Lumbar Sympathetic Nerve Activity Research Articles

Arcuate nucleus – a gateway for insulin's action on sympathetic activity

Landsberg and colleagues in the 1980s first showed that an increase in the level of circulating insulin results in an increase in sympathetic activity, both in humans and in rats, even under conditions where the level of plasma glucose is clamped (for review see Landsberg, 2001). Insulin levels increase as a consequence of insulin resistance, which is commonly associated with obesity and the metabolic syndrome (Landsberg, 2001; Lambert et al. 2010). These findings led to the hypothesis that the hyperinsulinaemia associated with obesity causes an increase in sympathetic activity, which could account, at least in part, for obesity-related hypertension (Landsberg, 2001). In support of this hypothesis, epidemiological studies carried out in the 1990s showed that obese subjects had higher levels of insulin and blood pressure. The correlation between hyperinsulinaemia and hypertension is likely to be causal, because an increase in insulin levels in obese subjects leads to an increase in sympathetic activity, whereas an acute reduction in insulin levels in patients with hyperinsulinaemia results in a small but significant decrease in both sympathetic activity and blood pressure (Landsberg, 2001). In these early studies sympathetic activity was assessed indirectly by measuring urinary excretion of noradrenaline, but more recently the findings have been confirmed using the methods of noradrenaline spillover and microneurography (Lambert et al. 2010). Thus, although several other factors such as increased levels of circulating leptin are also likely to be important, there is now very substantial evidence that hyperinsulinaemia is a causal factor in obesity-related hypertension, as a consequence of its stimulatory effect on sympathetic nerve activity. Where and how does insulin act in the brain to increase sympathetic activity? A study by Cassaglia et al. in a recent issue of The Journal of Physiology provides convincing evidence that an increased level of circulating insulin acts on neurons in the arcuate nucleus in the ventromedial hypothalamus, leading to increased sympathetic activity and baroreflex sensitivity (see Fig. 1). The authors found that in anaesthetized rats intravenous infusion of insulin caused a large (over 100%) increase in lumbar sympathetic nerve activity (LSNA) and in the gain of the baroreflex control of LSNA. These effects were reversed after inhibition by microinjection of the GABA receptor agonist muscimol into either the hypothalamic paraventricular nucleus (PVN) or the arcuate nucleus. Both of these nuclei contain a high density of insulin receptors. Cassaglia et al. found, however, that insulin increased sympathetic activity and baroreflex gain when injected locally into the arcuate nucleus, but had no effect when injected into the PVN. As the authors note, confirmation that the arcuate nucleus is the site at which circulating insulin triggers sympathoexcitation will require future experiments in which the effects of specific blockade of insulin receptors within the arcuate nucleus are tested. Figure 1 Postulated central pathways subserving the effect of insulin on sympathetic activity If insulin acts on receptors in the arcuate nucleus, how is it transported from the blood? It does not readily cross the blood–brain barrier, but may access neurons within the arcuate nucleus via receptor-mediated endocytosis. On the other hand, the arcuate nucleus is unusual in that it contains highly permeable capillaries (Ciofi, 2011) like those in circumventricular organs, and so insulin may directly activate receptors in this nucleus without requiring a specific transport mechanism. Although the results showed that the PVN is also an essential part of the neural pathway mediating the effects of insulin on the sympathetic outflow (see Fig. 1), insulin microinjection into the PVN had no effect on sympathetic activity even though the PVN contains a high density of insulin receptors, as mentioned above. As the authors point out, insulin has inhibitory effects on neurons, and so it is possible that the effects of insulin on PVN neurons may not be observable in anaesthesia, when the tonic activity of PVN-sympathetic neurons may be very low. Apart from the PVN, the arcuate nucleus also projects to other brain regions that regulate the sympathetic outflow, including the dorsomedial hypothalamus, midbrain periaqueductal grey, rostral ventrolateral medulla and the nucleus of the solitary tract, (as shown in Fig. 1). These pathways may also subserve, at least in part, sympathoexcitatory responses evoked from the arcuate nucleus. Furthermore, neurons in the arcuate nucleus are also influenced by other hormones apart from insulin, such as leptin and ghrelin, which may also contribute to obesity-related hypertension (Rahmouni et al. 2005). It is clear that much remains to be discovered about the central pathways and mechanisms by which circulating insulin can affect sympathetic outflow, both in the short and the long term. The present study, however, is an important step towards that goal.

Purinergic mechanisms mediating sympathoexcitation in the paraventricular nucleus (PVN) of hypothalamus

The PVN plays a key role in the control of autonomic function. Studies have shown that PVN neurons projecting to the rostroventrolateral medulla express P2X receptors. The aim of this study is to investigate the effects of ATP injected into the PVN on lumbar sympathetic nerve activity (LSNA) in an in situ artificially perfused rat preparation. Male Wistar rats (70 g) were anesthetized with halothane, decorticated and perfused with a Ringer's solution. Recordings of phrenic nerve (PNA) and LSNA were monitored. Three-barrelled glass pipettes was filled with L-glutamate (L-glu, 10 mM), ATP (10, 25 and 50 mM), PPADS (3 and 5 mM) for injection into the PVN (100 nL). Microinjection of ATP in three different concentration (10, 25 and 50 mM) into the PVN induced a dose-related increase in the LSNA (38±6 %, n=6; 72±7 %, n=5; 96±13 %, n=5, respectively). The sympathoexcitation induced by ATP (72±7 %, 25 mM) into the PVN was attenuated by previous injection of PPADS (3 mM, 10±3 %, n=5, p<0.05). Moreover, the increase in LSNA elicited by L-glu (67±19 %, n=6) was not affected by a previous injection of PPADS (5 mM) at the same PVN site. In conclusion our data suggests that exogenous ATP into the PVN elicits an increase in LSNA in a dose-dependent manner and this response is dependent of the activation of P2 receptors; however, the functional role of the ATP in the physiological context awaits further investigation. Sponsors: CAPES, CNPq and FAPESP.

The arcuate nucleus mediates the effect of insulin to increase lumbar sympathetic nerve activity and its baroreflex regulation

The arcuate nucleus mediates the effect of insulin to increase lumbar sympathetic nerve activity and its baroreflex regulation

Asymmetrical changes in lumbar sympathetic nerve activity following stimulation of the sciatic nerve in rat

Noxious stimulation of the leg increases hind limb blood flow (HBF) to the ipsilateral side and decreases to the contralateral in rat. Whether or not this asymmetrical response is due to direct control by sympathetic terminals or mediated by other factors such as local metabolism and hormones remains unclear. The aim of this study was to compare responses in lumbar sympathetic nerve activity, evoked by stimulation of the ipsilateral and contralateral sciatic nerve (SN). We also sought to determine the supraspinal mechanisms involved in the observed responses. In anesthetized and paralyzed rats, intermittent electrical stimulation (1 mA, 0.5 Hz) of the contralateral SN evoked a biphasic sympathoexcitation. Following ipsilateral SN stimulation, the response is preceded by an inhibitory potential with a latency of 50 ms ( N = 26). Both excitatory and inhibitory potentials are abolished following cervical C1 spinal transection ( N = 6) or bilateral microinjections of muscimol ( N = 6) in the rostral ventrolateral medulla (RVLM). This evidence is suggestive that both sympathetic potentials are supraspinally mediated in this nucleus. Blockade of RVLM glutamate receptors by microinjection of kynurenic acid ( N = 4) selectively abolished the excitatory potential elicited by ipsilateral SN stimulation. This study supports the physiological model that activation of hind limb nociceptors evokes a generalized sympathoexcitation, with the exception of the ipsilateral side where there is a withdrawal of sympathetic tone resulting in an increase in HBF.

Insulin acts in the arcuate nucleus to increase lumbar sympathetic nerve activity and baroreflex function in rats

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.

Sympathetic Response to Insulin Is Mediated by Melanocortin 3/4 Receptors in the Hypothalamic Paraventricular Nucleus

Hyperinsulinemia increases sympathetic nerve activity and contributes to cardiovascular dysfunction in obesity and diabetes. Neurons of the hypothalamic paraventricular nucleus (PVN) regulate sympathetic nerve activity through mono- and poly-synaptic connections to preganglionic neurons in the spinal cord. The purpose of the present study was to determine whether PVN neurons mediate the sympathetic response to insulin. Hyperinsulinemic-euglycemic clamps were performed in α-chloralose-anesthetized, male Sprague-Dawley rats (280-420 g) by an infusion of insulin (3.75 mU/kg per min) and 50% dextrose (0.75-2.0 mL/h) for 120 minutes. At 90 minutes, insulin significantly increased lumbar sympathetic nerve activity without any change in renal sympathetic nerve activity, heart rate, or blood glucose levels. Inhibition of the PVN with bilateral injection of the GABA(A) receptor agonist muscimol completely reversed the sympathoexcitatory response. However, direct injection of insulin into the PVN did not alter lumbar sympathetic nerve activity, and thereby suggests that insulin activates neurons upstream of the PVN. Interestingly, the sympathetic response to insulin was eliminated by PVN injection of the melanocortin 3/4 receptor antagonist SHU9119, but was unaffected by the angiotensin II type 1 receptor antagonist losartan. A final set of experiments suggests activation of PVN neurons during hyperinsulinemia increases glutamatergic drive to the rostral ventrolateral medulla. Collectively, these findings indicate that insulin activates a melanocortin-dependent pathway to the PVN that increases glutamatergic drive to the rostral ventrolateral medulla and alters cardiovascular function.

Hydrogen Sulfide in the RVLM and PVN has No Effect on Cardiovascular Regulation

Hydrogen sulfide (H2S) is now recognized as an important signaling molecule and has been shown to have vasodilator and cardio-protectant effects. More recently it has been suggested that H2S may also act within the brain to reduce blood pressure (BP). In the present study we have demonstrated the presence of the H2S-producing enzyme, cystathionine-β-synthase (CBS) in the rostral ventrolateral medulla (RVLM), and the hypothalamic paraventricular nucleus (PVN), brain regions with key cardiovascular regulatory functions. The cardiovascular role of H2S was investigated by determining the BP, heart rate (HR), and lumbar sympathetic nerve activity (LSNA) responses elicited by a H2S donor sodium hydrogen sulfide (NaHS) or inhibitors of CBS, microinjected into the RVLM and PVN. In anesthetized Wistar Kyoto rats bilateral microinjections of NaHS (0.2–2000 pmol/side) into the RVLM did not significantly affect BP, HR, or LSNA, compared to vehicle. Similarly, when the CBS inhibitors, amino-oxyacetate (AOA; 0.1–1.0 nmol/side) or hydroxylamine (HA; 0.2–2.0 nmol/side), were administered into the RVLM, there were no significant effects on the cardiovascular variables compared to vehicle. Microinjections into the PVN of NaHS, HA, and AOA had no consistent significant effects on BP, HR, or LSNA compared to vehicle. We also investigated the cardiovascular responses to NaHS microinjected into the RVLM and PVN in spontaneously hypertensive rats. Again, there were no significant effects on BP, HR, and LSNA. Together, these results suggest that H2S in the RVLM and PVN does not have a major role in cardiovascular regulation.

Tyrosine hydroxylase immunoreactivity as indicator of sympathetic activity: simultaneous evaluation in different tissues of hypertensive rats

Vasomotor control by the sympathetic nervous system presents substantial heterogeneity within different tissues, providing appropriate homeostatic responses to maintain basal/stimulated cardiovascular function both at normal and pathological conditions. The availability of a reproducible technique for simultaneous measurement of sympathetic drive to different tissues is of great interest to uncover regional patterns of sympathetic nerve activity (SNA). We propose the association of tyrosine hydroxylase immunoreactivity (THir) with image analysis to quantify norepinephrine (NE) content within nerve terminals in arteries/arterioles as a good index for regional sympathetic outflow. THir was measured in fixed arterioles of kidney, heart, and skeletal muscle of Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) (123 ± 2 and 181 ± 4 mmHg, 300 ± 8 and 352 ± 8 beats/min, respectively). There was a differential THir distribution in both groups: higher THir was observed in the kidney and skeletal muscle (∼3-4-fold vs. heart arterioles) of WKY; in SHR, THir was increased in the kidney and heart (2.4- and 5.3-fold vs. WKY, respectively) with no change in the skeletal muscle arterioles. Observed THir changes were confirmed by either: 1) determination of NE content (high-performance liquid chromatography) in fresh tissues (SHR vs. WKY): +34% and +17% in kidney and heart, respectively, with no change in the skeletal muscle; 2) direct recording of renal (RSNA) and lumbar SNA (LSNA) in anesthetized rats, showing increased RSNA but unchanged LSNA in SHR vs. WKY. THir in skeletal muscle arterioles, NE content in femoral artery, and LSNA were simultaneously reduced by exercise training in the WKY group. Results indicate that THir is a valuable technique to simultaneously evaluate regional patterns of sympathetic activity.

Insulin enhances the gain of arterial baroreflex control of muscle sympathetic nerve activity in humans

Recent animal studies indicate that insulin increases arterial baroreflex control of lumbar sympathetic nerve activity; however, the extent to which these findings can be extrapolated to humans is unknown. To begin to address this, muscle sympathetic nerve activity (MSNA) and arterial blood pressure were measured in 19 healthy subjects (27 ± 1 years) before, and for 120 min following, two common methodologies used to evoke sustained increases in plasma insulin: a mixed meal and a hyperinsulinaemic euglycaemic clamp. Weighted linear regression analysis between MSNA and diastolic blood pressure was used to determine the gain (i.e. sensitivity) of arterial baroreflex control of MSNA. Plasma insulin was significantly elevated within 30 min following meal intake (34 ± 6 uIU ml(1); P < 0.05) and remained above baseline for up to 120 min. Similarly, after meal intake, arterial baroreflex-MSNA gain for burst incidence and total MSNA was increased and remained elevated for the duration of the protocol (e.g. burst incidence gain: 3.29 ± 0.54 baseline vs. 5.64 ± 0.67 bursts (100 heart beats)(1) mmHg(1) at 120 min; P < 0.05). During the hyperinsulinaemic euglycaemic clamp, in which insulin was elevated to postprandial concentrations (42 ± 6 μIU ml(1); P < 0.05), while glucose was maintained constant, arterial baroreflex-MSNA gain was similarly enhanced (e.g. burst incidence gain: 2.44 ± 0.29 baseline vs. 4.74 ± 0.71 bursts (100 heart beats)(1) mmHg(1) at 120 min; P < 0.05). Importantly, during time control experiments, with sustained fasting insulin concentrations, the arterial baroreflex-MSNA gain remained unchanged. These findings demonstrate, for the first time in healthy humans, that increases in plasma insulin enhance the gain of arterial baroreflex control of MSNA.

Differential effects of insulin on sympathetic nerve activity in agouti obese mice

Hyperinsulinemia, which often coexists with obesity and type 2 diabetes, is a major risk factor for cardiovascular disease and thought to promote hypertension through the sympathetic effects of insulin. Here, we examined the effect of insulin on regional sympathetic nerve activity (SNA) in obesity. Glucose and insulin tolerance tests were performed to examine insulin sensitivity in agouti obese mice. We used also multifiber recording to compare the regional SNA response to intracerebroventricular (ICV) insulin between lean and agouti obese mice. Agouti obese mice have significantly elevated levels of blood glucose and plasma insulin associated with glucose intolerance and insulin resistance. In lean mice, ICV administration of insulin (20 and 100 microU) caused a dose-dependent increase in SNA subserving hindlimb, kidney and brown adipose tissue (BAT). Of note, the regional SNA responses to insulin were differentially altered in agouti obese mice. Whereas lumbar SNA response to insulin was intact in the obese mice, renal and BAT sympathetic activation to insulin were significantly attenuated in these agouti obese mice. Finally, we assessed the role of phosphoinositol-3 kinase (PI3K) signaling pathway in mediating sympathetic activation to insulin in obesity. Notably, ICV pretreatment with a PI3K inhibitor (LY294002) blocked the increase in lumbar SNA induced by ICV insulin in lean and agouti obese mice. Our data suggest a differential regulation by insulin of sympathetic outflow to peripheral tissues in obesity. Our findings also demonstrate the importance of PI3K in lumbar sympathetic activation to insulin in obesity.

Differential control of renal and lumbar sympathetic nerve activity during freezing behavior in conscious rats

The present study was designed to document changes in sympathetic nerve activity and cardiovascular function when conscious rats were challenged with a noise stressor to induce freezing behavior. The potential contribution of the arterial baroreceptors in regulating sympathetic nerve activity and cardiovascular adjustments during the freezing behavior was then examined. Wistar male rats were assigned to sham-operated (SO) and sinoaortic-denervated (SAD) groups and instrumented chronically with electrodes for measurements of renal (RSNA) and lumbar (LSNA) sympathetic nerve activity, electroencephalogram, electromyogram, and electrocardiogram and catheters for measurements of systemic arterial and central venous pressure. Both SO and SAD rats were exposed to 90 dB of white noise for 10 min, causing freezing behavior in both groups. In SO rats, freezing behavior was associated with an immediate and significant (P < 0.05) increase in RSNA, no changes in LSNA or mean arterial pressure, and a significant (P < 0.05) decrease in heart rate. SAD attenuated the magnitude of the immediate increase in RSNA and had no influence on the response in LSNA during freezing behavior compared with SO rats. Moreover, in SAD rats, mean arterial pressure increased significantly (P < 0.05) while heart rate did not change during the freezing behavior. These data indicate that freezing behavior evokes regionally different changes in sympathetic outflows, which may be involved in generating the patterned responses of cardiovascular function to stressful or threatening sensory stimulation. Moreover, it is suggested that the arterial baroreceptors are involved in generating the differential changes in RSNA and LSNA and thus the patterned changes in cardiovascular functions observed during freezing behavior in conscious rats.

Increased group I metabotropic glutamate receptor activity in paraventricular nucleus supports elevated sympathetic vasomotor tone in hypertension

The sympathetic nerve activity is elevated in cardiovascular diseases such as hypertension. Enhanced glutamatergic inputs in the paraventricular nucleus (PVN) of the hypothalamus contribute to heightened sympathetic outflow in spontaneously hypertensive rats (SHR). We determined the role of group I metabotropic glutamate receptors (mGluR) in the PVN in the control of sympathetic vasomotor tone in hypertension. Lumbar sympathetic nerve activity (LSNA), arterial blood pressure (ABP), and heart rate (HR) were recorded in anesthetized SHR and Wistar-Kyoto (WKY) rats. Bilateral microinjection of 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), a selective mGluR5 receptor antagonist, or (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), a selective mGluR1 receptor antagonist, into the PVN had no significant effect on LSNA and ABP in WKY rats. Strikingly, MPEP and LY367385 dose dependently decreased LSNA, ABP, and HR in SHR. The MPEP-induced decreases in LSNA and ABP were significantly greater than those inhibited by LY367385 in SHR. Furthermore, bilateral microinjection of (S)-3,5-dihydroxyphenylglycine (S-DHPG), a selective group I mGluR agonist, into the PVN caused a similar dose-dependent increase in LSNA, ABP, and HR in both groups. S-DHPG-induced responses were attenuated by MPEP or LY367385 alone and were abolished by a combination of MPEP and LY367385 in WKY rats and SHR. In addition, microinjection of the NMDA receptor antagonist attenuated the sympathoexcitatory responses induced by S-DHPG in both WKY rats and SHR. Collectively, this study provides important new evidence that the resting sympathetic vasomotor tone is maintained by tonic activation of group I mGluRs in the PVN in hypertension. Activation of NMDA receptors are involved in the sympathoexcitatory effect of group I mGluRs in the PVN.

The hypothalamic paraventricular nucleus is required for insulin's action to increase baroreflex gain of lumbar sympathetic nerve activity

During pregnancy, low brain insulin levels are associated with impaired baroreflex control of heart rate (HR) and sympathetic nerve activity (SNA), and intracerebral insulin infusion normalizes baroreflex gain (BRG). The specific site of insulin action is unknown, though a likely candidate is the paraventricular nucleus (PVN). To test this hypothesis, BRG of lumbar SNA (LSNA) and HR were obtained in chloralose anesthetized female rats before and at 30min intervals during iv infusion of insulin (15mU/kg/min, n=5) or saline (n=5). Insulin increased LSNA (P&lt;0.05; in % baseline) to 166±18 (30min), 224±43 (60min), 219±46 (90min), and 227±49 (120min). Similarly, insulin increased LSNA BRG (P&lt;0.05; in %LSNA/mmHg) from 2.6±0.8 to 2.9±0.7 (30min), 4.5±0.9 (60min), 4.3±0.5 (90min), and 4.7±0.6 (120min). Insulin did not affect basal HR or HR BRG. No changes in LSNA, HR or BRG were observed with saline infusion. In another group (n=4), the effect of insulin to increase LSNA and LSNA gain was reversed by blockade of PVN via bilateral microinjection of muscimol (M; 60nL of 1mM). LSNA (% baseline) increased to 223±37 after insulin and fell to 126±11 after M (P&lt;0.05). LSNA gain (%LSNA/mmHg) increased from 2.8±1.3 to 4.4±1.2, and decreased to 2.4±0.7 after M (P&lt;0.05). In summary, iv insulin increased LSNA and LSNA gain. This action was reversed by PVN blockade, suggesting that insulin acts via the PVN to improve BRG. Support: the EMBL/GenBank Data Libraries under Accession No. HL088552

Insulin Activates a NMDA Pathway to the RVLM to Elevate Sympathetic Nerve Activity

Hyperinsulinemia elevates lumbar sympathetic nerve activity (SNA) via activation of ionotropic glutamatergic receptors in the rostral ventrolateral medulla (RVLM). Therefore, the present study determined the contribution of NMDA vs non‐NMDA receptors. Male Sprague‐Dawley rats (250–300 g) were anesthetized with α‐chloralose and infused with insulin (3.75 mU/kg/min) and 50% dextrose (.25–.75 ml/hr) for 120 min. At 90 min, hyperinsulinemia significantly increased lumbar SNA (40±6%) with no change in arterial blood pressure (ABP, 0 min: 117±5 vs 90 min: 119±6 mmHg) or plasma glucose levels (0 min: 72±5 vs 90 min: 68±5 mg/dl). RVLM microinjection of the ionotropic glutamate receptor antagonist KYN significantly reduced lumbar SNA (−28±10%) and ABP (−8±2 mmHg, p&lt;0.05). Similarly, RVLM microinjection of the NMDA receptor antagonist AP5 significantly attenuated lumbar SNA (−30±8%) and ABP (−5±3 mmHg, P&lt;0.05). The decrease in lumbar SNA and ABP after injection of KYN vs AP5 was not statistically different. In contrast, microinjection of the non‐NMDA receptor antagonist NBQX into the RVLM had no significant effect on lumbar SNA (6±3%) or ABP (5±4 mmHg). In addition, direct microinjection of insulin into the RVLM did not significantly increase lumbar SNA or ABP. These findings indicate that hyperinsulinemia increases lumbar SNA through a glutamatergic NMDA projection to the RVLM.Supported by NIH HL090826, AHA 0630202N &amp; 0815372D

Increased Group I Metabotropic Glutamate Receptor Activity in Paraventricular Nucleus Contributes to Elevated Sympathetic Vasomotor Tone in Hypertension

Enhanced glutamatergic input in the paraventricular nucleus (PVN) contributes to elevated sympathetic outflow in hypertension. We determined the role of group I metabotropic glutamate receptors (mGluRs) in the PVN in the control of sympathetic outflow in spontaneously hypertensive rats (SHR). Lumbar sympathetic nerve activity (LSNA), arterial blood pressure (Bp), and heart rate (HR) were recorded from anesthetized SHR and WKY rats. Bilateral microinjection of MPEP (1–10 nmol), a mGluR5 antagonist, or LY367385 (10–100 nmol), a mGluR1 antagonist, into the PVN dose‐dependently decreased LSNA and Bp in SHR but not in WKY. MPEP‐induced decreases in LSNA and Bp were greater than that produced by LY367385 in SHR. Furthermore, microinjection of group I mGluR agonist DHPG (10–100 nmol) caused dose‐dependent increase in LSNA and Bp in both WKY and SHR. DHPG‐induced responses were significantly attenuated by MPEP or LY367385 and abolished by a combination of MPEP and LY367385 in both WKY and SHR. In addition, prior injection of NMDA receptor antagonist AP5 (1.4 nmol) into the PVN decreased LSNA and Bp in SHR and attenuated DHPG‐induced sympathoexcitation in both WKY and SHR. Thus, our study suggests that elevated sympathetic outflow is maintained by activation of group I mGluR receptors in the PVN in SHR. Increased group I mGluR receptor activity contributes to increased glutamatergic input via NMDA receptor in hypertension.

Diverse Changes in Sympathetic Nerve Activity during REM Sleep in Rats

This study aimed to investigate whether REM sleep evoked diverse changes in sympathetic outflows and, if so, the potential role of the diverse changes in sympathetic nerve activity in regulating systemic arterial pressure during REM sleep. Male Wistar rats were chronically instrumented with electrodes to measure renal (RSNA) and lumbar sympathetic nerve activity (LSNA), electroencephalogram, electromyogram and electrocardiogram and catheters to measure systemic arterial and central venous pressure. REM sleep resulted in diverse changes in sympathetic outflows; there was a step reduction in RNSA by 36.1 ± 2.7% (P&lt;0,05) which was associated with a step increase in LSNA by 15.3 ± 2% (P&lt;0.05). Systemic arterial pressure increased gradually (P&lt;0.05) during REM sleep while heart rate decreased in a step manner (P&lt;0.05) during REM sleep. In contrast, RSNA, LSNA, systemic arterial pressure and heart rate increased in an unidirectional manner associated with increases in physical activity levels in the order from NREM sleep, quiet awake, moving, and grooming state. It is suggested that the diverse changes in sympathetic outflows during REM sleep may be a unique way to increase systemic arterial pressure by balancing vascular resistances between muscles and vegetative organs without depending on the heart. JSPS Grant.

Age‐related changes in stress‐induced cardiovascular and lumbar sympathetic nerve activity responses

Central angiotensin II (AngII) signaling plays an important role in stress‐induced cardiovascular and sympathetic responses. However, the effects of age and high salt (HS) diet on central AngII‐mediated mechanisms are not known. We compared restraint stress‐induced BP, HR and lumbar sympathetic nerve activity (LSNA) responses in young and old rats under control conditions, after central infusion of losartan (15 μg/μL/hour), an AngII type‐1 receptor inhibitor, and after feeding rats with 8% HS diet for 10 days. BP, HR and LSNA were measured in young (5 mo) and old (27 mo) Fischer 344 x Brown Norway rats using radiotelemetry. Restraint stress was induced by placing animals in acrylic cylinder restrainers. Old rats had significantly higher resting BP and lower HR compared with young. Restraint stress induced rapid BP, HR and LSNA increases in both young and old; however, changes were significantly reduced in the old rats. Losartan significantly inhibited stress‐induced BP increases in young rats, while it had no effect on HR responses. In contrast, in the old rats, both BP and HR responses remained unaffected by losartan. HS diet feeding increased baseline BP both in young and old rats, with a more pronounced effect in old animals; however, it did not significantly alter restraint‐stress‐induced responses. In conclusion, resting BP increases with age, but hypertensive responses to stress diminish with age due to a decline in AngII‐mediated central mechanisms.

Diet‐Induced Obesity Causes Selective Insulin Resistance

Peripheral insulin resistance is a hallmark characteristic of the metabolic syndrome and obesity. However, whether obesity causes insulin resistance in brain is not known. To test this hypothesis, male Sprague‐Dawley rats (200–250g) were fed a moderately high‐fat diet (32% kcal as fat) or a low fat (LF) diet (10.6% kcal as fat). After 13 weeks, the body weights of rats fed the moderately high‐fat diet segregated into obesity‐prone (OP, 793±13 g) and obesity‐resistant (OR, 596±8 g) groups. The control group consisted of young adult rats (292±22 g, n=4) or rats fed LF (612±15 g, n=4). Rats were anesthetized with α‐chloralose and infused with insulin (3.75 mU/kg/min) and 50% dextrose (.25–.75 ml/hr) for 120 min. Baseline plasma insulin levels were significantly elevated in OP compared to OR and control rats (OP: 16±2 vs OR: 8±1 and control: 11±1 ng/ml, p&lt;0.05). As expected, glucose utilization was significantly reduced in OP rats (OP: 3.3±0.7 vs OR: 5.5±0.9 and control: 7.5±0.8 mg/kg/min, p&lt;0.05). Interestingly, hyperinsulinemia produced similar increases in lumbar sympathetic nerve activity across groups (OP: 127±6%; OR: 128±9%, control: 133±10% P&gt;0.05). Arterial blood pressure decreased significantly by 120 min in all groups (OP: 94±4 vs 84±3 mmHg; OR: 94±7 vs 87±6 mmHg, control: 100±7 vs 85±6, P&lt;0.05). These findings indicate that obesity causes peripheral but not central insulin resistance.Supported by NIH HL090826, AHA 0630202N &amp; 0815372D

Novel Methods to Investigate Multi‐fiber Nerve Recordings

We developed novel methods to investigate responses of neuronal populations within a multi‐fiber sympathetic nerve recording. We applied these methods to analyze the relationship between barosensitive RVLM medullospinal neurons marked by their response to systemic administration of the gastrointestinal hormone cholecystokinin (CCK) and splanchnic and lumbar sympathetic nerve activity (SNA). In addition to the standard analysis of Spike‐Triggered Averaging (STA) of SNA, we used Spike2 software to identify spike‐like structures within the sympathetic nerve recording (SND). We then calculated cross‐correlograms between apparent “spikes” in SND and RVLM neuronal discharges. Also, we calculated a STA of RVLM mean frequency triggered by the identified “spikes”. These novel analyses yielded consistent results with the standard STA of SNA and detected sympathetic nerve discharges driven only by the fast pathway. We find statistically significant peaks (p&lt;0.001) in the cross‐correlogram between CCK inhibited neurons and “spikes” driven by the fast pathway (n=5). The time‐delay between RVLM neuronal discharges and the fast burst of SNA in splanchnic nerves is 52 +/− 2ms. The corresponding delay for lumbar nerves is 78+/− 4ms. These novel analyses may serve as a tool to study differential behavior of populations of neurons that drive sympathetic vasomotor nerve activity. Supported by R01 NS39774, HL091440

Increased sympathoexcitation to angiotensin II (A II) and AT1 receptor mRNA in the paraventricular nucleus (PVN) of pregnant rats

Previously we reported that pressor and sympathetic nerve activity (SNA) responses due to AT1 receptor activation in the SFO are potentiated in term pregnant (P) compared to nonpregnant (NP) rats. The pathway requires ionotropic glutamate receptors (iGluRs) in the PVN and rostral ventrolateral medulla (RVLM) in both P and NP rats. AT1 receptors in the PVN and RVLM contribute to potentiated responses to SFO activation in P rats. Since the PVN is obligatory to this pathway, and PVN to RVLM projections are almost exclusively glutamatergic, we considered that the enhanced role of A II in the RVLM might be related to an A II dependent increase in excitatory drive from the PVN, which could then stimulate local production of A II in the RVLM. In sinoaortic denervated inactin anesthetized rats, increases in mean arterial pressure (MAP) and lumbar SNA (LSNA) due to microinjected A II (50 nl, 1.6 mM) in the PVN were greater in P (n = 4) compared to NP (n = 7) rats (ΔMAP: P= 25±5 mmHg; NP= 15±2 mmHg; ΔLSNA: P= 13±3%; NP= 6.5±2%). Real‐time RT‐PCR was used to assess relative expression of AT1 receptor mRNA (reference gene = GAPDH) in PVN tissue punches from 5 P and 5 NP rats. Expression of AT1 receptors in the PVN tended (P = 0.1) to be greater in P (2.4±0.82) compared to NP rats (1.0±0.22). These results are consistent with an increased role of PVN AT1 receptors in sympathoexcitation in pregnancy. (NIH HL 091164)