Linda S. Sorkin

Neural changes in acute arthritis in monkeys. I. Parallel enhancement of responses of spinothalamic tract neurons to mechanical stimulation and excitatory amino acids

Somatosensory neurons of the spinal cord, including projection neurons, become hyperexcitable to mechanical stimuli during the development of experimental arthritis in rats and cats and hence are suggested to participate in the generation of arthritic hyperalgesia in humans. The experiments described here show a potentiation of the responses of spinothalamic tract (STT) neurons in monkeys during the development of an acute arthritis. The results demonstrate that the responses of STT neurons to mechanical stimuli and to iontophoretically applied excitatory amino acids (EAAs), particularly those acting at non-N-methyl-D-aspartate (non-NMDA) receptors, become enhanced during the development of inflammation produced by intra-articular injection of kaolin and carrageenan. Since the enhancement of both responses follows a similar time course, the results of this work suggest a role for EAAs in the hyperalgesia associated with arthritis and hence may provide a possible pharmacologic target for alleviation and/or prevention of arthritic pain.

Neural changes in acute arthritis in monkeys. IV : Time-course of amino acid release into the lumbar dorsal horn

Extracellular levels of amino acids were measured during the development of experimental arthritis in anesthetized monkeys. Levels of glutamate, aspartate, glycine, serine, glutamine, taurine, cysteic acid and asparagine were each measured in consecutive 30 min samples before, during and for several hours after injection of kaolin and carrageenan into the articular capsule of one knee. Samples were obtained via a microdialysis probe placed in the lumbar dorsal horn ipsilateral to the injected knee and assayed using HPLC with fluorescence detection. Glutamate, aspartate, glycine and serine increased transiently following intra-articular injection of inflammatory agents. During this period glutamine levels decreased. A second phase of release then occurred which included more prolonged changes in amino acid levels that were sometimes of greater magnitude than those immediately following the injection. In animals which were later observed to have depletion of SP in the dorsal horn of the inflamed side, taurine levels increased starting after the Glu, Asp and Gly had plateaued at near baseline concentrations. Thus during the first stages of joint inflammation EAAs are released into the dorsal horn, followed by increased levels of IAAs, possibly representing activation of the descending endogenous analgesia system. This phase is followed by a semiacute response consisting in part of increased extracellular levels of SP and Tau. While SP is presumably part of an ascending nociceptive transmission system, Tau could be part of a second system aimed at reducing excessive neural activity including neural transmission resulting in intense maintained pain.

Raphe magnus stimulation-induced antinociception in the cat is associated with release of amino acids as well as serotonin in the lumbar dorsal horn.

Stimulation in the nucleus raphe magnus (NRM) inhibits transmission of nociceptive information within the spinal cord through activation of bulbospinal pathways. This study used microdialysis in combination with high pressure liquid chromatography to measure the release of serotonin (5HT) and several amino acids, including glutamate, aspartate and glycine, from the lumbar dorsal horn during electrical stimulation within the NRM in the alpha-chloralose anesthetized cat. Observed release of putative neurotransmitters was correlated with inhibition of nociceptive projection neurons recorded from sites within 800 microns rostral or caudal to the dialysis fiber. NRM stimulus parameters considered to preferentially activate myelinated fibers caused inhibition of nociceptive evoked activity, and increased the release of excitatory amino acids and glycine within the spinal cord, with no detectable release of 5HT. When pulse widths were lengthened and unmyelinated fibers were also activated, increases in 5HT in the spinal dialysate were observed as well. Strychnine administered through the dialysis fiber (0.02-1 mM) antagonized NRM-induced inhibition when 5HT release was not detected. Inhibition produced by stimulation that increased 5HT concentrations was relatively strychnine resistant. These results point to a raphe-spinal inhibitory pathway that is not dependent on 5HT, the activation of which results in the spinal release of glycine.

Amino acids and serotonin are released into the lumbar spinal cord of the anesthetized cat following intradermal capsaicin injections

Several amino acids including aspartate, glutamate and glycine and the monoamine serotonin were retrieved from the extracellular space of the dorsal horn of the lumbar spinal cord in the alpha-chloralose anesthetized cat in vivo using a transverse microdialysis probe. Neurotransmitter concentrations were determined using high pressure liquid chromatography in combination with fluorescence (amino acid) or electrochemical (serotonin) detection. Intradermal injection of 3% capsaicin into the hindleg either ipsilateral or contralateral to the dialysis probe was used to evoke release. Extracellular concentrations of aspartate, glutamate and serotonin increased significantly following capsaicin injection into the ipsilateral limb. An almost equal increase in serotonin and a less pronounced, but still significant, increase in aspartate accompanied contralateral capsaicin injection. Glutamate concentrations increased in the dialysate during contralateral capsaicin injection in about half of the animals. These data are consistent with the hypothesis that Asp and Glu are both neurotransmitters released from nociceptive primary afferent fibers and/or interneurons activated by these fibers. In addition, Asp is presumed to be released from intrinsic spinal or descending systems following nociceptive stimulation. Bilateral release of 5HT into the dorsal horn most likely results from non-topographic activation of descending endogenous analgesia pathways.

Neural changes in acute arthritis in monkeys. III. Changes in substance P, calcitonin gene-related peptide and glutamate in the dorsal horn of the spinal cord.

The effects of an experimentally induced arthritis on immunoreactivity of putative primary afferents neurotransmitter/neuromodulators were examined. Immunoreactive staining for substance P (SP), calcitonin gene-related peptide (CGRP) and glutamate (Glu) in the monkey dorsal horn was examined following inflammation of one knee joint induced by injection of 5% kaolin and 5% carrageenan. Spinal cords were examined at different time periods after induction of arthritis (2.5, 4, 6 and 8 h). Side to side differences in immunoreactivity were determined by a computer assisted quantitation system. A significant overall decrease in immunoreactivity of the lumbar versus the cervical dorsal horn was found for SP. The decrease for SP showed maximal changes of 68.3% at 4 h and 54.7% at 6 h. Immunoreactivity for CGRP was decreased 31.5% at 8 h and variable at other time points. Immunoreactivity for Glu, showed an ipsilateral increase of 31.4% at 4 h, 33.7% at 6 h, 39.9% at 8 h and a significant effect for lumbar versus cervical. Repetitive peripheral stimulation of the joint was shown to be important for changes in SP and Glu immunoreactivity. Without frequent peripheral stimulation in the early stages of the development of arthritis, SP showed no quantitative side to side differences. Increases in Glu immunoreactivity were present but not as prominent with minimal joint manipulation. These studies suggest that Glu may be involved in the aching pain of inflammation at rest whereas SP, CGRP and Glu may mediate pain induced by joint movement.

Microdialysis recovery of serotonin released in spinal cord dorsal horn

Methods for making and using hollow microdialysis fibers suitable for recovering extracellular substances from discrete regions of the spinal cord are described. After placement of the fiber, artificial cerebrospinal fluid was pushed through it at a low (4-5 microliters/min) rate. The perfusate was collected and samples analyzed on a high performance liquid chromatograph with an electrochemical detector. Serotonin, 5-hydroxyindole acetic acid and norepinephrine were recovered and identified. Single unit extracellular recordings were made during the perfusion and collection; thus simultaneous observation of neurotransmitter release and modulation of single cell activity is now possible.

Neural changes in acute arthritis in monkeys. II : Increased glutamate immunoreactivity in the medial articular nerve

Glutamate and other excitatory amino acids have been shown to play a key role in nociception and the hyperalgesia associated with the acute inflammatory response. In an effort to understand more fully the role of Glu in this process, we determined that there is Glu in a percentage of axons in the medial articular nerve (MAN) of monkeys, a source of preterminal afferent fibers innervating the knee joint. After induction of the experimental knee joint inflammation with a kaolin/carrageenan mixture, comparison was made of the percentage of Glu positive axons in the MAN on the side of the inflammation versus the contralateral MAN using post-embedding immunogold electron microscopic methods. A doubling in the percentage of Glu-containing axons was observed on the side of the experimental arthritis as compared to the MAN of the other side or of uninjected controls. Glu positive axons were unmyelinated or were included in the small, thinly myelinated group in control nerves. Following induction of the inflammation, axonal diameter measurements revealed an increase in Glu content primarily in the small, thinly myelinated axons, which correspond to the group III afferent fibers. These increases were observed in the anesthetized preparation only when injection of kaolin/carrageenan was combined with joint flexion and mechanical stimulation. The dramatic increase in percentages of fibers stainable for Glu in the MAN following the induction of inflammation suggests that Glu content is greatly increased in the afferent fibers and may be a major contributor to the enhanced responses of sensory neurons in inflammatory states such as arthritis.

Calcitonin gene-related peptide containing primary afferent fibers synapse on primate spinothalamic tract cells

Spinothalamic tract (STT) cells were identified by intracellular injection or retrograde labeling with horseradish peroxidase (HRP) in the primate spinal cord. Using immunohistochemical techniques, a population of primary afferents containing calcitonin gene-related peptide (CGRP) is demonstrated in synaptic contact with these neurons. Large glomerular type CGRP terminals with morphology considered typical of primary afferent fibers are occasionally observed in contact with STT profiles in laminae I and IIo; however, this morphological type targets STT profiles chiefly in the deeper dorsal horn (laminae III, IV and V). In contrast, the majority of CGRP terminals contacting STT profiles in the superficial dorsal horn (laminae I and IIo), are small, round or oblong shaped terminals. Thus, evidenced by these data, the absence of large, glomerular type terminals does not rule out primary afferent input.

Response properties of spinal neurons projecting to midbrain or midbrain-thalamus in the monkey☆

Response and receptive field properties were determined for 24 spinal neurons backfired from midbrain or midbrain-thalamus in the anesthetized monkey. Recording sites were located in laminae I (11 cells), IV (6), V (6) and VI (1) in the lumbosacral spinal cord. The mean axonal conduction velocity of cells projecting to midbrain was significantly less than that of cells projecting to midbrain-thalamus. Cells backfired only from midbrain had complex excitatory receptive fields whereas those projecting to midbrain-thalamus had excitatory receptive fields confined to a single limb. Cells in both populations had complex inhibitory receptive fields.

The effects of A- and C-fiber stimulation on patterns of neuropeptide immunostaining in the rat superficial dorsal horn

The present study determines the effects of sciatic nerve stimulation at intensities that activate A-fibers alone or both A- and C-fibers on immunostaining for substance P (SP), cholecystokinin-octapeptide (CCK-8), galanin (GAL), dynorphin (DYN) and vasoactive intestinal polypeptide (VIP) in the superficial dorsal horn of the rat spinal cord. The goal of this study is to provide a more precise spatial localization of the sites of release or accumulation of these compounds in relation to specific types of stimuli. Following A-fiber stimulation, there was no significant change in immunostaining for any of these compounds. However, A- and C-fiber stimulation resulted in major changes. For SP, CCK-8, GAL and DYN there was a large and significant loss of immunostaining in medial regions of the dorsal horn. This is the area where sciatic nerve primary afferent fibers terminate and the depletion is probably correlated with activity in these fibers. By contrast, VIP immunostaining is increased in the lateral part of the superficial cord, which is outside of the central sciatic afferent fiber terminations. This indicates that the increase is not in the fine sciatic sensory axons that are directly stimulated. As a final point, the fact that C-fiber but not A-fiber stimulation causes marked changes in the immunocytochemical distribution of all these compounds is further evidence, albeit indirect, that they are involved in nociceptive information processing.