Importantly, presympathetic responses to photoactivation of EGFP-VP neurons were almost completely blocked following bath application of the V1a receptor antagonist Figure 4C). No relationship between the uncaging distance

and magnitude or delay of the evoked response was observed. Although action potentials are necessary for axonal VP release, dendritic release can occur in a Ca2+-dependent but action potential-independent manner (Ludwig et al., 2002). Thus, to rule out a potential, but unlikely, contribution of VP released from an axon collateral within the PVN (Hatton et al., 1985 and Ludwig, 1998), experiments were repeated in the presence of 1 μM tetrodotoxin (TTX). Under these conditions, photolysis of NMDA TGF beta inhibitor onto EGFP-VP neurons still evoked a membrane depolarization in neighboring PVN-RVLM neurons

(n = 18), effects that were blocked by the V1a receptor antagonist (p < 0.01, n = 14; Figure 5). Conversely, following activation of EGFP-VP neurons, presympathetic responses were prevented by a 0 Ca2+/3 mM EGTA aCSF (Δ 1.2 ± 0.2 mV, n = 10, p > 0.3), without altering PVN-RVLM responses to direct uncaging of NMDA (Δ 5.5 ± 0.5 mV, p < 0.05, n = 4). These results support that Ca2+-dependent dendritic release of VP (Ludwig et al., 2002) stimulates the activity of neighboring selleck kinase inhibitor presympathetic PVN neurons via activation of V1a receptors. To rule out the possibility that PVN-RVLM neuronal responses were due to diffusion of caged NMDA beyond the photoactivated region in the EGFP-VP neurons, a subset of recorded PVN-RVLM neurons was dialyzed with the NMDAR blocker MK-801 (1 mM). Although intracellular MK801 significantly blocked the effect of direct photolysis of caged NMDA onto the recorded neurons (Δ 1.3 ± 0.9 mV; p > 0.2, n = 4), photolysis onto EGFP-VP neurons still evoked a depolarizing response from the same PVN-RVLM neurons (Δ 5.2 ± 1.4 mV; p < 0.05, n = 4) (Figure S5). Additional controls included laser stimulation without the presence of caged NMDA and bath application of caged NMDA alone, both of which failed to evoke neuronal responses (data not shown). Megestrol Acetate Finally, NMDA uncaging

onto EGFP-VP neurons (n = 33) failed to evoke changes in [Ca2+]I in the vast majority of Rhod-2-loaded astrocytes (∼87%). In the few responsive astrocytes, increases in [Ca2+]I occurred with a long delay (>40 s) (Figure S6). To further prove the neurosecretory-presympathetic neuronal crosstalk, we performed dual-patch recordings from identified EGFP-VP and PVN-RVLM neurons. In 13 out of 15 pairs tested, we found that evoked burst firing in VP neurons resulted in a significant membrane depolarization (p < 0.001; n = 13) and increase in firing discharge (p < 0.02; n = 13) in the neighboring presympathetic neurons (Figures 6B and 6F). These effects were largely blocked following bath application of the V1a antagonist (n = 7) or in pairs in which EGFP-VP neurons were dialyzed with BAPTA (n = 5) (Figures 6C and 6F).