Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus.

Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together.

Characterisation of One Class of Group III Sensory Neurons Innervating Abdominal Muscles of the Mouse.

Group III/IV striated muscle afferents are small diameter sensory neurons that play important roles in reflexes and sensation. To date, the morphological features of physiologically characterised group III/IV muscular afferents have not been identified. Here, the electrophysiological and morphological characteristics of sensory neurons innervating striated muscles of the mouse abdominal wall were investigated, ex vivo. Extracellular recordings were made from subcostal nerve trunks innervating the muscles. A distinctive class of mechanosensitive afferents was identified by a combination of physiological features including sensitivity to local compression, saturating response to graded stretch and, in most cases, absence of spontaneous firing. Studies were restricted to these distinctive units. These units had conduction velocities averaging 14 ±â€¯4 m/s (range: 8-20 m/s, n = 7); within the range of group III fibres in mice. Von Frey hairs were used to map receptive fields, which covered an area of 0.36 ±â€¯0.18 mm2 (n = 7). In 7 preparations, biotinamide filling of recorded nerve trunks revealed a single axon in the marked receptive field, with distinctive axonal branching and terminations meandering through the connective tissue sandwiched between two closely associated muscle layers. These axons were not immunoreactive for CGRP (n = 7) and were not activated by application of capsaicin (1 µM, n = 14). All of these afferents were strongly activated by a "metabolite mix" containing lactate, adenosine triphosphate and reduced pH. Responses to mechanical stimuli and to metabolites were additive. We have characterised a distinctive class of mechano- and chemo-sensitive group III afferent endings associated with connective tissue close to muscle fibres.

Distribution, projections, and association with calbindin baskets of motor neurons, interneurons, and sensory neurons in guinea-pig distal colon.

Normal gut function relies on the activity of the enteric nervous system (ENS) found within the wall of the gastrointestinal tract. The structural and functional organization of the ENS has been extensively studied in the guinea pig small intestine, but less is known about colonic circuitry. Given that there are significant differences between these regions in function, observed motor patterns and pathology, it would be valuable to have a better understanding of the colonic ENS. Furthermore, disorders of colonic motor function, such as irritable bowel syndrome, are much more common. We have recently reported specialized basket-like structures, immunoreactive for calbindin, that likely underlie synaptic inputs to specific types of calretinin-immunoreactive neurons in the guinea-pig colon. Based on detailed immunohistochemical analysis, we postulated the recipient neurons may be excitatory motor neurons and ascending interneurons. In the present study, we combined retrograde tracing and immunohistochemistry to examine the projections of circular muscle motor neurons, myenteric interneurons, and putative sensory neurons. We focused on neurons with immunoreactivity for calbindin, calretinin and nitric oxide synthase and their relationship with calbindin baskets. Retrograde tracing using indocarbocyanine dye (DiI) revealed that many of the nerve cell bodies surrounded by calbindin baskets belong to motor neurons and ascending interneurons. Unique functional classes of myenteric neurons were identified based on morphology, neuronal markers and polarity of projection. We provide evidence for three groups of ascending motor neurons based on immunoreactivity and association with calbindin baskets, a finding that may have significant functional implications.

New insights into neurogenic cyclic motor activity in the isolated guinea-pig colon.

BACKGROUND: The contents of the guinea pig distal colon consist of multiple pellets that move anally in a coordinated manner. This row of pellets results in continued distention of the colon. In this study, we have investigated quantitatively the features of the neurally dependent colonic motor patterns that are evoked by constant distension of the full length of guinea-pig colon. METHODS: Constant distension was applied to the excised guinea-pig by high-resolution manometry catheters or by a series of hooks. KEY RESULTS: Constant distension elicited regular Cyclic Motor Complexes (CMCs) that originated at multiple different sites along the colon and propagated in an oral or anal direction extending distances of 18.3±10.3 cm. CMCs were blocked by tetrodotoxin (TTX; 0.6 µ mol L-1 ), hexamethonium (100 µ mol L-1 ) or hyoscine (1 µ mol L-1 ). Application of TTX in a localized compartment or cutting the gut circumferentially disrupted the spatial continuity of CMCs. Localized smooth muscle contraction was not required for CMC propagation. Shortening the length of the preparations or disruption of circumferential pathways reduced the integrity and continuity of CMCs. CONCLUSIONS & INFERENCES: CMCs are a distinctive neurally dependent cyclic motor pattern, that emerge with distension over long lengths of the distal colon. They do not require changes in muscle tension or contractility to entrain the neural activity underlying CMC propagation. CMCs are likely to play an important role interacting with the neuromechanical processes that time the propulsion of multiple natural pellets and may be particularly relevant in conditions of impaction or obstruction, where long segments of colon are simultaneously distended.

Neurogenic and myogenic patterns of electrical activity in isolated intact mouse colon.

BACKGROUND: Relatively little is known about the electrical rhythmicity of the whole colon, where long neural pathways are preserved. METHODS: Smooth muscle electrical activity was recorded extracellularly from the serosa of isolated flat-sheet preparations consisting of the whole mouse colon (n=31). KEY RESULTS: Two distinct electrical patterns were observed. The first, long intense spike bursts, occurred every 349±256 seconds (0.2±0.2 cpm), firing action potentials for 31±11 seconds at 2.1±0.5 Hz. They were hexamethonium- and tetrodotoxin-sensitive, but persisted in nicardipine as 2 Hz electrical oscillations lacking action potentials. This pattern is called here neurogenic spike bursts. The second pattern, short spike bursts, occurred about every 30 seconds (2.0±0.6 cpm), with action potentials firing at about 1 Hz for 9 seconds (1.0±0.2 Hz, 9±4 seconds). Short spike bursts were hexamethonium- and tetrodotoxin-resistant but nicardipine-sensitive and thus called here myogenic spike bursts. Neurogenic spike bursts transiently delayed myogenic spike bursts, while blocking neurogenic activity enhanced myogenic spike burst durations. External stimuli significantly affected neurogenic but not myogenic spike bursts. Aboral electrical or mechanical stimuli evoked premature neurogenic spike bursts. Circumferential stretch significantly decreased intervals between neurogenic spike bursts. Lesioning the colon down to 10 mm segments significantly increased intervals or abolished neurogenic spike bursts, while myogenic spike bursts persisted. CONCLUSIONS & INFERENCES: Distinct neurogenic and myogenic electrical patterns were recorded from mouse colonic muscularis externa. Neurogenic spike bursts likely correlate with neurogenic colonic migrating motor complexes (CMMC) and are highly sensitive to mechanical stimuli. Myogenic spike bursts may correspond to slow myogenic contractions, whose duration can be modulated by enteric neural activity.

Computer vision-based diameter maps to study fluoroscopic recordings of small intestinal motility from conscious experimental animals.

BACKGROUND: When available, fluoroscopic recordings are a relatively cheap, non-invasive and technically straightforward way to study gastrointestinal motility. Spatiotemporal maps have been used to characterize motility of intestinal preparations in vitro, or in anesthetized animals in vivo. Here, a new automated computer-based method was used to construct spatiotemporal motility maps from fluoroscopic recordings obtained in conscious rats. METHODS: Conscious, non-fasted, adult, male Wistar rats (n=8) received intragastric administration of barium contrast, and 1-2 hours later, when several loops of the small intestine were well-defined, a 2 minutes-fluoroscopic recording was obtained. Spatiotemporal diameter maps (Dmaps) were automatically calculated from the recordings. Three recordings were also manually analyzed for comparison. Frequency analysis was performed in order to calculate relevant motility parameters. KEY RESULTS: In each conscious rat, a stable recording (17-20 seconds) was analyzed. The Dmaps manually and automatically obtained from the same recording were comparable, but the automated process was faster and provided higher resolution. Two frequencies of motor activity dominated; lower frequency contractions (15.2±0.9 cpm) had an amplitude approximately five times greater than higher frequency events (32.8±0.7 cpm). CONCLUSIONS & INFERENCES: The automated method developed here needed little investigator input, provided high-resolution results with short computing times, and automatically compensated for breathing and other small movements, allowing recordings to be made without anesthesia. Although slow and/or infrequent events could not be detected in the short recording periods analyzed to date (17-20 seconds), this novel system enhances the analysis of in vivo motility in conscious animals.

Hydrogen peroxide preferentially activates capsaicin-sensitive high threshold afferents via TRPA1 channels in the guinea pig bladder.

BACKGROUND AND PURPOSE: There is increasing evidence suggesting that ROS play a major pathological role in bladder dysfunction induced by bladder inflammation and/or obstruction. The aim of this study was to determine the effect of H2 O2 on different types of bladder afferents and its mechanism of action on sensory neurons in the guinea pig bladder. EXPERIMENTAL APPROACH: 'Close-to-target' single unit extracellular recordings were made from fine branches of pelvic nerves entering the guinea pig bladder, in flat sheet preparations, in vitro. KEY RESULTS: H2 O2 (300-1000 µM) preferentially and potently activated capsaicin-sensitive high threshold afferents but not low threshold stretch-sensitive afferents, which were only activated by significantly higher concentrations of hydrogen peroxide. The TRPV1 channel agonist, capsaicin, excited 86% of high threshold afferents. The TRPA1 channel agonist, allyl isothiocyanate and the TRPM8 channel agonist, icilin activated 72% and 47% of capsaicin-sensitive high threshold afferents respectively. The TRPA1 channel antagonist, HC-030031, but not the TRPV1 channel antagonist, capsazepine or the TRPM8 channel antagonist, N-(2-aminoethyl)-N-[[3-methoxy-4-(phenylmethoxy)phenyl]methyl]thiophene-2-carboxamide, significantly inhibited the H2 O2 -induced activation of high threshold afferents. Dimethylthiourea and deferoxamine did not significantly change the effect of H2 O2 on high threshold afferents. CONCLUSIONS AND IMPLICATIONS: The findings show that H2 O2 , in the concentration range detected in inflammation or reperfusion after ischaemia, evoked long-lasting activation of the majority of capsaicin-sensitive high threshold afferents, but not low threshold stretch-sensitive afferents. The data suggest that the TRPA1 channels located on these capsaicin-sensitive afferent fibres are probable targets of ROS released during oxidative stress.

Rotenone and elevated extracellular potassium concentration induce cell-specific fibrillation of α-synuclein in axons of cholinergic enteric neurons in the guinea-pig ileum.

BACKGROUND: Parkinson's disease is a progressive neurodegenerative disorder that results in the widespread loss of select classes of neurons throughout the nervous system. The pathological hallmarks of Parkinson's disease are Lewy bodies and neurites, of which α-synuclein fibrils are the major component. α-Synuclein aggregation has been reported in the gut of Parkinson's disease patients, even up to a decade before motor symptoms, and similar observations have been made in animal models of disease. However, unlike the central nervous system, the nature of α-synuclein species that form these aggregates and the classes of neurons affected in the gut are unclear. We have previously reported selective expression of α-synuclein in cholinergic neurons in the gut (J Comp Neurol. 2013; 521:657), suggesting they may be particularly vulnerable to degeneration in Parkinson's disease. METHODS: In this study, we used immunohistochemistry to detect α-synuclein oligomers and fibrils via conformation-specific antibodies after rotenone treatment or prolonged exposure to high [K+ ] in ex vivo segments of guinea-pig ileum maintained in organotypic culture. KEY RESULTS: Rotenone and prolonged raising of [K+ ] caused accumulation of α-synuclein fibrils in the axons of cholinergic enteric neurons. This took place in a time- and, in the case of rotenone, concentration-dependent manner. Rotenone also caused selective necrosis, indicated by increased cellular autofluorescence, of cholinergic enteric neurons, labeled by ChAT-immunoreactivity, also in a concentration-dependent manner. CONCLUSIONS & INFERENCES: To our knowledge, this is the first report of rotenone causing selective loss of a neurochemical class in the enteric nervous system. Cholinergic enteric neurons may be particularly susceptible to Lewy pathology and degeneration in Parkinson's disease.

High-resolution colonic motility recordings in vivo compared with ex vivo recordings after colectomy, in patients with slow transit constipation.

BACKGROUND: The pathogenesis of slow transit constipation (STC) remains poorly understood, with intrinsic and extrinsic abnormalities implicated. Here, we present high-resolution colonic manometry recordings from four STC patients recorded before total colectomy, and subsequently, ex vivo, after excision. METHODS: In four female, treatment-resistant STC patients (median age 35.5 years), a fiber-optic manometry catheter (72 sensors spaced at 1 cm intervals) was placed with the aid of a colonoscope, to the mid-transverse colon. Colonic manometry was recorded 2 h before and after a meal. After the colectomy, ex vivo colonic manometry was recorded in an organ bath. Ex vivo recordings were also made from colons from 4 patients (2 male; median age 67.5 years) undergoing anterior resection for nonobstructive carcinoma ('control' tissue). KEY RESULTS: A large increase in 'short single propagating contractions' was recorded in STC colon ex vivo compared to in vivo (ex vivo 61.3 ± 32.7 vs in vivo 2.5 ± 5/h). In STC patients, in vivo, the dominant frequency of contractile activity was 2-3 cycle per minute (cpm), whereas 1-cpm short-single propagating contractions dominated ex vivo. This same 1-cpm frequency was also dominant in control colons ex vivo. CONCLUSIONS & INFERENCES: In comparison to control adults, the colon of STC patients demonstrates significantly less propagating motor activity. However, once the STC colon is excised from the body it demonstrates a regular and similar frequency of propagating activity to control tissue. This paper provides interesting insights into the control of colonic motor patterns.

Sensory innervation of the guinea pig colon and rectum compared using retrograde tracing and immunohistochemistry.

BACKGROUND: Neurons in lumbar and sacral dorsal root ganglia (DRG) comprise extrinsic sensory pathways to the distal colon and rectum, but their relative contributions are unclear. In this study, sensory innervation of the rectum and distal colon in the guinea pig was directly compared using retrograde labeling combined with immunohistochemistry. METHODS: The lipophilic tracer, DiI, was injected in either the rectum or distal colon of anesthetized guinea pigs, then DRG (T6 to S5) and nodose ganglia were harvested and labeled using antisera for calcitonin gene-related peptide (CGRP) and transient receptor potential vanilloid 1(TRPV1). KEY RESULTS: More primary afferent cell bodies were labeled from the rectum than from the distal colon. Vagal sensory neurons, with cell bodies in the nodose ganglia comprised fewer than 0.5% of labeled sensory neurons. Spinal afferents to the distal colon were nearly all located in thoracolumbar DRG, in a skewed unimodal distribution (peak at L2); fewer than 1% were located in sacral ganglia. In contrast, spinal afferents retrogradely labeled from the rectum had a bimodal distribution, with one peak at L3 and another at S2. Fewer than half of all retrogradely labeled spinal afferent neurons were immunoreactive for CGRP or TRPV1 and these included the larger traced neurons, especially in thoracolumbar ganglia. CONCLUSIONS & INFERENCES: In the guinea pig, both the distal colon and the rectum receive a sensory innervation from thoracolumbar ganglia. Sacral afferents innervate the rectum but not the distal colon. Calcitonin gene-related peptide immunoreactivity was detectable in fewer than half of afferent neurons in both pathways.

Electrophysiological and morphological changes in colonic myenteric neurons from chemotherapy-treated patients: a pilot study.

BACKGROUND: Patients receiving anticancer chemotherapy experience a multitude of gastrointestinal side-effects. However, the causes of these symptoms are uncertain and whether these therapeutics directly affect the enteric nervous system is unknown. Our aim was to determine whether the function and morphology of myenteric neurons are altered in specimens of the colon from chemotherapy-treated patients. METHODS: Colon specimens were compared from chemotherapy-treated and non-treated patients following colorectal resections for removal of carcinoma. Intracellular electrophysiological recordings from myenteric neurons and immunohistochemistry were performed in whole mount preparations. KEY RESULTS: Myenteric S neurons from chemotherapy-treated patients were hyperexcitable; more action potentials (11.4 ± 9.4, p < 0.05) were fired in response to depolarising current pulses than in non-treated patients (1.4 ± 0.5). The rheobase and the threshold to evoke action potentials were significantly lower for neurons from chemotherapy-treated patients compared to neurons from non-treated patients (p < 0.01). Fast excitatory postsynaptic potential reversal potential was more positive in neurons from chemotherapy-treated patients (p < 0.05). An increase in the number of neurons with translocation of Hu protein from the cytoplasm to the nucleus was observed in specimens from chemotherapy-treated patients (103 ± 25 neurons/mm(2) , 37.2 ± 7.0%, n = 8) compared to non-treated (26 ± 5 neurons/mm(2) , 11.9 ± 2.7%, n = 12, p < 0.01). An increase in the soma size of neuronal nitric oxide synthase-immunoreactive neurons was also observed in these specimens. CONCLUSIONS & INFERENCES: This is the first study suggesting functional and structural changes in human myenteric neurons in specimens of colon from patients receiving anticancer chemotherapy. These changes may contribute to the causation of gastrointestinal symptoms experienced by chemotherapy-treated patients.

Activation of intestinal spinal afferent endings by changes in intra-mesenteric arterial pressure.

KEY POINTS: A major class of mechano-nociceptors to the intestine have mechanotransduction sites on extramural and intramural arteries and arterioles ('vascular afferents'). These sensory neurons can be activated by compression or axial stretch of vessels. Using isolated preparations we showed that increasing intra-arterial pressure, within the physiological range, activated mechano-nociceptors on vessels in intact mesenteric arcades, but not in isolated arteries. This suggests that distortion of the branching vascular tree is the mechanical adequate stimulus for these sensory neurons, rather than simple distension. The same rises in pressure also activated intestinal peristalsis in a partially capsaicin-sensitive manner indicating that pressure-sensitive vascular afferents influence enteric circuits. The results identify the mechanical adequate stimulus for a major class of mechano-nociceptors with endings on blood vessels supplying the gut wall; these afferents have similar endings to ones supplying other viscera, striated muscle and dural vessels. ABSTRACT: Spinal sensory neurons innervate many large blood vessels throughout the body. Their activation causes the hallmarks of neurogenic inflammation: vasodilatation through the release of the neuropeptide calcitonin gene-related peptide and plasma extravasation via tachykinins. The same vasodilator afferent neurons show mechanical sensitivity, responding to crushing, compression or axial stretch of blood vessels - responses which activate pain pathways and which can be modified by cell damage and inflammation. In the present study, we tested whether spinal afferent axons ending on branching mesenteric arteries ('vascular afferents') are sensitive to increased intravascular pressure. From a holding pressure of 5 mmHg, distension to 20, 40, 60 or 80 mmHg caused graded, slowly adapting increases in firing of vascular afferents. Many of the same afferent units showed responses to axial stretch, which summed with responses evoked by raised pressure. Many vascular afferents were also sensitive to raised temperature, capsaicin and/or local compression with von Frey hairs. However, responses to raised pressure in single, isolated vessels were negligible, suggesting that the adequate stimulus is distortion of the arterial arcade rather than distension per se. Increasing arterial pressure often triggered peristaltic contractions in the neighbouring segment of intestine, an effect that was mimicked by acute exposure to capsaicin (1 µm) and which was reduced after desensitisation to capsaicin. These results indicate that sensory fibres with perivascular endings are sensitive to pressure-induced distortion of branched arteries, in addition to compression and axial stretch, and that they contribute functional inputs to enteric motor circuits.

Quantitative immunohistochemical co-localization of TRPV1 and CGRP in varicose axons of the murine oesophagus, stomach and colorectum.

In the gastrointestinal (GI) tract of mammals, endings of spinal afferent neurons with cell bodies in dorsal root ganglia (DRG) detect many stimuli, including those that give rise to pain. Many of these sensory neurons express calcitonin gene-related peptide (CGRP) and TRPV1 in their cell bodies and axons. Indeed, CGRP and TRPV1 have been widely used as immunohistochemical markers of nociceptive spinal afferent axons. Although CGRP and TRPV1 often coexist in the same axons in the GI tract, their degree of coexistence along its length has yet to be quantified. In this study, we used double-labeling immunohistochemistry to quantify the coexistence of CGRP and TRPV1 in varicose axons of the murine oesophagus, stomach and colorectum. The great majority of CGRP-immunoreactive (IR) varicosities in myenteric ganglia of the lower esophagus (97±1%) and stomach (95±1%) were also TRPV1-immunoreactive. Similarly, the majority of TRPV1-IR varicosities in myenteric ganglia of the lower esophagus (95±1%) and stomach (91±1%) were also CGRP-IR. In the colorectum similar observations were made for an intensely immunoreactive population of CGRP-IR axons, of which most (91±1%) were also TRPV1-IR. Of the TRPV1-IR axons in the colorectum, most (96±1%) contained intense CGRP-IR. Another population of axons in myenteric ganglia of the colorectum had low intensity CGRP immunoreactivity; these showed negligible co-existence with TRPV1. Our observations reveal that in the myenteric plexus of murine oesophagus, stomach and colorectum, CGRP and TRPV1 are largely expressed together.

Human enteric neurons: morphological, electrophysiological, and neurochemical identification.

BACKGROUND: Access to tissue, difficulties with dissection, and poor visibility of enteric ganglia have hampered electrophysiological recordings of human enteric neurons. Here, we report a method to combine intracellular recording with simultaneous morphological identification of neurons in the intact myenteric plexus of human colon ex vivo. METHODS: Specimens of human colon were dissected into flat-sheet preparations with the myenteric plexus exposed. Myenteric neurons were impaled with conventional microelectrodes containing 5% 5,6-carboxyfluorescein in 20 mM Tris buffer and 1 M KCl. KEY RESULTS: Electrophysiological recordings identified myenteric neurons with S and AH type properties (n = 13, N = 7) which were dye filled and classified during the recording as Dogiel type I (n = 10), Dogiel type II (n = 2), or filamentous (n = 1) cells. This classification was confirmed after fixation, in combination with immunohistochemical characterization. CONCLUSIONS & INFERENCES: This method allows electrophysiological characterization with simultaneous identification of morphology. It can be used to identify recorded cells immediately after impalement and greatly facilitates recordings of human myenteric neurons in freshly dissected specimens of tissue. It can also be combined with immunohistochemical labeling of recorded cells.

Targeted electrophysiological analysis of viscerofugal neurons in the myenteric plexus of guinea-pig colon.

Enteric viscerofugal neurons are mechanosensory interneurons that form the afferent limb of intestino-intestinal reflexes involving prevertebral sympathetic neurons. Fast synaptic inputs to viscerofugal neurons arise from other enteric neurons, but their sources are unknown. We aimed to describe the origins of synaptic inputs to viscerofugal neurons by mapping the locations of their cell bodies within the myenteric plexus. Viscerofugal neuron somata were retrogradely traced with 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) from colonic nerve trunks and impaled with microelectrodes, in longitudinal muscle/myenteric plexus preparations of the guinea-pig distal colon (39 impalements, n=14). Thirty-eight viscerofugal neurons were uni-axonal and had the electrophysiological characteristics of myenteric S-neurons; one neuron was multipolar with AH-neuron electrophysiological characteristics. Depolarizing current pulses evoked either single- or multiple action potentials in viscerofugal neurons (range 1-25 spikes, 500 ms, 100-900 pA, 21 cells). Electrical stimulation of internodal strands circumferential to viscerofugal neurons evoked fast excitatory postsynaptic potentials (EPSPs) in 19/24 cells. Focal pressure-ejection of the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP, 10 µm) directly onto viscerofugal nerve cell bodies evoked large depolarizations and action potentials (23 ± 10 mV, latency 350 ± 230 ms, 21/22 cells). DMPP was then focally applied to multiple sites, up to 3mm from the recorded viscerofugal neuron, to activate other myenteric S-neurons. In a few sites in myenteric ganglia, DMPP evoked repeatable fast EPSPs in viscerofugal neurons (latency 300 ± 316 ms, 38/394 sites, 10 cells). The cellular sources of synaptic inputs to viscerofugal neurons were located both orally and aborally (19 oral, 19 aboral), but the amplitude of oral inputs was consistently greater than aboral inputs (13.1 ± 4.3 mV vs. 10.1 ± 4.8 mV, respectively, p<0.05, paired t-test, n=6). Most impaled viscerofugal neurons were nitric oxide synthase (NOS) immunoreactive (20/27 cells tested). Thus, the synaptic connections onto viscerofugal neurons within the myenteric plexus suggest that multiple enteric neural pathways feed into intestino-intestinal reflexes, involving sympathetic prevertebral ganglia.

Neurogenic and myogenic motor activity in the colon of the guinea pig, mouse, rabbit, and rat.

Gastrointestinal motility involves interactions between myogenic and neurogenic processes intrinsic to the gut wall. We have compared the presence of propagating myogenic contractions of the isolated colon in four experimental animals (guinea pig, mouse, rabbit, and rat), following blockade of enteric neural activity. Isolated colonic preparations were distended with fluid, with the anal end either closed or open. Spatiotemporal maps of changes in diameter were constructed from video recordings. Distension-induced peristaltic contractions were abolished by tetrodotoxin (TTX; 0.6 µM) in all animal species. Subsequent addition of carbachol (0.1-1 µM) did not evoke myogenic motor patterns in the mouse or guinea pig, although some activity was observed in rabbit and rat colon. These myogenic contractions propagated both orally and anally and differed from neurogenic propagating contractions in their frequency, extent of propagation, and polarity. Niflumic acid (300 µM), used to block myogenic activity, also blocked neural peristalsis and thus cannot be used to discriminate between these mechanisms. In all species, except the mouse colon, small myogenic "ripple" contractions were revealed in TTX, but in both rat and rabbit an additional, higher-frequency ripple-type contraction was superimposed. Following blockade of enteric nerve function, a muscarinic agonist can evoke propulsive myogenic peristaltic contractions in isolated rabbit and rat colon, but not in guinea pig or mouse colon. Marked differences between species exist in the ability of myogenic mechanisms to propel luminal content, but in all species there is normally a complex interplay between neurogenic and myogenic processes.

The presence of 5-HT in myenteric varicosities is not due to uptake of 5-HT released from the mucosa during dissection: use of a novel method for quantifying 5-HT immunoreactivity in myenteric ganglia.

BACKGROUND: Quantifying the relative abundance of different neurotransmitters in the myenteric plexus has proved challenging using conventional immunocytochemical approaches. Here, we present a new method of quantifying neurotransmitter content of an important enteric signalling molecule, serotonin (5-HT), in the myenteric plexus of guinea pig colon under different experimental conditions. METHODS: Sections of guinea pig distal colon were exposed to different conditions including changes in temperature, dissection protocol, stimulation with faecal pellet distension and exogenous 5-HT. Sections were fixed and immuno-labelled for 5-HT. 5-HT staining density was quantified within myenteric plexus ganglia using defined settings and an analysis approach that uses threshold settings allowing for variances in background and tissue staining intensities and which calculates the area of tissue containing 5-HT above these thresholds. KEY RESULTS: No differences were found in 5-HT immunoreactivity in the myenteric plexus when compared between tissues that were freshly fixed, undissected, or with mucosa and submucous plexus dissected away at either 4 or 37 °C. Increased myenteric plexus 5-HT density was observed in preparations repeatedly stimulated using faecal pellet stimulation prior to fixation. Furthermore, exogenous 5-HT also increased 5-HT density. CONCLUSIONS & INFERENCES: We demonstrate that quantitative differences in 5-HT immunoreactivity can be characterized using immunohistochemistry. This approach may be applied to measuring other neurotransmitter(s) within the enteric nervous system. While 5-HT is present in the guinea-pig enteric ganglia, this is not due to accumulation via in vitro handling and release from the mucosa, and furthermore, repeated colonic stimulation via distension increases 5-HT in the myenteric plexus.

Ascending excitatory neural pathways modulate slow phasic myogenic contractions in the isolated human colon.

BACKGROUND: In animal models, enteric reflex pathways have potent effects on motor activity; their roles have been much less extensively studied in human gut. The aim of this study was to determine if ascending excitatory interneuronal pathways can modulate spontaneous phasic contractions in isolated preparations of human colonic circular muscle. METHODS: Human colonic preparations were cut into T shapes, with vertical bar of the 'T' pharmacologically isolated. Electrical stimulation and the nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), were applied to the isolated region and circular muscle contractile activity was measured from the cross-bar of the T, more than 10 mm orally from the region of stimulation. KEY RESULTS: The predominant form of spontaneous muscle activity consisted of tetrodotoxin-resistant, large amplitude, slow phasic contractions (SPCs), occurring at average intervals of 124 ± 68 s. Addition of a high concentration of hexamethonium (1 mmol L(-1)) to the superfusing solution significantly increased the interval between SPCs to 278.1 ± 138.3 s (P < 0.005). Focal electrical stimulation more than 10 mm aboral to the muscle recording site advanced the onset of the next SPC, and this effect persisted in hexamethonium. However, the effect of electrical stimulation was blocked by tetrodotoxin (TTX, 1 µmol L(-1)). Application of the nicotinic agonist DMPP (1 mmol L(-1)) to the aboral chamber often stimulated a premature SPC (n = 4). CONCLUSIONS & INFERENCES: The major form of spontaneous contractility in preparations of human colonic circular muscle is SPCs, which are myogenic in origin. Activation of ascending excitatory neural pathways, which involve nicotinic receptors, can modulate the timing of SPCs and thus influence human colonic motility.

Identification and mechanosensitivity of viscerofugal neurons.

Enteric viscerofugal neurons are interneurons with cell bodies in the gut wall; they project to prevertebral ganglia where they provide excitatory synaptic drive to sympathetic neurons which control intestinal motility and secretion. Here, we studied the mechanosensitivity and firing of single, identified viscerofugal neurons in guinea-pig distal colon. Flat sheet preparations of gut were set up in vitro and conventional extracellular recordings made from colonic nerve trunks. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (1mM), was locally pressure ejected onto individual myenteric ganglia. In a few ganglia, DMPP promptly evoked firing in colonic nerves. Biotinamide filling of colonic nerves revealed that DMPP-responsive sites corresponded to viscerofugal nerve cell bodies. This provides a robust means to positively identify viscerofugal neuron firing. Of 15 single units identified in this way, none responded to locally-applied capsaicin (1 µM). Probing with von Frey hairs at DMPP-responsive sites reliably evoked firing in all identified viscerofugal neurons (18/18 units tested; 0.8-5 mN). Circumferential stretch of the preparation increased firing in all 14/14 units (1-5 g, p<0.05). Both stretch and von Frey hair responses persisted in Ca(2+)-free solution (6 mM Mg(2+), 1mM EDTA), indicating that viscerofugal neurons are directly mechanosensitive. To investigate their adequate stimulus, circular muscle tension and length were independently modulated (BAY K8644, 1 µM and 10 µM, respectively). Increases in intramural tension without changes in length did not affect firing. However, contraction-evoked shortening, under constant load, significantly decreased firing (p<0.001). In conclusion, viscerofugal neuron action potentials contribute to recordings from colonic nerve trunks, in vitro. They provide a significant primary afferent output from the colon, encoding circumferential length, largely independent of muscle tension. All viscerofugal neurons are directly mechanosensitive, although they have been reported to receive synaptic inputs. In short, viscerofugal neurons combine interneuronal function with length-sensitive mechanosensitivity.

Viscerofugal neurons recorded from guinea-pig colonic nerves after organ culture.

BACKGROUND: Enteric viscerofugal neurons provide cholinergic synaptic inputs to prevertebral sympathetic neurons, forming reflex circuits that control motility and secretion. Extracellular recordings of identified viscerofugal neurons have not been reported. METHODS: Preparations of guinea pig distal colon were maintained in organotypic culture for 4-6 days (n = 12), before biotinamide tracing, immunohistochemistry, or extracellular electrophysiological recordings from colonic nerves. KEY RESULTS: After 4-6 days in organ culture, calcitonin gene-related peptide and tyrosine hydroxylase immunoreactivity in enteric ganglia was depleted, and capsaicin-induced firing (0.4 µmol L(-1) ) was not detected, indicating that extrinsic sympathetic and sensory axons degenerate in organ culture. Neuroanatomical tracing of colonic nerves revealed that viscerofugal neurons persist and increase as a proportion of surviving axons. Extracellular recordings of colonic nerves revealed ongoing action potentials. Interestingly, synchronous bursts of action potentials were seen in 10 of 12 preparations; bursts were abolished by hexamethonium, which also reduced firing rate (400 µmol L(-1) , P < 0.01, n = 7). DMPP (1,1-dimethyl-4-phenylpiperazinium; 10(-4) mol L(-1) ) evoked prolonged action potential discharge. Increased firing preceded both spontaneous and stretch-evoked contractions (χ(2) = 11.8, df = 1, P < 0.001). Firing was also modestly increased during distensions that did not evoke reflex contractions. All single units (11/11) responded to von Frey hairs (100-300 mg) in hexamethonium or Ca(2+) -free solution. CONCLUSIONS & INFERENCES: Action potentials recorded from colonic nerves in organ cultured preparations originated from viscerofugal neurons. They receive nicotinic input, which coordinates ongoing burst firing. Large bursts preceded spontaneous and reflex-evoked contractions, suggesting their synaptic inputs may arise from enteric circuitry that also drives motility. Viscerofugal neurons were directly mechanosensitive to focal compression by von Frey hairs.