Characterization of spinal ganglion neurons in horse (Equus caballus). A morphometric, neurochemical and tracing study

Abstract

Spinal ganglion (SG) neurons have been widely described in rodents, and classified according to various criteria. On the basis of such studies, many features of rodent SG neurons have become benchmarks to classify these cells. However, these traits cannot be confirmed in all other species. In the present study, horse SG neurons were morphometrically and neurochemically characterized by detecting the neuronal markers calcitonin gene-related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (nNOS) and isolectin B4 (IB4) from Griffonia simplicifolia. Moreover, spinal cord staining and tracer studies were also performed injecting Fast Blue tracer in the ileo-cecal junction. The statistical analysis of the histograms related to the cross sectional area of dark and light SG neurons confirmed the presence of the categories of small and large neurons. The staining methods employed yielded the following results: (1) in all triple staining experiments performed, most SG stained neurons were triple-labeled; (2) SP-IR neurons showed the largest percentages of co-localization with the other markers studied; (3) CGRP-IR and IB4-labeled neurons were the SG neurons showing the largest percentages of single staining; (4) nNOS-IR neurons were more represented in horse SGs than in those from rodents; (5) IB4 was widely co-localized with both CGRP and SP. Retrograde tracer investigation combined with neurochemical evaluation showed that in horse, contrarily to rodents, IB4-labeled neurons are widely involved in visceral innervations. The results obtained from the observations of serial stained sections and from a critical analysis of triple-labeling experiments allowed us to conclude that (1) most stained SG neurons co-expressed IB4-nNOS-CGRP-SP neuronal markers, (2) IB4 is not indicated as a marker of non-peptidergic neurons in the horse, (3) horse IB4-labeled neurons are widely involved in visceral sensation, (4) differently from rodents, horse IB4-, CGRP- and SP-labeled fibers share the same spinal cord level terminations.