05 versus control; Figures 7C and 7D). In the presence of the V1a antagonist, however, U-50488 failed to affect the firing activity of presympathetic neurons (p > 0.6, n = 4; Figure S7B), Pazopanib supplier arguing against a direct effect of U50488 on the latter. No correlation between basal PVN-RVLM firing activity and the magnitude of the V1a antagonist effect was found in any of these different conditions (Pearson r = −0.02; p > 0.5). Dialysis of BAPTA into the recorded PVN-RVLM neurons prevented the effects of the V1a antagonist (baseline, 0.7 ±

0.1 Hz; V1a antagonist, 0.6 ± 0.1 Hz; p > 0.3, n = 6). A diffusible signal in the ECS could be influenced both by its half-life and the ECS tortuosity. Blockade of tissue aminopeptidase activity (amastatin

10 μM) increased the firing activity of presympathetic neurons (p < 0.01, n = 8; Figure 7E). The amastatin effect was not only blocked but also actually turned into an inhibitory effect in the presence of the V1a receptor blocker (p < 0.01 versus amastatin control, n = 7; Figure 7E). These results indicate that aminopeptidase blockade increased not only the availability and excitatory actions of endogenous VP but also of an unknown inhibitory signal, which was only unmasked when the VP excitatory effect was blocked. The identity of this inhibitory peptide signal was not further JAK inhibitor investigated in this study. Reducing the coefficient of diffusion in the ECS with 5% dextran (40 kDa)

(Min et al., 1998 and Piet to et al., 2004) also blocked the V1a antagonist effect on presympathetic firing discharge (−6.5% ± 8.8%; p > 0.6, n = 4). Taken together, these results indicate that tonically released VP within the PVN serves as a neurosecretory population signal, which acting in a diffusible manner, increased the activity of the presympathetic PVN neuronal population. We finally assessed whether dendritic release of VP serves as an interpopulation signal by which the integrated sympathoexcitatory output from the entire presympathetic neuronal population was modulated. To this end, we performed in vivo studies to directly monitor sympathoexcitatory outflow from the PVN. We found that direct microinjection of VP (8–32 pmol) onto the PVN elicited a dose-dependent sympathoexcitatory response, reflected by an increase in renal sympathetic nerve activity (RSNA; p < 0.02, n = 9; Figures 8A and 8B). These results indicate that the VP excitatory effect observed on presympathetic neurons in vitro translated into a systemic, population sympathoexcitatory response. It is well documented that a central osmotic challenge results in a robust PVN homeostatic response that involves an orchestrated activation of VP MNNs and presympathetic neurons, leading to increased plasma VP levels along with a concomitant increase in sympathetic outflow, respectively (Bourque, 2008 and Toney and Stocker, 2010).