Molecular Cloning and Functional Identification of Mouse Vesicular Glutamate Transporter 3 and Its Expression in Subsets of Novel Excitatory Neurons

Abstract

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H ؉ -dependent, was stimulated by Cl ؊ ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.

Vesicular glutamate transporter 3-immunoreactive pericellular baskets ensheath a distinct population of neurons in the lateral septum

“…As their target cells seem to be confined to the ventral LS and the formation of PB-like terminals was not reported for these afferents they are probably not a candidate for the widespread VGLUT3-innervation of the LS (Groenewegen et al, 1986;Morley, 1986;Risold and Swanson, 1997b). The expression of VGLUT3-mRNA in the substantia nigra pars compacta and ventral tegmental area has been described by Fremeau et al (2002), but was not found by others (Gras et al, 2002;Herzog et al, 2004;Schäfer et al, 2002). The TH-ir projections of the mesencephalon which form pericellular baskets intermingle with the VGLUT3-ir PBs in the LS.…”

“…Existence of VGLUT3-mRNA was repeatedly reported to be absent from the thalamus, and there are some controversial descriptions for the hypothalamus and ventral pallidum (Fremeau et al, 2002;Herzog et al, 2004;Schäfer et al, 2002). The thalamic afferents are probably represented by VGLUT2-ir as all thalamic subnuclei projecting to the LS express VGLUT2-ir and -mRNA (Fremeau et al, 2001;Herzog et al, 2001;Hur and Zaborszky, 2005;Kaneko et al, 2002;Moga et al, 1995;Staiger and Nürnberger, 1989).…”

“…In the mesencephalon, the interpeduncular nucleus (IP) displayed a high amount of VGLUT3-mRNA except in its central and lateral parts (Herzog et al, 2004;Schäfer et al, 2002). Projections from the IP originate primarily in the apical and lateral subnuclei.…”

“…In contrast, VGLUT3-mRNA was found in deeper brain areas like hypothalamus, substantia nigra and dorsal raphe (Fremeau et al, 2002;Gras et al, 2002;Herzog et al, 2004;Schäfer et al, 2002). Projection neurons located in these regions innervate the LS and their terminals form perisomatic and peridendritic plexus called pericellular baskets (Jakab and Leranth, 1995).…”

Vesicular glutamate transporter‐1 colocalizes with endogenous opioid peptides in axon terminals of the rat locus coeruleus

“…Three isoforms of VGLUT have so far been established. Two are recognized as definitive markers of glutamatergic neurons and their axon terminals (VGLUT1, VGLUT2) (Bellocchio et al, 1998(Bellocchio et al, , 2000Takamori et al, 2000;Fremeau et al, 2001Fremeau et al, , 2002Herzog et al, 2001;Hisano et al, 2002;Hisano, 2003) and a third is expressed by cholinergic and serotonergic neurons (VGLUT3) (Gras et al, 2002;Schafer et al, 2002). VGLUT1 mRNA expression in distinct regions of rat and human brain has been confirmed by in situ hybridization studies (Ni et al, 1994(Ni et al, , 1996Hisano et al, 2002;Hioki et al, 2003;Hisano, 2003).…”