Activation of the cannabinoid 2 (CB2) receptor inhibits murine mesenteric afferent nerve activity.

Abstract Cannabinoid 2 (CB2) receptors have both antinociceptive and antihypersensitivity effects, although the precise mechanisms of action are still unclear. In this study, the modulatory role of CB2 receptors on the mesenteric afferent response to the endogenous immunogenic agent bradykinin (BK) was investigated. Mesenteric afferent recordings were obtained from anaesthetized wild-type and CB2(-/-) mice using conventional extracellular recording techniques. Control responses to BK were obtained in all experiments prior to administration of either CB2 receptor agonist AM1241, or AM1241 plus the CB2 receptor antagonist AM630. Bradykinin consistently evoked activation of mesenteric afferents (n = 32). AM1241 inhibited the BK response in a dose dependent manner. In the presence of AM630 (10 mg kg(-1)), however, AM1241 (10 mg kg(-)1) had no significant effect on the BK response. Moreover, AM1241 had also no significant effect on the BK response in CB2(-/-) mice. Activation of the CB2 receptor inhibits the BK response in mesenteric afferents, demonstrating that the CB2 receptor is an important regulator of neuroimmune function. This may be a mechanism of action for the antinociceptive and antihypersensitive effects of CB2 receptor agonists.

Recording ionic events from cultured, DiI-labelled myenteric neurons in the guinea-pig proximal colon.

To date investigations of enteric neurons by patch clamping/calcium imaging have been limited by studying unidentified heterogeneous populations of neurons. In DiI-labelled colonic myenteric neurons, the feasibility of recording ionic events was determined by applying DiI either to the mucosa or the circular muscle, dispersing neurons after 48 h organotypic culture, and patch-clamping/calcium imaging labeled neurons after 3-7 days in culture. Myenteric neurons with diffuse DiI fluorescence were typically smooth and agranular. Neurons labeled after DiI was applied to circular muscle, fired in either a phasic or a tonic manner, and exhibited fast afterhyperpolarizations (100-300 ms duration) at the end of a depolarizing pulse. They expressed a fast inward current and at least three different outward currents. Action potentials elicited in DiI-labeled sensory neurons were followed by a prolonged afterhyperpolarization (AH, 4-6 s). The offset of a suprathreshold depolarizing step elicited a prolonged outward tail current that approximated the timecourse of the prolonged AH. In addition, in response to membrane depolarization in DiI-labeled neurons loaded with fura-2, robust Ca(2+) transients were recorded using the perforated patch technique. These results demonstrate that DiI labeling of cultured myenteric neurons is feasible, and patch clamp/Ca(2+) fluorescence recordings can be made from specific populations of cultured DiI-labeled colonic myenteric neurons.

Correlation of electrophysiology, neurochemistry and axonal projections of guinea-pig sphincter of Oddi neurones.

Sphincter of Oddi (SO) ganglia are comprised of two main types of neurones based either on their electrical or neurochemical properties. This study investigated whether any correlation exists between the electrical and neurochemical properties of these cells. SO neurones were characterized electrically as either Tonic or Phasic cells, labelled with neurobiotin, fixed, and processed for beta-nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-DA) staining and choline acetyltransferase immuno-reactivity to identify whether electrically characterized neurones were nitrergic or cholinergic. A total of 119 cells were analysed in this manner; 45% of cells were Tonic and 37% were Phasic. An equivalent number of Tonic (58.1%, 18/31) and Phasic cells (60%, 21/35) were choline acetyltransferase (ChAT) positive. Three of 34 Phasic cells were NADPH-DA positive, whereas 11/33 Tonic cells were NADPH-DA positive. In none of the preparations was ChAT immunoreactivity and NADPH-DA reactivity ever observed in the same neurone. Calretinin immunoreactivity was present in a subpopulation of both Tonic and Phasic neurones. No correlation was observed between the direction of axon projections and the electrophysiological or neurochemical properties of the cell. These results suggest that there is a lack of correlation between the electrical properties and the neurochemical content of SO neurones. Various explanations for these findings are discussed.

Serotonin and cholecystokinin activate different populations of rat mesenteric vagal afferents.

This electrophysiological study was performed to elucidate the interactions of serotonin (5-hydroxytryptamine, 5-HT) and cholecystokinin (CCK) on mesenteric afferents supplying the rat jejunum. 5-HT and CCK produced characteristic responses in multi-unit recordings of mesenteric afferents. Waveform analysis to extract single units from the whole nerve recording identified populations of single afferents that were sensitive to either 5-HT or CCK, but not both. Furthermore, devazepide (0.5 mg/kg) completely abolished the response to CCK without altering the response to 5-HT while granisetron (0.5 mg/kg) abolished the response to 5-HT with no effect on the response to CCK. These results suggest that there are discrete, noninteractive populations of jejunal afferents that possess either 5-HT3 or CCK-A receptors but not both.

Cilansetron acts at its site of absorption to antagonize the sensitivity of mesenteric afferent fibres to 5-hydroxytryptamine in the rat jejunum.

The present study compares the efficacy of cilansetron, a 5-hydroxytryptamine (5-HT3)-receptor antagonist, delivered via intravenous and intraluminal routes, on the sensitivity of mesenteric afferent fibres supplying the proximal jejunum. Waveform analysis was performed to extract 5-HT sensitive single units from electrophysiological recordings of whole afferent nerve discharge. Dose effects of intravenous cilansetron (0.2-20 microg/kg) on the afferent response to 5-HT (10 microg) were examined to determine the threshold dose of cilansetron (2 microg/kg). This dose applied intraluminally to the region of jejunum innervated by the afferents, resulted in a greater degree of antagonism of the 5-HT response than intravenous administration (47.8+/-7.9 vs. 76.9+/-4.7%, P = 0.008). We concluded that cilansetron is active at its site of absorption to antagonize 5-HT3 receptors on vagal mucosal afferent terminals.

Mesenteric afferent sensitivity to cholecystokinin and 5-hydroxytryptamine.

The present electrophysiological investigation examines the effect of CCK and 5-hydroxytryptamine on gastrointestinal afferent fibre discharge. 5-HT markedly stimulated mesenteric afferents. The response was transient (< 10s) and mediated by 5-HT3 receptors as demonstrated by the action of 2-methyl-5-HT and antagonism by granisetron. CCK was also a potent stimulus to mesenteric afferents causing a long-lasting (> 30s) increase in excitability. The response to CCK was mediated via the CCKA receptor as shown by the antagonistic action of devazepide. At doses of granisetron and devazepide which completely block the response to exogenous 5-HT and CCK, the afferent fibres still responded to both mechanical and chemical stimulation of the mucosa. Thus products of enteroendocrine cells can have profound effects on mucosal afferent sensitivity but do not play an obligatory role in afferent signal transduction.

Sensitivity to 5-hydroxytryptamine in different afferent subpopulations within mesenteric nerves supplying the rat jejunum.

1. This study was performed to elucidate the type of afferents that mediate the multiple actions of 5-hydroxytryptamine (5-HT) on mesenteric nerve discharge. Electrophysiological recordings were made from mesenteric afferents innervating the mid-jejunum of the urethane-anaesthetized rat. The discharge of single nerves within the whole nerve recording was monitored using waveform discrimination software. 2. Afferents responded to 5-HT in one of two ways: a short latency, transient excitation mediated by 5-HT3 receptors, or a delayed onset, more prolonged effect that was 5-HT2A receptor mediated. Afferents showing the 5-HT3-mediated response did not respond to luminal distension but were sensitive to intraluminal hydrochloric acid (150 mM) in twenty-eight of twenty-nine experiments. In eight experiments, the 5-HT3-mediated response was reversibly abolished by a 2 min exposure to intraluminal application of local anaesthetic (2 % Xylocaine). 3. Mechanosensitive afferents which responded to distension (< 10 cmH2O) did not show a 5-HT3-mediated response (P = 0.92, n = 14), and maintained this mechanosensitivity after luminal anaesthesia. Mechanosensitive afferents did show a secondary response to 5-HT that was significantly attenuated by atropine (100-200 microg kg-1), whereas hexamethonium (8 mg kg-1) had no effect. 4. In animals whose vagal afferent contribution to their mesenteric nerves had been eliminated by chronic truncal vagotomy, the 5-HT3-mediated response was absent in thirty-six of thirty-six nerve bundles. In contrast, mechanosensitivity to distension and the secondary response to 5-HT could still be evoked. 5. These results suggest that 5-HT stimulates mesenteric afferents by a direct action on 5-HT3 receptors that are present on vagal mucosal afferent terminals. The mucosal afferent response to luminal acid, however, was unaffected by treatment with granisetron (0.5 mg kg-1) indicating that endogenous 5-HT from enterochromaffin cells is not essential for transduction of this luminal signal. In contrast, mechanosensitivity in non-vagal afferents was modulated by 5-HT following an intestinal motor response which was influenced by cholinergic tone.

Plasticity in the mesenteric afferent response to cisplatin following vagotomy in the rat.

The aim of this study was to investigate the actions of the cytotoxic drug cisplatin on populations of mesenteric afferents supplying the rat jejunum. Extracellular whole mesenteric nerve discharge was monitored and the activity of individual single afferent units determined using waveform discriminator software. Baseline whole nerve discharge was 21.5 +/- 3.8 impulses s(-1). Nerve discharge began to increase approximately 10 min after cisplatin administration, reached a plateau around 30 min, and remained elevated at 60 min (35.3 +/- 5.7 impulses s(-1), p < 0.01). Granisetron reversed the increase in nerve activity indicating that the response to cisplatin was mediated by the release of endogenous 5-HT acting on 5-HT3 receptors. Single afferent units, selected by waveform analysis on the basis of their response to exogenous 5-HT, showed a similar time course of activation following cisplatin. In contrast, the discharge frequency of afferent units that were insensitive to 5-HT was unaffected by cisplatin or granisetron. The sensitivity of mesenteric afferent bundles to exogenous 5-HT was absent in chronically vagotomized animals. However, cisplatin elicited an increase in nerve discharge in vagotomized animals that was not different from control (34.6 +/- 8.9 impulses s(-1)) but this increase was unaffected by treatment with granisetron. Thus, after vagotomy there is a switch from 5-HT3 mediated activation of vagal afferents to a 5-HT3-independent activation of non-vagal (possibly splanchnic) afferents. Since this later mechanism of activation is absent in control animals, it appears that there is plasticity in the gastrointestinal afferent sensitivity to cisplatin.

5-HT is present in nerves of guinea pig sphincter of Oddi and depolarizes sphincter of Oddi neurons.

This study involved immunohistochemistry and intracellular electrophysiology to investigate serotonergic neurotransmission in the sphincter of Oddi (SO). 5-Hydroxytryptamine (HT)-positive neurons (14 cells/preparation) and nerve fibers were observed in the ganglionated plexus. Serotonergic nerve fibers, which persisted under 2- to 6-day organ culture, were densely distributed, with varicose endings encircling some SO neurons. When 5-HT was applied to SO neurons, it elicited three different responses: 1) a fast depolarization to 5-HT in 31 of 62 cells was mimicked by 2-methyl-5-HT and blocked by LY-278584 (1 microM); 2) a prolonged depolarization to 5-HT in 21 of 62 cells evoked an increase in input resistance and was attenuated by the 5-HT1P antagonist renzapride (1 microM) but not by the 5-HT4 antagonist SDZ-205557 (0.1-10 microM); and 3) an indirect depolarization blocked by TTX or atropine was observed in 32 of 62 cells. 5-HT superfusion elicited a dose-dependent monophasic depolarization (EC50 = 2 microM, n=14). In conclusion, 5-HT is present in nerves of the SO and elicits both 5-HT3 and 5-HT1P receptor-mediated depolarizations, supporting the concept that 5-HT plays a role in SO regulation.

Alpha-adrenoreceptor modulation of neurally evoked circular muscle responses of the guinea pig stomach.

The effects of alpha-adrenergic agonists on transmural-evoked motor responses were investigated in guinea pig gastric corpus in vitro, using preparations stripped of mucosa and orientated to record changes in circular muscle tension. Three tetrodotoxin-sensitive components to a 10 s burst of transmural stimulation could be distinguished: an initial 'on' contraction, an 'off' contraction and a transient relaxation. The 'on' response was blocked by atropine (0.1 microM), while the 'off' response and relaxation were unaffected at this dose. A submaximal dose of acetylcholine was used to assess the sensitivity of the preparation. The alpha 1 agonist L-phenylephrine decreased the amplitude of the 'off' response while simultaneously increasing both the 'on' response and the relaxation, although the response to acetylcholine was unchanged. These effects were dose-dependent and reversed by pretreatment with prazosin. In marked contrast, the alpha 2 agonist clonidine inhibited the 'on' response in a dose-dependent manner without affecting the 'off' response, the relaxation or the response to acetylcholine. Yohimbine reversed the effect of clonidine. We conclude that the inhibitory action of alpha-agonists involves both cholinergic and non-cholinergic pathways, with alpha 1 and alpha 2 adrenoceptors modulating different circuits within the enteric nervous system.

Inhibition of gastric mechanoreceptor discharge by cholecystokinin in the rat.

The aim of the present study was to investigate electrophysiologically the effect of systemic cholecystokinin (CCK) on the discharge of vagal gastric mechanoreceptors. Twenty-two single vagal afferent fibers were selected for the investigation of responses to intravenous CCK octapeptide (CCK-8) on the basis of a positive response to gastric distension. Resting discharge in these afferent fibers was 1.3 +/- 0.3 impulses.s-1 and increased to 9.2 +/- 0.9 impulses.s-1 during distension (P < 0.0001), CCK (20-100 pmol iv) caused a gastric relaxation of 2.1 +/- 0.2 cmH2O and inhibition of phasic motility. The discharge of 20/22 of vagal tension receptors closely followed the magnitude and time course of the fall in pressure. Mean discharge before and after CCK (50 pmol) was 7 +/- 0.9 and 3.9 +/- 0.8 impulses.s-1, respectively (P < 0.001, n = 22). Both the pressure response and the concomitant changes in afferent discharge were abolished by L-364,718 (1.2 mg/kg iv). Only two afferent units failed to show a decrease in firing following CCK (50 pmol), and at 500 pmol the discharge of these units was augmented. In conclusion, CCK (50 pmol) has predominantly indirect effects on gastric mechanoreceptors, which decrease their firing in association with gastric relaxation.

Ryanodine-sensitive stores regulate the excitability of AH neurons in the myenteric plexus of guinea-pig ileum.

Myenteric afterhyperpolarizing (AH) neurons are primary afferent neurons within the gastrointestinal tract. Stimulation of the intestinal mucosa evokes action potentials (AP) that are followed by a slow afterhyperpolarization (AHP(slow)) in the soma. The role of intracellular Ca(2+) ([Ca(2+)](i)) and ryanodine-sensitive Ca(2+) stores in modulating the electrical activity of myenteric AH neurons was investigated by recording membrane potential and bis-fura-2 fluorescence from 34 AH neurons. Mean resting [Ca(2+)](i) was approximately 200 nM. Depolarizing current pulses that elicited APs evoked AHP(slow) and an increase in [Ca(2+)](i), with similar time courses. The amplitudes and durations of AHP(slow) and the Ca(2+) transient were proportional to the number of evoked APs, with each AP increasing [Ca(2+)](i) by approximately 50 nM. Ryanodine (10 microM) significantly reduced both the amplitude and duration (by 60%) of the evoked Ca(2+) transient and AHP(slow) over the range of APs tested (1-15). Calcium-induced calcium release (CICR) was graded and proportional to the number of APs, with each AP triggering a rise in [Ca(2+)](i) of approximately 30 nM Ca(2+) via CICR. This indicates that CICR amplifies Ca(2+) influx. Similar changes in [Ca(2+)](i) and AHP(slow) were evoked by two APs in control and six APs in ryanodine. Thus, the magnitude of the change in bulk [Ca(2+)](i) and not the source of the Ca(2+) is the determinant of the magnitude of AHP(slow). Furthermore, lowering of free [Ca(2+)](i), either by reducing extracellular Ca(2+) or injecting high concentrations of Ca(2+) buffer, induced depolarization, increased excitability, and abolition of AHP(slow). In addition, activation of synaptic input to AH neurons elicited a slow excitatory postsynaptic potential (sEPSP) that was completely blocked in ryanodine. These results demonstrate the importance of [Ca(2+)](i) and CICR in sensory processing in AH neurons. Activity-dependent CICR may be a mechanism to grade the output of AH neurons according to the intensity of sensory input.

Direct and indirect actions of 5-hydroxytryptamine on the discharge of mesenteric afferent fibres innervating the rat jejunum.

1. This study was performed to elucidate the actions of 5-hydroxytryptamine (5-HT) on mesenteric afferent discharge and to determine the receptor-mechanisms responsible for these effects. The activity of mesenteric afferents innervating the mid-jejunum of urethane-anaesthetized rats was recorded with extracellular microelectrodes. The discharge of single nerves within the whole nerve recording was monitored using waveform discriminator software. 2. The intravenous injection of 5-HT produced a complex pattern of afferent activation with two distinct components which could be distinguished both in terms of the response characteristics and the receptors involved. Initially, in 64% of nerve bundles, there was a brief (2.0 +/- 0.1 s) but intense activation of afferent discharge with peak afferent firing increasing with incremental doses of 5-HT. The discharge frequency in seventeen single units from these bundles during the initial response to 10 micrograms 5-HT was 13.0 +/- 1.8 impulses s-1 from a baseline discharge of 1.0 +/- 0.1 impulses s-1. 3. This initial response was mimicked by the 5-HT3 receptor agonist, 2-methyl-5-HT, whereas 5-methoxytryptamine (5-MEOT, 10-100 micrograms) had no comparable effect. Similarly, the initial 4. 5-HT response was completely abolished by the 5-HT3 receptor antagonist, granisetron (0.5 mg kg-1). 5-HT also evoked, in approximately 35% of nerve bundles, a delayed response that single unit analysis showed to be mediated by an entirely different population of afferents from those activated during the initial response. This secondary response to 5-HT was characterized by a more prolonged (> 30 s) but less intense period of afferent activity which was coincident with an increase in intrajejunal pressure, and was mimicked by 5-MEOT (10-100 micrograms). The secondary response to 5-HT and the response to 5-MEOT were significantly attenuated by the 5-HT2A receptor antagonist, ketanserin (0.5 mg kg-1), which had no effect on the initial response. The initial response to 5-HT was unaffected by the L-type calcium channel inhibitor nifedipine (1 mg kg-1) or the N-type calcium channel inhibitor omega-conotoxin GVIA (25 micrograms kg-1). However, the secondary response to 5-HT was significantly reduced after treatment with nifedipine. 5. These results demonstrate that 5-HT activates different populations of afferent fibres innervating the rat jejunum. One population of afferents is activated directly via stimulation of 5-HT3 receptors, while another population responds to 5-HT with a time course consistent with secondary activation of mechanosensitive afferents following 5-HT2A-mediated contractile activity.

Neural control of the gallbladder: an intracellular study of human gallbladder neurons.

BACKGROUND/AIMS: Gallbladder neurons are important governors of gallbladder function. In animal models, gallbladder ganglia can be regulated both by neural and hormonal inputs. The purpose of this study was to demonstrate the feasibility of obtaining recordings from human gallbladder neurons. METHODS: Human gallbladders (n = 33) were bathed in oxygenated Krebs solution (37 degrees C) containing the vital fluorescent stain 4-Di-2-ASP to localize the ganglia. Cells were characterized using conventional intracellular recording techniques. RESULTS: The mean resting membrane potential of human gallbladder neurons was -51.2 +/- 1.8 mV (n = 11). Depolarizing current pulses elicited only 1-4 spikes regardless of the amplitude or duration of the stimulus. Afterspike hyperpolarizations had a mean duration of 144.5 +/- 19.2 ms (n = 10). Anodal break excitation was not recorded with hyperpolarizing current pulses. Fiber tract stimulation elicited fast excitatory postsynaptic potentials in all neurons tested. CONCLUSION: Intracellular recordings of human gallbladder neurons utilizing 4-Di-2-ASP are thus feasible, but are very problematic due to the density of connective tissue overlying the ganglia. As human and guinea pig gallbladder neurons have similar basic electrical properties, the guinea pig may be an appropriate model for further electrophysiological studies into gallbladder disease.

Role of 5-hydroxytryptamine in gastrointestinal chemosensitivity.

The present electrophysiological investigation examines the effect of 5-hydroxytryptamine (5-HT) on gastrointestinal afferent fiber discharge. 5-HT markedly and specifically stimulated vagal mucosal chemosensitive afferents. The response was mediated by 5-HT3 receptors as demonstrated by the action of 2-methyl-5-HT and antagonism by granisetron. At doses of granisetron that completely block the response to 5-HT, the afferent fibers still responded to both mechanical and chemical stimulation of the mucosa. This sensitivity of extrinsic afferents is in marked contrast to that reported for intrinsic afferents, suggesting fundamental differences in the organization of enteric and vagal reflexes.

Cholecystokinin depolarizes guinea pig sphincter of Oddi neurons by activating CCK-A receptors.

Motility studies indicate that cholecystokinin (CCK) acts through a neural mechanism in the sphincter of Oddi (SO) after meals. To evaluate its actions in SO ganglia, CCK was applied by microejection (0.1 mM) or superfusion (0.1 to 300 nM) while recording was carried out intracellularly from intact SO neurons. In tonic cells, microejection and superfusion of CCK caused a prolonged depolarization accompanied by action potentials. In phasic cells, microejection of CCK caused brief and/or prolonged depolarizations, but superfusion caused only prolonged depolarizations. In afterhyperpolarized cells, CCK did not cause a detectable change in the resting membrane potential. In low-Na+ Krebs solution, the prolonged depolarizations in both tonic and phasic cells were significantly reduced. Unsulfated CCK (100 nM) had no effect. CCK-induced depolarization was significantly reduced by a CCK-A, but not a CCK-B, receptor antagonist. It is concluded that CCK can act on CCK-A receptors to depolarize SO neurons. However, it is unlikely that hormonal CCK could mediate such an action because of the discrepancy between the sensitivity of SO neurons for CCK and the peak concentrations of CCK in the serum after a meal.

Sensitivity of vagal mucosal afferents to cholecystokinin and its role in afferent signal transduction in the rat.

1. Extracellular recordings from rat mesenteric paravascular nerve bundles were made in order to characterize the responses of different populations of afferents supplying the small intestine to intravenous cholecystokinin (CCK; in the form of sulphated CCK8). 2. Approximately 70% of mesenteric nerve bundles contained CCK-sensitive afferent fibres. Responsive afferents had low spontaneous discharge (1.6 +/- 0.3 impulses s-1) and showed a 14-fold increase in firing at the peak of the response to 50 pmol CCK with the overall response lasting several minutes. The onset of the response occurred after a latency of (3.9 +/- 0.1 s) following i.v. administration of CCK, which corresponds largely to the circulation delay in these animals. The threshold dose of CCK was < 5 pmol. 3. The response to 100 pmol CCK was completely abolished by devazepide (0.5 mg kg-1) and by chronic subdiaphragmatic vagotomy performed 10-14 days prior to experimentation, indicating that CCK sensitivity was via CCKA receptors and exclusively mediated via vagal afferents rather than splanchnic or enteric afferents. 4. Evidence that CCK-sensitive afferents had mucosal receptive fields was indicated by the lack of any response to luminal distension and the sensitivity of the CCK response to luminal anaesthesia. Furthermore, CCK-sensitive afferents responded to luminal hydrochloric acid (50 mM) in a slowly adapting manner. The response to acid was significantly reduced (P < 0.005), but not abolished, by devazepide at a time when the response to exogenous CCK had been completely eliminated. 5. The exquisite sensitivity of some vagal mucosal afferents to CCK suggests that they may play a physiological role in the reflex and behavioural consequences of CCK release from the small intestine, possibly acting in a paracrine fashion. However, this sensitivity to CCK represents only one aspect of the broad chemosensitivity of these mucosal afferents and is not an obligatory component of the signal transduction pathway.

Diverse ionic currents and electrical activity of cultured myenteric neurons from the guinea pig proximal colon.

The aim of this study was to perform a patch-clamp analysis of myenteric neurons from the guinea pig proximal colon. Neurons were enzymatically dispersed, cultured for 2-7 days, and recorded from using whole cell patch clamp. The majority of cells fired phasically, whereas about one-quarter of the neurons fired in a tonic manner. Neurons were divided into three types based on the currents activated. The majority of tonically firing neurons lacked an A-type current, but generated a large fast transient outward current that was associated with the rapid repolarizing phase of an action potential. The fast transient outward current was dependent on calcium entry and was blocked by tetraethylammonium. Cells that expressed both an A-type current and a fast transient outward current were mostly phasic. Depolarization of these cells to suprathreshold potentials from less than -60 mV failed to trigger action potentials, or action potentials were only triggered after a delay of >50 ms. However, depolarizations from more positive potentials triggered action potentials with minimal latency. Neurons that expressed neither the A-type current or the fast transient outward current were all phasic. Sixteen percent of neurons were similar to AH/type II neurons in that they generated a prolonged afterhyperpolarization following an action potential. The current underlying the prolonged afterhyperpolarization showed weak inward rectification and had a reversal potential near the potassium equilibrium potential. Thus cultured isolated myenteric neurons of the guinea pig proximal colon retain many of the diverse properties of intact neurons. This preparation is suitable for further biophysical and molecular characterization of channels expressed in colonic myenteric neurons.

Identification of the cholinergic neurons in guinea-pig sphincter of Oddi ganglia.

The muscular tone of the sphincter of Oddi (SO) can be up- or down-regulated by neurons that lie within ganglia in the wall of the tissue. Previous studies have demonstrated that neurons in the ganglia of the guinea-pig SO can be classified into two major populations, one of which expresses tachykinins and enkephalin and another which expresses nitric oxide synthase. Although results of previous pharmacological studies indicate that acetylcholine is released in the SO, the neurons that express this neurotransmitter have not previously been identified. This study was conducted to establish which neurons in the ganglia of the guinea-pig SO are cholinergic by examining the distribution of choline acetyltransferase (ChAT) immunoreactivity, since the enzyme, ChAT is necessary for acetylcholine synthesis. Choline acetyltransferase immunoreactivity was intense and widespread in the ganglionated plexus of the SO. ChAT-immunoreactive nerve fibers were present in ganglia, interganglionic fiber bundles and in the circular muscle layer. Neurons that were immunoreactive for ChAT comprised about 69% of the population and most of these neurons were also tachykinin-immunoreactive. Co-expression of ChAT and nitric oxide synthase was not observed in nerve cell bodies or nerve fibers. Data from this study support the concept that SO ganglia are largely made up of two populations of neurons, one excitatory and the other inhibitory, on the basis of their chemical coding. The excitatory neurons are cholinergic and co-express tachykinin and opiate peptides and the inhibitory neurons are ChAT-negative and express nitric oxide synthase.