Two proteins previously known as Na(+)-dependent phosphate transporters have been identified recently as vesicular glutamate transporters (VGLUT1 and VGLUT2). Together, VGLUT1 and VGLUT2 are operating at most central glutamatergic synapses. In this study, we characterized a third vesicular glutamate transporter (VGLUT3), highly homologous to VGLUT1 and VGLUT2. Vesicles isolated from endocrine cells expressing recombinant VGLUT3 accumulated l-glutamate with bioenergetic and pharmacological characteristics similar, but not identical, to those displayed by the type-1 and type-2 isoforms. Interestingly, the distribution of VGLUT3 mRNA was restricted to a small number of neurons scattered in the striatum, hippocampus, cerebral cortex, and raphe nuclei, in contrast to VGLUT1 and VGLUT2 transcripts, which are massively expressed in cortical and deep structures of the brain, respectively. At the ultrastructural level, VGLUT3 immunoreactivity was concentrated over synaptic vesicle clusters present in nerve endings forming asymmetrical as well as symmetrical synapses. Finally, VGLUT3-positive neurons of the striatum and raphe nuclei were shown to coexpress acetylcholine and serotonin transporters, respectively. Our study reveals a novel class of glutamatergic nerve terminals and suggests that cholinergic striatal interneurons and serotoninergic neurons from the brainstem may store and release glutamate.

Before their exocytotic release during stimulation of nerve terminals, nonpeptide neurotransmitters are loaded into synaptic vesicles by specific transporters. Recently, a protein initially identified as brain-specific Na(+)-dependent inorganic phosphate transporter I (BNPI) has been shown to represent a vesicular glutamate transporter (VGLUT1). In this study, we investigated whether a highly homologous "differentiation-associated Na(+)-dependent inorganic phosphate transporter" (DNPI) is involved in glutamatergic transmission. Vesicles isolated from BON cells expressing recombinant DNPI accumulated l-glutamate with bioenergetical and pharmacological characteristics identical to those displayed by VGLUT1 and by brain synaptic vesicles. Moreover, DNPI localized to synaptic vesicles, at synapses exhibiting classical excitatory features. DNPI thus represents a novel vesicular glutamate transporter (VGLUT2). The distributions of each VGLUT transcript in brain were highly complementary, with only a partial regional and cellular overlap. At the protein level, we could only detect either VGLUT1- or VGLUT2-expressing presynaptic boutons. The existence of two VGLUTs thus defines distinct subsets of glutamatergic neurons.

Summary The dorsal raphe nucleus (DRN) in the midbrain is a key center for serotonin (5-hydroxytryptamine; 5-HT) expressing neurons. Serotonergic neurons in the DRN have been theorized to encode punishment by opposing the reward signaling of dopamine neurons. Here, we show that DRN neurons encode reward, but not punishment, through 5-HT and glutamate. Optogenetic stimulation of DRN Pet-1 neurons reinforces mice to explore the stimulation-coupled spatial region, shifts sucrose preference, drives optical self-stimulation, and directs sensory discrimination learning. DRN Pet-1 neurons increase their firing activity during reward tasks and this activation can be used to rapidly change neuronal activity patterns in the cortnassociated with 5-HT, they also release glutamate, and both neurotransmitters contribute to reward signaling. These experiments demonstrate the ability of DRN neurons to organize reward behaviors and might provide insights into the underlying mechanisms of learning facilitation and anhedonia treatment.

Recent data indicate that 'classical' neurotransmitters can also act as co-transmitters. This notion has been strengthened by the demonstration that three vesicular glutamate transporters (vesicular glutamate transporter 1 (VGLUT1), VGLUT2 and VGLUT3) are present in central monoamine, acetylcholine and GABA neurons, as well as in primarily glutamatergic neurons. Thus, intriguing questions are raised about the morphological and functional organization of neuronal systems endowed with such a dual signalling capacity. In addition to glutamate co-release, vesicular synergy - a process leading to enhanced packaging of the 'primary' transmitter - is increasingly recognized as a major property of the glutamatergic co-phenotype. The behavioural relevance of this co-phenotype is presently the focus of considerable interest.