Non-technical summary Though the pancreatic hormone insulin is known to act in the brain to increase sympathetic nerve activity and baroreflex control of sympathetic nerve activity, its specific site of action had yet to be identified. We show that a region in the hypothalamus, the arcuate nucleus, is the site at which insulin's effects are initiated. This new information may lead to a greater understanding of the role of insulin in the brain in adverse cardiovascular complications, like hypertension and heart attacks, which are associated with insulin-resistant states, such as obesity and diabetes.Abstract Although the central effects of insulin to activate the sympathetic nervous system and enhance baroreflex gain are well known, the specific brain site(s) at which insulin acts has not been identified. We tested the hypotheses that (1) the paraventricular nucleus of the hypothalamus (PVN) and the arcuate nucleus (ArcN) are necessary brain sites and (2) insulin initiates its effects directly in the PVN and/or the ArcN. In α-chloralose anaesthetised female Sprague-Dawley rats, mean arterial pressure (MAP), heart rate (HR) and lumbar sympathetic nerve activity (LSNA) were recorded continuously, and baroreflex gain of HR and LSNA were measured before and during a hyperinsulinaemic-euglycaemic clamp. After 60 min, intravenous infusion of insulin (15 mU kg −1 min −1 ), but not saline, significantly increased (P < 0.05) basal LSNA (to 228 ± 28% control) and gain of baroreflex control of LSNA (from 3.8 ± 1.1 to 7.4 ± 2.4% control mmHg −1 ). These effects were reversed (P < 0.05) by local inhibition (bilateral microinjection of musimol) of the PVN (LSNA to 124 ± 8.8% control; LSNA gain to 3.9 ± 1.7% control mmHg −1 ) or of the ArcN (LSNA in % control: from 100 ± 0 to 198 ± 24 (insulin), then 133 ± 23 (muscimol) LSNA gain in % control mmHg −1 : from 3.9 ± 0.3 to 8.9 ± 0.9 (insulin), then 5.1 ± 0.5 (muscimol)). While insulin receptor immunoreactivity was identified in neurons in pre-autonomic PVN subnuclei, microinjection of insulin (0.6, 6 and 60 nU) into the PVN failed to alter LSNA or LSNA gain. However, ArcN insulin increased (P < 0.05) basal LSNA (in % control to 162 ± 19, 0.6 nU; 193 ± 19, 6 nU; and 205 ± 28, 60 nU) and LSNA baroreflex gain (in % control mmHg −1 from 4.3 ± 1.2 to 6.9 ± 1.0, 0.6 nU; 7.7 ± 1.2, 6 nU; and 7.8 ± 1.3, 60 nU). None of the treatments altered MAP, HR, or baroreflex control of HR. Our findings identify the ArcN as the site at which insulin acts to activate the sympathetic nervous system and increase baroreflex gain, via a neural pathway that includes the PVN. Abbreviations ArcN, arcuate nucleus; BRG, baroreflex gain; HR, heart rate; LSNA, lumbar sympathetic nerve activity; MAP, mean arterial pressure; PVN, paraventricular nucleus; SNA, sympathetic nerve activity.

Sympathetic vasoconstriction of cerebral vessels has been proposed to be a protective mechanism for the brain, limiting cerebral perfusion and microcirculatory pressure during transient increases in arterial pressure. To furnish direct neural evidence for this proposition, we aimed to develop a method for recording cerebral sympathetic nerve activity (SNA) from the superior cervical ganglion (SCG). We hypothesized that SNA recorded from the SCG increases during imposed hypertension, but not during hypotension. Lambs (n = 11) were anesthetized (alpha-chloralose, 20 mg.kg(-1).h(-1)) and ventilated. SNA was measured using 25-microm tungsten microelectrodes inserted into the SCG. Arterial blood pressure (AP) was pharmacologically raised (adrenaline, phenylephrine, or ANG II, 1-50 microg/kg iv), mechanically raised (intravascular balloon in the thoracic aorta), or lowered (sodium nitroprusside, 1-50 microg/kg iv). In response to adrenaline (n = 10), mean AP increased 135 +/- 10% from baseline (mean +/- SE), and the RMS value of SNA (Square Root of the Mean of the Squares, SNA(RMS)) increased 255 +/- 120%. In response to mechanically induced hypertension, mean AP increased 43 +/- 3%, and SNA(RMS) increased 53 +/- 13%. Generally, (9 of 10 animals), SNA(RMS) did not increase, as AP was lowered with sodium nitroprusside. Using a new model for direct recording of cerebral SNA from the SCG, we have demonstrated that SNA increases in response to large induced rises, but not falls, in AP. These findings furnish direct support for the proposed protective role for sympathetic nerves in the cerebral circulation.

Key pointsr Neuropeptide Y (NPY) acts in the brain to decrease sympathetic nerve activity (SNA); however, the specific site is unknown.r We identify the paraventricular nucleus of the hypothalamus (PVN) as a site of action, since nanoinjection of NPY into the PVN dose-dependently decreases SNA, whereas PVN injection of NPY Y1 and Y5 receptor antagonists increases SNA.r NPY may directly inhibit PVN presympathetic neurons, since these neurons express Y1 receptors and, in patch-clamp experiments, are inhibited by NPY.r Our data also indicate that identified PVN presympathetic neurons that are inhibited by NPY are also excited by α-melanocyte-stimulating hormone.r These results identify endogenous PVN NPY as a novel and potent inhibitory neuromodulator that may contribute to changes in SNA that occur in states associated with altered energy balance, such as obesity and pregnancy.Abstract Neuropeptide Y (NPY), a brain neuromodulator that has been strongly implicated in the regulation of energy balance, also acts centrally to inhibit sympathetic nerve activity (SNA); however, the site and mechanism of action are unknown. In chloralose-anaesthetized female rats, nanoinjection of NPY into the paraventricular nucleus of the hypothalamus (PVN) dose-dependently suppressed lumbar SNA (LSNA) and its baroreflex regulation, and these effects were blocked by prior inhibition of NPY Y1 or Y5 receptors. Moreover, PVN injection of Y1 and Y5 receptor antagonists in otherwise untreated rats increased basal and baroreflex control of LSNA, indicating that endogenous NPY tonically inhibits PVN presympathetic neurons. The sympathoexcitation following blockade of PVN NPY inhibition was eliminated by prior PVN nanoinjection of the melanocortin 3/4 receptor inhibitor SHU9119. Moreover, presympathetic neurons, identified immunohistochemically using cholera toxin b neuronal tract tracing from the rostral ventrolateral medulla (RVLM), express NPY Y1 receptor immunoreactivity, and patch-clamp recordings revealed that both NPY and α-melanocyte-stimulating hormone (α-MSH) inhibit and stimulate, respectively, PVN-RVLM neurons. Collectively, these data suggest that PVN NPY inputs converge with α-MSH to influence presympathetic neurons. Together these results identify endogenous NPY as a novel and potent inhibitory neuromodulator within the PVN that may contribute to changes in SNA that occur in states associated with altered energy balance, such as obesity and pregnancy. P. A. Cassaglia and Z. Shi contributed equally to this work.

While leptin is known to increase sympathetic nerve activity (SNA), we tested the hypothesis that leptin also enhances baroreflex control of SNA and HR. Using α-chloralose anesthetized male rats, mean arterial pressure (MAP), HR, lumbar SNA (LSNA), splanchnic SNA (SSNA), and renal SNA (RSNA) were recorded before and for 2 hr after lateral cerebroventricular (LV) leptin or aCSF administration. Baroreflex function was assessed using a four parameter sigmoidal fit of HR and SNA responses to slow ramp (3-5 min) changes in MAP, induced by iv infusion of nitroprusside and phenylephrine. Leptin (3 μg) increased (P<0.05) basal LSNA, SSNA, RSNA, HR and MAP, and the LSNA, SSNA, RSNA, and HR baroreflex maxima. Leptin also increased gain of baroreflex control of LSNA and RSNA, but not of SSNA or HR. The elevations in HR were eliminated by pretreatment with methscopalamine, to block parasympathetic nerve activity; however, after cardiac sympathetic blockade with atenolol, leptin still increased basal HR and MAP and the HR baroreflex maximum and minimum. Leptin (1.5 μg) also increased LSNA and enhanced LSNA baroreflex gain and maximum, but did not alter MAP, HR, or the HR baroreflex. LV aCSF had no effects. Finally, to test if leptin acts in the brainstem, leptin (3 μg) was infused into the 4th ventricle; however, no significant changes were observed. In conclusion, leptin acts in the forebrain to differentially influence baroreflex control of LSNA, RSNA, SSNA and HR, with the latter action mediated via suppression of parasympathetic nerve activity.