European Journal zyxwvutsrq of Neuroscience, Vol. zyxwvutsrq 7, pp. 1342-1352, zyxwvuts 1995 zyxwvutsr @ European Neuroscience Association Regional Distribution and Developmental Variation of the Glycine Transporters GLYTI and GLYT2 in the Rat CNS z Francisco Zafra, Jesus Gomeza, Luis Olivares, Carmen Aragon and Cecilio Gimenez Centro de Biologia Molecular 'Severo Ochoa', Facultad de Ciencias, Universidad Autonoma de Madrid, 28049 Madrid, Spain Keywords: amino acid neurotransmitters,development, mRNA distribution Abstract 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 GLYTl and GLYT2 transcripts and proteins in different regions and developmental stages of the rat brain were analysed by Northern, Western and in situ hybridizationtechniques. 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 GLTYI and GLYT2 transporters respectively. High levels of GLYTI 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 GLYTl 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. Introduction zyxwvutsrqp Glycine is thought to have two roles in neurotransmission. In the spinal cord and brainstem glycine is an important inhibitory neurotransmitter that binds to postsynaptic strychnine-sensitive glycine receptors. This receptor is a chloride channel that, after opening, produces inhibitory postsynaptic potentials. In addition, glycine potentiates the action of glutamate, the main excitatory neurotransmitter in the brain, on postsynaptic N-methy1-D-aspartate (NMDA) receptors. The re-uptake of neurotransmitter amino acids into presynaptic nerve endings or neighbouring fine glial processes provides one way of clearing the extracellular space of the neuroactive substances, and zyxwvut so constitutes an efficient mechanism by which the postsynaptic action can be terminated (Henn and Hamberger, 197 1 ; Iversen, 197 1 ; Schousboe, 198 1 ; Kanner and Shuldiner, 1987). Specific high-affinity transport systems have been identified in nerve terminals and glial cells for several amino acid neurotransmitters including glycine (Johnston and Iversen, 1971; Logan and Snyder, 1972; Balcar and Johnston, 1973; Bennet et al., 1973; Kuhar and Zarbin, 1978; Sershen and Lajtha, 1979; Zafra and Gimtnez, 1986; Fedele and Foster, 1992). In the past few years, some neurotransmitter transporters have been purified from mammalian brains (Radian et al., 1986; Danbolt er al., 1990; Mpez-Corcuera et al., 1991) and, more recently, cDNA clones encoding transporters for GABA, catecholamines, serotonin, glycine, glutamate and proline have also been isolated (Guastella et al., 1990; Blakely et al., 1991b; Hoffman et al., 1991, 1992; Kilty et al., 1991; Pacholczyk et al., 1991; Shimada et al., 1991; Usdin er al., 1991; Borden et al., 1992; Fremau et al., 1992; Giros et al., 1992; Kanai and Hediger, 1992; Pines et al., 1992; Liu et al., 1992, 1993; Smith et al., 1992; Storck er al., 1992). Having studied these proteins using cDNA cloning technique, transporters can now been classified into families and subfamilies based on topology and sequence relatedness (Liu et al., 1992; Uhl and Harting, 1992; Amara and Kuhar, 1993). To date, two different glycine transporters have been cloned: GLYTl (Guastella et al., 1992; Liu et al., 1992; Smith et al., 1992), which presents three isoforms produced by alternative splicing and/or alternative promoter usage (Borowsky et al., 1993; Kim et al., 1994) [termed GLYTIa, GLYTlb and GLYTlc, according to Liu et al. (1993) and Kim et al. (1994)], and GLYT2 (Liu et al., 1993). GLYT2 is present specifically in the brainstem and spinal cord, brain areas where strychnine-sensitive glycine receptors are better represented. The present work was undertaken to study regional, cellular and developmental variations in the expression of mRNA and protein for the two high-affinity Na+-dependent uptake systems for glycine, GLYTl and GLYT2. Materials and methods Preparation of glutathione-S-transferase (GST)-glycine transporter fusion proteins The 228 bp of the C-terminus of GLYTl and 570 bp of the zy N- terminus of GLYT2 were synthesized by PCR using as templates the Correspondence to: Dr. Cecilio Girninez Received 30 June 1994, revised 24 August 1994, accepted 23 January 1995