Distribution Of Glycine Receptors Research Articles

Distribution of glycine receptors on the surface of the mature calyx of Held nerve terminal.

The physiological functions of glycine receptors (GlyRs) depend on their subcellular locations. In axonal terminals of the central neurons, GlyRs trigger a slow facilitation of presynaptic transmitter release; however, their spatial relationship to the release sites is not known. In this study, we examined the distribution of GlyRs in the rat glutamatergic calyx of Held nerve terminal using high-resolution pre-embedding immunoelectron microscopy. We performed a quantitative analysis of GlyR-associated immunogold (IG) labeling in 3D reconstructed calyceal segments. A variable density of IG particles and their putative accumulations, inferred from the frequency distribution of inter-IG distances, indicated a non-uniform distribution of the receptors in the calyx. Subsequently, increased densities of IG particles were found in calyceal swellings, structures characterized by extensive exocytosis of glutamate. In swellings as well as in larger calyceal stalks, IG particles did not tend to accumulate near the glutamate releasing zones. On the other hand, GlyRs in swellings (but not in stalks) preferentially occupied membrane regions, unconnected to postsynaptic cells and presumably accessible by ambient glycine. Furthermore, the sites with increased GlyR concentrations were found in swellings tightly juxtaposed with GABA/glycinergic nerve endings. Thus, the results support the concept of an indirect mechanism underlying the modulatory effects of calyceal GlyRs, activated by glycine spillover. We also suggest the existence of an activity-dependent mechanism regulating the surface distribution of α homomeric GlyRs in axonal terminals of central neurons.

Afferent regulation of glycine receptor distribution in the gerbil LSO.

Synaptic activity plays an important role in many aspects ofneuronal development, particularly the expression of proteins. In this study, the influence of inhibitory and excitatory afferents on the development of glycine receptor density in the lateral superior olive (LSO) of Mongolian gerbils was investigated. Afferent activity was manipulated by removing one or both cochleas at postnatal day 7, prior to the onset of sound-evoked responses. Due to the anatomy of the LSO, these manipulations result in either excitatory denervation, inhibitory denervation, or both. The density of glycine receptors in the LSO was determined at 21 days postnatal. Glycine receptors were either labeled with tritiated strychnine (3H-SN) or with an antibody directed against gephyrin, a protein closely associated with the receptor complex. Antibody binding was used to quantify the differential glycine receptor density between the medial limb (high frequency area) and the lateral limb (low frequency area) of the LSO. 3H-SN was used to quantify the amount of glycine receptors in each part of the LSO in control and experimental animals. In addition, changes in neuron density and neuron cross-sectional area were quantified following cochlear ablations. In control animals, the amount of glycine receptors is about 2- to 3-fold higher in the high-frequency than in the low-frequency region. In bilaterally ablated animals, the same density of glycine receptors was measured in the high- and low-frequency region. Unilateral ablations had no significant effect on glycine receptor distribution, either ipsi- or contralateral to the ablation. The neuron cross-sectional area decreased about 30% in the ipsilateral LSO of unilaterally ablated animals and in bilaterally ablated animals. However, alterations of soma density and cross-sectional area were similar in the high- and low-frequency projection region. These results suggest that the distribution of glycine receptors is only changed when excitatory and inhibitory afferents have been denervated.

The distribution of glycine receptors and interactions with the cytoskeleton

Abstract In the central nervous system, mechanisms of postsynaptic clustering of neurotransmitter receptors are best understood at glycinergic inhibitory synapses. The glycine receptor is a pentameric protein composed of three α and two β transmembrane proteins: the ligand-binding α subunit and a structural β subunit. At glycinergic synapses, these receptors form clusters at the postsynaptic membrane directly opposite to the presynaptic release sites. Anchoring of glycine receptors in the neuronal membrane depends on a peripheral membrane protein named gephyrin. Gephyrin binds to the glycine receptor via the β subunit and links the receptor to the subsynaptic cytoskeleton. Gephyrin may be involved in the anchoring of other neurotransmitter receptors such as the γ-aminobutyric acid (GABAA) receptor.

Inhibitory neurotransmission in rat spinal cord: co-localization of glycine- and GABA A-receptors at GABAergic synaptic contacts demonstrated by triple immunofluorescence staining

Synaptic inhibition in rat spinal cord is mediated by the amino acids γ-aminobutyric acid (GABA) and glycine. Most spinal cord neurons respond to both neurotransmitters, suggesting co-expression of GABA A- and strychnine-sensitive glycine-receptors in individual cells. While the distribution of glycine-receptors has been extensively characterized, much less is known about the cellular localization of GABA A-receptors in spinal cord neurons. In the present study, the distribution of GABA A-receptors was analyzed immunohistochemically with a subunit-specific antiserum recognizing the α1-subunit. Their co-localization with glycine-receptors and their apposition to GABAergic axon terminals were assessed by confocal laser microscopy in sections processed for double- and triple-immunofluorescence staining, using a monoclonal antibody against the 93 kDa glycine-receptor-associated protein, gephyrin, and an antiserum to glutamic acid decar☐ylase. Staining for the GABA A-receptor α1-subunit decorated the soma and dendrites of numerous neurons in laminae III–VIII and X of the spinal cord, revealing their morphology in clear detail. By contrast, laminae II and IX contained little immunoreactivity for these GABA A-receptors. Double-immunofluorescence staining showed that most GABA A-receptor-positive cells in layers III–VIII and X also exhibited a prominent glycine-receptor immunoreactivity. Both types of receptors had very similar distribution patterns in the cell membrane and were frequently co-localized in sites apposed to GABAergic axon terminals. These results indicate that GABA A- and glycine-receptors may co-exist within single postsynaptic densities, suggesting a possible synergism in the action of GABA and glycine in spinal cord neurons.

Synaptic connections involving immunoreactive glycine receptors in the turtle retina.

The distribution of glycine receptors in the turtle retina was studied with the aid of a monoclonal antibody that detects the 93-kD protein associated with the strychnine-sensitive glycine receptor. Light microscopically, receptors were found in the inner plexiform layer and, more sparsely, in the innermost parts of the inner nuclear layer. No receptors were seen to be associated with photoreceptor cells, horizontal cells, or any other structures in the distal inner nuclear layer or outer plexiform layer. Ultrastructurally, glycine receptors were found on the inner face of postsynaptic membranes of processes from amacrine and presumed ganglion cells and always involved amacrine cell processes as the presynaptic element. Such glycine receptor immunoreactive synapses onto amacrine cell processes were distributed throughout the inner plexiform layer with a peak density near the middle. On the other hand, output synapses onto ganglion cell processes displaying immunoreactive glycine receptor sites showed a bimodal distribution in the inner plexiform layer. Glycine receptor immunoreactivity was not detected on bipolar cells, but presumed glycine-utilizing processes (i.e. those presynaptic to immunoreactive glycine receptors) were occasionally found to be postsynaptic in bipolar cell dyads. The majority of the synaptic input to the presumed glycine-utilizing amacrine cell processes was from other amacrine processes, some of which were themselves glycine utilizing. The observations suggest that glycinergic synapses in the turtle retina are, to a large extent, engaged in processing interamacrine signals.

Distribution of glycine receptors on the membrane of a central neuron: an immunoelectron microscopy study

The distribution of glycine receptors on the plasmalemma of the teleost Mauthner cell was examined with light and electron microscopy using monoclonal antibodies raised against the glycine receptors of the rat. These immunoglobulins recognize a peptide of 93 kDa associated with the intracellular side of the receptor complex. With indirect immunofluorescence, labeling appeared as small clusters over the whole surface of the neuron. This distribution indicates that, unlike other neurons, the Mauthner cell can express glycine receptors at the dendritic and at the somatic level as well. Vestibular or reticular neurons lying in the proximity of the Mauthner cell were also stained. At the ultrastructural level, the immunoenzymatic reaction product, localized in front of the presynaptic active zones, slightly overextends the limits of the synaptic complex. This observation may account for vesicular exocytosis at the border of the presynaptic grid. More generally, inside and outside the axon-cap, stained postsynaptic membranes were apposed to terminals identified as unmyelinated club endings and some small vesicle boutons, respectively. Each of them could establish classical flat synapses or spinelike contacts, the latter being described here for the first time. Patches of membrane remained unstained, however, particularly along the lateral and ventral dendrites of the cell; they corresponded to the contact sites of excitatory afferents. In order to enhance the penetration of immunoreactive substances, a particular fixation procedure was used; its effect on the shape of the synaptic vesicles is described. Our results support the notion that glycine is responsible for both somatic and remote dendritic Cl- -dependent inhibition of the Mauthner cell. The first one is produced by the so-called PHP exhibiting neurons, which correspond to cells of recurrent collateral network and vestibulovestibular pathway. In contrast, cells giving rise to the more distal inhibitory synapses on both dendrites are not yet characterized.

The distribution of glycine receptors in the human brain. A light microscopic autoradiographic study using [ 3H]strychnine

Glycine receptors were localized autoradiographically in postmortem human brain material using [ 3H]strychinine as a ligand. Slide mounted tissue sections were labeled in vitro by incubation with [ 3H]strychnine and autoradiograms obtained using [ 3H]Ultrofilm. Receptor densities were quantified by computer assisted microdensitometry. No specific binding of [ 3H]strychnine was observed in any of the forebrain areas studied. Low densities were seen in the midbrain except for dorsal and lateral parts of the periaqueductal grey matter and the oculomotor nuclei. In pons, medulla oblongata and upper cervical cord high densities of [ 3H]strychnine binding sites were associated with some nuclei including the motor and sensory trigeminal nuclei, the facial and the hypoglossal nuclei. The highest densities of grains were associated with the substantia gelatinosa of the trigeminal nucleus in the medulla oblongata. A peculiar spotty distribution of [ 3H]strychnine binding sites were found in the gracilis and cuneatus nuclei. The distribution of glycine receptors in the human brain is comparable to that seen in the rat brain, although densities are much higher in the rat. The distribution of glycine receptors in the human brain provides an anatomical substrate for the understanding of the effects of drugs acting in these receptors, particularly strychnine.

Distribution of glycine receptors at central synapses: an immunoelectron microscopy study.

The distribution of receptors for a neurotransmitter was investigated cytochemically for the first time in the central nervous system, at synapses established on cells of the ventral horn of the rat cervical spinal cord. Three monoclonal antibodies (mAb's) raised against glycine receptors were used. Immunofluorescent staining already showed discontinuous labeling at the surface of neurons, and immunoenzymatic electron microscopy further revealed that the antigenic determinants were confined to the postsynaptic membrane and concentrated at the level of the synaptic complex. More specifically, one mAb directed against the receptive subunit of the oligomeric receptor recognized an epitope on the extracellular side of the plasma membrane, whereas two other mAb's bound to the cytoplasmic face. Epitopes for the last two mAb's were more accurately localized with protein A-colloidal gold, using an intermediate rabbit anti-mouse immunoglobulin serum. (a) In addition to the presence of gold particles in areas facing the presynaptic active zone (visualized with ethanolic phosphotungstic acid), the labeling extended beyond this zone for approximately 50-60 nm, which corresponds to the width of one presynaptic dense projection. (b) The distances between the mid membrane and the gold particles were different for the two mAb's (with means of 21.7 +/- 8.5 nm and 29.8 +/- 10.4 nm, respectively). The data suggest that one of the recognized epitopes is close to the plasma membrane, whereas the second protrudes into the cytoplasm. Our results indicate that the receptor is a transmembrane protein which has a restricted spatial distribution on the postsynaptic neuronal surface.