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After the neurons had been identified by epicardial application of capsaicin, the effects of epicardial application of saline, capsaicin, bradykinin, adenosine and H2O2 were randomly tested on the same neuron. Successive applications of chemicals were separated by at least 15 min for the complete recovery of neuronal activity and MAP. Protocol 2. In order to exclude the possibility that the increased activity of the capsaicin-sensitive neurons in the PVN was secondary to the elevated arterial pressure induced by epicardial application of the chemicals, the response of the PVN neurons to transient and mild elevation in MAP induced by mild aortic coarctation was determined in rats with VD and BD. After a capsaicin-sensitive neuron had been identified, mild aortic coarctation and sham aortic coarctation were carried out randomly while measuring the responses in the same neuron. Protocol 3. The rats were randomly divided into four groups Selleckchem PLX 4720 to determine the roles of vagal and baroreceptor afferents. The effects of epicardial application of capsaicin on the activity of capsaicin-sensitive neurons in the PVN were determined and compared among the rats without denervation (intact), the rats with VD and BD, VD alone and BD alone. A 30 min stabilization period was allowed between the denervation and the epicardial application of capsaicin. Protocol 4. The effects of electrical stimulation of the vagal afferents on the activity of capsaicin-sensitive Entinostat order neurons in the PVN were determined. Bilateral VD was carried out and the central ends of vagi were placed on silver electrodes. A 30 min stabilization period was allowed between the Reelin VD and the bilaterally simultaneous vagal stimulation (VS). Epicardial application of capsaicin was carried out 30 s after the beginning of the stimulation (0.5 ms, 8 Hz and 4 V). Protocol 5. To determine the effects of endogenous chemicals, the neuronal responses to LAD ligation-induced myocardial ischaemia and sham ligation were randomly determined in the same capsaicin-sensitive or -insensitive neurons of rats with VD and BD. The duration of myocardial ischaemia was no more than 90 s in order to avoid severe myocardial damage. The intervals between coronary ligation and sham ligation were at least 60 min to allow for complete recovery. Capsaicin, bradykinin, adenosine, H2O2, urethane, ��-chloralose and Pontamine Sky Blue were obtained from Sigma Chemical Co. Student's paired t test was used for comparisons of PVN firing (spikes per second) between the values before chemicals and the values after chemicals in Figs 3, 5 and 7. Unpaired student's t test was used for comparisons between the sham and myocardial ischaemia values in Fig. 7. One-way ANOVA followed by the Newman�CKeuls test for post hoc analysis was used for multiple comparisons in Figs 3�C5. All data were expressed as means ��s.e.m. A value of P