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There has been a gradual shift in the delivery of physiology laboratory classes over the last 30 years. For many, wet-lab demonstrations using animal tissues have been reduced or replaced with student-led investigations where students are both subjects and researchers. Despite these changes, expectations remain that physiology courses should include a practical component to encourage deeper and higher-order learning. Wet-lab tissue experiments and student-based group research formats can be expensive to run, associated with various ethical constraints, and, as discovered in these times of COVID-19, difficult to operate while adhering to physical distancing. We address the proposition that online and/or remote delivery of laboratory classes using digital technologies may provide a solution to both financial and ethical constraints of on-campus laboratory classes. Our discussions, as an international group of 10 physiologists from the United States, the United Kingdom, Canada, and Australia, revealed that although some of the financial and ethical constraints of using animal tissues and student-led investigations were addressed by the introduction of online alternatives, the construction and maintenance of online delivery modes could also be expensive and ethical issues, not previously considered, included digital equity and student data security. There was also a collective perception that if face-to-face laboratory classes were changed to an entirely virtual mode there was a risk that some intended learning outcomes would not be met. It was concluded that the “ideal” approach is likely a hybrid model whereby student attendance in face-to-face, on-campus classes is supported with interactive digital content either developed in house or obtained through third-party providers.

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.

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.

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.

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.

In visceral organs of mammals, most noxious (painful) stimuli as well as innocuous stimuli are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRGs). One of the major unresolved questions is the location, morphology, and neurochemistry of the nerve endings of spinal afferents that actually detect these stimuli in the viscera. In the upper gastrointestinal (GI) tract, there have been many anterograde tracing studies of vagal afferent endings, but none on spinal afferent endings. Recently, we developed a technique that now provides selective labeling of only spinal afferents. We used this approach to identify spinal afferent nerve endings in the upper GI tract of mice. Animals were anesthetized, and injections of dextran-amine were made into thoracic DRGs (T8-T12). Seven days post surgery, mice were euthanized, and the stomach and esophagus were removed, fixed, and stained for calcitonin gene-related peptide (CGRP). Spinal afferent axons were identified that ramified extensively through many rows of myenteric ganglia and formed nerve endings in discrete anatomical layers. Most commonly, intraganglionic varicose endings (IGVEs) were identified in myenteric ganglia of the stomach and varicose simple-type endings in the circular muscle and mucosa. Less commonly, nerve endings were identified in internodal strands, blood vessels, submucosal ganglia, and longitudinal muscle. In the esophagus, only IGVEs were identified in myenteric ganglia. No intraganglionic lamellar endings (IGLEs) were identified in the stomach or esophagus. We present the first identification of spinal afferent endings in the upper GI tract. Eight distinct types of spinal afferent endings were identified in the stomach, and most of them were CGRP immunoreactive. J. Comp. Neurol. 524:3064-3083, 2016. © 2016 Wiley Periodicals, Inc.

Toll-like receptors (TLRs) are expressed in enteric neurons, glia, gastrointestinal (GI) smooth muscle and mucosa, yet their functional roles in the GI tract are not fully understood. TLRs have been linked to many of the undesirable central effects of chronic opioid administration including hyperalgesia and dependence via activation of central microglia. Opioid-induced bowel dysfunction (OIBD) remains a primary reason for the reduction or withdrawal of opioid analgesics. Morphine-induced inhibition of colonic motility was assessed in vivo by GI transit studies and in vitro using isolated colons from wildtype (WT) and TLR deficient mice. Morphine slowed movement of ingested content in WT but this retardation effect was attenuated in TLR4 −/− and TLR2/4 −/− . In isolated colons, morphine reduced amplitude and frequency colonic migrating motor contractions in both WT and TLR2/4 −/− . Electrical field stimulation elicited distal colon relaxation that was potentiated by morphine in WT but not in TLR2/4 −/− . Inhibitory junction potentials were of similar amplitude and kinetics in WT and TLR2/4 −/− distal colon and not altered by morphine. Enteric nerve density and proportion of nitrergic nerves were similar in WT and TLR2/4 −/− distal colon. These data suggest an involvement of TLRs in opioid pharmacodynamics and thus a potential interventional target for OIBD.

Autonomic neurons and chromaffin cells, which constitute the autonomic nervous system, are derived from a common progenitor from the neural crest, and its development is controlled by a network of transcription factors, including the master regulator, Phox2b, and its downstream, Gata3. Anti-Phox2b and anti-Gata3 antibodies were applied to a total of 77 autonomic nervous system tumors, including 35 paragangliomas, 21 pheochromocytomas, 9 neuroblastomas, 4 ganglioneuroblastomas, and 8 ganglioneuromas, as well as their potential morphologic mimics, including tumors of the small round cell tumor group, neuroendocrine carcinomas of lung and gastrointestinal tract (carcinoid tumors/neuroendocrine tumors, large cell neuroendocrine carcinomas, and small cell carcinomas), Merkel cell carcinomas, benign and malignant tumors of thyroid, parathyroid, and adrenal cortex, and malignant melanomas. A variety of nonendocrine/neuroendocrine carcinomas were also studied. Gata3 expression was seen in 89% of paragangliomas, 95% of pheochromocytomas, and all neuroblastomas, ganglioneuroblastomas, and ganglioneuromas, as well as in all parathyroid tumors, a majority of urothelial and mammary carcinomas, and a subset of squamous cell carcinomas, but all other tumors were negative. Phox2b expression was seen in all neuroblastomas, ganglioneuroblastomas, and ganglioneuromas and in 40% of paragangliomas, but pheochromocytomas and all other tumors were negative. Gata3 is a highly reliable marker for paragangliomas, pheochromocytomas, and neuroblastic tumors to distinguish from their simulators. This is an additional utility for this marker, which is used for the diagnosis of urothelial and mammary carcinomas. Phox2b is also highly specific, but its low sensitivity to paragangliomas and pheochromocytomas would limit the utility only to neuroblastic tumors.

Intramuscular interstitial cells of Cajal (ICC-IM) are closely associated with enteric motor nerve terminals and electrically coupled to smooth muscle cells within the gastric musculature. Previous studies investigating the role of ICC-IM in motor neurotransmission have used indiscriminate electric field stimulation of neural elements within the gastric wall. To determine the role of ICC-IM in transduction of vagally-mediated motor input to gastric muscles electrical and mechanical responses to selective electrical vagal stimulation (EVS) were recorded from gastric fundus and antral regions of wild type and W/WV mice, which lack most ICC-IM. EVS evoked inhibitory junction potentials (IJPs) in wild type muscles that were attenuated or abolished by L-NNA. IJPs were rarely evoked in W/WV muscles by EVS, and not affected by L-NNA. EVS evoked relaxation of wild type stomachs, but the predominant response of W/WV stomachs was contraction. EVS applied after pre-contraction with bethanechol caused relaxation of wild type gastric tissues and these were inhibited by the nitric oxide synthase inhibitor L-NNA. Relaxation responses were of smaller amplitude in W/WV muscles and L-NNA did not attenuate relaxation responses in W/WV fundus muscles. These data suggest an important role for ICC-IM in vagally-mediated nitrergic relaxation in the proximal and distal stomach.

In mature animals, neurons and interstitial cells of Cajal (ICC) are essential for organized intestinal motility. We investigated motility patterns, and the roles of neurons and myenteric ICC (ICC-MP), in the duodenum and colon of developing mice in vitro. Spatiotemporal mapping revealed regular contractions that propagated in both directions from embryonic day (E)13.5 in the duodenum and E14.5 in the colon. The propagating contractions, which we termed ripples, were unaffected by tetrodotoxin and were present in the intestine of embryonic Ret null mutant mice, which lack enteric neurons. Neurally mediated motility patterns were first observed in the duodenum at E18.5. To examine the possible role of ICC-MP, three approaches were used. First, intracellular recordings from the circular muscle of the duodenum did not detect slow wave activity at E16.5, but regular slow waves were observed in some preparations of E18.5 duodenum. Second, spatiotemporal mapping revealed ripples in the duodenum of E13.5 and E16.5 W/W(v) embryos, which lack KIT+ ICC-MP and slow waves. Third, KIT-immunoreactive cells with the morphology of ICC-MP were first observed at E18.5. Hence, ripples do not appear to be mediated by ICC-MP and must be myogenic. Ripples in the duodenum and colon were abolished by cobalt chloride (1 mm). The L-type Ca(2+) channel antagonist nicardipine (2.5 microm) abolished ripples in the duodenum and reduced their frequency and size in the colon. Our findings demonstrate that prominent propagating contractions (ripples) are present in the duodenum and colon of fetal mice. Ripples are not mediated by neurons or ICC-MP, but entry of extracellular Ca(2+) through L-type Ca(2+) channels is essential. Thus, during development of the intestine, the first motor patterns to develop are myogenic.

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.

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.

Idiopathic constipation is higher in women of reproductive age than postmenopausal women or men, suggesting that female steroid hormones influence gastrointestinal motility. How female hormones affect motility is unclear. Colonic motility is regulated by ion channels in colonic myocytes. Voltage-dependent K+ channels serve to set the excitability of colonic muscles. We investigated regulation of Kv4.3 channel expression in response to acute or chronic changes in female hormones. Patch clamp experiments and quantitative PCR were used to compare outward currents and transcript expression in colonic myocytes from male, non-pregnant, pregnant and ovariectomized mice. Groups of ovariectomized mice received injections of oestrogen or progesterone to investigate the effects of hormone replacement. The capacitance of colonic myocytes from non-pregnant females was larger than in males. Net outward current density in male and ovariectomized mice was higher than in non-pregnant females and oestrogen-treated ovariectomized mice. Current densities in late pregnancy were lower than in female controls. Progesterone had no effect on outward currents. A-type currents were decreased in non-pregnant females compared with ovariectomized mice, and were further decreased by pregnancy or oestrogen replacement. Kv4.3 transcripts did not differ significantly between groups; however, expression of the potassium channel interacting protein KChIP1 was elevated in ovariectomized mice compared with female controls and oestrogen-treated ovariectomized mice. Delayed rectifier currents were not affected by oestrogen. In the mouse colon, oestrogen suppresses A-type currents, which are important for regulating excitability. These observations suggest a possible link between female hormones and altered colonic motility associated with menses, pregnancy and menopause.

Interstitial cells of Cajal (ICC) are specialized cells in smooth muscle organs that generate and propagate pacemaker activity, receive inputs from motor neurons, and serve as mechanosensors. In the gastrointestinal tract, development and maintenance of the ICC phenotype have been linked to intracellular signaling via Kit, but its role in development of ICC during embryogenesis is controversial. Here we have studied the development of functional ICC-MY during the late gestational period in mice. Blocking Kit with a neutralizing antibody before and after development of spontaneous electrical activity (E17 to P0) caused loss of ICC-MY networks and pacemaker activity. ICC-MY and pacemaker activity developed normally in W/+ and W(V)/+ heterozygotes, but failed to develop between E17 to P0 in W/W(V) embryos with compromised Kit function. Muscles treated with Kit neutralizing antibody or the tyrosine kinase inhibitor, imatinib mesylate (STI571), from E17-P0 for 3 days caused loss of functionally developed ICC-MY networks, but ICC-MY and pacemaker activity recovered within 9 days after discontinuing treatment with neutralizing antibody or imatinib mesylate. These data suggest that Kit signaling is an important factor in lineage decision and in the development of functional ICC in late gestation. ICC-MY demonstrate significant plasticity in gastrointestinal tissues. Manipulation of the ICC phenotype might provide useful therapies in gastrointestinal disease where the Kit-positive cell population is either lost or amplified.

The excitability of smooth muscles is regulated, in part, by background K+ conductances that determine resting membrane potential. However, the K+ conductances so far described in gastrointestinal (GI) muscles are not sufficient to explain the negative resting potentials of these cells. Here we describe expression of two-pore K+ channels of the TASK family in murine small and large intestinal muscles. TASK-2, cloned from murine intestinal muscles, resulted in a pH-sensitive, time-dependent, non-inactivating K+ conductance with slow activation kinetics. A similar conductance was found in native intestinal myocytes using whole-cell patch-clamp conditions. The pH-sensitive current was blocked by local anaesthetics. Lidocaine, bupivacaine and acidic pH depolarized circular muscle cells in intact muscles and decreased amplitude and frequency of slow waves. The effects of lidocaine were not blocked by tetraethylammonium chloride, 4-aminopyridine, glibenclamide, apamin or MK-499. However, depolarization by acidic pH was abolished by pre-treatment with lidocaine, suggesting that lidocaine-sensitive K+ channels were responsible for pH-sensitive changes in membrane potential. The kinetics of activation, sensitivity to pH, and pharmacology of the conductance in intestinal myocytes and the expression of TASK-1 and TASK-2 in these cells suggest that the pH-sensitive background conductance is encoded by TASK genes. This conductance appears to contribute significantly to resting potential and may regulate excitability of GI muscles.

Autonomic neurotransmission is thought to occur via a loose association between nerve varicosities and smooth muscle cells. In the gastrointestinal tract ultrastructural studies have demonstrated close apposition between enteric nerves and intramuscular interstitial cells of Cajal (ICC-IM) in the stomach and colon and ICC in the deep muscular plexus (ICC-DMP) of the small intestine. In the absence of ICC-IM, postjunctional neural responses are compromised. Although membrane specializations between nerves and ICC-IM have been reported, the molecular identity of these specializations has not been studied. Here we have characterized the expression and distribution of synapse-associated proteins between nerve terminals and ICC-IM in the murine stomach. Transcripts for the presynaptic proteins synaptotagmin, syntaxin, and SNAP-25 were detected. Synaptotagmin and SNAP-25-immunopositive nerve varicosities were concentrated in varicose regions of motor nerves and were closely apposed to ICC-IM but not smooth muscle. W/W(V) mice were used to examine the expression and distribution of synaptic proteins in the absence of ICC-IM. Transcripts encoding synaptotagmin, syntaxin, and SNAP-25 were detected in W/W(V) tissues. In the absence of ICC-IM, synaptotagmin and SNAP-25 were localized to nerve varicosities. Reverse transcriptase polymer chain reaction (RT-PCR) and immunohistochemistry demonstrated the expression of postsynaptic density proteins PSD-93 and PSD-95 in the stomach and expression levels of PSD-93 and PSD-95 were reduced in W/W(V) mutants. These data support the existence of synaptic specializations between enteric nerves and ICC-IM in gastric tissues. In the absence of ICC-IM, components of the synaptic vesicle docking and fusion machinery is trafficked and concentrated in enteric nerve terminals.

Intracellular recordings were made from isolated bundles of the circular muscle layer of mouse and guinea-pig gastric fundus. These preparations displayed an ongoing discharge of membrane noise (unitary potentials), similar to that recorded from similar preparations made from the circular layer of the antrum. Bundles of muscle from the fundus of W/WV mice, which lack intramuscular interstitial cells of Cajal (ICCIM) lacked the discharge of membrane noise observed in wild-type tissues. When the membrane potential was changed by passing depolarizing or hyperpolarizing current pulses, the discharge of membrane noise was little changed. The membrane noise was unaffected by adding chloride channel blockers; however, agents which buffered the internal concentration of calcium ions reduced the discharge of membrane noise. Treatment of tissues with CCCP, which interferes with the uptake of calcium ions by mitochondria, also reduced the membrane noise and caused membrane hyperpolarization. Similar observations were made on bundles of tissue isolated from the circular layer of the guinea pig antrum. Together the observations indicate that membrane noise is generated by a pathway located in ICCIM. The properties of this pathway appear to vary dramatically within a given organ. The lack of voltage sensitivity of the discharge of membrane noise in the fundus provides a possible explanation for the lack of rhythmic electrical activity in this region of the stomach.

Phase advancement of electrical slow waves and regulation of pacemaker frequency was investigated in the circular muscle layer of the gastric antra of wild-type and W/W(V) mice. Slow waves in the murine antrum of wild-type animals had an intrinsic frequency of 4.4 cycles min(-1) and were phase advanced and entrained to a maximum of 6.3 cycles min(-1) using 0.1 ms pulses of electrical field stimulation (EFS) (three pulses delivered at 3-30 Hz). Pacing of slow waves was blocked by tetrodotoxin (TTX) and atropine, suggesting phase advancement was mediated via intrinsic cholinergic nerves. Phase advancement and entrainment of slow waves via this mechanism was absent in W/W(V) mutants which lack intramuscular interstitial cells of Cajal (ICC-IM). These data suggest that neural regulation of slow wave frequency and regulation of smooth muscle responses to slow waves are mediated via nerve-ICC-IM interactions. With longer stimulation parameters (1.0-2.0 ms), EFS phase advanced and entrained slow waves in wild-type and W/W(V) animals. Pacing with 1-2 ms pulses was not inhibited by TTX or atropine. These data suggest that stimulation with longer pulse duration is capable of directly activating the pacemaker mechanism in ICC-MY networks. In summary, intrinsic excitatory neurons can phase advance and increase the frequency of antral slow waves. This form of regulation is mediated via ICC-IM. Longer pulse stimulation can directly activate ICC-MY in the absence of ICC-IM.

Ca+/calmodulin-dependent protein kinase II (CaM kinase II) is regulated by calcium oscillations, autophosphorylation, and its subunit composition. All four subunit isoforms were detected in gastric fundus and proximal colon smooth muscles by RT-PCR, but only the γ and δ isoforms are expressed in myocytes. Relative γ and δ message levels were quantitated by real-time PCR. CaM kinase II protein and Ca2+/calmodulin-stimulated (total) activity levels are higher in proximal colon smooth muscle lysates than in fundus lysates, but Ca2+/calmodulin-independent (autonomous) activity is higher in fundus lysates. CaM kinase II in fundus lysates is relatively unresponsive to Ca2+/calmodulin. Alkaline phosphatase decreased CaM kinase II autonomous activity in fundus lysates and restored its responsiveness to Ca2+/calmodulin. Acetylcholine (ACh) increased autonomous CaM kinase II activity in fundus and proximal colon smooth muscles in a time- and dose-dependent manner. KN-93 enhanced ACh-induced fundus contractions but inhibited proximal colon contractions. The different properties of CaM kinase II from fundus and proximal colon smooth muscles suggest differential regulation of its autophosphorylation and activity in tonic and phasic gastrointestinal smooth muscles.

Studies of W/W(V) mice, which lack intramuscular interstitial cells of Cajal (IC-IM), have suggested that IC-IM act as mediators of enteric motor neurotransmission in the gastrointestinal tract. We have studied Sl/Sl(d) mice, which lack the ability to make membrane-bound stem cell factor, to determine the consequences of inappropriate stem cell factor expression on IC-IM populations and on enteric motor neurotransmission. IC-IM were found within the circular and longitudinal muscles of the gastric fundus of wild-type mice. IC-IM were intimately associated with motor nerve terminals and nerve varicosities formed synaptic structures with these cells. IC-IM were also connected with neighbouring smooth muscle cells via gap junctions. Immunohistochemistry and electron microscopy showed that IC-IM were absent from fundus muscles of Sl/Sl(d) mice, but the density of excitatory and inhibitory nerves was not significantly different than in wild-type muscles. Loss of IC-IM was associated with decreased membrane noise (unitary potentials) and significant reductions in post-junctional excitatory and inhibitory enteric nerve responses. Reductions in neural responses were not due to defects in smooth muscle cells as responses to exogenous ACh and K(+)-induced depolarization were normal in Sl/Sl(d) mice. Responses to neurally released ACh were revealed in Sl/Sl(d) mice by inhibiting ACh breakdown with the acetylcholinesterase inhibitor neostigmine. Inhibitory nerve stimulation elicited inhibitory junction potentials (IJPs) and relaxations in wild-type mice. IJPs were reduced in amplitude and relaxation responses were absent in Sl/Sl(d) mice. These observations suggest that membrane-bound stem cell factor is essential for development of IC-IM and that the close, synaptic-like relationship between nerve terminals and IC-IM may be the primary site of innervation by enteric motor neurons in gastric muscles.

When intracellular recordings were made from the antral region of murine stomach, cells with three different patterns of electrical activity were detected. One group of cells generated follower potentials, the second group generated pacemaker potentials and the third group generated slow waves that consisted of primary and secondary components. Slow waves recorded in different regions of the gastric antrum had similar amplitudes but different characteristic shapes. At the greater curvature, slow waves had large initial components. Midway between the greater and lesser curvature, the amplitude of the initial component was reduced and at the lesser curvature an initial component was difficult to detect. When the distributions of myenteric (ICC-MY) and intramuscular interstitial cells of Cajal (ICC-IM) were determined, using an antibody to Kit, ICC-MY were found to be present at the greater curvature but were greatly reduced in density at the lesser curvature. In contrast, ICC-IM were found in the circular layer of each region. When recordings were made from the antrum of W/W(V) mice, which lack ICC-IM, incomplete slow waves were detected and their amplitudes fell from the greater to the lesser curvature. Again, a corresponding fall in the density of ICC-MY was detected. The observations indicate that the contribution of ICC-MY and ICC-IM to the generation of slow waves varies in different regions of the mouse gastric antrum.

PGE(2) has been linked to the production of gastric arrhythmias such as tachygastria. The interstitial cells of Cajal (ICC) generate electrical rhythmicity in gastrointestinal muscles, and may therefore be a target for PGE(2) in gastric muscles. We cultured ICC from the murine gastric antrum, verified that cells were Kit immunoreactive, and measured spontaneous slow waves. These events were caused by spontaneous inward (pacemaker) currents that were not blocked by nifedipine. Forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP) reduced the frequency of pacemaker currents in ICC and of slow waves in intact antral muscles. The effects of forskolin and 8-Br-cAMP were not blocked by inhibitors of protein kinase A, suggesting that cAMP has direct effects on pacemaker activity. PGE(2) mimicked the effects of forskolin and 8-Br-cAMP on ICC, but increased slow-wave frequency in intact muscles. Therefore, the chronotropic effects of specific prostaglandin EP receptor agonists were examined. Butaprost and ONO-AE1-329, EP(2) and EP(4) receptor agonists, mimicked the effects of forskolin and 8-Br-cAMP on ICC and intact muscles. Sulprostone (EP(3)EP(1) agonist), GR63799, and ONO-AE-248 (EP(3) agonists) enhanced the frequencies of pacemaker currents in ICC and slow waves in intact muscles. The effects of sulprostone were not blocked by SC-19220, an EP(1) receptor antagonist. These observations suggest that the positive chronotropic effects of PGE(2) in intact muscles are mediated by EP(3) receptor stimulation. The effects of PGE(2) in intact muscles may be dependent upon the relative expression of EP receptors and/or proximity of receptors to sources of PGE(2).

Aims To describe the clinicopathological and immunophenotypic features of 26 cases of extra-medullary myeloid tumour (EMMT)/granulocytic sarcoma, which remains poorly recognized and is frequently confused with malignant lymphoma, and to discuss the main diagnostic problems experienced by the referring pathologist. Methods and results Haematoxylin and eosin (H & E) sections of 26 cases of EMMT were re-examined. Immunostains for myeloperoxidase, lysozyme, neutrophil elastase, LCA, CD79a, CD20, CD43, CD45RO, CD3, CD30, CD15, CD68, MAC387, VS38C, MIC2, and the Leder stain for naphthol-ASD-chloroacetate esterase were performed on all cases. Clinical and follow-up data were obtained through a questionnaire to the referring pathologist or from the notes of the patients where available. In the 10 cases with known myeloproliferative disease, the initial diagnosis was correct in 10 whereas all cases presenting with EMMT without a previous history of myeloproliferative disorder had an initial incorrect diagnosis. The most common suggested diagnosis was that of a non-Hodgkin's lymphoma. The morphology of the tumours varied from well differentiated which included all stages of myeloid differentiation to poorly differentiated or blastic showing little or no evidence of myeloid differentiation. The proportion of positive cells for each stain varied. Chloroacetate esterase, myeloperoxidase and CD15 stained a large proportion of cells of the majority of the well differentiated tumours and a smaller proportion of the poorly differentiated/blastic tumours with very focal staining of some of the cases. Lysozyme and CD43 were the most sensitive of the markers staining a large proportion of cells of the majority of the tumours in both groups. Neutrophil elastase was the least sensitive of the markers of myeloid differentiation. CD79a, CD20, CD3 and CD30 were negative in all cases. CD43 was positive in all cases. CD68 stained a substantial number of cells in the majority of tumours. A smaller proportion of the tumours stained with MAC387. Four of the tumours showed positivity for MIC2. One tumour was positive for VS38C. Conclusion This series documents continuing difficulties in the diagnosis of EMMT. Even well differentiated tumours are frequently mistakenly diagnosed as malignant lymphomas when they present without any history of antecedent myeloproliferative disorder. Careful evaluation of morphology for evidence of myeloid differentiation and a high index of suspicion when confronted with a less differentiated neoplasm are required to avoid this important diagnostic error. We suggest that a panel which includes chloroacetate-esterase, myeloperoxidase, lysozyme and CD43, together with other B- and T-lineage markers, in particular CD79a and CD3 should be used to confirm the diagnosis.

In the central nervous system, bidirectional signaling between glial cells and neurons (‘neuroimmune communication’) facilitates the development of persistent pain. Spinal glia can contribute to heightened pain states by a prolonged release of neurokine signals that sensitize adjacent centrally projecting neurons. Although many persistent pain conditions are disproportionately common in females, whether specific neuroimmune mechanisms lead to this increased susceptibility remains unclear. This review summarizes the major known contributions of glia and neuroimmune interactions in pain, which has been determined principally in male rodents and in the context of somatic pain conditions. It is then postulated that studying neuroimmune interactions involved in pain attributed to visceral diseases common to females may offer a more suitable avenue for investigating unique mechanisms involved in female pain. Further, we discuss the potential for primed spinal glia and subsequent neurogenic inflammation as a contributing factor in the development of peripheral inflammation, therefore, representing a predisposing factor for females in developing a high percentage of such persistent pain conditions.

This is the accepted version of the following article: [Spencer, N. J., Nicholas, S. J., Sia, T. C., Staikopoulos, V., Kyloh, M. and Beckett, E. A. (2013), By what mechanism does ondansetron inhibit colonic migrating motor complexes: does it require endogenous serotonin in the gut wall?. Neurogastroenterology & Motility, 25: 677–685.], which has been published in final form at [http://dx.doi.org/10.1111/nmo.12136]. In addition, authors may also transmit, print and share copies with colleagues, provided that there is no systematic distribution of the submitted version, e.g. posting on a listserve, network or automated delivery., 5-HT3 antagonists, such as ondansetron (Zofran), retard colonic transit and provide effective relief of symptoms of chronic diarrhea and diarrhea-predominant irritable bowel syndrome (IBS), but the mechanism by which ondansetron retards transit is unclear. What is clear is that the frequency of colonic migrating motor complexes (CMMCs) is reduced by ondansetron, which could account for reduced transit. Our aim was to determine whether an acute depletion of 5-HT from enteric neurons would inhibit spontaneous CMMCs; and determine whether the sensitivity of ondansetron to reduce CMMC frequency would change in a 5-HT-depleted preparation., Australian National Health & Medical Research Council

OBJECTIVE To examine the role of pH-sensitive K+ channels in setting the resting membrane potential in murine bladder smooth muscle, as bladder contractility is influenced by the resting membrane potential, which is mainly regulated by background K+ conductances. MATERIALS AND METHODS Using conventional microelectrode recordings, isometric tension measurements, patch-clamp recordings, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry, we assessed bladder smooth muscle cells and tissues. RESULTS Acidic pH (pH 6.5) depolarized the resting membrane potential of murine bladder smooth muscles and increased muscle tone and contractility. The pH-induced changes were not abolished by neuronal blockers or classical K+-channel antagonists. Lidocaine (1 mm) and bupivacaine (100 µm) mimicked the effects of acidifying the external solution, and in the presence of lidocaine no further increase in contractility was induced by reducing the pH to 6.5. Voltage-clamp experiments on freshly dispersed bladder myocytes showed that pH 6.5 decreased the outward current. Pre-treatment of bladder myocytes with the classical K+ antagonists tetraethylammonium (10 mm), 4-aminopyridine (5 mm), glibenclamide (10 µm) or apamin (300 nm) did not inhibit the effects of low pH on outward current. However, treatment with lidocaine (1 mm) abolished the effects of acidic pH on outward current. RT-PCR showed the expression of the acid-sensitive K+ channel (TASK)-1 and TASK-2 gene transcripts in murine bladder, and immunohistochemistry and Western blot analysis showed TASK-1 and TASK-2 channel expression and distribution in smooth muscle tissues and cells. CONCLUSION TASK channels are expressed in bladder smooth muscle and contribute to the basal K+ conductances responsible for resting membrane potential.

Background: It has recently become apparent that the lymph pump is an electrical entity thatrivals the heart in complexity. Many interesting currents have been demonstrated by voltage clamping isolated lymphatic smooth muscle cells, but until now the role of these currents in the intact syncitium has not been studied. Methods and Results: Intracellular microelectrode recordings were made from smooth muscle of sheep mesenteric lymphatics to investigate the electrophysiological basis of lymphatic pumping. Approximately 50% of the vessels exhibited spontaneous electrical activity, varying from regular oscillations in membrane potential to spike complexes. Spike complexes generally consisted of one or more action potentials superimposed on a slower depolarization or ‘plateau’ phase and were often preceded by a slow diastolic depolarization or ‘pre-potential’. Norepinephrine (5 uM) induced depolarizing events in quiescent preparations. Both agonist-induced oscillations and spike complexes were attenuated or completely abolished by 2-aminoethoxydiphenyl borate (2-APB); 10–100 uM). Cesium (1 mM) reduced the frequency of spontaneous firing by approximately 30% by flattening the pre-potential phase. In addition to having a negative inotropic effect, 10 mM Cs+ also caused gradual membrane depolarization and prolonged the plateau. 1 uM nifedipine abolished spontaneous events while tetrodotoxin (TTX; 0.5–1 uM) decreased the amplitude and maximum dV/dt of the spike upstroke or stopped activity completely. Spontaneously active segments of lymphatic vessel were inhibited by the chloride channel blocker, anthracene-9-carboxylic acid (9-AC; 250 uM-1 mM) suggesting that I Cl(Ca) plays a significant role in the generation of spontaneous activity in this tissue. Penitrem-A (0.1 uM) did not affect resting membrane potential but increased action potential amplitude and prolonged the plateau, suggesting that calcium-activated potassium current does not make a significant contribution to resting membrane conductance but is important in membrane repolarization following calcium influx during the action potential. In contrast 4-aminopyridine (4-AP; 5 uM) caused significant membrane depolarization, suggesting the existence of an active 4-AP-sensitive current at rest. Conclusions: These results demonstrate that the currents found in isolated voltage-clamped cells from sheep mesenteric lymphatics do play a significant role in the shaping of spontaneous electrical activity of the intact syncitium.

Interstitial cells of Cajal (ICC) have been identified in specific areas throughout the smooth musculature of the gastrointestinal (GI) tract. Located within the circular and longitudinal muscle layers of the gastric fundus lies a specific type of ICC, termed “intramuscular” ICC or IC-IM. The principal function of this cell type is to act as “mediators of excitatory and inhibitory enteric neurotransmission.” The functional role of these cells has been investigated using W/ Wvmutant mice that specifically lack IC-IM, resulting in disrupted enteric neurotransmission. The aim of the present study was to investigate differential gene expression in W/ Wvmutant mice, from the tunica muscularis of the gastric fundus using a mouse cDNA microarray containing 1,081 known genes. Verification of the microarray data was attained using real-time “quantitative” PCR (qPCR). Of the 1,081 arrayed genes, 36 demonstrated differential expression by >2-fold in the W/ Wvmice. An agreement rate of 50% (7 of 14 tested) was obtained using qPCR. Of the seven confirmed changes in expression, several were indicative of a supersensitive phenotype, observed in denervation models. Expression of several putative neurotransmitter receptors including P2Y, the receptor for the inhibitory neurotransmitter ATP, was upregulated. The functional role of the P2Y receptor was also investigated using electrophysiological recordings. These results offer a new insight into the molecular changes that occur in W/ Wvfundic smooth muscle and may also provide novel information with regard to the importance of IC-IM in enteric neurotransmission.

Interstitial cells of Cajal (ICC) are interposed between enteric neurons and smooth muscle cells in gastrointestinal muscles. The role of intramuscular ICC (IC-IM) in mediating enteric excitatory neural inputs was studied using gastric fundus muscles of wild-type animals andW/Wvmutant mice, which lack IC-IM. Excitatory motor neurons, labeled with antibodies to vesicular acetylcholine transporter or substance-P, were closely associated with IC-IM. Immunocytochemistry showed close contacts between enteric neurons and IC-IM. IC-IM also formed gap junctions with smooth muscle cells. Electrical field stimulation yielded fast excitatory junction potentials in the smooth muscle that were blocked by atropine. Neural responses were greatly reduced in muscles ofW/Wvanimals. Loss of cholinergic responses inW/Wvmuscles seemed to be caused by the loss of close synaptic contacts between motor neurons and IC-IM, because these muscles were not less responsive to exogenous acetylcholine than were wild-type muscles.W/Wvmuscles also responded to excitatory nerve stimulation when the breakdown of acetylcholine was blocked by neostigmine. The density of cholinergic nerve bundles within the muscles was not significantly different in wild-type andW/Wvmuscles, and similar amounts of14[C]choline were released from preloaded wild-type andW/Wvmuscles in response to nerve stimulation. The impact of losing IC-IM on gastric compliance was also evaluated in intact stomachs. Pressure increased as a function of fluid volume and infusion rate in wild-type animals, butW/Wvanimals showed little basal tone and minimal increases in pressure with fluid infusions. These data suggest that IC-IM play a major role in receiving cholinergic excitatory inputs from the enteric nervous system in the murine fundus.