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J Kirsch

Publications and source records attributed to J Kirsch.

At least 37 records · Page 2Linked to original sources
PubMed1998

Localization of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb of the rat.

The tubulin-binding protein gephyrin is essential for the formation of postsynaptic glycine-receptor clusters in cultured spinal neurons. In addition, there is increasing evidence that gephyrin can also be present at nonglycinergic synapses. Here we analyzed immunocytochemically the subcellular localization of gephyrin in the main olfactory bulb of the rat and compared its distribution with that of gamma-aminobutyric acid (GABA) and of two major GABA(A)-receptor subunits. Gephyrin was selectively localized to the postsynaptic side of symmetric synaptic junctions, where the presynaptic terminals contained GABA. Moreover, gephyrin colocalized extensively with the alpha1 and gamma2 subunits of the GABA(A) receptor. In contrast, gephyrin was not detected at presumed glutamatergic synapses. These results indicate that gephyrin is not uniquely associated with glycine receptors, but can also be found at distinct GABAergic synapses. Thus, they raise the possibility that gephyrin is involved in anchoring certain GABA(A)-receptor subtypes in the postsynaptic membrane.

Animals↗
PubMed1998

CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species.

B cells and monocytes endocytose DNA into an acidified intracellular compartment. If this DNA contains unmethylated CpG dinucleotides in particular base contexts (CpG motifs), these leukocytes are rapidly activated. We now show that both B cell and monocyte-like cell line responses to DNA containing CpG motifs (CpG DNA) are sensitive to endosomal acidification inhibitors; they are completely blocked by bafilomycin A, chloroquine, and monensin. The specificity of these inhibitors is demonstrated by their failure to prevent responses to LPS, PMA, or ligation of CD40 or IgM. Acidification of endosomal CpG DNA is coupled to the rapid generation of intracellular reactive oxygen species. The CpG DNA-induced reactive oxygen species burst is linked to the degradation of IkappaB and the activation of NFkappaB, which induces leukocyte gene transcription and cytokine secretion. These studies demonstrate a novel pathway of leukocyte activation triggered by CpG motifs.

Animals↗
PubMed1998

Glycine-receptor activation is required for receptor clustering in spinal neurons.

The ability of nerve cells to receive up to several thousands of synaptic inputs from other neurons provides the anatomical basis for information processing in the vertebrate brain. The formation of functional synapses involves selective clustering of neurotransmitter receptors at presumptive postsynaptic regions of the neuronal plasma membrane. Receptor-associated proteins are believed to be crucial for this process. In spinal neurons, synaptic targeting of the inhibitory glycine receptor (GlyR) depends on the expression of the anchoring protein gephyrin. Here we show that the competitive GlyR antagonist strychnine and L-type Ca2+-channel blockers inhibit the accumulation of GlyR and gephyrin at postsynaptic membrane areas in cultured rat spinal neurons. Our data are consistent with a model in which GlyR activation that results in Ca2+ influx is required for the clustering of gephyrin and GlyR at developing postsynaptic sites. Similar activity-driven mechanisms may be of general importance in synaptogenesis.

Animals↗
PubMed1998

Simultaneous in vivo spectral editing and water suppression.

Water suppression is typically performed in vivo by exciting the longitudinal magnetization in combination with dephasing, or by using frequency-selective coherence generation. MEGA, a frequency-selective refocusing technique, can be placed into any pulse sequence element designed to generate a Hahn spin-echo or stimulated echo, to dephase transverse water coherences with minimal spectral distortions. Water suppression performance was verified in vivo using stimulated echo acquisition mode (STEAM) localization, which provided water suppression comparable with that achieved with four selective pulses in 3,1-DRYSTEAM. The advantage of the proposed method was exploited for editing J-coupled resonances. Using a double-banded pulse that selectively inverts a J-coupling partner and simultaneously suppresses water, efficient metabolite editing was achieved in the point resolved spectroscopy (PRESS) and STEAM sequences in which MEGA was incorporated. To illustrate the efficiency of the method, the detection of gamma-aminobutyric acid (GABA) was demonstrated, with minimal contributions from macromolecules and overlying singlet peaks at 4 T. The estimated occipital GABA concentration was consistent with previous reports, suggesting that editing for GABA is efficient when based on MEGA at high field strengths.

Brain Chemistry↗
PubMed1998

First one in, last one out: the role of gabaergic transmission in generation and degeneration.

This paper is the result of discussions between scientists working in widely separated areas, united by an interest in the hippocampus. The discussions focused on the possible role of GABA in the development and maturation of the hippocampus and in neurodegeneration in Alzheimer's disease (AD). GABA neurons are among the first to differentiate in the hippocampus and the properties of GABA neurotransmission in the developing hippocampus are distinct from those in the adult. GABAergic transmission may play a role in the clustering and maturation of GABA receptors, as well as of receptors for other neurotransmitters. The development and maturation of synaptic connections involves changes in the organization of the cytoskeleton, and mechanical force generation is probably required to establish appropriate points of contact. This generation of force may require coupling of specific receptors to the cytoskeleton through specialized proteins. In AD, much of the developmental process is progressively unraveled in the hippocampus, as afferent fibers, most notably from entorhinal excitatory neurons and from basal forebrain cholinergic cells, degenerate. This denervation undoubtedly has consequences for receptor systems, dendritic morphology and the underlying cytoskeleton. GABA neurons remain in the AD hippocampus, and may actually contribute to abnormal firing and degeneration of remaining pyramidal neurons. This attempt to bring together data from different areas of research has allowed the development of a scheme which identifies significant specific gaps in our knowledge, which could be readily filled by focused experimental work.

Animals↗
PubMed1998

Molecular mechanisms that underlie structural and functional changes at the postsynaptic membrane during synaptic plasticity.

The synaptic plasticity that is addressed in this review follows neurodegeneration in the brain and thus has both structural as well as functional components. The model of neurodegeneration that has been selected is the kainic acid lesioned hippocampus. Degeneration of the CA3 pyramidal cells results in a loss of the Schaffer collateral afferents innervating the CA1 pyramidal cells. This is followed by a period of structural plasticity where new synapses are formed. These are associated with changes in the numbers and shapes of spines as well as changes in the morphometry of the dendrites. It is suggested that this synaptogenesis is responsible for an increase in the ratio of NMDA to AMPA receptors mediating excitatory synaptic transmission at these synapses. Changes in the temporal and spatial properties of these synapses resulted in an altered balance between LTP and LTD. These properties together with a reduction in the inhibitory drive increased the excitability of the surviving CA1 pyramidal cells which in turn triggered epileptiform bursting activity. In this review we discuss the insights that may be gained from studies of the underlying molecular machinery. Developments in one of the collections of the cogs in this machinery has been summarized through recent studies characterizing the roles of neural recognition molecules in synaptic plasticity in the adult nervous systems of vertebrates and invertebrates. Such investigations of neural cell adhesion molecules, cadherins and amyloid precursor protein have shown the involvement of these molecules on the morphogenetic level of synaptic changes, on the one hand, and signal transduction effects, on the other. Further complex cogs are found in the forms of the low-density lipoprotein receptor (LDL-R) family of genes and their ligands play pivotal roles in the brain development and in regulating the growth and remodelling of neurones. Evidence is discussed for their role in the maintenance of cognitive function as well as Alzheimer's. The molecular mechanisms responsible for the clustering and maintenance of transmitter receptors at postsynaptic sites are the final cogs in the machinery that we have reviewed. Postsynaptic densities (PSD) from excitatory synapses have yielded many cytoskeletal proteins including actin, spectrin, tubulin, microtubule-associated proteins and calcium/calmodulin-dependent protein kinase II. Isolated PSDs have also been shown to be enriched in AMPA, kainate and NMDA receptors. However, recently, a new family of proteins, the MAGUKs (for membrane-associated guanylate kinase) has emerged. The role of these proteins in clustering different NMDA receptor subunits is discussed. The MAGUK proteins are also thought to play a role in synaptic plasticity mediated by nitric oxide (NO). Both NMDA and non-NMDA receptors are highly clustered at excitatory postsynaptic sites in cortical and hippocampal neurones but have revealed differences in their choice of molecular components. Both GABAA and glycine (Gly) receptors mediate synaptic inhibition in the brain and spinal cord. Whilst little is known about how GABAA receptors are localized in the postsynaptic membrane, considerable progress has been made towards the elucidation of the molecular mechanisms underlying the formation of Gly receptors. It has been shown that the peripheral membrane protein gephyrin plays a pivotal role in the formation of Gly receptor clusters most likely by anchoring the receptor to the subsynaptic cytoskeleton. Evidence for the distribution as well as function of gephyrin and Gly receptors is discussed. Postsynaptic membrane specializations are complex molecular machinery subserving a multitude of functions in the proper communication between neurones. Despite the fact that only a few key players have been identified it will be a fascinating to watch the story as to how they contribute to structural and functional plasticity unfold.

Animals↗
PubMed1998

Choice of NSAID and management strategy in rheumatoid arthritis and osteoarthritis. The impact on costs and outcomes in the UK.

OBJECTIVE: Although nonsteroidal anti-inflammatory drugs (NSAIDs) are an effective therapy for rheumatoid arthritis, they are associated with significant adverse effects, the management of which imposes additional costs on the healthcare system. Prescribing NSAIDs which have a lower risk of major adverse effects as the first-line NSAID for patients with rheumatoid arthritis and osteoarthritis may be expected to lead to an improvement in clinical outcomes and reduce overall treatment costs. This analysis examines data from a published randomised controlled trial of 5 NSAIDs to explore these hypotheses. DESIGN AND SETTING: Data from a clinical trial comparing 5 NSAIDs were combined with published cost data to construct 2 clinical decision models, reflecting alternative approaches to the management of major and minor adverse effects in the UK. INTERVENTIONS: The 5 NSAIDs evaluated in the analysis were nabumetone, diclofenac, ibuprofen, piroxicam and naproxen, although only the results for ibuprofen and nabumetone are reported. MAIN OUTCOME MEASURES AND RESULTS: The total cost of care per patient receiving nabumetone was estimated to be between 25 pounds sterling (Pound) and 41 Pounds more expensive than ibuprofen. In a hypothetical cohort of 100,000 patients, there were between 690 and 821 more major adverse effects using ibuprofen than nabumetone. The cost per life-year gained (LYG) from using nabumetone rather than ibuprofen ranged between 1880 Pounds and 2517 Pounds (1995 values), depending upon the management of adverse effects. CONCLUSIONS: These results indicate that: (i) prescribing the newer, currently more expensive, NSAIDs will not necessarily lead to cost savings; (ii) the management of adverse effects can have a significant impact on costs; and (iii) the additional cost may be justifiable in terms of the mortality and morbidity gains associated with the new lower-risk NSAIDs.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1997

Development of adult-type inhibitory glycine receptors in the central auditory system of rats.

Inhibitory synaptic activity is crucial for many aspects of acoustic information processing and mainly mediated by glycine and gamma-aminobutyric acid, the two principal inhibitory neurotransmitters in the auditory system. Glycine exerts its inhibitory action via binding to postsynaptic receptors existing in various isoforms. Here we have investigated the spatiotemporal distribution of adult-type, strychnine-sensitive glycine receptors (GlyRs) in the rat auditory system by using a specific antibody against the ligand-binding alpha1 GlyR subunit. In adults, alpha1 GlyRs were found at all relay stations of the auditory pathway except for the medial geniculate body and the auditory cortex. In most brainstem nuclei, labeling was characterized by dense clusters of heavily immunoreactive puncta outlining the somata and proximal dendrites, indicative of a powerful glycinergic inhibition. No alpha1 immunoreactivity was seen in the auditory system of fetal rats, consistent with results obtained by others in the spinal cord. At birth, labeling was weak and restricted to defined nuclei of the cochlear nuclear complex and the superior olivary complex. By postnatal day 8, labeling was seen in all brainstem nuclei. At the first appearance of immunoreactivity, alpha1 GlyRs were diffusely distributed on the neuronal surface, yet they became clustered with age, finally densely incrusting the somata and proximal dendrites between the 3rd and 4th postnatal week, when the mature pattern of immunoreactivity was established. We never observed an overexpression of alpha1 GlyRs or a transient appearance in areas that are devoid of the receptor in adults. The late formation of glycinergic synapses harboring the adult-type GlyRs in the auditory system, at a time when internuclear connections have already formed, indicates that alpha1 GlyRs do not participate in early synaptogenesis.

Aging↗
PubMed1997

Analysis of the promoter region of the murine gephyrin gene.

The peripheral membrane protein gephyrin is essential for the postsynaptic localization of the inhibitory glycine receptor and is thought to anchor the receptor complex to the subsynaptic cytoskeleton. Since gephyrin transcripts are also found in non-neuronal tissues, we analyzed the 5'-flanking region of the gephyrin gene to identify its promoter region. This region contains several SP1 consensus sites, which may account for the widespread expression of gephyrin mRNA. Indeed, reporter gene constructs encompassing at least the two SP1 consensus sites upstream from the transcription start site were efficiently transcribed in both a fibroblast and a neuronal cell line. Our data are consistent with the widespread expression of the gephyrin gene in most mammalian tissues.

Animals↗
PubMed1996

The glycine binding site of the N-methyl-D-aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits. Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis. Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity. These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins. Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly. These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.

Amino Acid Sequence↗
PubMed1996

[Noise field screening. Results of a television field study for detection of visual field defects].

White noise field campimetry is able to transform usually negative scotomas immediately into perceivable visual field defects. As this method needs a monitor, the obvious solution was to evaluate the suitability of the noise field stimulus broadcasted to home TV sets. Therefore, in cooperation with the "Süddeutscher Rundfunk" (SDR) as well as several health insurance companies ("AOK Baden-Württemberg" and other "RVO-Kassen"), approximately 300,000 viewers were invited to take this test after receiving brief information and instruction. There were 531 calls for questionnaires to document the subjects' findings and the results of a subsequent ophthalmological examination. In most cases this clarified the perceived noise field defect. Out of 127 evaluable questionnaires, 78 cases did not show any relevant ophthalmological pathology; this held true especially for lesions of the visual pathway. However, in 49 persons the ophthalmologists detected pathological findings; in 20 of these, the scotomas were previously unknown to the physicians. Glaucomatous optic neuropathy and macular degeneration were most frequently diagnosed as causing the white noise field defects. Finally, some preliminary estimates of the costs and benefit of this study are presented.

Adolescent↗
PubMed1996

Expression of glycine receptor alpha subunits and gephyrin in cultured spinal neurons.

The inhibitory glycine receptor is a pentameric membrane protein composed of alpha and beta subunits. In the postsynaptic membrane, the glycine receptor and the copurifying peripheral membrane protein gephyrin are clustered underneath glycine-releasing nerve terminals. Here, we describe the expression of gephyrin and the neonatal and adult glycine receptor alpha subunit isoforms alpha1 and alpha2 during in vitro differentiation of rat spinal neurons. Analysis by immunoassays and the reverse transcriptase-polymerase chain reaction showed that gephyrin and alpha subunit mRNA and protein levels exhibited a marked increase from 1 to 5 days in vitro, i.e. prior to the formation of functional synaptic contacts. Using confocal and standard immunofluorescence, we determined the number of immunoreactive cells and the cellular localization of the alpha subunits and gephyrin. At 3 days in vitro, glycine receptor immunoreactivity revealed by the monoclonal antibody mAb4a was found in < 10% of cells and was mainly localized intracellularly; in contrast, gephyrin was detected in in vitro, gephyrin was essentially localized at the neuronal surface. At this stage, the number of glycine receptor-positive cells approached that of gephyrin-containing neurons (50%), and glycine receptor antigen was found both intracellularly and at the periphery of the cells. The antibody mAb2b, which binds exclusively to the alpha1 subunit, revealed aggregates at the surface of a few neurons. At 10 days in vitro, glycine receptor and gephyrin staining was localized in clusters at the periphery of the soma and the neurites. This quantitative analysis corroborates temporal differences in the cellular distribution of gephyrin and glycine receptor alpha subunits, the former being accumulated first at the neuronal surface.

Animals↗
PubMed1995

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↗
PubMed1995

Modulation by zinc ions of native rat and recombinant human inhibitory glycine receptors.

1. The effect of the divalent cation Zn2+ on inhibitory glycine receptor (GlyR) currents was investigated in rat embryonic spinal cord neurons and Xenopus oocytes expressing recombinant GlyRs. 2. In cultured spinal neurons, Zn2+ potentiated glycine-induced whole-cell currents about 3-fold when applied extracellularly at concentrations of 0.5-10 microM. In contrast, higher concentrations (> 100 microM) of Zn2+ decreased the glycine response. 3. A similar biphasic modulation of glycine-induced currents by Zn2+ was also found with recombinant homo- and hetero-oligomeric GlyRs generated in Xenopus oocytes. Dose-response analysis showed that both the potentiating and inhibitory effects of Zn2+ result from changes in apparent agonist affinity. 4. Analysis of chimeric constructs of the GlyR alpha 1- and beta-subunits revealed that the positive and negative modulatory effects of Zn2+ are mediated by different regions of the alpha 1-subunit. 5. Our data indicate the existence of distinct high- and low-affinity Zn2+ binding sites on the ligand-binding alpha-subunits of the GlyR. These sites may be implicated in the regulation of synaptic efficacy within glycinergic pathways.

Amino Acid Sequence↗
PubMed1995

Targeting of glycine receptor subunits to gephyrin-rich domains in transfected human embryonic kidney cells.

In adult spinal neurons inhibitory glycine receptors (GlyR) are localized at postsynaptic membrane specializations underlying glycinergic nerve terminals. The peripheral membrane protein gephyrin has been shown to be essential for the formation of postsynaptic GlyR clusters. Here, we coexpressed GlyR polypeptides and gephyrin in 293 cells and observed rerouting of hetero-oligomeric GlyR and its beta, but not of alpha subunits to intracellular gephyrin aggregates. A GlyR chimeric alpha 1/beta protein was also accumulated at these gephyrin aggregates, indicating that colocalization with gephyrin depends on cytoplasmic domains of the beta subunit. gamma-Aminobutyric acid type-A receptor (GABAAR) subunits were not targeted to intracellular gephyrin aggregates with the exception of the GABAAR beta 3 subunit, which partially colocalized with gephyrin. These data show that gephyrin alters the subcellular localization of the GlyR beta and, to some extent, GABAAR beta 3 subunits. Thus, gephyrin-binding subunits might target hetero-oligomeric ion channels to a gephyrin matrix underlying the differentiating postsynaptic membrane.

Animals↗
PubMed1995

Identification of a gephyrin binding motif on the glycine receptor beta subunit.

The tubulin-binding protein gephyrin copurifies with the inhibitory glycine receptor (GlyR) and is essential for its postsynaptic localization. Here we have analyzed the interaction between the GlyR and recombinant gephyrin and identified a gephyrin binding site in the cytoplasmic loop between the third and fourth transmembrane segments of the beta subunit. GlyR alpha subunits and GABAA receptor proteins failed to bind recombinant gephyrin. However, insertion of an 18 residue segment of the GlyR beta subunit into the GABAA receptor beta 1 subunit conferred gephyrin binding both in an overlay assay and in transfected mammalian cells. These results indicate that beta subunit expression is essential for the formation of a postsynaptic GlyR matrix.

Amino Acid Sequence↗