The bladder wall is innervated by a complex network of afferent nerves that detect bladder stretch during filling. Sensory signals, generated in response to distension, are relayed to the spinal cord and brain to evoke physiological and painful sensations and regulate urine storage and voiding. Hyperexcitability of these sensory pathways is a key component in the development of chronic bladder hypersensitivity disorders including interstitial cystitis/bladder pain syndrome and overactive bladder syndrome. Despite this, the full array of ion channels that regulate bladder afferent responses to mechanical stimuli have yet to be determined. Here, we investigated the role of low-voltage-activated T-type calcium (Ca V 3) channels in regulating bladder afferent responses to distension. Using single-cell reverse-transcription polymerase chain reaction and immunofluorescence, we revealed ubiquitous expression of Ca V 3.2, but not Ca V 3.1 or Ca V 3.3, in individual bladder-innervating dorsal root ganglia neurons. Pharmacological inhibition of Ca V 3.2 with TTA-A2 and ABT-639, selective blockers of T-type calcium channels, dose-dependently attenuated ex-vivo bladder afferent responses to distension in the absence of changes to muscle compliance. Further evaluation revealed that Ca V 3.2 blockers significantly inhibited both low- and high-threshold afferents, decreasing peak responses to distension, and delayed activation thresholds, thereby attenuating bladder afferent responses to both physiological and noxious distension. Nocifensive visceromotor responses to noxious bladder distension in vivo were also significantly reduced by inhibition of Ca V 3 with TTA-A2. Together, these data provide evidence of a major role for Ca V 3.2 in regulating bladder afferent responses to bladder distension and nociceptive signalling to the spinal cord.

The distal colon and rectum (colorectum) are innervated by spinal and vagal afferent pathways. The central circuits into which vagal and spinal afferents relay colorectal nociceptive information remain to be comparatively assessed. To address this, regional colorectal retrograde tracing and colorectal distension (CRD)-evoked neuronal activation were used to compare the circuits within the dorsal vagal complex (DVC) and dorsal horn (thoracolumbar [TL] and lumbosacral [LS] spinal levels) into which vagal and spinal colorectal afferents project. Vagal afferent projections were observed in the nucleus tractus solitarius (NTS), area postrema (AP), and dorsal motor nucleus of the vagus (DMV), labeled from the rostral colorectum. In the NTS, projections were opposed to catecholamine and pontine parabrachial nuclei (PbN)-projecting neurons. Spinal afferent projections were labeled from rostral through to caudal aspects of the colorectum. In the dorsal horn, the number of neurons activated by CRD was linked to pressure intensity, unlike in the DVC. In the NTS, 13% ± 0.6% of CRD-activated neurons projected to the PbN. In the dorsal horn, at the TL spinal level, afferent input was associated with PbN-projecting neurons in lamina I (LI), with 63% ± 3.15% of CRD-activated neurons in LI projecting to the PbN. On the other hand, at the LS spinal level, only 18% ± 0.6% of CRD-activated neurons in LI projected to the PbN. The collective data identify differences in the central neuroanatomy that support the disparate roles of vagal and spinal afferent signaling in the facilitation and modulation of colorectal nociceptive responses.

Visceral pain following infection or inflammation is a major clinical problem. Although we have knowledge of how peripheral endings of colonic afferents change in disease, their central projections have been overlooked. With neuroanatomical tracing and colorectal distension (CRD), we sought to identify colonic afferent central terminals (CACTs), the dorsal horn (DH) neurons activated by colonic stimuli in the thoracolumbar (T10-L1) DH, and determine how they are altered by postinflammatory chronic colonic mechanical hypersensitivity. Retrograde tracing from the colon identified CACTs in the DH, whereas immunohistochemistry for phosphorylated MAP kinase ERK 1/2 (pERK) identified DH neurons activated by CRD (80 mmHg). In healthy mice, CACTs were located primarily in DH laminae I (LI) and V (LV) and projected down middle and lateral DH collateral pathways. CRD evoked pERK immunoreactivity in DH neurons, the majority of which were located in LI and LV, the same regions as CACTs. In postinflammatory mice, CACTs were significantly increased in T12-L1 compared with healthy mice. Although CACTs remained abundant in LI, they were more widespread and were now present in deeper laminae. After CRD, significantly more DH neurons were pERK-IR postinflammation (T12-L1), with abundant expression in LI and deeper laminae. In both healthy and postinflammatory mice, many pERK neurons were in close apposition to CACTs, suggesting that colonic afferents can stimulate specific DH neurons in response to noxious CRD. Overall, we demonstrate that CACT density and the number of responsive DH neurons in the spinal cord increase postinflammation, which may facilitate aberrant central representation of colonic nociceptive signaling following chronic peripheral hypersensitivity.

Dietary, microbial, and inflammatory factors modulate the gut-brain axis and influence physiological processes ranging from metabolism to cognition. The gut epithelium is a principal site for detecting such agents, but precisely how it communicates with neural elements is poorly understood. Serotonergic enterochromaffin (EC) cells are proposed to fulfill this role by acting as chemosensors, but understanding how these rare and unique cell types transduce chemosensory information to the nervous system has been hampered by their paucity and inaccessibility to single-cell measurements. Here, we circumvent this limitation by exploiting cultured intestinal organoids together with single-cell measurements to elucidate intrinsic biophysical, pharmacological, and genetic properties of EC cells. We show that EC cells express specific chemosensory receptors, are electrically excitable, and modulate serotonin-sensitive primary afferent nerve fibers via synaptic connections, enabling them to detect and transduce environmental, metabolic, and homeostatic information from the gut directly to the nervous system.

Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. While pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene-related GPCRs Mrgpra3 and Mrgprc11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intracolonic administration of individual TGR5, MrgprA3, or MrgprC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Coadministration of these agonists as an "itch cocktail" augmented hypersensitivity to colorectal distension and changed mouse behavior. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5, as well as the human ortholog MrgprX1, and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders.

Chronic pelvic pain (CPP) is the most debilitating symptom of gynaecological disorders such as endometriosis. However, it remains unclear how sensory neurons from pelvic organs affected by endometriosis, such as the female reproductive tract, detect and transmit nociceptive events and how these signals are processed within the central nervous system (CNS). Using a previously characterized mouse model of endometriosis, we investigated whether the increased pain sensitivity occurring in endometriosis could be attributed to (i) changes in mechanosensory properties of sensory afferents innervating the reproductive tract, (ii) alterations in sensory input from reproductive organs to the spinal cord or (iii) neuroinflammation and sensitization of spinal neural circuits. Mechanosensitivity of vagina-innervating primary afferents was examined using an ex vivo single-unit extracellular recording preparation. Nociceptive signalling from the vagina to the spinal cord was quantified by phosphorylated MAP kinase ERK1/2 immunoreactivity. Immunohistochemistry was used to determine glial and neuronal circuit alterations within the spinal cord. We found that sensory afferents innervating the rostral, but not caudal portions of the mouse vagina, developed mechanical hypersensitivity in endometriosis. Nociceptive signalling from the vagina to the spinal cord was significantly enhanced in mice with endometriosis. Moreover, mice with endometriosis developed microgliosis, astrogliosis and enhanced substance P neurokinin-1 receptor immunoreactivity within the spinal cord, suggesting the development of neuroinflammation and sensitization of spinal circuitry in endometriosis. These results demonstrate endometriosis-induced neuroplasticity occurring at both peripheral and central sites of sensory afferent pathways. These findings may help to explain the altered sensitivity to pain in endometriosis and provide a novel platform for targeted pain relief treatments for this debilitating disorder.