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Differential regulation of opioid binding sites in rat spinal cord in an experimental model of chronic pain
DIFFERENTIAL REGULATION OF OPIOID BINDING SITES IN RAT SPINAL CORD IN AN EXPERIMENTAL MODEL OF CHRONIC PAIN. C.W. Stevensl, K.C. Kajander2 G.J.Bennett2, and V.S. Seybold’, ‘Dept. Cell Bio. and Neuroanatomy, Univ. of Minnesot Minneapolis, MN, ZNAB, NIDR, NM, Bethesda, MD 20892. !
Poster 167 RED Mon-Tues Exhibit Hall Abs No
210
’ AIM OF INVESTIGATION: This study quantified changes in mu, delta, and kappa opioid binding sites in laminae I-II, V, and X of the rat spinal cord at 2,5, and 10 days after the unilateral loose ligation of the sciatic nerve Guidelines for the ethical treatment of animals were followed for all procedures. Spinal segment METHODS: L4 was obtained from sham and experimental rats (N=8 per group) at 2,5, and 10 days after ligation. Serial, cryostat sections (101.Q of freshly-frozen tissues were processed for receptor autoradiography using uitiated sufentanil(0.3 nM), DPDPE (5nM). or U-69593 (3nM) to label mu, delta, or kappa opioid sites, respectively. Autoradiograms were analyzed by computerized densitometry. Areas of the emulsion covering laminae I-II, V, and X were quantified on each side of each spinal cord. Data were analysed by paired t-tests of specific binding of the side contralateral compared to the side unilateral to the nerve ligation. The effect of unilateral loose ligation of the sciatic nerve on opioid binding sites are shown in the table below. The direction of significant changes of the ligated side with respect to the control side is indicated by the arrows. Dashed lines indicate no significant differences were found. CONCLUSION: These results demonstrate that in this model of chronic pain there is an initial increase in binding sites in laminae I-II for all ligands at 2 days post-ligation. These changes may be related to an immediate increase in the activity of small diameter primary afferent fibers. The decreases seen at later days, especially for the kappa sites in lamina V, parallel increases in levels of prodynorphin mRNA and dynorphin peptide previously noted in this paradigm. The temporal changes underscore the complexity in central reorganization of activity within spinal circuitry during the development of peripheral neuropathy.
Poster 168 RED Mon-Tues Exhibit Hall
CHANGES IN CALCITONIN GENERBLATED PBPTIDE AND SUBSTANCE P BINDING SITES IN RAT SPINAL CORD FOLLGWING DORSAL -MY. VS. Sevbold, L.M. Aanonsen* and M.G. Gamy*, Dept. of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN, USA
Abs No
II
211
The purpose of this study was to determine whether INVESTIGATION: dorsal rhizotomy causes changes in the density of substance (SP) and calcitonm gene-related peptide (CGRP) binding sites in the spinal cord, and the time course over which these changes occur. METHODS: Quantitative receptor autoradiography was used to determine the amount of [t2QSP and -CGRP bound within the dorsal horn of the spinal cord following dorsal rhizotomy. Across all animals, unilateral rhizotomy minimaBy included lesion of left dorsal roots L2-L6. Groups of animals (8/ up) were sacrificed at 1,2,4 and 8 days post-surgery. A group of unoperated animals served as a control. [t p I] Bolton-Hunter-SP (0.05 nM) and [ 12sHCGRP (0.1 nM) were used to label SP and CGRP binding sites, respectively, on tissue sections (10 urn) of spinal segment L4 from each animal. Incubation of adjacent tissue sections in radiolabeled peptide with 0.1 @I peptide was used to determine nonspecific binding. The density of autoradiographic grains within the emulsion overlying laminae I+II, V and X of each tissue section was quantified by computerized image processing. BESULTS: The amount of [12sIJSP and [1251]CGBP that was specifically bound to the tissue increased in laminae 1+11ipsilateral to the lesions following dorsal rhizotomy. The amount of [*29YjSPbound was increased 2 and 4 days after rhizotomy, but no difference occurred 8 days post-lesion. In contrast, the amount of [t~IJCGBP bound did not increase until 4 days post-lesion, and remained elevated at 8 days. ParalIel changes in the amount of [1251KKiRP bound occurred in lamina V, but the amount of [WISP bound was not changed in this region at any time point. No change in the amount of [rsI]SP and [*aIjCGRP bound was observed in lamina X at any time point studied. CONCLUSION: These data indicate that SP and CGBP binding sites increase following decreases in SP and CGRP release after removal of primary afferent neurons from the spinalcord by dorsal rhizotomy. The different time courses of the changes indicate that SP and CGBP receptors am regulated differently. These studies were supported by USPHS grants NS 17702, DA05309 and DAO7234.
Poster 167 RED Mon-Tues Exhibit Hall Abs No
210
’ AIM OF INVESTIGATION: This study quantified changes in mu, delta, and kappa opioid binding sites in laminae I-II, V, and X of the rat spinal cord at 2,5, and 10 days after the unilateral loose ligation of the sciatic nerve Guidelines for the ethical treatment of animals were followed for all procedures. Spinal segment METHODS: L4 was obtained from sham and experimental rats (N=8 per group) at 2,5, and 10 days after ligation. Serial, cryostat sections (101.Q of freshly-frozen tissues were processed for receptor autoradiography using uitiated sufentanil(0.3 nM), DPDPE (5nM). or U-69593 (3nM) to label mu, delta, or kappa opioid sites, respectively. Autoradiograms were analyzed by computerized densitometry. Areas of the emulsion covering laminae I-II, V, and X were quantified on each side of each spinal cord. Data were analysed by paired t-tests of specific binding of the side contralateral compared to the side unilateral to the nerve ligation. The effect of unilateral loose ligation of the sciatic nerve on opioid binding sites are shown in the table below. The direction of significant changes of the ligated side with respect to the control side is indicated by the arrows. Dashed lines indicate no significant differences were found. CONCLUSION: These results demonstrate that in this model of chronic pain there is an initial increase in binding sites in laminae I-II for all ligands at 2 days post-ligation. These changes may be related to an immediate increase in the activity of small diameter primary afferent fibers. The decreases seen at later days, especially for the kappa sites in lamina V, parallel increases in levels of prodynorphin mRNA and dynorphin peptide previously noted in this paradigm. The temporal changes underscore the complexity in central reorganization of activity within spinal circuitry during the development of peripheral neuropathy.
Poster 168 RED Mon-Tues Exhibit Hall
CHANGES IN CALCITONIN GENERBLATED PBPTIDE AND SUBSTANCE P BINDING SITES IN RAT SPINAL CORD FOLLGWING DORSAL -MY. VS. Sevbold, L.M. Aanonsen* and M.G. Gamy*, Dept. of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN, USA
Abs No
II
211
The purpose of this study was to determine whether INVESTIGATION: dorsal rhizotomy causes changes in the density of substance (SP) and calcitonm gene-related peptide (CGRP) binding sites in the spinal cord, and the time course over which these changes occur. METHODS: Quantitative receptor autoradiography was used to determine the amount of [t2QSP and -CGRP bound within the dorsal horn of the spinal cord following dorsal rhizotomy. Across all animals, unilateral rhizotomy minimaBy included lesion of left dorsal roots L2-L6. Groups of animals (8/ up) were sacrificed at 1,2,4 and 8 days post-surgery. A group of unoperated animals served as a control. [t p I] Bolton-Hunter-SP (0.05 nM) and [ 12sHCGRP (0.1 nM) were used to label SP and CGRP binding sites, respectively, on tissue sections (10 urn) of spinal segment L4 from each animal. Incubation of adjacent tissue sections in radiolabeled peptide with 0.1 @I peptide was used to determine nonspecific binding. The density of autoradiographic grains within the emulsion overlying laminae I+II, V and X of each tissue section was quantified by computerized image processing. BESULTS: The amount of [12sIJSP and [1251]CGBP that was specifically bound to the tissue increased in laminae 1+11ipsilateral to the lesions following dorsal rhizotomy. The amount of [*29YjSPbound was increased 2 and 4 days after rhizotomy, but no difference occurred 8 days post-lesion. In contrast, the amount of [t~IJCGBP bound did not increase until 4 days post-lesion, and remained elevated at 8 days. ParalIel changes in the amount of [1251KKiRP bound occurred in lamina V, but the amount of [WISP bound was not changed in this region at any time point. No change in the amount of [rsI]SP and [*aIjCGRP bound was observed in lamina X at any time point studied. CONCLUSION: These data indicate that SP and CGBP binding sites increase following decreases in SP and CGRP release after removal of primary afferent neurons from the spinalcord by dorsal rhizotomy. The different time courses of the changes indicate that SP and CGBP receptors am regulated differently. These studies were supported by USPHS grants NS 17702, DA05309 and DAO7234.