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.

Specificity testing should be performed under conditions identical to or closely similar to those of the immunocytochemical procedure. This paper describes a new model system that meets this requirement for postembedding immunocytochemistry of amino acids at the light- and electron microscopic levels. Test conjugates, obtained by reacting different amino acids with brain macromolecules in the presence of glutaraldehyde, were freeze-dried and embedded in an epoxy resin (Durcupan) exactly as for brain tissue. One section from each of the embedded amino acid conjugates and from a brain protein-glutaraldehyde conjugate (without amino acid) were piled on top of each other and embedded anew. Transverse semithin (0.5 micron) and ultrathin sections were cut through the stack. These test sections, in which all the different conjugates were represented, were then processed in the same drops of sera as the tissue sections to permit identical conditions for testing and immunocytochemistry. After immunogold labelling for electron microscopy, a quantitative expression of crossreactivity was obtained by computer-assisted calculation of gold particle densities over the different conjugates. The antisera used in the present study (glutamate antiserum 13, taurine antiserum 20, and GABA antiserum 26) showed highly selective labelling of the respective amino acid conjugates and produced distinct labelling patterns in simultaneously processed cerebellar sections.

The coexistence of immunoreactivities for tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) and/or gamma-aminobutyric acid (GABA) was revealed in various brain regions in colchicine-injected and untreated rats, using the peroxidase-antiperoxidase method. Consecutive 40 micron thick Vibratome sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for TH and GAD or GABA in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell incubated in two different antisera. In the olfactory bulb, retina, diencephalon, mesencephalic central grey and cerebral cortex, many TH-like immunoreactive neurons also showed GAD-like or GABA-like immunoreactivity, whereas in the substantia nigra, ventral tegmental area and locus ceruleus none of TH-like immunoreactive neurons showed either GAD-like or GABA-like immunoreactivity. In the olfactory bulb, retina and cerebral cortex, the majority of the TH-like immunoreactive neurons were also GAD-like or GABA-like immunoreactive. In the diencephalon of colchicine-injected rats, at least one-third of the TH-like immunoreactive neurons were GAD-like immunoreactive. Using serial 0.5 micron thick plastic-embedded sections, it was shown that immunoreactivities for three antigens, GAD, GABA and TH could occur in the same neurons in the olfactory bulb. These observations indicate the possible coexistence of two classical transmitters. GABA and catecholamine, in various brain regions of the rat.

Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

Polyclonal antibodies were raised against the GABA transporter (GABA-Tp) purified from rat brain tissue (Radian et al., 1986) and used for immunocytochemical localization of the antigen in several rat brain areas, including the cerebellum, hippocampus, substantia nigra, and cerebral cortex. Light microscopic studies with the peroxidase-antiperoxidase and biotin-avidin-peroxidase techniques suggested that GABA-Tp is localized in the same types of axons and terminals that contain endogenous GABA, as judged by comparison with parallel sections incubated with antibodies against glutaraldehyde-conjugated GABA. However, as expected from biochemical results, different neurons differed in their relative contents of GABA-Tp and GABA; thus, GABA-Tp was relatively low in striatonigral and Purkinje axon terminals and relatively high in nerve plexus around the bases of cerebellar Purkinje cells and hippocampal pyramidal and granule cells. The GABA-Tp antiserum did not produce detectable labeling of nerve cell bodies. Electron microscopic studies supported the light microscopic observations and provided direct evidence of cellular co-localization of GABA-Tp and GABA (as visualized by the peroxidase-antiperoxidase technique and postembedding immunogold labeling, respectively). The ultrastructural studies indicated the presence of GABA-Tp also in glial processes but not in glial cell bodies. The relative intensity of the neuronal and glial staining varied among regions: glial staining predominated over neuronal staining in the substantia nigra, whereas the converse was true in the cerebellum and hippocampus. The present immunocytochemical data demonstrate directly what has previously been inferred from biochemical and autoradiographic evidence: that the mechanisms for high-affinity GABA uptake is selectively and differentially localized in GABAergic neurons and in glial cells.