Furthermore, the EGFP-positive fibers in the liver appear to be osmosensitive as they exhibit robust increases in pERK staining after intake of 1 ml water in transgenic mice ( Figure 5D). In Ca2+-imaging experiments a striking 53% of the acutely isolated thoracic EGFP-positive neurons responded to the 230 mOsm stimulus (41/77 neurons), a significantly

higher proportion than found in EGFP-negative thoracic neurons (15%, 21/140 neurons) and of nonselected neurons in wild-type mice (31%, 62/217 neurons), p < 0.01 and p < 0.05 chi-square test ( Figure 5E). Thus, osmosensitive neurons are enriched within the population of EGFP-positive thoracic DRGs many of which innervate the liver. However, though very supportive, these results did not clarify whether cells that give rise to this website pERK-positive fibers in vivo and cells that are osmosensitive in cultures constitute the same subpopulation of thoracic DRG neurons. To test directly whether thoracic neurons with an osmosensitive current are hepatic osmoreceptors, we retrogradely labeled hepatic sensory neurons by injecting click here an Alexa Fluor-488 conjugated dextran amine into the liver. Three to four days after tracer injection into the liver, the thoracic ganglia were isolated and whole-cell patch-clamp recordings made from identified hepatic afferent neurons (Figure 6A). As a control for tracer leakage, DRGs were cultured from nearby spinal segments, but no labeled neurons were found. Strikingly,

almost all the hepatic afferent neurons 91.3% (21/23 cells) possessed a fast inward current in response to local perfusion of a 260 mOsm hypo-osmotic

solution (Figures 6A and 6B, green bars). The proportion too of osmosensitive neurons among the labeled hepatic afferent neurons (21/23 cells) was significantly higher than that found in randomly selected neurons from T7–T13 DRGs (25/37 cells, p < 0.05; Student’s unpaired t test; Figure 6B, black bars). In contrast, identified hepatic afferent neurons from Trpv4−/− mice only infrequently exhibited an inward current to hypo-osmotic stimulation 31.6% (6/19 cells), significantly different from wild-type neurons 91.3% (21/23 cells) chi-square test p < 0.01. Interestingly, the remaining osmosensitive currents found in Trpv4−/− mice were kinetically indistinguishable from those found in wild-type mice ( Figure S2). However, closer inspection of osmosensitive and nonosmosensitive neurons revealed that these two classes of neurons were also different in other respects. Identified hepatic sensory neurons that had lost their osmosensitivity in Trpv4−/− mice were significantly smaller (23.9 ± 0.8 μm versus 35 ± 1.6 μm, p < 0.001, Student’s paired t test) and had wider action potentials (3.35 ± 0.18 ms versus 1.60 ± 0.11 ms, p < 0.001, Student’s paired t test) than neurons that retain their osmosensitivity. Moreover, the small TRPV4-dependent osmosensitive neurons were more excitable as they fired an average of 2.9 ± 0.