Adrenal Sympathetic Nerve Activity Research Articles

Hepatic glycogenolysis and hypometabolism induced by chemogenetic stimulation of C1 neurons.

The precise regulation of blood glucose levels is indispensable for maintaining physiological functions. C1 neurons determine the outflow of the autonomic nervous and endocrine systems to maintain blood glucose levels in the body. In contrast, activation of C1 neurons induces a decrease in activity, suggesting that hypoactivity also participates in maintaining blood glucose levels. To examine this, we evaluated both glycogenolysis and hypometabolism induced by the selective activation of C1 neurons. We used DbhCre/0 mice expressing receptors for chemogenetic tools in C1 neurons, owing to microinjection of the viral vector. C1 neurons were activated by intraperitoneal injection of clozapine N-oxide (CNO). The chemogenetic activation of C1 neurons significantly decreased body temperature, oxygen consumption, and carbon dioxide production. On the other hand, the blood glucose level was increased by the activation of C1 neurons 2 h after CNO administration, even in the fasting state. In this situation, an increase in glucagon and corticosterone levels was observed, while hepatic glycogen content decreased significantly. Plasma insulin level was not changed by the activation of C1 neurons despite the blood glucose level increased. Furthermore, adrenal sympathetic nerve activity was significantly increased by the activation of C1 neurons, and plasma catecholamine levels increased significantly. In conclusion, the selective activation of C1 neurons using chemogenetic tools induced an increase in blood glucose level, probably because of hepatic glycogenolysis and hypometabolism. KEY POINTS: Chemogenetic activation of C1 neurons in medulla oblongata decreased body temperature. Oxygen consumption and carbon dioxide production were decreased by chemogenetic activation of C1 neurons in medulla oblongata. Blood glucose level was increased by chemogenetic activation of C1 neurons in medulla oblongata. Chemogenetic activation of C1 neurons in medulla oblongata increased glucagon, corticosterone, and catecholamine levels in plasma. Increase in blood glucose level by activation of C1 neurons occurred due to combined effect of hepatic glycogenolysis and hypometabolism. Abstract figure legend We investigated the effects of selectively activating C1 neurons using chemogenetic tools on blood glucose levels. The activation of C1 neurons resulted in an increase in blood glucose levels. This increase is due to the activation of hepatic glycogenolysis through the release of humoral factors, including adrenaline, glucagon, and corticosterone. Additionally, the decrease in energy expenditure, as evidenced by a reduction in body temperature, oxygen consumption, and carbon dioxide production, suggests that activation of C1 neurons may induce hypometabolism, which could also contribute to an increase in blood glucose levels. These responses indicate that C1 neurons may serve as a switch for energy conservation to maintain blood glucose levels, presumably by being activated in a stressful situation. This article is protected by copyright. All rights reserved.

Revisiting differential control of sympathetic outflow by the rostral ventrolateral medulla.

The rostral ventrolateral medulla (RVLM) is an important brain region involved in both resting and reflex regulation of the sympathetic nervous system. Anatomical evidence suggests that as a bilateral structure, each RVLM innervates sympathetic preganglionic neurons on both sides of the spinal cord. However, the functional importance of ipsilateral versus contralateral projections from the RVLM is lacking. Similarly, during hypotension, the RVLM is believed to rely primarily on withdrawal of tonic gamma aminobutyric acid (GABA) inhibition to increase sympathetic outflow but whether GABA withdrawal mediates increased activity of functionally different sympathetic nerves is unknown. We sought to test the hypothesis that activation of the ipsilateral versus contralateral RVLM produces differential increases in splanchnic versus adrenal sympathetic nerve activities, as representative examples of functionally different sympathetic nerves. We also tested whether GABA withdrawal is responsible for hypotension-induced increases in splanchnic and adrenal sympathetic nerve activity. To test our hypothesis, we measured splanchnic and adrenal sympathetic nerve activity simultaneously in Inactin-anesthetized, male Sprague-Dawley rats during ipsilateral or contralateral glutamatergic activation of the RVLM. We also produced hypotension (sodium nitroprusside, i.v.) before and after bilateral blockade of GABAA receptors in the RVLM (bicuculline, 5mM 90nL). Glutamate (100mM, 30nL) injected into the ipsilateral or contralateral RVLM produced equivalent increases in splanchnic sympathetic nerve activity, but increased adrenal sympathetic nerve activity by more than double with ipsilateral injections versus contralateral injections (p < 0.05; n = 6). In response to hypotension, increases in adrenal sympathetic nerve activity were similar after bicuculline (p > 0.05), but splanchnic sympathetic nerve activity responses were eliminated (p < 0.05; n = 5). These results provide the first functional evidence that the RVLM has predominantly ipsilateral innervation of adrenal nerves. In addition, baroreflex-mediated increases in splanchnic but not adrenal sympathetic nerve activity are mediated by GABAA receptors in the RVLM. Our studies provide a deeper understanding of neural control of sympathetic regulation and insight towards novel treatments for cardiovascular disease involving sympathetic nervous system dysregulation.

Slow but Steady-The Responsiveness of Sympathoadrenal System to a Hypoglycemic Challenge in Ketogenic Diet-Fed Rats.

The sympathoadrenal counterregulatory response to hypoglycemia is critical for individuals with type 1 diabetes due to impaired ability to produce glucagon. Ketogenic diets (KD) are an increasingly popular diabetes management tool; however, the effects of KD on the sympathoadrenal response are largely unknown. Here, we determined the effects of KD-induced ketosis on the sympathoadrenal response to a single insulin-induced hypoglycemic challenge. We investigated how a 3 week KD feeding regimen affected the main components of the sympathoadrenal counterregulatory response: adrenal sympathetic nerve activity (ASNA), adrenal gland activity, plasma epinephrine, and brainstem glucose-responsive C1 neuronal activation in anesthetized, nondiabetic male Sprague-Dawley rats. Rats on KD had similar blood glucose (BG) levels and elevated ketone body β-hydroxybutyrate (BHB) levels compared to the control Chow diet group. All KD rats responded to hypoglycemia with a robust increase in ASNA, which was initiated at significantly lower BG levels compared to Chow-fed rats. The delay in hypoglycemia-induced ASNA increase was concurrent with rapid disappearance of BHB from cerebral and peripheral circulation. Adrenal gland activity paralleled epinephrine and ASNA response. Overall, KD-induced ketosis was associated with initiation of the sympathoadrenal response at lower blood glucose levels; however, the magnitude of the response was not diminished.

Ketogenic Diet‐Induced Ketosis Delays the Onset, but Preserves the Magnitude of The Sympathoadrenal Response to Hypoglycemia in Rats

The sympathoadrenal counterregulatory response to hypoglycemia is critical for individuals with type 1 diabetes due to impaired ability to produce glucagon. Ketogenic diets (KD) are an increasingly popular diabetes management tool, however the effects of KD on the sympathoadrenal response to hypoglycemia, and hence safety, are largely unknown. We propose that the KD could delay the sympathoadrenal response to hypoglycemia due to the availability of an additional energy substrate to the brain in the form of ketone bodies, but the magnitude of the response will not be affected. The aim of this study was to investigate the effects of KD on the main peripheral components of the sympathoadrenal counterregulatory response: adrenal sympathetic nerve activity (ASNA), adrenal gland activity and plasma epinephrine in non-diabetic male rats. Methods Male Sprague-Dawley rats fed KD or chow diet (CHOW) for 3 weeks were anaesthetised and ASNA recorded. Insulin (5 U/kg; iv) induced hypoglycemia and the sympathoadrenal counterregulatory response. Blood glucose and β-hydroxybutyrate (BHB, ketone body) were measured every 15 min post-insulin for 2 hours. Plasma epinephrine was determined via ELISA. Adrenal gland activity was assessed by immunohistochemistry for Fos. Results Rats on KD, compared to the control chow diet (CHOW) group, had similar blood glucose (BG) levels, and had elevated ketone body β-hydroxybutyrate (BHB) levels. None of the KD rats developed clonic convulsions post-insulin, while >40% of the CHOW rats did. CHOW rats had either a strong ASNA response without convulsions, or a failed ASNA response with convulsions. All KD rats responded to hypoglycemia with a robust increase in ASNA, which was initiated at lower BG levels compared to convulsion-free CHOW rats. Both adrenal gland activity and epinephrine levels positively correlated with ASNA response. Conclusions KD-induced ketosis was associated with initiation of the sympathoadrenal response at lower blood glucose levels, however the magnitude of the response was not diminished. Furthermore, KD appeared to be protective against sympathoadrenal failure and severe hypoglycemia-induced neuroglycopenia.

Hypothalamic MC4R regulates glucose homeostasis through adrenaline-mediated control of glucose reabsorption via renal GLUT2 in mice.

Melanocortin 4 receptor (MC4R) mutation is the most common cause of known monogenic obesity in humans. Unexpectedly, humans and rodents with MC4R deficiency do not develop hyperglycaemia despite chronic obesity and insulin resistance. To explain the underlying mechanisms for this phenotype, we determined the role of MC4R in glucose homeostasis in the presence and absence of obesity in mice. We used global and hypothalamus-specific MC4R-deficient mice to investigate the brain regions that contribute to glucose homeostasis via MC4R. We performed oral, intraperitoneal and intravenous glucose tolerance tests in MC4R-deficient mice that were either obese or weight-matched to their littermate controls to define the role of MC4R in glucose regulation independently of changes in body weight. To identify the integrative pathways through which MC4R regulates glucose homeostasis, we measured renal and adrenal sympathetic nerve activity. We also evaluated glucose homeostasis in adrenaline (epinephrine)-deficient mice to investigate the role of adrenaline in mediating the effects of MC4R in glucose homeostasis. We employed a graded [13C6]glucose infusion procedure to quantify renal glucose reabsorption in MC4R-deficient mice. Finally, we measured the levels of renal glucose transporters in hypothalamus-specific MC4R-deficient mice and adrenaline-deficient mice using western blotting to ascertain the molecular mechanisms underlying MC4R control of glucose homeostasis. We found that obese and weight-matched MC4R-deficient mice exhibited improved glucose tolerance due to elevated glucosuria, not enhanced beta cell function. Moreover, MC4R deficiency selectively in the paraventricular nucleus of the hypothalamus (PVH) is responsible for reducing the renal threshold for glucose as measured by graded [13C6]glucose infusion technique. The MC4R deficiency suppressed renal sympathetic nerve activity by 50% in addition to decreasing circulating adrenaline and renal GLUT2 levels in mice, which contributed to the elevated glucosuria. We further report that adrenaline-deficient mice recapitulated the increased excretion of glucose in urine observed in the MC4R-deficient mice. Restoration of circulating adrenaline in both the MC4R- and adrenaline-deficient mice reversed their phenotype of improved glucose tolerance and elevated glucosuria, demonstrating the role of adrenaline in mediating the effects of MC4R on glucose reabsorption. These findings define a previously unrecognised function of hypothalamic MC4R in glucose reabsorption mediated by adrenaline and renal GLUT2. Taken together, our findings indicate that elevated glucosuria due to low sympathetic tone explains why MC4R deficiency does not cause hyperglycaemia despite inducing obesity and insulin resistance. Graphical abstract.

Activation of µ-opioid receptors in the rostral ventrolateral medulla blocks the sympathetic counterregulatory response to glucoprivation.

Activation of neurons in the rostral ventrolateral medulla (RVLM) following glucoprivation initiates sympathoadrenal activation, adrenaline release, and increased glucose production. Here, we aimed to determine the role of RVLM µ-opioid receptors in the counterregulatory response to systemic glucoprivation. Experiments were performed in pentobarbital sodium anesthetized male Sprague-Dawley rats ( n = 30). Bilateral activation of RVLM µ-opioid receptors with [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) (8 mM, 50 nl) depressed adrenal sympathetic nerve activity for ~60 min ( n = 6; Δ49.9 ± 5.8%, P < 0.05). The counterregulatory response to glucoprivation (measured by adrenal sympathetic efferent nerve activity) induced by 2-deoxyglucose (2-DG) ( n = 6; Δ63.6 ± 16.5%, P < 0.05) was completely blocked 60 min after DAMGO microinjections ( n = 6; Δ10.2 ± 3.5%, P < 0.05). Furthermore, DAMGO pretreatment attenuated the increase in blood glucose levels after 2-DG infusion ( n = 6; 6.1 ± 0.7mmol/l vs. baseline 5.2 ± 0.3mmol/l, P > 0.05) compared with 2-DG alone ( n = 6; 7.6 ± 0.4mmol/l vs. baseline 6.0 ± 0.4mmol/l, P < 0.05). Thus, activation of RVLM µ-opioid receptors attenuated the neural efferent response to glucoprivation and reduced glucose production.

β-eudesmol, an oxygenized sesquiterpene, affects efferent adrenal sympathetic nerve activity via transient receptor potential ankyrin 1 in rats

The autonomic nervous system innervates various peripheral tissue functions. Various external stimuli affect autonomic nerve activity, however, there is little information about the involvement of sensory receptors in the responses. The TRPA1 is a calcium-permeable non-selective cation channel which plays a crucial role in the susceptibility to various stimuli. β-Eudesmol, an oxygenated sesquiterpene found in hop essential oil and beer, activates the TRPA1. Intragastric administration of β-eudesmol decreased efferent adrenal sympathetic nerve activity (ASNA) in rats, whereas subcutaneous administration did not. ASNA suppression by β-eudesmol was not observed in TRPA1 knockout rats. The β-eudesmol derived ASNA suppression was partially, but significantly, eliminated by subdiaphragmatic vagotomy in rats, suggesting the afferent vagal nerve from the gastrointestinal tract to the brain is involved in the effect of β-eudesmol on ASNA. Our results indicate that β-eudesmol suppresses ASNA, partly through TRPA1 and the afferent vagus nerve. These findings introduce the physiological significance of the TRPA1 in the control of ASNA.

Anaphylactic hypotension causes renal and adrenal sympathoexcitaion and induces c-fos in the hypothalamus and medulla oblongata.

What is the central question of this study? Whether anaphylaxis affects sympathetic outflows to the brown adipose tissue (BAT) and adrenal gland and whether anaphylaxis affects some brain areas in association with sympathetic regulation. What is the main finding and its importance? Sympathoexcitatory responses to anaphylaxis occurred regionally in the kidney and adrenal gland, but not in the thermogenesis-related BAT. Further, anaphylactic hypotension also caused increase in c-fos immunoreactivity in the hypothalamic and medullary areas. Moreover, catecholaminergic neurons of the brainstem cause adrenal sympathoexcitation in a baroreceptor-independent manner. We previously reported that sympathetic nerve activity (SNA) to the kidney and the hindlimb increases during anaphylactic hypotension in anaesthetized rats. Based on this evidence, we examined effects of anaphylactic hypotension on SNA to the brown adipose tissue (BAT), and the adrenal gland and kidney in anaesthetized rats. We demonstrated that adrenal and renal SNA, but not BAT-SNA, were stimulated. In addition, the effects of anaphylaxis on neural activities of the hypothalamic and medullary nuclei, which are candidates for relaying efferent SNA to the peripheral organs, were investigated via immunohistochemical staining of c-fos. Anaphylaxis increased c-fos expression in the neurons of the paraventricular nucleus (PVN) of the hypothalamus and in those of the nucleus tractus solitarii (NTS) and rostral ventrolateral medulla (RVLM) of the medulla oblongata; c-fos was expressed in γ-aminobutyric acid (GABA)-ergic neurons of the NTS and in the catecholaminergic neurons of the RVLM. In addition, c-fos expression in the rostral NTS and mid NTS during anaphylaxis was reduced by sinoaortic baroreceptor denervation; however, increased c-fos expression in the caudal NTS and RVLM or adrenal sympathoexcitation were not affected by sinoaortic baroreceptor denervation. These results indicated that anaphylactic hypotension activates the hypothalamic PVN and the medullary NTS and RVLM independently of the baroreflex pathway. Further, it stimulated efferent SNA to the adrenal gland and kidney to restore blood pressure.

Activation of μ‐opioid receptors in the rostral ventrolateral medulla blocks the sympathetic counter‐regulatory response to glucoprivation

Intensive insulin therapy is the gold standard for managing serum glucose levels in patients with insulin‐deficient diabetes mellitus. Hypoglycaemia‐associated autonomic failure (HAAF) is a clinical syndrome characterised by the absence of a glucose counter‐regulatory response to hypoglycaemia or glucoprivation. The precise physiologic mechanisms underlying HAAF remain unknown. Recent data from human studies suggests activation of the endogenous opioid system during antecedent hypoglycaemia may contribute to unawareness of hypoglycaemia.Here, we aimed to determine whether or not activation of rostral ventrolateral medullary (RVLM) μ‐opioid receptors results in downstream sympathoinhibition, resulting in attenuated glucose production, and an impaired counter‐regulatory response following glucoprivation. Experiments were performed in sodium pentobarbital anaesthetised (65 mg/kg) male Sprague‐Dawley rats (250–350g).Bilateral activation of RVLM μ‐opioid receptors with DAMGO (8 mM, 50 nl) caused depression of adrenal sympathetic nerve activity (ASNA), that lasted at least 60 minutes (Δ−56.9 ± 9.8% vs saline: Δ1.7 ± 4.4%, P&lt;0.05). In parallel, blood glucose levels were also reduced (Δ−0.8 ± 0.2 mM vs saline: Δ0.2 ± 0.3 mM, P&lt;0.05). The counter‐regulatory response to glucoprivation (measured by ASNA efferent activity) induced by 2‐deoxyglucose was completely attenuated 60 minutes following DAMGO microinjections (Δ−4.4 ± 0.7% vs saline+2‐DG: Δ59.6 ± 8.0%, P&lt;0.05). Bilateral μ‐opioid receptor activation also caused a significant decrease in mean arterial pressure (Δ−28.7 ± 6.1 mmHg vs saline: Δ−9.9 ± 7.3 mmHg, P&lt;0.05) and heart rate (Δ−40.4 ± 14.9 bpm vs saline: Δ−11.1 ± 9.7 bpm, P&lt;0.05).In conclusion, activation of RVLM μ‐opioid receptors reproduces features of HAAF (attenuated neural efferent response to glucoprivation). The mechanism underlying HAAF may include activation of RVLM μ‐opioid receptors to facilitate sympathoinhibition. Coupled with this sympathoinhibition, the reduced blood glucose levels may contribute to HAAF by increasing the threshold to elicit counter‐regulatory response.Support or Funding InformationNational Health and Medical Research Council of Australia, The University of Sydney, The Heart Research InstituteThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NaCl and osmolarity produce different responses in organum vasculosum of the lamina terminalis neurons, sympathetic nerve activity and blood pressure.

Changes in extracellular osmolarity stimulate thirst and vasopressin secretion through a central osmoreceptor; however, central infusion of hypertonic NaCl produces a greater sympathoexcitatory and pressor response than infusion of hypertonic mannitol/sorbitol. Neurons in the organum vasculosum of the lamina terminalis (OVLT) sense changes in extracellular osmolarity and NaCl. In this study, we discovered that intracerebroventricular infusion or local OVLT injection of hypertonic NaCl increases lumbar sympathetic nerve activity, adrenal sympathetic nerve activity and arterial blood pressure whereas equi-osmotic mannitol/sorbitol did not alter any variable. In vitro whole-cell recordings demonstrate the majority of OVLT neurons are responsive to hypertonic NaCl or mannitol. However, hypertonic NaCl stimulates a greater increase in discharge frequency than equi-osmotic mannitol. Intracarotid or intracerebroventricular infusion of hypertonic NaCl evokes a greater increase in OVLT neuronal discharge frequency than equi-osmotic sorbitol. Collectively, these novel data suggest that subsets of OVLT neurons respond differently to hypertonic NaCl versus osmolarity and subsequently regulate body fluid homeostasis. These responses probably reflect distinct cellular mechanisms underlying NaCl- versus osmo-sensing. Systemic or central infusion of hypertonic NaCl and other osmolytes readily stimulate thirst and vasopressin secretion. In contrast, central infusion of hypertonic NaCl produces a greater increase in arterial blood pressure (ABP) than equi-osmotic mannitol/sorbitol. Although these responses depend on neurons in the organum vasculosum of the lamina terminalis (OVLT), these observations suggest OVLT neurons may sense or respond differently to hypertonic NaCl versus osmolarity. The purpose of this study was to test this hypothesis in Sprague-Dawley rats. First, intracerebroventricular (icv) infusion (5μl/10min) of 1.0m NaCl produced a significantly greater increase in lumbar sympathetic nerve activity (SNA), adrenal SNA and ABP than equi-osmotic sorbitol (2.0osmoll-1 ). Second, OVLT microinjection (20nl) of 1.0m NaCl significantly raised lumbar SNA, adrenal SNA and ABP. Equi-osmotic sorbitol did not alter any variable. Third, in vitro whole-cell recordings demonstrate that 50% (18/36) of OVLT neurons display an increased discharge to both hypertonic NaCl (+7.5mm) and mannitol (+15mm). Of these neurons, 56% (10/18) displayed a greater discharge response to hypertonic NaCl vs mannitol. Fourth, in vivo single-unit recordings revealed that intracarotid injection of hypertonic NaCl produced a concentration-dependent increase in OVLT cell discharge, lumbar SNA and ABP. The responses to equi-osmotic infusions of hypertonic sorbitol were significantly smaller. Lastly, icv infusion of 0.5m NaCl produced significantly greater increases in OVLT discharge and ABP than icv infusion of equi-osmotic sorbitol. Collectively, these findings indicate NaCl and osmotic stimuli produce different responses across OVLT neurons and may represent distinct cellular processes to regulate thirst, vasopressin secretion and autonomic function.

Perifornical hypothalamic pathway to the adrenal gland: Role for glutamatergic transmission in the glucose counter-regulatory response.

Adrenaline is an important counter-regulatory hormone that helps restore glucose homeostasis during hypoglycaemia. However, the neurocircuitry that connects the brain glucose sensors and the adrenal sympathetic outflow to the chromaffin cells is poorly understood. We used electrical microstimulation of the perifornical hypothalamus (PeH) and the rostral ventrolateral medulla (RVLM) combined with adrenal sympathetic nerve activity (ASNA) recording to examine the relationship between the RVLM, the PeH and ASNA. In urethane-anaesthetised male Sprague-Dawley rats, intermittent single pulse electrical stimulation of the rostroventrolateral medulla (RVLM) elicited an evoked ASNA response that consisted of early (60±3ms) and late peaks (135±4ms) of preganglionic and postganglionic activity. In contrast, RVLM stimulation evoked responses in lumbar sympathetic nerve activity that were almost entirely postganglionic. PeH stimulation also produced an evoked excitatory response consisting of both preganglionic and postganglionic excitatory peaks in ASNA. Both peaks in ASNA following RVLM stimulation were reduced by intrathecal kynurenic acid (KYN) injection. In addition, the ASNA response to systemic neuroglucoprivation induced by 2-deoxy-d-glucose was abolished by bilateral microinjection of KYN into the RVLM. This suggests that a glutamatergic pathway from the perifornical hypothalamus (PeH) relays in the RVLM to activate the adrenal SPN and so modulate ASNA. The main findings of this study are that (i) adrenal premotor neurons in the RVLM may be, at least in part, glutamatergic and (ii) that the input to these neurons that is activated during neuroglucoprivation is also glutamatergic.

Organum Vasculosum of the Lamina Terminalis Detects NaCl to Elevate Sympathetic Nerve Activity and Blood Pressure.

High-salt diet elevates NaCl concentrations in the cerebrospinal fluid to increase sympathetic nerve activity (SNA) in salt-sensitive hypertension. The organum vasculosum of the lamina terminalis (OVLT) resides along the rostral wall of the third ventricle, lacks a complete blood-brain barrier, and plays a pivotal role in body fluid homeostasis. Therefore, the present study used a multifaceted approach to examine whether OVLT neurons of Sprague-Dawley rats are intrinsically sensitive to changes in extracellular NaCl concentrations and mediate the sympathoexcitatory responses to central NaCl loading. Using in vitro whole-cell recordings, step-wise increases in extracellular NaCl concentrations (2.5-10 mmol/L) produced concentration-dependent excitation of OVLT neurons. Additionally, these excitatory responses were intrinsic to OVLT neurons because hypertonic NaCl evoked inward currents, despite pharmacological synaptic blockade. In vivo single-unit recordings demonstrate that the majority of OVLT neurons (72%, 13/19) display concentration-dependent increases in neuronal discharge to intracarotid (50 μL/15 s) or intracerebroventricular infusion (5 μL/10 minutes) of hypertonic NaCl. Microinjection of hypertonic NaCl (30 nL/60 s) into the OVLT, but not adjacent areas, increased lumbar SNA, adrenal SNA, and arterial blood pressure in a concentration-dependent manner. Renal SNA decreased and splanchnic SNA remained unaffected. Finally, local inhibition of OVLT neurons with the GABAA receptor agonist muscimol (24 nL/10 s) significantly attenuated the sympathoexcitatory and pressor responses to intracerebroventricular infusion of 0.5 mol/L or 1.0 mol/L NaCl. Collectively, these findings indicate that OVLT neurons detect changes in extracellular NaCl concentrations to selectively alter SNA and raise arterial blood pressure.

Blockade of orexin receptors reduces glutamatergic excitation of adrenal premotor neurons in the rostral ventrolateral medulla

Excitation of adrenal sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM) elicits epinephrine release as part of the glucose counter‐regulatory response to hypoglycemia. It is known that this response is partly mediated by excitatory inputs from hypothalamic orexin neurons. Although some orexin neurons co‐express glutamate, whether orexin and glutamate interact in order to facilitate adrenal sympathetic premotor neurons is unknown. In pentobarbitone‐anesthetized male Sprague‐Dawley rats, we show tested whether glutamatergic activation of adrenal premotor neurons depends on activation of orexin receptors (Ox‐R). Microinjection of glutamate (50 mM/50 nl) into the RVLM, where adrenal premotor neurons are found, increased adrenal sympathetic nerve activity (ASNA). The increase in ASNA was reduced following non‐selective blockade of orexin receptors. RVLM orexin receptors were blocked by microinjecting a combination of SB334867 (Ox1‐R antagonist) and TCS OX2 29 (Ox2‐R antagonist) into the RVLM. The reduction in ASNA in response to glutamate microinjection into the RVLM was dose dependent (0.1–10 mM/50 nl), and the response was abolished with 1 mM of the combined antagonists (P&lt;0.05). The results suggest that the counter‐regulatory release of epinephrine during hypoglycemia depends on the interaction between glutamate and orexin receptors located on the adrenal sympathetic premotor neurons.Support or Funding InformationNHMRC AustraliaAustin Medical Research Foundation

Effects of intracerebroventricular injection of nesfatin‐1 on autonomic nerve activities in rats and mice

Nesfatin‐1, one of neuropeptides, reduces appetite and enhances energy expenditure in animals. Previous our experiment showed that intracerebroventricular (ICV) administration of nesfatin‐1 suppressed food intake and stimulated sympathetic nerve outflows to the kidney and the white adipose tissue through the hypothalamic ERK signals (Tanida M., et al., Diabetes 2015). However, it has not been examined whether central nesfatin‐1 affects efferent sympathetic nerve activities to the adrenal gland or the brown adipose tissue regulating cardiovascular function or thermogenesis, respectively, and efferent vagal nerve outflow. Here, we investigated effects of ICV injection of nesfatin‐1 on efferent adrenal sympathetic nerve activity (ASNA) and brown adipose tissue sympathetic nerve activity (BAT‐SNA), and efferent gastric vagal nerve activity (GVNA) in anesthetized rats. In addition, to check species difference in nesfatin‐1 action on autonomic nerves, sympathetic response to ICV injection of nesfatin‐1 was determined in anesthetized mice.Electrophysiological recordings of autonomic nerve activities in anesthetized rats and mice were performed within the scope of our previous studies (Tanida M., et al., Diabetes 2015, Tanida M., Neuroscience 2013). All animal care and handling procedures were approved by the Animal Research Committees of Kanazawa Medical University. 7–10days after ICV surgery, rats and mice were anesthetized with intraperitoneal injections of a urethane (750 mg/kg) and a‐chloralose (75 mg/kg) mixture. ASNA, BAT‐SNA and GVNA in rats, and sympathetic nerve to the kidney in mice were measured by electrophysiological system.In the present study, we found that ICV injection of nesfatin‐1 (20, 200 pmol) activated ASNA and BAT‐SNA in dose‐dependent manner while the GVNA was suppressed by nesfatin‐1. In addition, pretreatment with the melanocortin‐3/4 receptor antagonist, SHU9119, attenuated the increase in ASNA induced by nesfatin‐1. These data suggest that hypothalamic nesfatin‐1 stimulates not only renal sympathetic nerve but also sympathetic nerve activities to the adrenal gland and the brown adipose tissue via the hypothalamic melanocortin system to regulate cardiovascular and thermogenic actions. Similar with sympathetic responses of rats to nesfatin‐1, nesfatin‐1 injection to the mice also activated renal sympathetic outflow, indicating that sympathoexcitation of the kidney was also induced in mice.

Cerebrospinal Fluid Hypernatremia Elevates Sympathetic Nerve Activity and Blood Pressure via the Rostral Ventrolateral Medulla.

Elevated NaCl concentrations of the cerebrospinal fluid increase sympathetic nerve activity (SNA) in salt-sensitive hypertension. Neurons of the rostral ventrolateral medulla (RVLM) play a pivotal role in the regulation of SNA and receive mono- or polysynaptic inputs from several hypothalamic structures responsive to hypernatremia. Therefore, the present study investigated the contribution of RVLM neurons to the SNA and pressor response to cerebrospinal fluid hypernatremia. Lateral ventricle infusion of 0.15 mol/L, 0.6 mol/L, and 1.0 mol/L NaCl (5 µL/10 minutes) produced concentration-dependent increases in lumbar SNA, adrenal SNA, and arterial blood pressure, despite no change in splanchnic SNA and a decrease in renal SNA. Ganglionic blockade with chlorisondamine or acute lesion of the lamina terminalis blocked or significantly attenuated these responses, respectively. RVLM microinjection of the gamma-aminobutyric acid (GABAA) agonist muscimol abolished the sympathoexcitatory response to intracerebroventricular infusion of 1 mol/L NaCl. Furthermore, blockade of ionotropic glutamate, but not angiotensin II type 1, receptors significantly attenuated the increase in lumbar SNA, adrenal SNA, and arterial blood pressure. Finally, single-unit recordings of spinally projecting RVLM neurons revealed 3 distinct populations based on discharge responses to intracerebroventricular infusion of 1 mol/L NaCl: type I excited (46%; 11/24), type II inhibited (37%; 9/24), and type III no change (17%; 4/24). All neurons with slow conduction velocities were type I cells. Collectively, these findings suggest that acute increases in cerebrospinal fluid NaCl concentrations selectively activate a discrete population of RVLM neurons through glutamate receptor activation to increase SNA and arterial blood pressure.

Abstract P191: Increases in Cerebrospinal Fluid NaCl Concentration Excites Neurons of the Organum Vasculosum of the Lamina Terminalis to Elevate Sympathetic Outflow and Blood Pressure

Accumulating evidence suggests salt-sensitive hypertension is mediated partly by an increase in cerebrospinal fluid (CSF) NaCl concentration and elevation in sympathetic nerve activity (SNA). Increased NaCl concentration or osmolality is sensed by specialized neurons in the organum vasculosum of the lamina terminalis (OVLT). The present study investigated the contribution of these neurons to the SNA and arterial blood pressure (ABP) responses during acute increases in CSF NaCl concentrations. Male Sprague-Dawley rats were anesthetized with Inactin (120mg/kg, IV) and prepared for SNA and ABP recordings. Lateral ventricle infusion of 1M NaCl (5uL over 10 min) increased CSF [Na+] by 5±1 mM and elevated mean ABP (9±1 mmHg), lumbar SNA (125±3%) and adrenal SNA (121±5%) but decreased renal SNA (-9±1%, n=8) and did not alter splanchnic SNA (102±3%). Inhibition of the OVLT with injection of the GABAA agonist muscimol (2.5mM per 20nL, n=5) significantly attenuated the NaCl-induced increase in ABP (2±1 mmHg), lumbar SNA (102±1%), adrenal SNA (103±2%) and decrease in renal SNA (-3±1%). In vivo single-unit recordings demonstrate that lateral ventricular infusion of 1M NaCl (5uL per 10 min) significantly increased the firing rate in 75% (3/4) of OVLT neurons from 1.2±0.4Hz to 5.1±1.2Hz (P&lt;0.05). Furthermore, direct injection of NaCl (100nL over 20s, n=3) into the OVLT produced dose-dependent increases in mean ABP (0.15M: 0±1mmHg; 0.5M: 2±1mmHg; 1.0M: 4±1mmHg; 2.0M: 7±1mmHg) and lumbar SNA (0.15M: 101±2%; 0.5M: 107±1%; 1.0M: 112±3%; 2.0M: 118±4%). Altogether, these findings suggest that increases in CSF NaCl concentrations excite OVLT neurons to elevate lumbar and adrenal SNA and ABP.

Hypothalamic Modulation of Adrenal Sympathetic Nerve Activity and the Glucose Counter‐Regulatory Response: Involvement of Medullary and Spinal Glutamate Receptors

The rostral ventrolateral medulla (RVLM) and perifornical hypothalamus (PeF) control adrenaline release during the glucose counter-regulatory response (GCRR) but the circuitry is not well-defined. In anesthetised rats, we measured ASNA after RVLM or PeF electrical stimulation (0.5Hz, 50-400µA, 1ms). We gave intrathecal kynurenate (KYN; 5µmol/10μl) or vehicle before stimulation and 2-deoxyglucose (2-DG) iv or into PeF after KYN or vehicle into RVLM. Hexamethonium (hex; 20-40mg/kg iv) discriminated pre- and post-ganglionic ASNA responses. In other rats, we injected the tracer CTB into the adrenal medulla and immunoperoxidase stained cord sections for CTB+orexin. After RVLM stimulation, ASNA showed pre- or post-ganglionic early peaks (63±3 ms; 56±6 ms) and pre- or post-ganglionic late peaks (132±7 ms; 139±4 ms). Intrathecal KYN reduced all peaks (P<0.05). After PeF stimulation, ASNA consisted of pre- or post-ganglionic early peaks (84±1 ms; 91±1 ms) and pre- or post-ganglionic late peaks (133±2 ms; 143±2 ms). Hex reduced or abolished all RVLM-evoked peaks but only the PeF-evoked early peak (P<0.05). KYN in RVLM (5nmol/100nl) reduced (P<0.05) ASNA after iv and PeF 2-DG. Orexin terminals apposed sympathoadrenal neurons in the spinal cord. These data implicate a monosynaptic PeF-spinal pathway, long latency pre-ganglionic responses and orexin input in control of adrenaline release. Glutamatergic circuits including PeF, RVLM and spinal cord may underpin the GCRR.

Severe hemorrhage attenuates cardiopulmonary chemoreflex control of regional sympathetic outputs via NTS adenosine receptors.

Selective stimulation of inhibitory A1 and facilitatory A2a adenosine receptor subtypes located in the nucleus of the solitary tract (NTS) powerfully inhibits cardiopulmonary chemoreflex (CCR) control of regional sympathetic outputs via different mechanisms: direct inhibition of glutamate release and facilitation of an inhibitory neurotransmitter release, respectively. However, it remains unknown whether adenosine naturally released into the NTS has similar inhibitory effects on the CCR as the exogenous agonists do. Our previous study showed that adenosine is released into the NTS during severe hemorrhage and contributes to reciprocal changes of renal (decreases) and adrenal (increases) sympathetic nerve activity observed in this setting. Both A1 and A2a adenosine receptors are involved. Therefore, we tested the hypothesis that, during severe hemorrhage, CCR control of the two sympathetic outputs is attenuated by adenosine naturally released into the NTS. We compared renal and adrenal sympathoinhibitory responses evoked by right atrial injections of 5HT3 receptor agonist phenylbiguanide (2-8 μg/kg) under control conditions, during hemorrhage, and during hemorrhage preceded by blockade of NTS adenosine receptors with bilateral microinjections of 8-(p-sulfophenyl) theophylline (1 nmol/100 nl) in urethane/chloralose anesthetized rats. CCR-mediated inhibition of renal and adrenal sympathetic activity was significantly attenuated during severe hemorrhage despite reciprocal changes in the baseline activity levels, and this attenuation was removed by bilateral blockade of adenosine receptors in the caudal NTS. This confirmed that adenosine endogenously released into the NTS has a similar modulatory effect on integration of cardiovascular reflexes as stimulation of NTS adenosine receptors with exogenous agonists.

Orexinergic Activation of Medullary Premotor Neurons Modulates the Adrenal Sympathoexcitation to Hypothalamic Glucoprivation

Glucoprivation activates neurons in the perifornical hypothalamus (PeH) and in the rostral ventrolateral medulla (RVLM), which results in the release of adrenaline. The current study aimed to establish 1) whether neuroglucoprivation in the PeH or in the RVLM elicits adrenaline release in vivo and 2) whether direct activation by glucoprivation or orexin release in the RVLM modulates the adrenaline release. Neuroglucoprivation in the PeH or RVLM was elicited by microinjections of 2-deoxy-D-glucose or 5-thio-D-glucose in anesthetized, euglycemic rats. Firstly, inhibition of neurons in the PeH abolished the increase in adrenal sympathetic nerve activity (ASNA) to systemic glucoprivation. Secondly, glucoprivation of neurons in the PeH increased ASNA. Thirdly, in vivo or in vitro glucoprivation did not affect the activity of RVLM adrenal premotor neurons. Finally, blockade of orexin receptors in the RVLM abolished the increase in ASNA to neuroglucoprivation in the PeH. The evoked changes in ASNA were directly correlated to levels of plasma metanephrine but not to normetanephrine. These findings suggest that orexin release modulates the activation of adrenal presympathetic neurons in the RVLM.

Orexinergic activation of medullary premotor neurons mediates the adrenal sympathoexcitation to hypothalamic glucoprivation (1126.2)

Glucoprivation activates neurons in the perifornical hypothalamus (PeH) and in the rostral ventrolateral medulla (RVLM), which results in release of adrenaline. The current study aimed to establish in vivo (i) whether neuroglucoprivation in the PeH or in the RVLM elicits adrenaline release; and (ii) whether direct activation of RVLM neurons by glucoprivation or the orexinergic drive to the RVLM mediates the release of adrenaline. Neuroglucoprivation in the PeH or RVLM was elicited by microinjections of 2-deoxy-D-glucose or 5-thio-D-glucose in anesthetized, euglycemic, rats. We found that inhibition of neurons in the PeH abolished the increase in adrenal sympathetic nerve activity (ASNA) to systemic glucoprivation. Secondly, glucoprivation of neurons in the PeH elicited an increase in ASNA. Thirdly, in vivo or in vitro glucoprivation did not affect the activity of RVLM adrenal premotor neurons. Finally, blockade of orexin receptors in the RVLM abolished the increase in ASNA to neuroglucoprivation in the PeH. The evoked changes in ASNA were directly correlated to levels of plasma metanephrine, but not to normetanephrine. These findings suggest that glucoprivation in the PeH elicits orexin release in the RVLM, capable of activating adrenal medullary presympathetic neurons in the RVLM. Grant Funding Source: Supported by NHMRC & ARC - Australia