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.

Disengaging spinal afferent nerve communication with the brain in live mice.

Our understanding of how abdominal organs (like the gut) communicate with the brain, via sensory nerves, has been limited by a lack of techniques to selectively activate or inhibit populations of spinal primary afferent neurons within dorsal root ganglia (DRG), of live animals. We report a survival surgery technique in mice, where select DRG are surgically removed (unilaterally or bilaterally), without interfering with other sensory or motor nerves. Using this approach, pain responses evoked by rectal distension were abolished by bilateral lumbosacral L5-S1 DRG removal, but not thoracolumbar T13-L1 DRG removal. However, animals lacking T13-L1 or L5-S1 DRG both showed reduced pain sensitivity to distal colonic distension. Removal of DRG led to selective loss of peripheral CGRP-expressing spinal afferent axons innervating visceral organs, arising from discrete spinal segments. This method thus allows spinal segment-specific determination of sensory pathway functions in conscious, free-to-move animals, without genetic modification.

Quantification of CGRP-immunoreactive myenteric neurons in mouse colon.

Quantitative data of biological systems provide valuable baseline information for understanding pathology, experimental perturbations, and computational modeling. In mouse colon, calcitonin gene-related peptide (CGRP) is expressed by myenteric neurons with multiaxonal (Dogiel type II) morphology, characteristic of intrinsic primary afferent neurons (IPANs). Analogous neurons in other species and gut regions represent 5-35% of myenteric neurons. We aimed to quantify proportions of CGRP-immunopositive (CGRP+) myenteric neurons. Colchicine-treated wholemount preparations of proximal, mid, and distal colon were labeled for HuC/D, CGRP, nitric oxide synthase (NOS), and peripherin (Per). The pan-neuronal markers (Hu+/Per+) co-labeled 94% of neurons. Hu+/Per- neurons comprised ∼6%, but Hu-/Per+ cells were rare. Thus, quantification was based on Hu+ myenteric neurons (8576 total; 1225 ± 239 per animal, n = 7). CGRP+ cell bodies were significantly larger than the average of all Hu+ neurons (329 ± 13 vs. 261 ± 12 µm2 , p < .0001). CGRP+ neurons comprised 19% ± 3% of myenteric neurons without significant regional variation. NOS+ neurons comprised 42% ± 2% of myenteric neurons overall, representing a lower proportion in proximal colon, compared to mid and distal colon (38% ± 2%, 44% ± 2%, and 44% ± 3%, respectively). Peripherin immunolabeling revealed cell body and axonal morphology in some myenteric neurons. Whether all CGRP+ neurons were multiaxonal could not be addressed using peripherin immunolabeling. However, of 118 putatively multiaxonal neurons first identified based on peripherin immunoreactivity, all were CGRP+ (n = 4). In conclusion, CGRP+ myenteric neurons in mouse colon were comprehensively quantified, occurring within a range expected of a putative IPAN marker. All Per+ multiaxonal neurons, characteristic of Dogiel type II/IPAN morphology, were CGRP+.

The gut-brain axis: spatial relationship between spinal afferent nerves and 5-HT-containing enterochromaffin cells in mucosa of mouse colon.

Cross talk between the gastrointestinal tract and brain is of significant relevance for human health and disease. However, our understanding of how the gut and brain communicate has been limited by a lack of techniques to identify the precise spatial relationship between extrinsic nerve endings and their proximity to specific cell types that line the inner surface of the gastrointestinal tract. We used an in vivo anterograde tracing technique, previously developed in our laboratory, to selectively label single spinal afferent axons and their nerve endings in mouse colonic mucosa. The closest three-dimensional distances between spinal afferent nerve endings and axonal varicosities to enterochromaffin (EC) cells, which contain serotonin (5-hydroxytryptamine; 5-HT), were then measured. The mean distances (± standard deviation) between any varicosity along a spinal afferent axon or its nerve ending, and the nearest EC cell, were 5.7 ± 6.0 µm (median: 3.6 µm) and 26.9 ± 18.6 µm (median: 24.1 µm), respectively. Randomization of the spatial location of EC cells revealed similar results to this actual data. These distances are ∼200-1,000 times greater than those between pre- and postsynaptic membranes (15-25 nm) that underlie synaptic transmission in the vertebrate nervous system. Our findings suggest that colonic 5-HT-containing EC cells release substances to activate centrally projecting spinal afferent nerves likely via diffusion, as such signaling is unlikely to occur with the spatial fidelity of a synapse.NEW & NOTEWORTHY We show an absence of close physical contact between spinal afferent nerves and 5-HT-containing EC cells in mouse colonic mucosa. Similar relative distances were observed between randomized EC cells and spinal afferents compared with actual data. This spatial relationship suggests that substances released from colonic 5-HT-containing EC cells are unlikely to act via synaptic transmission to neighboring spinal afferents that relay sensory information from the gut lumen to the brain.

Identification of a novel distension-evoked motility pattern in the mouse uterus.

The dynamic changes in uterine contractility in response to distension are incompletely understood. Rhythmic, propagating contractions of nonpregnant uterine smooth muscle occur in the absence of nerve activity (i.e., myogenic), events that decline during pregnancy and reemerge at parturition. We therefore sought to determine how myogenic contractions of the nonpregnant uterus are affected by distension, which might provide mechanistic clues underlying distension-associated uterine conditions such as preterm birth. Uteri isolated from nulliparous adult female mice in proestrus were video imaged to generate spatiotemporal maps, and myoelectrical activity simultaneously recorded using extracellular suction electrodes. Motility patterns were examined under basal conditions and following ramped intraluminal distension with fluid to 5 and 10 cmH2O. Intraluminal distension caused pressure-dependent changes in the frequency, amplitude, propagation speed, and directionality of uterine contractions, which reversed upon pressure release. Altered burst durations of underlying smooth muscle myoelectric events were concurrently observed, although action potential spike intervals were unchanged. Voltage-gated sodium channel blockade [tetrodotoxin (TTX); 0.6 µM] attenuated both the amplitude of contractions and burst duration of action potentials, whereas all activity was abolished by L-type calcium channel blockade (nifedipine; 1 µM). These data suggest that myogenic motility patterns of the nonpregnant mouse uterus are sensitive to changes in intraluminal pressure and, at high pressures, may be modulated by voltage-gated sodium channel activity. Future studies may investigate whether similar distension-evoked changes occur in the pregnant uterus and the possible pathophysiological role of such activity in the development of preterm birth.

Morphological identification of thoracolumbar spinal afferent nerve endings in mouse uterus.

Major sensory innervation to the uterus is provided by spinal afferent nerves, whose cell bodies lie predominantly in thoracolumbar dorsal root ganglia (DRG). While the origin of the cell bodies of uterine spinal afferents is clear, the identity of their sensory endings has remained unknown. Hence, our major aim was to identify the location, morphology, and calcitonin gene-related peptide (CGRP)-immunoreactivity of uterine spinal afferent endings supplied by thoracolumbar DRG. We also sought to determine the degree of uterine afferent innervation provided by the vagus nerve. Using an anterograde tracing technique, nulliparous female C57BL/6 mice were injected unilaterally with biotinylated dextran into thoracolumbar DRG (T13-L3). After 7-9 days, uterine horns were stained to visualize traced nerve axons and endings immunoreactive to CGRP. Whole uteri from a separate cohort of animals were injected with retrograde neuronal tracer (DiI) and dye uptake in nodose ganglia was examined. Anterogradely labeled axons innervated each uterine horn, these projected rostrally or caudally from their site of entry, branching to form varicose endings in the myometrium and/or vascular plexus. Most spinal afferent endings were CGRP-immunoreactive and morphologically classified as "simple-type." Rarely, uterine nerve cell bodies were labeled in nodose ganglia. Here, we provide the first detailed description of spinal afferent nerve endings in the uterus of a vertebrate. Distinct morphological types of spinal afferent nerve endings were identified throughout multiple anatomical layers of the uterine wall. Compared to other visceral organs, uterine spinal afferent endings displayed noticeably less morphological diversity. Few neurons in nodose ganglia innervate the uterus.

Sensory nerve endings arising from single spinal afferent neurons that innervate both circular muscle and myenteric ganglia in mouse colon: colon-brain axis.

There is considerable interest in understanding how contents within the gut wall (including microbiome) can activate sensory nerve endings in the gut that project to the central nervous system. However, we have only recently begun to understand the location and characteristics of extrinsic spinal afferent nerve endings that innervate the lower gastrointestinal (GI) tract. Our aim is to identify the nerve endings in the mouse distal colon that arise from single spinal afferent neurons. C57BL/6 mice were anaesthetised and single dorsal root ganglia (DRG) between lumbosacral L6-S1 were injected with dextran biotin. Mice recovered for 7 days. Animals were then euthanized and whole colons removed, fixed and stained for calcitonin-gene-related-peptide (CGRP). Single spinal afferent nerve axons were identified entering the distal colon that ramified along many rows of myenteric ganglia, often giving rise to varicose nerve endings. These same axons bifurcated in the circular muscle giving rise to 4-5 groups of branching-type intramuscular endings, where each group of endings was separated by ~ 370 µm in the rostro-caudal axis and projected 1.2 mm around the circumference. As spinal afferent axons bifurcated, their axons often showed dramatic reductions in diameter. Here, we identified in the distal colon, the characteristics of nerve endings that arise from single colorectal-projecting axons with cell bodies in DRG. These findings suggest that a population of sensory neurons in DRG can potentially detect sensory stimuli simultaneously via different morphological types of endings that lie in both colonic smooth muscle and myenteric ganglia.

Identification of spinal afferent nerve endings in the colonic mucosa and submucosa that communicate directly with the spinal cord: The gut-brain axis.

The major sensory nerve pathway between the colon and central nervous system (spinal cord and brain) that underlies the gut-brain axis, is via spinal afferent neurons, with cell bodies in dorsal root ganglia (DRG). Our aim was to identify the sensory nerve endings in the colon that arise from single colorectal-projecting DRG neurons. C57BL/6 mice were anesthetized and lumbosacral L6-S1 DRG injected with dextran biotin. Mice recovered for 7 days. The whole colon was then removed and stained to visualize single axons and nerve endings immunoreactive to calcitonin gene-related peptide (CGRP). Single axons arising from DRG were identified in the distal colon and their morphological features and CGRP immunoreactivity characterized. After entering the colon, single axons ramified rostrally or caudally along many rows of myenteric ganglia with little circumferential displacement, giving off varicose endings in multiple ganglia. Nerve endings arising from two classes of colorectal-projecting DRG neuron were identified. One class was peptidergic neurons that had nerve endings in circular muscle, myenteric ganglia, and submucosa. Another class of nonpeptidergic neurons innervated mucosal crypts, myenteric ganglia, and submucosa. Different morphological types of nerve endings which innervate different anatomical layers of colon can arise from the same axon and sensory neuron in DRG. These findings suggest single peptidergic and nonpeptidergic sensory neurons in DRG are potentially capable of detecting sensory stimuli from different anatomical layers of the colon, via different types of nerve endings.

Spinal Glial Adaptations Occur in a Minimally Invasive Mouse Model of Endometriosis: Potential Implications for Lesion Etiology and Persistent Pelvic Pain.

Glial adaptations within the central nervous system are well known to modulate central sensitization and pain. Recently, it has been suggested that activity of glial-related proinflammatory cytokines may potentiate peripheral inflammation, via central neurogenic processes. However, a role for altered glial function has not yet been investigated in the context of endometriosis, a chronic inflammatory condition in women associated with peripheral lesions, often manifesting with persistent pelvic pain. Using a minimally invasive mouse model of endometriosis, we investigated associations between peripheral endometriosis-like lesions and adaptations in central glial reactivity. Spinal cords (T13-S1) from female C57BL/6 mice with endometriosis-like lesions (ENDO) were imaged via fluorescent immunohistochemistry for the expression of glial fibrillary acidic protein (GFAP; astrocytes) and CD11b (microglia) in the dorsal horn (n = 5). Heightened variability ( P = .02) as well as an overall increase ( P = .04) in the mean area of GFAP immunoreactivity was found in ENDO versus saline-injected control animals. Interestingly, spinal levels showing the greatest alterations in GFAP immunoreactivity appeared to correlate with the spatial location of lesions within the abdominopelvic cavity. A subtle but significant increase in the mean area of CD11b immunostaining was also observed in ENDO mice compared to controls ( P = .02). This is the first study to describe adaptations in nonneuronal, immune-like cells of the central nervous system attributed to the presence of endometriosis-like lesions.

Lesion development is modulated by the natural estrous cycle and mouse strain in a minimally invasive model of endometriosis.

Many rodent models of endometriosis are invasive, involving surgery to implant donor endometrial tissue into recipient animals. Moreover, few studies have compared and contrasted lesions between rodent strains and estrous stages without exogenous hormone manipulation. This is despite extensive data demonstrating that genetic and hormonal factors can influence endometriosis progression. Here, we have refined a minimally invasive model of endometriosis using naturally cycling mice (donor and recipient matched for cycle phase) to investigate lesion development in two different strains (C57BL/6 and BALB/c), induced in estrous stages of high and low estrogen (proestrus or estrus, respectively), and with varying amounts of donor endometrial tissue (7.5-40 mg), injected intraperitoneally. The overall probability of developing endometriosis-like lesions was higher in proestrus than estrus, and increased with greater masses of donor tissue. Similarly, the total number of lesions (0-3) increased from 7.5 to 40 mg, and was significantly greater in proestrus C57BL/6 mice but not BALB/cs. The dominant lesion type also differed between mouse strains; C57BL/6 mice were more likely to develop dense-type lesions, whereas BALB/c mice developed a greater proportion of cystic type. These data further support a role for estrogen in the development of endometriosis, and that genetic variance can influence the degree and characteristics of lesions. Our minimally invasive model would be beneficial for studies with outcome measurements particularly sensitive to incisional injury, such as pain, or alterations to sex hormones, including fertility.

A composite fibre optic catheter for monitoring peristaltic transit of an intra-luminal bead.

A fibre optic motion sensor has been developed for monitoring the proximity and direction of motion of a ferrous bead travelling axial to the sensor. By integrating an array of these sensors into our previously developed fibre optic manometry catheters we demonstrate simultaneous detection of peristaltic muscular activity and the associated motion of ferrous beads through a colonic lumen. This allows the motion of solid content to be temporally and spatially related to pressure variations generated by peristaltic contractions without resorting to videoflouroscopy to track the motion of a radio opaque bolus. The composite catheter has been tested in an in-vitro animal preparation consisting of excised sections of rabbit colon. Cut-away image of the fibre optic motion sensor showing the location of the fibre Bragg gratings and the rare earth magnet.

Uterine Contractility in the Nonpregnant Mouse: Changes During the Estrous Cycle and Effects of Chloride Channel Blockade.

Mechanisms involved in the generation of spontaneous uterine contractions are not fully understood. Kit-expressing interstitial cells of Cajal are pacemakers of contractile rhythm in other visceral organs, and recent studies describe a role for Ca(2+)-activated Cl(-) currents as the initiating conductance in these cells. The existence and role of similar specialized pacemaker cells in the nonpregnant uterus remains undetermined. Spontaneous contractility patterns were characterized throughout the estrous cycle in isolated, nonpregnant mouse uteri using spatiotemporal mapping and tension recordings. During proestrus, estrus, and diestrus, contraction origin predominated in the oviduct end of the uterus, suggesting the existence of a dominant pacemaker site. Propagation speed of contractions during estrus and diestrus were significantly slower than in proestrus and metestrus. Five major patterns of activity were predominantly exhibited in particular stages: quiescent (diestrus), high-frequency phasic (proestrus), low-frequency phasic (estrus), multivariant (metestrus), and complex. Kit-immunopositive cells reminiscent of pacemaking ICCs were not consistently observed within the uterus. Niflumic acid (10 µM), anthracene-9-carboxylic acid (0.1-1 mM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (10 µM) each reduced the frequency of spontaneous contractions, suggesting involvement of Cl(-) channels in generating spontaneous uterine motor activity. It is unlikely that this conductance is generated by the Ca(2+)-activated Cl(-) channels, anoctamin-1 and CLCA4, as immunohistochemical labeling did not reveal protein expression within muscle or pacemaker cell networks. In summary, these results suggest that spontaneous uterine contractions may be generated by a Kit-negative pacemaker cell type or uterine myocytes, likely involving the activity of a yet-unidentified Cl(-) channel.

Neurally mediated propagating discrete clustered contractions superimposed on myogenic ripples in ex vivo segments of human ileum.

Narrow muscle strips have been extensively used to study intestinal contractility. Larger specimens from laboratory animals have provided detailed understanding of mechanisms that underlie patterned intestinal motility. Despite progress in animal tissue, investigations of motor patterns in large, intact specimens of human gut ex vivo have been sparse. In this study, we tested whether neurally dependent motor patterns could be detected in isolated specimens of intact human ileum. Specimens (n = 14; 7-30 cm long) of terminal ileum were obtained with prior informed consent from patients undergoing colonic surgery for removal of carcinomas. Preparations were set up in an organ bath with an array of force transducers, a fiberoptic manometry catheter, and a video camera. Spontaneous and distension-evoked motor activity was recorded, and the effects of lidocaine, which inhibits neural activity, were studied. Myogenic contractions (ripples) occurred in all preparations (6.17 ± 0.36/min). They were of low amplitude and formed complex patterns by colliding and propagating in both directions along the specimen at anterograde velocities of 4.1 ± 0.3 mm/s and retrogradely at 4.9 ± 0.6 mm/s. In five specimens, larger amplitude clusters of contractions were seen (discrete clustered contractions), which propagated aborally at 1.05 ± 0.13 mm/s and orally at 1.07 ± 0.09 mm/s. These consisted of two to eight phasic contractions that aligned with ripples. These motor patterns were abolished by addition of lidocaine (0.3 mM). The ripples continued unchanged in the presence of this neural blocking agent. These results demonstrate that both myogenic and neurogenic motor patterns can be studied in isolated specimens of human small intestine.

Characterization of primary afferent spinal innervation of mouse uterus.

The primary afferent innervation of the uterus is incompletely understood. The aim of this study was to identify the location and characteristics of primary afferent neurons that innervate the uterine horn of mice and correlate the different morphological types of putative primary afferent nerve endings, immunoreactive to the sensory marker, calcitonin gene related peptide (CGRP). Using retrograde tracing, injection of 5-10 µL of 1,1'-didodecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI) into discrete single sites in each uterine horn revealed a biomodal distribution of sensory neurons in dorsal root ganglia (DRG) with peak labeling occurring between T13-L3 and a second smaller peak between L6-S1. The mean cross sectional area of labeled cells was 463 µm(2) ± s.e.m. A significantly greater proportion of labeled neurons consisted of small cell bodies (<300 µm(2)) in the sacral spinal cord (S2) compared with peak labeling at the lumbar (L2) region. In both sections and whole mount preparations, immunohistochemical staining for CGRP revealed substantial innervation of the uterus by CGRP-positive nerve fibers located primarily at the border between the circular and longitudinal muscle layers (N = 4). The nerve endings were classified into three distinct types: "single," "branching," or "complex," that often aligned preferentially in either the circular or longitudinal axis of the smooth muscles. Complex endings were often associated with mesenteric vessels. We have identified that the cell bodies of primary afferent neurons innervating the mouse uterus lie primarily in DRG at L2 and S1 spinal levels. Also, the greatest density of CGRP immunoreactivity lies within the myometrium, with at least three different morphological types of nerve endings identified. These findings will facilitate further investigations into the mechanisms underlying sensory transduction in mouse uterus.