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

Abstract

Hibberd TJ, Kestell GR, Kyloh MA, Brookes SJ, Wattchow DA, Spencer NJ. Identification of different functional types of spinal afferent neurons innervating the mouse large intestine using a novel CGRP␣ transgenic reporter mouse. Am J Physiol Gastrointest Liver Physiol 310: G561-G573, 2016. First published January 28, 2016 doi:10.1152/ajpgi.00462.2015.-Spinal afferent neurons detect noxious and physiological stimuli in visceral organs. Five functional classes of afferent terminals have been extensively characterized in the colorectum, primarily from axonal recordings. Little is known about the corresponding somata of these classes of afferents, including their morphology, neurochemistry, and electrophysiology. To address this, we made intracellular recordings from somata in L 6/S1 dorsal root ganglia and applied intraluminal colonic distensions. A transgenic calcitonin gene-related peptide-␣ (CGRP␣)-mCherry reporter mouse, which enabled rapid identification of soma neurochemistry and morphology following electrophysiological recordings, was developed. Three distinct classes of low-threshold distension-sensitive colorectal afferent neurons were characterized; an additional group was distension-insensitive. Two of three low-threshold classes expressed CGRP␣. One class expressing CGRP␣ discharged phasically, with inflections on the rising phase of their action potentials, at low frequencies, to both physiological (Ͻ30 mmHg) and noxious (Ͼ30 mmHg) distensions. The second class expressed CGRP␣ and discharged tonically, with smooth, briefer action potentials and significantly greater distension sensitivity than phasically firing neurons. A third class that lacked CGRP␣ generated the highest-frequency firing to distension and had smaller somata. Thus, CGRP␣ expression in colorectal afferents was associated with lower distension sensitivity and firing rates and larger somata, while colorectal afferents that generated the highest firing frequencies to distension had the smallest somata and lacked CGRP␣. These data fill significant gaps in our understanding of the different classes of colorectal afferent somata that give rise to distinct functional classes of colorectal afferents. In healthy mice, the majority of sensory neurons that respond to colorectal distension are low-threshold, wide-dynamic-range afferents, encoding both physiological and noxious ranges. colon; afferent; nociceptor; pain; sensory neuron IN MAMMALS, SPINAL AFFERENT neurons, with somata in dorsal root ganglia (DRG), encode noxious and physiological sensory stimuli in visceral organs. Most recordings from spinal afferents have been obtained via extracellular recording of nerve trunks containing a mix of different types of efferent and afferent nerve axons (12,27,33). In the large intestine of mice, extracellular recordings have been used to generate a classification scheme consisting of approximately five different functional types of colorectal afferents (5, 16). Numerous studies also report characteristics of colorectal-projecting spinal afferent som...

Visceral pain – Novel approaches for optogenetic control of spinal afferents

“…Recently, we generated a transgenic mouse in which the fluorescent reporter mCherry was knocked into the CGRPα locus (Hibberd et al, 2016). This had also been performed using different reporter genes by earlier investigators (McCoy et al, 2012).…”

“…CGRPα is highly expressed in DRG neurons, making it particularly a useful marker (Mulderry et al, 1988). This mouse has proved very useful for the visualization of peptidergic spinal afferent neurons in live DRG and visceral organs (Hibberd et al, 2016). However, the morphological resolution of CGRPα expressing axons was low in visceral organs and precise morphological features of spinal afferents could not be determined (Spencer et al, 2016b).…”

“…However, the morphological resolution of CGRPα expressing axons was low in visceral organs and precise morphological features of spinal afferents could not be determined (Spencer et al, 2016b). Nevertheless, the reporter (mCherry) signal enabled recordings from individual varicosities of spinal afferent axon terminals within live visceral organs, such as the gut wall) (Spencer et al, 2016b), and was also useful in reporting the functional properties of the peptidergic and non-peptidergic sensory nerve cell bodies in live tissue (Hibberd et al, 2016).…”

“…This contrasts with light delivery in peripheral visceral organs, where multiple functional classes of axons can express the same genes and may cause inadvertent coactivation. The gut wall for example, contains both spinal afferent nerve endings (Mulderry et al, 1988, Hibberd et al, 2016 and enteric neurons that express CGRP (Furness et al, 2004).…”

“…Therefore, photostimulation of the gut wall could indiscriminately activate spinal afferents and enteric nerves (Mulderry et al, 1988, Hibberd et al, 2016. Also, delivering light to any periphery organ would require illumination of a larger spatial field compared with individual DRGs, which in mice are approximately 1mm in diameter.…”