Vesicular glutamate transporters (VGLUT1, ‐2, and ‐3) mediate the accumulation of transmitter glutamate into synaptic vesicles in glutamatergic neurons. VGLUT1 and VGLUT2 are more reliable glutamatergic neuron markers, since VGLUT3 also exists in other neuron types. To study whether the dopaminergic neuron uses glutamate as a cotransmitter, we analyzed VGLUTs expression in dopamine neurons of adult male rats by in situ hybridization and immunohistochemistry. In the ventral midbrain, in situ hybridization analysis revealed no VGLUT1 mRNA expression, a widespread but discrete pattern of VGLUT2 mRNA expression, and a highly limited expression of VGLUT3 mRNA. Reverse‐transcriptase polymerase chain reaction analysis detected full‐length VGLUT2 gene transcripts in the ventral midbrain. Using in situ hybridization combined with tyrosine hydroxylase (TH) immunostaining, only VGLUT2 signals were detectable in some TH‐labeled neurons of A10 dopamine neuron groups, with the highest incidence (20%) in the rostral linear nucleus of the ventral tegmental area. In the forebrain, VGLUT2 signals were demonstrated in half of the A11 TH‐labeled neurons in the hypothalamus. Double‐label immunostaining for VGLUT2 and vesicular monoamine transporter 2 or TH showed that double‐labeled varicosities are rarely observed in any target regions examined of A10 and A11 dopamine neuron groups. These results indicate that VGLUT2 is expressed in subsets of A10 and A11 dopamine neurons, which might release dopamine and glutamate separately from different varicosities in the majority of their single axons. J. Comp. Neurol. 498:581–592, 2006. © 2006 Wiley‐Liss, Inc.

Abstract:We have isolated a human cDNA encoding a protein, designated DNPI, that shows 82% amino acid identity and 92% similarity to the human brain-specific Na ϩ -dependent inorganic phosphate (Na ϩ /P i ) cotransporter (BNPI), which is localized exclusively to neuron-rich regions. Expression of DNPI mRNA in Xenopus oocytes resulted in a significant increase in Na ϩ -dependent P i transport, indicating that DNPI is a novel Na ϩ /P i cotransporter. Northern blot analysis shows that DNPI mRNA is expressed predominantly in brain, where the highest levels are observed in medulla, substantia nigra, subthalamic nucleus, and thalamus, all of which express BNPI mRNA at low levels. In contrast, DNPI mRNA is expressed at low levels in cerebellum and hippocampus, where BNPI mRNA is expressed at high levels. No hybridizing signal for DNPI mRNA is observed in the glia-rich region of corpus callosum. In other regions examined, both mRNAs are moderately or highly expressed. These results indicate that BNPI and DNPI, which coordinate Na ϩ -dependent P i transport in the neuron-rich regions of the brain, may form a new class within the Na ϩ /P i cotransporter family. Key Words: cDNA cloning-Phosphate transport-Structurally related family-Neuron. J. Neurochem. 74, 2622Neurochem. 74, -2625Neurochem. 74, (2000.Inorganic phosphate (P i ) is essential for various cellular metabolic functions and signal transduction, and its homeostasis in the body is maintained primarily by the kidney (Murer and Biber, 1996). The cells in the proximal tubules of the kidney reabsorb P i in the glomerular filtrate through complex Na ϩ -dependent P i (Na ϩ /P i ) cotransport systems that are driven by the transmembrane electrochemical gradient for Na ϩ . Several cDNAs encoding distinct Na ϩ /P i cotransporters, which are classified into type 1 (NaP i -1-related), type 2 (NaP i -2-related), and type 3 (viral receptor-related) on the basis of molecular structure, have been identified in kidney and some other tissues (Murer and Biber, 1996;Werner et al., 1998). Amino acid comparison of the proteins shows weak overall homology (ϳ20% identity) between these types.A distinct type of the brain-specific Na ϩ /P i cotransporter (BNPI), which has ϳ30% amino acid identity to the type 1 proteins, has been described (Ni et al., 1994(Ni et al., , 1996. Northern blot analysis revealed that BNPI mRNA is expressed predominantly in brain, and in situ hybridization analysis revealed high levels of mRNA expression in certain neuron-rich regions of amygdala, cerebral cortex, and hippocampus. On the other hand, this mRNA was detected at low levels in substantia nigra, subthalamic nuclei, and thalamus, and no hybridization signals were detected in caudate nucleus and hypothalamus, suggesting that additional related proteins may be present in these regions to complement P i transport in brain.Rat pancreatic AR42J cells (Rosewicz et al., 1992) share the feature of pluripotency of the common precursor cells of the pancreas. Treatment of the cells with activin A converts them...

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