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ABSTRACT: Gephyrin is a multifunctional protein responsible for the clustering of glycine receptors (GlyR) and -aminobutyric acid type A receptors (GABAAR). GlyR and GABAAR are heteropentameric chloride ion channels that facilitate fast-response, inhibitory neurotransmission in the mammalian brain and spinal cord. We investigated the immunohistochemical distribution of gephyrin and the major GABAAR and GlyR subunits in the human light microscopically in the rostral and caudal one-thirds of the pons, in the middle and caudal one-thirds of the medulla oblongata, and in the first cervical segment of the spinal cord. The results demonstrate a widespread pattern of immunoreactivity for GlyR and GABAAR subunits throughout these regions, including the spinal trigeminal nucleus, abducens nucleus, facial nucleus, pontine reticular formation, dorsal motor nucleus of the vagus nerve, hypoglossal nucleus, lateral cuneate nucleus, and nucleus of the solitary tract. The GABAAR 1 and GlyR 1 and β subunits show high levels of immunoreactivity in these nuclei. The GABAAR subunits 2, 3, β2,3, and 2 present weaker levels of immunoreactivity. Exceptions are intense levels of GABAAR 2 subunit immunoreactivity in the inferior olivary complex and high levels of GABAAR 3 subunit immunoreactivity in the locus coeruleus and raphe nuclei. Gephyrin immunoreactivity is highest in the first segment of the cervical spinal cord and hypoglossal nucleus. Our results suggest that a variety of different inhibitory receptor subtypes is responsible for inhibitory functions in the human brainstem and cervical spinal cord and that gephyrin functions as a clustering molecule for major subtypes of these inhibitory neurotransmitter receptors. J. Comp. Neurol. 518:305–328, 2010. © 2009 Wiley-Liss, Inc.
The Journal of Comparative Neurology 01/2010; 518(3):305 - 328. · 3.81 Impact Factor
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ABSTRACT: Gephyrin is a multifunctional protein responsible for the clustering of glycine receptors (GlyR) and gamma-aminobutyric acid type A receptors (GABA(A)R). GlyR and GABA(A)R are heteropentameric chloride ion channels that facilitate fast-response, inhibitory neurotransmission in the mammalian brain and spinal cord. We investigated the immunohistochemical distribution of gephyrin and the major GABA(A)R and GlyR subunits in the human light microscopically in the rostral and caudal one-thirds of the pons, in the middle and caudal one-thirds of the medulla oblongata, and in the first cervical segment of the spinal cord. The results demonstrate a widespread pattern of immunoreactivity for GlyR and GABA(A)R subunits throughout these regions, including the spinal trigeminal nucleus, abducens nucleus, facial nucleus, pontine reticular formation, dorsal motor nucleus of the vagus nerve, hypoglossal nucleus, lateral cuneate nucleus, and nucleus of the solitary tract. The GABA(A)R alpha(1) and GlyR alpha(1) and beta subunits show high levels of immunoreactivity in these nuclei. The GABA(A)R subunits alpha(2), alpha(3), beta(2,3), and gamma(2) present weaker levels of immunoreactivity. Exceptions are intense levels of GABA(A)R alpha(2) subunit immunoreactivity in the inferior olivary complex and high levels of GABA(A)R alpha(3) subunit immunoreactivity in the locus coeruleus and raphe nuclei. Gephyrin immunoreactivity is highest in the first segment of the cervical spinal cord and hypoglossal nucleus. Our results suggest that a variety of different inhibitory receptor subtypes is responsible for inhibitory functions in the human brainstem and cervical spinal cord and that gephyrin functions as a clustering molecule for major subtypes of these inhibitory neurotransmitter receptors.
The Journal of Comparative Neurology 01/2010; 518(3):305-28. · 3.81 Impact Factor
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ABSTRACT: To identify subunit variants of the GABAA-receptor antisera were developed against specific cDNA-derived peptide sequences of the α1-and α3–subunits of rat brain. The α1-subunit antiserum selectively recognized a protein of 50±1 kDa in rat and bovine GABAA – receptor preparations, while the α3–subunit antiserum interacted with a protein doublet of 59±2 kDa and 61±3 kDa. The α-subunit immunoreactivity resides in a large population of GABAA-receptors as shown by immunoprecipitation of 63+6% of [3H]flumazenil binding sites with the α1-subunit antiserum. In contrast, only 24±3% of receptor binding sites were precipitated with the α3–subunit antiserum. Co-precipitation studies suggest that the α1- and α3–subunit immunoreactivities do not share the same receptor population while the γ2–subunit immunoreactivity is associated with the α1-subunit immunoreactivity.
09/2008; 11(1-4):407-424.
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ABSTRACT: Gamma-aminobutyric acid(A) (GABA(A)) receptors (GABA(A)R) are inhibitory heteropentameric chloride ion channels comprising a variety of subunits and are localized at postsynaptic sites within the central nervous system. In this study we present the first detailed immunohistochemical investigation on the regional, cellular, and subcellular localisation of alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits of the GABA(A)R in the human substantia nigra (SN). The SN comprises two major regions, the SN pars compacta (SNc) consisting of dopaminergic projection neurons, and the SN pars reticulata (SNr) consisting of GABAergic parvalbumin-positive projection neurons. The results of our single- and double-labeling studies demonstrate that in the SNr GABA(A) receptors contain alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and are localized in a weblike network over the cell soma, dendrites, and spines of SNr parvalbumin-positive nonpigmented neurons. By contrast, GABA(A)Rs on the SNc dopaminergic pigmented neurons contain predominantly alpha(3) and gamma(2) subunits; however there is GABA(A)R heterogeneity in the SNc, with a small subpopulation (6.5%) of pigmented SNc neurons additionally containing alpha(1) and beta(2,3) GABA(A)R subunits. Also, in the SNr, parvalbumin-positive terminals are adjacent to GABA(A)R on the soma and proximal dendrites of SNr neurons, whereas linear arrangements of substance P-positive terminals are adjacent to GABA(A) receptors on all regions of the dendritic tree. These results show marked GABA(A)R subunit hetereogeneity in the SN, suggesting that GABA exerts quite different effects on pars compacta and pars reticulata neurons in the human SN via GABA(A) receptors of different subunit configurations.
The Journal of Comparative Neurology 03/2008; 506(6):912-29. · 3.81 Impact Factor
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ABSTRACT: γ-Aminobutyric acidA (GABAA) receptors (GABAAR) are inhibitory heteropentameric chloride ion channels comprising a variety of subunits and are localized at postsynaptic sites within the central nervous system. In this study we present the first detailed immunohistochemical investigation on the regional, cellular, and subcellular localisation of α1, α2, α3, β2,3, and γ2 subunits of the GABAAR in the human substantia nigra (SN). The SN comprises two major regions, the SN pars compacta (SNc) consisting of dopaminergic projection neurons, and the SN pars reticulata (SNr) consisting of GABAergic parvalbumin-positive projection neurons. The results of our single- and double-labeling studies demonstrate that in the SNr GABAA receptors contain α1, α3, β2,3, and γ2 subunits and are localized in a weblike network over the cell soma, dendrites, and spines of SNr parvalbumin-positive nonpigmented neurons. By contrast, GABAARs on the SNc dopaminergic pigmented neurons contain predominantly α3 and γ2 subunits; however there is GABAAR heterogeneity in the SNc, with a small subpopulation (6.5%) of pigmented SNc neurons additionally containing α1 and β2,3 GABAAR subunits. Also, in the SNr, parvalbumin-positive terminals are adjacent to GABAAR on the soma and proximal dendrites of SNr neurons, whereas linear arrangements of substance P-positive terminals are adjacent to GABAA receptors on all regions of the dendritic tree. These results show marked GABAAR subunit hetereogeneity in the SN, suggesting that GABA exerts quite different effects on pars compacta and pars reticulata neurons in the human SN via GABAA receptors of different subunit configurations. J. Comp. Neurol. 506:912–929, 2008. © 2007 Wiley-Liss, Inc.
The Journal of Comparative Neurology 02/2008; 506(6):912 - 929. · 3.81 Impact Factor
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ABSTRACT: 1. The aim of this study was to define the biophysical properties contributed by the gamma2 subunit to native single GABAA receptors. 2. Single-channel activity was recorded from neurones of wild-type (gamma2+/+) mice and compared with that from mice which were heterozygous (gamma2+/-) or homozygous (gamma2-/-) for a targeted disruption in the gamma2 subunit gene of the GABAA receptor. Unitary currents were evoked by low concentrations of GABA (0.5-5 microM) in membrane patches from acutely isolated dorsal root ganglion (DRG) neurones (postnatal day 0) and by 1 microM GABA in patches from embryonic hippocampal neurones which were cultured for up to 3 weeks. 3. GABAA receptors from DRG and hippocampal neurones of gamma2+/+ and gamma2+/- mice displayed predominantly a conductance state of 28 pS and less frequently 18 and 12 pS states. In gamma2-/- mice, conductance states mainly of 12 pS and less frequently of 24 pS were found. 4. The mean open duration of the 28 pS state in gamma2+/+ GABAA receptors (1.5-2.6 ms) was substantially longer than for the 12 pS state of gamma2-/- GABAA receptors (0.9-1.2 ms) at all GABA concentrations. For gamma2+/+ and gamma2-/- channels, the mean open duration was increased at higher GABA concentrations. 5. Open duration frequency distributions of 28 and 12 pS receptors revealed the existence of at least three exponential components. Components with short mean durations declined and components with long mean durations increased in relative frequency at higher GABA concentration indicating at least two binding sites of GABA per 28 and 12 pS receptor. 6. Shut time frequency distributions revealed at least four exponential components of which two were identified as intraburst components in 28 pS and one in 12 pS GABAA receptors. 7. The mean burst duration and the mean number of openings per burst increased in 28 and 12 pS GABAA receptors with increasing GABA concentration. At least two burst types were identified: simple bursts consisting of single openings and complex bursts of five to six openings in 28 pS but only two to three openings in 12 pS GABAA receptors. 8. We conclude that the gamma2 subunit enhances the efficacy of GABA by determining open conformations of high conductance and long lifetime, and by prolonging the time receptors remain in the activated bursting state.
The Journal of Physiology 09/2000; 527 Pt 1:11-31. · 4.72 Impact Factor
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ABSTRACT: The regional and cellular localisation of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the human basal ganglia using receptor autoradiography and immunohistochemical staining for five GABA(A) receptor subunits (alpha(1), alpha(2), alpha(3), beta(2, 3), and gamma(2)) and other neurochemical markers. The results demonstrated that GABA(A) receptors in the striatum showed considerable subunit heterogeneity in their regional distribution and cellular localisation. High densities of GABA(A) receptors in the striosome compartment contained the alpha(2), alpha(3), beta(2, 3), and gamma(2) subunits, and lower densities of receptors in the matrix compartment contained the alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. Also, six different types of neurons were identified in the striatum on the basis of GABA(A) receptor subunit configuration, cellular and dendritic morphology, and chemical neuroanatomy. Three types of alpha(1) subunit immunoreactive neurons were identified: type 1, the most numerous (60%), were medium-sized aspiny neurons that were immunoreactive for parvalbumin and alpha(1), beta(2,3), and gamma(2) subunits; type 2 (38%) were medium-sized to large aspiny neurons immunoreactive for calretinin and alpha(1), alpha(3), beta(2,3), and gamma(2) subunits; and type 3 (2%) were large sparsely spiny neurons immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits. Type 4 neurons were calbindin-positive and immunoreactive for alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. The remaining neurons were immunoreactive for choline acetyltransferase (ChAT) and alpha(3) subunit (type 5) or were neuropeptide Y-positive with no GABA(A) receptor subunit immunoreactivity (type 6). The globus pallidus contained three types of neurons: types 1 and 2 were large neurons and were immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and for parvalbumin alone (type 1) or for both parvalbumin and calretinin (type 2); type 3 neurons were medium-sized and immunoreactive for calretinin and alpha(1), beta(2, 3), and gamma(2) subunits. These results show that the subunit composition of GABA(A) receptors displays considerable regional and cellular variation in the human striatum but are more homogeneous in the globus pallidus.
The Journal of Comparative Neurology 01/2000; 415(3):313-40. · 3.81 Impact Factor
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ABSTRACT: Adenosine, an endogenous inhibitory neuromodulator in the central nervous system, exerts anticonvulsant activity that is largely based on the inhibition of the release of excitatory amino acids. As a novel approach to treat pharmacoresistant partial epilepsies, the grafting of adenosine-releasing cells is foreseen to provide a local and sustained source of adenosine. The feasibility of this cell-based therapy was investigated in the present study by the intraventricular implantation of synthetic polymers that release adenosine. Kindled rats with a ventricular implant of an adenosine-releasing polymer showed a profound reduction of seizure activity. This was demonstrated not only by a 75% reduction of grade 5 seizures but also by a reduction of the amplitude and duration of afterdischarges in electroencephalographic (EEG) recordings. Kindled control rats that were implanted with bovine serum albumin (BSA)-containing polymers or were sham operated, continued to show their presurgery seizure pattern. Adenosine displayed antiepileptic activity when released in an amount of 20-50 ng per day. This finding sets the target for the required amount of adenosine to be released from future adenosine-releasing cells for antiepileptic therapy. The present results clearly support the feasibility of a novel therapy for epilepsy based on adenosine-releasing cells.
Experimental Neurology 12/1999; 160(1):164-74. · 4.70 Impact Factor
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ABSTRACT: Patients with panic disorders show a deficit of GABAA receptors in the hippocampus, parahippocampus and orbitofrontal cortex. Synaptic clustering of GABAA receptors in mice heterozygous for the gamma2 subunit was reduced, mainly in hippocampus and cerebral cortex. The gamma2 +/- mice showed enhanced behavioral inhibition toward natural aversive stimuli and heightened responsiveness in trace fear conditioning and ambiguous cue discrimination learning. Implicit and spatial memory as well as long-term potentiation in hippocampus were unchanged. Thus gamma2 +/- mice represent a model of anxiety characterized by harm avoidance behavior and an explicit memory bias for threat cues, resulting in heightened sensitivity to negative associations. This model implicates GABAA-receptor dysfunction in patients as a causal predisposition to anxiety disorders.
Nature Neuroscience 10/1999; 2(9):833-9. · 15.53 Impact Factor
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ABSTRACT: The subunit architecture of gamma-aminobutyric acid, type B (GABA(B)), receptors in situ is largely unknown. The GABA(B) receptor variants, characterized by the constituents GBR1a and GBR1b, were therefore analyzed with regard to their subunit composition as well as their regional and subcellular distribution in situ. The analysis was based on the use of antisera recognizing selectively GBR1a, GBR1b, and GBR2. Following their solubilization, GBR1a and GBR1b were both found by immunoprecipitation to occur as heterodimers associated with GBR2. Furthermore, monomers of GBR1a, GBR1b, or GBR2 were not detectable, suggesting that practically all GABA(B) receptors are heterodimers in situ. Finally, there was no evidence for an association of GBR1a with GBR1b indicating that these two constituents represent two different receptor populations. A size determination of solubilized GABA(B) receptors by sucrose density centrifugation revealed two distinct peaks of which one corresponded to dimeric receptors, and the higher molecular weight peak pointed to the presence of yet unknown receptor-associated proteins. The distribution and relative abundance of GBR2 immunoreactivity corresponded in all brain regions to that of the sum of GBR1a and GBR1b, supporting the view that most if not all GBR1 proteins are associated with GBR2. However, GBR1a was present preferentially at postsynaptic densities, whereas GBR1b may be mainly attributed to presynaptic or extrasynaptic sites. Thus, GBR1a and GBR1b are both associated with GBR2 to form heterodimers at mainly different subcellular locations where they are expected to subserve different functions.
Journal of Biological Chemistry 10/1999; 274(38):27323-30. · 4.77 Impact Factor
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ABSTRACT: GABAB (gamma-aminobutyric acid)-receptors have been implicated in central nervous system (CNS) functions, e.g. cognition and pain perception, and dysfunctions including spasticity and absence epilepsy. To permit an analysis of the two known GABAB-receptor splice variants GABAB-R1a (GB1a) and GABAB-R1b (GB1b), their distribution pattern has been differentiated in the rat brain, using Western blotting and immunohistochemistry with isoform-specific antisera. During postnatal maturation, the expression of the two splice variants was differentially regulated with GB1a being preponderant at birth. In adult brain, GB1b-immunoreactivity (-IR) was predominant, and the two isoforms largely accounted for the pattern of GABAB-receptor binding sites in the brain. Receptor heterogeneity was pronounced in the hippocampus, where both isoforms occurred in CA1, but only GB1b in CA3. Similarly, in the cerebellum, GB1b was exclusively found in Purkinje cells in a zebrin-like pattern. The staining was most pronounced in Purkinje cell dendrites and spines. Using electron microscopy, over 80% of the spine profiles in which a synaptic contact with a parallel fibre was visible contained GB1b-IR at extrasynaptic sites. This subcellular localization is unrelated to GABAergic inputs, indicating that the role of GABAB-receptors in vivo extends beyond synaptic GABAergic neurotransmission and may, in the cerebellum, involve taurine as a ligand.
European Journal of Neuroscience 04/1999; 11(3):761-8. · 3.63 Impact Factor
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ABSTRACT: In the process of drug discovery, different classes of genetically-modified animals are valuable tools for the identification of drug targets, distinction of target isoforms, differentiation of signaling pathways, generation of disease models and toxicological testing. The analysis is refined by methodologies which permit the inducible regulation of gene expression in a time- and tissue-specific manner. Finally, multiplexing techniques and knockout embryonic stem (ES) cell libraries will help to link DNA sequence information to biological function.
Current opinion in drug discovery & development 04/1999; 2(2):134-41. · 4.20 Impact Factor
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ABSTRACT: Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [3H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [3H]CGP 61594 labeled with high affinity (K(D) = 23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [3H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [3H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [3H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors.
Neuropharmacology 03/1999; 38(2):233-42. · 4.81 Impact Factor
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ABSTRACT: The distribution of gamma-aminobutyric acidA (GABAA) receptors was investigated in the basal ganglia in the baboon brain by using receptor autoradiography and the immunohistochemical localisation of the α1 and β2,3 subunits of the GABAA receptor by light and electron microscopy. In the caudate-putamen, the α1 subunit was distributed in high densities in the matrix compartment, and the β2,3 subunits were more homogeneously distributed; the globus pallidus showed lower levels of the α1 and β2,3 subunits. Four types of α1 subunit immunoreactive neurons were identified in the baboon striatum: the most numerous (75%) were type 1 medium-sized aspiny neurons; type 2 (2%) were large aspiny neurons with an indented nuclear membrane located in the ventral striatum; type 3 neurons were the least numerous (1%) and were comprised of large neurons in the ventromedial regions of the striatum; and type 4 (22%) neurons were medium to large aspiny neurons located in striosomes. At the ultrastructural level, α1 and β2,3 subunit immunoreactivity was localised in the neuropil of the striatum in both symmetrical and asymmetrical synaptic contacts. In the globus pallidus, α1 and β2,3 subunits were localised on large neurons and were found in three types of synaptic terminals: type 1 terminals were small and established symmetrical synapses; type 2 terminals were large; and type 3 terminals formed small synaptic terminals with subjunctional dense bodies. These results show that the subunit composition of GABAA receptors varies between the striosome and the matrix compartments in the striatum and that there is receptor subunit homogeneity in the globus pallidus. J. Comp. Neurol. 397:297–325, 1998. © 1998 Wiley-Liss, Inc.
The Journal of Comparative Neurology 12/1998; 397(3):297 - 325. · 3.81 Impact Factor
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ABSTRACT: The distribution of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the basal ganglia in the baboon brain by using receptor autoradiography and the immunohistochemical localisation of the alpha1 and beta2,3 subunits of the GABA(A) receptor by light and electron microscopy. In the caudate-putamen, the alpha1 subunit was distributed in high densities in the matrix compartment, and the beta2,3 subunits were more homogeneously distributed; the globus pallidus showed lower levels of the alpha1 and beta2,3 subunits. Four types of alpha1 subunit immunoreactive neurons were identified in the baboon striatum: the most numerous (75%) were type 1 medium-sized aspiny neurons; type 2 (2%) were large aspiny neurons with an indented nuclear membrane located in the ventral striatum; type 3 neurons were the least numerous (1%) and were comprised of large neurons in the ventromedial regions of the striatum; and type 4 (22%) neurons were medium to large aspiny neurons located in striosomes. At the ultrastructural level, alpha1 and beta2,3 subunit immunoreactivity was localised in the neuropil of the striatum in both symmetrical and asymmetrical synaptic contacts. In the globus pallidus, alpha1 and beta2,3 subunits were localised on large neurons and were found in three types of synaptic terminals: type 1 terminals were small and established symmetrical synapses; type 2 terminals were large; and type 3 terminals formed small synaptic terminals with subjunctional dense bodies. These results show that the subunit composition of GABA(A) receptors varies between the striosome and the matrix compartments in the striatum and that there is receptor subunit homogeneity in the globus pallidus.
The Journal of Comparative Neurology 09/1998; 397(3):297-325. · 3.81 Impact Factor
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ABSTRACT: Amino acids in the alpha- and gamma-subunits contribute to the benzodiazepine binding site of GABA(A)-receptors. We show that the mutation of a conserved histidine residue in the N-terminal extracellular segment (alpha1H101R, alpha2H101R, alpha3H126R, and alpha5H105R) results not only in diazepam-insensitivity of the respective alphaxbeta2,3gamma2-receptors but also in an increased potentiation of the GABA-induced currents by the partial agonist bretazenil. Furthermore, Ro 15-4513, an inverse agonist at wild-type receptors, acts as an agonist at all mutant receptors. This conserved molecular switch can be exploited to identify the pharmacological significance of specific GABA(A)-receptor subtypes in vivo.
FEBS Letters 08/1998; 431(3):400-4. · 3.54 Impact Factor
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ABSTRACT: The ability of neurons to display more than a single GABA(A)-receptor subtype per cell requires intricate targeting mechanisms. Analysis by confocal laser scanning microscopy revealed that the alpha2- and alpha5-subunits differed strikingly in their subcellular distribution in hippocampal pyramidal cells and olfactory bulb granule cells, while the distribution of the gamma2-subunit was rather uniform. In mutant mice lacking the alpha5-subunit gene due to a chromosomal deletion, the absence of the alpha5-subunit was accompanied by a corresponding decrease of the gamma2-subunit immunoreactivity. In striking contrast, the subcellular distribution of the alpha2-subunit was unchanged in these mutant mice. These findings indicate that the assembly of distinct GABA(A)-receptor subtypes in the same neuron is regulated independently. Furthermore, the alpha-subunit is a prime candidate for providing domains which direct subcellular targeting.
Neuroscience Letters 07/1998; 249(2-3):99-102. · 2.11 Impact Factor
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M Honer,
D Benke,
B Laube,
J Kuhse,
R Heckendorn,
H Allgeier,
C Angst,
H Monyer,
P H Seeburg,
H Betz, H Mohler
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ABSTRACT: The binding site for the co-agonist glycine on N-methyl-D-aspartate (NMDA) receptors has been mapped to the NR1 subunit whereas binding of the principal agonist glutamate is mediated by the NR2 subunits. Using the novel glycine site antagonist and photoaffinity label CGP 61594, distinct contributions of the NR2 subunit variants to the glycine antagonist binding domains of NMDA receptor subtypes are demonstrated. High affinity sites for CGP 61594 were exclusively displayed by NR1/2B receptors, as shown by their co-distribution with the NR2B subunit, by subunit-selective immunoprecipitation and by functional analysis of NR1/2B receptors expressed in Xenopus oocytes (inhibitory potency, IC50 = 45 +/- 11 nM). Other NMDA receptor subtypes are clearly distinguished by reduced inhibitory potencies for CGP 61594, being low for NR1/2A and NR1/2D receptors (IC50 = 430 +/- 105 nM and 340 +/- 61 nM, respectively) and intermediate for NR1/2C receptors (IC50 = 164 +/- 27 nM). Glycine antagonist sites with low and intermediate affinity for [3H]CGP 61594 were detected also in situ by radioligand binding in brain areas predominantly expressing the NR2A and NR2C subunits, respectively. Thus, [3H]CGP 61594 is the first antagonist radioligand that reliably distinguishes the glycine site of NMDA receptor subtypes. [3H]CGP 61594 is a promising tool to identify the NR2 subunit domains that contribute to differential glycine antagonist sites of NMDA receptor subtypes.
Journal of Biological Chemistry 06/1998; 273(18):11158-63. · 4.77 Impact Factor
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ABSTRACT: The rules governing the assembly of GABA(A) receptors in vivo were assessed in subunit mutant mice. The transcription of individual subunit genes was regulated independently. The lack of a particular subunit did not result in a molecular rescue by an enhanced transcription of other subunits. In addition, the availability of an alpha- and beta-subunit was essential for receptor formation. Finally, highly selective recognition processes directed the subcellular targeting of receptors. The loss of a particular receptor subtype (alpha5) did not lead to a subcellular redistribution of the remaining subtype (alpha2) present in the same cell.
Life Sciences 02/1998; 62(17-18):1611-5. · 2.53 Impact Factor
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ABSTRACT: The mechanisms governing the assembly of alpha-, beta- and gamma-subunits to form GABAA-receptors are poorly understood. Here, we report that the alpha-subunit is essential for receptor assembly. In mice homozygous for a deletion on chromosome 7 spanning the alpha 5- and gamma 3-subunit genes, zolpidem-insensitive benzodiazepine binding sites, corresponding to GABAA-receptors containing the alpha 5-subunit, were absent in the hippocampus. This loss of alpha 5-GABAA-receptor binding was also apparent as a 21% decrease in the total number of benzodiazepine binding sites in the hippocampus. In addition, immunoreactivity for the beta 2,3- and gamma 2-subunit was decreased exclusively in neurons which normally express the alpha 5-subunit, such as olfactory bulb granule cells and hippocampal pyramidal cells. In other brain regions of the mutants, the beta 2,3- and gamma 2-subunit staining was unaffected. Controls included two lines of mice homozygous for a shorter chromosomal deletion, that either included or excluded the gamma 3-subunit gene. These two lines were indistinguishable with regard to numbers of benzodiazepine binding sites and levels alpha 5-, beta 2,3- and gamma 2-subunit immunoreactivity, indicating that the lack of gamma 3-subunit gene did not contribute to the observed deficit in receptor formation. These results demonstrate that the absence of the alpha 5-subunit gene prevents the formation of the entire respective receptor complex in adult mouse brain. Thus, the alpha-subunit, unlike the gamma 2-subunit, might play a major role in the assembly or targeting of GABAA-receptor complexes.
Neuroscience 01/1998; 81(4):1043-53. · 3.38 Impact Factor
