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GRK 1482 Jahrbuch 2011-2014

Publications [1] Bourque CW. Central mechanisms of osmosensation and systemic osmoregulation. Nat Rev Neurosci. 2008, 9(7):519-31. [2] Bourque CW, Ciura S, Trudel E, Stachniak TJE and Sharif- Naeini R. Neurophysiological characterization of mam- malian osmosensitive neurons. Exp Physiol. 2007, 92.3 pp 499-505. [3] Lechner SG, Markworth S, Poole K, St. John Smith E, LapatsinaL,FrahmS,MayM,PischkeS,SuzukiM,Ibanez- Tallon I, Luft FC, Jordan J and Lewin GR. The Molecular and Cellular Identity of Peripheral Osmo- receptors. Neuron. 2011, 332-344. [4] CostaM,BrookesSJ,HennigGW.Anatomyandphysiology oftheentericnervoussystem.Gut.2000,47Suppl4:iv15-9. [5] Liu M, Seino S, Kirchgessner AL. Identification and cha- racterization of glucoresponsive neurons in the enteric nervous system. J Neurosci. 1999, 1; 19(23):10305-17. [6] Michel K, ...Berghe P et al. Fast calcium and voltage- sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge. J Physiol. 2011, 589:5941 - 5947. PhD FELLOWS GRK Progress Report 2011-2014 | Page 57 Aim The aim of this study is to investigate the role of the enteric neurons and glia in detection and response to osmolality chan- ges within the gut. More in detail, since the ENS is made up of two plexi with different location within the gut wall, we want to investigate the possible differences in the responses of the neu- rons of the submucous plexus versus the myenteric neurons. Our aim is also to study osmosensitivity relating to region de- pendency. Methods and Results Tissue samples are obtained from male guinea pigs. Samples from the small or the large intes-tine were quickly removed and the submucous or the myenteric plexus are further prepared for the electrophysiological experiments. We use a Neuro imaging technique to detect signals from the enteric ganglia loaded with the fluorescent dye Di-8-ANEPPS. This dye is incorporated into the neuronal membrane (see figure 1) by pressure ejection and is voltage sensitive. Recordings are performed with an ultra- fast CCD based Neuro imaging system which detects signals with a high temporal (1.6 kHz) and spatial (up to 4 µm2 per pixel) resolution. Signals are expressed as relative changes in fluorescence intensity (∆F/F) [6]. Myenteric or submucous neurons are then briefly exposed to various hypo - or hyperosmolar solutions, obtained by adjusting the NaCl concentration of the Krebs HEPES buffered solution (or adding Mannitol). These solutions are applied ia a local per- fusion system. This allows us to accurately adjust application volume and rate of osmotic solutions. Preliminary data showed a direct sensitivity of the enteric neu- rons to hypo- and hyperosmolar solutions. Responses could be recorded both in the small and in the large intestine in submu- cous as well in myenteric neurons. Preliminary experiments to reliably control osmolality at the neuronal level are in progress. These data are necessary in order to have an extremely accurate measure of osmolality changes over time that the neurons undergo during experi- ments. Outlook After showing the feasibility and reproducibility of the neuro- imaging method we now expect to describe and characterize responsiveness of enteric neurons from the two plexi of the dif- ferent gut regions to various osmotic changes. We estimate to find different sensitivity to osmotic changes due to submucous neurons being located closer to the luminal compartment than myenteric neurons. Figure 1: Example of dye loaded ganglion of the jejunal myenteric ple- xus stained with fluorescent dye Di-8-ANEPPS. Individual neurons can be seen due to the dye being incorporated into the cell membrane. Solutions of different osmolality are micro-ejected onto the neurons with a local perfusion system. Supervisors Prof. Dr. Michael Schemann I TUM I Human Biology Dr. Gemma Mazzuoli I TUM I Human Biology Prof. Dr. Martin Klingenspor | TUM | Molecular Nutritional Medicine Start of project: June 2012 Academic background: Studies of Neuroscience at the Ludwig-Maximilians-Universität München