Neurochemical characterization of extrinsic innervation of the guinea pig rectum

Abstract

The presence of markers for parasympathetic, sympathetic, and glutamatergic or peptidergic sensory innervation was investigated by using in vitro tracing with biotinamide, combined with immunohistochemistry, to characterise quantitatively extrinsic axons to myenteric ganglia of the guinea pig rectum. Of biotinamide-filled varicose axons, 3.6 +/- 1.3% were immunoreactive for tyrosine hydroxylase (TH) and 16.0 +/- 4.8% for vesicular acetylcholine transporter (VAChT). TH and vesicular monoamine transporter (VMAT1) showed high coexistence (83-100%), indicating that varicosities lacking TH immunoreactivity also lacked VMAT1. VAChT was detectable in 77% of choline acetyltransferase (ChAT)-immunoreactive varicosities. Calcitonin gene-related peptide (CGRP) was detected in 5.3 +/- 1.6% of biotinamide-labeled varicosities, the vesicular glutamate transporter (VGluT) 1 in 2.8 +/- 0.8%, and VGluT2 in 11.3 +/- 4.2% of varicosities of extrinsic origin. Varicosities from the same axon showed consistent immunoreactivity. A novel type of nerve ending was identified, with branching, flattened lamellar endings, similar to the intraganglionic laminar endings (IGLEs) of the proximal gut. Rectal IGLEs were frequently immunoreactive for VGluT1 and VGluT2. Thus most varicose axons of extrinsic origin, which innervate rectal myenteric ganglia, lack detectable levels of immunoreactivity for TH, VMAT1, VAChT, ChAT, VGluT1/2, or CGRP, under conditions in which these markers are readily detectable in other axons. Although some unlabeled varicosities may belong to afferent axons that lack detectable CGRP or VGluT1/2 in the periphery, this suggests that a large proportion of axons do not release any of the major autonomic or sensory transmitters. We speculate that this may vary under particular circumstances, for example, inflammation or obstruction of the gut.

Sensory innervation of the guinea pig colon and rectum compared using retrograde tracing and immunohistochemistry

“…For example, a large population of low threshold mechanoreceptors innervates the rectum 4 ; these are much sparser in the colonic/splanchnic pathway 14 . In contrast, splanchnic afferents innervating the colon are largely medium-to-high threshold mechanonociceptors 18 which are not as abundant in pelvic pathways 19 .…”

“…In the guinea pig, anterograde tracing of lumbar colonic and rectal nerves has been used to characterise autonomic and sensory neurons to the distal colon and rectum 14,15 . Surprisingly, in both distal colon and rectum, a substantial proportion of extrinsic axons lacked any of the common markers (calcitonin gene-related peptide, vesicular acetylcholine transporter, tyrosine hydroxylase, vasoactive intestinal polypeptide, nitric oxide synthase, somatostatin, and vesicular glutamate transporters 1 and 2) that have previously been used to define autonomic and extrinsic sensory neurons 14,15 .…”

“…Surprisingly, in both distal colon and rectum, a substantial proportion of extrinsic axons lacked any of the common markers (calcitonin gene-related peptide, vesicular acetylcholine transporter, tyrosine hydroxylase, vasoactive intestinal polypeptide, nitric oxide synthase, somatostatin, and vesicular glutamate transporters 1 and 2) that have previously been used to define autonomic and extrinsic sensory neurons 14,15 . Retrograde tracing from the distal colon and rectum was used to analyse sympathetic and parasympathetic efferent pathways and the presence of key markers (tyrosine hydroxylase, nitric oxide synthase and choline acetyltransferase) in their nerve cell bodies 16 .…”

Extrinsic primary afferent signalling in the gut

“…In the guinea pig, these rIGLEs are abundant in the rectum but are increasingly sparse further up the distal colon. 41 Each afferent has endings in multiple enteric ganglia. Electrophysiologically, they are typically silent at rest, but are powerfully activated by distension, with instantaneous firing frequencies up to 50 Hz.…”

“…Dye fills of extrinsic nerve trunks to the colon and internal anal sphincter label arrays of axons within circular and/or longitudinal muscle layers, particularly in the rectum. 41 In mutant mice that lack enteric ganglia in the distal bowel, stretch-sensitive mechanoreceptors that transduce mechanical stimuli from intramuscular endings are present. 74,75 In the smooth muscle of the internal anal sphincter, sacral afferents form arrays of intramuscular endings that are sensitive to both distension and to light von Frey hairs.…”