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The role of metabotropic glutamate receptors in the processing of somatosensory information was studied in dorsal horn neurons of the rat spinal cord. Activation of metabotropic glutamate receptors by local iontophoresis of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid resulted in an increased response of dorsal horn neurons to ionotropic glutamate receptor agonists (N-methyl-D-aspartate and kainic acid) applied by iontophoresis. Greater amounts of 1S,3R-1-amino-cyclopentane-1,3-dicarboxylic acid, ejected at high iontophoresis currents, directly excited dorsal horn neurons. Application of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid also led to a significant increase in responses to innocuous (brush, pressure) but not in responses to noxious (pinch, squeeze) mechanical stimulation. The excitatory effects of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid were selectively blocked by (S)-4-carboxy-3-hydroxyphenyl-glycine, an antinociceptive phenylglycine derivative which is a selective group 1 metabotropic glutamate receptor antagonist, confirming the involvement of these receptors. In wide dynamic range neurons, wind-up, the progressive potentiation of C-fibre-evoked responses during a train of stimuli, was increased by iontophoretic application of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid or decreased by iontophoresis of (S)-4-carboxy-3-hydroxyphenyl-glycine without significant change in the C-fibre input. The results suggest an interaction between metabotropic and ionotropic glutamate receptors in spinal dorsal horn neurons. Metabotropic glutamate receptors proved to be involved in the frequency-dependent potentiation of C-fibre responses possibly via modulation of ionotropic glutamate receptors. The long-lasting effects of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid on wind-up and on responses to peripheral mechanical stimuli strongly support the view that metabotropic glutamate receptors in these neurons may play a significant role in spinal synaptic plasticity, and therefore, may contribute to the central sensitization during mechanical hyperalgesia.

Substance P, a putative neurotransmitter or neuromodulator of nociception or pain in the spinal cord, exhibits both antinociceptive and hyperalgesic properties. Investigators have shown that the N-terminal metabolite of substance P, substance P(1-7), produces naloxone-reversible antinociception when given supraspinally and systemically in mice and hyperalgesia when injected intrathecally in rats. The goal of our investigation was to identify the receptors mediating these actions of substance P(1-7) at the initial site of release of substance P, i.e. in the spinal cord. Thirty minutes after intrathecal injection, substance P(1-7) produced naloxone-reversible antinociception in a dose-dependent manner in the abdominal stretch assay. When administered with naloxone, substance P(1-7) produced hyperalgesia 5 and 10 min after injection, which was inhibited by dizocilpine (MK-801), a phencyclidine ligand and non-competitive antagonist of N-methyl-D-aspartate. Antinociception was inhibited by the mu-selective opioid antagonist beta-funaltrexamine, but not by the mu 1-selective opioid antagonist naloxonazine or the delta-selective antagonist naltrindole, indicating a mu 2-opioid receptor-mediated effect. These findings suggest that the N-terminal portion of substance P may modulate nociception or pain, as demonstrated in the acetic acid abdominal stretch (writhing) assay, via activation of two different receptor systems. Substance P(1-7)-induced hyperalgesia is mediated by a phencyclidine-sensitive mechanism and antinociception involves activity at mu-opioid, most likely mu 2, receptors.

Sensitization to the behavioral effects of intrathecal kainate in mice depends on an accumulation of the N-terminus of substance P in the spinal cord and may reflect similar synaptic activity as that underlying pain transmission. The purpose of this study was to determine whether kainate sensitization, like pain, is sensitive to inhibition by phencyclidine ligands. Doses that selectively inhibit the behavioral response to a single injection of N-methyl-D-aspartate, but not kainate, were established for two non-competitive antagonists, dizocilpine (MK-801) and phencyclidine, as well as two competitive antagonists, D-amino-5-phosphonovaleric acid and (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, of N-methyl-D-aspartate. Using these doses, we found that 1 nmol of MK-801 or 3 nmol of phencyclidine blocked sensitization to four injections of 25 pmol of kainate administered at 2 min intervals. In contrast, 1.48 nmol of D-amino-5-phosphonovaleric acid and 0.5 nmol of (+/-)-3)2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid failed to alter sensitization to kainate, indicating that activation of N-methyl-D-aspartate receptors is not necessary for kainate sensitization. Haloperidol (1 nmol), a sigma receptor ligand, also failed to inhibit sensitization to kainate, suggesting that the actions of MK-801 and phencyclidine were not produced by a non-selective effect at sigma sites. Together, these data suggest that MK-801 and phencyclidine inhibit behavioral sensitization to kainate via phencyclidine receptors that are not linked to the N-methyl-D-aspartate receptor complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrathecal injection of mice with substance P or its C-terminal fragments evokes a well documented behavioral syndrome characterized by caudally-directed biting and scratching. We have previously shown that repeated injections of substance P result in naloxone-sensitive desensitization to this substance P-induced behavior, possibly through interactions of N-terminal fragments of substance P with mu opiate binding sites. The present investigation tests the hypothesis that substance P metabolites play a role in the development of desensitization to substance P by using the biting and scratching behavioral paradigm. While substance P-induced behaviors are produced by as little as 1 pmol of substance P, repeated injections of 7.5 pmol at 60-s intervals was found to be the minimum dose capable of causing desensitization. The C-terminal peptides, substance P3-11 and substance P5-11, elicited substance P-like behaviors, but repeated injection of these compounds did not result in desensitization to this behavior. In contrast to C-terminal fragments, intrathecal injection of N-terminal fragments, (substance P1-4, substance P1-7 and substance P1-9), did not elicit any overt substance P-like behaviors when administered alone, but when co-administered with substance P, decreased the magnitude of substance P-induced behaviors in a dose-related fashion. Various peptidase inhibitors significantly inhibited the catabolism of co-administered substance P. Co-administration of substance P with peptidase inhibitors enhanced and prolonged the substance P-induced behavioral episode, but also prevented the development of substance P-induced desensitization. Together these results support the hypothesis that the accumulation of endogenously generated N-terminal metabolites of substance P mediate desensitization to substance P-induced behaviors in the spinal cord. Substance P metabolism may therefore decrease ongoing substance P activity both by the hydrolysis of the C-terminal portion of substance P as well as by the production of N-terminal metabolites that are capable of inhibiting the effects of substance P.

The pontine nuclei form the key relay nuclei in the cerebropontocerebellar pathway. Although a great deal of information is available regarding the anatomy of this region, the identity of the neurotransmitter(s) contained in the neurons of the pontine gray are not known. The aim of the present investigation is to utilize immunohistochemical techniques to determine whether glutamate, a putative excitatory transmitter, and the enzymes responsible for its metabolism, are found in pontine neurons. Both glutaminase, an enzyme which converts glutamine to glutamate, and aspartate aminotransferase, an enzyme which is involved in the interconversion between glutamate and aspartate, have been proposed to be markers of neurons which use excitatory amino acids as neurotransmitters. The present study utilizes a monoclonal antibody against carbodiimide-fixed glutamate and polyclonal antisera against glutaminase and aspartate aminotransferase in conjunction with the indirect peroxidase technique or the peroxidase-labeled biotin-avidin procedure to localize glutamatergic neurons in the pontine nuclei of the rat. Numerous neurons in all subdivisions of the pontine nuclei were found to contain carbodiimide-fixed glutamate-like immunoreactivity, glutaminase-like immunoreactivity or aspartate aminotransferase-like immunoreactivity. Horseradish peroxidase was injected into the cerebellum of four rats for use with a combined retrograde transport-immunohistochemical procedure. Double-labeled neurons were observed in all subdivisions of the pontine nuclei, indicating that pontine neurons which contain glutamate-like immunoreactivity project to the cerebellum. Based on the hypothesis that increased levels of glutamate, glutaminase and aspartate aminotransferase reflect a transmitter role for glutamate, the present data raise the possibility that glutamate may be a major neurotransmitter of pontocerebellar fibers.

Tubulin and glutamate were immunohistochemically localized in the hippocampus and amygdala of rats using monoclonal antibodies to gamma-Glu-Glu (Glu-1) and glutaraldehyde-fixed glutamate (Glu-2), respectively. Glu-2 was shown to be selectively immunoreactive for glutaraldehyde-fixed Glu using enzyme-linked immunoassays and inhibition enzyme-linked immunoassays. Glu-1 was immunoreactive with tubulin on immunoblots of brain homogenates. However, only tubulin with a glutamate carboxy-terminal appeared to be immunoreactive with Glu-1 since tubulin from Chinese hamster ovary cells was not immunoreactive on immunoblots unless the tubulin was first treated with carboxypeptidase. Intense immunocytochemical staining by Glu-1 of hippocampus and amygdala was co-localized in the same neurons as the immunocytochemical staining for glutaraldehyde-fixed Glu produced by Glu-2. The distribution of immunostaining in the brain by Glu-1 was very similar to the distribution of immunostaining produced by Glu-2. The major difference was that glutamate-like immunoreactivity, visualized by Glu-2 staining, was intense in the nuclei of neurons, while nuclei were unstained by Glu-1. The distribution of immunostaining by these monoclonal antibodies was very similar to that reported in previous studies using other antibodies to Glu. All granule cells in the area dentata of the hippocampus exhibited intense immunoreactivity with both antibodies. Immunoreactivity was also observed in the stratum lucidum of CA3, the zone of termination of mossy fiber axons of granule cells. The majority of pyramidal cells in CA1, and many pyramidal cells in CA3 of the hippocampus were immunoreactive. In addition, it appeared that all of the pyramidal cells in the subiculum exhibited immunoreactivity. Light, diffuse immunoreactivity was observed in the neuropil of the hippocampus and subiculum. Most perikarya in the amygdala were characterized by light to moderate Glu-1 immunoreactivity and moderate to intense Glu-2 immunoreactivity. Fairly intense Glu-1 and Glu-2 immunoreactivity was seen in some neurons of the lateral nucleus, basolateral nucleus, lateral subdivision of the central nucleus, and the periamygdaloid cortex. The morphology of immunostained neurons in the lateral and basolateral nuclei indicates that the majority of these cells correspond to the pyramidal class 1 neurons described in previous Golgi studies.(ABSTRACT TRUNCATED AT 400 WORDS)