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.

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Presynaptic inhibition of primary muscle spindle (group Ia) afferent terminals in motor nuclei of the spinal cord plays an important role in regulating motor output and is produced by a population of GABAergic axon terminals known as P boutons. Despite extensive investigation, the cells that mediate this control have not yet been identified. In this work, we use immunocytochemistry with confocal microscopy and EM to demonstrate that P boutons can be distinguished from other GABAergic terminals in the ventral horn of rat and mouse spinal cord by their high level of the glutamic acid decarboxylase (GAD) 65 isoform of GAD. By carrying out retrograde labeling from lamina IX in mice that express green fluorescent protein under the control of the GAD65 promoter, we provide evidence that the cells of origin of the P boutons are located in the medial part of laminae V and VI. Our results suggest that P boutons represent the major output of these cells within the ventral horn and are consistent with the view that presynaptic inhibition of proprioceptive afferents is mediated by specific populations of interneurons. They also provide a means of identifying P boutons that will be important in studies of the organization of presynaptic control of Ia afferents.GABA ͉ Ia afferent ͉ presynaptic inhibition T he inhibitory transmitter GABA is used by many neurons in the spinal cord and generates both postsynaptic inhibition at axo-dendritic and axo-somatic synapses and presynaptic inhibition at axo-axonic synapses (1). GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD), and Abs against GAD have been used to identify GABAergic axonal boutons in the spinal cord (2, 3). More recently, two forms of the enzyme have been identified, and based on their molecular weights these forms have been named GAD65 and GAD67 (4). Both forms are present in the ventral horn of the rat spinal cord but have a very different distribution (5, 6). The majority of GABAergic boutons throughout the ventral horn show strong GAD67 immunoreactivity and a low level of GAD65. However, there are clusters of boutons in lamina IX that contain very high levels of GAD65. We have suggested (6) that these clusters may correspond to the P boutons that form axo-axonic synapses with terminals of group Ia afferents (7-10). In this work, we have used a variety of approaches to confirm this hypothesis and to identify the cells of origin of these boutons. MethodsAll experiments were approved by the Ethical Review Process Applications Panel of the University of Glasgow or the Animal Care and Protection Committee at the University of Debrecen and were performed in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and the European Communities Council Directives. Immunocytochemical reactions were performed on free-floating 60-m transverse Vibratome sections that had been treated with 50% ethanol for 30 min to enhance Ab penetration. Abs were diluted in PBS with 0.3% Triton X-100 (except on sections used for EM).Primary Afferent Labeling. Transgang...