Glycine is the major inhibitory neurotransmitter in the spinal cord and brainstem and is also required for the activation of NMDA receptors. The extracellular concentration of this neuroactive amino acid is regulated by at least two glycine transporters (GLYT1 and GLYT2). To study the localization and properties of these proteins, sequence- specific antibodies against the cloned glycine transporters have been raised. Immunoblots show that the 50–70 kDa band corresponding to GLYT1 is expressed at the highest concentrations in the spinal cord, brainstem, diencephalon, and retina, and, in a lesser degree, to the olfactory bulb and brain hemispheres, whereas it is not detected in peripheral tissues. Pre-embedding light and electron microscopic immunocytochemistry show that GLYT1 is expressed in glial cells around both glycinergic and nonglycinergic neurons except in the retina, where it is expressed by amacrine neurons, but not by glia. The expression of a 90–110 kDa band corresponding to GLYT2 is restricted to the spinal cord, brain-stem, and cerebellum; in addition, very low levels occur in the diencephalon. GLYT2 is found in presynaptic elements of neurons thought to be glycinergic. However, in the cerebellum, GLYT2 is expressed both in terminal boutons and in glial elements. The physiological consequences of the regional and cellular distributions of these two proteins as well as the possibility of the existence of an unidentified neuronal form of GLYT1 are discussed.

The high-affinity glycine transporter in neurons and glial cells is the primary means of inactivating synaptic glycine. Previous molecular cloning studies have indicated heterogeneity of glycine transporters in the CNS. Here the distribution of glycine transporter GLYT1 and GLYT2 transcripts and proteins in different regions and developmental stages of the rat brain were analysed by Northern, Western and in situ hybridization techniques. Sequence-specific riboprobes and two specific antibodies raised against fusion proteins were used, containing either 76 or 193 amino acids of the C or N terminus of the GLYT1 and GLYT2 transporters respectively. High levels of GLYT1 transcripts were found in the spinal cord, brainstem and cerebellum, and moderate levels in forebrain regions such as the cortex or hippocampus. GLYT2 transcripts are restricted to the spinal cord, brainstem and cerebellum. The onset of both GLYT1 and GLYT2 expression in the brainstem occurred in late fetal life, and full expression of these proteins was observed before weaning. There was a stepwise increase in the levels of mRNA and protein for these two transporters, reaching a maximum by the second postnatal week, followed by a slight decrease until adult values were reached by the fourth postnatal week. These data reveal interesting parallelism between the distribution of different glycine transporters and glycine receptor subunits, and suggest discrete roles for distinct glycine transporters.

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