In vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation.

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca(2+) concentration ([Ca(2+)](i)). The degranulatory EC(50) for the mast cell secretagogue compound 48/80 (C48/80; 10 microg/ml) and the neuropeptides CGRP (2.10(-8) M) and substance P (SP; 3.10(-8) M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 microg/ml) and CGRP and SP (both 10(-7) M) to Fluo-4-loaded MMC induced a transient rise in [Ca(2+)](i) after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 microg/ml), indicating involvement of G(i) proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca(2+)](i), indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.

Ca2+ involvement in the action potential generation of myenteric neurones in the rat oesophagus.

Intracellular recordings were used to study the physiological behaviour of rat oesophageal myenteric neurones, which are embedded in striated muscle. Injection of depolarizing pulses evoked action potentials with a clear 'shoulder' in all neurones. This shoulder disappeared under low Ca2+/high Mg2+ conditions. Tetrodotoxin (TTX; 1 micromol L-1) did not impede spike firing, whereas under combined TTX and low Ca2+/high Mg2+ conditions the action potentials were completely abolished, indicating that TTX- resistant action potentials are mediated by a Ca2+ current. Further experiments with omega-conotoxin GVIA (100 nmol L-1) revealed that these Ca2+ currents enter the cell via N-type voltage-activated Ca2+ channels (see also accompanying paper). Tetraethylammonium (10 mmol L-1) caused broadening of the action potentials, which probably resulted from prolonged Ca2+ influx due to blockade of the delayed rectifier K+ channel. Although Ca2+ appears to be involved in the spike generation of all rat oesophageal myenteric neurones, only a minority (14%) shows a slow afterhyperpolarization. Thus, no strict correlation exists between the presence of a shoulder and a slow afterhyperpolarization. Furthermore, morphological identification of 25 of the impaled neurones revealed that there was no strict correlation between morphology and electrophysiological behaviour. Consequently, rat oesophageal myenteric neurones appear to differ in several aspects from myenteric neurones in smooth muscle regions of the gastrointestinal tract.

Immunohistochemical localization of voltage-activated calcium channels in the rat oesophagus.

Voltage-activated calcium channels play an important role in the physiology of the enteric nervous system. To determine which types of voltage-activated calcium channels are present in the rat oesophagus, an immunohistochemical study was performed using specific antibodies for the alpha1 subunits of Cav2.1 (P/Q-type), Cav2.2 (N-type), Cav1.2 and Cav1.3 (L-type) calcium channels. All myenteric cell bodies showed Cav2.2 immunoreactivity, whereas labelling for this N-type channel was absent in nerve fibres. Cav1.2 immunoreactivity was found on nerve fibres in the myenteric plexus and on fibres innervating the striated muscle of the rat oesophagus, whereas no labelling was detected on neuronal somata. Immunoreactivity against Cav1.3 was not detected in the myenteric plexus or at the level of the striated muscle. Labelling for Cav2.1 was absent at the level of the myenteric plexus, but present in the striated muscle layer at the level of the motor endplates. Comparison with recent literature data from rat small intestine reveals region-specific distribution patterns of the various subtypes of voltage-activated calcium channels within the enteric nervous system. In addition, the present immunohistochemical data corroborate our physiological data (see accompanying paper), which indicate that the Cav2.2 (N-type) channel is the predominant channel involved in the generation of the calcium-dependent action potential evoked by intrasomatic depolarizing current pulses in all rat oesophageal myenteric neurones.

Excitatory synaptic inputs on myenteric Dogiel type II neurones of the pig ileum.

The synaptic input on myenteric Dogiel type II neurones (n = 63) obtained from the ileum of 17 pigs was studied by intracellular recording. In 77% of the neurones, electrical stimulation of a fibre tract evoked fast excitatory postsynaptic potentials (fEPSPs) with an amplitude of 6 +/- 5 mV (mean +/- S.D.) and lasting 49 +/- 29 ms. The nicotinic nature of the fEPSPs was demonstrated by superfusing hexamethonium (20 microM). High-frequency stimulation (up to 20 Hz, 3 seconds) did not result in a rundown of the fEPSPs, and did not evoke slow excitatory or inhibitory postsynaptic potentials. The effects of neurotransmitters, possibly involved in these excitatory responses, were investigated. Pressure microejection of acetylcholine (10 mM in pipette) resulted in a fast nicotinic depolarisation in 67%(18/27) of the neurones (13 +/- 9 mV, duration 7.0 +/- 7.2 seconds) as did 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) application (10 mM; 14 +/- 10 mV, duration 4.1 +/- 2.8 seconds) in 76% of the cells. The fast nicotinic response to acetylcholine was sometimes (6/27) followed by a slow muscarinic depolarisation (8 +/- 4 mV; duration 38.7 +/- 10.8 seconds). Immunostaining revealed 5-hydroxytryptamine hydrochloride (5-HT)- and calcitonin gene-related peptide (CGRP)-positive neuronal baskets distributed around and in close vicinity to Dogiel type II neuronal cell bodies. Microejection of 5-HT (10 mM) resulted in a fast nicotinic-like depolarisation (12 +/- 6 mV, duration 3.0 +/- 1.3 seconds) in 4 of 8 neurones tested, whereas microejection of CGRP (20 mM) gave rise to a slow muscarinic-like depolarisation (6 +/- 2 mV, duration 56.0 +/- 27.5 seconds) in 8 of 12 neurones tested. In conclusion, myenteric Dogiel type II neurones in the porcine ileum receive diverse synaptic input. Mainly with regard to the prominent presence of nicotinic responses, these neurones behave contrary to their guinea pig counterparts.

Electrophysiological features of morphological Dogiel type II neurons in the myenteric plexus of pig small intestine.

By intracellular recording, 99 myenteric neurons with Dogiel type II morphology were electrophysiologically characterized in the porcine ileum and further subdivided into three groups based on their different types of afterhyperpolarization (AHP). In response to a depolarizing current injection, a fast AHP (fAHP; duration 34 +/- 11 ms; amplitude -11 +/- 6 mV; mean +/- SD) immediately followed every action potential in all neurons. In 32% of the neurons, this fAHP was the sole type of hyperpolarization recorded. Statistical analysis revealed the presence of two neuronal subpopulations that displayed either a long-lasting medium AHP (mAHP; duration after a single spike 773 +/- 753 ms; 51% of neurons) or a slow AHP (sAHP; 4, 205 +/- 1,483 ms; 17%). Slow AHP neurons also differed from mAHP neurons in the delayed onset of the AHP (mAHP 0 ms; sAHP 100-200 ms), as well as in maximum amplitude values and in the time to reach this amplitude (t(max); 148 +/- 11 ms vs. 628 +/- 108 ms). Medium AHP neurons further differed from the sAHP neurons in the occurrence of the AHP following subthreshold current injection and in their resting membrane potential (mAHP, -53 +/- 8 mV; sAHP, -62 +/- 10 mV). Medium AHP and sAHP behaved similarly in that a higher number of spikes increased their amplitude and duration, but not t(max). The majority of neurons fired multiple spikes (up to 25) in response to a 500-ms current injection (81/99) and showed a clear TTX-resistant shoulder on the repolarizing phase of the action potential (77/99), irrespective of the presence of sAHP or mAHP. These results demonstrate that the porcine Dogiel type II neurons differ in various essential electrophysiological properties from their morphological counterparts in the guinea pig ileal myenteric plexus. The most striking interspecies differences were the low occurrence of sAHP (17% vs. 80-90% in guinea pig) with relatively small amplitude (-5 vs. -20 mV), the high occurrence of mAHPs (unusual in guinea pig) and the ability to fire long spike trains (up to 25 spikes vs. 1-3 in guinea pig). In fact, Dogiel type II neurons in porcine ileum combine distinct electrophysiological features considered typical of either S-type or sAHP-type neurons in guinea pig. It can therefore be concluded that in spite of a similar morphology, Dogiel type II neurons do not behave electrophysiologically in a universal way in large and small mammals.

Species-dependent features of Dogiel type II neurones in the mammalian enteric nervous system.

Although autonomic gastrointestinal reflex movements, which occur in all mammalian species, have been described almost a century ago, little was known on the mechanisms underlying this behaviour. Recently, however, intrinsic primary afferent neurones, functioning as the first relay in the reflex arches embedded in the intestinal wall, have been identified in the guinea pig ileum. In guinea pig, such neurones display a Dogiel type II morphology and behave electrophysiologically as slow AHP neurones. In other gastrointestinal regions, in both guinea pig and rat, Dogiel type II cells are also encountered, but the strong correlation with slow AHP neuronal features seems less strict. In large mammals, a correlation of the cellular morphology with intracellular el ectrophysiological recordings has only been obtained in the pig small intestine. Surprisingly, in these experiments aberrant electrophysiological behaviour of Dogiel type II neurones is even more striking since the majority of these cells display electrophysiological features considered typical of S neurones. Furthermore, in those rare cases in which a slow afterhyperpolarization (AHP) could be recorded in porcine Dogiel type II cells, its amplitudes were negligible. This has led us to the conclusion that the differences in electrophysiological behaviour of neurones with comparable morphology in different species are most probably due to the modulating influence of the neurotransmitter substances present. This seems to be the most likely hypothesis in view of the considerable differences in neurotransmitter content of neurones with comparable functions throughout the species.