Immunocytochemical localization of candidates for vesicular glutamate transporters in the rat cerebral cortex.

Brain-specific Na(+)-dependent inorganic phosphate cotransporter (BNPI) was recently reported to serve as a vesicular glutamate transporter (VGluT), and was renamed VGluT1 (Bellocchio et al. [ 2000] Science 289:957-960; Takamori et al. [2000] Nature 407:189-194). Ahead of these reports, cDNA encoding another brain-specific inorganic phosphate transporter, which showed 82% amino acid identity to VGluT1, was cloned and designated differentiation-associated Na(+)-dependent inorganic phosphate cotransporter (DNPI; Aihara et al. [2000] J Neurochem 74:2622-2625). In the present study, we produced a specific antibody against a C-terminal portion of DNPI, and studied the immunohistochemical localization of DNPI in the rat cerebral cortex in comparison with that of VGluT1. DNPI immunoreactivity was enriched in neuropil of layers I and IV and to a lesser extent in the upper portion of layer VI of the cerebral neocortex, whereas VGluT1 immunoreactivity was distributed more evenly in neuropil of the neocortex. Electron microscopic observation revealed that both DNPI and VGluT1 immunoreactivities were mainly located on synaptic vesicles in nerve terminals which made asymmetrical contacts in the neocortex. Furthermore, neither DNPI nor VGluT1 immunoreactivity in the neocortex was colocalized with gamma aminobutyric acid (GABA)ergic axon terminal markers, immunoreactivity for glutamic acid decarboxylase or vesicular GABA transporter. Neuronal depletion in the ventrobasal thalamic nuclei produced by the kainic acid injection resulted in a clear reduction of DNPI immunoreactivity in layers I, IV, and VI of the somatosensory cortex. These results indicate that DNPI is located on the membrane of synaptic vesicles in thalamocortical axon terminals, and that it may be a candidate for VGluT of thalamocortical glutamatergic neurons. Copyright 2001 Wiley-Liss, Inc.