Identification of neuropeptides in pelvic and pudendal nerve afferent pathways to the sacral spinal cord of the cat

Abstract

The distribution of several neuropeptides, including vasoactive intestinal polypeptide (VIP), substance P, somatostatin, leucine enkephalin, methionine enkephalin, and cholecystokinin, in sacral afferent pathways of the cat was examined by immunohistochemical techniques. Certain peptides (substance P, somatostatin, and leucine enkephalin could be demonstrated in normal dorsal root ganglion cells; however, topical administration or injections of colchicine solution into ganglia 36-56 hours prior to removal markedly increased the number of cells labeled and the intensity of staining. Other peptides (VIP, cholecystokinin, and methionine enkephalin were only detected in significant numbers of cells following intraganglionic injections of colchicine. The distribution of peptides in dorsal root ganglion cells projecting to the pelvic nerve (visceral) and the pudendal nerve (somatic) was examined by retrograde dye labeling combined with immunohistochemistry. Fluorescent dyes were applied to the cut ends of the nerves 2 weeks prior to removal. A considerably higher percentage of pelvic nerve afferent neurons than pudendal nerve afferent neurons exhibited peptide immunoreactivity; e.g., VIP (42% vs. 10%), cholecystokinin (29% vs. 12%), substance P (24% vs. 21%), leucine enkephalin (30% vs. 24%), and methionine enkephalin (10% vs. 3%). Somatostatin was present in only a small percentage of either type of afferent neuron (0.3-2%). The total percentage of peptide-containing pelvic afferent neurons exceeded 100% (137%), suggesting that more than one peptide is present in some visceral afferent neurons. This has been confirmed in preliminary experiments. The peptide-containing cells were in general less than 40 μm in average diameter; however, a significant percentage of substance P and cholecystokinin neurons ranged from 40 to 60 μm in average diameter. VIP cells had the smallest average diameter (30 μm) whereas somatostatin cells had the largest average diameter (36μm). Statistical analysis of cell sizes revealed that substance P cells projecting to the pelvic nerve were smaller than substance P cells sending axons into the pudendal nerve. On the other hand, VIP cells in the two afferent pathways were not significantly different in size. Sacral visceral and somatic afferent neurons contain a wide spectrum of neuropeptides, some of which (e.g., VIP and cholecystokinin) seem to be preferentially distributed in the visceral afferent systems. The high percentage of visceral afferent neurons containing VIP is consistent with previous reports of the high concentration of VIP in the sacral spinal cord and provides further support for the view that VIP may be an important transmitter in afferent pathways from the pelvic organs. The demonstration of enkephalins in visceral and somatic afferent pathways raises the possibility that these substances, which are generally thought to have inhibitory functions, may be released by primary afferent pathways in the spinal cord and mediate feedback inhibition by activation of opioid inhibitory receptors on afferent terminals.