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.

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.

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.

Central and peripheral autonomic mechanisms involved in the circulatory actions of methyldopa.

Intracisternal (i.c.) and intravenous (i.v.) administration of methyldopa in conscious rabbits produced closely similar changes in hemodynamics, heart rate, and falls in plasma norepinephrine levels. Two weeks after giving i.c. 6-hydroxydopamine (6-OHDA), when there is widespread destruction of central noradrenergic neurons, the effects of i.c. methyldopa virtually were abolished. This suggests that noradrenergic neurons are the major central site of biotransformation into active metabolites. The circulatory and norepinephrine effects of i.v. methyldopa were attenuated but not completely abolished after giving i.c. 6-OHDA. Hence, in the rabbit about 70% of the action of methyldopa was central and about 30% was peripheral in the human therapeutic range of methyldopa concentrations. Preliminary lesion experiments suggest that the A5 nucleus plays an important role in the bradycardia. Two weeks after giving 5,6-dihydroxytryptamine (5,6-DHT) to destroy serotonergic (5HT) neurons the effects of i.c. methyldopa on mean arterial pressure (MAP) and heart rate were attenuated to approximately 50% of control effects. Therefore, some of the central effects of methyldopa apparently are mediated through 5HT pathways. We also compared the effects of i.c. methyldopa with those of i.c. clonidine (an alpha 2-adrenergic receptor agonist) and with the effects of transmitter release from the endings of noradrenergic and 5HT neurons during the first few hours after either 6-OHDA or 5,6-DHT administration. Our findings suggest that after biotransformation of methyldopa its active metabolites increase the activity of the bulbospinal noradrenergic neurons that control MAP and heart rate and reduce the activity of bulbospinal 5HT neurons.

Interactions of endogenous opioid and excitatory amino acid inputs to the caudal ventrolateral medulla of the rat.

This study investigated the cardiovascular consequences of interactions between endogenous opioid and excitatory amino acid inputs to the caudal ventrolateral medulla of the anaesthetised rat. Drugs were injected bilaterally into the functionally identified depressor region of the caudal ventrolateral medulla. The opioid antagonist, naloxone (2.5-8.0 nmol/side) elicited a dose-dependent decrease in blood pressure and a bradycardia. The NMDA-receptor antagonist, 2-amino, 5-phosphonovaleric acid (2-APV; 1.25-500 pmol/side), dose-dependently increased blood pressure but had little effect on heart rate. After the maximum dose of naloxone, the pressor response to both 1.25 and 25 pmol/side of 2-APV was attenuated by 89 and 66%, respectively. By contrast, the pressor response, elicited by injection of the GABA agonist, muscimol (1 pmol/side), was not affected. After 2-APV (500 pmol/side), the depressor response to 2.5 nmol/side of naloxone was enhanced by 84%, although this effect was lost when a larger dose of naloxone (5 nmol/side) was used. 2-Amino,5-phosphonovaleric acid also potentiated the depressor response to a submaximal dose of the GABA antagonist, bicuculline (2 pmol/side). The results suggest firstly that, in the caudal ventrolateral medulla, excitatory amino acid inputs are functionally less important when tonic opioid effects are blocked. This interaction appears to be pharmacologically specific. Secondly, tonic inhibitory inputs, whether due to opioids or to GABA, are functionally more effective after excitatory amino acid inputs are antagonized.

Inhibition by morphine of the cardiovascular and behavioral responses evoked by centrally administered substance P in conscious rats.

The effect of endogenous opioid receptor stimulation on the central cardiovascular and behavioral actions of substance P (SP) was examined in conscious rats. SP (55 pmol) injected intracerebroventricularly (i.c.v.) elicited increases in mean arterial pressure, heart rate, and stereotyped behavioral activation such as exploring and grooming, which were considered to be parts of the cardiovascular defense reaction. Intravenous (i.v.) pretreatment with morphine (2.5 and 5.0 mg/kg) attenuated the cardiovascular and behavioral responses produced by SP i.c.v. dose-dependently. The i.v. pretreatment with naloxone (10 mg/kg) had no effect on the central SP-induced response. Pressor responses elicited by i.c.v. injection of corticotropin-releasing factor or angiotensin II were also attenuated by pretreatment with i.v. morphine (5.0 mg/kg). Our results showed that endogenous opioid receptor stimulation antagonizes the central cardiovascular and behavioral actions of SP. Morphine may not influence the primary site of action of SP but does influence the central neural pathway which conveys the SP-induced sympathetic activation signal.

Distribution of activated neurons in the rabbit brain following a volume load.

Immunohistochemical detection of the protein, Fos, was used to identify neurons in the brain activated following a volume load. The plasma expanders, Haemaccel and 6% dextran, were infused intravenously in conscious rabbits for 60 min. Compared to control animals both stimuli significantly increased right atrial pressure but had no effect on blood pressure. Heart rate was significantly elevated with dextran only. Volume expansion with Haemaccel also reduced renal sympathetic nerve activity by about 50% from the pre-infusion resting level. Ninety minutes after the start of the infusion, the rabbits were perfusion fixed and the distribution of Fos-positive cell nuclei was examined. Following Haemaccel infusion there were significant increases in the number of Fos-positive cell nuclei in the organum vasculosum of the lamina terminalis, parvocellular paraventricular nucleus and in specific rostrocaudal levels of the nucleus tractus solitarius and ventrolateral medulla. Following dextran similar effects were observed in the medulla but Fos-positive cell nuclei were not significantly elevated above controls in the forebrain. After Haemaccel or dextran areas such as the supraoptic nucleus, the magnocellular paraventricular nucleus, the bed nucleus of the stria terminalis, diagonal band of Broca and amygdala either did not produce Fos or were not consistently different from the control group. The results suggest that specific brain regions, that are known to be important in cardiovascular control, are activated by a volume load. These areas are likely to play an important role in the reflex responses initiated by that particular stimulus.

Fos production in retrogradely labelled neurons of the lamina terminalis following intravenous infusion of either hypertonic saline or angiotensin II.

The lamina terminalis consists of neurons which are activated by both osmotic and angiotensinergic stimuli and which project axons to many sites including regions of the hypothalamus responsible for vasopressin production. Combination of retrograde neuronal tracing procedures with the identification of Fos protein following discrete stimuli shows populations of neurons, projecting to the supraoptic nuclei, which are preferentially activated by intravenous infusion of either hypertonic saline or angiotensin II. Following infusion of hypertonic saline, the greatest percentage of neurons both labelled with cholera toxin-gold and having elevated levels of Fos protein occurred in that part of the lamina terminalis called the organum vasculosum lamina terminalis. Conversely, angiotensin infusion resulted in greatest numbers of Fos and cholera toxin-gold-labelled neurons in the subfornical organ with fewer double-labelled cells represented in the other components of the lamina terminalis, the median preoptic nucleus and the organum vasculosum lamina terminalis. While these data do not support more than a general separation of the functions examined among neurons of the lamina terminalis, they do highlight a discrete group of osmoresponsive neurons in the dorsal cap of the organum vasculosum lamina terminalis. These cells, by virtue of their response to infusions of hypertonic saline and their axonal connections to regions of the hypothalamus responsible for vasopressin production, are likely candidates for cerebral osmoreceptors.

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.

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.

Localization of the main noradrenergic neuron groups in the pons and medulla of the rabbit and the importance of cathodal lesions for prolonged survival.

Methods for stereotaxically localizing the major noradrenergic (NA) cell groups (i.e. A1, A2, A5 and A6 + A7) in the rabbit are described. Using a modified Kopf head holder we used surface landmarks including the obex for making lesions of the A1 and A2 cells in the medulla. Localization of the pontine cell groups was done by mapping intracerebral structures including the facial nerve for A5 and the motor nucleus of the trigeminal nerve for A6 + A7. In the initial experiments we made A1 lesions by passing anodal currents through stainless steel electrodes, which was associated with pulmonary oedema, neurological complications and a high mortality. This syndrome was probably related to toxic effects of ferric ion deposition, and disappeared when cathodal currents were employed. We have now made 106 bilateral cathodal lesions in the different groups, with a 20% intraoperative mortality. But virtually all survivors remained indefinitely in clinically good condition for the 2-4 weeks duration of our experiments. In 65 of these rabbits we achieved greater than 75% of NA cell destruction (average 84%). From the cardiovascular viewpoint 'non-specific' damage by the lesions was relatively small, except after A2 lesions where there was some impairment in the baroreceptor-heart rate reflex, though a considerable amount of residual function remained.

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 that project to the rostral ventrolateral medulla are activated by haemorrhage.

The retrogradely-transported tracer, rhodamine-tagged microspheres, was injected into the pressor region of the rostral ventrolateral medulla (RVLM) to identify 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 a hypotensive haemorrhage. Compared to controls, Fos production was increased by approximately 3-fold in the paraventricular nucleus (P<0.009) and there was a significant increase in the number of retrogradely-labelled cells that expressed Fos. These represented 5% of the retrogradely-labelled cell population. The results suggest that a small subpopulation of PVN neurons projecting to the RVLM are activated by haemorrhage and may be involved in the reflex responses initiated by that stimulus.

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.

Cardiovascular role of A1 catecholaminergic neurons in the rabbit. Effect of chronic lesions on responses to methyldopa, clonidine and 6-OHDA induced transmitter release.

We confirmed the findings of previous investigators that bilateral anodal lesions of the A1 region were associated with hypertension, bradycardia, pulmonary edema and a high mortality. All these sequelae (except the bradycardia) no longer occurred after cathodal lesions and these were therefore used to investigate the role of the catecholaminergic (CA) neurons of the A1 region in circulatory regulation. Conscious rabbits were studied 2-4 weeks after A1 lesions or sham-operation, when resting mean arterial pressure (MAP) and heart rate (HR) were closely similar in both groups. We tested for differences in MAP and HR responses between lesioned and sham-operated groups: to intracisternal (i.c.) alpha-methyldopa (MD) and to clonidine; and to the acute effects of i.c. 6-hydroxydopamine (6-OHDA) which elicits central CA release. Since these tests depend on the integrity of the central CA neurons, response differences between lesioned and sham-operated groups denote participation by the CA neurons of the A1 region in the central circulatory pathways. The bradycardia responses in the above tests were all smaller in lesioned than sham-operated rabbits, but there were no differences in MAP responses. Electrical stimulation of the region under alfathesin anaesthesia produced depressor responses at low frequencies and pressor responses at high frequencies. From the results in conscious rabbits CA neurons of the A1 region mainly influence the pathways regulating HR, rather than blood pressure. The changes in MAP during electrical stimulation are thus probably mediated through non-CA neurons.

Angiotensin II-induced noradrenaline release from anterior hypothalamus in conscious rats: a brain microdialysis study.

Interaction with aminergic transmitter substances has been implicated in the central actions of angiotensin II (ANG II). We used the novel technique of brain microdialysis in conscious rats to investigate whether ANG II influences the release of endogenous catecholamines (CA) from the anterior hypothalamus (AH). Intracerebroventricular (i.c.v.) administration of ANG II (1 ng and 100 ng) increased mean arterial pressure. ANG II at 1 ng had no effect on the release of noradrenaline (NA) from the AH but ANG II at 100 ng significantly increased NA release. Intracerebroventricular pretreatment with the ANG II-receptor antagonist sarilesin (Sar1, Ile8-ANG II; 3 micrograms) prevented the ANG II-induced NA release. The release of the intraneuronal NA and dopamine (DA) metabolites 3,4-dihydroxyphenylethyl glycol (DOPEG) and 3,4-dihydroxyphenylacetic acid (DOPAC) from the AH was not altered by i.c.v. ANG II. Our results provide the first in vivo evidence for NA release from the hypothalamus induced by periventricular ANG II receptor stimulation. They support the hypothesis that hypothalamic noradrenergic mechanisms are involved in the central actions of this peptide.

Cardiovascular role of the major noradrenergic cell groups in the rabbit: analysis based on 6-hydroxydopamine-induced transmitter release.

We examined the role of the noradrenergic (NA) neurons of the A1, A2, A1 + A2, A5 and A6 + A7 regions on mean arterial pressure (MAP) and heart rate (HR), by comparing the acute responses of chronically lesioned and sham-operated rabbits to intracisternal 6-hydroxydopamine (6-OHDA, 600 micrograms/kg) which induces central release of transmitter. We studied rabbits (1) with intact arterial baroreceptors (non-denervated) and (2) after sino-aortic denervation (SAD). The acute transmitter release response consisted of an early fall in MAP (observed in SAD rabbits) and a late rise in MAP (observed in both non-denervated and SAD rabbits). Medullary lesions had no effect on either MAP component, but A5 and A6 + A7 lesions attenuated both pressor and depressor responses. Normally the transmitter release-induced MAP responses are modified by baroreceptor feedback. The 6-OHDA-induced HR changes were vagal in non-denervated rabbits and were sympathetically mediated in SAD rabbits. In non-denervated rabbits, A1, A2 and A1 + A2 lesions affected mainly the early vagal component, whilst A6 + A7 lesions affected the late vagal component. In SAD rabbits the early bradycardia was due to sympathetic inhibition and the late tachycardia due to sympathetic excitation; A1 + A2 lesions and A5 lesions attenuated the sympathetic bradycardia. We conclude that the various components of the MAP and HR responses are mediated through distinctive NA pathways; the deficits of a given lesion could be due to either to loss of NA cell bodies or of NA fibers of passage.

Intravenous angiotensin II induces Fos-immunoreactivity in circumventricular organs of the lamina terminalis.

Conscious rats were infused intravenously with either angiotensin II (30-55 pmol/kg/min), isotonic saline or phenylephrine for 2 h, then killed. Fos was identified by immunohistochemistry in the brains. Fos expression occurred in many neurons of the subfornical organ and organum vasculosum of the lamina terminalis (OVLT) with angiotensin infusion but not with isotonic NaCl or phenylephrine. Fos immunoreactivity was induced in cells in several medullary, hypothalamic and limbic structures with infusions of angiotensin II or phenylephrine at pressor doses. The results suggest that blood-borne angiotensin II at physiological levels causes angiotensin receptive neurons in the subfornical organ and OVLT to express Fos. Activation of baroreceptor pathways may also induce Fos expression at several other sites.

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.

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.