Disinhibition of RVLM neural circuits and regulation of sympathetic nerve discharge at peak hyperthermia.

Hyperthermia is a potent activator of visceral sympathetic nerve discharge (SND), and the functional integrity of the rostral ventral lateral medulla (RVLM) is critically important for sustaining sympathoexcitation at peak hyperthermia. However, RVLM mechanisms mediating SND activation to acute heat stress are not well understood. Because RVLM GABA is tonically inhibitory to sympathetic nerve outflow, it is plausible to hypothesize that disinhibition of RVLM sympathetic neural circuits, via withdrawal of GABAergic tone, may affect SND regulation at peak hyperthermia. The effect of RVLM bicuculline (BIC; GABAA receptor antagonist, 100-200 pmol) microinjections on the level of renal SND in anesthetized rats was determined after internal body temperature (Tc) had been increased to 41.5°C. Temperature-control experiments involved RVLM BIC (100-200 pmol) microinjections, with Tc maintained at 38°C. As expected, acute heating significantly increased renal SND from control levels. Bilateral RVLM BIC microinjections at 41.5°C produced immediate and significant increases in renal SND above heating-induced levels of activation. Bilateral RVLM BIC microinjections at 38°C increased renal SND to similar levels as produced by RVLM BIC microinjections after Tc had been increased to 41.5°C (heating + RVLM BIC). These results demonstrate that a considerable level of RVLM GABAergic inhibition is sustained at peak hyperthermia, an interesting physiological response profile based on the significance of SND activation to cardiovascular regulation during heat stress.

Neuronal nitric oxide synthase inhibition and regional sympathetic nerve discharge: implications for peripheral vascular control.

Neuronal nitric oxide (NO) synthase (nNOS) inhibition with systemically administered S-methyl-l-thiocitrulline (SMTC) elevates mean arterial pressure (MAP) and reduces rat hindlimb skeletal muscle and renal blood flow. We tested the hypothesis that those SMTC-induced cardiovascular effects resulted, in part, from increased sympathetic nerve discharge (SND). MAP, HR, and lumbar and renal SND (direct nerve recordings) were measured in 9 baroreceptor (sino-aortic)-denervated rats for 20min each following both saline and SMTC (0.56mg/kg i.v.). SMTC increased MAP (peak ΔMAP: 50±8mmHg, p<0.01) compared to saline. Lumbar and renal SND were not different between saline and SMTC conditions at any time (p>0.05). The ΔSND between saline and SMTC conditions for the lumbar and renal nerves were not different from zero (peak ΔSND, lumbar: 2.0±6.8%; renal: 9.7±9.0%, p>0.05 versus zero for both). These data support that SMTC-induced reductions in skeletal muscle and renal blood flow reported previously reflect peripheral nNOS-derived NO vascular control as opposed to increased sympathetic vasoconstriction.

Inhibition of RVLM synaptic activation at peak hyperthermia reduces visceral sympathetic nerve discharge.

Hyperthermia is an environmental stressor that produces marked increases in visceral sympathetic nerve discharge (SND) in young rats. The brainstem in rats contains the essential neural circuitry for mediating visceral sympathetic activation; however, specific brainstem sites involved remain virtually unknown. The rostral ventral lateral medulla (RVLM) is a key central nervous system region involved in the maintenance of basal SND and in mediating sympathetic nerve responses evoked from supraspinal sites. In the present study we tested the hypothesis that inhibition of RVLM synaptic activation at peak hyperthermia (internal body temperature, Tc, increased to 41.5 degrees C) would affect heating-induced visceral sympathetic activation. Experiments were completed in chloralose-urethane anesthetized, baroreceptor-intact and sinoaortic-denervated, 3-6 month-old Sprague-Dawley rats. Bilateral inhibition of RVLM synaptic activation produced by muscimol microinjections (400 and 800 pmol) at 41.5 degrees C resulted in immediate and significant reductions in peak heating-induced renal and splenic sympathoexcitation. Interruption of RVLM synaptic activation and axonal transmission by lidocaine microinjections (40 nmol) at 41.5 degrees C produced significant reductions in hyperthermia-induced sympathetic activation to similar levels produced by RVLM muscimol microinjections. The total amount of SND inhibited by RVLM muscimol and lidocaine microinjections was significantly more during hyperthermia (41.5 degrees C) than normothermia (38 degrees C). These findings demonstrate that maintenance of sympathetic activation at peak hyperthermia is dependent on the integrity of RVLM neural circuits.

Central nervous system administration of interleukin-6 produces splenic sympathoexcitation.

Interleukin-6 (IL-6) is a multifunctional cytokine that has been shown to play a pivotal role in centrally-mediated physiological responses including activation of the hypothalamic-pituitary-adrenal axis. Cerebral spinal fluid (CSF) concentrations of IL-6 are elevated in multiple pathophysiological conditions including Alzheimer's disease, autoimmune disease, and meningitis. Despite this, the effect of IL-6 on central regulation of sympathetic nerve discharge (SND) remains unknown which limits understanding of sympathetic-immune interactions in health and disease. In the present study we determined the effect of intracerebroventricular (i.c.v, lateral ventricle) administration of IL-6 on splenic SND in urethane-chloralose-anesthetized rats. A second goal was to determine if icv injected IL-6 enters the brain parenchyma and acts as a volume transmission signal to access areas of the brain involved in regulation of sympathetic nerve outflow. i.c.v administration of IL-6 (10 ng, 100 ng, and 400 ng) significantly and progressively increased splenic SND from control levels in baroreceptor-denervated Sprague-Dawley rats. Administration of 100-ng and 400-ng IL-6 resulted in significantly higher SND responses when compared to those elicited with a 10-ng dose. Sixty minutes following icv administration, fluorescently labeled IL-6 was not distributed throughout the parenchyma of the brain but was localized to the periventricular areas of the ventricular system. Brain sections counter-stained for the IL-6 receptor (IL-6R) revealed that IL-6 and the IL-6R were co-localized in periventricular areas adjoining the third ventricle. These results demonstrate that icv IL-6 administration increases splenic SND, an effect likely achieved via signaling mechanisms originating in the periventricular cells.

Aging alters regulation of visceral sympathetic nerve responses to acute hypothermia.

Hypothermia produced by acute cooling prominently alters sympathetic nerve outflow. Skin sympathoexcitatory responses to skin cooling are attenuated in aged compared with young subjects, suggesting that advancing age influences sympathetic nerve responsiveness to hypothermia. However, regulation of skin sympathetic nerve discharge (SND) is only one component of the complex sympathetic nerve response profile to hypothermia. Whether aging alters the responsiveness of sympathetic nerves innervating other targets during acute cooling is not known. In the present study, using multifiber recordings of splenic, renal, and adrenal sympathetic nerve activity, we tested the hypothesis that hypothermia-induced changes in visceral SND would be attenuated in middle-aged and aged compared with young Fischer 344 (F344) rats. Colonic temperature (Tc) was progressively reduced from 38 degrees C to 31 degrees C in young (3 to 6 mo), middle-aged (12 mo), and aged (24 mo) baroreceptor-innervated and sinoaortic-denervated (SAD), urethane-chloralose anesthetized, F344 rats. The following observations were made. 1) Progressive hypothermia significantly (P < 0.05) reduced splenic, renal, and adrenal SND in young baroreceptor-innervated F344 rats. 2) Reductions in splenic, renal, and adrenal SND to progressive hypothermia were less consistently observed and, when observed, were generally attenuated in baroreceptor-innervated middle-aged and aged compared with young F344 rats. 3) Differences in splenic, renal, and adrenal SND responses to reduced Tc were observed in SAD young, middle-aged, and aged F344 rats, suggesting that age-associated attenuations in SND responses to acute cooling are not the result of age-dependent modifications in arterial baroreflex regulation of SND. These findings demonstrate that advancing chronological age alters the regulation of visceral SND responses to progressive hypothermia, modifications that may contribute to the inability of aged individuals to adequately respond to acute bouts of hypothermia.

Hypothermia-enhanced splenic cytokine gene expression is independent of the sympathetic nervous system.

Splenic nerve denervation abrogates enhanced splenic cytokine gene expression responses to acute heating, demonstrating that hyperthermia-induced activation of splenic sympathetic nerve discharge (SND) increases splenic cytokine gene expression. Hypothermia alters SND responses; however, the role of the sympathetic nervous system in mediating splenic cytokine gene expression responses to hypothermia is not known. The purpose of the present study was to determine the effect of hypothermia on the relationship between the sympathetic nervous system and splenic cytokine gene expression in anesthetized F344 rats. Gene expression analysis was performed using a microarray containing 112 genes, representing inflammatory cytokines, chemokines, cytokine/chemokine receptors and housekeeping genes. A subset of differentially expressed genes was verified by real-time RT-PCR analysis. Splenic SND was decreased significantly during cooling (core temperature decreased from 38 to 30 degrees C) in splenic-intact rats but remained unchanged in sham-cooled splenic-intact rats (core temperature maintained at 38 degrees C). Hypothermia upregulated the transcripts of several genes, including, chemokine ligands CCL2, CXCL2, CXCL10, and CCL20, and interleukins IL-1alpha, IL-1beta, and IL-6. Gene expression responses to hypothermia were similar for the majority of cytokine genes in splenic-intact and splenic-denervated rats. These results suggest that hypothermia-enhanced splenic cytokine gene expression is independent of splenic SND.

Central angiotensin II-enhanced splenic cytokine gene expression is mediated by the sympathetic nervous system.

We tested the hypothesis that central angiotensin II (ANG II) administration would activate splenic sympathetic nerve discharge (SND), which in turn would alter splenic cytokine gene expression. Experiments were completed in sinoaortic nerve-lesioned, urethane-chloralose-anesthetized, splenic nerve-intact (splenic-intact) and splenic nerve-lesioned (splenic-denervated) Sprague-Dawley rats. Splenic cytokine gene expression was determined using gene-array and real-time RT-PCR analyses. Splenic SND was significantly increased after intracerebroventricular administration of ANG II (150 ng/kg, 10 microl), but not artificial cerebrospinal fluid (aCSF). Splenic mRNA expression of IL-1beta, IL-6, IL-2, and IL-16 genes was increased in ANG II-treated splenic-intact rats compared with aCSF-treated splenic-intact rats. Splenic IL-1beta, IL-2, and IL-6 gene expression responses to ANG II were significantly reduced in splenic-denervated compared with splenic-intact rats. Splenic gene expression responses did not differ significantly in ANG II-treated splenic-denervated and aCSF-treated splenic-intact rats. Splenic blood flow responses to intracerebroventricular ANG II administration did not differ between splenic-intact and splenic-denervated rats. These results provide experimental support for the hypothesis that ANG II modulates the immune system through activation of splenic SND, suggesting a novel relation between ANG II, efferent sympathetic nerve outflow, and splenic cytokine gene expression.

Hyperthermia-enhanced splenic cytokine gene expression is mediated by the sympathetic nervous system.

Whole body hyperthermia (WBH) has been used in experimental settings as an adjunct to radiochemotherapy for the treatment of various malignant diseases. The therapeutic effect of WBH has been hypothesized to involve activation of the immune system, although the effect of hyperthermia-induced activation of sympathetic nerve discharge (SND) on splenic immune function is not known. We tested the hypothesis that heating-induced splenic sympathoexcitation would alter splenic cytokine gene expression as determined using gene array and real-time RT-PCR analyses. Experiments were performed in splenic-intact and splenic-denervated anesthetized Sprague-Dawley rats (n=32). Splenic SND was increased during heating (internal temperature increased from 38 degrees to 41 degrees C) in splenic-intact rats but remained unchanged in nonheated splenic-intact rats. Splenic interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and growth-regulated oncogene 1 (GRO 1) mRNA expression was higher in heated than in nonheated splenic-intact rats. Splenic IL-1beta, IL-6, and GRO 1 mRNA expression was reduced in heated splenic-denervated compared with heated splenic-intact rats, but did not differ between heated splenic-denervated and nonheated splenic-intact rats. These results support the hypothesis that hyperthermia-induced activation of splenic SND enhances splenic cytokine gene expression.

Central Tempol alters basal sympathetic nerve discharge and attenuates sympathetic excitation to central ANG II.

In the present study, we established dose-response relationships between central administration of 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol, a superoxide dismutase mimetic) and the level of renal sympathetic nerve discharge (SND) and tested the hypothesis that intracerebroventricular (icv) Tempol pretreatment would attenuate centrally mediated changes in SND produced by icv ANG II administration. Urethane-chloralose-anesthetized, baroreceptor-denervated, normotensive rats were used. We found that icv Tempol administration produced dose-dependent sympathoinhibitory, hypotensive, and bradycardic responses. Mean arterial pressure and SND values were significantly increased after icv ANG II (150 ng/kg) administration, and these responses were abrogated after icv pretreatment with Tempol (75 micromol/kg) or losartan. Brain superoxide levels tended to be higher in ANG II-treated rats compared with rats treated with Tempol and ANG II. Tempol pretreatment did not prevent increases in SND level that were produced by acute heat stress, which indicates specificity in the effect of Tempol in reducing sympathoexcitation. These results demonstrate that icv Tempol administration influences central sympathetic neural circuits in a dose-dependent manner and attenuates SND responses to central ANG II infusion.

Forebrain and brain stem neural circuits contribute to altered sympathetic responses to heating in senescent rats.

Acute heating in young rats increases visceral sympathetic nerve discharge (SND); however, renal and splanchnic SND responses to hyperthermia are attenuated in senescent compared with young Fischer 344 (F344) rats (Kenney MJ and Fels RJ. Am J Physiol Regul Integr Comp Physiol 283: R513-R520, 2002). Central mechanisms by which aging alters visceral SND responses to heating are unknown. We tested the hypothesis that forebrain neural circuits are involved in suppressing sympathoexcitatory responses to heating in chloralose-anesthetized, senescent F344 rats. Renal and splanchnic SND responses to increased (38 degrees C-41 degrees C) internal temperature were determined in midbrain-transected (MT) and sham-MT young (3-mo-old), mature (12-mo-old), and senescent (24-mo-old) F344 rats and in cervical-transected (CT) and sham-CT senescent rats. Renal SND remained unchanged during heating in MT and sham-MT senescent rats but was increased in CT senescent rats. Splanchnic SND responses to heating were higher in MT vs. sham-MT senescent rats and in CT vs. MT senescent rats. SND responses to heating were similar in MT and sham-MT young and mature rats. Mean arterial pressure (MAP) was increased during heating in MT but not in sham-MT senescent rats, whereas heating-induced increases in MAP were higher in sham-MT vs. MT young rats. These data suggest that in senescent rats suppression of splanchnic SND to heating involves forebrain and brain stem neural circuits, whereas renal suppression is mediated solely by brain stem neural circuits. These results support the concept that aging alters the functional organization of pathways regulating SND and arterial blood pressure responses to acute heating.

Central interleukin-1beta antibody increases renal and splenic sympathetic nerve discharge.

We tested the hypothesis that intracerebroventricular (lateral ventricle) administration of interleukin-1beta (IL-1beta) antibody increases the level of sympathetic nerve discharge (SND) in alpha-chloralose-anesthetized rats. Mean arterial pressure (MAP), heart rate (HR), and SND (splenic and renal) were recorded before (Preinfusion), during (25 min), and for 45 min after infusion of IL-1beta antibody (15 microg, 50 microl icv) in baroreceptor-intact (intact) and sinoaortic-denervated (SAD) rats. The following observations were made. First, intracerebroventricular infusion of IL-1beta antibody (but not saline and IgG) significantly increased MAP and the pressor response was higher in SAD compared with intact rats. Second, renal and splenic SND were significantly increased during and after intracerebroventricular IL-1beta antibody infusion and sympathoexcitatory responses were higher in SAD compared with intact rats. Third, intracerebroventricular administration of a single dose of IL-1beta antibody (15 microg, 5 microl for 2 min) significantly increased splenic and renal SND in intact rats. These results suggest that under the conditions of the present experiments central neural IL-1beta plays a role in the tonic regulation of SND and arterial blood pressure.

Role of paraventricular nucleus in regulation of sympathetic nerve frequency components.

Autospectral and coherence analyses were used to determine the role of and interactions between paraventricular nucleus (PVN) nitric oxide, gamma-aminobutyric acid (GABA), and the N-methyl-D-aspartic acid (NMDA)-glutamate receptor in regulation of sympathetic nerve discharge (SND) frequency components in anesthetized rats. Four observations were made. First, PVN microinjection of bicuculline (BIC) (GABA(A) receptor antagonist), but not single PVN injections of NMDA (excitatory amino acid) or N(G)-monomethyl-L-arginine (L-NMMA; a nitric oxide synthase inhibitor), altered SND frequency components. Second, combined PVN microinjections of L-NMMA and NMDA changed the SND bursting pattern; however, the observed pattern change was different from that produced by PVN BIC and not observed after sinoaortic denervation. Third, PVN microinjection of kynurenic acid prevented and reversed BIC-induced changes in the SND bursting pattern. Finally, vascular resistance (renal and splenic) was significantly increased after PVN BIC microinjection despite the lack of change in the level of renal and splenic SND. These data demonstrate that the PVN contains the neural substrate for altering SND frequency components and suggest complex interactions between specific PVN neurotransmitters and between PVN neurotransmitters and the arterial baroreceptor reflex in SND regulation.

Sympathoexcitation to intravenous interleukin-1beta is dependent on forebrain neural circuits.

We investigated the contributions of forebrain, brain stem, and spinal neural circuits to interleukin (IL)-1beta-induced sympathetic nerve discharge (SND) responses in alpha-chloralose-anesthetized rats. Lumbar and splenic SND responses were determined in spinal cord-transected (first cervical vertebra, C1), midbrain-transected (superior colliculus), and sham-transected rats before and for 60 min after intravenous IL-1beta (285 ng/kg). The observations made were the following: 1) lumbar and splenic SND were significantly increased after IL-1beta in sham C1-transected rats but were unchanged after IL-1beta in C1-transected rats; 2) intrathecal administration of DL-homocysteic acid (10 ng) increased SND in C1-transected rats; 3) lumbar and splenic SND were significantly increased after IL-1beta in sham- but not midbrain-transected rats; and 4) midbrain transection did not alter the pattern of lumbar and splenic SND, demonstrating the integrity of brain stem sympathetic neural circuits after decerebration. These results demonstrate that an intact forebrain is required for mediating lumbar and splenic sympathoexcitatory responses to intravenous IL-1beta, thereby providing new information about the organization of neural circuits responsible for mediating sympathetic-immune interactions.

Sympathetic nerve regulation to heating is altered in senescent rats.

Renal and splanchnic sympathetic nerve discharge (SND) responses to increased (38-41 degrees C) internal temperature were determined in anesthetized young (3-6 mo old), mature (12 mo old), and senescent (24 mo old) Fischer 344 (F344) rats. We hypothesized that SND responses would be altered in senescent and mature rats as demonstrated by attenuated sympathoexcitatory responses to heating and by the absence of hyperthermia-induced SND pattern changes. The following observations were made. 1) Renal and splanchnic SND responses were significantly increased during heating in young and mature but not in senescent rats. 2) At 41 degrees C, renal and splanchnic SND responses were higher in young compared with senescent rats, and renal SND was higher in mature than in senescent rats. 3) Heating changed the SND bursting pattern in young, but not in mature or senescent, rats. 4) SND responses to heating did not differ between baroreceptor-innervated (BRI) and sinoaortic-denervated (SAD) senescent rats but were higher in SAD compared with BRI young rats. These results demonstrate an attenuated responsiveness of sympathetic neural circuits to heating in senescent F344 rats.