Synaptic cell adhesion molecule Cdh6 identifies a class of sensory neurons with novel functions in colonic motility.

Intrinsic sensory neurons are an essential part of the enteric nervous system (ENS) and play a crucial role in gastrointestinal tract motility and digestion. Neuronal subtypes in the ENS have been distinguished by their electrophysiological properties, morphology, and expression of characteristic markers, notably neurotransmitters and neuropeptides. Here we investigated synaptic cell adhesion molecules as novel cell type markers in the ENS. Our work identifies two Type II classic cadherins, Cdh6 and Cdh8, specific to sensory neurons in the mouse colon. We show that Cdh6+ neurons demonstrate all other distinguishing classifications of enteric sensory neurons including marker expression of Calcb and Nmu, Dogiel type II morphology and AH-type electrophysiology and I H current. Optogenetic activation of Cdh6+ sensory neurons in distal colon evokes retrograde colonic motor complexes (CMCs), while pharmacologic blockade of rhythmicity-associated current I H disrupts the spontaneous generation of CMCs. These findings provide the first demonstration of selective activation of a single neurochemical and functional class of enteric neurons, and demonstrate a functional and critical role for sensory neurons in the generation of CMCs.

Anatomical distribution of CGRP-containing lumbosacral spinal afferent neurons in the mouse uterine horn.

Sensory stimuli from the uterus are detected by spinal afferent neurons whose cell bodies arise from thoracolumbar and lumbosacral dorsal root ganglia (DRG). Using an in vivo survival surgical technique developed in our laboratory to remove select DRG from live mice, we recently quantified the topographical distribution of thoracolumbar spinal afferents innervating the mouse uterine horn, revealed by loss of immunoreactivity to calcitonin gene-related peptide (CGRP). Here, we used the same technique to investigate the distribution of lumbosacral uterine spinal afferents, in which L5-S1 DRG were unilaterally removed from adult female C57BL/6J mice (N = 6). Following 10-12 days recovery, CGRP immunoreactivity was quantified along the length of uterine horns using fluorescence immunohistochemistry. Relative to myometrial thickness, overall CGRP density in uterine tissues ipsilateral to L5-S1 DRG removal was reduced compared to the DRG-intact, contralateral side (P = 0.0265). Regionally, however, myometrial CGRP density was unchanged in the cranial, mid, and caudal portions. Similarly, CGRP-expressing nerve fiber counts, network lengths, junctions, and the proportion of area occupied by CGRP immunoreactivity were unaffected by DRG removal (P ≥ 0.2438). Retrograde neuronal tracing from the caudal uterine horn revealed fewer spinal afferents here arise from lumbosacral than thoracolumbar DRG (P = 0.0442) (N = 4). These data indicate that, unlike thoracolumbar DRG, lumbosacral spinal afferent nerves supply relatively modest sensory innervation across the mouse uterine horn, with no regional specificity. We conclude most sensory information between the mouse uterine horn and central nervous system is likely relayed via thoracolumbar spinal afferents.

Modification of Neurogenic Colonic Motor Behaviours by Chemogenetic Ablation of Calretinin Neurons.

How the enteric nervous system determines the pacing and propagation direction of neurogenic contractions along the colon remains largely unknown. We used a chemogenetic strategy to ablate enteric neurons expressing calretinin (CAL). Mice expressing human diphtheria toxin receptor (DTR) in CAL neurons were generated by crossing CAL-ires-Cre mice with Cre-dependent ROSA26-DTR mice. Immunohistochemical analysis revealed treatment with diphtheria toxin incurred a 42% reduction in counts of Hu-expressing colonic myenteric neurons (P = 0.036), and 57% loss of CAL neurons (comprising ∼25% of all Hu neurons; P = 0.004) compared to control. As proportions of Hu-expressing neurons, CAL neurons that contained nitric oxide synthase (NOS) were relatively spared (control: 15 ± 2%, CAL-DTR: 13 ± 1%; P = 0.145), while calretinin neurons lacking NOS were significantly reduced (control: 26 ± 2%, CAL-DTR: 18 ± 5%; P = 0.010). Colonic length and pellet sizes were significantly reduced without overt inflammation or changes in ganglionic density. Interestingly, colonic motor complexes (CMCs) persisted with increased frequency (mid-colon interval 111 ± 19 vs. 189 ± 24 s, CAL-DTR vs. control, respectively, P < 0.001), decreased contraction size (mid-colon AUC 26 ± 24 vs. 59 ± 13 gram/seconds, CAL-DTR vs. control, respectively, P < 0.001), and lacked preferential anterograde migration (P < 0.001). The functional effects of modest calretinin neuron ablation, particularly increased neurogenic motor activity frequencies, differ from models that incur general enteric neuron loss, and suggest calretinin neurons may contribute to pacing, force, and polarity of CMCs in the large bowel.

Distinct patterns of myogenic motor activity identified in isolated human distal colon with high-resolution manometry.

BACKGROUND: Colonic high-resolution manometry (HRM) has been used to reveal discrete, propagating colonic motor patterns. To help determine mechanisms underlying these patterns, we used HRM to record contractile activity in human distal colon ex vivo. METHODS: Surgically excised segments of descending (n = 30) or sigmoid colon (n = 4) were immersed in oxygenated Krebs solution at 36°C (n = 34; 16 female; 67.6 ± 12.4 years; length: 24.7 ± 3.5 cm). Contractility was recorded by HRM catheters. After 30 minutes of baseline recording, 0.3 mM lidocaine and/or 1 mM hexamethonium were applied. Ascending neural pathways were activated by electrical field stimulation (EFS; 10 Hz, 0.5 ms, 50 V, 5-s duration) applied to the anal end before and after drug application. RESULTS: Spontaneous propagating contractions were recorded in all specimens (0.1-1.5 cycles/minute). Most contractions occurred synchronously across all recording sites. In five specimens, rhythmic antegrade contractions propagated across the full length of the preparation. EFS evoked local contractions at the site of stimulation (latency: 5.5 ± 2.4 seconds) with greater amplitude than spontaneous contractions (EFS; 29.3 ± 26.9 vs 12.1 ± 14.8 mm Hg; P = .02). Synchronous or retrograde propagating motor patterns followed EFS; 71% spanned the entire preparation length. Hexamethonium and lidocaine modestly and only temporarily inhibited spontaneous contractions, whereas TTX increased the frequency of contractile activity while inhibiting EFS-evoked contractions. CONCLUSIONS AND INFERENCES: Our study suggests that the propagated contractions recorded in the organ bath have a myogenic origin which can be regulated by neural input. Once activated at a local site, the contractions do not require the propulsion of fecal content to sustain long-distance propagation.

Visceral pain - Novel approaches for optogenetic control of spinal afferents.

Painful stimuli arising within visceral organs are detected by peripheral nerve endings of spinal afferents, whose cell bodies are located in dorsal root ganglia (DRG). Recent technical advances have made it possible to reliably expose and inject single DRG with neuronal tracers or viruses in vivo. This has facilitated, for the first time, unequivocal identification of different types of spinal afferent endings in visceral organs. These technical advances paved the way for a very exciting series of in vivo experiments where individual DRG are injected to facilitate opsin expression (e.g. Archaerhodopsin). Organ-specific expression of opsins in sensory neurons may be achieved by retrograde viral transduction. This means activity of target-specific populations of sensory neurons, within single DRG, can be modulated by optogenetic photo-stimulation. Using this approach we implanted micro light-emitting diodes (micro-LEDs) adjacent to DRG of interest, thereby allowing focal DRG-specific control of visceral and/or somatic afferents in conscious mice. This is vastly different from broad photo-illumination of peripheral nerve endings, which are dispersed over much larger surface areas across an entire visceral organ; and embedded deep within multiple anatomical layers. Focal DRG photo-stimulation also avoids the potential that wide-field illumination of the periphery could inadvertently activate other closely apposed organs, or co-activate different classes of axons in the same organ (e.g. enteric and spinal afferent endings in the gut). It is now possible to selectively control nociceptive and/or non-nociceptive pathways to specific visceral organs in vivo, using wireless optogenetics and micro-LEDs implanted adjacent to DRG, for targeted photo-stimulation.

Identifying unique subtypes of spinal afferent nerve endings within the urinary bladder of mice.

Spinal afferent neurons are responsible for the transduction and transmission of noxious (painful) stimuli and innocuous stimuli that do not reach conscious sensations from visceral organs to the central nervous system. Although the location of the nerve cell bodies of spinal afferents is well known to reside in dorsal root ganglia (DRG), the morphology and location of peripheral nerve endings of spinal afferents that transduce sensory stimuli into action potentials is poorly understood. The individual nerve endings of spinal afferents that innervate the urinary bladder have never been unequivocally identified in any species. We used an anterograde tracing technique developed in our laboratory to selectively label only spinal afferents. Mice were anesthetized and unilateral injections of dextran-amine made into lumbosacral DRGs (L5-S2). Seven to nine days postsurgery, mice were euthanized, the urinary bladder removed, then fresh-fixed and stained for immunoreactivity to calcitonin-gene-related-peptide (CGRP). Four distinct morphological types of spinal afferent ending in the bladder were identified. Three types existed in the detrusor muscle and one major type in the sub-urothelium and urothelium. Most nerve endings were located in detrusor muscle where the three types could be identified as having: "branching", "simple", or "complex" morphology. The majority of spinal afferent nerve endings were CGRP-immunoreactive. Single spinal afferent axons bifurcated many times upon entering the bladder and developed varicosities along their axon terminal endings. We present the first morphological identification of spinal afferent nerve endings in the mammalian urinary bladder.