Recordings from human myenteric neurons using voltage-sensitive dyes
Research highlights
▶ Fast voltage-sensitive dye recordings in human myenteric plexus. ▶ Di-8-ANEPPS revealed signals in intact plexus and primary cell cultures. ▶ Recordings of synaptic potentials and responses to exogenous drug application.
Introduction
The enteric nervous system (ENS) primarily consists of two ganglionated plexi which are embedded in the gut wall. The ENS acts as an autonomous nervous system which controls the main gut functions. This is achieved by the submucous plexus which mainly regulates mucosal functions while the myenteric plexus, located between the two muscle layers, coordinates smooth muscle activity.
Electrophysiological properties of myenteric neurons have been studied with conventional microelectrode techniques, mainly in small laboratory animals (Wood, 1970, Wood, 1973, Ohkawa and Prosser, 1972, Nishi and North, 1973, Hirst et al., 1974, Furukawa et al., 1986, Brookes et al., 1988, Browning and Lees, 1996, Cornelissen et al., 2001). Such studies have advanced our knowledge on basic functions of the ENS in guinea-pigs, cats, mice, rats and pig, but did also highlight species-specific ENS functions. There has been much less progress in the neurobiology of the human ENS as the basis to understand pathophysiology of functional, structural and inflammatory gastrointestinal diseases. This is mainly due to technical obstacles preventing the routine application of conventional microelectrode techniques to human intestinal tissue. In pioneering studies Obaid et al., 1992, Obaid et al., 1999 and Neunlist et al. (1999), adapted multi-site optical imaging systems in combination with a voltage-sensitive dye (VSD) to record from enteric neurons in the guinea-pig. With this technique, we were able to also routinely record from human submucous neurons with a high spatial and temporal resolution that revealed fast changes in membrane potential such as action potentials and fast excitatory postsynaptic potentials (EPSPs) (Schemann et al., 2002, Schemann et al., 2005, Michel et al., 2005, Breunig et al., 2007, Buhner et al., 2009).
However, recordings from the human myenteric ganglia remained a challenge because of their limited visibility and dye accessibility. So far only two studies on the electrophysiology of human myenteric neurons have been published. They used conventional intracellular recordings to investigate basic properties of cultured myenteric neurons (Maruyama, 1981) or myenteric neurons from freshly dissected human colon (Brookes et al., 1987). Both studies had limitations: the number of studied neurons was rather small with 2 and 43, respectively. Many years required to record from such small number of neurons are one explanation why such studies have not been followed up. In addition, the study by Maruyama used cultured fetal myenteric neurons which very likely do not reflect behaviour of mature enteric neurons.
We have already demonstrated that in principle it is feasible to record from human myenteric neurons using voltage-sensitive dye imaging (Schemann et al., 2002). However, we were only successful in one tissue and reliable recordings of reproducible signals from human myenteric neurons remained a challenge. Therefore, it was the aim of our study to establish a protocol that allows the successful use of VSD imaging for recordings of human myenteric neurons. The protocols were tested in freshly dissected human tissues as well as in primary cultures of human myenteric neurons. We investigated the usefulness of several voltage-sensitive dyes both in human as well as in guinea-pig myenteric plexus. As visibility of intact, non-cultured, human myenteric ganglia is a limitation we used several vital dyes to pre-stain the ganglia. Myenteric plexus preparations from guinea-pigs were used to test whether these dyes alter electrophysiological behaviour of myenteric neurons which would exclude their use in human tissue.
Section snippets
Guinea-pig myenteric plexus preparations
For the experiments we used male Dunkin Hartley guinea-pigs (Charles River Laboratories, Kisslegg, Germany; Harlan GmbH, Borchen, Germany). After killing the animals by cervical dislocation followed by exsanguination, the ileum was quickly removed and dissected in ice cold Krebs solution to obtain longitudinal muscle myenteric plexus preparations. The procedures were according to the German animal protection law. During preparation the tissue was constantly perfused with ice cold Carbogen (5% CO
Visualization (pre-staining) of the human myenteric plexus
Guinea-pig myenteric ganglia can be easily identified in living tissue by conventional light microscopy. In contrast, myenteric ganglia cannot be visualized in freshly dissected, non-stained vital human tissue preparations. This however is crucial to load ganglia by intraganglionic pressure ejection. In initial experiments we found that bulk loading of the tissues by long term dye incubation did not lead to any specific staining, very likely because of limited dye penetration and accessibility
Discussion
This is the first report on reproducible recordings from human myenteric neurons with voltage-sensitive dye imaging paving the way for further in depth studies on the neurobiology of human myenteric neurons.
We described a protocol that improves visibility of human myenteric ganglia in intact tissue by pre-staining with 4-Di-2-ASP. Of all the tested vital dyes 4-Di-2-ASP was the only dye that had no negative effect on the electrophysiological behaviour of guinea-pig myenteric neurons. These
Acknowledgements
We thank Prof. Dr. Peter Fromherz for the gift of ANINNE-6-Plus. This work was supported by DFG (International Research Training Group 1373).
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These authors contributed equally.