Stimulation of extrinsic sympathetic nerves differentially affects neurogenic motor activity in guinea pig distal colon.

The speed of pellet propulsion through the isolated guinea pig distal colon in vitro significantly exceeds in vivo measurements, suggesting a role for inhibitory mechanisms from sources outside the gut. The aim of this study was to investigate the effects of sympathetic nerve stimulation on three different neurogenic motor behaviors of the distal colon: transient neural events (TNEs), colonic motor complexes (CMCs), and pellet propulsion. To do this, segments of guinea pig distal colon with intact connections to the inferior mesenteric ganglion (IMG) were set up in organ baths allowing for simultaneous extracellular suction electrode recordings from smooth muscle, video recordings for diameter mapping, and intraluminal manometry. Electrical stimulation (1-20 Hz) of colonic nerves surrounding the inferior mesenteric artery caused a statistically significant, frequency-dependent inhibition of TNEs, as well as single pellet propulsion, from frequencies of 5 Hz and greater. Significant inhibition of CMCs required stimulation frequencies of 10 Hz and greater. Phentolamine (3.6 µM) abolished effects of colonic nerve stimulation, consistent with a sympathetic noradrenergic mechanism. Sympathetic inhibition was constrained to regions with intact extrinsic nerve pathways, allowing normal motor behaviors to continue without modulation in adjacent extrinsically denervated regions of the same colonic segments. The results demonstrate differential sensitivities to sympathetic input among distinct neurogenic motor behaviors of the colon. Together with findings indicating CMCs activate colo-colonic sympathetic reflexes through the IMG, these results raise the possibility that CMCs may paradoxically facilitate suppression of pellet movement in vivo, through peripheral sympathetic reflex circuits.

Mechanisms underlying initiation of propulsion in guinea pig distal colon.

Colonic motor complexes (CMCs) are a major neurogenic activity in guineapig distal colon. The identity of the enteric neurons that initiate this activity is not established. Specialized intrinsic primary afferent neurons (IPANs) are a major candidate. We aimed to test this hypothesis. To do this, segments of guineapig distal colon were suspended vertically in heated organ baths and propulsive forces acting on a pellet inside the lumen were recorded by isometric force transducer while pharmacological agents were applied to affect IPAN function. In the absence of drugs, CMCs acted periodically on the pellet, generating peak propulsive forces of 12.7 ± 5 g at 0.56 ± 0.22 cpm, lasting 49 ± 17 s (215 preparations; n = 60). Most but not all CMCs were abolished by nicotinic receptor blockade to inhibit fast excitatory synaptic transmission (50/62 preparations; n = 25). Remarkably, CMCs inhibited by hexamethonium were restored by a pharmacological strategy that aimed to enhance IPAN excitability. Thus, CMCs were restored by increased smooth muscle tension (using BAY K8644, bethanechol or carbachol) and by IPAN excitation using phorbol dibutyrate; NK3 receptor agonist, senktide; and partially by αCGRP. The IPAN inhibitor, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one (DCEBIO), decreased CMC frequency. CGRP, but not NK3-receptor antagonists, decreased CMC frequency in naive preparations. Finally, CMCs were blocked by tetrodotoxin, and this was not reversed by any drugs listed above. These results support a major role for IPANs that does not require fast synaptic transmission, in the periodic initiation of neurogenic propulsive contractions. Endogenous CGRP plays a role in determining CMC frequency, whereas further unidentified signaling pathways may determine their amplitude and duration.NEW & NOTEWORTHY The colonic motor complex (CMC) initiates propulsion in guinea pig colon. Here, CMCs evoked by an intraluminal pellet were restored during nicotinic receptor blockade by pharmacological agents that directly or indirectly enhance intrinsic primary afferent neuron (IPAN) excitability. IPANs are the only enteric neuron in colon that contain CGRP. Blocking CGRP receptors decreased CMC frequency, implicating their role in CMC initiation. The results support a role for IPANs in the initiation of CMCs.

Characterization of alternating neurogenic motor patterns in mouse colon.

BACKGROUND: Colonic motor complexes (CMCs) have been widely recorded in the large intestine of vertebrates. We have investigated whether in the smooth muscle, a single unified pattern of electrical activity, or different patterns of electrical activity give rise to the different neurogenic patterns of motility underlying CMCs in vitro. METHODS: To study differences of the CMCs between proximal and distal colon, we used a novel combination of techniques to simultaneously record muscle diameter and force at multiple sites along the whole mouse colon ex vivo. In addition, electrical activity of smooth muscle was recorded by suction electrodes. KEY RESULTS: Two distinct types of CMCs were distinguished; CMCs that propagated along the entire colon (complete CMC) and CMCs which were restricted to the proximal colon (incomplete CMC). The two types of CMC often occurred in the same preparations. Incomplete CMCs had longer bursts of smooth muscle action potentials than complete CMCs and propagated more slowly. Interestingly, both types of CMC were associated with similar frequency bursts of smooth muscle action potentials at ~2.4 Hz. In the most proximal colon, an additional firing frequency was detected close to ~7 Hz generating multiple peaks within each CMC. CONCLUSIONS & INFERENCES: We report distinct characteristics underlying complete and incomplete CMCs in isolated mouse colon. Recognizing these distinct patterns of motility will be important for future interpretation of analysis of murine colonic motility recordings. The identification of alternating patterns of motor activity in proximal colon, but not distal colon may reflect specific neural mechanisms for fecal pellet formation.

Intrinsic sensory neurons provide direct input to motor neurons and interneurons in mouse distal colon via varicose baskets.

Connections from intrinsic primary afferent neurons (IPANs), to ascending motor and interneurons have been described in guinea pig colon. These mono- and polysynaptic circuits may underlie polarized motor reflexes evoked by local gut stimulation. There is a need to translate findings in guinea pig to mouse, a species increasingly used in enteric neuroscience. Here, mouse distal colon was immunolabeled for CGRP, a marker of putative IPANs. This revealed a combination of large, intensely immunofluorescent axons in myenteric plexus and circular muscle, and thinner varicose axons with less immunofluorescence. The latter formed dense, basket-like varicosity clusters (CGRP+ baskets) that enveloped myenteric nerve cell bodies. Immunolabeling after 4-5 days in organ culture caused loss of large CGRP+ axons, but not varicose CGRP+ fibers and CGRP+ baskets. Baskets were characterized further by triple labeling with CGRP, nitric oxide synthase (NOS) and calretinin (CALR) antibodies. Approximately half (48%) of nerve cell bodies inside CGRP+ baskets lacked both NOS and CALR, while two overlapping populations containing NOS and/or CALR comprised the remainder. Quantitative analysis revealed CGRP+ varicosities were most abundant in baskets, followed by CALR+ varicosities, with a high degree of colocalization between the two markers. Few NOS+ varicosities occurred in baskets. Significantly higher proportions of CALR+ and CGRP+ varicosities colocalized in baskets than in circular muscle. In conclusion, CGRP+ baskets in mouse colon are formed by intrinsic enteric neurons with a neurochemical profile consistent with IPANs and have direct connections to both excitatory and inhibitory neurons.