Glycine receptor subunits expression in the developing rat retina.

BACKGROUND AND METHODS: Glycine receptor (GlyR) consists of two α (1-4) and three ß subunits. Considerable evidence indicates that the adult retina expresses the four types of α subunits; however, the proportion of these subunits in adult and immature retina is almost unknown. In this report we have studied mRNA and the protein expression of GlyR subunits in the retina during postnatal rat development by Real-Time qRT-PCR and western blot. RESULTS: mRNA and protein expression indicated a gradual increase of the α1, α3, α4 and ß GlyR subunits during postnatal ages tested. The mRNA ß subunit showed higher expression levels (∼3 fold) than those observed for the α1 and α3 subunits. Very interestingly, the α2 GlyR subunit had the highest expression in the retina, even in the adult. CONCLUSIONS: These results revealed the expression of GlyR at early postnatal ages, supporting its role in retina development. In addition, our results indicated that the adult retina expressed a high proportion of the α2 subunit, suggesting the expression of monomeric and/or heteromeric receptors. A variety of studies are needed to further characterize the role of the specific subunits in both adult and immature retina.

A microdeletion at Xq22.2 implicates a glycine receptor GLRA4 involved in intellectual disability, behavioral problems and craniofacial anomalies.

BACKGROUND: Among the 21 annotated genes at Xq22.2, PLP1 is the only known gene involved in Xq22.2 microdeletion and microduplication syndromes with intellectual disability. Using an atypical microdeletion, which does not encompass PLP1, we implicate a novel gene GLRA4 involved in intellectual disability, behavioral problems and craniofacial anomalies. CASE PRESENTATION: We report a female patient (DGDP084) with a de novo Xq22.2 microdeletion of at least 110 kb presenting with intellectual disability, motor delay, behavioral problems and craniofacial anomalies. While her phenotypic features such as cognitive impairment and motor delay show overlap with Pelizaeus-Merzbacher disease (PMD) caused by PLP1 mutations at Xq22.2, this gene is not included in our patient's microdeletion and is not dysregulated by a position effect. Because the microdeletion encompasses only three genes, GLRA4, MORF4L2 and TCEAL1, we investigated their expression levels in various tissues by RT-qPCR and found that all three genes were highly expressed in whole human brain, fetal brain, cerebellum and hippocampus. When we examined the transcript levels of GLRA4, MORF4L2 as well as TCEAL1 in DGDP084's family, however, only GLRA4 transcripts were reduced in the female patient compared to her healthy mother. This suggests that GLRA4 is the plausible candidate gene for cognitive impairment, behavioral problems and craniofacial anomalies observed in DGDP084. Importantly, glycine receptors mediate inhibitory synaptic transmission in the brain stem as well as the spinal cord, and are known to be involved in syndromic intellectual disability. CONCLUSION: We hypothesize that GLRA4 is involved in intellectual disability, behavioral problems and craniofacial anomalies as the second gene identified for X-linked syndromic intellectual disability at Xq22.2. Additional point mutations or intragenic deletions of GLRA4 as well as functional studies are needed to further validate our hypothesis.

Differential alterations in the expression of neurotransmitter receptors in inner retina following loss of photoreceptors in rd1 mouse.

Loss of photoreceptors leads to significant remodeling in inner retina of rd1 mouse, a widely used model of retinal degeneration. Several morphological and physiological alterations occur in the second- and third-order retinal neurons. Synaptic activity in the excitatory bipolar cells and the predominantly inhibitory amacrine cells is enhanced. Retinal ganglion cells (RGCs) exhibit hyperactivity and aberrant spiking pattern, which adversely affects the quality of signals they can carry to the brain. To further understand the pathophysiology of retinal degeneration, and how it may lead to aberrant spiking in RGCs, we asked how loss of photoreceptors affects some of the neurotransmitter receptors in rd1 mouse. Using Western blotting, we measured the levels of several neurotransmitter receptors in adult rd1 mouse retina. We found significantly higher levels of AMPA, glycine and GABAa receptors, but lower levels of GABAc receptors in rd1 mouse than in wild-type. Since GABAa receptor is expressed in several retinal layers, we employed quantitative immunohistochemistry to measure GABAa receptor levels in specific retinal layers. We found that the levels of GABAa receptors in inner plexiform layer of wild-type and rd1 mice were similar, whereas those in outer plexiform layer and inner nuclear layer combined were higher in rd1 mouse. Specifically, we found that the number of GABAa-immunoreactive somas in the inner nuclear layer of rd1 mouse retina was significantly higher than in wild-type. These findings provide further insights into neurochemical remodeling in the inner retina of rd1 mouse, and how it might lead to oscillatory activity in RGCs.

Disturbed neuronal ER-Golgi sorting of unassembled glycine receptors suggests altered subcellular processing is a cause of human hyperekplexia.

Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) α1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR α1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment and cis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/ß2-3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR α1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/ß2-3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding.

Glycine modulates membrane potential, cell volume, and phagocytosis in murine microglia.

Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1-4). In BV-2 cells, glycine (5 mM) led to a rapid Na(+)-dependent depolarization of membrane potential (V mem). Furthermore, glycine increased CV by about 9%. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13%. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na(+) with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs.

Distribution of the glycine receptor ß-subunit in the mouse CNS as revealed by a novel monoclonal antibody.

Inhibitory glycine receptors (GlyRs) are composed of homologous α- (α1-4) and ß-subunits. The ß-subunits (GlyRß) interact via their large cytosolic loops with the postsynaptic scaffolding protein gephyrin and are therefore considered essential for synaptic localization. In situ hybridization studies indicate a widespread distribution of GlyRß transcripts throughout the mammalian central nervous system (CNS), whereas GlyRα mRNAs and proteins display more restricted expression patterns. Here we report the generation of a monoclonal antibody that specifically recognizes rodent GlyRß (mAb-GlyRß) and does not exhibit crossreactivity with any of the GlyRα1-4 subunits. Immunostaining with this antibody revealed high densities of punctate GlyRß immunoreactivity at inhibitory synapses in mouse spinal cord, brainstem, midbrain, and olfactory bulb but not in the neocortex, cerebellum, or hippocampus. This contrasts the abundance of GlyRß transcripts in all major regions of the rodent brain and suggests that GlyRß protein levels are regulated posttranscriptionally. When mAb-GlyRß was used in double-labeling experiments with GlyRα1-, α2-, α3-, or α4-specific antibodies to examine the colocalization of GlyRß with these GlyR subunits in the mouse retina, >90% of the GlyRα1-3 clusters detected were found to be GlyRß-immunoreactive. A subset (about 50%) of the GlyRα4 puncta in the inner plexiform layer, however, was found to lack GlyRß and gephyrin immunostaining. These GlyRα4-only clusters were apposed to bassoon immunoreactivity and hence synaptically localized. Their existence points to a gephyrin-independent synaptic localization mechanism for a minor subset of GlyRs.

Incompatibility between a pair of residues from the pre-M1 linker and Cys-loop blocks surface expression of the glycine receptor.

Regulation of cell membrane excitability can be achieved either by modulating the functional properties of cell membrane-expressed single channels or by varying the number of expressed channels. Whereas the structural basis underlying single channel properties has been intensively studied, the structural basis contributing to surface expression is less well characterized. Here we demonstrate that homologous substitution of the pre-M1 linker from the ß subunit prevents surface expression of the α1 glycine receptor chloride channel. By investigating a series of chimeras comprising α1 and ß subunits, we hypothesized that this effect was due to incompatibility between a pair of positively charged residues, which lie in close proximity to each other in the tertiary structure, from the pre-M1 linker and Cys-loop. Abolishing either positive charge restored surface expression. We propose that incompatibility (electrostatic repulsion) between this pair of residues misfolds the glycine receptor, and in consequence, the protein is retained in the cytoplasm and prevented from surface expression by the quality control machinery. This hypothesis suggests a novel mechanism, i.e. residue incompatibility, for explaining the mutation-induced reduction in channel surface expression, often present in the cases of hereditary hyperekplexia.

Intrinsically photosensitive ganglion cells of the primate retina express distinct combinations of inhibitory neurotransmitter receptors.

Intrinsically-photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and function as irradiance detectors, responsible for crucial non-image forming visual functions. In addition to their intrinsic photosensitivity, ipRGCs are also activated by synaptic inputs originating at the classical photoreceptors, rods and cones. Little is known about inhibition through these retinal pathways, despite ipRGCs receiving massive synaptic inputs from inhibitory amacrine interneurons. We performed a wide anatomical screening for neurotransmitter receptors possibly involved in the inhibitory modulation of ipRGCs in the macaque retina. We investigated both subtypes of primate ipRGCs described so far and report that outer-stratifying (M1) cells possess mainly GlyR α2 and GABA(A)R α3 subunits, while inner-stratifying (M2) cells are overall subject to less inhibitory modulation. Our results suggest that M1 and M2 ipRGC subtypes are modulated via distinct inhibitory intraretinal circuits.

Monaural conductive hearing loss alters the expression of the GluA3 AMPA and glycine receptor α1 subunits in bushy and fusiform cells of the cochlear nucleus.

The impact of conductive hearing loss (CHL), the second most common form of hearing loss, on neuronal plasticity in the central auditory pathway is unknown. After short-term (1 day) monaural earplugging, the GluA3 subunits of the AMPA receptor (AMPAR) are upregulated at auditory nerve synapses on the projection neurons of the cochlear nucleus; glycine receptor α1 (GlyRα1) subunits are downregulated at inhibitory synapses in the same neuronal population. These data suggest that CHL affects receptor trafficking at synapses. We examined the impact of 7 days of CHL on the general expression of excitatory and inhibitory receptors by quantitative biochemistry and immunohistochemistry, using specific antibodies to detect AMPAR subunits (GluA1, GluA2, GluA2/3, and GluA4), GlyRα1, and the GABA(A) receptor subunits ß2/3. Following monaural earplugging and an elevation of the hearing threshold by approximately 35 dB, the immunolabeling of the antibody for the GluA2/3 subunits but not the GluA2 subunit increased on bushy cells (BCs) and fusiform cells (FCs) of the ipsilateral ventral and dorsal cochlear nuclei. These same cell types showed a downregulation of the GlyRα1 subunit. Similar results were observed in the contralateral nuclei. The expression levels of GABA(A) ß2/3 were unchanged. These findings suggest that, following longer periods of monaural conductive hearing loss, the synthesis and subsequent composition of specific glutamate and glycine receptors in projection neurons and their synapses are altered; these changes may contribute to abnormal auditory processing.

Effects of inhibitory amino acids on expression of GABAA Rα and glycine Rα1 in hypoxic rat cortical neurons during development.

Recent studies suggest that GABA and glycine are protective to mature but toxic to immature cortical neurons during prolonged hypoxia. Since the action of these inhibitory amino acids is mediated by GABA and glycine receptors, the expression of these receptors is a critical factor in determining neuronal response to GABA(A) and glycine in hypoxia. Therefore, we asked whether in rat cortical neurons, 1) hypoxia alters the expression of the GABA and glycine receptors; 2) inhibitory amino acids change the course of GABA and glycine receptor expression; and 3) there are any differences between the immature and mature neurons. In cultured rat cortical neurons from day 4 (four days in vitro or DIV 4) to day 20 (DIV 20), we observed that 1) GABA(A)Rα and GlyRα1 underwent differential changes in expression during the development in vitro; 2) hypoxia for 3 days decreased GABA(A)Rα and GlyRα1 density in the neurons in-between DIV 4 and DIV 20, but did not induce a major change in immature (DIV 4) and mature (DIV 20) neurons; 3) during normoxia GABA, glycine and taurine decreased GABA(A)Rα and GlyRα1 density in the immature neurons, but had a tendency to increase the density in the mature neurons, except for taurine; 4) under hypoxia, all these amino acids decreased GABA(A)Rα and GlyRα1 density in most groups of the immature neurons with a slight effect on the mature neurons; and 5) δ-opioid receptor activation with DADLE increased GABA(A)Rα and GlyRα1 density in both the immature and mature neurons under normoxia and in the mature neurons under hypoxic condition. These data suggest that inhibitory amino acids differentially regulate the expression of GABA(A) and glycine receptors in rat cortical neurons under normoxic and hypoxic conditions with major differences between the immature and mature neurons.

Maternal voluntary drinking in C57BL/6J mice: advancing a model for fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) remain the most common preventable cause of behavioural abnormalities and cognitive deficits, yet little is known about the biological mechanisms involved in FASD pathology. Maternal voluntary ethanol consumption in mice may be a useful model for establishing the biological basis of moderate ethanol exposure phenotypes, which make up the majority of FASD cases. We have employed a two-bottle choice paradigm of maternal ethanol consumption throughout gestation and the early postnatal period in C57BL/6J mice. We assessed the efficacy of this model to produce a range of FASD-relevant phenotypes and evaluated gene expression changes in the adult offspring. Results showed stable maternal consumption and lack of maternal care differences between ethanol-consuming and water-only dams. Ethanol-exposed offspring showed delays in neonatal reflex and coordination development. Further, ethanol-exposed adolescent mice showed decreased activity in a novel environment that appeared to be the result of novelty-induced anxiety, and acquisition learning deficits. Evaluation of the neurotransmitter-associated genes Gabra6, Glra1, and Grin2c revealed significant down-regulation of Glra1 and Grin2c in the brains of ethanol-exposed young adult males. These results suggest that this model is able to produce a range of behavioural phenotypes consistent with prenatal ethanol exposure and may be used to evaluate resulting long-term genetic changes. Given the range of genetic resources available for inbred mouse strains, the model described here may prove to be a useful tool in evaluating the molecular basis of FASD.

Ultrastructure, synaptic organization, and molecular components of bushy cell networks in the anteroventral cochlear nucleus of the rhesus monkey.

Bushy cells (BCs) process auditory information in the ventral cochlear nucleus (VCN). Yet, most neuroanatomical findings come from studies in cats and rodents, and the ultrastructural morphological features of BCs in humans and higher nonhuman primates are unknown. In this study, we combined histological, immunocytochemical, and ultrastructural methods to examine the morphology and synaptic organization of BCs in the rhesus monkey VCN. We observed that BCs were organized in a complex neural network that appears to interconnect the cells. The fine structure of BC somata and dendrites, as well as their synaptic inputs, are similar to those in other mammals. We found that BCs received numerous endbulb-like VGLUT1- and VGLUT2-immunopositive endings. In addition, they expressed glutamate AMPA (GluR2/3 and GluR4), NMDA (NR1), delta1/2 receptor subunits, and the α1 subunit of the glycine receptor. These receptor types and subunits mediate fast excitatory synaptic transmission from the cochlea and inhibitory neurotransmission from noncochlear inputs. Parvalbumin immunostaining and semithin sections showed that BC dendrites are oriented toward neighboring BC somas to form neuronal clusters. Within the cluster, the incoming inputs established multiple, divergent synaptic contacts. Thus, BCs were connected by specialized dendrosomatic and somasomatic membrane junctions. Our results indicate that the cytoarchitectural organization of BCs is well conserved between primates and other mammalian species.

Differential expression of glycine receptor subunit messenger RNA in the rat following spinal cord injury.

STUDY DESIGN: Spinal cord injury (SCI) results in alterations in the regulation of many genes that may influence neuronal death and the subsequent loss of motor function and neuropathic pain. The subtype expression mRNA levels of glycine receptors (GlyRs) after SCI are unknown. METHODS: Using real-time reverse transcription PCR, this study analyzed changes in the mRNA abundance of the four major GlyR subunits (α13 and ß) at 6, 24 h and 3, 7 and 10 days after SCI in adult male rats. SCI was induced at the T9 level by transection. RESULTS: Our results show a complicated temporal and spatial pattern of alteration in GlyR mRNA expression levels after SCI. Temporal and spatial variations with different degrees and direction (up or downregulation) of expression alteration were observed. The greatest variation was seen in GlyRα1, whereas GlyRα2 was downregulated in all regions following SCI. CONCLUSION: This study shows that alteration in GlyR expression starts as early as 6 h after SCI. The most significant points of this research are temporal elevation of GlyRα1 and continuous reduction of GlyRα2. Alterations in GlyR expression within the spinal cord may have a key role in the development of pathological pain. Therefore, control of GlyR expression could represent a novel therapeutic avenue for the development of new painkiller agents in SCI.

The inhibitory neurotransmitter glycine modulates macrophage activity by activation of neutral amino acid transporters.

Glycine, an important inhibitory neurotransmitter in the mammalian central nervous system (CNS), has been shown to modulate peripheral immune cell responses. In that respect, glycine levels are increased in several neuroinflammatory disorders, such as amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). In this study, we show that glycine modulates macrophage effector functions implicated in CNS inflammation and in other, related inflammatory conditions. We demonstrate that glycine does not affect the production of reactive oxygen species but stimulates myelin phagocytosis and the production of the proinflammatory mediators nitric oxide (NO) and tumor necrosis factor (TNF)-alpha by rat macrophages. These effects of glycine are not mediated by the glycine receptor (GlyR) or by glycine transporters (GlyTs), as neither the GlyR antagonist strychnine nor the antagonist of GlyT1 (ALX5407) reverses the observed effects. In contrast, 2-aminoisobutyric acid, a substrate of neutral amino acid transporters (NAATs), inhibits the glycine-mediated enhancement of myelin phagocytosis as well as of NO and TNF-alpha production. In conclusion, our findings demonstrate that glycine modulates macrophage function through activation of NAATs. Glycine may thereby influence immunological processes in inflammatory diseases involving macrophage activation and demyelination, including MS and related conditions associated with altered glycine levels.

Brain derived neurotrophic factor and neurotrophic factor 3 modulate neurotransmitter receptor expressions on developing spiral ganglion neurons.

Cochlear spiral ganglion neurons (SGN) provide the only pathway for transmitting sound evoked activity from the hair cells to the central auditory system. Neurotrophic factor 3 (NT-3) and brain derived neurotrophic factor (BDNF) released from hair cells and supporting cells exert a profound effect on SGN survival and neural firing patterns; however, it is unclear what the effects NT-3 and BDNF have on the type of neurotransmitter receptors expressed on SGN. To address this question, the whole-cell patch clamp recording technique was used to determine what effect NT-3 and BDNF had on the function and expression of glutamate, GABA and glycine receptors (GlyR) on SGN of cochlea from postnatal C57 mouse. Receptor currents induced by the agonist of each receptor were recorded from SGN cultured with or without BDNF or NT-3. NT-3 and BDNF exerted different effects. NT-3, and to a lesser extent BDNF, enhanced the expression of GABA receptors and had comparatively little effect on glutamate receptors. Absence of BDNF and NT-3 resulted in the emergence of glycine-induced currents; however, GlyR currents were absent from the short term cultured SGN. In contrast, NT-3 and BDNF suppressed GlyR expression on SGN. These results indicate that NT-3 and BDNF exert a profound effect on the types of neurotransmitter receptors expressed on postnatal SGN, results that may have important implications for neural development and plasticity.

Multiplexed labeling of viable cells for high-throughput analysis of glycine receptor function using flow cytometry.

Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed cells that expressed functionally distinct glycine receptor chloride channels and analyzed responses to glycine in terms of chloride-sensitive YFP expression. Here, data provided by flow cytometry can be used to discriminate between functional and nonfunctional mutations in the glycine receptor, a process accelerated by the use of multiplexing. Further, this data correlates to data generated using a microscopy-based technique. The present study demonstrates multiplexed labeling of live cells, to enable cell populations to be subject to further cell culture and experimentation, and compares the results with those obtained using live cell microscopy.

Synaptic homeostasis in a zebrafish glial glycine transporter mutant.

Truncated escape responses characteristic of the zebrafish shocked mutant result from a defective glial glycine transporter (GlyT1). In homozygous GlyT1 mutants, irrigating brain ventricles with glycine-free solution rescues normal swimming. Conversely, elevating brain glycine levels restores motility defects. These experiments are consistent with previous studies that demonstrate regulation of global glycine levels in the CNS as a primary function of GlyT1. As GlyT1 mutants mature, their ability to mount an escape response naturally recovers. To understand the basis of this recovery, we assay synaptic transmission in primary spinal motor neurons by measuring stimulus-evoked postsynaptic potentials. At the peak of the motility defect, inhibitory synaptic potentials are both significantly larger and more prolonged indicating a prominent role for GlyT1 in shaping fast synaptic transmission. However, as GlyT1 mutants naturally regain their ability to swim, the amplitude of inhibitory potentials decreases to below wild-type levels. In parallel with diminishing synaptic potentials, the glycine concentration required to evoke the mutant motility defect increases 61-fold during behavioral recovery. Behavioral recovery is also mirrored by a reduction in the levels of both glycine receptor protein and transcript. These results suggest that increased CNS glycine tolerance and reduced glycine receptor expression in GlyT1 mutants reflect compensatory mechanisms for functional recovery from excess nervous system inhibition.

Spinal antiallodynia action of glycine transporter inhibitors in neuropathic pain models in mice.

Neuropathic pain is refractory against conventional analgesics, and thus novel medicaments are desired for the treatment. Glycinergic neurons are localized in specific brain regions, including the spinal cord, where they play an important role in the regulation of pain signal transduction. Glycine transporter (GlyT)1, present in glial cells, and GlyT2, located in neurons, play roles in modulating glycinergic neurotransmission by clearing synaptically released glycine or supplying glycine to the neurons and thus could modify pain signal transmission in the spinal cord. In this study, we demonstrated that i.v. or intrathecal administration of GlyT1 inhibitors, cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl methyl)amino methylcarboxylic acid (ORG25935) or sarcosine, and GlyT2 inhibitors, 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)-methyl]benzamide (ORG25543) and (O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-L-serine) (ALX1393), or knockdown of spinal GlyTs by small interfering RNA of GlyTs mRNA produced a profound antiallodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice. The antiallodynia effect is mediated through spinal glycine receptor alpha3. These results established GlyTs as the target molecules for the development of medicaments for neuropathic pain. However, these manipulations to stimulate glycinergic neuronal activity were without effect during the 4 days after nerve injury, whereas manipulations to inhibit glycinergic neuronal activity protected against the development of allodynia in this phase. The results implied that the timing of medication with their inhibitors should be considered, because glycinergic control of pain was reversed in the critical period of 3 to 4 days after surgery. This may also provide important information for understanding the underlying molecular mechanisms of the development of neuropathic pain.

The effects of the local anesthetics lidocaine and procaine on glycine and gamma-aminobutyric acid receptors expressed in Xenopus oocytes.

BACKGROUND: The voltage-dependent sodium channel is the primary site of action for local anesthetics (LAs). Although systemically administered low-dose LAs have been shown to exert antihyperalgesic effects, the molecular targets responsible for these effects are not fully known and their functional effects on inhibitory neurotransmitter receptors associated with antinociception have not been sufficiently studied. METHODS: We examined the effects of lidocaine and procaine (0.1 microM to 3 or 10 mM) on recombinant human alpha1 glycine, alpha1beta2gamma2S gamma-aminobutyric acid type A (GABA(A)), and rho1 GABA(C) receptors expressed in Xenopus laevis oocytes, using a two-electrode voltage-clamp system. We also evaluated the effects of LAs on two mutant glycine receptors, alpha1(S267C) and alpha1(S267Q), in an effort to clarify the interaction between LAs and glycine receptors. RESULTS: Low concentrations of both lidocaine and procaine enhanced glycine receptor function, whereas high concentrations of lidocaine and procaine inhibited glycine receptor function. Lidocaine (10 microM) produced a significant leftward shift in the glycine concentration-response curve, indicating an increase in the apparent affinity for glycine. This enhancement was not altered in the mutant receptors. Both lidocaine and procaine at high concentrations inhibited GABA(A) receptor currents, whereas neither lidocaine nor procaine affected GABA(C) receptor function. CONCLUSIONS: Lidocaine and procaine enhanced glycine receptor function at low concentrations and inhibited the functions of glycine and GABA(A) receptors at high concentrations. The mechanism of the LA-induced enhancement of glycine receptor function probably differs from that of general anesthetics. These findings may explain the pharmacological effects of LAs, such as antinociception and convulsion.

Expression of heteromeric glycine receptor-channels in rat spinal cultures and inhibition by neuroactive steroids.

Ionotropic glycine receptors were studied in cultured spinal cord neurons prepared from 17-day-old rat embryos, using whole-cell patch clamp electrophysiology. Glycine receptors of 3-17 days in vitro were characterized via subtype-specific channel blockade by micromolar picrotoxin and cyanotriphenylborate, as well as nanomolar strychnine. Potentiation by nanomolar tropisetron indicated coexpression of beta with alpha subunits. The neuroactive steroids pregnenolone sulfate and dehydroepiandrosterone sulfate, as well as alphaxalone and its 3beta epimer betaxalone inhibited the chloride current with IC(50) values of 19, 46, 16 and 208 microM, respectively, with no potentiation. Reverse transcription polymerase chain reaction and immunocytochemistry demonstrated mRNAs and proteins of alpha1, alpha2, alpha3 and beta subunits in rat spinal cord cultures. In conclusion, neuroactive steroids, both positive and negative modulators of gamma-aminobutyric-acid(A) receptors, inhibited heteromeric glycine receptors at micromolar concentrations.