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

Brain-specific Na+-dependent inorganic phosphate cotransporter (BNPI) was r ecently reported to serve as a vesicular glutamate transporter (VGluT), and was renamed VGluT1 (Bellocchio et al. [ 2000] Science 289:957-960; Takamor i et al. [2000] Nature 407:189-194). Ahead of these reports, cDNA encoding another brain-specific inorganic phosphate transporter, which showed 82% am ino acid identity to VGluT1, was cloned and designated differentiation-asso ciated Na+-dependent inorganic phosphate cotransporter (DNPI; Aihara et al. [2000] J Neurochem 74:2622-2625). In the present study, we produced a spec ific antibody against a C-terminal portion of DNPI, and studied the immunoh istochemical localization of DNPI in the rat cerebral cortex in comparison with that of VGluT1. DNPI immunoreactivity was enriched in neuropil of laye rs I and TV and to a lesser extent in the upper portion of layer VI of the cerebral neocortex, whereas VGluT1 immunoreactivity was distributed more ev enly in neuropil of the neocortex. Electron microscopic observation reveale d that both DNPI and VGluT1 immunoreactivities were mainly located on synap tic vesicles in nerve terminals which made asymmetrical contacts in the neo cortex. Furthermore, neither DNPI nor VGluT1 immunoreactivity in the neocor tex 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 produce d by the kainic acid injection resulted in a clear reduction of DNPI immuno reactivity in layers I, IV, and VI of the somatosensory cortex. These resul ts indicate that DNPI is located on the membrane of synaptic vesicles in th alamocortical axon terminals, and that it may be a candidate for VGluT of t halamocortical glutamatergic neurons. (C) 2001 Wiley-Liss, Inc.