Active peer-mentored learning can improve student understanding of physiological concepts in an undergraduate journal club

One of the most ubiquitous active learning modalities in the biological sciences at third level is the journal club. Journal club can promote several beneficial learning outcomes for students such as gaining critical reading skills to evaluate the scientific literature, improving scientific literacy, serving as an introduction to new concepts and techniques and improving communication skills. However, it can be difficult for instructors who facilitate journal club to gauge student audiences' understanding of topics being related by presenters. At the University of Nevada, Reno School of Medicine, international life sciences undergraduate students enrolled in our research program undergo a 12-month placement in selected research laboratories within the medical school in order to develop an understanding of basic medical scientific research and physiological concepts. As such, an integral component of this program is participation in regular journal club sessions which we had assumed helped students to develop such an understanding. However as we had never empirically assessed if this was the case or not, the aim of the current study was to determine if student understanding could be improved by complementing the standard journal club with peer-mentored workshop presentations. Data from this case study suggest that by allowing students to undergo peer-mentored learning in conjunction with journal club, student understanding of physiological concepts, as well as student confidence in presenting and communication, increases

The effect of high [K(+)]o on spontaneous Ca(2+) waves in freshly isolated interstitial cells of Cajal from the rabbit urethra.

Interstitial cells of Cajal (ICC) act as putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous global Ca(2+) waves that can be increased in frequency by raising external [K(+)]. The purpose of this study was to elucidate the mechanism of this response. Intracellular [Ca(2+)] was measured in fluo-4-loaded smooth muscle cells (SMCs) and ICC using a Nipkow spinning disk confocal microscope. Increasing [K(+)]o to 60 mmol/L caused an increase in [Ca(2+)]i accompanied by contraction in SMCs. Raising [K(+)]o did not cause contraction in ICC, but the frequency of firing of spontaneous calcium waves increased. Reducing [Ca(2+)]o to 0 mmol/L abolished the response in both cell types. Nifedipine of 1 μmol/L blocked the response of SMC to high [K(+)]o, but did not affect the increase in firing in ICC. This latter effect was blocked by 30 μmol/L NiCl2 but not by the T-type Ca(2+) channel blocker mibefradil (300 nmol/L). However, inhibition of Ca(2+) influx via reverse-mode sodium/calcium exchange (NCX) using either 1 μmol/L SEA0400 or 5 μmol/L KB-R7943 did block the effect of high [K(+)]o on ICC. These data suggest that high K(+) solution increases the frequency of calcium waves in ICC by increasing Ca(2+) influx through reverse-mode NCX

Tonic inhibition of murine proximal colon is due to nitrergic suppression of Ca2+ signaling in interstitial cells of Cajal

Spontaneous excitability and contractions of colonic smooth muscle cells (SMCs) are normally suppressed by inputs from inhibitory motor neurons, a behavior known as tonic inhibition. The post-junctional cell(s) mediating tonic inhibition have not been elucidated. We investigated the post-junctional cells mediating tonic inhibition in the proximal colon and whether tonic inhibition results from suppression of the activity of Ano1 channels, which are expressed exclusively in interstitial cells of Cajal (ICC). We found that tetrodotoxin (TTX), an inhibitor of nitric oxide (NO) synthesis, L-NNA, and an inhibitor of soluble guanylyl cyclase, ODQ, greatly enhanced colonic contractions. Ano1 antagonists, benzbromarone and Ani9 inhibited the effects of TTX, L-NNA and ODQ. Ano1 channels are activated by Ca2+ release from the endoplasmic reticulum (ER) in ICC, and blocking Ca2+ release with a SERCA inhibitor (thapsigargin) or a store-operated Ca(2+ )entry blocker (GSK 7975 A) reversed the effects of TTX, L-NNA and ODQ. Ca2+ imaging revealed that TTX, L-NNA and ODQ increased Ca2+ transient firing in colonic ICC. Our results suggest that tonic inhibition in the proximal colon occurs through suppression of Ca2+ release events in ICC. Suppression of Ca2+ release in ICC limits the open probability of Ano1 channels, reducing the excitability of electrically-coupled SMCs

Ca2+‐activated Cl− channels (TMEM16A) underlie spontaneous electrical activity in isolated mouse corpus cavernosum smooth muscle cells

Abstract Penile detumescence is maintained by tonic contraction of corpus cavernosum smooth muscle cells (CCSMC), but the underlying mechanisms have not been fully elucidated. The purpose of this study was to characterize the mechanisms underlying activation of TMEM16A Ca2+‐activated Cl− channels in freshly isolated murine CCSMC. Male C57BL/6 mice aged 10–18 weeks were euthanized via intraperitoneal injection of sodium pentobarbital (100 mg.kg−1). Whole‐cell patch clamp, pharmacological, and immunocytochemical experiments were performed on isolated CCSM. Tension measurements were performed in whole tissue. TMEM16A expression in murine corpus cavernosum was confirmed using immunocytochemistry. Isolated CCSMC developed spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarizations (STDs) in current clamp mode of the whole cell, perforated patch clamp technique. STICs reversed close to the predicted Cl− equilibrium potential and both STICs and STDs were blocked by the TMEM16A channel blockers, Ani9 and CaCC(inh)‐A01. These events were also blocked by GSK7975A (ORAI inhibitor), cyclopiazonic acid (CPA, sarcoplasmic reticulum [SR] Ca2+‐ATPase blocker), tetracaine (RyR blocker), and 2APB (IP3R blocker), suggesting that they were dependent on Ca2+ release from intracellular Ca2+ stores. Nifedipine (L‐type Ca2+ channel blocker) did not affect STICs, but reduced the duration of STDs. Phenylephrine induced transient depolarizations and transient inward currents which were blocked by Ani9. Similarly, phenylephrine induced phasic contractions of intact corpus cavernosum muscle strips and these events were also inhibited by Ani9. This study suggests that contraction of CCSM is regulated by activation of TMEM16A channels and therefore inhibition of these channels could lead to penile erection

Inhibitory Neural Regulation of the Ca2+ Transients in Intramuscular Interstitial Cells of Cajal in the Small Intestine

Gastrointestinal motility is coordinated by enteric neurons. Both inhibitory and excitatory motor neurons innervate the syncytium consisting of smooth muscle cells (SMCs) interstitial cells of Cajal (ICC) and PDGFRα+ cells (SIP syncytium). Confocal imaging of mouse small intestines from animals expressing GCaMP3 in ICC were used to investigate inhibitory neural regulation of ICC in the deep muscular plexus (ICC-DMP). We hypothesized that Ca2+ signaling in ICC-DMP can be modulated by inhibitory enteric neural input. ICC-DMP lie in close proximity to the varicosities of motor neurons and generate ongoing Ca2+ transients that underlie activation of Ca2+-dependent Cl− channels and regulate the excitability of SMCs in the SIP syncytium. Electrical field stimulation (EFS) caused inhibition of Ca2+ for the first 2–3 s of stimulation, and then Ca2+ transients escaped from inhibition. The NO donor (DEA-NONOate) inhibited Ca2+ transients and Nω-Nitro-L-arginine (L-NNA) or a guanylate cyclase inhibitor (ODQ) blocked inhibition induced by EFS. Purinergic neurotransmission did not affect Ca2+ transients in ICC-DMP. Purinergic neurotransmission elicits hyperpolarization of the SIP syncytium by activation of K+ channels in PDGFRα+ cells. Generalized hyperpolarization of SIP cells by pinacidil (KATP agonist) or MRS2365 (P2Y1 agonist) also had no effect on Ca2+ transients in ICC-DMP. Peptidergic transmitter receptors (VIP and PACAP) are expressed in ICC and can modulate ICC-DMP Ca2+ transients. In summary Ca2+ transients in ICC-DMP are blocked by enteric inhibitory neurotransmission. ICC-DMP lack a voltage-dependent mechanism for regulating Ca2+ release, and this protects Ca2+ handling in ICC-DMP from membrane potential changes in other SIP cells

Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility

Microtubules position ion channels close to each other to ensure autoregulation of vascular tone in cerebral arteries.</jats:p

Excitatory Neuronal Responses of Ca 2+

Interstitial cells of Cajal (ICC) regulate smooth muscle excitability and motility in the gastrointestinal (GI) tract. ICC in the deep muscular plexus (ICC-DMP) of the small intestine are aligned closely with varicosities of enteric motor neurons and thought to transduce neural responses. ICC-DMP generate Ca2+ transients that activate Ca2+ activated Cl-channels and generate electrophysiological responses. We tested the hypothesis that excitatory neurotransmitters regulate Ca2+ transients in ICC-DMP as a means of regulating intestinal muscles. High-resolution confocal microscopy was used to image Ca2+ transients in ICC-DMP within murine small intestinal muscles with cell-specific expression of GCaMP3. Intrinsic nerves were stimulated by electrical field stimulation (EFS). ICC-DMP exhibited ongoing Ca2+ transients before stimuli were applied. EFS caused initial suppression of Ca2+ transients, followed by escape during sustained stimulation, and large increases in Ca2+ transients after cessation of stimulation. Basal Ca2+ activity and the excitatory phases of Ca2+ responses to EFS were inhibited by atropine and neurokinin 1 receptor (NK1) antagonists, but not by NK2 receptor antagonists. Exogenous ACh and substance P (SP) increased Ca2+ transients, atropine and NK1 antagonists decreased Ca2+ transients. Neurokinins appear to be released spontaneously (tonic excitation) in small intestinal muscles and are the dominant excitatory neurotransmitters. Subcellular regulation of Ca2+ release events in ICC-DMP may be a means by which excitatory neurotransmission organizes intestinal motility patterns

Clustering of Ca2+ transients in interstitial cells of Cajal defines slow wave duration

Interstitial cells of Cajal (ICC) in the myenteric plexus region (ICC-MY) of the small intestine are pacemakers that generate rhythmic depolarizations known as slow waves. Slow waves depend on activation of Ca2+-activated Cl- channels (ANO1) in ICC, propagate actively within networks of ICC-MY, and conduct to smooth muscle cells where they generate action potentials and phasic contractions. Thus, mechanisms of Ca2+ regulation in ICC are fundamental to the motor patterns of the bowel. Here, we characterize the nature of Ca2+ transients in ICC-MY within intact muscles, using mice expressing a genetically encoded Ca2+ sensor, GCaMP3, in ICC. Ca2+ transients in ICC-MY display a complex firing pattern caused by localized Ca2+ release events arising from multiple sites in cell somata and processes. Ca2+ transients are clustered within the time course of slow waves but fire asynchronously during these clusters. The durations of Ca2+ transient clusters (CTCs) correspond to slow wave durations (plateau phase). Simultaneous imaging and intracellular electrical recordings revealed that the upstroke depolarization of slow waves precedes clusters of Ca2+ transients. Summation of CTCs results in relatively uniform Ca2+ responses from one slow wave to another. These Ca2+ transients are caused by Ca2+ release from intracellular stores and depend on ryanodine receptors as well as amplification from IP3 receptors. Reduced extracellular Ca2+ concentrations and T-type Ca2+ channel blockers decreased the number of firing sites and firing probability of Ca2+ transients. In summary, the fundamental electrical events of small intestinal muscles generated by ICC-MY depend on asynchronous firing of Ca2+ transients from multiple intracellular release sites. These events are organized into clusters by Ca2+ influx through T-type Ca2+ channels to sustain activation of ANO1 channels and generate the plateau phase of slow waves