Terence K. Smith

Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections

SummaryThe distribution of nerve cells with immunoreactivity for the calcium-binding protein, calbindin, has been studied in the small intestine of the guinea-pig, and the projections of these neurons have been analysed by tracing their processes and by examining the consequences of nerve lesions. The immunoreactive neurons were numerous in the myenteric ganglia; there were 3500±100 reactive nerve cells per cm2 of undistended intestine, which is 30% of all nerve cells. In contrast, reactive nerve cells were extremely rare in submucous ganglia. The myenteric nerve cells were oval in outline and gave rise to several long processes; this morphology corresponds to Dogiels type-II classification. Processes from the cell bodies were traced through the circular muscle in perforating nerve fibre bundles. Other processes ran circumferentially in the myenteric plexus. Removal of the myenteric plexus, allowing time for subsequent fibre degeneration, showed that reactive nerve fibres in the submucous ganglia and mucosa came from the myenteric cell bodies. Operations to sever longitudinal or circumferential pathways in the myenteric plexus indicated that most reactive nerve terminals in myenteric ganglia arise from myenteric cell bodies whose processes run circumferentially for 1.5 mm, on average. It is deduced that the calbindin-reactive neurons are multipolar sensory neurons, with the sensitive processes in the mucosa and with other processes innervating neurons of the myenteric plexus.

Distension-evoked ascending and descending reflexes in the circular muscle of guinea-pig ileum : an intracellular study

Reflex responses evoked by distension of the guinea-pig small intestine were recorded from the circular muscle with intracellular microelectrodes. For this purpose a mechanically stable preparation that allowed the intestinal wall to be distended within 9 mm of the recording site was developed. A segment of intestine was opened along the mesenteric border and pinned mucosa uppermost over a balloon set in the base of an organ bath, so that inflation of the balloon could distend the intestinal wall without simultaneously pushing against the mucosa. Compound excitatory junction potentials (EJPs) and compound inhibitory junction potentials (IJPs) were recorded at sites up to 40 mm oral and anal to the distending stimulus, respectively. The compound EJPs recorded orally had amplitudes of up to 24 mV and declined to baseline during distensions that exceeded 10-15 s. Distensions at intervals of less than 20 s evoked successively smaller oral compound EJPs; after four distensions in 30 the amplitude of the compound EJP had fallen to less than 10%. The amplitude of the oral compound EJP was reduced by hyoscine (1 microM), but the extent of the reduction depended on the degree of distension; responses to mild stimuli were blocked, whereas those to strong stimuli were only slightly reduced. The amplitude of the hyoscine-resistant component of the compound EJP was markedly reduced by antagonists of substance P receptors in the muscle. In the presence of muscarinic and substance P receptor antagonists, a transient compound IJP could be detected on the oral side of the stimulus. The compound IJPs recorded anal to the distension had amplitudes up to 22 mV but the potential returned to baseline during prolonged distension. In the presence of hyoscine (1 microM) some inhibitory activity continued throughout prolonged stimuli. Compound IJP amplitudes were not significantly reduced by repeated distensions separated by more than 6 s. At anal sites a transient depolarization (off-response) was recorded immediately following the termination of a distension in some preparations. The off-response was unaffected by hyoscine and was more readily observed after the further addition of substance P antagonists. The compound IJPs were almost completely blocked by apamin (0.2 microM). The compound EJPs and IJPs recorded orally were blocked by hexamethonium (100 microM), but the amplitudes of compound IJPs recorded anally were significantly reduced by hexamethonium (100-200 microM) only at recording sites greater than 15 mm from the centre of the balloon. The off-response was reduced by hexamethonium at all sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Reflex changes in circular muscle activity elicited by stroking the mucosa: an electrophysiological analysis in the isolated guinea-pig ileum

A preparation of isolated small intestine from the guinea-pig was studied in which reflex responses of the circular muscle were recorded intracellularly when sensory receptors in the mucosa were stimulated mechanically. This preparation was used to examine the properties of mucosa to muscle reflexes that involve non-cholinergic motor neurons innervating the circular muscle. Reproducible stimulation of the mucosa was achieved by stroking with a motor-driven brush. Gentle brush-strokes applied to the mucosa typically evoked inhibitory junction potentials anal to the stimulus and excitatory junction potentials at recording sites oral to the stimulus. Both events were rapid in onset and up to 25 mV in amplitude. The reflexes were blocked by tetrodotoxin (0.5 microM). Junction potentials declined in amplitude with distance from the stimulus, the amplitude of the excitation 15 mm oral to the stimulus was half that at 5 mm from the stimulus, whereas the amplitude of the inhibitory potential at 40-45 mm was about 60% of that at 5-10 mm anal to the stimulus. Hexamethonium (100-200 microM) blocked the ascending excitation but only slightly reduced the descending inhibition. Ascending excitation was blocked by antagonists for substance P receptors in the muscle, and inhibition was substantially reduced by apamin (0.2 microM), both before and after hexamethonium. Both responses were abolished by removal of the mucosa from the stimulus site and when lesions were made through the myenteric plexus between the stimulation and recording sites, but persisted when similar lesions were made through the submucous plexus. It is concluded that there are neurons with mechanoreceptive nerve endings in the mucosa. Stimulation of such sensory neurons leads to activation of pathways in the myenteric plexus that excite motor neurons to the muscle both oral and anal to the stimulation site. The demonstration that mucosa to muscle reflexes can be consistently evoked in the small intestine in vitro provides an opportunity for close analysis of the reflex pathways. Such analysis is not so readily achieved when reflexes are initiated by distension that, by moving the intestine, can dislodge the recording electrode.

Mechanosensory S‐neurons rather than AH‐neurons appear to generate a rhythmic motor pattern in guinea‐pig distal colon

Simultaneous intracellular recordings were made from myenteric neurons and circular muscle (CM) cells in isolated, stretched segments of guinea‐pig distal colon. We have shown previously that maintained stretch generates a repetitive and coordinated discharge of ascending excitatory and descending inhibitory neuronal reflex pathways in the distal colon. In the presence of nifedipine (1–2 μm) to paralyse the muscle, simultaneous recordings were made from 25 pairs of AH (after‐hyperpolarization)‐neurons and CM cells separated by 100–500 μm. In all 25 AH‐neurons, proximal process potentials (PPPs) were never recorded, even though at the same time, all recordings from neighbouring CM cells showed an ongoing discharge of inhibitory junction potentials (IJPs) anally, or excitatory junction potentials (EJPs) orally. In fact, 24 of 25 AH‐neurons were totally silent, while in one AH‐cell, some spontaneous fast excitatory postsynaptic potentials (FEPSPs) were recorded. All 10 electrically silent AH‐cells that were injected with neurobiotin were found to be multipolar Dogiel type II neurons. In contrast, when recordings were made from myenteric S‐neurons, two distinct electrical patterns of electrical activity were recorded. Recordings from 25 of 48 S‐neurons showed spontaneous FEPSPs, the majority of which (22 of 25) showed periods when discrete clusters of  FEPSPs (mean duration 88 ms) could be temporally correlated with the onset of EJPs or anal IJPs in the CM. Nine S‐neurons were electrically quiescent. The second distinct electrical pattern in 14 S‐neurons consisted of bursts, or prolonged trains of action potentials, which could be reduced to proximal process potentials (PPPs) in six of these 14 neurons during membrane hyperpolarization. Unlike FEPSPs, PPPs were resistant to a low Ca2+–high Mg2+ solution and did not change in amplitude during hyperpolarizing pulses. Mechanosensory  S‐neurons were found to be uniaxonal or pseudounipolar filamentous neurons, with morphologies consistent with interneurons. No slow EPSPs were ever recorded from AH‐ or S‐type neurons when IJPs or EJPs occurred in the CM. In summary, we have identified a population of mechanosensory S‐neurons in the myenteric plexus of the distal colon which appear to be largely stretch sensitive, rather than muscle‐tension sensitive, since they generate ongoing trains of action potentials in the presence of nifedipine. No evidence was found to suggest that in paralysed preparations, the repetitive firing in ascending excitatory or descending inhibitory nerve pathways was initiated by myenteric AH‐neurons, or slow synaptic transmission.

Synchronous movements of the longitudinal and circular muscle during peristalsis in the isolated guinea‐pig distal colon

1 Peristalsis, which involves enteric nervous reflexes, is the co‐ordinated movements of the longitudinal (LM) and circular (CM) muscle layers that propel intraluminal contents down the bowel. Although the movements of the CM during peristalsis are reasonably clear the relative movements of the LM are poorly understood. 2 We studied the oral and anal movements of the LM and CM during a peristaltic wave in isolated segments of guinea‐pig distal colon. Dissection techniques were used to prevent mechanical interactions between the LM and CM; also, the colonic segment was passed through a partition to prevent mechanical disturbances created by a peristaltic wave in the bulk of the colon from influencing the end from which recordings were made. 3 Peristalsis was generated by slowly filling the lumen of the colon with fluid. At threshold, the LM and CM synchronously contracted oral (ascending excitation) to, and relaxed anal (descending inhibition) to, a peristaltic wave. The anal relaxation was followed by a contraction (descending excitation) of both muscle layers. 4 Atropine (1 μm) in the recording chamber reduced both the oral (LM by 40 % and CM by 27 %) and anal (LM by 36 % and CM by 36 %) contractile responses as well as the anal relaxation response in both muscle layers. Hexamethonium (300 μm) almost blocked the oral contractile responses of the LM and CM but had no affect on the anal responses of either muscle layer. 5 N ω‐nitro‐L‐arginine (L‐NA; 100 μm) reduced the oral contractile response of the LM and CM by 50 %, the anal contractile response of the LM by 30 %, and the anal relaxation response of the LM and CM by about 30 %. The anal contractile response of the CM was unaffected by L‐NA. 6 Apamin (0.5 μm) also reduced the evoked anal relaxation of both the LM and CM by about 50 %. Further addition of L‐NA nearly abolished the relaxation response in the LM, but did not cause any further reduction in the relaxation response of the CM observed in apamin alone. 7 It is concluded, that the LM and CM exhibit synchronous movements during peristalsis in the colon. Also, peristalsis consists of activation of ascending excitatory, and descending inhibitory and excitatory nervous pathways to the LM and CM, which are cholinergic and non‐cholinergic, respectively. Nitric oxide is an important neuromodulator within the intrinsic nervous pathways.

Purinergic and cholinergic neuro‐neuronal transmission underlying reflexes activated by mucosal stimulation in the isolated guinea‐pig ileum

1 We present evidence that adenosine triphosphate (ATP) plays a major role in excitatory neuro‐neuronal transmission in ascending and descending reflex pathways to the longitudinal (LM) and circular muscle (CM). 2 A partitioned bath was used for the pharmacological isolation of a segment of guinea‐pig ileum (∼6 cm in length), allowing drugs to be selectively applied to an intermediate region between the region where mucosal stimulation was applied and that where mechanical recordings were made. 3 Brush stroking the mucosa (3 strokes) elicited a synchronous contraction of the LM and CM both above (ascending excitation) and below (descending excitation) the site of stimulation. All reflexes were abolished when tetrodotoxin (1 μm) was applied to the intermediate chamber. 4 Hexamethonium (300 μm) added to the intermediate chamber abolished the ascending contraction in 15 % of oral preparations (from 26 preparations, 18 animals) and the descending contraction in 13 % of anal preparations studied (from 53 preparations, 48 animals). In the remaining 85 % of oral preparations, hexamethonium usually attenuated the oral contraction of the LM and CM. However, in the remaining 87 % of anal preparations, hexamethonium had no effect on the anal contraction of the LM and CM. 5 Oral and anal reflexes that were hexamethonium resistant were either abolished or attenuated by the further addition of the P2 purinergic receptor antagonist pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS, 10 μm) or α,β‐methylene ATP (50–100 μm) to the intermediate chamber. 6 1,1‐Dimethyl‐4‐phenyl‐piperazinium iodide (DMPP, 20 μm) or α,β‐methylene ATP (50–100 μm) stimulated both ascending and descending excitatory pathways, when applied to the intermediate chamber. 7 In conclusion, ascending and descending neuro‐neuronal transmission in excitatory nervous pathways to the LM and CM is complex and clearly involves neurotransmitter(s) other than acetylcholine (ACh). We suggest mucosal stimulation releases ACh and ATP in both ascending and descending excitatory reflex pathways that synapse with excitatory motoneurons to the LM and CM.

Does the guinea-pig ileum obey the ‘law of the intestine’?

1 We report the first simultaneous mechanical reflex responses of the longitudinal muscle (LM) and circular muscle (CM) layers of the guinea‐pig ileum following mucosal stimulation and distension in vitro. 2 Dissection techniques were used to prevent mechanical interaction between the LM and CM layers both oral and anal to a stimulus site. 3 All graded stimuli produced graded contractions of both the LM and CM orally and anally to the stimulus. Contractions occurred synchronously in the LM and CM and under no circumstances were inhibitory responses recorded in either muscle layer, despite the presence of ongoing cholinergic tone in both the LM and CM. Contractions were abolished by tetrodotoxin (1.6 μm). 4 Local brush stroking of the mucosa evoked a peristaltic wave which readily conducted distally over 13 cm, without the presence of fluid in the lumen. No descending relaxation was observed. 5 Apamin (300 nm) disrupted evoked peristaltic waves and significantly increased the rate‐of‐rise of the LM and CM contractions anal to a stimulus, and the LM oral to a stimulus. 6 N ω‐nitro‐L‐arginine (100 μm), a nitric oxide synthesis inhibitor, had no overall significant effect on the characteristics of the LM and CM contractions, although on occasion an enhancement in their peak amplitude was noted. 7 It is suggested that the guinea‐pig ileum does not conform to the ‘law of the intestine’ as postulated by Bayliss & Starling (1899) . Rather, local physiological stimulation of the ileum elicits a contraction both orally and anally to a stimulus, which occurs synchronously in both the CM and LM layers. Apamin‐sensitive inhibitory neurotransmission modulates the rate‐of‐rise of the anal contraction of the CM, possibly to generate distal propulsion.

Motoneurones of the submucous plexus regulate electrical activity of the circular muscle of canine proximal colon.

The hypothesis that the circular muscle of the canine proximal colon receives motor input from neurones in the submucous plexus was tested. Circular muscle cells were impaled with micro‐electrodes and submucous plexus neurones were stimulated by electrical field stimulation and microejection of acetylcholine (ACh). In the presence of atropine to block the direct muscarinic effects, microejection of ACh onto the submucosa where intact submucous ganglia were suspended evoked: (i) an inhibitory junction potential (i.j.p.) that reduced the amplitude, duration and rate of rise of the subsequent slow wave; (ii) a slow wave of increased duration following the initial inhibitory response. These responses were enhanced by increasing the volume of ACh administered. Responses to ACh were blocked by hexamethonium, 10(‐4) M; d‐tubocurarine, 10(‐4) M; or tetrodotoxin (TTX), 10(‐6) M, suggesting they were neural in origin. Both inhibitory and excitatory responses were the result of non‐cholinergic and non‐adrenergic nerves. The transmitters mediating these effects are unknown. Removal of the longitudinal muscle, myenteric plexus, and the serosal portion of the circular muscle had no apparent effect on the responses to application of ACh to submucosal ganglia. In these preparations the responses to field stimulation were identical to those produced by ACh. The submucous plexus also provides cholinergic input to the circular muscle. When ACh was discretely applied to the submucosa cholinergic responses were elicited at the muscle cell which were significantly reduced by hexamethonium or TTX. These findings suggest that the cholinergic responses were the result of ACh release by neurones at the effector and not by overflow of the exogenous ACh. Cholinergic responses were also elicited in preparations in which the myenteric plexus had been removed. Slow waves in circular muscle of the proximal colon yield excitation‐contraction coupling in the absence of Ca2+ action potentials. Therefore the influence of submucous neurones on electrical slow waves has direct consequences on motor activity. Reduction in the amplitude and duration of slow wave by i.j.p.s. results in reduction in the amplitude and duration of phasic contractions. Excitatory inputs enhance contractions. The data support a new concept: motoneurones emanating from submucous ganglia innervate the circular muscle and provide inhibitory and excitatory inputs to regulate slow wave activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Induction and organization of Ca2+ waves by enteric neural reflexes.

The motility of the gastrointestinal tract consists of local, non-propulsive mixing (pendular or segmental) and propulsive (peristaltic) movements. It is generally considered that mixing movements are produced by intrinsic pacemakers which generate rhythmic contractions, and peristalsis by intrinsic excitatory and inhibitory neural reflex pathways,,, but the relationship between mixing and peristalsis is poorly understood. Peristalsis is compromised in mice lacking interstitial cells of Cajal, suggesting that these pacemaker cells may also be involved in neural reflexes. Here we show that mixing movements within longitudinal muscle result from spontaneously generated waves of elevated internal calcium concentration which originate from discrete locations (pacing sites), spread with anisotropic conduction velocities in all directions, and terminate by colliding with each other or with adjacent neurally suppressed regions. Excitatory neural reflexes control the spread of excitability by inducing new pacing sites and enhancing the overall frequency of pacing, whereas inhibitory reflexes suppress the ability of calcium waves to propagate. We provide evidence that the enteric nervous system organizes mixing movements to generate peristalsis, linking the neural regulation of pacemakers to both types of gut motility.