Activation of intrinsic afferent pathways in submucosal ganglia of the guinea pig small intestine

Abstract

The enteric nervous system contains intrinsic primary afferent neurons that allow mucosal stimulation to initiate reflexes without CNS input. We tested the hypothesis that submucosal primary afferent neurons are activated by 5-hydroxytryptamine (5-HT) released from the stimulated mucosa. Fast and/or slow EPSPs were recorded in submucosal neurons after the delivery of exogenous 5-HT, WAY100325 (a 5-HT(1P) agonist), mechanical, or electrical stimuli to the mucosa of myenteric plexus-free preparations (+/- extrinsic denervation). These events were responses of second-order cells to transmitters released by excited primary afferent neurons. After all stimuli, fast and slow EPSPs were abolished by a 5-HT(1P) antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, and by 1.0 microM tropisetron, but not by 5-HT(4)-selective antagonists (SB204070 and GR113808A) or 5-HT(3)-selective antagonists (ondansetron and 0.3 microM tropisetron). Fast EPSPs in second-order neurons were blocked by hexamethonium, and most slow EPSPs were blocked by an antagonist of human calcitonin gene-related peptide (hCGRP(8-37)). hCGRP(8-37) also inhibited the spread of excitation in the submucosal plexus, assessed by measuring the uptake of FM2-10 and induction of c-fos. In summary, data are consistent with the hypothesis that 5-HT from enterochromaffin cells in response to mucosal stimuli initiates reflexes by stimulating 5-HT(1P) receptors on submucosal primary afferent neurons. Second-order neurons respond to these cholinergic/CGRP-containing cells with nicotinic fast EPSPs and/or CGRP-mediated slow EPSPs. Slow EPSPs are necessary for excitation to spread within the submucosal plexus. Because some second-order neurons contain also CGRP, primary afferent neurons may be multifunctional and also serve as interneurons.

Fig. 1.

Diagram showing the mucosa–submucosa preparation used to study the effects of mucosal stimulation on the activity of submucosal neurons. The myenteric plexus is removed to permit submucosal responses to be analyzed in the absence of confounding effects from myenteric neurons. A central segment of mucosa was left intact (facing up) for stimulation but was elsewhere stripped away to permit the submucosal plexus to be visualized. The submucosal ganglia that were selected for recording were those that were adjacent to the mucosal edge and positioned on a connective that appeared to run toward the site of stimulation.

Fig. 2.

Responses of impaled submucosal neurons to the mucosal application of 5-HT. 5-HT was applied to the intact central strip of mucosa (see Fig. 1) by ejection from a micropipette (↑). The records also show electrotonic potentials to intracellular injection of depolarizing or hyperpolarizing current pulses. A, Depolarizing current was injected into cells until the mucosa was stimulated with 5-HT. After stimulation, hyperpolarizing current was injected. A response consisted of a train of fast excitatory potentials (open arrow). B, Mucosal 5-HT elicits a prolonged depolarizing response superimposed on which are fast excitatory potentials (open arrow). During the slow depolarization the neuron is irritable and discharges action potentials that arise from the fast excitatory events (↘). C, Mucosal 5-HT elicits a prolonged depolarizing response with no associated fast excitatory potentials. The amplitude of the electrotonic potentials is increased during the slow response, indicating that input resistance is increased.

Fig. 3.

Fast and slow excitatory potentials recorded in submucosal neurons after the mucosal application of 5-HT are blocked by tetrodotoxin (TTX), ω-conotoxin (Ω-CTX), and low Ca²⁺/high Mg²⁺-containing media. A, Fast excitatory potentials evoked by the mucosal application of 5-HT (↑) are blocked by TTX. B, A prolonged (slow) excitatory potential evoked by the mucosal application of 5-HT (↑) is blocked by TTX. C, Both the prolonged (slow) excitatory potential and the superimposed fast excitatory potential are inhibited by Ω-CTX. The inhibition of fast potentials is incomplete.D, Fast excitatory potentials and action potentials are abolished by superfusion with Ca²⁺/high Mg²⁺-containing media. Calibration: 20 sec, 20 mV.

Fig. 4.

Mucosally applied 5-HT does not exert direct effects on impaled submucosal neurons. A, The impaled neuron does not respond to the mucosal application of 5-HT (↑).B, The same neuron does respond to the direct application of 5-HT to its surface (↑). The response is biphasic and consists of an initial fast response, during which input resistance is decreased, and a following prolonged (slow) response, during which input resistance is increased. C, Neither component of the direct response to 5-HT is inhibited by TTX.

Fig. 5.

Processes of neurons that project to the mucosal site of stimulation make close contacts with impaled submucosal neurons. After intracellular records were obtained from impaled submucosal neurons, the cells were marked by intracellular injection of Neurobiotin. The preparations were then fixed, and DiI-coated beads were inserted into the mucosa at the site that had been stimulated. After time was allowed for retrograde flow of DiI, Neurobiotin was demonstrated with FITC-labeled streptavidin. A, The site of stimulation is marked by the red fluorescence of DiI. Labeled axons (▴) leading to labeled nerve cell bodies (→) can be seen at the periphery of the site. B, At higher magnification, DiI can be seen to have labeled a number of neurites and nerve cell bodies. C, A neuron marked by the intracellular injection of Neurobiotin (filter set for FITC fluorescence). D, The same cell visualized with a filter that passes both the green fluorescence of FITC and thered fluorescence of DiI. The impaled neuron has not been labeled by DiI, but it is contacted by a DiI-labeled varicose nerve fiber. Scale bars: A, B, 100 μm;C, D, 25 μm.

Fig. 6.

Fast EPSPs evoked in a submucosal neuron by focal electrical stimulation directed at the mucosa. A, The fast EPSP is blocked by TTX. B, The fast EPSP is blocked by hexamethonium and thus is cholinergic and mediated by nicotinic receptors. C, A histogram is plotted showing the stimulus–response delay for fast EPSPs evoked in submucosal neurons by electrical stimulation of the mucosa.

Fig. 7.

Fast and slow EPSPs evoked in submucosal neurons by the mucosal application of 5-HT are mediated by 5-HT_1Preceptors. A, Fast EPSPs evoked by the mucosal application of 5-HT are inhibited by the 5-HT_1P antagonist 5-HTP-DP. B, A slow EPSP evoked by the mucosal application of 5-HT is inhibited by 5-HTP-DP. C, Application of the 5-HT_1P agonist WAY100325 to the mucosa evokes fast EPSPs in a submucosal neuron.

Fig. 8.

Responses of submucosal neurons to mucosal applications of 5-HT are not inhibited by antagonism of 5-HT₃ receptors but are antagonized by high concentrations of tropisetron. Responses of four cells (A–D) from different animals are illustrated. A, Fast EPSPs and an associated burst of action potentials are evoked by mucosal application of 5-HT (↑). These responses are not inhibited by 0.1 μm tropisetron (T).B, In another cell, fast EPSPs and associated action potentials are evoked by mucosal application of 5-HT (↑). These responses are not inhibited by 1 μm ondansetron (O), but they are abolished by tropisetron (10 μm). C, A slow EPSP is evoked in a submucosal neuron by mucosal 5-HT (↑). This response is blocked by 10 μm tropisetron. D, Mucosal 5-HT (↑) elicits a prolonged slow EPSP superimposed on which are fast EPSPs and associated action potentials. Neither the fast nor the slow EPSPs are inhibited by ondansetron (O), but they are reversibly inhibited by a high concentration of tropisetron (3 μm). After the washout of tropisetron (W), both fast and slow EPSPs return. Fast but not slow EPSPs are abolished by hexamethonium (C6).

Fig. 9.

Fast and slow EPSPs evoked in submucosal neurons by mechanical stimulation of the mucosa are mediated by 5-HT_1P receptors. A, A prolonged slow EPSP is evoked in a submucosal neuron by application of a mechanical stimulus to the mucosa (underline). The neuron becomes excited during the response and discharges a burst of action potentials. B, In a different preparation from another animal, a small slow EPSP and bursts of fast EPSPs are evoked by the delivery of a mechanical stimulus (underline) to the mucosa. Both slow and fast EPSPs are reversibly inhibited by 5-HTP-DP. Both slow and fast EPSPs recover after the washout of 5-HTP-DP. The responses are then reversibly inhibited by tropisetron (1 μm) and again recover after the washout of the drug.

Fig. 10.

Slow EPSPs evoked by the mucosal application of 5-HT are mediated by CGRP. A, The delivery of 5-HT (↑) to the mucosa elicits a slow EPSP in a submucosal neuron. This response is mimicked by the delivery of CGRP (↑) directly to the surface of the same neuron. B, CGRP evokes a slow depolarizing response when applied to a submucosal neuron. This effect is blocked by hCGRP_8–37. C, The application of 5-HT (↑) to the mucosa evokes a slow EPSP during which the neuron becomes excitable and discharges action potentials. The response of the cell to the mucosal application of 5-HT is blocked by hCGRP_8–37.

Fig. 11.

A subset of the slow EPSPs evoked in submucosal neurons by mucosally applied 5-HT is not mediated by CGRP.A, The application of 5-HT (↑) to the mucosa elicits a slow EPSP in a submucosal neuron. B, This response is not inhibited by hCGRP_8–37. C, The same neuron is unresponsive to the direct application of CGRP to its surface.

Fig. 12.

Analysis of the uptake of the activity probe FM2-10 suggests that CGRP-mediated neurotransmission is necessary for the spread of excitation in the submucosal plexus after the delivery of a mechanical stimulus (stroking) to the mucosa. The intact mucosal surface was gently stroked to activate submucosal primary afferent neurons counted in surviving preparations of mucosa–submucosa. FM2-10 was present and used as a neuronal activity probe. The number of submucosal neurons taking up FM2-10 was determined in nonstroked and stroked regions. Under control conditions, the number of neurons taking up FM2-10 was greatly increased by stroking the mucosa. The effect of stroking was blocked by the CGRP antagonist hCGRP_8–37.

Fig. 13.

Analysis of Fos immunoreactivity supports the idea that CGRP-mediated neurotransmission is required for the spread of excitation in the submucosal plexus after the delivery of a mechanical stimulus to the mucosa. Puffs of N₂ were used to excite submucosal neurons as described previously (Kirchgessner et al., 1992). A, There is no Fos immunoreactivity in the nuclei of the neurons in a submucosal ganglion of a nonstimulated preparation.B, Much Fos immunoreactivity is found in a submucosal ganglion after the mucosa had been stimulated under control conditions with N₂ puffs for 30 min. C, No Fos-immunoreactive nuclei can be found in submucosal ganglia when the mucosa was stimulated with N₂ puffs for 30 min (as inB) but in the presence of the CGRP antagonist hCGRP_8–37. Scale bar, 50 μm.

Fig. 14.

Top. A subset of the submucosal neurons that are induced to take up FM2-10 in response to the mucosal application of 5-HT mucosa contain CGRP. 5-HT was applied to the mucosa in the presence of the activity probe FM2-10. Active neurons were located and illuminated in the presence of DAB to photoconvert FM2-10 fluorescence to an insoluble DAB reaction product. The preparations were then fixed, and CGRP immunoreactivity was demonstrated.A, CGRP immunofluorescence. B, DAB reaction product visualized in the same field with bright-light and interference contrast optics. The arrows point to doubly labeled cells that contain both CGRP immunoreactivity and the FM2-10 → DAB photoconversion reaction product. Scale bar, 20 μm.

Fig. 15.

Sympathetic nerves were absent in loops of intestine subjected to chronic external denervation. TH immunoreactivity was used as a marker for the sympathetic innervation, which in turn served as an indicator of the completeness of extrinsic denervation. The dense array of varicose TH-immunoreactive nerve fibers can be seen in both the submucosal (A) and myenteric (C) plexuses of control loops of gut from the operated animals. In contrast, the denervated loops of intestine from the same animals contain no TH-immunoreactive nerve fibers in either the submucosal (B) or myenteric (D) plexuses. Notice that the perivascular TH-immunoreactive fibers are also lacking in the submucosa of the denervated loops of gut. The same results, showing that the extrinsic innervation was eliminated, were obtained in three of three operated guinea pigs. Scale bar, 100 μm.