, 2011). We also found that VP excitatory effects on PVN-RVLM neurons were blocked by flufenamic acid (FFA; 200 μM) (n = 9; Figure S2F), a relatively specific blocker of TRPM4/TRPM5 channels (Ullrich et al., 2005). In the presence of FFA, PVN-RVLM neurons were still capable of displaying a burst of action potentials in response to a puff of 20 μM NMDA (n = 3; data not shown), indicating that FFA effects were not due to nonspecific effects on overall neuronal function or due to changes in PVN-RVLM responsiveness

to NMDA. Further studies, however, are needed to precisely identify the molecular identity of the CAN channel underlying VP actions in presympathetic neurons. Selleck LY2835219 To directly probe for a crosstalk between MNNs and presympathetic neurons, we developed an approach using transgenic EGFP-VP rats

(Ueta et al., 2005) that received an injection of a fluorescent retrograde tracer in the RVLM (Figure S3). Our approach consisted of selectively http://www.selleckchem.com/products/crenolanib-cp-868596.html activating individual VP neurons using laser photolysis of caged NMDA while simultaneously monitoring the electrical activity of neighboring presympathetic neurons in acute hypothalamic slices. To validate this approach, we show that laser photolysis of caged NMDA onto restricted somatodendritic regions of patched EGFP-VP neurosecretory neurons induced reproducible inward currents along with a concurrent high-frequency burst of action potentials (Figure 4A), previously shown to efficiently evoke dendritic release of peptides from MNNs in brain slices (Kombian et al., 1997). Moreover, photolysis of caged NMDA in the somata of Fluo-5F-loaded EGFP-VP neurons increased [Ca2+]i levels, which rapidly propagated into dendritic compartments (Figure S4). To test the hypothesis that dendritic VP release acts as a crosstalk signal between neurosecretory and presympathetic neurons, we then obtained patch recordings from PVN-RVLM neurons and assessed their responses to photolysis

of caged NMDA in neighboring EGFP-VP neurons. On average, three different EGFP-VP neurons were photoactivated per patched PVN-RVLM neuron. The mean distance between the somata of presympathetic and the photoactivated VP neurons was 111.6 ± 7.9 μm. Photolysis of caged NMDA at the somata of individual Mephenoxalone EGFP-VP neurons consistently evoked an excitatory response in neighboring PVN-RVLM neurons, characterized by a burst of activity, which was underlain by a membrane depolarization (n = 38 EGFP-VP neurons/11 PVN-RVLM neurons, p < 0.001; Figure 4B). Responses in presympathetic neurons occurred with a mean latency of 3.5 ± 1.0 s following photolysis in neighboring EGFP-VP neurons. In a few cases (n = 4), stimulation of an EGFP-VP neuron failed to evoke a response in PVN-RVLM neurons, which were, however, responsive to other EGFP-VP neurons in the same preparation. Direct photolysis of caged NMDA onto the recorded neurons (EGFP-VP or presympathetic) resulted in an almost instantaneous effect (p < 0.