Glycine receptor distribution in mouse CNS: autoradiographic localization of [3H]strychnine binding sites

Biochemical and electrophysiological studies of mammalian CNS indicate that the amino-acid, glycine, is a major inhibitory neurotransmitter whose location is, for the large part, confined to the spinal cord and brain stem. In this study, autoradiographs of C57BL/6J mouse brain sections labeled with [3H] strychnine, a potent antagonist of glycine, were used to map the distribution of glycine receptors in the CNS. Autoradiographs showed highly localized areas of grain density, which confirmed the gross distributions described in homogenate binding studies and gave a more precise regional localization of glycine receptors in this animal. The highest overall labeling was observed in the spinal cord and medulla; areas of highest grain density included the dorsal horn of the spinal cord, the cranial nerve nuclei, the dorsal column nuclei and nuclei of the medullary reticular formation. A decrease in overall grain density was observed rostrally throughout the midbrain and pons; in caudal regions, however, dense labeling was observed over the trigeminal, vestibular and facial nuclei and over the major nuclei of the auditory system. In more rostral areas, the interpeduncular nucleus and the substantia nigra were also clearly delineated, as were certain thalamic nuclei. The cerebellum, cortex, hippocampus and olfactory bulbs showed only very low levels of grain density. In summary, it appears that high concentrations of glycine receptors in the brain and spinal cord may be preferentially localized to neurons involved in the processing of information originating from exteroceptive sensory mechanoreceptors.     

Glycine receptor distribution in mouse CNS: autoradiographic localization of [H]strychnine binding sites

Biochemical and electrophysiological studies of mammalian CNS indicate that the amino acid, glycine, is a major inhibitory neurotransmitter whose location is, for the large part, confined to the spinal cord and brain stem. In this study, autoradiographs of C57BL/6J mouse brain sections labeled with [³H] strychnine, a potent antagonist of glycine, were used to map the distribution of glycine receptors in the CNS. Autoradiographs showed highly localized areas of grain density, which confirmed the gross distributions described in homogenate binding studies and gave a more precise regional localization of glycine receptors in this animal. The highest overall labeling was observed in the spinal cord and medulla; areas of highest grain density included the dorsal horn of the spinal cord, the cranial nerve nuclei, the dorsal column nuclei and nuclei of the medullary reticular formation. A decrease in overall grain density was observed rostrally throughout the midbrain and pons; in caudal regions, however, dense labeling was observed over the trigeminal, vestibular and facial nuclei and over the major nuclei of the auditory system. In more rostral areas, the interpeduncular nucleus and the substantia nigra were also clearly delineated, as were certain thalamic nuclei. The cerebellum, cortex, hippocampus and olfactory bulbs showed only very low levels of grain density. In summary, it appears that high concentrations of glycine receptors in the brain and spinal cord may be preferentially localized to neurons involved in the processing of information originating from exteroceptive sensory mechanoreceptors.     

Conservation of antigenic epitopes of the inhibitory glycine receptor in rodent and goldfish CNS.

Monoclonal antibodies against the inhibitory glycine receptor of rat spinal cord were used to identify corresponding receptor polypeptides in goldfish CNS. Both Western blot analysis and quantitative receptor immunoassays revealed crossreacting antigens in goldfish brain membranes. A polypeptide of 46 kDa molecular weight is immunologically related to the 48 kDa alpha subunit of the mammalian receptor. Similarly, a large receptor-associated protein of 93 kDa is present both in goldfish and mammals. Throughout the goldfish CNS, glycine-displaceable [3H]strychnine binding codistributes with the alpha subunit protein as determined immunologically. Glycine receptor contents were highest in goldfish medulla oblongata, medium in optic tectum and mesencephalon, whereas little or no receptor was detected in cerebellum, olfactory bulb, and spinal cord. Immunohistochemistry confirmed that the alpha subunit antigen and the 93 kDa protein were located in the plasma membrane of neurons and concentrated in small clusters found on the soma and dendrites. These data indicate that immunological properties and cellular distribution of glycine receptors are conserved from fish to mammals.     

Solubilization of the glycine receptor from rat spinal cord

Glycine receptors, as detected by glycine-displaceable [3H]strychnine binding, were solubilized from a membrane fraction of rat spinal cord by the non-ionic detergent Triton X-100. The solubilized material retained its high affinity for [3H]strychnine and exhibited the typical pharmacological properties of the membrane-bound glycine receptor. On sucrose density gradients, the solubilized receptor had a sedimentation coefficient of 8.3 +/- 0.4 S. Gel exclusion chromatography on Sepharose 6 B in the presence of phosphatidylcholine gave a Stokes radius of 7.3 +/- 0.3 nm.     

Glycine high affinity uptake and strychnine binding associated with glycine receptors in the frog central nervous system

Accumulation of [3H]glycine into synaptosomal fractions occurs by high affinity systems in cerebral cortex, optic tectum, brain stem and spinal cord of the frog. Specific [3H]strychnine binding which appears associated with postsynaptic glycine receptors is also demonstrable in these regions. By contrast, only very low levels of strychnine binding and high affinity glycine uptake occur in higher centers of the rat central nervous system. The relative potencies of small neutral amino acids in competing for [3H]strychnine binding are similar in frog brain and spinal cord. No evidence for a high affinity accumulation of [3H]taurine by synaptosomal fractions of frog spinal cord can be demonstrated. These observations favor glycine rather than taurine as an inhibitory transmitter in frog spinal cord. Moreover, these findings suggest that glycine may have a synaptic role in higher brain centers in the frog.     

Age-dependent changes in brain glycine concentration and strychnine-induced seizures in the rat

Glycine levels and receptor binding were measured in the medulla and spinal cord of 2-month, 10-month, and 24-month-old Fischer 344 rats. The behavioral response to the administration of the glycine antagonist, strychnine, was also evaluated in 2- and 24-month-old animals to investigate the relevance of these parameters to the susceptibility to seizures. Significant reductions in glycine in both the spinal cord and medulla occurred from 2 to 24 months of age. The glycine precursors, serine and threonine, were decreased only in the spinal cord. [3H]Strychnine binding was also decreased by 38% and 34% in the medulla and spinal cord, respectively, of 24-month-old rats compared to 2-month-olds. [3H]GABA binding was similarly reduced while no age-related changes in [3H]diazepam binding in the spinal cord were detected. Comparison of 2- and 24-month-old animals after systemic injection of 1.75 mg/kg strychnine showed that senescent animals have a higher incidence of seizures and mortality compared to young animals. Decreases in glycinergic neurotransmission may lower strychnine seizure threshold in the aged animal.     

Putative glycine receptors in Hydra: a biochemical and behavioural study

Glycine acts as an inhibitory transmitter in the lower brain stem and spinal cord of vertebrate species, while very few data are yet available to support a similar role in invertebrate nervous systems. Here we report the identification and characterization of glycine receptors in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa) by biochemical and behavioural studies. Saturation experiments revealed the occurrence of one population of binding sites of nanomolar affinity (KD = 33 nm) and low capacity (Bmax = 79 fmol/mg protein) for [(3)H]strychnine. The addition of glycine or taurine (0.1 microm-1 mm) produced a dose-dependent inhibition of [(3)H]strychnine binding. Beta-alanine (0.1-1 mm) did not significantly affect [(3)H]strychnine binding. The pharmacological properties of these receptors compare with those of vertebrate glycine receptors. Stimulation of Hydra polyps by reduced glutathione resulted in a significant increase in the duration of mouth opening in the presence of glycine, taurine or beta-alanine. The enhancement of the response was related both to amino acid (10-100 microm) and to glutathione concentration (1-10 microm). The effects of glycine or its agonists were suppressed by strychnine (1-10 microm). D-serine, a glycine agonist at the vertebrate NMDA receptor, produced opposite effects to those of glycine. The effects of d-serine were suppressed by 5,7-dichlorokynurenic acid but not by strychnine. In vitro, [(3)H]strychnine binding was not displaced by d-serine. These results indicate a dual action of glycine in Hydra tissues. The hypothesis that NMDA receptors may also be present in this elementary nervous system is proposed.     

Cloning and functional characterization of two glycine receptor alpha-subunits from the perch retina

Glycine receptors are ligand-gated ion channel proteins mediating synaptic inhibition in the spinal cord, retina and brain of vertebrates. We have cloned and functionally characterized two glycine receptor alpha-subunits from the perch (Roccus americana) retina. Based on sequence homology with the mammalian counterparts, we termed these subunits alpha 1 and alpha 3. RT-PCR revealed the presence of both subunits in retina and brain, whereas alpha1 was predominant in spinal cord. A short splice variant of alpha1 was detected in the brain but not in the retina. Functional expression of the perch subunits in HEK-293 cells yielded robust glycine-gated currents sensitive to strychnine. The perch receptors displayed a high efficacy for taurine and GABA and thus differ from the mammalian counterparts. Because the retina is a rich source for taurine, this finding could be of physiological importance. The structural features of the ligand binding domain strongly support the notion of increased glycine/GABA discrimination in higher vertebrates.     

Glycine receptor reduction within segmental gray matter in a rat model of neuropathic pain

Abstract

Glycine is an amino acid neurotransmitter found in the spinal cord and is closely associated with interneurons that modulate afferent activity. We have previously shown that low segmental glycine concentrations or blockade of normal glycinergic activity lowers the threshold for pain thresholds. In addition, intrathecal glycine infusion increases the pain threshold in animal models of neuropathic pain. However, the role of the glycine receptor in neuropathic pain is not clear and is the basis for the current study. Using a unilateral sciatic nerve constriction injury model of neuropathic pain, the strychnine sensitive glycine receptor population was studied using immunohistochemical techniques. Glycine receptors are reduced in number in the dorsal horn bilaterally in injured animals. Glycine and related compounds are potentially valuable agents for treating chronic pain conditions in humans. A better understanding of glycine-receptor interactions should prove valuable as these compounds are studied in greater depth. [Neural Res 1998; 20: 161–168]     

The glycine receptor in the mutant mouse spastic (spa): strychnine binding characteristics and pharmacology

There is a marked deficit in the binding of the glycine receptor antagonist strychnine to the CNS of the mutant mouse spastic. The characteristics and pharmacology of [3H]strychnine binding to washed homogenates of spastic and littermate control spinal cord and brainstem were investigated to determine the nature of this defect. The maximal binding of [3H]strychnine to spastic homogenates is approximately 20% the value obtained from littermate control homogenates; the affinity of [3H]strychnine binding is approximately 25% lower than littermate control values. The pharmacology of [3H]strychnine binding has the same rank order of potency in spastic and littermate control mice; however, there are small differences in the potency of several compounds in spastic vs littermate control animals. These results indicate that the glycine receptor alteration seen in the spastic mutant mouse is primarily due to a decrease in receptor number. They also suggest that the pharmacological characteristics of the glycine receptor in spastic are different from littermate control. The data do not permit a distinction between whether the strychnine binding sites found in spastic represent the same population seen in littermate control animals or are a subpopulation that is spared by the mutation.     

Distribution and lateral mobility of glycine receptors on cultured spinal cord neurons

Strychnine is one of the most potent antagonists of glycine-mediated inhibitory conductances in the mammalian spinal cord. In order to examine the distribution of glycine receptors (GlyRs) on neuronal cells, 2 novel fluorescent strychnine derivatives were synthesized and characterized chemically, spectroscopically, and biologically. Both compounds retain their biological activity after derivatization and are potent inhibitors of 3H-strychnine binding to bovine spinal cord membranes and membranes from rat spinal cord cultures. Using these fluorescent strychnine analogs, the cellular distribution and lateral mobility of GlyRs on cultured rat spinal cord neurons were studied by digital fluorescence imaging and photobleach recovery microscopy. On these neurons, even in the absence of observable synaptic contact and early in development GlyRs are predominantly localized to cell bodies with sparse labeling of neuritic processes. Although GlyRs are confined to the neuronal cell body, approximately 50% of the receptors are very mobile, with lateral diffusion coefficients of 1.15 +/- 0.05 x 10(-9) cm2/sec, a value which is characteristic of unrestricted protein lateral diffusion. However, the remaining fraction of these receptors are immobile on the neuronal cell body. More than 70% of the GlyRs distributed on neuronal processes are immobile, while 30% are laterally mobile, with diffusion rates of 5.50 +/- 0.1 x 10(-10) cm2/sec. The results indicate that even early in development GlyRs are expressed and segregated to the cell body, where they are confined within a domain that restricts their redistribution.     

Specific [H]strychnine binding associated with glycine receptors in bovine retina

Saturable, specific [³H]strychnine binding can be demonstrated in homogenates of bovine retina. Scatchard plots revealed only one set of binding sites with a dissociation constant (K_d) of about 60 nM and a maximal number of binding sites of about 1.5 pmol/mg protein. The structural specificity of [³H]strychnine binding sites in bovine retina parallels the properties found for [³H]strychnine binding sites in the spinal cord of several vertebrates. Thus, the data do not give any evidence that specific [³H]strychnine binding in bovine retina labels taurine rather than glycine receptors and favors glycine rather than taurine as inhibitory neurotransmitter in bovine retina. The subcellular distribution of specific [³H]strychnine binding in bovine retina parallels that of sodium-dependent, high-affinity uptake of glycine and taurine. All 3 parameters are mainly found in the P₂ fractions of bovine retina homogenates, containing conventional synaptosomes, most abundant in the inner plexiform layer, but can also be found in the P₁ fractions, containing large synaptosomes from the photoreceptor cell layer.     

Central effects of systemic lidocaine mediated by glycine spinal receptors: an iontophoretic study in the rat spinal cord

The objective of this investigation was to demonstrate the possible interactions of systemic lidocaine (lido) with inhibitory receptors in the spinal cord. In the lumbar dorsal horn of anesthetized and curarized rats, 60 physiologically identified, wide dynamic range (WDR) neurons, were recorded extracellularly. Glutamate, glycine and its selective antagonist, strychnine, were iontophoretically applied onto the neurons either singularly or concurrently. The effects of systemic lido on the drug-induced frequency changes and the interaction with the glycine receptors, using strychnine as a probe, were studied. It was consistently found that (i) lido (3–4 mg/kg) inhibited the excitatory responses to iontophoretic glutamate, (ii) this inhibition was significantly antagonized by concurrent iontophoretic strychnine, (iii) iontophoretic glycine induced comparable glutamate inhibition that was reversed by strychnine. In contrast, no effect on glutamate-induced excitations was observed when lido was applied by micropressure or a different local anesthetic was systemically administered. The results suggest that central inhibitory effects of lido could by mediated by spinal strychnine-sensitive glycine receptors, activated by lido itself or possibly by its glycine residue-bearing metabolites.     

Distribution of glycine receptor immunoreactivity in the spinal cord of the rat: cytochemical evidence for a differential glycinergic control of lamina I and V nociceptive neurons

In this study we characterized the distribution of glycine receptor immunoreactivity in the spinal cord of the rat by using monoclonal antisera directed against the purified glycine receptor. There was dense, punctate glycine receptor immunoreactive staining in all regions of the gray matter ventral to the substantia gelatinosa. The densest staining was found in laminae III and IV of the dorsal horn. There were also distinct, tributarylike bands of punctate staining that extended well into the white matter of the lateral and ventral funiculi. The only consistent cell body staining was found in small neurons of the ventral horn. The labelled neurons were distributed among larger, unlabelled motoneurons. In general, the pattern of glycine receptor immunoreactivity was similar at all levels of the spinal cord and was comparable to that seen with binding of a tritiated glycine receptor antagonist, strychnine, to sections of rat spinal cord (Zarbin et al.: J. Neurosci. 1:532-547, '81). Two important exceptions, however, were observed. In contrast to the high levels of strychnine binding reported in the substantia gelatinosa, we found almost no glycine receptor immunoreactivity in laminae I and II of the superficial dorsal horn of the spinal cord or of the trigeminal nucleus caudalis. There was also a notable absence of antibody staining in the intermediolateral cell column of the thoracic cord. The presence of dense glycine receptor immunoreactivity in the region of lamina V and its absence in the superficial dorsal horn are discussed in terms of a possible differential glycinergic control of nociceptive neurons of laminae I and V.     

Binding of [H]glycine, [H]β-alanine and [H]strychnine in cultured rat spinal cord and brain stem

Cultures of rat brain stem and spinal cord were used to visualize binding sites for [³H]glycine, [³H]β-alanine and their antagonist [³H]strychnine by light microscopic autoradiography. In spinal cord cultures, all radio-ligands were bound mainly to large neurones, probably motoneurones whereas in brain stem cultures, both medium-sized and large neurones were labelled. In contrast, glial cells did not show binding sites for any of the compounds studied, suggesting that glial elements may not possess receptors for glycine and β-alanine.     

Autoradiographic localization of thyrotropin-releasing hormone (TRH) receptors in human spinal cord

Thyrotropin-releasing hormone (TRH) exerts many effects upon spinal cord function in animals, and may also play a role in human spinal cord function. We have used the technique of quantitative autoradiography to anatomically localize specific receptors for TRH within human spinal cord. Highest concentrations of TRH receptors were localized within lamina II, the substantia gelatinosa. A moderate density of TRH receptors was found in lamina IX, the motor neurons of the anterior horn. Low levels of TRH receptors were noted throughout the remainder of the gray matter of the human spinal cord, and no TRH receptors were localized within white matter. This anatomic distribution of TRH receptors within the human spinal cord is consistent with the localization of endogenous TRH and the effects of exogenously applied TRH in animal studies. These results suggest that any effects of TRH on human spinal cord function may be mediated by TRH receptors.     

Distribution of neuromedin U binding sites in the rat CNS revealed by in vitro receptor autoradiography

Neuromedin U (NMU), a neuropeptide implicated in feeding, inflammation, pain control and anxiety-related behaviours, is widely distributed in peripheral organs and the CNS. These effects are thought to be mediated by its receptors NMU(1) and NMU(2). Since its precise sites of interaction in the CNS were to date unknown, we studied the distribution of in vitro binding sites for (125)I-NMU-23 in the rat CNS by receptor autoradiography. High-density specific binding was found in discrete areas of the brain and spinal cord, namely in the limbic system (hippocampal formation, septohippocampal nucleus, indusium griseum, hypothalamus, amygdaloid nuclei), superior colliculus, dorsal raphé, and substantia gelatinosa of the spinal cord. Our findings provide further supportive evidence for a multifunctional role for the peptide in the brain and spinal cord.     

The glycine receptor deficiency of the mutant mouse spastic: evidence for normal glycine receptor structure and localization

Homozygotes of the mutant mouse spastic exhibit reduced binding of 3H-strychnine to homogenates from various regions of the CNS compared with unaffected littermates (White and Heller, 1982). Here we report evidence that the spastic mutation coincides with a reduced concentration and an unaltered structure of the glycine receptor in spinal cord. Scatchard analysis of 3H-strychnine binding revealed a single binding site with a Bmax of 267 +/- 62 fmol/mg protein for spastic and of 864 +/- 220 fmol/mg protein for control mice; no difference was found for the corresponding KD values. Also Ki values of glycine for 3H-strychnine binding and displacement of 3H-strychnine by beta-alanine and taurine were indistinguishable for both preparations. Photoaffinity labeling of synaptic membranes with 3H-strychnine identified an Mr = 48,000 polypeptide in both control and spastic mouse membranes. Tryptic digestion of these membranes produced radiolabeled peptide fragments of identical molecular weights, suggesting that the proteolytic cleavage sites around the antagonist binding site are conserved in the mutant glycine receptor protein. Glycine receptors from both control and mutant mice were purified by affinity chromatography on aminostrychnine agarose. SDS/PAGE revealed three polypeptides of Mr = 48,000, 58,000, and 93,000 in both receptor preparations. Monoclonal antibodies directed against different subunits of the glycine receptor were applied to an enzyme-linked immunosorbent assay. The same pattern of immunoreactivity was obtained for glycine receptor from spinal cord of spastic homozygotes, control mice, and rats, suggesting conservation of the antigenic epitopes in the mutant receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABA, not glycine, mediates inhibition of latent respiratory motor pathways after spinal cord injury

Previous work has shown that latent respiratory motor pathways known as crossed phrenic pathways are inhibited via a spinal inhibitory process; however, the underlying mechanisms remain unknown. The present study investigated whether spinal GABA-A and/or glycine receptors are involved in the inhibition of the crossed phrenic pathways after a C2 spinal cord hemisection injury. Under ketamine/xylazine anesthesia, adult, female, Sprague-Dawley rats were hemisected at the C2 spinal cord level. Following 1 week post injury, rats were anesthetized with urethane, vagotomized, paralyzed and ventilated. GABA-A receptor (bicuculline and Gabazine) and glycine receptor (strychnine) antagonists were applied directly to the cervical spinal cord (C3-C7), while bilateral phrenic nerve motor output was recorded. GABA-A receptor antagonists significantly increased peak phrenic amplitude bilaterally and induced crossed phrenic activity in spinal-injured rats. Muscimol, a specific GABA-A receptor agonist, blocked these effects. Glycine receptor antagonists applied directly to the spinal cord had no significant effect on phrenic motor output. These results indicate that phrenic motor neurons are inhibited via a GABA-A mediated receptor mechanism located within the spinal cord to inhibit the expression of crossed phrenic pathways.