rearing-long-term-maintenance-eristalis-tenax-hoverflies-for-research

Rearing and Long-Term Maintenance of <em>Eristalis tenax</em> Hoverflies for Research Studies

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are ...

Flinders University

The neuronal mechanism by which distension of the colon triggers peristalsis and the propulsion of colonic contents is incompletely understood. In this study, we used video imaging and spatiotemporal mapping techniques to investigate the neuroneuronal mechanisms underlying peristalsis in isolated guinea pig distal colon. In direct contrast to previous studies, we found that hexamethonium (100 muM-1 mM) or mecamylamine (20 muM) never abolished peristalsis or fecal pellet propulsion, although a temporary blockade of peristalsis was common, giving the impression perhaps that peristalsis was blocked permanently. During the initiation of peristalsis, the intraluminal propulsive force applied to an inserted fecal pellet was significantly reduced by hexamethonium 100 muM, even though, once initiated, the propagation velocity of fecal pellets was never reduced by nicotinic antagonists. In the presence of hexamethonium or mecamylamine, further addition of PPADS (10 muM), ondansetron (1 muM), and SR 142801 (300 nM) had no inhibitory effect on the propagation velocity of fecal pellets. In these preparations, antagonists for nicotinic, purinergic (P2), serotonergic (5-HT3), or tachykinergic (NK3) receptors always abolished responses to the agonists for these receptors, confirming that when peristalsis occurred, nicotinic, P2, 5-HT3, and NK3 receptors were blocked. Tetrodotoxin abolished nonnicotinic peristalsis. In summary, nicotinic transmission contributes to excitatory neuroneuronal transmission underlying peristalsis and fecal pellet propulsion but is not required for peristalsis, nor fecal pellet propulsion, as once thought. These observations could be explained by an excitatory nonnicotinic neuroneuronal pathway that can generate peristalsis and induce normal fecal pellet propagation velocities but does not require nicotinic, P2, 5-HT3, or NK3 receptors.

Peristalsis and fecal pellet propulsion do not require nicotinic, purinergic, 5-HT3, or NK3 receptors in isolated guinea pig distal colon.

Endothelin peptides and their endogenous receptors play a major role in nociception in a variety of different organs. They also play an essential role in the development of the enteric nervous system. Mice with deletions of the endothelin-3 gene (lethal spotted mice, ls/ls) develop congenital aganglionosis. However, little is known about how nociception might be affected in the aganglionic rectum of mice deficient in endothelin-3. In this study we investigated changes in spinal afferent innervation and visceral pain transmission from the aganglionic rectum in ls/ls mice. Electromyogram recordings from anaesthetized ls/ls mice revealed a deficit in visceromotor responses arising from the aganglionic colorectum in response to noxious colorectal distension. Loss of visceromotor responses (VMRs) in ls/ls mice was selective, as no reduction in VMRs was detected after stimulation of the bladder or somatic organs. Calcitonin gene related peptide (CGRP) immunoreactivity, retrograde neuronal tracing and extracellular afferent recordings from the aganglionic rectum revealed decreased colorectal spinal innervation, combined with a reduction in mechanosensitivity of rectal afferents. The sensory defect in ls/ls mice is primarily associated with changes in low threshold wide dynamic range rectal afferents. In conclusion, disruption of endothelin 3 gene expression not only affects development and function of the enteric nervous system, but also specific classes of spinal rectal mechanoreceptors, which are required for visceral nociception from the colorectum.

Loss of visceral pain following colorectal distension in an endothelin-3 deficient mouse model of Hirschsprung's disease.

In patients with irritable bowel syndrome, visceral pain is evoked more readily following distension of the colorectum. However, the identity of extrinsic afferent nerve pathway that detects and transmits visceral pain from the colorectum to the spinal cord is unclear. In this study, we identified which extrinsic nerve pathway(s) underlies nociception from the colorectum to the spinal cord of rodents. Electromyogram recordings were made from the transverse oblique abdominal muscles in anesthetized wild type (C57BL/6) mice and acute noxious intraluminal distension stimuli (100-120 mmHg) were applied to the terminal 15 mm of colorectum to activate visceromotor responses (VMRs). Lesioning the lumbar colonic nerves in vivo had no detectable effect on the VMRs evoked by colorectal distension. Also, lesions applied to the right or left hypogastric nerves failed to reduce VMRs. However, lesions applied to both left and right branches of the rectal nerves abolished VMRs, regardless of whether the lumbar colonic or hypogastric nerves were severed. Electrical stimulation applied to either the lumbar colonic or hypogastric nerves in vivo, failed to elicit a VMR. In contrast, electrical stimulation (2-5 Hz, 0.4 ms, 60 V) applied to the rectum reliably elicited VMRs, which were abolished by selective lesioning of the rectal nerves. DiI retrograde labeling from the colorectum (injection sites 9-15 mm from the anus, measured in unstretched preparations) labeled sensory neurons primarily in dorsal root ganglia (DRG) of the lumbosacral region of the spinal cord (L6-S1). In contrast, injection of DiI into the mid to proximal colon (injection sites 30-75 mm from the anus, measured in unstretched preparations) labeled sensory neurons in DRG primarily of the lower thoracic level (T6-L2) of the spinal cord. The visceral pain pathway activated by acute noxious distension of the terminal 15 mm of mouse colorectum is transmitted predominantly, if not solely, through rectal/pelvic afferent nerve fibers to the spinal cord. The sensory neurons of this spinal afferent pathway lie primarily in the lumbosacral region of the spinal cord, between L6 and S1.

Identification of the visceral pain pathway activated by noxious colorectal distension in mice.

The mechanisms underlying distension-evoked peristalsis in the colon are incompletely understood. It is well known that, following colonic distension, 5-hydroxytryptamine (5-HT) is released from enterochromaffin (EC) cells in the intestinal mucosa. It is also known that exogenous 5-HT can stimulate peristalsis. These observations have led some investigators to propose that endogenous 5-HT release from EC cells might be involved in the initiation of colonic peristalsis, following distension. However, because no direct evidence exists to support this hypothesis, the aim of this study was to determine directly whether release of 5-HT from EC cells was required for distension-evoked colonic peristalsis. Real-time amperometric recordings of 5-HT release and video imaging of colonic wall movements were performed on isolated segments of guinea pig distal colon, during distension-evoked peristalsis. Amperometric recordings revealed basal and transient release of 5-HT from EC cells before and during the initiation of peristalsis, respectively. However, removal of mucosa (and submucosal plexus) abolished 5-HT release but did not inhibit the initiation of peristalsis nor prevent the propagation of fecal pellets or intraluminal fluid. Maintained colonic distension by fecal pellets induced repetitive peristaltic waves, whose intrinsic frequency was also unaffected by removal of the submucosal plexus and mucosa, although their propagation velocities were slower. In conclusion, the mechanoreceptors and sensory neurons activated by radial distension to initiate peristalsis lie in the myenteric plexus and/or muscularis externa, and their activation does not require the submucosal plexus, release of 5-HT from EC cells, nor the presence of the mucosa. The propagation of peristalsis and propulsion of liquid or solid content along the colon is entrained by activity within the myenteric plexus and/or muscularis externa and does not require sensory feedback from the mucosa, nor neural inputs arising from submucosal ganglia.

Mechanisms underlying distension-evoked peristalsis in guinea pig distal colon: is there a role for enterochromaffin cells?

The functional role of the different classes of visceral afferents that innervate the large intestine is poorly understood. Recent evidence suggests that low-threshold, wide-dynamic-range rectal afferents play an important role in the detection and transmission of visceral pain induced by noxious colorectal distension in mice. However, it is not clear which classes of spinal afferents are activated during naturally occurring colonic motor patterns or during intense contractions of the gut smooth muscle. We developed an in vitro colorectum preparation to test how the major classes of rectal afferents are activated during spontaneous colonic migrating motor complex (CMMC) or pharmacologically induced contraction. During CMMCs, circular muscle contractions increased firing in low-threshold, wide-dynamic-range muscular afferents and muscular-mucosal afferents, which generated a mean firing rate of 1.53 ± 0.23 Hz (n = 8) under isotonic conditions and 2.52 ± 0.36 Hz (n = 17) under isometric conditions. These low-threshold rectal afferents were reliably activated by low levels of circumferential stretch induced by increases in length (1-2 mm) or load (1-3 g). In a small proportion of cases (5 of 34 units), some low-threshold muscular and muscular-mucosal afferents decreased their firing rate during the peak of the CMMC contractions. High-threshold afferents were never activated during spontaneous CMMC contractions or tonic contractions induced by bethanechol (100 μM). High-threshold rectal afferents were only activated by intense levels of circumferential stretch (10-20 g). These results show that, in the rectal nerves of mice, low-threshold, wide-dynamic-range muscular and muscular-mucosal afferents are excited during contraction of the circular muscle that occurs during spontaneous CMMCs. No activation of high-threshold rectal afferents was detected during CMMCs or intense contractile activity in naïve mouse colorectum.

Firing patterns and functional roles of different classes of spinal afferents in rectal nerves during colonic migrating motor complexes in mouse colon.

Recent studies have shown that endogenous serotonin is not required for colonic peristalsis in vitro, nor gastrointestinal (GI) transit in vivo. However, antagonists of 5-Hydroxytryptamine (5-HT) receptors can inhibit peristalsis and GI-transit in mammals, including humans. This raises the question of how these antagonists inhibit GI-motility and transit, if depletion of endogenous 5-HT does not cause any significant inhibitory changes to either GI-motility or transit? We investigated the mechanism by which 5-HT3 and 5-HT4 antagonists inhibit distension-evoked peristaltic contractions in guinea-pig distal colon. In control animals, repetitive peristaltic contractions of the circular muscle were evoked in response to fixed fecal pellet distension. Distension-evoked peristaltic contractions were unaffected in animals with mucosa and submucosal plexus removed, that were also treated with reserpine (to deplete neuronal 5-HT). In control animals, peristaltic contractions were blocked temporarily by ondansetron (1-10 μM) and SDZ-205-557 (1-10 μM) in many animals. Interestingly, after this temporary blockade, and whilst in the continued presence of these antagonists, peristaltic contractions recovered, with characteristics no different from controls. Surprisingly, similar effects were seen in mucosa-free preparations, which had no detectable 5-HT, as detected by mass spectrometry. In summary, distension-evoked peristaltic reflex contractions of the circular muscle layer of the guinea-pig colon can be inhibited temporarily, or permanently, in the same preparation by selective 5-HT3 and 5-HT4 antagonists, depending on the concentration of the antagonists applied. These effects also occur in preparations that lack any detectable 5-HT. We suggest caution should be exercised when interpreting the effects of 5-HT3 and 5-HT4 antagonists; and the role of endogenous 5-HT, in the generation of distension-evoked colonic peristalsis. 

5-HT3 and 5-HT4 antagonists inhibit peristaltic contractions in guinea-pig distal colon by mechanisms independent of endogenous 5-HT.

There are many hypotheses accounting for detrusor overactivity; however, the exact mechanisms are still incompletely understood. We used a model of bladder outlet obstruction in male guinea pigs as a way to produce detrusor overactivity. The objective was to determine whether changes in voiding of obstructed guinea pigs correlates with specific changes in contractile activity of their isolated bladders in vitro.

Sarah Nicholas

Centre for Neuroscience

Flinders University

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

Sarah Nicholas

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Centre for Neuroscience

Flinders University

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

Centre for Neuroscience