Control of sympathetic outflow by pre-sympathetic paraventricular nucleus (PVN) neurons is partially mediated through a monosynaptic pathway that innervates the rostral ventrolateral medulla (RVLM). Here we investigated the role of the thapsigargin (TG), the endoplasmic reticulum (ER) Ca2+ ATPase inhibitor, in regulating the in vitro excitability of PVN neurons projecting to RVLM (PVN-RVLM) under brain slice preparation. In current-clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +200 pA injections and averaged 22 ± 2 Hz (n=7) and was significantly greater (P < 0.05) in the presence of TG (0.5 μM) (32 ± 4 Hz, n=5). Next, the effect of Ca2+ store depletion from the ER in regulating SNA and blood pressure was assessed by bilateral PVN microinjection of TG in anesthetized rats. PVN injection of TG (0.15, 0.3 0.75 and 1.5 nmol/100nl) increased the renal sympathetic nerve activity (SNA) and mean arterial pressure (MAP) in a dose-dependent manner. Maximum increase in renal SNA and MAP elicited by PVN TG (0.75 nmol/100nl; n=3) were 105 ± 31% and 18 ± 11 mmHg respectively. Pre-treatment of CyPPA (5 nmol/100 nl), a SK channel activator/opener (n=3), remarkably attenuated the PVN TG evoked sympathoexcitatory and pressor responses (7 ± 7% for renal SNA and 4 ± 7 mmHg for MAP). PVN injection of CyPPA did not significantly alter the baseline of renal SNA and MAP. Our data indicate that inhibition of ER function by TG contributes to the enhanced in vitro excitability of PVN-RVLM neurons and increased in vivo renal SNA and MAP. The effect of TG on the excitability of pre-sympathetic PVN neurons and sympathetic outflow through the Ca2+ store depletion from the ER may involve the down-regulation of SK channel function. Support: NSDP2010008 (JZ) and AHA10SDG2640130 (CQH).