levels and a retardation in the developmental changes in the relative amounts of NR2B and NR2C mRNAs. The correspondence between NMDA receptor activity and the amount of NR1 and NR2A subunit proteins was excellent, the rank order being K25 > K10 + NMDA > K10 at 9 days in vitro. Because the increase in subunit mRNA was not always paralleled by an increase in subunit protein, the control of NMDA receptor expression involves critically, in addition to gene transcription, regulation of translational and/or posttranslational events.
Affinity Labels↗
Regionally selective and age-dependent alterations in benzodiazepine receptor binding in the genetically dystonic hamster.
Previous pharmacological studies have indicated that impairment of GABAergic transmission may be involved in the pathophysiology of dystonia in the mutant dtsz hamster, i.e., a genetic animal model for idiopathic dystonia. In the present experiments, the kinetic constants of [3H]flumazenil binding to the benzodiazepine site of the GABAA receptor were calculated from equilibrium binding measurements in various brain regions of genetically dystonic hamsters and age-matched controls. Because dystonia in mutant dtsz hamsters is transient and disappears after approximately 60-70 days of age, [3H]flumazenil binding was studied at the age of maximum severity of dystonia (30-40 days) and after disappearance of the disease, to examine which neurochemical changes were related to dystonia. In mutant hamsters with the maximum severity of dystonia, receptor affinity of [3H]flumazenil was increased in olfactory bulb, striatum, tectum, and cerebellum, as exemplified by significantly decreased dissociation constants (KD) in these regions. An increased number of binding sites (Bmax) were seen in striatum and frontal cortex but not in the other eight regions studied in this regard. All these changes in [3H]flumazenil binding disappeared in parallel with dystonia, implicating a causal relationship between altered benzodiazepine receptor binding and dystonia in mutant dtsz hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗
GABAA receptor subunit immunoreactivity in primate visual cortex: distribution in macaques and humans and regulation by visual input in adulthood.
Subunit proteins that make up functional GABAA receptors were localized immunocytochemistry in the primary visual cortex (area 17) of adult monkeys and humans. Immunoreactivity for the alpha 1, beta 2/3, and gamma 2 subunits is greatest in layers (II-III, IVA and IVC) of monkey area 17 that contain the highest density of GABA neurons and terminals. Immunostaining for each subunit is unevenly distributed in layers II and III, where patches of immunoreactivity correspond to regions of intense cytochrome oxidase (CO) staining, and in layer IVA, where intense immunoreactivity forms a honeycomb pattern identical to the CO staining pattern. Immunoreactivity for the subunits is localized principally within the neuropil, which, by simultaneous comparison with the distribution of microtubule-associated protein immunostaining, was found to include bundles of thin dendrites and zones of numerous dendritic segments. In addition, gamma 2 immunostaining surrounds the somata of a subpopulation of GABAergic neurons, immunoreactive for the calcium-binding protein parvalbumin. All three subunits are present in the somata and processes of neurons that occupy the white matter subjacent to monkey area 17. In human visual cortex, the alpha 1, beta 2/3, and gamma 2 subunits are distributed in a manner similar to that found in monkeys, with relatively intense immunostaining in layers IVC and IVA. In layer IVC, vertical stripes of intense receptor immunostaining (20-30 microns wide) alternate with wider stripes of pale immunostaining (30-60 microns wide). In the upper and lower halves of IVC beta, these stripes form lattices similar to those in layers IVC and IVA of monkeys. Following monocular deprivation by intravitreal injections of TTX in adult monkeys, immunoreactivity for each subunit in layer IVC consists of alternating intensely and lightly stained stripes. Comparison with the pattern of CO staining indicates that intense immunostaining for alpha 1, beta 2/3, and gamma 2 occurs in normal-eye stripes while abnormally light immunostaining is present in deprived-eye stripes. For all three subunits, immunoreactivity in deprived-eye stripes is reduced within 5 d of monocular deprivation and remains abnormally low for deprivations that extend to at least 30 d. These findings indicate that each of several GABAA receptor subunits adopt similar laminar and compartmental distributions in monkey and human area 17 and are likely to be expressed by the same neurons. The deprivation-dependent reduction in immunoreactivity for alpha 1, beta 2/3, and gamma 2 subunits suggests that all are regulated by visually driven activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Adult↗
Developmental regulation of voltage-gated K+ channel and GABAA receptor expression in Bergmann glial cells.
Bergmann glial cells are closely associated with neurons: during development they provide guiding structures for migrating granule cells and in the adult cerebellum they display intimate interactions with Purkinje cells. In this study, we have addressed the question of whether such changes in neuronal-glial interactions during development are accompanied by variations in the membrane properties of Bergmann glial cells. We used a mouse cerebellum slice preparation to study membrane currents of the Bergmann glial cells at various stages of development in situ using the patch-clamp technique. The distinct morphology of Bergmann glial cells was revealed by Lucifer yellow injections during recording. While Bergmann glial cells in mice of postnatal day 20 (P20) to P30 have thick processes with arborized, irregularly shaped leaf-like appendages, the processes of cells from younger mice (P5-P7) are thinner and smoother. This morphological maturation is accompanied by a variation in voltage-gated currents. In cells from P5 to P7, delayed outward- and inward-rectifying K+ currents were recorded, while older Bergmann glial cells were characterized by, large, voltage- and time-independent K+ currents. In addition, application of GABA induces two effects, a rapid activation of a Cl- conductance and a longer-lasting decrease in the (resting) K+ conductance. Both effects were mediated by benzodiazepine-insensitive GABAA receptors. Responses in cells of P5-P7 mice were large as compared to the small or even undetectable responses in P20-P30 cells. These GABAA receptors were characterized immunohistochemically in mice and rat brain sections with five subunit-specific antibodies. Bergmann glial cells exhibit a distinct but transient immunoreactivity for the GABAA receptor alpha 2-, alpha 3-, and delta-subunits. Staining is maximal between P7 and P10 and decreases gradually thereafter. In contrast, antibodies to the alpha 1- and beta 2,3-subunits fail to decorate Bergmann glial cells, although they yield a prominent staining of both the Purkinje cells and the granule cells. These changes in the Bergmann glial cell membrane properties and GABAA receptor expression suggest a transition between functional states during development of the Bergmann glial cells.
Aging↗
Stoichiometry of a recombinant GABAA receptor deduced from mutation-induced rectification.
Ligand-gated ion channels generally display a heterooligomeric subunit structure. The present report describes an electrophysiological method that provides criteria indicating the subunit stoichiometry of a recombinant GABAA receptor composed of alpha 3, beta 2 and gamma 2 subunits. Our results exclude the stoichiometries 3 alpha 1 beta 1 gamma, 1 alpha 3 beta 1 gamma, 1 alpha 1 beta 3 gamma and suggest that the possible subunit stoichiometries for this receptor are 2 alpha 1 beta 2 gamma, 2 alpha 2 beta 1 gamma or 1 alpha 2 beta 2 gamma, of which the alpha subunit composition 2 alpha 1 beta 2 gamma may be favoured. The method is based on the quantification of the outward rectification of the GABA-evoked current induced by point mutation of charged amino acids located near the ion channel pore.
Amino Acid Sequence↗
Cholinergic amacrine cells of the rat retina express the delta-subunit of the GABAA-receptor.
Antibodies directed against the delta-subunit of the GABAA-receptor were applied to cryostat sections of rat retinae. Two narrow bands of the inner plexiform layer were strongly immunoreactive. Some cell bodies in both the amacrine- and ganglion-cell layer were weakly immunoreactive. The position of the labelled bands and the distribution of the cell bodies was strongly reminiscent of the cholinergic amacrine cells. In order to show directly that cholinergic amacrine cells express the delta-subunit of the GABAA-receptor, double immunofluorescence with an antibody against choline acetyltransferase (ChAT) and with antibodies against the delta-subunit was performed on the same cryostat sections. This showed the labelled cells to be cholinergic amacrine cells.
Animals↗
Structure, pharmacology and function of GABA-A receptors in cochlear outer hair cells.
There is evidence that the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is released from some efferent olivocochlear nerve endings terminating at outer hair cells (OHCs). Using monoclonal antibodies against postsynaptic GABAA receptor from bovine cerebral cortex we confirm the presence of GABA and benzodiazepine bindings sites of alpha- and beta-subunits of GABAA receptors at the basal pole of isolated OHCs. Whole-cell recording with viable OHCs revealed that the application of 10(-3)-10(-8) M GABA to the cell surface was followed by a concentration-dependent hyperpolarization of the outer cell membrane. Hyperpolarization was increased in the presence of 2.5 x 10(-5) M chlorazepate, a benzodiazepine derivative. Electrophysiological effects caused by GABA alone or in combination with chlorazepate were specifically inhibited by 10(-6) M of the GABA-receptor antagonist picrotoxin. Moreover, 10(-5)-10(-7) M GABA caused reversible slow elongation of the cylindrical hair cell body in OHCs examined. These neurotransmitter-induced motile responses were specifically blocked by 10(-4) M picrotoxin. The results suggest that a subpopulation of OHCs express alpha- and beta-subunits of GABAA receptors which both form a GABA/benzodiazepine-receptor complex at the basal pole of isolated OHCs. These receptors are thought to allow GABA which is released from efferent auditory nerve terminals to bind to the cell surface of OHCs, resulting in GABAA-receptor activation. This probably gates a GABAA-receptor-associated chloride channel in the postsynaptic OHC membrane, allowing hyperpolarization and elongation of the cell.
Animals↗
Differential regulation of N-methyl-D-aspartate receptor subunit messenger RNAs in kindling-induced epileptogenesis.
N-methyl-D-aspartate-receptors are implicated in several neuropathological conditions including epilepsy. As a model of complex partial seizures, rapid hippocampal kindling was chosen to investigate changes in the expression of messenger RNAs encoding the N-methyl-D-aspartate-receptor subunits NR1, NR2A and NR2B both during and in the period immediately following the induction of the kindled state. The study demonstrates a cell-specific, time-dependent modulation of the N-methyl-D-aspartate-receptor subunit messenger RNAs almost entirely restricted to the granule cells of the dentate gyrus. In partially kindled animals (10 stimulations), while the NR1 subunit messenger RNA remained unaltered after a period of 2 h, the NR2A and NR2B subunit messenger RNAs were bilaterally reduced in dentate gyrus granule cells by around 50% below control values. In fully kindled animals (40 stimulations), a progressive reduction in NR1 subunit messenger RNA levels in the dentate gyrus was observed, being maximal after 4 h (-67%). At the same time point, NR2A and NR2B transcript levels were transiently increased by 102% and 46% above control values, respectively. These data point to a differential regulation of N-methyl-D-aspartate-receptor subunit messenger RNAs. No alterations were detected in pyramidal cells. Long-term maintenance of the kindled state was not associated with alterations in N-methyl-D-aspartate-receptor subunit messenger RNAs since control levels of messenger RNA were attained by 12 h and persisted for at least five days. The early changes in messenger RNAs described in this study indicate that the expression of N-methyl-D-aspartate-receptor subunits is under independent regulatory control. This phenomenon may contribute to epileptogenesis and to kindling-associated plasticity by mediating a structural reorganization of N-methyl-D-aspartate-receptors, leading to an altered excitability of dentate gyrus granule cells.
Animals↗