Fluorescent molecules as tools to study Ca2+ signaling, mitochondrial dynamics and synaptic function in enteric neurons.

The enteric nervous system (ENS) controls different tasks in the gastrointestinal tract. One of the ganglionated networks, the myenteric plexus is situated between longitudinal and circular muscle layers of the gut wall and basically controls intestinal motility. Several studies using micro-electrode recordings have generated invaluable information on neuronal subtypes, membrane conductances and simple synaptic inputs. Besides physiological recordings immunohistochemical techniques have also advanced the understanding of the organization of the ENS. Antibodies against several epitopes have identified a variety of neuronal subtypes, each with its characteristic combination of neurotransmitters and neuropeptides. In order to understand how the integrated plexus functions, especially in view of interactions between different cells, it is important to use optical techniques. They allow studying several cells simultaneously and provide both spatial and temporal information with a reasonably adequate resolution. Fluorescence is the method of choice, since due to the spectral properties; several probes can be combined to obtain information about different events within the cell. We used Ca2+ indicators both as general markers for activity and also as a tool to study the intrinsic Ca2+ handling characteristics of myenteric neurons. This enabled us to visualize neuronal activity in cultures and in multi-layered intestinal preparations. Using this method, the mechanisms underlying acetylcholine, substance P and serotonin signaling were dissected out as well as the Ca2+ removal mechanisms in enteric neurons. Further, we used neuronal retrograde tracers and combined this with Ca2+ imaging techniques to study specific signaling in a functionally identified population of neurons. Mitochondria are important organelles, which provide the bulk of the energy and play an important role in buffering cytosolic Ca2+. We investigated the role of mitochondria in myenteric neurons and found that Ca2+ mitochondrial buffering actually sets membrane excitability, especially in the primary afferent neurons of the intestine. Furthermore, mitochondria were also studied in processes of enteric neurons in culture, where they modify the Ca2+ signals in nerve boutons. In order to provide energy and Ca2+ buffer capacity to these distant regions, mitochondria need to be transported. The characteristics of this axonal transport were investigated using MitoTracker green. Last, we also visualized synaptic transmission directly, by using markers, which are trapped during vesicle recycling. The kinetics of release were studied optically both in culture and tissue preparations and were found to be remarkably similar to central nervous system synapses. In this report we summarize the results of different fluorescent imaging techniques to investigate important cellular and subcellular characteristics of enteric neurons. Because optical methods provide both spatial and temporal information they can be used to study activity and patterns in neuronal networks. Imaging is applicable to multi-layered intestinal preparations; therefore it provides a useful and promising tool to further expand our understanding of gastro-intestinal motility control and enteric neuroscience in general.