Glutamate, the major excitatory neurotransmitter in brain, is almost exclusively intracellular due to the action of the glutamate transporters in the plasma membranes. To study the localization and properties of these proteins, we have raised antibodies specifically recognizing parts of the sequences of two cloned rat glutamate transporters, GLT-1 (Pines et al., 1992) and GLAST (Storck et al., 1992). On immunoblots the antibodies against GLT-1 label a broad heterogeneous band with maximum density at around 73 kDa, while the antibody against GLAST labels a similarly broad band at around 66 kDa in the cerebellum and a few kilodaltons lower in other brain regions. GLT-1 is expressed at the highest concentrations in the hippocampus, lateral septum, cerebral cortex, and striatum, while GLAST is preferentially expressed in the molecular layer of the cerebellum. However, both transporters are present throughout the brain, and have roughly parallel distributions in the cerebral hemispheres and brainstem. Preembedding light and electron microscopical immunocytochemistry shows that both GLT-1 and GLAST are restricted to astrocytes, which appear to express both proteins concomitantly, but in different proportions in different parts of the brain. Nerve terminal labeling was not observed. Both the amino and carboxyl terminals of GLT- 1 and GLAST are located intracellularly, indicating an even number of transmembrane segments. Antibodies against a synthetic peptide corresponding to amino acid residues 2–11 of the proposed sequence of GLT-1 recognize the native rat brain GLT-1 protein, confirming that the translation initiation site is at the first ATG.

The role of transporters in shaping the glutamate concentration in the extracellular space after synaptic release is controversial because of their slow cycling and because diffusion alone gives a rapid removal. The transporter densities have been measured electrophysiologically, but these data are from immature brains and do not give precise information on the concentrations of the individual transporter subtypes. Here we show by quantitative immunoblotting that the numbers of the astroglial glutamate transporters GLAST (EAAT1) and GLT (EAAT2) are 3200 and 12,000 per m 3 tissue in the stratum radiatum of adult rat hippocampus (CA1) and 18,000 and 2800 in the cerebellar molecular layer, respectively. The total astroglial cell surface is 1.4 and 3.8 m 2 /cm 3 in the two regions, respectively, implying average densities of GLAST and GLT molecules in the membranes around 2300 and 8500 m Ϫ2 in the former and 4700and 740 m Ϫ2 in the latter region. The total concentration of glial glutamate transporters in both regions corresponds to three to five times the estimated number of glutamate molecules in one synaptic vesicle from each of all glutamatergic synapses. However, the role of glial glutamate transporters in limiting synaptic spillover is likely to vary between the two regions because of differences in the distribution of astroglia. Synapses are completely ensheathed and separated from each other by astroglia in the cerebellar molecular layer. In contrast, synapses in hippocampus (stratum radiatum) are only contacted by astroglia and are often found side by side without intervening glial processes.

A transporter thought to mediate accumulation of GABA into synaptic vesicles has recently been cloned (McIntire et al., 1997). This vesicular GABA transporter (VGAT), the first vesicular amino acid transporter to be molecularly identified, differs in structure from previously cloned vesicular neurotransmitter transporters and defines a novel gene family. Here we use antibodies specific for N- and C-terminal epitopes of VGAT to localize the protein in the rat CNS. VGAT is highly concentrated in the nerve endings of GABAergic neurons in the brain and spinal cord but also in glycinergic nerve endings. In contrast, hippocampal mossy fiber boutons, which although glutamatergic are known to contain GABA, lack VGAT immunoreactivity. Post-embedding immunogold quantification shows that the protein specifically associates with synaptic vesicles. Triple labeling for VGAT, GABA, and glycine in the lateral oliva superior revealed a higher expression of VGAT in nerve endings rich in GABA, with or without glycine, than in others rich in glycine only. Although the great majority of nerve terminals containing GABA or glycine are immunopositive for VGAT, subpopulations of nerve endings rich in GABA or glycine appear to lack the protein. Additional vesicular transporters or alternative modes of release may therefore contribute to the inhibitory neurotransmission mediated by these two amino acids.

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.