Glycinergic Synapses Research Articles

Gene structure and glial expression of the glycine transporter GlyT1 in embryonic and adult rodents

Na+/Cl(-)-dependent glycine transporters are crucial for the termination of neurotransmission at glycinergic synapses. Two different glycine transporter genes, GlyT1 and GlyT2, have been described. Several isoforms differing in their 5' ends originate from the GlyT1 gene. We have determined the genomic structure of the murine GlyT1 gene to elucidate the genetic basis underlying the different isoforms. Analysis of cDNA 5'-ends revealed that the GlyT1a and 1b/1c mRNAs are transcribed from two different promoters. During murine embryonic development GlyT1 mRNAs were detectable by RNase protection assays as early as embryonic day E9 and reached maximal levels between E13 and E15. In situ hybridization revealed GlyT1 expression in the developing spinal cord mainly in the ventral part of the ventricular zone at E12. At later stages (E15) transcripts were also found in the lateral half of the basal and intermediate gray matter. In contrast, the second glycine transporter gene GlyT2 displayed a completely different expression pattern. At E11 it is expressed in the mantle zone, and at later stages throughout the ventral horns. In the adult rat brain and spinal cord, GlyT1 hybridization signals were found exclusively in glial cells. Our data indicate that GlyT1 is an early marker of neural development and encodes glia-specific transporter proteins.

Glycinergic synapses in the rod pathway of the rat retina: cone bipolar cells express the alpha 1 subunit of the glycine receptor

Glycine receptors (GlyRs) and their role in retinal circuitry were analyzed immunocytochemically in the rat retina. Specific antibodies against the alpha 1 subunit of the GlyR and against the GlyR-associated protein gephyrin, respectively, were used. In the inner plexiform layer (IPL), both antibodies produced a punctate label that was shown by electron microscopy to occur at synapses. Gephyrin-like immunoreactivity (-LI) was more widely distributed, indicating that gephyrin might also occur at nonglycinergic synapses. At the ultrastructural level, gephyrin-LI was found at the cytoplasmic face of postsynaptic membranes of amacrine and ganglion cells, but was never detected in bipolar cell axons. Immunoreactivity for the alpha 1 subunit was concentrated in the cleft of conventional synapses made by amacrine cell processes onto ganglion cell dendrites and cone bipolar axons. The latter synapses differ from other glycinergic synapses since they are not labeled by the antibody against gephyrin used in this study. In order to identify the type of bipolar cell involved in these synapses, the distribution of the alpha 1 subunit was compared with that of recoverin-immunoreactive cone bipolar cells and with that of parvalbumin-immunoreactive All-amacrine cells. Double-label immunofluorescence showed that, in the outer part of the IPL, 75% of the alpha 1-immunoreactive puncta were colocalized with recoverin-positive bipolar cell axons and 71% of the alpha 1-immunoreactive puncta were colocalized with parvalbumin-positive All-amacrine processes. Hence, the alpha 1 subunit of the GlyR is present at the chemical synapses established by All-amacrine cells with OFF-cone bipolar cells and OFF-ganglion cells. These synapses play a key role in the transmission of scotopic signals through the OFF-channel of the rod pathway.

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.

Strychnine blockade of the non-reciprocal inhibition of trigeminal motoneurons induced by stimulation of the parvocellular reticular formation

Stimulation of a region within the parvocellular medullary reticular formation (PcRF) that contains somas of premotor interneurons produces short latency inhibitory synaptic potentials (IPSPs) in cat trigeminal motoneurons. The present study was undertaken to determine whether glycinergic synapses are responsible for these IPSPs. The intravenous administration of strychnine, an established glycine antagonist, abolished these PcRF-IPSPs. This effect appears to be specific for glycinergic inhibitory synapses because the short lasting component of the IPSP produced by inferior alveolar nerve (IAN) stimulation was also abolished, whereas, in contrast, the long lasting non-glycinergic component of this IPSP was not suppressed. These results indicate that a glycinergic system in the reticular formation is responsible for the non-reciprocal postsynaptic inhibition of trigeminal motoneurons.

GABA‐ergic and glycinergic pathways in the inner plexiform layer of the goldfish retina

GABA-ergic and glycinergic circuitry in the inner plexiform layer of the goldfish retina was evaluated by electron microscopic autoradiography of 3H-GABA and 3H-glycine uptake, combined with retrograde horseradish peroxidase (HRP) labeling of ganglion cells. GABA-ergic and glycinergic synapses were found on labeled ganglion cells throughout the inner plexiform layer. This reinforces the idea that physiological evidence of GABA-ergic and glycinergic influence on a variety of ganglion cells in goldfish and carp often reflects direct inputs. Double-labeled synapses are presented as evidence of direct type Ab amacrine cell input to on-center ganglion cells. At least one population of type Aa sustained-off GABA-ergic amacrine cell is proposed, on the basis of profuse GABA-ergic inputs onto bipolar cells in sublamina a. Similar GABA-labeled profiles are shown to synapse onto HRP-labeled probable off-center ganglion cells. Thus GABA-ergic amacrine cells not only provide the predominant feedback to depolarizing (on-center) and hyperpolarizing (off-center) bipolar cells but also provide feed-forward inputs to on- and off-center ganglion cells. Large-caliber GABA-ergic dendrites present in both sublaminae a and b resemble those expected of a previously described bistratified, transient amacrine cell. These processes synapse onto HRP-labeled ganglion cell profiles in both sublaminae. Two morphologies of glycinergic amacrine cell are proposed on the basis of light microscopic autoradiography, 1) the previously described small pyriform cell and 2) a multipolar cell. The differential connectivity of the glycinergic neurons described, however, remains indistinguishable. Whereas abundant glycinergic inputs to ganglion cells occur throughout the inner plexiform layer, contacts between glycinergic profiles and bipolar cells are extremely rare. Therefore, interpreting the meaning of glycinergic input to ganglion cells will require further study of amacrine cell circuitry.

The mammalian glycine receptor: biology and structure of a neuronal chloride channel protein

Glycine is a major inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates and invertebrates. The postsynaptic receptor for this amino acid is an oligomeric glycoprotein which, upon binding of glycine, transiently forms an anion-selective transmembrane channel. Agonist-mediated receptor activation is antagonized by strychnine, a high-affinity ligand of the glycine receptor (GlyR). Biochemical and immunological data show that affinity-purified preparations of the mammalian GlyR contain three polypeptides of M(r) 48,000, 58,000 and 93,000. These polypeptides have different functional properties and/or topologies in the postsynaptic membrane of the glycinergic synapse. The primary sequence of the M(r) 48,000 subunit deduced by cDNA cloning exhibits structural and amino-acid homology to nicotinic acetylcholine and GABA(a) receptor proteins, indicating a common evolutionary relationship between the different neurotransmitter-gated ion channels of excitable membranes. Monoclonal antibodies against the GlyR allow its histochemical localization in different regions of the CNS. GlyR deficiencies have been implicated in the pathogenesis of spasticity and spinal cord degeneration in mouse and man.

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.