Embryonic rat spinal cord neurons change expression of glycine receptor subtypes during development in vitro

The expression of functional glycine receptors (GlyRs) by embryonic rat spinal cord neurons during development in vitro was investigated using whole-cell patch-clamp recordings. Functional GlyRs were expressed by most neurons within 1 day in vitro, and by all neurons from 4 days onward. However, the extent to which responses to glycine were blocked by the antagonist strychnine differed significantly between the first few days and 8 days in culture. Responses to glycine by neurons during the first few days in culture exhibited significantly less blockade by strychnine than those in neurons after 1 week in culture. Responses to glycine at both ages reflected an increased conductance to chloride ions, ruling out involvement of N-methyl-D -aspartate type glutamate receptors, and were not due to cross activation of γ-aminobutyric acid receptors. Monoclonal antibody 4a, which recognizes multiple subtypes of rat GlyR α subunits, labeled most neurons as early as 1 day in vitro, confirming that neurons express some form of GlyR α subunits by the first day in culture. These results show that rat spinal cord neurons express GlyRs early in their differentiation in vitro, and they suggest that individual neurons express as functional, cell-surface GlyRs a strychnine-insensitive isoform of the GlyR, possibly the previously described α2^* subunit. In addition, these results indicate that the expression of GlyR isoforms changes from predominantly a strychnine-insensitive isoform to other, strychnine-sensitive isoform(s) GlyR during development in vitro. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 579–592, 1997     

Agonist binding to purified glycine receptor reconstituted into giant liposomes elicits two types of chloride currents.

Using 'inside-out' membrane patches obtained from reconstituted giant liposomes containing purified glycine receptor from rat spinal cord, we have detected chloride currents elicited in response to the presence of the agonists glycine or beta-alanine. Regardless of the agonist employed, two different patterns of single channel currents could be detected, which differ in their main conductance, complexity of substates and opening frequency. In agreement with the expectations of glycine receptor heterogeneity suggested recently at the mRNA and cDNA level, our results indicate the existence of functionally different glycine receptors in the adult rat spinal cord.     

Substance P Receptor Expression and Cellular Responses to Substance P in Prenatal Rat Spinal Cord Cells

Abstract

Substance P receptors (SPRs) are expressed by prenatal rat spinal cord neurons and glial cells early in their differentiation, and SPRs may mediate developmental influences in the developing spinal cord. In order to understand better early SPR expression, we quantified SPR mRNA in the rat spinal cord during prenatal development using a cDNA probe for the rat SPR in nuclease protection assays. SPR mRNA was present in the rat spinal cord at E14, the earliest stage examined, and the presence of specific binding sites for radiolabeled SP suggested that SPRs were expressed at the protein level as well. Comparisons of samples from rats at different prenatal ages showed that the relative abundance of SPR mRNA declined by about 75% from E14 through the remainder of prenatal development. Assays of the hydrolysis of phosphatidyl inositol performed on prenatal spinal cord cells in culture revealed that SP caused a small but significant stimulation. These results show that expression of SPRs is an early molecular event in the development of the rat spinal cord in vivo and that SPRs on young spinal cord cells can mediate functional responses at early developmental stages.     

Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.     

Purinergic signalling in a latent stem cell niche of the rat spinal cord

The ependyma of the spinal cord harbours stem cells which are activated by traumatic spinal cord injury. Progenitor-like cells in the central canal (CC) are organized in spatial domains. The cells lining the lateral aspects combine characteristics of ependymocytes and radial glia (RG) whereas in the dorsal and ventral poles, CC-contacting cells have the morphological phenotype of RG and display complex electrophysiological phenotypes. The signals that may affect these progenitors are little understood. Because ATP is massively released after spinal cord injury, we hypothesized that purinergic signalling plays a part in this spinal stem cell niche. We combined immunohistochemistry, in vitro patch-clamp whole-cell recordings and Ca²⁺ imaging to explore the effects of purinergic agonists on ependymal progenitor-like cells in the neonatal (P1–P6) rat spinal cord. Prolonged focal application of a high concentration of ATP (1 mM) induced a slow inward current. Equimolar concentrations of BzATP generated larger currents that reversed close to 0 mV, had a linear current–voltage relationship and were blocked by Brilliant Blue G, suggesting the presence of functional P2X7 receptors. Immunohistochemistry showed that P2X7 receptors were expressed around the CC and the processes of RG. BzATP also generated Ca²⁺ waves in RG that were triggered by Ca²⁺ influx and propagated via Ca²⁺ release from internal stores through activation of ryanodine receptors. We speculate that the intracellular Ca²⁺ signalling triggered by P2X7 receptor activation may be an epigenetic mechanism to modulate the behaviour of progenitors in response to ATP released after injury.     

Postnatal Development of Synaptic Glycine Receptors in Normal and Hyperglycinemic Rats

The postnatal development of glycine synaptic receptors has been studied. Strychnine binding to the synaptic membrane fraction is very low at birth, increases thereafter, and reaches adult values at the 15th day in the brain, and at the 30th day in the spinal cord. Throughout postnatal development, there are more glycine receptors in the spinal cord than in the brain. The development of receptors in the spinal cord displays a pattern similar to that reported previously for the glycine reuptake system in spinal cord slices and in the activity of spinal cord glycine synthase. In rats with experimental hyperglycinemia strychnine binding to spinal cord glycine receptors increases much more rapidly, reaching a level 1.5 times the control value by day 10. When the hyperglycinemia was induced after the 10th postnatal day, however, no effect on the glycine receptors was observed. This increased number of receptors could be explained by an effect of glycine on the synaptic stabilisation process. No changes in the KD for strychnine were observed either during postnatal development or in hyperglycinemic rats. The KD remained approximately 10 nM in the spinal cord and 50 nM in the brain. Results are discussed with respect to the ontogeny of glycinergic synapses and the pathogenesis of nonketotic hyperglycinemia.     

Glial Regulation of α7-Type Nicotinic Acetylcholine Receptor Expression in Cultured Rat Cortical Neurons

Abstract: Primary embryonic cortical cultures were used as an in vitro model to evaluate the influence of glia on developmental expression of α7-type nicotinic acetylcholine receptors in rat brain. In cells cultured in serum-containing medium without mitotic inhibitors, specific ¹²⁵I-α-bungarotoxin binding to α7-type nicotinic receptors was maximal 4–8 days after plating. Treatment with 5'-fluorodeoxyuridine (80 µ M ) from 1 to 3 days in vitro significantly reduced glial proliferation and concomitantly increased ¹²⁵I-α-bungarotoxin binding, whereas plating onto a glial bed layer decreased binding. There was no significant binding to pure glial cultures. Treatment-induced changes in neuronal binding resulted from alterations in receptor density, with no change in affinity. 5'-Fluorodeoxyuridine treatment also increased cellular expression of α7 receptor mRNA but had no effect on N -[³H]methylscopolamine binding to muscarinic receptors. Glial conditioned medium decreased ¹²⁵I-α-bungarotoxin binding in both control and 5'-fluorodeoxyuridine-treated cultures, suggesting the release of a soluble factor that inhibits α7-type nicotinic receptor expression. An additional mechanism of glial regulation may involve removal of glutamate from the surrounding medium, as added glutamate (200 µ M ) increased ¹²⁵I-α-bungarotoxin binding in astrocyte-poor cultures but not in those that were astrocyte enriched. These results suggest that glia may serve a physiological role in regulating α7-type nicotinic receptors in developing brain.     

Glycine Binding to Rat Cortex and Spinal Cord: Binding Characteristics and Pharmacology Reveal Distinct Populations of Sites

Glycine is the principal inhibitory neurotransmitter in posterior regions of the brain. In addition, glycine serves as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) acidic amino acid receptor subtype. The studies presented here characterize [3H]glycine binding to washed membranes prepared from rat spinal cord and cortex, areas enriched in glycine inhibitory and NMDA receptors, respectively, in an attempt to define the glycine recognition sites on the two classes of receptors. Specific binding for [3H]glycine was seen in both cortex and spinal cord. Saturation analyses in cortex were best fitted by a two-site model with respective equilibrium dissociation constants (KD values) of 0.24 and 5.6 microM and respective maximal binding constants (Bmax values) of 3.4 and 26.7 pmol/mg of protein. Similar analyses in spinal cord were best fitted by a one-site model with a KD of 5.8 microM and Bmax of 20.2 pmol/mg of protein. Na+ had no effect on [3H]glycine binding to cortical membranes but increased the binding to spinal cord membranes by greater than 15-fold. This Na+-dependent binding may reflect glycine binding to the recognition site of the high-affinity, Na+-dependent glycine uptake system. Several short-chain, neutral amino acids displaced [3H]glycine binding from both cortical and spinal cord membranes. The most potent displacers of [3H]glycine binding to cortical membranes were D-serine and D-alanine, followed by the L-isomers of serine and alanine and beta-alanine. In contrast, D-serine and D-alanine were similar in potency to L-serine in spinal cord membranes. Compounds active at receptors for the acidic amino acids had disparate effects on the binding of [3H]glycine. At 10 microM, NMDA resulted in a 25% increase, whereas D- and L-2-amino-5-phosphonovaleric acid at 100 microM resulted in a 30% decrease, in [3H]glycine binding to cortical membranes. Kynurenic acid was the most potent of the acidic amino acid-related compounds at displacing [3H]glycine binding. In cortical membranes, kynurenic acid displacement was resolved into a high- and a low-affinity component; the high-affinity component displaced the high-affinity component of [3H]glycine binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

TETANUS TOXIN AND AMINO ACID LEVELS IN CAT SPINAL CORD

Abstract— —The levels of the depressant amino acids found in appreciable amounts in cord extracts—α-alanine, cystathionine, GABA, glycine and serine—were not significantly influenced by tetanus toxin. This supports the view that the antagonism of spinal inhibition by the toxin is the result of an interference with transmitter release rather than a reduction in the amount of transmitter available for release.
The marked increase in aspartic acid levels found in the spinal cord after treatment with tetanus toxin may reflect the association of aspartic acid with the increased activity of spinal excitatory interneurones or the involvement of aspartic acid as a glycine precursor in spinal tissue.     

THE OXIDATION OF GLYCINE BY d-AMINO ACID OXIDASE IN EXTRACTS OF MAMMALIAN CENTRAL NERVOUS TISSUE

Abstract— Glycine was a substrate for d -amino acid oxidase purified from extracts of cat spinal cord and sheep cerebellum. d -Aspartate and N -methyl- d -aspartate were oxidized at a rate similar to that of glycine by the purified sheep cerebellum extract; d -α-alanine and d -serine were oxidized appreciably faster than glycine, while GABA and d -glutamate were not oxidized at a measurable rate. p -Mercuribenzoate and kojate inhibited the oxidation of glycine by the purified sheep cerebellum extract.
d -Amino acid oxidase activity was higher in the grey than in the white matter of cat spinal cord, while the reverse was true for the cerebral cortex; the activity in the cord and cerebral cortex was much lower than that in the cerebellum.     

Action of precursors of noradrenaline synthesis on membrane receptors of chick embryo spinal neurons

Effects of noradrenaline precursors on glycine and N-methyl-D-aspartate (NMDA) receptors in spinal cord neurons recently isolated from chick embryo were investigated using whole cell patch-clamp and concentration clamp techniques. Both L-alanine and L-DOPA were found to be glycine agonists capable of potentiating NMDA response, while L-tyrosine does not activate glycine but can potentiate NMDA response. Lastly, L-phenylalanine and dopamine do not interact with either glycine or NMDA receptors.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 665–670, September–October, 1990.     

Studies on the Biosynthesis of Intermediate Filament Proteins in the Rat CNS

Abstract: The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell-free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isoated by immunoaffinity chromatography and then analyzed by two-dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population was found to synthesize two neurofilament proteins (MW 145K, p15.4, and MW 70K, pl 5.3) and three isomers of GFA (α, β, and γ) that differ in isoelectric point. Wheat germ homogenate containing messenger RNA extracted from free cord polysomes synthesized two proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the α, β, and γ isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Our data are consistent with the conclusion that synthesis of neurofilament proteins requires multiple messenger RNAs. Also, synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthcsized by the rough microsomes.     

Immunoblot identification of glial fibrillary acidic protein in rat sciatic nerve,brain, and spinal cord during development

The appearance of the glial fibrillary acidic protein (GFAP) during embryonic and postnatal development of the rat brain and spinal cord and in rat sciatic nerve during postnatal development was examined by the immunoblot technique. Cytoskeletal proteins were isolated from the central and peripheral nervous system and separated by SDS slab gel electrophoresis or two-dimensional gel electrophoresis. Proteins from the acrylamide gels were transferred to nitrocellulose sheets which were treated with anti-bovine GFAP serum and GFAP was identified by the immunoblot technique. GFAP was present in the embryonic rat brain and spinal cord at 14 and 16 days of gestation respectively. The appearance of GFAP at this stage of neural development suggests that the synthesis of GFAP may be related to the proliferation of radial glial cells from which astrocytes are derived. It is also feasible that GFAP provides structural support for the radial glial cell processes analogous to its role in differentiated astrocytes. GFAP was found to be present in rat sciatic nerves at birth and at all subsequent stages of development. These results indicate that some cellular elements in the rat sciatic nerve, such as Schwann cells, are capable of synthesizing GFAP which is immunochemically indistinguishable from its counterpart in the central nervous system. Thus it appears that GFAP is present both in the central and peripheral nervous system of the rat when the glial cells synthesizing GFAP are still undergoing differentiation.     

Toll-like receptor 3 contributes to spinal glial activation and tactile allodynia after nerve injury

Toll-like receptors (TLRs) play an essential role in innate immune responses and in the initiation of adaptive immune responses. Microglia, the resident innate immune cells in the CNS, express TLRs. In this study, we show that TLR3 is crucial for spinal cord glial activation and tactile allodynia after peripheral nerve injury. Intrathecal administration of TLR3 antisense oligodeoxynucleotide suppressed nerve injury-induced tactile allodynia, and decreased the phosphorylation of p38 mitogen-activated protein kinase, but not extracellular signal-regulated protein kinases 1/2, in spinal glial cells. Antisense knockdown of TLR3 also attenuated the activation of spinal microglia, but not astrocytes, caused by nerve injury. Furthermore, down-regulation of TLR3 inhibited nerve injury-induced up-regulation of spinal pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α. Conversely, intrathecal injection of the TLR3 agonist polyinosine–polycytidylic acid induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Indeed, TLR3-deficient mice did not develop tactile allodynia after nerve injury or polyinosine–polycytidylic acid injection. Our results indicate that TLR3 has a substantial role in the activation of spinal glial cells and the development of tactile allodynia after nerve injury. Thus, blocking TLR3 in the spinal glial cells might provide a fruitful strategy for treating neuropathic pain.     

Glutamate-, kainate- and NMDA-evoked membrane currents in identified glial cells in rat spinal cord slice

The effect of L-glutamate, kainate and N-methyl-D-aspartate (NMDA) on membrane currents of astrocytes, oligodendrocytes and their respective precursors was studied in acute spinal cord slices of rats between the ages of postnatal days 5 and 13 using the whole-cell patch-clamp technique. L-glutamate (10(-3) M), kainate (10(-3) M), and NMDA (2x10(-3) M) evoked inward currents in all glial cells. Kainate evoked larger currents in precursors than in astrocytes and oligodendrocytes, while NMDA induced larger currents in astrocytes and oligodendrocytes than in precursors. Kainate-evoked currents were blocked by the AMPA/kainate receptor antagonist CNQX (10(-4) M) and were, with the exception of the precursors, larger in dorsal than in ventral horns, as were NMDA-evoked currents. Currents evoked by NMDA were unaffected by CNQX and, in contrast to those seen in neurones, were not sensitive to Mg2+. In addition, they significantly decreased during development and were present when synaptic transmission was blocked in a Ca2+-free solution. NMDA-evoked currents were not abolished during the block of K+ inward currents in glial cells by Ba2+; thus they are unlikely to be mediated by an increase in extracellular K+ during neuronal activity. We provide evidence that spinal cord glial cells are sensitive to the application of L-glutamate, kainate and transiently, during postnatal development, to NMDA.     

Molecular biology of glycinergic neurotransmission

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem of vertebrates. Glycine is accumulated into synaptic vesicles by a proton-coupled transport system and released to the synaptic cleft after depolarization of the presynaptic terminal. The inhibitory action of glycine is mediated by pentameric glycine receptors (GlyR) that belong to the ligand-gated ion channel superfamily. The synaptic action of glycine is terminated by two sodium- and chloride-coupled transporters, GLYT1 and GLYT2, located in the glial plasma membrane and in the presynaptic terminals, respectively. Dysfunction of inhibitory glycinergic neurotransmission is associated with several forms of inherited mammalian myoclonus. In addition, glycine could participate in excitatory neurotransmission by modulating the activity of the NMDA subtype of glutamate receptor. In this article, we discuss recent progress in our understanding of the molecular mechanisms that underlie the physiology and pathology of glycinergic neurotransmission.     

Sensitive Immunoassay Shows Selective Association of Peripheral and Integral Membrane Proteins of the Inhibitory Glycine Receptor Complex

The inhibitory glycine receptor of mammalian spinal cord is a ligand-gated chloride channel that, on affinity purification, contains two subunits of 48-kilodalton (kD) and 58-kD molecular mass in addition to an associated 93-kD protein. Ligand-binding 48-kD subunit and 93-kD protein were quantified in the CNS of the adult rat using a newly developed dot receptor assay (detection limit less than or equal to 1 fmol/assay) which employs monoclonal antibodies specific for glycine receptor polypeptides. The 93-kD protein was found to codistribute at a fixed stoichiometry with the 48-kD subunit throughout the CNS of the rat. Moreover, the 93-kD protein cofractionated with the ligand-binding subunit on solubilization and affinity chromatography or immunoprecipitation. However, both proteins were separated on sucrose gradient centrifugation of detergent extracts of spinal cord membranes in accord with earlier observations on purified receptor. These data prove that the 93-kD polypeptide is selectively associated with the membrane core of the strychnine-sensitive glycine receptor. The regional distribution of glycine receptor polypeptides was also determined in the CNS of the spastic rat mutant. In contrast to hereditary spasticity in mouse and cattle, no reduction of glycine receptors was found in the spastic rat.     

Glycinergic transmission

Inhibition in the mature central nervous system is mediated by activation of γ-aminobutyric acid (GABA_A) and glycine receptors. Both receptors belong to the same superfamily of ligand-gated ion channels and share common transmembrane topology and structural and functional features. Glycine receptors are pentameric ligand-gated anion channels composed of two different subunits, named α und β, that assemble with a fixed stoichiometric ratio of two α to three β subunits. Four genes encoding the α subunits exist, whereas only one gene encoding the β subunit has been detected. Ligand binding occurs at the interface of α and β subunits. The β subunit, which is unable to form homo-oligomeric receptors, is responsible for assembly and channel properties. Moreover, this subunit carries a binding motif for the cytoplasmic protein gephyrin, which is believed to mediate synaptic clustering and anchoring at inhibitory synapses by interacting with the subsynaptic cytoskeleton. Synaptic gephyrin appears to restrict the mobility of glycine receptors diffusing in the plane of the plasma membrane, thereby generating dynamic plasma membrane domains contributing to the plasticity of inhibitory synapses. Glycine receptors are well established as playing important roles in controlling motor functions and sensory signaling in vision and audition and those in the dorsal horn of the spinal cord are now considered to be new targets for pain therapies. Like GABA_A receptors, glycine receptors have been shown to be depolarizing during development. The functional meaning of the developmental switch from excitatory to inhibitory glycine receptor action remains to be elucidated.     

Gliosis alters expression and uptake of spinal glial amino acid transporters in a mouse neuropathic pain model

Gliosis is strongly implicated in the development and maintenance of persistent pain states following chronic constriction injury of the sciatic nerve. Here we demonstrate that in the dorsal horn of the spinal cord, gliosis is accompanied by changes in glial amino acid transporters examined by immunoblot, immunohistochemistry and RT-PCR. Cytokines, proinflammatory mediators and microglia increase up to postoperative day (pd) 3 before decreasing on pd 7. Then, spinal glial fibrillary acidic protein increases on pd 7, lasting until pd 14 and later. Simultaneously, the expression of glial amino acid transporters for glycine and glutamate (GlyT1 and GLT1) is reduced on pd 7 and pd 14. Consistent with a reduced expression of GlyT1 and GLT1, high performance liquid chromatography reveals a net increase in the concentration of glutamate and glycine on pd 7 and pd 14 in tissue from the lumbar spinal cord of neuropathic mice. In this study we have confirmed that microglial activation precedes astrogliosis. Such a glial cytoskeletal rearrangement correlates with a marked decrease in glycine and glutamate transporters, which might, in turn, be responsible for the increased concentration of these neurotransmitters in the spinal cord. We speculate that these phenomena might contribute, via over-stimulation of NMDA receptors, to the changes in synaptic functioning that are responsible for the maintenance of persistent pain.