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.

By using immunofluorescence methodology, extensive galanin (GAL) and GAL message-associated peptide (GMAP)-positive terminal networks were observed in the hippocampal formation. The majority of the GAL/GMAP fibers were dopamine beta-hydroxylase- (DBH) positive, that is, they were noradrenergic. This finding was established with GAL/GMAP-DBH double-staining and with 6-hydroxy-dopamine treatment, which totally abolished all fibers in which GAL/GMAP and DBH coexisted. Also, reserpine treatment caused a marked depletion of GAL. No evidence for GAL/GMAP coexistence with 5-hydroxytryptamine was obtained. In the ventral hippocampus, GAL/GMAP-, DBH-negative fibers were seen in the stratum oriens, the anterior stratum radiatum, along the granule cell layer and in the strata oriens and alveus. In the locus coeruleus (LC), around 80% of the GMAP-positive neurons contained neuropeptide tyrosine (NPY), and about 40% of the NPY-positive neurons expressed GMAP. GAL-R1 receptor mRNA was expressed in Barrington's nucleus (close to the LC), but was not detected in the hippocampal formation/dorsal cortical areas. GAL-R2 receptor mRNA was found in the granule cell layer in the dentate gyrus. The present results show that most, but not all, immunohistochemically detectable GAL/GMAP in the hippocampal formation/dorsal cortex is present in noradrenergic nerve terminals originating in the LC, which has a robust GAL/GMAP synthesis. The functional role of GAL may be related to noradrenaline, possibly by a presynaptic action. However, the presence of GAL in other systems and of GAL-R2 receptor mRNA in granule cells also indicates other targets.

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Galanin is a neuropeptide with a wide variety of biological functions, including that of a strong endogenous anticonvulsant. No nonpeptide ligands, capable of activating galanin receptors, are available today. Based on known pharmacophores of galanin, a combinatorial library was designed, synthesized, and screened at the rat hippocampal galanin receptor. A low molecular weight galanin receptor agonist, 7-((9-fluorenylmethoxycarbonyl)cyclohexylalanyllysyl)amino-4-methylcoumarin (galnon) was found to displace 125 I-galanin with micromolar affinity at Bowes cellular and rat hippocampal membranes. Autoradiographic binding assay on rat spinal cord sections confirmed the ability of galnon to displace 125 I-galanin from its binding sites. Galnon inhibited adenylate cyclase activity, suggesting an agonist action at galanin receptors. When injected i.p. galnon reduced the severity and increased the latency of pentylenetetrazole-induced seizures in mice and reversed the proconvulsant effects of the galanin receptor antagonist M35, injected into a lateral ventricle. Intrahippocampal injection of galnon also shortened the duration of self-sustaining status epilepticus in rats, confirming its agonist properties in vivo. Pretreatment of rats with antisense peptide nucleic acid targeted to galanin receptor type 1 mRNA abolished the effect of galnon, suggesting mediation of its anticonvulsant properties through this receptor subtype. These findings introduce a systemically active nonpeptide galanin agonist anticonvulsant.

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Galanin can normally be detected only in a few dorsal root ganglion (DRG) neurons, but it is dramatically upregulated after peripheral nerve injury in both rat and monkey. Galanin is stored in large dense core vesicles, which after axotomy are often found close to the membrane of afferent nerve endings in the dorsal horn. In the monkey there is an increase in galanin in many nerve terminals in the superficial dorsal horn after axotomy, but such an increase is more difficult to detect in the rat. Galanin is also present in local dorsal horn neurons, where it is upregulated by peripheral inflammation. Both galanin-R1 and galanin-R2 receptor mRNAs are expressed in rat DRGs, mainly in, respectively, large and small DRG neurons. Galanin-R1 receptor mRNA is downregulated in DRG neurons after axotomy, and a small decrease in galanin-R2 receptor mRNA levels can also be seen. After peripheral tissue inflammation galanin-R1 receptor mRNA levels decrease and galanin-R2 receptor mRNA levels increase. The present results show that galanin and galanin receptors are present in sensory and local dorsal horn neurons and are regulated by nerve injury and inflammation. Galanin may therefore be involved in processing of pain information, primarily exerting analgesic effects. Whereas local dorsal horn neurons represent a defense system against inflammatory pain, we have proposed that a second defense system, against neuropathic pain, is intrinsic to DRG neurons.

Using in situ hybridization with riboprobes the distribution of alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptor mRNAs were studied in normal rat dorsal root ganglia and after unilateral peripheral nerve injury (total nerve transection) or inflammation. The most common adrenoceptor mRNA was of the alpha(2C) subtype (almost 80% of all neuron profiles) followed by the alpha(2A) subtype (almost 20%), whereas alpha(2B)-adrenoceptor mRNA was only found in small numbers of neuron profiles. The most dramatic effect of peripheral nerve injury was observed for the alpha(2A)-adrenoceptor mRNA, which increased to 45% of all neuron profiles. In contrast, alpha(2C) adrenoceptor mRNA showed a small decrease in this situation. Carrageenan-induced peripheral inflammation did not affect the percentage of alpha(2A)- or alpha(2C)-adrenoceptor mRNA-positive profiles. These findings suggest that, if any of the alpha(2) adrenoceptor, the alpha(2A) subtype represents the most likely candidate in DRG neurons to be involved in sympathetically maintained pain.

The effect of unilateral transection of the sciatic nerve on expression of immunoreactive galanin (GAL), galanin-message-associated peptide (GMAP) and neuropeptide tyrosine (NPY) in dorsal root ganglia (DRGs) was studied in wild-type mice and in leukemia inhibitory factor (LIF)-deficient mice. In normal and contralateral DRGs small numbers of weakly fluorescent GAL- and GMAP-positive neuronal cell bodies and numerous positive fibers were observed. No NPY-positive cell bodies but a few fibers surrounding blood vessels were seen. In LIF deficient mice hardly any GAL- or GMAP-positive neurons or fibers were seen, nor was NPY-like immunoreactivity present in cell bodies. After axotomy there was a dramatic upregulation of all three peptides in wild-type DRG neurons, whereby 50-60% of the neuron profiles, encompassing both small and large profiles, were GAL- and GMAP-immunoreactive (IR). About one third of all neuron profiles, mainly large ones, were NPY-positive. In LIF-deficient mice this upregulation was much less pronounced. Thus GAL- and GMAP-IR neuron profiles were reduced by 65-70% compared with the wild-type mice. The number of NPY-positive neuron profiles was reduced to half but this difference was not significant. There was also an ipsilateral decrease in fluorescence intensity for all three peptide immunoreactivities in the LIF-deficient mice as compared with wild-type mice after axotomy. There was no apparent difference in size between, respectively, GAL- and GMAP-positive profiles when comparing LIF-deficient and wild-type mice before or after axotomy. There were, however, no small NPY-IR profiles in the LIF-deficient group. The present results suggests that LIF is important for the dramatic upregulation of GAL and GMAP seen after axotomy. It may also be important for the normal expression of galanin in mouse DRGs, since wild-type mice seemed to have somewhat more positive cell bodies and more fluorescent fibers. LIF seems to be less important for the control of NPY synthesis, but may be involved in NPY regulation in small-sized neurons.