Experiments were done in urethane-anesthetized, barodenervated, male Wistar rats. Chemical stimulation of the hypothalamic paraventricular nucleus (PVN) by unilateral microinjections of Nmethyl-D-aspartic acid (NMDA) elicited increases in mean arterial pressure (MAP) and greater splanchnic nerve activity (GSNA). The increases in the MAP and GSNA induced by chemical stimulation of the PVN were significantly exaggerated by bilateral microinjections of D-AP7 and NBQX (ionotropic glutamate receptor antagonists) into the medial subnucleus of the nucleus tractus solitarius (mNTS). These results were confirmed by single unit recordings; i.e., excitation of mNTS barosensitive neurons caused by chemical stimulation of the ipsilateral PVN was blocked by application of D-AP7 and NBQX to these neurons. Bilateral microinjections of D-AP7 and NBQX into the mNTS elicited pressor responses which were significantly attenuated by inhibition of PVN neurons by bilateral microinjections of muscimol. Unilateral microinjections of fluorogold into the mNTS resulted in bilateral retrograde labeling of the PVN neurons. Unilateral microinjections of biotinylated dextran amine into the PVN resulted in anterograde labeling of axons and terminals in the mNTS bilaterally and the labeled terminals exhibited vesicular glutamate transporter-2 immunoreactivity. These results indicated that; 1) a tonically active glutamatergic bilateral projection from the PVN to the mNTS exists, 2) bilateral blockade of ionotropic glutamate receptors in the mNTS exaggerates the increases in MAP and GSNA to the chemical stimulation of the PVN, and 3) this projection may serve as a restraint mechanism for excitatory cardiovascular effects of PVN stimulation.

Arakawa H, Chitravanshi VC, Sapru HN. The hypothalamic arcuate nucleus: a new site of cardiovascular action of angiotensin-(1-12) and angiotensin II. Am J Physiol Heart Circ Physiol 300: H951-H960, 2011. First published December 24, 2010 doi:10.1152/ajpheart.01144.2010.-The hypothalamic arcuate nucleus (ARCN) has been reported to play a significant role in cardiovascular regulation. It has been hypothesized that the ARCN may be one of the sites of cardiovascular actions of angiotensins (ANGs). Experiments were carried out in urethaneanesthetized, artificially ventilated, adult male Wistar rats. The ARCN was identified by microinjections of N-methyl-D-aspartic acid (NMDA; 10 mM). Microinjections (50 nl) of ANG-(1-12) (1 mM) into the ARCN elicited increases in mean arterial pressure (MAP), heart rate (HR), and greater splanchnic nerve activity (GSNA). The tachycardic responses to ANG-(1-12) were attenuated by bilateral vagotomy. The cardiovascular responses elicited by ANG-(1-12) were attenuated by microinjections of ANG II type 1 receptor (AT1R) antagonists but not ANG type 2 receptor (AT2R) antagonist. Combined inhibition of ANG-converting enzyme (ACE) and chymase in the ARCN abolished ANG-(1-12)-induced responses. Microinjections of ANG II (1 mM) into the ARCN also increased MAP and HR. Inhibition of ARCN by microinjections of muscimol (1 mM) attenuated the pressor and tachycardic responses to intravenously administered ANG-(1-12) and ANG II (300 pmol/kg each). These results indicated that 1) microinjections of ANG-(1-12) into the ARCN elicited increases in MAP, HR, and GSNA; 2) HR responses were mediated via both sympathetic and vagus nerves; 3) AT1Rs, but not AT2Rs, in the ARCN mediated ANG-(1-12)-induced responses; 4) both ACE and chymase were needed to convert ANG-(1-12) to ANG II in the ARCN; and 5) ARCN plays a role in mediating the cardiovascular responses to circulating ANGs. blood pressure; heart rate; microinjection; N-methyl-D-aspartic acid; sympathetic nerve activity THE HYPOTHALAMIC ARCUATE NUCLEUS (ARCN) may play a significant role in cardiovascular regulation (10, 38). Consistent with this notion, we (31) have recently reported that chemical stimulation of the ARCN elicited increases in mean arterial pressure (MAP), heart rate (HR), and sympathetic nerve activity (SNA). These reports have provided a basis for investigations on different neurotransmitters and neuromodulators in the ARCN that may play a role in the regulation of cardiovascular function in normal and pathological states.A new endogenous angiotensin (ANG), ANG-(1-12), has recently been identified (30,44). Intravenous administration of this peptide has been reported to elicit an immediate pressor response in the rat, and this effect was blocked by prior administration of an ANG-converting enzyme (ACE) inhibitor or an ANG II type 1 receptor (AT 1 R) antagonist (30). These data indicated that in the periphery, ANG-(1-12) exerts its actions via a rapid conversion to ANG II (30). High concentrations of ANG-(1-12) have been reported in th...

Experiments were carried out in pentobarbital-anesthetized, vagotomized, paralyzed, and artificially ventilated male Wistar rats with a pneumothorax. Blood pressure, heart rate, and phrenic nerve activity were recorded. Extracellular neuronal recordings were made by glass micropipettes in a midline area in the commissural subnucleus of the nucleus tractus solitarius. At the end of the experiment, the recording sites were marked. Chemoreceptors were stimulated by tracheal administration of N2 or injection of CO2-saturated saline (50 microliters) near the carotid body. The baroreceptors were stimulated by an intravenous injection of phenylephrine (1-3 micrograms). Neurons that were excited by chemoreceptor stimulation but not baroreceptor stimulation were found in the commissural subnucleus of nucleus tractus solitarius and were designated as chemoreceptor-sensitive neurons. They exhibited a continuous discharge (9.9 +/- 1.3 spikes/s) that showed no apparent relation to either phrenic nerve bursts or arterial pulses. Such neurons were not found in the rostral portions of the nucleus tractus solitarius. These results confirm and extend our previous reports in which a discrete chemoreceptor projection site was identified in the commissural subnucleus of the rat.

G protein-coupled estrogen receptor (GPER) has been identified in several brain regions including cholinergic neurons of nucleus ambiguus, which are critical for the parasympathetic cardiac regulation. Using calcium imaging and electrophysiological techniques, microinjection into nucleus ambiguus and blood pressure measurement we examined the in vitro and in vivo effects of GPER activation in nucleus ambiguus neurons. G-1, a GPER selective agonist, produced a sustained increase in cytosolic Ca2+ concentration in a concentration-dependent manner in retrogradely-labeled cardiac vagal neurons of nucleus ambiguus. The increase in cytosolic Ca2+ produced by G-1 was abolished by pretreatment with G36, a GPER antagonist. G-1 depolarized cultured cardiac vagal neurons of nucleus ambiguus. The excitatory effect of G-1 was also identified by whole-cell visual patch-clamp recordings in nucleus ambiguus neurons, in medullary slices. To validate the physiological relevance of our in vitro studies, we carried out in vivo experiments. Microinjection of G-1 into the nucleus ambiguus elicited a decrease in heart rate; the effect was blocked by prior microinjection of G36. Systemic injection of G-1, in addition to a previously reported decrease in blood pressure, also reduced the heart rate. The G-1-induced bradycardia was prevented by systemic injection of atropine, a muscarinic antagonist, or by bilateral microinjection of G36 into the nucleus ambiguus. Our results indicate that GPER-mediated bradycardia occurs via activation of cardiac parasympathetic neurons of the nucleus ambiguus and support the involvement of GPER in the modulation of cardiac vagal tone.

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The presence of endomorphin-like immunoreactivity has been reported in the nucleus tractus solitarius (NTS). It was hypothesized that endomorphins may play a role in cardiovascular regulation in the medial subnucleus of the NTS (mNTS). Endomorphin-2 (E-2, 0.1-4 mmol/l) was microinjected (100 nl) into the mNTS of urethane-anesthetized, artificially ventilated, adult male Wistar rats. E-2 (0.2 mmol/l) elicited decreases in mean arterial pressure (40 +/- 3.5 mmHg) and heart rate (50 +/- 7.0 beats/min). These responses were blocked by prior microinjections of naloxonazine (1 mmol/l) into the mNTS. Responses to microinjections of E-2 into the mNTS were abolished by prior combined microinjections of d-2-amino-7-phosphonoheptanoic acid (an NMDA receptor antagonist, 5 mmol/l) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (a non-NMDA receptor antagonist, 2 mmol/l) into the mNTS. These results were confirmed by extracellular neuronal recordings. Blockade of GABA receptors in the mNTS by prior combined microinjections of gabazine (a GABA(A) receptor antagonist, 2 mmol/l) and 2-hydroxysaclofen (a GABA(B) receptor antagonist, 100 mmol/l) also blocked the responses to E-2. It was concluded that 1) the depressor and bradycardic responses to microinjections of E-2 into the mNTS are mediated via micro(1)-opioid receptors as well as ionotropic glutamate receptors, 2) GABAergic neurons in the mNTS, which may inhibit the release of glutamate from nerve terminals, are inhibited by E-2 via micro(1)-opioid receptors, and 3) disinhibition caused by the inhibition of GABAergic neurons by E-2 may result in an increase in the glutamate release from nerve terminals, which, in turn, may elicit depressor and bradycardic responses.

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