Objective: This study was undertaken to examine the neurofilament and neurochemical composition of subpopulations of primary afferent neurons innervating the levator ani muscle by combining retrograde tracing and triple labeling immunofluorescence in the female squirrel monkey.

Study design: Cholera toxin B subunit (CTB) was injected unilaterally into the levator ani muscle of 3 monkeys to identify primary sensory neurons in the dorsal root ganglia (DRG) and their central projections in the spinal cord. L7-S2 DRG were processed for dual or triple labeling immunofluorescence 3 days after injection to examine labeling of the 200 kD neurofilament marker RT97 (a marker of myelinated neurons), calcitonin gene-related peptide (CGRP; a marker of peptidergic neurons), isolectin B4 (IB4; a marker of small, unmyelinated neurons), and nerve growth factor receptor (TrkA) in CTB-positive neurons.

Results: RT97-negative (C-fiber) neurons were more numerous (74% of total CTB-labeled neurons) and smaller in size than RT97-positive (A-fiber) afferent neurons (26% of CTB-labeled neurons). IB4 labeling was almost exclusively found in RT97-negative afferent neurons. Approximately 43% of all CTB-labeled DRG neurons expressed CGRP, and the majority of these were small. The distribution and sizes of CTB-labeled TrkA-positive DRG neurons were similar to those of CTB-labeled CGRP-positive DRG neurons.

Conclusion: The levator ani muscle is innervated by 3 major subpopulations of primary afferent neurons consisting of cells with large, neurofilament-rich soma and A fibers (putative proprioceptive neurons) and those with small, peptidergic or nonpeptidergic, neurofilament-poor soma and C fibers (putative nociceptive, mechanoreceptive, ergoreceptive, and thermoreceptive neurons). Future investigation is needed to elucidate the relationship between primary sensory neuron subpopulations and changes in neuropeptide and neurotrophin expression on experimental levator ani nerve damage, childbirth, and aging.