Identification of Different Types of Spinal Afferent Nerve Endings That Encode Noxious and Innocuous Stimuli in the Large Intestine Using a Novel Anterograde Tracing Technique

Abstract

In mammals, sensory stimuli in visceral organs, including those that underlie pain perception, are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRG). One of the major challenges in visceral organs has been how to identify the different types of nerve endings of spinal afferents that transduce sensory stimuli into action potentials. The reason why spinal afferent nerve endings have been so challenging to identify is because no techniques have been available, until now, that can selectively label only spinal afferents, in high resolution. We have utilized an anterograde tracing technique, recently developed in our laboratory, which facilitates selective labeling of only spinal afferent axons and their nerve endings in visceral organs. Mice were anesthetized, lumbosacral DRGs surgically exposed, then injected with dextran-amine. Seven days post-surgery, the large intestine was removed. The characteristics of thirteen types of spinal afferent nerve endings were identified in detail. The greatest proportion of nerve endings was in submucosa (32%), circular muscle (25%) and myenteric ganglia (22%). Two morphologically distinct classes innervated myenteric ganglia. These were most commonly a novel class of intraganglionic varicose endings (IGVEs) and occasionally rectal intraganglionic laminar endings (rIGLEs). Three distinct classes of varicose nerve endings were found to innervate the submucosa and circular muscle, while one class innervated internodal strands, blood vessels, crypts of lieberkuhn, the mucosa and the longitudinal muscle. Distinct populations of sensory endings were CGRP-positive. We present the first complete characterization of the different types of spinal afferent nerve endings in a mammalian visceral organ. The findings reveal an unexpectedly complex array of different types of primary afferent endings that innervate specific layers of the large intestine. Some of the novel classes of nerve endings identified must underlie the transduction of noxious and/or innocuous stimuli from the large intestine.

Identification of different functional types of spinal afferent neurons innervating the mouse large intestine using a novel CGRPα transgenic reporter mouse

“…They fire to small amounts of stretch and continue to encode without saturating to distensions into the noxious range (64). Consistent with HF afferents, rectal IGLEs are present in the murine colorectum (55). Furthermore, rectal IGLEs lack CGRP, and their somata are in the lumbosacral DRG.…”

“…Similar strategies have been employed for mouse colorectal afferents (53,54). However, the high density of afferents in the colorectum, which have been shown to have numerous sites of innervation in the colon, is a significant barrier to this approach in this species (55). Functional intracellular electrophysiology performed in vitro with a target organ attached is an alternative that has been used successfully in cutaneous afferent studies (46,49) and, more recently, proven using the gut (2,35).…”

“…Furthermore, rectal IGLEs lack CGRP, and their somata are in the lumbosacral DRG. Indeed, rectal IGLEs are among the few afferent ending classes that arise from L 6 that lack CGRP (55). Thus, rectal IGLEs should be considered a candidate peripheral ending for HF afferents.…”

Optogenetic activation of mechanically insensitive afferents in mouse colorectum reveals chemosensitivity

“…In contrast, all four MSA classes respond to blunt probing of their RF; in addition, muscular and muscular-mucosal afferents also respond to circumferential stretch and mucosal and muscularmucosal afferents also respond to mucosal stroking. The terms "mucosal" and "muscular" are suggestive of histological localization of RFs in the colorectum, for which histological support exists (36). However, they have not yet been extensively validated by studies that couple functional characterization with afferent ending localization.…”

“…Colorectal afferents that respond only to punctate probing of their RF were previously denoted "serosal" afferents, suggestive of a RF in the serosa. However, a recent morphological study of the mouse colorec-tum failed to identify sensory endings in the colorectal serosa (36), prompting the use of "probing only" to represent this class of afferent here.…”