Peter O. Bayguinov

Localized Release of Serotonin (5-Hydroxytryptamine) by a Fecal Pellet Regulates Migrating Motor Complexes in Murine Colon

BACKGROUND & AIMS The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of fecal matter through a rhythmic sequence of electrical activity and/or contractions along the large bowel. CMMCs have largely been studied in empty preparations; we investigated whether local reflexes generated by a fecal pellet modify the CMMC to initiate propulsive activity. METHODS Recordings of CMMCs were made from the isolated murine large bowel, with or without a fecal pellet. Transducers were placed along the colon to record muscle tension and propulsive force on the pellet and microelectrodes were used to record electrical activity from either side of a fecal pellet, circular muscle cells oral and anal of a pellet, and in colons without the mucosa. RESULTS Spontaneous CMMCs propagated in both an oral or anal direction. When a pellet was inserted, CMMCs increased in frequency and propagated anally, exerting propulsive force on the pellet. The amplitude of slow waves increased during the CMMC. Localized mucosal stimulation/circumferential stretch evoked a CMMC, regardless of stimulus strength. The serotonin (5-hydroxytryptamine-3) receptor antagonist ondansetron reduced the amplitude of the CMMC, the propulsive force on the pellet, and the response to mucosal stroking, but increased the apparent conduction velocity of the CMMC. Removing the mucosa abolished spontaneous CMMCs, which still could be evoked by electrical stimulation. CONCLUSIONS The fecal pellet activates local mucosal reflexes, which release serotonin (5-hydroxytryptamine) from enterochromaffin cells, and stretch reflexes that determine the site of origin and propagation of the CMMC, facilitating propulsion.

Ca2+ imaging of activity in ICC-MY during local mucosal reflexes and the colonic migrating motor complex in the murine large intestine.

Colonic migrating motor complexes (CMMCs) are neurally mediated, cyclical contractile and electrical events, which typically propagate along the colon every 2–3 min in the mouse. We examined the interactions between myenteric neurons, interstitial cells of Cajal in the myenteric region (ICC‐MY) and smooth muscle cells during CMMCs using Ca2+ imaging. CMMCs occurred spontaneously or were evoked by stimulating the mucosa locally, or by brushing it at either end of the colon. Between CMMCs, most ICC‐MY were often quiescent; their lack of activity was correlated with ongoing Ca2+ transients in varicosities on the axons of presumably inhibitory motor neurons that were on or surrounded ICC‐MY. Ca2+ transients in other varicosities initiated intracellular Ca2+ waves in adjacent ICC‐MY, which were blocked by atropine, suggesting they were on the axons of excitatory motor neurons. Following TTX (1 μm), or blockade of inhibitory neurotransmission with Nω‐nitro‐l‐arginine (l‐NA, a NO synthesis inhibitor, 10 μm) and MRS 2500 (a P2Y1 antagonist, 1 μm), ongoing spark/puff like activity and rhythmic intracellular Ca2+ waves (38.1 ± 2.9 cycles min−1) were observed, yet this activity was uncoupled, even between ICC‐MY in close apposition. During spontaneous or evoked CMMCs there was an increase in the frequency (62.9 ± 1.4 cycles min−1) and amplitude of Ca2+ transients in ICC‐MY and muscle, which often had synchronized activity. At the same time, activity in varicosites along excitatory and inhibitory motor nerve fibres increased and decreased respectively, leading to an overall excitation of ICC‐MY. Atropine (1 μm) reduced the evoked responses in ICC‐MY, and subsequent addition of an NK1 antagonist (RP 67580, 500 nm) completely blocked the responses to stimulation, as did applying these drugs in reverse order. An NKII antagonist (MEN 10,376, 500 nm) had no effect on the evoked responses in ICC‐MY. Following TTX application, carbachol (1 μm), substance P (1 μm) and an NKI agonist (GR73632, 100 nm) produced the fast oscillations superimposed on a slow increase in Ca2+ in ICC‐MY, whereas SNP (an NO donor, 10 μm) abolished all activity in ICC‐MY. In conclusion, ICC‐MY, which are under tonic inhibition, are pacemakers whose activity can be synchronized by excitatory nerves to couple the longitudinal and circular muscles during the CMMC. ICC‐MY receive excitatory input from motor neurons that release acetylcholine and tachykinins acting on muscarinic and NK1 receptors, respectively.

Ca2+ transients in myenteric glial cells during the colonic migrating motor complex in the isolated murine large intestine.

Non‐technical summary  A ganglionated neural plexus, the myenteric plexus, within the colon generates a propagating contraction called the colonic migrating motor complex (CMMC) that underlies faecal pellet propulsion. Neurons in the myenteric plexus are surrounded by a network of enteric glia cells (EGCs), which were traditionally thought to be the glue that held the neurons together. Using imaging techniques, we demonstrate that 36% of EGCs respond with prolonged Ca2+ transients following their activation by excitatory nerve fibres that generate the CMMC, suggesting that EGCs are innervated and are activated during the CMMC.

Heterogeneities in ICC Ca2+ Activity Within Canine Large Intestine

BACKGROUND & AIMS In human and canine colon, both slow (slow waves, 2-8/min) and fast (myenteric potential oscillations [MPOs]; 16-20/min) electrical rhythms in the smooth muscle originate at the submucosal and myenteric borders, respectively. We used Ca(2+) imaging to investigate whether interstitial cells of Cajal (ICCs) at these borders generated distinct rhythms. METHODS Segments of canine colon were pinned with submucosal or myenteric surface uppermost or cut in cross section. Tissues were loaded with a Ca(2+) indicator (fluo-4), and activity was monitored at 36.5 +/- 0.5 degrees C using an electron multiplying charge coupled device (EMCCD). RESULTS Rhythmic, biphasic Ca(2+) transients (5-8/min), similar in waveform to electrical slow waves, propagated without decrement as a wave front (2-5 mm/s) through the ICC-SM network lying along the submucosal surface of the circular muscle (CM). In contrast, rhythmic intracellular Ca(2+) waves (approximately 16/min) and spontaneous reductions in Ca(2+) were observed in ICCs at the myenteric border (ICC-MY). Normally, intracellular Ca(2+) waves were unsynchronized between adjacent ICC-MY, although excitatory nerve activity synchronized activity. In addition, spontaneous reductions in Ca(2+) were observed that inhibited Ca(2+) waves. N omega-nitro-L-arginine (100 micromol/L; nitric oxide antagonist) blocked the reductions in Ca(2+) and increased the frequency (approximately 19/min) of intracellular Ca(2+) waves within ICC-MY. CONCLUSIONS ICC-SMs form a tightly coupled network that is able to generate and propagate slow waves. In contrast, Ca(2+) transients in ICC-MYs, which are normally not synchronized, have a similar duration and frequency as MPOs. Like MPOs, their activity is inhibited by nitrergic nerves and synchronized by excitatory nerves.

ICC‐MY coordinate smooth muscle electrical and mechanical activity in the murine small intestine

Background  Animals carrying genetic mutations have provided powerful insights into the role of interstitial cells of Cajal (ICC) in motility. One classic model is the W/WV mouse which carries loss‐of‐function mutations in c‐kit alleles, but retains minimal function of the tyrosine kinase. Previous studies have documented loss of slow waves and aberrant motility in the small intestine of W/WV mice where myenteric ICC (ICC‐MY) are significantly depleted.

Calcium activity in different classes of myenteric neurons underlying the migrating motor complex in the murine colon

The spontaneous colonic migrating motor complex (CMMC) is a cyclical contractile and electrical event that is the primary motor pattern underlying fecal pellet propulsion along the murine colon. We have combined Ca2+ imaging with immunohistochemistry to determine the role of different classes of myenteric neurons during the CMMC. Between CMMCs, myenteric neurons usually displayed ongoing but uncoordinated activity. Stroking the mucosa at the oral or anal end of the colon resulted in a CMMC (latency: ∼6 to 10 s; duration: ∼28 s) that consisted of prolonged increases in activity in many myenteric neurons that was correlated to Ca2+ transients in and displacement of the muscle. These neurons were likely excitatory motor neurons. Activity in individual neurons during the CMMC was similar regardless of whether the CMMC occurred spontaneously or was evoked by anal or oral mucosal stimulation. This suggests that convergent interneuronal pathways exist which generate CMMCs. Interestingly, Ca2+ transients in a subset of NOS +ve neurons were substantially reduced during the CMMC. These neurons are likely to be inhibitory motor neurons that reduce their activity during a complex (disinhibition) to allow full excitation of the muscle. Local stimulation of the mucosa evoked synchronized Ca2+ transients in Dogiel Type II (mitotracker/calbindin‐positive) neurons after a short delay (∼1–2 s), indicating they were the sensory neurons underlying the CMMC. These local responses were observed in hexamethonium, but were blocked by ondansetron (5‐HT3 antagonist), suggesting Dogiel Type II neurons were activated by 5‐HT release from enterochromaffin cells in the mucosa. In fact, removal of the mucosa yielded no spontaneous CMMCs, although many neurons (NOS +ve and NOS −ve) exhibited ongoing activity, including Dogiel Type II neurons. These results suggest that spontaneous or evoked 5‐HT release from the mucosa is necessary for the activation of Dogiel Type II neurons that generate CMMCs.

Colonic elongation inhibits pellet propulsion and migrating motor complexes in the murine large bowel.

The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pattern. Although the CMMC spontaneously propagates along an empty colon it is responsible for faecal pellet propulsion in the murine large bowel. Unlike the peristaltic reflex, the CMMC is an ‘all or none’ event that appears to be dependent upon Dogiel Type II/AH neurons for its regenerative slow propagation down the colon. A reduction in the amplitude of CMMCs or an elongated colon have both been thought to underlie slow transit constipation, although whether these phenomena are related has not been considered. In this study we examined the mechanisms by which colonic elongation might affect the CMMC using video imaging of the colon, tension and electrophysiological recordings from the muscle and Ca2+ imaging of myenteric neurons. As faecal pellets were expelled from the murine colon, it shortened by up to ∼29%. Elongation of the colon resulted in a linear reduction in the velocity of a faecal pellet and the amplitude of spontaneous CMMCs. Elongation of the oral end of a colonic segment reduced the amplitude of CMMCs, whereas elongation of the anal end of the colon evoked a premature CMMC, and caused the majority of CMMCs to propagate in an anal to oral direction. Dogiel Type II/AH sensory neurons and most other myenteric neurons responded to oral elongation with reduced amplitude and frequency of spontaneous Ca2+ transients, whereas anal elongation increased their amplitude and frequency in most neurons. The inhibitory effects of colonic elongation were reduced by blocking nitric oxide (NO) production with l‐NA (100 μm) and soluble guanylate cyclase with 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 10 μm); whereas, l‐arginine (1–2 mm) enhanced the inhibitory effects of colonic elongation. In conclusion, polarized neural reflexes can be triggered by longitudinal stretch. The dominant effect of elongation is to reduce CMMCs primarily by inhibiting Dogiel Type II/AH neurons, thus facilitating colonic accommodation and slow transit.

Polarized intrinsic neural reflexes in response to colonic elongation

Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despite this, transit through the shorter colon is at least 10 times slower. Recently we demonstrated that elongating a segment of colon releases nitric oxide (NO) to inhibit peristalsis. The aims of this study were to determine if colonic elongation was physiologically significant, and whether elongation activated polarized intrinsic neural reflexes. Video imaging monitored fecal pellet evacuation from isolated guinea‐pig colons full of pellets. Recordings were made from the circular muscle (CM) and longitudinal muscle (LM) in flat sheet preparations using either intracellular microelectrode or Ca2+ imaging techniques. Full colons were 158.1 ± 6.1% longer than empty colons. As each pellet was expelled, the colon shortened and pellet velocity increased exponentially (full 0.34, empty 1.01 mm s−1). In flat sheet preparations, maintained circumferential stretch generated ongoing peristaltic activity (oral excitatory and anal inhibitory junction potentials) and Ca2+ waves in LM and CM. Colonic elongation (140% of its empty slack length) applied oral to the recording site abolished these activities, whereas anal elongation significantly increased the frequency and amplitude of ongoing peristaltic activity. Oral elongation inhibited the excitation produced by anal elongation; this inhibitory effect was reversed by blocking NO synthesis. Pelvic nerve stimulation elicited polarized responses that were also suppressed by NO released during colonic elongation. In conclusion, longitudinal stretch excites specific mechosensitive ascending and descending interneurons, leading to activation of polarized reflexes. The dominance of the descending inhibitory reflex leads to slowed emptying of pellets in a naturally elongated colon.

Excitatory Synaptic Feedback from the Motor Layer to the Sensory Layers of the Superior Colliculus

Neural circuits that translate sensory information into motor commands are organized in a feedforward manner converting sensory information into motor output. The superior colliculus (SC) follows this pattern as it plays a role in converting visual information from the retina and visual cortex into motor commands for rapid eye movements (saccades). Feedback from movement to sensory regions is hypothesized to play critical roles in attention, visual image stability, and saccadic suppression, but in contrast to feedforward pathways, motor feedback to sensory regions has received much less attention. The present study used voltage imaging and patch-clamp recording in slices of rat SC to test the hypothesis of an excitatory synaptic pathway from the motor layers of the SC back to the sensory superficial layers. Voltage imaging revealed an extensive depolarization of the superficial layers evoked by electrical stimulation of the motor layers. A pharmacologically isolated excitatory synaptic potential in the superficial layers depended on stimulus strength in the motor layers in a manner consistent with orthodromic excitation. Patch-clamp recording from neurons in the sensory layers revealed excitatory synaptic potentials in response to glutamate application in the motor layers. The location, size, and morphology of responsive neurons indicated they were likely to be narrow-field vertical cells. This excitatory projection from motor to sensory layers adds an important element to the circuitry of the SC and reveals a novel feedback pathway that could play a role in enhancing sensory responses to attended targets as well as visual image stabilization.

Single-trial imaging of spikes and synaptic potentials in single neurons in brain slices with genetically encoded hybrid voltage sensor

Genetically encoded voltage sensors expand the optogenetics toolkit into the important realm of electrical recording, enabling researchers to study the dynamic activity of complex neural circuits in real time. However, these probes have thus far performed poorly when tested in intact neural circuits. Hybrid voltage sensors (hVOS) enable the imaging of voltage by harnessing the resonant energy transfer that occurs between a genetically encoded component, a membrane-tethered fluorescent protein that serves as a donor, and a small charged molecule, dipicrylamine, which serves as an acceptor. hVOS generates optical signals as a result of voltage-induced changes in donor-acceptor distance. We expressed the hVOS probe in mouse brain by in utero electroporation and in transgenic mice with a neuronal promoter. Under conditions favoring sparse labeling we could visualize single-labeled neurons. hVOS imaging reported electrically evoked fluorescence changes from individual neurons in slices from entorhinal cortex, somatosensory cortex, and hippocampus. These fluorescence signals tracked action potentials in individual neurons in a single trial with excellent temporal fidelity, producing changes that exceeded background noise by as much as 16-fold. Subthreshold synaptic potentials were detected in single trials in multiple distinct cells simultaneously. We followed signal propagation between different cells within one field of view and between dendrites and somata of the same cell. hVOS imaging thus provides a tool for high-resolution recording of electrical activity from genetically targeted cells in intact neuronal circuits.