Carla Cirillo1, Jan Tack, Pieter Vanden Berghe. 1. Laboratory for Enteric Neuroscience (LENS), TARGID, KU Leuven, O&N 1 Herestraat 49-box 701, Leuven 3000, Belgium.
Abstract
BACKGROUND: Most direct understanding of enteric nerve (patho)physiology has been obtained by electrode and imaging techniques in animal models and human surgical samples. Until now, neuronal activity recordings from a more accessible human tissue source have remained a true challenge. OBJECTIVES: To record nerve activity in human intestinal biopsies using imaging techniques. DESIGN: Submucous plexus was isolated from duodenal biopsies. Enteric nerves were functionally and morphologically examined using calcium (Ca(2+)) imaging and immunohistochemistry. Exogenous application of high-K(+) solution, the nicotinic cholinergic receptor agonist (1,1-dimethyl-4-phenylpiperazinium; DMPP) or serotonin (5-HT), and electrical stimulation of interganglionic fibre tracts were used to activate the neurons, and intracellular Ca(2+) concentrations ([Ca(2+)](i)) were monitored. Enteric ganglia were stained with neuronal and glial markers. RESULTS: Using high-K(+) solution, 146 neurons were identified in 70 ganglia (44 biopsies from 29 subjects). The exogenous application of DMPP or 5-HT caused a transient [Ca(2+)](i) increase, respectively, in 68% and 63% of the neurons identified by high-K(+). Electrical stimulation evoked responses in 57% of the neurons; these responses were totally or partly suppressed by tetrodotoxin or zero-Ca(2+) solution, respectively. Immunohistochemical analysis showed both isolated neurons and ganglia interconnected by typical interganglionic fibre bundles. The average number of ganglia was 7.7±6.0 per biopsy and each ganglion contained on average 4.5±1.2 neurons. CONCLUSION: In this study, for the first time, live recordings were performed of nerve activity in intestinal biopsies. This novel approach is of key importance to study living neurons in both health and disease and to test newly developed compounds in an in-vitro human tissue model.
BACKGROUND: Most direct understanding of enteric nerve (patho)physiology has been obtained by electrode and imaging techniques in animal models and human surgical samples. Until now, neuronal activity recordings from a more accessible human tissue source have remained a true challenge. OBJECTIVES: To record nerve activity in human intestinal biopsies using imaging techniques. DESIGN: Submucous plexus was isolated from duodenal biopsies. Enteric nerves were functionally and morphologically examined using calcium (Ca(2+)) imaging and immunohistochemistry. Exogenous application of high-K(+) solution, the nicotinic cholinergic receptor agonist (1,1-dimethyl-4-phenylpiperazinium; DMPP) or serotonin (5-HT), and electrical stimulation of interganglionic fibre tracts were used to activate the neurons, and intracellular Ca(2+) concentrations ([Ca(2+)](i)) were monitored. Enteric ganglia were stained with neuronal and glial markers. RESULTS: Using high-K(+) solution, 146 neurons were identified in 70 ganglia (44 biopsies from 29 subjects). The exogenous application of DMPP or 5-HT caused a transient [Ca(2+)](i) increase, respectively, in 68% and 63% of the neurons identified by high-K(+). Electrical stimulation evoked responses in 57% of the neurons; these responses were totally or partly suppressed by tetrodotoxin or zero-Ca(2+) solution, respectively. Immunohistochemical analysis showed both isolated neurons and ganglia interconnected by typical interganglionic fibre bundles. The average number of ganglia was 7.7±6.0 per biopsy and each ganglion contained on average 4.5±1.2 neurons. CONCLUSION: In this study, for the first time, live recordings were performed of nerve activity in intestinal biopsies. This novel approach is of key importance to study living neurons in both health and disease and to test newly developed compounds in an in-vitro human tissue model.
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