The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn

Abstract

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid-lumbar spinal cord of the rat. The great majority (94-100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2-immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin- and enkephalin-containing boutons (including somatostatin-immunoreactive boutons that lacked calcitonin gene-related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present. We also investigated VGLUT expression in central terminals of primary afferents. Myelinated afferents were identified with cholera toxin B subunit; most of those in lamina I were VGLUT2-immunoreactive, whereas all those in deeper laminae were VGLUT1-immunoreactive, and some (in laminae III-VI) appeared to contain both transporters. However, peptidergic primary afferents that contained substance P or somatostatin (most of which are unmyelinated), as well as nonpeptidergic C fibres (identified with Bandeiraea simplicifolia isolectin B4) showed low levels of VGLUT2-immunoreactivity, or were not immunoreactive with either VGLUT antibody. As all primary afferents are thought to be glutamatergic, this raises the possibility that unmyelinated afferents, most of which are nociceptors, express a different vesicular glutamate transporter.

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 and VGlut1 immunoreactivity are widely distributed in the central nervous system and are found in some overlapping regions [34,53,63]. Neurons synthesizing VGlut2 have been identified in the ventral medulla, a region that projects to the lumbosacral spinal cord and is involved in regulating pelvic function [42,60].…”