Effects of central administration of resistin on renal sympathetic nerve activity in rats fed a high-fat diet: a comparison with leptin.

Similar to leptin, resistin acts centrally to increase renal sympathetic nerve activity (RSNA). In high-fat fed animals, the sympatho-excitatory effects of leptin are retained, in contrast to the reduced actions of leptin on dietary intake. In the present study, we investigated whether the sympatho-excitatory actions of resistin were influenced by a high-fat diet. Further, because resistin and leptin combined can induce a greater sympatho-excitatory response than each alone in rats fed a normal chow diet, we investigated whether a high-fat diet (22%) could influence this centrally-mediated interaction. Mean arterial pressure (MAP), heart rate (HR) and RSNA were recorded before and for 3 hours after i.c.v. saline (control; n=5), leptin (7 µg; n=4), resistin (7 µg; n=5) and leptin and resistin combined (n=6). Leptin alone and resistin alone significantly increased RSNA (71±16%, 62±4%, respectively). When leptin and resistin were combined, there was a significantly greater increase in RSNA (195±41%) compared to either hormone alone. MAP and HR responses were not significantly different between hormones. When the responses in high-fat fed rats were compared to normal chow fed rats, there were no significant differences in the maximum RSNA responses. The findings indicate that sympatho-excitatory effects of resistin on RSNA are not altered by high-fat feeding, including the greater increase in RSNA observed when resistin and leptin are combined. Our results suggest that diets rich in fat do not induce resistance to the increase in RSNA induced by resistin alone or in combination with leptin.

Identification of CNS neurons with polysynaptic connections to both the sympathetic and parasympathetic innervation of the submandibular gland.

Coordinated modulation of sympathetic and parasympathetic nervous activity is required for physiological regulation of tissue function. Anatomically, whilst the peripheral sympathetic and parasympathetic pathways are separate, the distribution of premotor neurons in higher brain regions often overlaps. This co-distribution would enable coordinated regulation and might suggest individual premotor neurons could project to both sympathetic and parasympathetic outflows. To investigate this one submandibular gland was sympathectomized. One of two isogenic strains of the pseudorabies virus, expressing different fluorophores, was injected into the cut sympathetic nerve and the other into the submandibular gland. Independent labeling of the peripheral sympathetic and parasympathetic pathways was observed. Dual-labeled neurons were observed in many CNS regions known to be involved in regulating salivary function. We propose these observations highlight a common pattern of organization of the CNS, providing the anatomical framework for the fine control of organ function required for homeostatic regulation and the coordination of organ responses to enable complex behaviors.

Microglia are selectively activated in endocrine and cardiovascular control centres in streptozotocin-induced diabetic rats.

Type 1 and 2 diabetes are associated with dysfunction in multiple hormone systems, as well as increased sympathetic nerve activity, which may contribute to the development of diabetic complications. In other pathologies, such as myocardial infarction, increased sympathetic drive is associated with neuroinflammation and microglial activation in the hypothalamic paraventricular nucleus (PVN), a brain region that regulates sympathetic drive and multiple endocrine responses. In the present study, we used immunohistochemistry to study microglial and neuronal activation in the PVN and related brain regions in streptozotocin (STZ)-induced diabetic rats. As expected, STZ treatment was associated with elevated blood glucose within 1 week. STZ injections also caused neuronal activation in the PVN and superoptic nucleus (SON) but not in the nucleus tractus solitarius (NTS), which was evident by 6 weeks. STZ-treated rats showed increased plasma osmolarity, which would be expected to activate PVN and SON neurones. There was no apparent increase in histochemical markers of microglial activation, including phospho-p38, phospho-extracellular signal regulated kinase, P2X4 receptor or interleukin 1-ß even at 10 weeks after STZ-treatment. However, we did see a significant increase in the percentage of microglia with an activated morphology in the PVN, SON and NTS, although not in surrounding hypothalamic, brainstem or cortical regions. These morphological changes included a significant reduction in microglial process length and were evident by 8 weeks but not 6 weeks. The delayed onset of microglial changes compared to neuronal activation in the PVN and SON suggests the over-excitation of neurones as a mechanism of microglial activation. This delayed microglial activation may, in turn, contribute to the endocrine dysregulation and the elevated sympathetic nerve activity reported in STZ-treated rats.

An investigation of the mechanisms of hydrogen sulfide-induced vasorelaxation in rat middle cerebral arteries.

Hydrogen sulfide (H(2)S) is an endogenous mediator with peripheral vasorelaxant effects; however, the mechanism of H(2)S-induced vasorelaxation in cerebral blood vessels has not been extensively studied. Vasorelaxation studies were performed on middle cerebral arteries from male Sprague Dawley rats using wire myography. Immunofluorescence staining was used to detect the presence of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE). CSE was present in the endothelium and smooth muscle of middle cerebral arteries. The CSE substrate, L-cysteine, induced vasorelaxation that was sensitive to the CSE inhibitor DL-propargylglycine. This relaxation was independent of endothelium, suggesting that H(2)S was produced in the vascular smooth muscle. The H(2)S donor, sodium hydrogen sulfide (NaHS; 0.1-3.0 mM) produced concentration-dependent relaxation, which was unaffected by endothelium removal. Nifedipine (3 µM) significantly reduced the maximum relaxation elicited by NaHS. Inhibiting potassium (K(+)) conductance with 50 mM K(+) significantly attenuated NaHS-induced relaxation, however, selective blockers of ATP sensitive (K(ATP)), calcium sensitive (K(Ca)), voltage dependent (K(V)), or inward rectifier (K(ir)) channels alone or in combination did not affect the response to NaHS. 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 300 µM) caused a significant rightward shift of the NaHS concentration-response curve, but this effect could not be explained by inhibition of Cl(-) channels or Cl(-)/HCO (3)(-) exchange, as selective blockade of these mechanisms had no effect. These findings suggest endogenous H(2)S can regulate cerebral vascular function. The H(2)S-mediated relaxation of middle cerebral arteries is DIDS sensitive and partly mediated by inhibition of L-type calcium channels, with an additional contribution by K channels but not K(ATP), K(Ca), K(V), or K(ir) subtypes.

Central resistin enhances renal sympathetic nerve activity via phosphatidylinositol 3-kinase but reduces the activity to brown adipose tissue via extracellular signal-regulated kinase 1/2.

Resistin is an adipokine, originally identified in adipose tissue, and its plasma levels are elevated in obesity. Characteristics of obesity include impaired metabolic regulation and cardiovascular dysfunction, such as increased sympathetic nerve activity (SNA) to the kidney and skeletal muscle vasculature. Resistin can affect energy homeostasis through central mechanisms that include reduced food intake and reduced thermogenesis, and can also increase lumbar SNA via a central action. The present study investigated: (i) the effect of centrally-administered resistin on SNA targeting the kidney and (ii) the intracellular signalling pathways mediating the changes in SNA innervating the kidney and brown adipose tissue (BAT) induced by resistin. Intracerebroventricular resistin (7 µg) injected into overnight fasted, anaesthetised rats induced a significant increase in renal SNA by approximately 40%. This response was prevented when phosphatidylinositol 3-kinase (PI3K) was inhibited by i.c.v. administration of LY294002 (5 µg). Resistin reduced BAT SNA and this response was delayed by 150 min when extracellular-regulated kinase (ERK)1/2 was inhibited by i.c.v. administration of U0126. The findings indicate that resistin increases renal SNA via PI3K and reduces BAT SNA via ERK1/2.

Centrally administered resistin enhances sympathetic nerve activity to the hindlimb but attenuates the activity to brown adipose tissue.

Resistin, an adipokine, is believed to act in the brain to influence energy homeostasis. Plasma resistin levels are elevated in obesity and are associated with metabolic and cardiovascular disease. Increased muscle sympathetic nerve activity (SNA) is a characteristic of obesity, a risk factor for diabetes and cardiovascular disease. We hypothesized that resistin affects SNA, which contributes to metabolic and cardiovascular dysfunction. Here we investigated the effects of centrally administered resistin on SNA to muscle (lumbar) and brown adipose tissue (BAT), outputs that influence cardiovascular and energy homeostasis. Overnight-fasted rats were anesthetized, and resistin (7 µg) was administered into the lateral cerebral ventricle (intracerebroventricular). The lumbar sympathetic nerve trunk or sympathetic nerves supplying BAT were dissected free, and nerve activity was recorded. Arterial blood pressure, heart rate, body core temperature, and BAT temperature were also recorded. Responses to resistin or vehicle were monitored for 4 h after intracerebroventricular administration. Acutely administered resistin increased lumbar SNA but decreased BAT SNA. Mean arterial pressure and heart rate, however, were not significantly affected by resistin. BAT temperature was significantly reduced by resistin, and there was a concomitant fall in body temperature. The findings indicate that resistin has differential effects on SNA to tissues involved in metabolic and cardiovascular regulation. The decreased BAT SNA and the increased lumbar SNA elicited by resistin suggest that it may contribute to the increased muscle SNA and reduced energy expenditure observed in obesity and diabetes.

Activation of NADPH-diaphorase-positive projections to the rostral ventrolateral medulla following cardiac mechanoreceptor stimulation in the conscious rat.

Stimulation of cardiac mechanoreceptors during volume expansion elicits reflex compensatory changes in sympathetic nerve activity (SNA). The hypothalamic paraventricular nucleus (PVN) and nucleus of the tractus solitarius (NTS) are autonomic regions known to contribute to this reflex. Both of these nuclei project to the rostral ventrolateral medulla (RVLM), critical in the tonic generation of SNA. Recent reports from our laboratory show that these pathways 1) are activated following cardiac mechanoreceptor stimulation, and 2) produce nitric oxide, known to influence SNA. The aims of the present study were to determine whether 1) the activated neurons within the PVN and NTS were nitrergic and 2) these neurons projected to the RVLM. Animals were prepared, under general anesthesia, by microinjection of a retrogradely transported tracer into the pressor region of the RVLM and the placement of a balloon at the right venoatrial junction. In conscious rats, the balloon was inflated to stimulate the cardiac mechanoreceptors or was left uninflated. Balloon inflation elicited a significant increase in Fos-positive neurons in the parvocellular PVN (sevenfold) and NTS (fivefold). In the PVN, 51% of nitrergic neurons and 61% of RVLM-projecting nitrergic neurons were activated. In the NTS, these proportions were 8 and 18%, respectively. The data suggest that nitrergic neurons within the PVN and, to a lesser extent, in the NTS, some of which project to the RVLM, may contribute to the central pathways influencing SNA elicited by cardiac mechanoreceptor stimulation.

Right atrial stretch activates neurons in autonomic brain regions that project to the rostral ventrolateral medulla in the rat.

Activation of the cardiac mechanoreceptors results in changes in sympathetic nerve activity and plays an important role in the responses elicited by elevated blood volume. Stimulation of the reflex influences several key autonomic regions, namely the paraventricular nucleus (PVN), the nucleus of the tractus solitarius (NTS) and the caudal ventrolateral medulla (CVLM). Neurons in these regions project directly to the rostral ventrolateral medulla (RVLM), a critical region in the generation of sympathetic vasomotor tone. The aim of the present experiments was to determine whether neurons in the PVN, NTS and CVLM that are activated by cardiac mechanoreceptor stimulation also project to the RVLM. Animals were prepared, under general anesthesia, by microinjection of a retrogradely transported tracer into the pressor region of the RVLM, and the placement of a balloon-tipped cannula at the junction of the right atrium and the superior vena cava. On the experimental day, in conscious rats, the balloon was inflated to stimulate cardiac mechanoreceptors (n = 9), or left uninflated (control, n = 8). Compared with controls, there was a significantly increased number of Fos-immunoreactive neurons (a marker of activation) in both the PVN (2.5-fold) and NTS (two-fold), but this was not seen in the CVLM. Compared with controls, a significant number of the neurons in the PVN (8%) and NTS (4.0%) that projected to the RVLM were activated. The data suggest that subgroups of RVLM-projecting neurons located in the PVN and NTS are involved in the central reflex pathway activated by cardiac mechanoreceptor stimulation.

Fos, RVLM-projecting neurons, and spinally projecting neurons in the PVN following hypertonic saline infusion.

Hypertonic saline (HTS; 1.7 M) infused intravenously into conscious rats increases the production of Fos, a marker of cell activation, in the hypothalamic paraventricular nucleus (PVN). The parvocellular PVN contains subpopulations of neurons. However, which subpopulations are activated by HTS is unknown. We determined whether PVN neurons that innervate the rostral ventrolateral medulla (RVLM) or the spinal cord (important autonomic sites) expressed Fos following HTS. Experiments were performed 24-96 h after chronic implantation of an intravenous cannula. HTS significantly increased the number of Fos-positive cells. In the parvocellular PVN, the maximum number of Fos-positive cells occurred rostral of the anterior-posterior level at which the number of neurons that projected to the medulla or spinal cord peaked. Compared with controls, HTS did not significantly increase the number of double-labeled neurons. These findings demonstrate that an elevation in plasma osmolality activates PVN neurons but not the subgroups of PVN neurons with projections to the RVLM or to the spinal cord.

Efferent neural projections of angiotensin receptor (AT1) expressing neurones in the hypothalamic paraventricular nucleus of the rat.

Angiotensin II acts within the hypothalamic paraventricular nucleus (PVN) to help mediate a number of autonomic and endocrine responses. Evidence is sparse in regard to the particular neuronal cell groups that exhibit angiotensin II type 1 receptors within the PVN, and does not exist in relation to specified efferent neuronal populations in the nucleus. In the present experiments, retrogradely transported neuronal tracers were utilized in conjunction with immunohistochemistry using a well characterized polyclonal antibody raised against a decapeptide sequence at the carboxy terminus of the AT1 receptor, to determine whether it is preferentially distributed amongst different efferent populations within the PVN. The AT1 receptor is not associated with neurones in the PVN that project axons to the spinal cord, dorsomedial or ventrolateral medulla but coexists strongly with neurones in the anterior parvocellular division of the nucleus which direct axons to the median eminence. Such neurones often contain corticotropin releasing factor. These findings highlight the role that angiotensin II and AT1 receptors in the PVN may play in the mediation of responses to stress.

Hypothalamic paraventricular nucleus and cardiovascular regulation.

1. The hypothalamic paraventricular nucleus (PVN) is an important integrative site within the brain composed of magnocellular and parvocellular neurons. It is known to influence sympathetic nerve activity. 2. The parvocellular PVN contains neurons that project to the intermediolateral cell column of the thoraco-lumbar spinal cord (IML). This defines the PVN as an autonomic 'premotor nucleus', one of only five present within the brain. 3. Another projection arising from the PVN is a prominent innervation of the pressor region of the rostral ventrolateral medulla (RVLM), also a premotor nucleus. The distribution of the PVN neurons projecting to the RVLM is similar to that of the PVN neurons that project to the IML. 4. It has been found that up to 30% of spinally projecting neurons in the PVN also send collaterals to the RVLM. Thus, there are neurons in the PVN that can: (i) directly influence sympathetic nerve activity (via PVN-IML connections); (ii) indirectly influence sympathetic nerve activity (via PVN-RVLM connections); and (iii) both directly and indirectly influence sympathetic nerve activity (via neurons with collaterals to the IML and RVLM). 5. In the rat, results of studies using the protein Fos to identify activated neurons in the brain suggest that neurons in the PVN with projections to the IML or RVLM may be activated by decreases in blood volume. 6. In conclusion, the PVN can influence sympathetic nerve activity. Within the PVN are neurons with anatomical connections that enable them to affect sympathetic nerve activity either directly, indirectly or via both mechanisms (via collaterals). Studies that have examined the role of specific subgroups within the PVN suggest that PVN neurons with connections to the IML or to the RVLM may play a role in the reflex changes in sympathetic nerve activity that are involved in blood volume regulation.

Blockade of central angiotensin II does not affect the reduction in renal sympathetic nerve activity following a volume load in heart failure rabbits.

1. We investigated the role in sympathetic nerve regulation of endogenous angiotensin (Ang)II in the brain in heart failure by examining the effect of losartan on the resting mean arterial blood pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) and the reflex reduction in RSNA elicited by acute volume expansion in conscious rabbits. 2. Heart failure was induced with doxorubicin (1 mg/kg, i.v., twice weekly for 6 weeks). On the experimental day, MAP, HR and RSNA were recorded in conscious rabbits before and after losartan (10 microg; n = 6) or Ringer's (control; n = 5) injected in the fourth brain ventricle. Animals were then administered an acute volume load with the plasma expander Haemaccel (Hoechst Marion Roussel, Lane Cove, NSW, Australia; 2 mL/min, i.v., for 30 min). 3. Losartan did not significantly affect the resting basal levels of MAP, HR and RSNA. There was no significant difference between losartan- and Ringer's-treated animals. 4. Volume expansion in the control group elicited a significant reduction of 40% in RSNA. In the losartan-treated group, a similar reduction (42%) was observed. There was no significant difference between the treatments. The administration of losartan did not significantly affect MAP and HR during volume expansion compared with the control group. 5. We conclude that AngII in the brainstem does not play a major role in the maintenance of resting MAP, HR and RSNA or in the reflex reduction in RSNA elicited by volume expansion in the conscious rabbit with doxorubicin-induced heart failure.

Role of brain angiotensin II in renal nerve inhibition elicited by volume expansion in the conscious rabbit.

Losartan (10 microg/25 microl) or vehicle was injected into the fourth brain ventricle prior to volume expansion (VE) with Haemaccel (2 ml/min for 30 min). RSNA was reduced by a maximum of 45% in response to the VE following vehicle and by 33% following losartan. There was no significant difference between the treatments in RSNA, nor in the blood pressure and heart rate responses. We conclude that endogenous angiotensin II does not make a major contribution to the reflex reduction in RSNA initiated by VE.

Harmane produces hypotension following microinjection into the RVLM: possible role of I(1)-imidazoline receptors.

The beta-carboline, harmane (0.1 - 1.0 nmol) produces dose dependent hypotension when microinjected unilaterally into the rostral ventrolateral medulla (RVLM) of the anaesthetized rat. The potency of harmane on blood pressure is similar to that of the imidazoline, clonidine. The hypotensive effects of both clonidine and harmane are reversed by microinjection of the relatively I(1)-receptor selective antagonist efaroxan (20 nmol). These results are consistent with harmane acting at an I(1)-receptor in the RVLM. This is the first report of an endogenous ligand for I(1)-receptors that has central effects on blood pressure.

Effects of leptin on spinally projecting neurons in the PVN of the hypothalamus.

The study examined whether intravenous (i.v.) leptin increased Fos-production in spinally projecting neurons in the hypothalamic paraventricular nucleus (PVN). We combined (i) rhodamine tagged microspheres injected into the upper thoracic spinal cord and (ii) Fos (marker of neuronal activation) immunohistochemistry. Effects of recombinant murine leptin were compared to vehicle (containing lipopolysaccharide a contaminant present in the leptin solution). Following leptin, 10% of the spinally projecting neurons contained Fos. However, vehicle elicited similar effects and there was no significant difference between the groups.

Volume expansion does not activate neuronal projections from the NTS or depressor VLM to the RVLM.

We investigated whether a monosynaptic connection from the nucleus tractus solitarius (NTS) or the depressor ventrolateral medulla (VLM) to the pressor region of the rostral VLM (RVLM) constituted part of the reflex pathway activated by cardiopulmonary baroreceptors. Volume expansion in the conscious rabbit, which elicits renal nerve inhibition predominantly via cardiac mechanoreceptors, was used as the stimulus. The protein Fos was used as a marker of neuronal activation. The retrogradely transported tracer rhodamine-tagged microspheres, previously injected into the pressor region of the RVLM, identified medullary neurons that projected to that region. Volume expansion significantly increased the number of Fos-positive cell nuclei in the NTS and in the depressor VLM. Neurons that projected to the RVLM were found throughout the depressor region of the VLM and in the NTS but were not activated by volume expansion. Thus, although the central reflex pathways activated by volume expansion include the NTS and the depressor region of the VLM, we could not find evidence for a monosynaptic connection between those regions and the RVLM.

Sympathetic response to stimulation of the pontine A5 region in conscious rabbits.

Studies in anaesthetized animals have shown that the pontine A5 noradrenergic region plays an important role in the sympathetic control of arterial pressure (AP). The aim of this study was to develop, in conscious rabbits, a technique for microinjections into the A5 region and examine the effects of stimulation of this region on renal sympathetic nerve activity (RSNA). In preliminary mapping experiments on four anaesthetized rabbits, electrical stimulation of the A5 region induced a pressor response ranging between 25 and 75 mmHg while unilateral injection of glutamate (100 nmol) did not change AP. The mapping experiments were used to enable guide cannulae implantation for subsequent microinjections into the A5 region. In six conscious rabbits, unilateral injection of glutamate (100 nmol) caused a consistent increase in RSNA (+45%) but did not change AP. In another eight rabbits, bilateral injection of glutamate (0.3, 3, 30 nmol) into the A5 region dose-dependently increased RSNA by 13%, 30% and 40%, respectively. In four rabbits, angiotensin II (0.3, 3, 30 pmol) injected bilaterally into the A5 region increased RSNA by 5%, 22% and 28%, respectively. In all animals the increase in RSNA was mainly mediated by increasing amplitude of sympathetic synchronized bursts while their frequency remained unchanged. However, both glutamate and angiotensin II did not change AP indicating that the sympathoexcitatory response to the A5 stimulation might be relatively confined to the renal bed. Using a novel microinjection technique developed for conscious rabbits, we found that the A5 region may provide an important excitatory and possibly selective input to the renal sympathetic preganglionic neurons.

Volume expansion fails to normally activate neural pathways in the brain of conscious rabbits with heart failure.

Immunohistochemical detection of the protein, Fos, was used to identify neurons in the brain activated following a volume load in conscious rabbits with doxorubicin-induced congestive cardiomyopathy. The plasma expander, Haemaccel, was infused intravenously into rabbits for 60 min and significantly increased right atrial pressure, blood pressure and heart rate. The rabbits were perfusion fixed 90 min after the start of the infusion and the distribution of Fos-positive cell nuclei was examined. Compared to control rabbits with heart failure, there was a small significant increase in the number of Fos-positive cell nuclei in the organum vasculosum of the lamina terminalis following volume expansion. In other regions of the brain that were studied in detail, there were no significant increases in Fos production. These included the parvocellular paraventricular nucleus (PVN) of the hypothalamus, the midbrain periaqueductal gray, the nucleus tractus solitarius (NTS), area postrema and the ventrolateral medulla (VLM). In the supraoptic nucleus and the magnocellular PVN, no Fos-positive cell nuclei were present as expected. The median preoptic nucleus, the bed nucleus of the striae terminalis and the diagonal band of Broca contained some Fos but there was no marked difference between volume expanded and control animals. In the anterior cortical and medial subnuclei of the amygdala there was a high concentration of Fos but there was no consistent difference between the two groups. The present findings in heart failure rabbits suggest that most brain regions are not activated sufficiently by the stimulus to elicit Fos expression. The results are in accord with findings showing that sympathetic reflexes initiated by volume expansion are attenuated in heart failure.

Neurons in the hypothalamic paraventricular nucleus that project to the rostral ventrolateral medulla are not activated by hypotension.

The retrogradely-transported tracer, rhodamine-tagged microspheres was injected into the pressor region of the rostral ventrolateral medulla (RVLM) to enable detection of paraventricular neurons in the hypothalamus that project to the RVLM. The protein, Fos, was detected immunohistochemically and used to highlight neurons that were activated by hypotension (-16+/-5 mmHg) induced by diazoxide (30 mg/kg s.c.). Compared to controls, Fos production was increased by three-fold in the parvocellular paraventricular nucleus but there was no significant increase in the number of retrogradely-labelled cells that expressed Fos. The results suggest paraventricular nucleus (PVN) neurons projecting to the RVLM are not activated by hypotension.

Neurons in the hypothalamic paraventricular nucleus send collaterals to the spinal cord and to the rostral ventrolateral medulla in the rat.

The hypothalamic paraventricular nucleus (PVN) projects to the rostral ventrolateral medulla (RVLM) and to the intermediolateral cell column (IML) of the spinal cord. The present study determined whether the same neurons can innervate both regions. In each rat, two retrogradely-transported tracers, microspheres tagged with fluorescein or rhodamine, were injected into the left lower thoracic/upper lumbar IML (fluorescein) and into the pressor region of the left RVLM (rhodamine). In the PVN over 90% of the neurons labelled with either tracer were found ipsilateral to the injection site. Double labelled cells averaged almost one-third of the spinally-projecting cells in four of the five animals. In the remaining animal, there were few double-labelled cells. The results suggest that a population of PVN neurons innervates both the lower-thoracic/upper lumbar IML and the RVLM.