Coexpressed δ-, µ-, and κ-Opioid Receptors Modulate Voltage-Gated Ca2+ Channels in Gastric-Projecting Vagal Afferent Neurons.

Opioid analgesics are frequently associated with gastrointestinal side effects, including constipation, nausea, dysphagia, and reduced gastric motility. Though it has been shown that stimulation of opioid receptors expressed in enteric motor neurons contributes to opioid-induced constipation, it remains unclear whether activation of opioid receptors in gastric-projecting nodose ganglia neurons contributes to the reduction in gastric motility and emptying associated with opioid use. In the present study, whole-cell patch-clamp recordings were performed to determine the mechanism underlying opioid receptor-mediated modulation of Ca2+ currents in acutely isolated gastric vagal afferent neurons. Our results demonstrate that CaV2.2 channels provide the majority (71% ± 16%) of Ca2+ currents in gastric vagal afferent neurons. Furthermore, we found that application of oxycodone, U-50488, or deltorphin II on gastric nodose ganglia neurons inhibited Ca2+ currents through a voltage-dependent mechanism by coupling to the Gα i/o family of heterotrimeric G-proteins. Because previous studies have demonstrated that the nodose ganglia expresses low levels of δ-opioid receptors, we also determined the deltorphin II concentration-response relationship and assessed deltorphin-mediated Ca2+ current inhibition following exposure to the δ-opioid receptor antagonist ICI 174,864 (0.3 µM). The peak mean Ca2+ current inhibition following deltorphin II application was 47% ± 24% (EC50 = 302.6 nM), and exposure to ICI 174,864 blocked deltorphin II-mediated Ca2+ current inhibition (4% ± 4% versus 37% ± 20%). Together, our results suggest that analgesics targeting any opioid receptor subtype can modulate gastric vagal circuits. SIGNIFICANCE STATEMENT: This study demonstrated that in gastric nodose ganglia neurons, agonists targeting all three classical opioid receptor subtypes (µ, δ, and κ) inhibit voltage-gated Ca2+ channels in a voltage-dependent mechanism by coupling to Gαi/o. These findings suggest that analgesics targeting any opioid receptor subtype would modulate gastric vagal circuits responsible for regulating gastric reflexes.

Regional Heterogeneity in Intestinal Epithelial Barrier Permeability and Mesenteric Perfusion After Thoracic Spinal Cord Injury.

BACKGROUND: Spinal cord injury (SCI) disrupts intestinal barrier function, thereby increasing antigen permeation and leading to poor outcomes. Despite the intestinal tract's anatomic and physiologic heterogeneity, studies following SCI have not comprehensively addressed intestinal pathophysiology with regional specificity. AIMS AND METHODS: We used an experimental model of high thoracic SCI to investigate (1) regional mucosal oxidative stress using dihydroethidium labeling; (2) regional paracellular permeability to small- and large-molecular probes via Ussing chamber; (3) regional intestinal tight junction (TJ) protein expression; and (4) hindgut perfusion via the caudal mesenteric artery. RESULTS: Dihydroethidium staining was significantly elevated within duodenal mucosa at 3-day post-SCI. Molar flux of [14C]-urea was significantly elevated in duodenum and proximal colon at 3-day post-SCI, while molar flux of [3H]-inulin was significantly elevated only in duodenum at 3-day post-SCI. Barrier permeability was mirrored by a significant increase in the expression of pore-forming TJ protein claudin-2 in duodenum and proximal colon at 3-day post-SCI. Claudin-2 expression remained significantly elevated in proximal colon at 3-week post-SCI. Expression of the barrier-forming TJ protein occludin was significantly reduced in duodenum at 3-day post-SCI. Caudal mesenteric artery flow was unchanged by SCI at 3 days or 3 weeks despite significant reductions in mean arterial pressure. CONCLUSION: These data show that T3-SCI provokes elevated mucosal oxidative stress, altered expression of TJ proteins, and elevated intestinal barrier permeability in the proximal intestine. In contrast, mucosal oxidative stress and intestinal barrier permeability were unchanged in the hindgut after SCI. This regional heterogeneity may result from differential sensitivity to reduced mesenteric perfusion, though further studies are required to establish a causal link. Understanding regional differences in intestinal pathophysiology is essential for developing effective treatments and standards of care for individuals with SCI.

Mesenteric vascular dysregulation and intestinal inflammation accompanies experimental spinal cord injury.

Cervical and high thoracic spinal cord injury (SCI) drastically impairs autonomic nervous system function. Individuals with SCI at thoracic spinal level 5 (T5) or higher often present cardiovascular disorders that include resting systemic arterial hypotension. Gastrointestinal (GI) tissues are critically dependent upon adequate blood flow and even brief periods of visceral hypoxia triggers GI dysmotility. The aim of this study was to test the hypothesis that T3-SCI induces visceral hypoperfusion, diminished postprandial vascular reflexes, and concomitant visceral inflammation. We measured in vivo systemic arterial blood pressure and superior mesenteric artery (SMA) and duodenal blood flow in anesthetized T3-SCI rats at 3 days and 3 wk postinjury either fasted or following enteral feeding of a liquid mixed-nutrient meal (Ensure). In separate cohorts of fasted T3-SCI rats, markers of intestinal inflammation were assayed by qRT-PCR. Our results show that T3-SCI rats displayed significantly reduced SMA blood flow under all experimental conditions (P < 0.05). Specifically, the anticipated elevation of SMA blood flow in response to duodenal nutrient infusion (postprandial hyperemia) was either delayed or absent after T3-SCI. The dysregulated SMA blood flow in acutely injured T3-SCI rats coincides with abnormal intestinal morphology and elevation of inflammatory markers, all of which resolve after 3 wk. Specifically, Icam1, Ccl2 (MCP-1), and Ccl3 (MIP-1α) were acutely elevated following T3-SCI. Our data suggest that arterial hypotension diminishes mesenteric blood flow necessary to meet mucosal demands at rest and during digestion. The resulting GI ischemia and low-grade inflammation may be an underlying pathology leading to GI dysfunction seen following acute T3-SCI.

Plasticity in the brainstem vagal circuits controlling gastric motor function triggered by corticotropin releasing factor.

Stress impairs gastric emptying, reduces stomach compliance and induces early satiety via vagal actions. We have shown recently that the ability of the anti-stress neuropeptide oxytocin (OXT) to modulate vagal brainstem circuits undergoes short-term plasticity via alterations in cAMP levels subsequent to vagal afferent fibre-dependent activation of metabotropic glutamate receptors. The aim of the present study was to test the hypothesis that the OXT-induced gastric response undergoes plastic changes in the presence of the prototypical stress hormone, corticotropin releasing factor (CRF). Whole cell patch clamp recordings showed that CRF increased inhibitory GABAergic synaptic transmission to identified corpus-projecting dorsal motor nucleus of the vagus (DMV) neurones. In naive brainstem slices, OXT perfusion had no effect on inhibitory synaptic transmission; following exposure to CRF (and recovery from its actions), however, re-application of OXT inhibited GABAergic transmission in the majority of neurones tested. This uncovering of the OXT response was antagonized by pretreatment with protein kinase A or adenylate cyclase inhibitors, H89 and di-deoxyadenosine, respectively, indicating a cAMP-mediated mechanism. In naive animals, OXT microinjection in the dorsal vagal complex induced a NO-mediated corpus relaxation. Following CRF pretreatment, however, microinjection of OXT attenuated or, at times reversed, the gastric relaxation which was insensitive to l-NAME but was antagonized by pretreatment with a VIP antagonist. Immunohistochemical analyses of vagal motoneurones showed an increased number of oxytocin receptors present on GABAergic terminals of CRF-treated or stressed vs. naive rats. These results indicate that CRF alters vagal inhibitory circuits that uncover the ability of OXT to modulate GABAergic currents and modifies the gastric corpus motility response to OXT.

Vagal afferent fibres determine the oxytocin-induced modulation of gastric tone.

Oxytocin (OXT) inputs to the dorsal vagal complex (DVC; nucleus of the tractus solitarius (NTS) dorsal motor nucleus of the vagus (DMV) and area postrema) decrease gastric tone and motility. Our first aim was to investigate the mechanism(s) of OXT-induced gastric relaxation. We demonstrated recently that vagal afferent inputs modulate NTS-DMV synapses involved in gastric and pancreatic reflexes via group II metabotropic glutamate receptors (mGluRs). Our second aim was to investigate whether group II mGluRs similarly influence the response of vagal motoneurons to OXT. Microinjection of OXT in the DVC decreased gastric tone in a dose-dependent manner. The OXT-induced gastric relaxation was enhanced following bethanechol and reduced by l-NAME administration, suggesting a nitrergic mechanism of gastroinhibition. DVC application of the group II mGluR antagonist EGLU induced a gastroinhibition that was not dose dependent and shifted the gastric effects of OXT to a cholinergic-mediated mechanism. Evoked and miniature GABAergic synaptic currents between NTS and identified gastric-projecting DMV neurones were not affected by OXT in any neurones tested, unless the brainstem slice was (a) pretreated with EGLU or (b) derived from rats that had earlier received a surgical vagal deafferentation. Conversely, OXT inhibited glutamatergic currents even in naive slices, but their responses were unaffected by EGLU pretreatment. These results suggest that the OXT-induced gastroinhibition is mediated by activation of the NANC pathway. Inhibition of brainstem group II mGluRs, however, uncovers the ability of OXT to modulate GABAergic transmission between the NTS and DMV, resulting in the engagement of an otherwise silent cholinergic vagal neurocircuit.

Plasticity of colonic enteric nervous system following spinal cord injury in male and female rats.

BACKGROUND: Neurogenic bowel is a dysmotility disorder following spinal cord injury (SCI) that negatively impacts quality of life, social integration, and physical health. Colonic transit is directly modulated by the enteric nervous system. Interstitial Cells of Cajal (ICC) distributed throughout the small intestine and colon serve as specialized pacemaker cells, generating rhythmic electrical slow waves within intestinal smooth muscle, or serve as an interface between smooth muscle cells and enteric motor neurons of the myenteric plexus. Interstitial Cells of Cajal loss has been reported for other preclinical models of dysmotility, and our previous experimental SCI study provided evidence of reduced excitatory and inhibitory enteric neuronal count and smooth muscle neural control. METHODS: Immunohistochemistry for the ICC-specific marker c-Kit was utilized to examine neuromuscular remodeling of the distal colon in male and female rats with experimental SCI. KEY RESULTS: Myenteric plexus ICC (ICC-MP) exhibited increased cell counts 3 days following SCI in male rats, but did not significantly increase in females until 3 weeks after SCI. On average, ICC-MP total primary arborization length increased significantly in male rats at 3-day, 3-week, and 6-week time points, whereas in females, this increase occurred most frequently at 6 weeks post-SCI. Conversely, circular muscle ICC (ICC-CM) did not demonstrate post-SCI changes. CONCLUSIONS AND INFERENCES: These data demonstrate resiliency of the ICC-MP in neurogenic bowel following SCI, unlike seen in other related disease states. This plasticity underscores the need to further understand neuromuscular changes driving colonic dysmotility after SCI in order to advance therapeutic targets for neurogenic bowel treatment.

Spinal cord injury-mediated changes in electrophysiological properties of rat gastric nodose ganglion neurons.

In preclinical rodent models, spinal cord injury (SCI) manifests as gastric vagal afferent dysfunction both acutely and chronically. However, the mechanism that underlies this dysfunction remains unknown. In the current study, we examined the effect of SCI on gastric nodose ganglia (NG) neuron excitability and on voltage-gated Na+ (NaV) channels expression and function in rats after an acute (i.e. 3-days) and chronic (i.e. 3-weeks) period. Rats randomly received either T3-SCI or sham control surgery 3-days or 3-weeks prior to experimentation as well as injections of 3% DiI solution into the stomach to identify gastric NG neurons. Single cell qRT-PCR was performed on acutely dissociated DiI-labeled NG neurons to measure NaV1.7, NaV1.8 and NaV1.9 expression levels. The results indicate that all 3 channel subtypes decreased. Current- and voltage-clamp whole-cell patch-clamp recordings were performed on acutely dissociated DiI-labeled NG neurons to measure active and passive properties of C- and A-fibers as well as the biophysical characteristics of NaV1.8 channels in gastric NG neurons. Acute and chronic SCI did not demonstrate deleterious effects on either passive properties of dissociated gastric NG neurons or biophysical properties of NaV1.8. These findings suggest that although NaV gene expression levels change following SCI, NaV1.8 function is not altered. The disruption throughout the entirety of the vagal afferent neuron has yet to be investigated.

Feasibility Study of Bariatric Surgery in a Rat Model of Spinal Cord Injury to Achieve Beneficial Body Weight Outcome.

Approximately two thirds of spinal cord injury (SCI) persons become overweight or obese. Obesity increases the risk of developing type 2 diabetes and limits self-help techniques. Weight-loss surgery (WLS), including vertical sleeve gastrectomy (VSG), is regarded as highly effective in the long-term treatment of obesity and remission of associated type 2 diabetes. Given the increased risk of obesity post-SCI, WLS offers an attractive intervention strategy. Alterations in the physiology of energy homeostasis after SCI necessitate that SCI persons should not be regarded as similar to able-bodied persons. Because of current knowledge gaps, it is unknown whether an obese phenotype with SCI will respond to WLS similarly to the neurally intact obese phenotype. Therefore, this study tested the hypothesis that the VSG procedure is well tolerated and effective in an animal model of high-thoracic (T3) SCI. In Wistar male rats, subsequent to a 2-week recovery period after T3-SCI, but not control laminectomy surgery, daily consumption of a high-fat diet (HFD; 60% kcal from fat) was elevated over 4 weeks preceding VSG. After a 2-week recovery period post-VSG, HFD consumption in T3-SCI rats over a 4-week monitoring period returned to levels comparable to control. Body weight was significantly reduced in T3-SCI rats and remained reduced whereas control rats regained body weight. Further, no adverse complications directly attributable to the VSG procedure were identified. Thus, this rodent model is a viable tool for addressing fundamental questions regarding the mechanisms leading to obesity post-SCI and the development of translational strategies.

Altered physiology of gastrointestinal vagal afferents following neurotrauma.

The adaptability of the central nervous system has been revealed in several model systems. Of particular interest to central nervous system-injured individuals is the ability for neural components to be modified for regain of function. In both types of neurotrauma, traumatic brain injury and spinal cord injury, the primary parasympathetic control to the gastrointestinal tract, the vagus nerve, remains anatomically intact. However, individuals with traumatic brain injury or spinal cord injury are highly susceptible to gastrointestinal dysfunctions. Such gastrointestinal dysfunctions attribute to higher morbidity and mortality following traumatic brain injury and spinal cord injury. While the vagal efferent output remains capable of eliciting motor responses following injury, evidence suggests impairment of the vagal afferents. Since sensory input drives motor output, this review will discuss the normal and altered anatomy and physiology of the gastrointestinal vagal afferents to better understand the contributions of vagal afferent plasticity following neurotrauma.

Diminished enteric neuromuscular transmission in the distal colon following experimental spinal cord injury.

Neurogenic bowel following spinal cord injury (SCI) leads to decreased colonic motility, remodeling of the neuromuscular compartment and results in chronic evacuation difficulties. The distal colon of the rat serves a dual role for fluid absorption and storage that is homologous to the descending colon of humans. Dysmotility of the descending colon is one component of neurogenic bowel. We investigated the integrity of the enteric neuromuscular transmission responsible for the generation of excitatory and inhibitory junction potentials (EJPs and IJPs, respectively) in the distal colon of rats. We previously demonstrated a chronic reduction in colonic enteric neurons from rats with acute and chronic high-thoracic (T3) SCI and hypothesized that neurogenic bowel following T3-SCI results from diminished enteric neuromuscular transmission. Immunohistochemical labeling for myenteric neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT) neurons demonstrated a significant loss of presumptive nitric oxide (NO) and acetylcholine (ACh) immunoreactive neurons in both 3-day and 3-week injured animals. Colonic neuromuscular transmission in response to transmural electrical stimulation of the colon was significantly reduced 3-days and 3-weeks following SCI in male rats. Specifically, cholinergic-mediated excitatory junction potentials (EJPs) and nitrergic-mediated slow inhibitory junction potentials (IJPs) were significantly reduced while ATP-mediated fast IJPs remained unaffected. We conclude that a reduction in excitatory and inhibitory enteric neuromuscular transmission contributes to neurogenic bowel observed following SCI, and that these loss-of-function changes involve enteric-mediated cholinergic and nitrergic pathways.

Recommendations for evaluation of bladder and bowel function in pre-clinical spinal cord injury research.

Objective: In order to encourage the inclusion of bladder and bowel outcome measures in preclinical spinal cord injury (SCI) research, this paper identifies and categorizes 1) fundamental, 2) recommended, 3) supplemental and 4) exploratory sets of outcome measures for pre-clinical assessment of bladder and bowel function with broad applicability to animal models of SCI.Methods: Drawing upon the collective research experience of autonomic physiologists and informed in consultation with clinical experts, a critical assessment of currently available bladder and bowel outcome measures (histological, biochemical, in vivo functional, ex vivo physiological and electrophysiological tests) was made to identify the strengths, deficiencies and ease of inclusion for future studies of experimental SCI.Results: Based upon pre-established criteria generated by the Neurogenic Bladder and Bowel Working Group that included history of use in experimental settings, citations in the literature by multiple independent groups, ease of general use, reproducibility and sensitivity to change, three fundamental measures each for bladder and bowel assessments were identified. Briefly defined, these assessments centered upon tissue morphology, voiding efficiency/volume and smooth muscle-mediated pressure studies. Additional assessment measures were categorized as recommended, supplemental or exploratory based upon the balance between technical requirements and potential mechanistic insights to be gained by the study.Conclusion: Several fundamental assessments share reasonable levels of technical and material investment, including some that could assess bladder and bowel function non-invasively and simultaneously. Such measures used more inclusively across SCI studies would advance progress in this high priority area. When complemented with a few additional investigator-selected study-relevant supplemental measures, they are highly recommended for research programs investigating the efficacy of therapeutic interventions in preclinical animal models of SCI that have a bladder and/or bowel focus.

Gastric vagal afferent neuropathy following experimental spinal cord injury.

Dramatic impairment of gastrointestinal (GI) function accompanies high-thoracic spinal cord injury (T3-SCI). The vagus nerve contains mechano- and chemosensory fibers as well as the motor fibers necessary for the central nervous system (CNS) control of GI reflexes. Cell bodies for the vagal afferent fibers are located within the nodose gangla (NG) and the majority of vagal afferent axons are unmyelinated C fibers that are sensitive to capsaicin through activation of transient receptor potential vanilloid-1 (TRPV1) channels. Vagal afferent fibers also express receptors for GI hormones, including cholecystokinin (CCK). Previously, T3-SCI provokes a transient GI inflammatory response as well as a reduction of both gastric emptying and centrally-mediated vagal responses to GI peptides, including CCK. TRPV1 channels and CCK-A receptors (CCKar) expressed in vagal afferents are upregulated in models of visceral inflammation. The present study investigated whether T3-SCI attenuates peripheral vagal afferent sensitivity through plasticity of TRPV1 and CCK receptors. Vagal afferent response to graded mechanical stimulation of the stomach was significantly attenuated by T3-SCI at 3-day and 3-week recovery. Immunocytochemical labeling for CCKar and TRPV1 demonstrated expression on dissociated gastric-projecting NG neurons. Quantitative assessment of mRNA expression by qRT-PCR revealed significant elevation of CCKar and TRPV1 in the whole NG following T3-SCI in 3-day recovery, but levels returned to normal after 3-weeks. Three days after injury, systemic administration of CCK-8 s showed a significantly diminished gastric vagal afferent response in T3-SCI rats compared to control rats while systemic capsaicin infusion revealed a significant elevation of vagal response in T3-SCI vs control rats. These findings demonstrate that T3-SCI provokes peripheral remodeling and prolonged alterations in the response of vagal afferent fibers to the physiological signals associated with digestion.

Vagally mediated effects of glucagon-like peptide 1: in vitro and in vivo gastric actions.

Glucagon-like peptide-1 (GLP-1) is a neuropeptide released following meal ingestion that, among other effects, decreases gastric tone and motility. The central targets and mechanism of action of GLP-1 on gastric neurocircuits have not, however, been fully investigated. A high density of GLP-1 containing neurones and receptors are present in brainstem vagal circuits, suggesting that the gastroinhibition may be vagally mediated. We aimed to investigate: (1) the response of identified gastric-projecting neurones of the dorsal motor nucleus of the vagus (DMV) to GLP-1 and its analogues; (2) the effects of brainstem application of GLP-1 on gastric tone; and (3) the vagal pathway utilized by GLP-1 to induce gastroinhibition. We conducted our experiments using whole-cell recordings from identified gastric-projecting DMV neurones and microinjection in the dorsal vagal complex (DVC) of anaesthetized rats while monitoring gastric tone. Perfusion with GLP-1 induced a concentration-dependent excitation of a subpopulation of gastric-projecting DMV neurones. The GLP-1 effects were mimicked by exendin-4 and antagonized by exendin-9-39. In an anaesthetized rat preparation, application of exendin-4 to the DVC decreased gastric tone in a concentration-dependent manner. The gastroinhibitory effects of exendin-4 were unaffected by systemic pretreatment with the pro-motility muscarinic agonist bethanechol, but were abolished by systemic administration of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), or by bilateral vagotomy. Our data indicate that GLP-1 activates selective receptors to excite DMV neurones mainly and that the gastroinhibition observed following application of GLP-1 in the DVC is due to the activation of an inhibitory non-adrenergic, non-cholinergic input to the stomach.

Investigating neurogenic bowel in experimental spinal cord injury: where to begin?

The devastating losses following traumatic spinal cord injury (SCI) encompass the motor, sensory and autonomic nervous systems. Neurogenic bowel is a slow transit colonic dysfunction marked by constipation, rectal evacuation difficulties, decreased anorectal sensation, fecal incontinence or some combination thereof. Furthermore, neurogenic bowel is one of the most prevalent comorbidities of SCI and is recognized by afflicted individuals and caregivers as a lifelong physical and psychological challenge that profoundly affects quality of life. The restoration of post-injury control of movement has received considerable scientific scrutiny yet the daily necessity of voiding the bowel and bladder remains critically under-investigated. Subsequently, physicians and caregivers are rarely presented with consistent, evidence-based strategies to successfully address the consequences of dysregulated voiding reflexes. Neurogenic bowel is commonly believed to result from the interruption of the supraspinal control of the spinal autonomic circuits regulating the colon. In this mini-review, we discuss the clinical challenges presented by neurogenic bowel and emerging pre-clinical evidence that is revealing that SCI also initiates functional remodeling of the colonic wall concurrent with a decrease in local enteric neurons. Since the enteric input to the colonic smooth muscle is the final common pathway for functional contractions of the colon, changes to the neuromuscular interface must first be understood in order to maximize the efficacy of therapeutic interventions targeting colonic dysfunction following SCI.

Gastrointestinal dysfunction after spinal cord injury.

The gastrointestinal tract of vertebrates is a heterogeneous organ system innervated to varying degrees by a local enteric neural network as well as extrinsic parasympathetic and sympathetic neural circuits located along the brainstem and spinal axis. This diverse organ system serves to regulate the secretory and propulsive reflexes integral to the digestion and absorption of nutrients. The quasi-segmental distribution of the neural circuits innervating the gastrointestinal (GI) tract produces varying degrees of dysfunction depending upon the level of spinal cord injury (SCI). At all levels of SCI, GI dysfunction frequently presents life-long challenges to individuals coping with injury. Growing attention to the profound changes that occur across the entire physiology of individuals with SCI reveals profound knowledge gaps in our understanding of the temporal dimensions and magnitude of organ-specific co-morbidities following SCI. It is essential to understand and identify these broad pathophysiological changes in order to develop appropriate evidence-based strategies for management by clinicians, caregivers and individuals living with SCI. This review summarizes the neurophysiology of the GI tract in the uninjured state and the pathophysiology associated with the systemic effects of SCI.

Purinergic receptor expression and function in rat vagal sensory neurons innervating the stomach.

The nodose ganglion (NG) is the main parasympathetic ganglion conveying sensory signals to the CNS from numerous visceral organs including digestive signals such as gastric distension or the release the gastrointestinal peptides. The response characteristics of NG neurons to ATP and ADP and pharmacological interrogation of purinergic receptor subtypes have been previously investigated but often in NG cells of undetermined visceral origin. In this study, we confirmed the presence of P2X3 and P2Y1 receptors and characterized P2X and P2Y responses in gastric-innervating NG neurons. Application of ATP-evoked large inward currents and cytosolic Ca2+ increases in gastric-innervating NG neurons. Despite the expression of P2Y1 receptors, ADP elicited only minor modulation of voltage-gated Ca2+ channels. Considering the sensitivity of NG neurons to comorbidities associated with disease or neural injury, purinergic modulation of gastric NG neurons in disease- or injury-states is worthy of further investigation.

Anatomical and Functional Changes to the Colonic Neuromuscular Compartment after Experimental Spinal Cord Injury.

A profound reduction in colorectal transit time accompanies spinal cord injury (SCI), yet the colonic alterations after SCI have yet to be understood fully. The loss of descending supraspinal input to lumbosacral neural circuits innervating the colon is recognized as one causal mechanism. Remodeling of the colonic enteric nervous system/smooth muscle junction in response to inflammation, however, is recognized as one factor leading to colonic dysmotility in other pathophysiological models. We investigated the alterations to the neuromuscular junction in rats with experimental high-thoracic (T3) SCI. One day to three weeks post-injury, both injured and age-matched controls underwent in vivo experimentation followed by tissue harvest for histological evaluation. Spontaneous colonic contractions were reduced significantly in the proximal and distal colon of T3-SCI rats. Histological evaluation of proximal and distal colon demonstrated significant reductions of colonic mucosal crypt depth and width. Markers of intestinal inflammation were assayed by qRT-PCR. Specifically, Icam1, Ccl2 (MCP-1), and Ccl3 (MIP-1α) mRNA was acutely elevated after T3-SCI. Smooth muscle thickness and collagen content of the colon were increased significantly in T3-SCI rats. Colonic cross sections immunohistochemically processed for the pan-neuronal marker HuC/D displayed a significant decrease in colonic enteric neuron density that became more pronounced at three weeks after injury. Our data suggest that post-SCI inflammation and remodeling of the enteric neuromuscular compartment accompanies SCI. These morphological changes may provoke the diminished colonic motility that occurs during this same period, possibly through the disruption of intrinsic neuromuscular control of the colon.

5-Hydroxytryptamine2C receptors on pudendal motoneurons innervating the external anal sphincter.

The aim of this study was to determine the localization of 5-hydroxytryptamine2C (5-HT2C) receptors on the motoneurons innervating the external anal sphincter (EAS) of male rats. Motoneurons were retrogradely labeled after percutaneous intramuscular injection of Fluorogold (FG) into the EAS. Using fluorescent immunohistochemistry, FG-positive EAS motoneurons that were immunoreactive for the 5-HT2C receptor (5-HT2C-IR) were targeted for specific examination with widefield microscopy or confocal laser scanning microscopy with spectral separation. Widefield microscopy revealed distributions of FG-positive EAS motoneurons in the L5-S1 gray matter corresponding to the dorsomedial cell group. 5-HT2C-IR positive cells were distributed in the intermediolateral cell column and the ventral horn. Ventral horn 5-HT2C-IR labeling included the dorsomedial cell group as well as the dorsolateral, ventromedial and ventrolateral areas. Confocal analysis of FG-positive EAS motoneurons and 5-HT2C-IR positive motoneuron profiles adjacent to EAS motoneurons that were not labeled with FG but presumably innervate the bulbospongiosus muscle confirmed that EAS motoneurons were immunopositive for the 5-HT2C receptor. These data suggest that previously identified descending serotonergic immunopositive fibers observed terminating on EAS motoneurons might mediate their input through the activation of 5-HT2C receptors.

Descending spinal projections from the rostral gigantocellular reticular nuclei complex.

Electrophysiological and physiological studies have suggested that the ventral medullary gigantocellular reticular nuclei (composed of the gigantocellular ventralis and pars alpha nuclei as well as the adjacent lateral paragigantocellular nucleus; abbreviated Gi-LPGi complex) provide descending control of pelvic floor organs (Mackel [1979] J. Physiol. (Lond.) 294:105-122; Hubscher and Johnson [1996] J. Neurophysiol. 76:2474-2482; Hubscher and Johnson [1999] J. Neurophysiol. 82:1381-1389; Johnson and Hubscher [1998] Neuroreport 9:341-345). Specifically, this complex of paramedian reticular nuclei has been implicated in the inhibition of sexual reflexes. In the present study, an anterograde fluorescent tracer was used to investigate direct descending projections from the Gi-LPGi complex to retrogradely labeled pudendal motoneurons (MN) in the male rat. Our results demonstrated that, although a high density of arborizations from Gi-LPGi fibers appears to be in close apposition to pudendal MNs, this relationship also applies to other MNs throughout the entire spinal cord. The Gi-LPGi also projects to spinal autonomic regions, i.e., both the intermediolateral cell column and the sacral parasympathetic nucleus, as well as to regions of the intermediate gray, which contain interneurons involved in the organization of pelvic floor reflexes. Lastly, throughout the length of the spinal cord, numerous neurons located primarily in laminae VII-X, were retrogradely labeled with Fluoro-Ruby after injections into the Gi-LPGi. The diffuse descending projections and arborizations of this pathway throughout the spinal cord suggest that this brainstem area is involved in the direct, descending control of a variety of spinal activities. These results are in contrast with our observations of the discrete projections of the caudal nucleus raphe obscurus, which target the autonomic and somatic MNs involved specifically in sexual and eliminative functions (Hermann et al. [1998] J. Comp. Neurol. 397:458-474).

TNF alpha-p55 receptors: medullary brainstem immunocytochemical localization in normal and vagus nerve-transected rats.

Tumor necrosis factor alpha (TNF(alpha)) is a potent modulator of autonomic reflex mechanisms that control the stomach. Evidence suggests that TNF(alpha) action directly on vago-vagal reflex control circuits causes the autonomic misregulation of digestion manifested as gastrointestinal stasis, nausea, and emesis associated with illness. Neurophysiological studies indicated that TNF(alpha) may have effects on vagal afferents in the solitary nucleus, as well as neurons of the solitary nucleus (NST) and dorsal motor nucleus (DMN) of the vagus. The aim of this study was to determine the location of the TNFR1 receptor (p55) in the medulla using immunocytochemical methods. We devised a technique for localizing the p55 receptor using heat-induced antigen recovery in fixed tissue sections. This protocol allowed us to demonstrate that dense p55-immunoreactivity (p55-ir) is constitutively present on central (but not peripheral) vagal afferents in the solitary tract (ST) and nucleus; p55-ir is also present on afferents entering the spinal trigeminal nucleus. Unilateral supra-nodose vagotomy eliminated p55-ir from ipsilateral central vagal afferents. Virtually all neurons in the brainstem appeared to express p55-ir at a low level, i.e., just above background. However, vagotomy caused a dramatic up-regulation of p55-ir in vagal motor neurons. This increase in p55-ir in axotomized neurons may play a pivotal role in the connection between the occurrence of the injury and the initiation of apoptotic processes resulting in elimination of damaged neurons.