Data was collected from at least four animals per group. The number and area of pERK-stained fibers increased significantly following intake of 1 ml water compared to a sham drinking control or to ingestion of 1 ml near-isotonic PBS (p < 0.01 compared to sham, p < 0.05 compared to PBS; Figures 1B and 1C). The absence of significantly increased pERK staining after intake of 1 ml PBS suggested that liver nerve fibers detect the decreased osmolality in the hepatic circulation and not stretch or pressure changes in the vessels. Members of the TRP ion channel family have been reported to be involved in osmosensation (Clapham et al., 2005 and Everaerts
et al., 2010). We thus let mice drink 1 ml of BAY 73-4506 water doped with 100 μM Ruthenium red (RR) a broad blocker of TRP channels and observed
no increase in pERK positive fibers compared to controls (Figures 1B and 1C). The liver is innervated by vagal afferents and thoracic DRG neurons (Berthoud, 2004, Carobi and Magni, 1985 and Magni and Carobi, 1983). We therefore analyzed the osmosensitivity of acutely isolated vagal nodose neurons and DRG neurons from different spinal levels (cervical C4–C7, thoracic T7–T13, and lumbar L1–L6) using Fura-2-based Ca2+ imaging. Repeated stimulation with hypo-osmotic solutions (230 mOsm for 20 s) induced robust and reversible increases in the intracellular calcium concentration [Ca2+]i in neurons from all levels with little evidence of tachyphylaxis (Figure 2A). CHIR-99021 nmr Interestingly, the incidence of osmosensitive neurons was low in all ganglia tested (lumbar DRG 9.1%, cervical DRG 11.2%, and nodose 9.9%) with the notable exception of the thoracic ganglia, where 31% of the neurons showed responses to hypo-osmotic stimuli (Figure 2B). Most cell types increase their volume when exposed to hypo-osmotic fluids (Hoffmann et al., 2009); we thus examined the temporal correlation between cell swelling and changes in [Ca2+]i. Cell
swelling was quantified by measuring changes in the area of the fluorescence signal after background subtraction and conversion into binary images (Figure 2C). This analysis revealed that significant increases in [Ca2+]i occurred concurrent with detectable changes in cell volume (Figure 2D; see Movie S1 available online). Interestingly, not all whatever cells that exhibited cell swelling also showed a calcium response (Movie S1). We next investigated the mechanism underlying the increase in [Ca2+]i. Removal of extracellular calcium almost completely abolished the Ca2+-response to hypo-osmotic stimulation, whereas depletion of intracellular calcium stores by a 30 min incubation with 5 μM thapsigargin neither changed the proportion (32% of the neurons responded), nor the magnitude of the Ca2+-response (Figure 3A). Blocking voltage-gated calcium channels with 100 μM Cd2+ did not affect Ca2+-signals (Figure 3A).