Several indices of peptidergic, primary afferent neural transmission in rat at the level of the lumbar spinal cord exhibited differential changes over time in response to adjuvant-induced inflammation of the hindpaw. The indices were measurements of the production of messenger RNA encoding the precursors for substance P and calcitonin gene-related peptide in dorsal root ganglia, the storage of substance P and calcitonin gene-related peptide in the dorsal spinal cord and the release of the peptides evoked by application of capsaicin to the dorsal spinal cord. A 47% decrease in the content of immunoreactive substance P in the dorsal half of the lumbar spinal cord, as determined by radioimmunoassay, was measured at 6 h following the injection of complete Freund's adjuvant into the hindpaw. Decreased content of immunoreactive SP persisted for four days, but was no longer present at eight days after the adjuvant injection. The content of immunoreactive calcitonin gene-related peptide in the dorsal spinal cord was decreased by 29% at one day following the injection of adjuvant into the rat hindpaw and 43% at two days; the content then increased to a level greater than that of control animals at eight days. The amount of messenger RNA encoding preprotachykinin and prepro-calcitonin gene-related peptide in L4-L6 dorsal root ganglia was determined from northern blot analysis of the total messenger RNA extracted from the dorsal root ganglia. Each species of messenger RNA had increased compared to the control animals at two days following the injection of adjuvant into the rat hindpaws and remained elevated after eight days. Thus, an increase in the messenger RNAs encoding substance P and calcitonin gene-related peptide in the dorsal root ganglia preceeded the recovery of the content of the peptides in the spinal cord. Morphometric studies of calcitonin gene-related peptide-immunoreactive perikarya in the L4 dorsal root ganglia indicated that the increase in messenger RNA occurred in neurons of the size that normally express calcitonin gene-related protein. Radioimmunoassay of the superfusate of the dorsal half of the lumbar spinal cord was used to measure the release of immunoreactive substance P and immunoreactive calcitonin gene-related protein in vitro. Although the basal release of immunoreactive substance P and immunoreactive calcitonin-gene related protein from the dorsal spinal cord was constant throughout the time points examined, changes occurred in the release of peptide evoked by 10 μM capsaicin. The capsaicin-evoked release of immunoreactive substance P was decreased at 6 h and eight days post-injection of adjuvant. In contrast, at four days after the injection of adjuvant into the rat hindpaw, the capsaicin-evoked release of immunoreactive calcitonin gene-related protein from the dorsal half of the spinal cord was increased. Thus, the basal release and capsaicin-releasable pool of immunoreactive substance P and immunoreactive calcitonin gene-related protein were largely maintained in spite of the persistent, decreased content of the peptides in the spinal cord. In total, these data illustrate the time course of the plasticity that occurs presynaptically in response to adjuvant-induced inflammation in primary afferent neurons containing substance P and calcitonin gene-related protein. The changes support the hypothesis that substance P and calcitonin gene-related protein neurotransmission have a role in generating and maintaining the hyperalgesia and edema that accompany peripheral inflammation.
Bibliographical noteFunding Information:
Acknowledgements--The authors are grateful to Lia Abrahams, Ami Mariash, Ameena Mogh6, Ann Parsons and Cheryl Stucky for skilled technical assistance during the execution of the studies. The cDNA probe for rat fl-PPT mRNA was provided by Dr J. Krause (Washington University), the probe for rat prepro-somatostatin mRNA was provided by Dr J. Dixon (Purdue University) and the probe for rat GAPDH mRNA was provided by Dr F. Boyd (University of Minnesota). The work was supported by a grant from NIH NS 17702. M. T. Galeazza is supported by NIDA training grant T32DA07234. M. G. Garry was supported by NRSA training grant F32DE05606.
- 2-[ N-morpholino]ethanesulphonic acid
- bovine serum albumin
- calcitonin gene-related peptide
- complete Freund's adjuvant
- dorsal root ganglia
- ethylene diaminetetra-acetate
- excitatory amino acids
- fluoroscein isothiocyanate
- glyceraldehyde phosphate dehydrogenase
- sodium dodecyl sulphate
- standard saline citrate
- substance P