Recordings from human myenteric neurons using voltage-sensitive dyes

Abstract

Voltage-sensitive dye (VSD) imaging became a powerful tool to detect neural activity in the enteric nervous system, including its routine use in submucous neurons in freshly dissected human tissue. However, VSD imaging of human myenteric neurons remained a challenge because of limited visibility of the ganglia and dye accessibility. We describe a protocol to apply VSD for recordings of human myenteric neurons in freshly dissected tissue and myenteric neurons in primary cultures. VSD imaging of guinea-pig myenteric neurons was used for reference. Electrical stimulation of interganglionic fiber tracts and exogenous application of nicotine or elevated KCl solution was used to evoke action potentials. Bath application of the VSDs Annine-6Plus, Di-4-ANEPPS, Di-8-ANEPPQ, Di-4-ANEPPDHQ or Di-8-ANEPPS revealed no neural signals in human tissue although most of these VSD worked in guinea-pig tissue. Unlike methylene blue and FM1–43, 4-Di-2-ASP did not influence spike discharge and was used in human tissue to visualize myenteric ganglia as a prerequisite for targeted intraganglionic VSD application. Of all VSDs, only intraganglionic injection of Di-8-ANEPPS by a volume controlled injector revealed neuronal signals in human tissue. Signal-to-noise ratio increased by addition of dipicrylamine to Di-8-ANEPPS (0.98 ± 0.16 vs. 2.4 ± 0.62). Establishing VSD imaging in primary cultures of human myenteric neurons led to a further improvement of signal-to-noise ratio. This allowed us to routinely record spike discharge after nicotine application. The described protocol enabled reliable VSD recordings from human myenteric neurons but may also be relevant for the use of other fluorescent dyes in human tissues.

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.