Cellular localization of three vesicular glutamate transporter mRNAs and proteins in rat spinal cord and dorsal root ganglia

Abstract

Glutamate is transported into synaptic vesicles by vesicular glutamate transporter (VGLUT) proteins. Three different VGLUTs, VGLUT1, VGLUT2, and VGLUT3, have recently been characterized, and they are considered to represent the most specific marker so far for neurons using glutamate as transmitter. We analyzed the cellular localization of VGLUT1-3 in the rat spinal cord and dorsal root ganglia (DRGs) in control rats and after dorsal rhizotomy. Using in situ hybridization, VGLUT1 mRNA containing neurons were shown in the dorsomedial part of the intermediate zone, whereas VGLUT2 mRNA-expressing neurons were present in the entire intermediate zone, both populations most likely representing interneurons. VGLUT3 mRNA could not be detected in the spinal cord. In the ventral horn, a dense plexus of VGLUT1-immunoreactive (ir) nerve terminals was present, with large varicosities abutting on presumed motoneurons. In the dorsal horn a similarly dense plexus was seen, except in laminae I and II. A very dense plexus of VGLUT2-ir fibers was distributed in the entire gray matter of the spinal cord, with many fibers lying close to presumed motoneurons. Few VGLUT3-ir fibers were distributed in the white and gray matter, including lamina IX. However, a dense VGLUT3-ir plexus was seen in the sympathetic intermedio-lateral column (IML). Multiple-labeling immunohistochemistry revealed that the VGLUT1-, VGLUT2-, and VAChT-containing varicosities in lamina IX all represent separate entities. There was no colocalization of VGLUT3 with VAChT or 5-HT in varicose fibers of the ventral horn, but some VGLUT3-ir fibers in the IML were 5-HT-positive. Lesioning of the dorsal roots resulted in an almost complete disappearance of VGLUT1-ir fibers around motoneurons and a less pronounced decrease in the remaining gray matter, whereas the density of VGLUT2- and VAChT-ir fibers appeared unaltered after lesion. Many VGLUT1-ir neurons were observed in DRGs; they were almost all large and did not colocalize calcitonin gene-related peptide (CGRP), and there was no overlap between these markers in fibers in the superficial dorsal horn. VGLUT2 was, at most, seen in a few DRG neurons. Taken together, these results suggest that the VGLUTs mRNAs are present in distinct subsets of neuronal populations at the spinal level. VGLUT1 is mainly present in primary afferents from large, CGRP-negative DRG neurons, VGLUT2 has mainly a local origin, and VGLUT3 fibers probably have a supraspinal origin.

Target selection of proprioceptive and motor axon synapses on neonatal V1‐derived Ia inhibitory interneurons and Renshaw cells

“…This antibody labels a single band of appropriate molecular weight (≈62 kDa) in Western blots of synaptic vesicle fractions of rat brains (Synaptic Systems, Goettingen, Germany). In our spinal cord sections the laminar distribution of immunolabeled puncta (high densities in LIII-LV, LVII, and LIX; weaker densities in LI and LII and LVIII) is identical to that previously published by us Mentis et al, 2006) and other authors using this or similar antibodies raised in different species (Todd et al, 2003;Oliveira et al, 2003).…”

“…This antibody labels a single band of appropriate molecular weight (≈62 kDa) in Western blots of synaptic vesicle fractions of rat brains (Synaptic Systems, Goettingen, Germany). In our spinal cord sections the laminar distribution of immunolabeled puncta (high densities in LIII-LV, LVII, and LIX; weaker densities in LI and LII and LVIII) is identical to that previously published by us Mentis et al, 2006) and other authors using this or similar antibodies raised in different species (Todd et al, 2003;Oliveira et al, 2003).Vesicular acetylcholine transporter (VAChT) antibodies: We used two polyclonal antibodies raised in guinea pig and goat, respectively, each against a similar, but not identical, sequence in the C-terminus (see Table 1). By Western blot the guinea pig antibody reacted with a single band in rat brain homogenates that corresponded to the size predicted for VAChT (Chemicon, Temecula, CA).…”

“…VGLUT1-IR was used as a marker of sensory afferent synapses (Todd et al, 2003;Oliveira et al, 2003;Alvarez et al, 2004;Mentis et al, 2006) …”

Spinal neurons activated in response to pudendal or pelvic nerve stimulation in female rats

“…The present study examined the relationship of VGlut2, VGlut3 transporters and NKI receptors with fos-I nuclei as both glutamate and NKI receptors have been shown to regulate pelvic reflexes [64,69]. Glutamate is the major excitatory neurotransmitter and recent studies have documented the distribution of glutamate transporters in the lumbosacral cord [2,34,38,51,57,63]. Studies have suggested that VGlut1 and VGlut2 expression is restricted to glutaminergic neurons; however, VGlut3 expression is found not only in glutaminergic neurons but also neurons that contain acetylcholine and serotonin [28].…”

“…VGlut2 also innervates the parasympathetic preganglionic neurons of the lumbosacral spinal cord [38]. In contrast, the distribution of VGlut3 has been less well studied, and existing data suggests that VGlut3 exhibits a more restricted expression pattern compared to VGlut1 and VGlut2 [50,51]. VGlut3 has been shown in some cholinergic and serotonergic neurons in the brain and pelvic function is modulated by serotonergic and cholinergic supraspinal and spinal pathways [27,28,43].…”