Rostroventrolateral Medulla Research Articles

Orexin 1 receptors are a novel target to modulate panic responses and the panic brain network

BackgroundAlthough the hypothalamic orexin system is known to regulate appetitive behaviors and promote wakefulness and arousal (Sakurai, 2007 [56]), this system may also be important in adaptive and pathological anxiety/stress responses (Suzuki et al., 2005 [4]). In a recent study, we demonstrated that CSF orexin levels were significantly higher in patients experiencing panic attacks compared to non-panicking depressed subjects (Johnson et al., 2010 [9]). Furthermore, genetically silencing orexin synthesis or blocking orexin 1 receptors attenuated lactate-induced panic in an animal model of panic disorder. Therefore, in the present study, we tested if orexin (ORX) modulates panic responses and brain pathways activated by two different panicogenic drugs. MethodsWe conducted a series of pharmacological, behavioral, physiological and immunohistochemical experiments to study the modulation by the orexinergic inputs of anxiety behaviors, autonomic responses, and activation of brain pathways elicited by systemic injections of anxiogenic/panicogenic drugs in rats. ResultsWe show that systemic injections of two different anxiogenic/panicogenic drugs (FG-7142, an inverse agonist at the benzodiazepine site of the GABAA receptor, and caffeine, a nonselective competitive adenosine receptor antagonist) increased c-Fos induction in a specific subset of orexin neurons located in the dorsomedial/perifornical (DMH/PeF) but not the lateral hypothalamus. Pretreating rats with an orexin 1 receptor antagonist attenuated the FG-7142-induced anxiety-like behaviors, increased heart rate, and neuronal activation in key panic pathways, including subregions of the central nucleus of the amygdala, bed nucleus of the stria terminalis, periaqueductal gray and in the rostroventrolateral medulla. ConclusionOverall, the data here suggest that the ORX neurons in the DMH/PeF region are critical to eliciting coordinated panic responses and that ORX1 receptor antagonists constitute a potential novel treatment strategy for panic and related anxiety disorders. The neural pathways through which ORX1 receptor antagonists attenuate panic responses involve the extended amygdala, periaqueductal gray, and medullary autonomic centers.

Activation of brainstem neurons by underwater diving in the rat

The mammalian diving response is a powerful autonomic adjustment to underwater submersion greatly affecting heart rate, arterial blood pressure, and ventilation. The bradycardia is mediated by the parasympathetic nervous system, arterial blood pressure is mediated via the sympathetic system and still other circuits mediate the respiratory changes. In the present study we investigate the cardiorespiratory responses and the brainstem neurons activated by voluntary diving of trained rats, and, compare them to control and swimming animals which did not dive. We show that the bradycardia and increase in arterial blood pressure induced by diving were significantly different than that induced by swimming. Neuronal activation was calculated after immunohistochemical processing of brainstem sections for Fos protein. Labeled neurons were counted in the caudal pressor area, the medullary dorsal horn, subnuclei of the nucleus tractus solitarii (NTS), the nucleus raphe pallidus (RPa), the rostroventrolateral medulla, the A5 area, the nucleus locus coeruleus, the Kölliker–Fuse area, and the external lateral and superior lateral subnuclei of the parabrachial nucleus. All these areas showed significant increases in Fos labeling when data from voluntary diving rats were compared to control rats and all but the commissural subnucleus of the NTS, A5 area, and RPa were significantly different from swimming rats. These data provide a substrate for more precise experiments to determine the role of these nuclei in the reflex circuits driving the diving response.

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

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

Inhibition of the caudal pressor area reduces cardiorespiratory chemoreflex responses

The caudal pressor area (CPA) is a brainstem area located close to the spinal cord. The activation of the CPA increases sympathetic activity and mean arterial pressure (MAP) by mechanisms dependent on the commissural nucleus of the solitary tract (commNTS) and rostroventrolateral medulla, however, the signals that activate the CPA to produce these responses are still unknown. Therefore, in the present study, we investigated the activity of glutamatergic and GABAergic mechanisms from the CPA and commNTS in rats exposed to hypoxia and the effects of the inhibition of CPA neurons on cardiorespiratory responses to peripheral chemoreceptor activation with i.v. sodium cyanide (NaCN). Male Sprague–Dawley rats (250–280 g, n=5–8/group) were used. In conscious rats, most of the commNTS neurons (66±11%) and part of the CPA neurons (36±7%) activated by hypoxia (8% O2) were glutamatergic (contained VGLUT2mRNA). Small part of the neurons activated during hypoxia was GABAergic (contained GAD-67mRNA) in the commNTS (9±4%) or the CPA (6±2%). In urethane anesthetized rats, the inhibition of CPA neurons with bilateral injections of muscimol (GABA-A agonist, 2 mM) reduced baseline MAP, splanchnic sympathetic nerve discharge (SND) and phrenic nerve discharge (PND). Muscimol into the CPA also reduced by around 50% the pressor and sympathoexcitatory responses and the increase in PND to peripheral chemoreceptor activation with NaCN (50 μg/kg i.v.), without changing sympathetic baroreflex responses. These data suggest that CPA mechanisms facilitate cardiorespiratory responses to peripheral chemoreflex activation. Immunohistochemistry results also suggest that at least part of the CPA mechanisms activated by hypoxia is glutamatergic.

Effect of intracerebroventricular benzamil on cardiovascular and central autonomic responses to DOCA-salt treatment

DOCA-salt treatment increases mean arterial pressure (MAP), while central infusion of benzamil attenuates this effect. The present study used c-Fos immunoreactivity to assess the role of benzamil-sensitive proteins in the brain on neural activity following chronic DOCA-salt treatment. Uninephrectomized rats were instrumented with telemetry transmitters for measurement of MAP and with an intracerebroventricular (ICV) cannula for benzamil administration. Groups included rats receiving DOCA-salt treatment alone, rats receiving DOCA-salt treatment with ICV benzamil, and appropriate controls. At study completion, MAP in vehicle-treated DOCA-salt rats reached 142 ± 4 mmHg. In contrast DOCA-salt rats receiving ICV benzamil had lower MAP (124 ± 3 mmHg). MAP in normotensive controls was 102 ± 3 mmHg. c-Fos immunoreactivity was quantified in the supraoptic nucleus (SON) and across subnuclei of the hypothalamic paraventricular nucleus (PVN), as well as other cardiovascular regulatory sites. Compared with vehicle-treated normotensive controls, c-Fos expression was increased in the SON and all subnuclei of the PVN, but not in other key autonomic nuclei, such as the rostroventrolateral medulla. Moreover, benzamil treatment decreased c-Fos immunoreactivity in the SON and in medial parvocellular and posterior magnocellular neurons of the PVN in DOCA-salt rats but not areas associated with regulation of sympathetic activity. Our results do not support the hypothesis that DOCA-salt increases neuronal activity (as indicated by c-Fos immunoreactivity) of other key regions that regulate sympathetic activity. These results suggest that ICV benzamil attenuates DOCA-salt hypertension by modulation of neuroendocrine-related PVN nuclei rather than inhibition of PVN sympathetic premotor neurons in the PVN and rostroventrolateral medulla.

Rostroventrolateral medullary neurons modulate glucose homeostasis in the rat

Several lines of evidence support the view that the premotor sympathetic input to the adrenal gland arises from the rostroventrolateral medulla (RVLM). The aim of this study was to determine whether RVLM neurons play a role in glucose homeostasis. We identified RVLM neurons that control epinephrine secretion by searching for medullospinal neurons that responded to neuroglucoprivation induced by systemic 2-deoxyglucose (2-DG) administration. We tested the effect of disinhibition of the RVLM on arterial blood pressure and plasma glucose concentration. RVLM medullospinal barosensitive neurons (n = 17) were either unaffected or slightly inhibited by 2-DG. In contrast, we found a group (n = 6) of spinally projecting neurons that were excited by 2-DG administration. These neurons were not barosensitive and had spinal conduction velocities in the unmyelinated range (<1 m/s). These neurons may mediate epinephrine secretion and participate in the counterregulatory responses to neuroglucoprivation. To test the hypothesis that activation of the RVLM leads to adrenomedullary activation and subsequent hyperglycemia, we applied the GABA(A) antagonist bicuculline to the RVLM and measured blood pressure, heart rate, and blood glucose in rats with intact adrenals or after bilateral adrenalectomy. Disinhibition of the RVLM resulted in hypertension, tachycardia, and hyperglycemia (4.9 ± 0.3 to 14.7 ± 0.9 mM, n = 5, P < 0.05). Adrenalectomy significantly reduced the hyperglycemic response but did not alter the cardiovascular responses. These data suggest that the RVLM is a key component of the neurocircuitry that is recruited in the counterregulatory response to hypoglycemia.

Potent hyperglycemic and hyperinsulinemic effects of thyrotropin-releasing hormone microinjected into the rostroventrolateral medulla and abnormal responses in type 2 diabetic rats

We identified ventrolateral medullary nuclei in which thyrotropin-releasing hormone (TRH) regulates glucose metabolism by modulating autonomic activity. Immunolabeling revealed dense prepro-TRH-containing fibers innervating the rostroventrolateral medulla (RVLM) and nucleus ambiguus (Amb), which contain, respectively, pre-sympathetic motor neurons and vagal motor neurons. In anesthetized Wistar rats, microinjection of the stable TRH analog RX77368 (38–150 pmol) into the RVLM dose-dependently and site-specifically induced hyperglycemia and hyperinsulinemia. At 150 pmol, blood glucose reached a peak of 180±18 mg% and insulin increased 4-fold. The strongest hyperglycemic effect was induced when RX77368 was microinjected into C1 area containing adrenalin cells. Spinal cord transection at cervical-7 abolished the hyperglycemia induced by RVLM RX77368, but not the hyperinsulinemic effect. Bilateral vagotomy prevented the rise in insulin, resulting in a prolonged hyperglycemic response. The hyperglycemic and hyperinsulinemic effects of the TRH analog in the RVLM was peptide specific, since angiotensin II or a substance P analog at the same dose had weak or no effects. Microinjection of RX77368 into the Amb stimulated insulin secretion without influencing glucose levels. In conscious type 2 diabetic Goto–Kakizaki (GK) rats, intracisternal injection of RX77368 induced a remarkably amplified hyperglycemic effect with suppressed insulin response compared to Wistar rats. RX77368 microinjected into the RVLM of anesthetized GK rats induced a significantly potentiated hyperglycemic response and an impaired insulin response, compared to Wistar rats. These results indicate that the RVLM is a site at which TRH induces sympathetically-mediated hyperglycemia and vagally-mediated hyperinsulinemia, whereas the Amb is mainly a vagal activating site for TRH. Hyperinsulinemia induced by TRH in the RVLM is not secondary to the hyperglycemic response. The potentiated hyperglycemic and suppressed hyperinsulinemic responses in diabetic GK rats indicate that an unbalanced “sympathetic-over-vagal” activation by TRH in brainstem RVLM contributes to the pathophysiology of impaired glucose homeostasis in type 2 diabetes.

Central CO2chemoreception in cardiorespiratory control

Central CO₂chemoreception in cardiorespiratory control

Serotonin neurons of the caudal raphe nuclei contribute to sympathetic recovery following hypotensive hemorrhage

Serotonin is thought to contribute to the syncopal-like response that develops during severe blood loss by inhibiting presympathetic neurons of the rostroventrolateral medulla (RVLM). Here, we tested whether serotonin cells activated during hypotensive hemorrhage, i.e., express the protein product of the immediate early gene c-Fos, are critical for the normal sympathetic response to blood loss in unanesthetized rats. Serotonin-immunoreactive cells of the raphe obscurus and raphe magnus, parapyramidal cells of the B3 region, subependymal cells of the ventral parapyramidal region, and cells of the ventrolateral periaqueductal gray region were activated by hypotensive hemorrhage, but not by hypotension alone. In contrast to findings in anesthetized animals, lesion of hindbrain serotonergic cells sufficient to produce >80% loss of serotonin nerve terminal immunoreactivity in the RVLM accelerated the sympatholytic response to blood loss, attenuated recovery of sympathetic activity after termination of hemorrhage, and exaggerated metabolic acidosis. Hindbrain serotonin lesion also attenuated ventilatory and sympathetic responses to stimulation of central chemoreceptors but increased spontaneous arterial baroreflex sensitivity and decreased blood pressure variability. A more global neurotoxic lesion that also eliminated tryptophan hydroxylase-immunoreactive cells of the ventrolateral periaqueductal gray region had no further effect on the sympatholytic response to blood loss. Together, the data indicate that serotonin cells of the caudal hindbrain contribute to compensatory responses following blood loss that help maintain oxygenation of peripheral tissue in the unanesthetized rat. This effect may be related to facilitation of chemoreflex responses to acidosis.

Importance of angiotensinergic mechanisms for the pressor response to l-glutamate into the rostral ventrolateral medulla

Pressor responses to l-glutamate into the rostroventrolateral medulla (RVLM) are reduced by lesions of the anteroventral third ventricle (AV3V) region, a main site related to central angiotensinergic pressor mechanisms. Therefore, similar to AV3V lesions, in the present study we investigated if the blockade of central angiotensinergic mechanisms with losartan or ZD 7155 might affect pressor responses to l-glutamate into the RVLM. Male Holtzman rats (280–320 g, n = 4–8/group) with cannulas implanted into the RVLM and lateral ventricle (LV) were used. Injections of l-glutamate (5 nmol/100 nl) or angiotensin II (200 ng/100 nl) into the RVLM increased MAP (54 ± 5 and 26 ± 3 mm Hg, respectively). Losartan (100 µg/1 µl) or ZD 7155 (50 µg/1 µl) injected into the LV reduced the pressor responses to l-glutamate into the RVLM (22 ± 5 and 26 ± 7 mm Hg, respectively), without changing the pressor responses to angiotensin II into the RVLM. Losartan (10 µg/100 nl) or ZD 7155 (5 µg/100 nl) into the RVLM reduced the pressor response to l-glutamate (5 ± 3 and 33 ± 4 mm Hg, respectively) or angiotensin II (5 ± 3 and 6 ± 2 mm Hg, respectively) into the RVLM. Previous injection of angiotensin II (50 ng/100 nl) into the RVLM increased the pressor response to l-glutamate into the RVLM (from 44 ± 5 to 68 ± 7 mm Hg). The results suggest that angiotensinergic mechanisms directly in the RVLM and outside the RVLM (probably forebrain) are important for the pressor responses to l-glutamate into the RVLM.

Induction of c-Fos in ‘panic/defence’-related brain circuits following brief hypercarbic gas exposure

Inspiration of air containing high concentrations of carbon dioxide (CO(2); hypercarbic gas exposure) mobilizes respiratory, sympathetic and hypothalamic-pituitary-adrenal axis responses and increases anxiety-like behaviour in rats and humans. Meanwhile the same stimulus induces panic attacks in the majority of panic disorder patients. However, little is known about the neural circuits that regulate these acute effects. In order to determine the effects of acute hypercarbic gas exposure on forebrain and brainstem circuits, conscious adult male rats were placed in flow cages and exposed to either atmospheric air or increasing environmental CO(2) concentrations (from baseline concentrations up to 20% CO(2)) during a 5 min period. The presence of immunoreactivity for the protein product of the immediate-early gene c-fos was used as a measure of functional cellular responses. Exposing rats to hypercarbic gas increased anxiety-related behaviour and increased numbers of c-Fos-immunoreactive cells in subcortical regions of the brain involved in: (1) the initiation of fear- or anxiety-associated behavioural responses (i.e. the dorsomedial hypothalamus, perifornical nucleus and dorsolateral and ventrolateral periaqueductal gray); (2) mobilization of the hypothalamic-pituitary-adrenal axis (i.e. the dorsomedial hypothalamus, perifornical nucleus and paraventricular hypothalamic nucleus); and (3) initiation of stress-related sympathetic responses (i.e. the dorsomedial hypothalamus, dorsolateral periaqueductal grey and rostroventrolateral medulla). These findings have implications for understanding how the brain senses changes in environmental CO(2) concentrations and the neural mechanisms underlying the subsequent adaptive changes in stress-related physiology and behaviour.

Gastric leptin: a novel role in cardiovascular regulation

Gastric-derived leptin affects satiety and gastrointestinal function via vagal mechanisms and has been shown to interact with the gut hormone cholecystokinin (CCK). CCK selectively inhibits splanchnic sympathetic nerve discharge (SND) and the activity of a subset of presympathetic vasomotor neurons in the rostroventrolateral medulla (RVLM). The present study sought to examine the effects of gastric leptin on arterial pressure (AP), heart rate (HR), SND, and RVLM neuronal activity to determine whether its effects on cardiovascular regulation are dependent on CCK(1) receptors and vagal afferent transmission. To mimic gastric leptin, leptin (15-30 microg/kg) was administered close to the coeliac artery in anesthetized, artificially ventilated Sprague-Dawley rats. Within 5 min, leptin selectively decreased the activity of RVLM neurons also inhibited by CCK (-27 +/- 4%; P < 0.001; n = 15); these inhibitory effects were abolished following administration of the CCK(1) receptor antagonist lorglumide. Leptin significantly decreased AP and HR (-10 +/- 2 mmHg, P < 0.001; and -8 +/- 2 beats/min, P < 0.01; n = 35) compared with saline (-1 +/- 2 mmHg, 3 +/- 2 beats/min; n = 30). In separate experiments, leptin inhibited splanchnic SND compared with saline (-9 +/- 2% vs. 2 +/- 3%, P < 0.01; n = 8). Bilateral cervical vagotomy abolished the sympathoinhibitory, hypotensive, and bradycardic effects of leptin (P < 0.05; n = 6). Our results suggest that gastric leptin may exert acute sympathoinhibitory and cardiovascular effects via vagal transmission and CCK(1) receptor activation and may play a separate role to adipose leptin in short-term cardiovascular regulation.

Increased vasopressin transmission from the hypothalamic paraventricular nucleus (PVN) to the rostroventrolateral medulla (RVLM) augments cardiorespiratory outflow in CIH‐conditioned rats

Conditioning with chronic intermittent hypoxia (CIH) causes hypertension associated with elevated sympathetic nerve activity and increased cardiorespiratory sensitivity to hypoxia. Hypoxia sensed by the carotid bodies drives cardiorespiratory regulating sites, including the PVN via the nucleus tractus solitarius. Our hypothesis is that CIH‐conditioning alters the balance of excitatory and inhibitory influences on CR control via the PVN. Male SD rats were exposed to CIH for 8 h/day for 10 days. Blood pressure (BP), heart rate (HR), diaphragm (DEMG) and genioglossus (GGEMG) activity were recorded in anesthetized, ventilated and vagotomized rats. The PVN was disinhibited by injecting bicuculline (BIC, a GABAA receptor antagonist, 100 pmol) before and after blocking AVP receptors in the RVLM with SR49059 (200 pmol/side, bilateral). In control rats, BIC increased BP, HR, minute DEMG and GGEMG. Following RVLM blockade, these variables were significantly decreased (p &lt; 0.05). Although more pronounced, similar responses were observed in CIH‐conditioned rats; however, it required a higher dose of blocker (400 pmol/side, bilateral) to blunt the effect (p &lt; 0.05). These finding suggests that AVP modulates cardiorespiratory output via the RVLM and CIH‐conditioned rats show augmented AVP transmission, suggesting AVP as a candidate neuropeptide that controls sympathetic tone and respiratory output during CIH.Supported by: HL 87620.

Mechanisms of the ventrolateral medulla on the control of sympathetic activity by central chemoreflex in rats

In the absence of inputs from carotid bodies and cardiopulmonary receptors hypercapnia markedly increases sympathetic nerve discharge (SND) to the heart and blood vessels. In the present study, splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane anaesthetized sino‐aortic denervated and vagotomized rats. Hypercapnia (end‐expiratory CO2 from 5% to 10%) increased sSND (δ= 103 ± 7%). Bilateral injection of muscimol (GABA‐A agonist ‐ 2 mM in 30 nl) into the retrotrapezoid nucleus (RTN) eliminated PND and reduced the effect of CO2 on sSND (δ= 56 ± 8%). Injection of AP‐5 (NMDA glutamate receptor antagonist ‐ 100 mM in 50 nl) or DNQX (non NMDA glutamate receptor antagonist ‐ 100 mM in 50 nl) bilaterally into rostroventrolateral medulla (RVLM) reduced PND, but did not change the effect of CO2 on sSND. Bilateral injection of MCPG (metabotropic glutamate receptor antagonist ‐ 100 mM in 50 nL) into the RVLM reduced PND and the effect of CO2 on sSND (δ= 41 ± 7%). In conclusion, RVLM metabotropic receptors and RTN are involved in the increase in sSND produced by CO2.

Functional Neuroanatomy of the Noradrenergic Locus Coeruleus: Its Roles in the Regulation of Arousal and Autonomic Function Part I: Principles of Functional Organisation

The locus coeruleus (LC) is the major noradrenergic nucleus of the brain, giving rise to fibres innervating extensive areas throughout the neuraxis. Recent advances in neuroscience have resulted in the unravelling of the neuronal circuits controlling a number of physiological functions in which the LC plays a central role. Two such functions are the regulation of arousal and autonomic activity, which are inseparably linked largely via the involvement of the LC. The LC is a major wakefulness-promoting nucleus, resulting from dense excitatory projections to the majority of the cerebral cortex, cholinergic neurones of the basal forebrain, cortically-projecting neurones of the thalamus, serotoninergic neurones of the dorsal raphe and cholinergic neurones of the pedunculopontine and laterodorsal tegmental nucleus, and substantial inhibitory projections to sleep-promoting GABAergic neurones of the basal forebrain and ventrolateral preoptic area. Activation of the LC thus results in the enhancement of alertness through the innervation of these varied nuclei. The importance of the LC in controlling autonomic function results from both direct projections to the spinal cord and projections to autonomic nuclei including the dorsal motor nucleus of the vagus, the nucleus ambiguus, the rostroventrolateral medulla, the Edinger-Westphal nucleus, the caudal raphe, the salivatory nuclei, the paraventricular nucleus, and the amygdala. LC activation produces an increase in sympathetic activity and a decrease in parasympathetic activity via these projections. Alterations in LC activity therefore result in complex patterns of neuronal activity throughout the brain, observed as changes in measures of arousal and autonomic function.

The acute effects of close arterial leptin administration on cardiovascular regulation and RVLM presympathetic vasomotor neurons

The gastric source of leptin may exert its effects directly by paracrine activation of vagal afferents, different to the mode of action for adipose leptin. Evidence is emerging for an interactive relationship between leptin and the gastrointestinal hormone cholecystokinin (CCK). The aim of this study was to examine the acute effects of close arterial injection of leptin (10–30 μg/kg) on arterial pressure (AP), heart rate (HR) and firing rate (FR) of presympathetic vasomotor neurons in the rostroventrolateral medulla (RVLM). We recorded AP, HR and RVLM neuronal discharge in anesthetised, paralysed, male Sprague‐Dawley rats. The effects of baroreflex activation, von Bezold‐Jarisch reflex activation (phenylbiguanide, PBG; 5 μg/kg i.v.) and CCK (1 μg/kg, i.v.) were examined. Close arterial leptin administration produced a significant decrease in HR (369 + 12 bpm to 357 + 14 bpm; n=6; P &lt; 0.05; paired t‐test) and AP (107 + 3 mmHg to 85 + 6 mmHg; n=6; P &lt; 0.05) within 5 minutes of administration. All RVLM neurons studied were inhibited by increases in AP and PBG. The FR of RVLM neurons inhibited by CCK (n=3) was significantly attenuated by leptin administration (from 10.6 + 5 to 8.7 + 5 spikes/s) despite the fall in AP, whereas the FR of those not inhibited by CCK (n=3) was either increased or unaffected. These results suggest that leptin may be exerting its cardiovascular effects via a CCK‐dependent mechanism.

C‐Fos immunoreactivity (CFI) analysis of sympathetic and non‐sympathetic neural pathways in chronic DOCA‐salt hypertension: effect of benzamil hydrochloride

Intracerebroventricular (ICV) infusion of benzamil attenuates DOCA‐salt hypertension. We examined which brain regions may be involved in this response. Uninephrectomized rats were instrumented with an ICV catheter connected to a minipump delivering either benzamil (16 nmol/day) or vehicle. DOCA (50 mg) or vehicle was implanted subcutaneously, creating four groups: benzamil and DOCA (n=7), benzamil but no DOCA (n=7), DOCA but no benzamil (n=6), or neither benzamil nor DOCA (n=6). Rats drank 0.9% NaCl / 0.2% KCl for 33 days. We analyzed CFI in 5 distinct regions of the hypothalamic paraventricular nucleus (PVN), as well as in supraoptic nucleus (SON), rostroventrolateral medulla (RVLM), and the nucleus of the tractus solitarious (NTS). DOCA increased CFI in all regions of the PVN and in the SON but had no effect in the RVLM or NTS. Benzamil attenuated CFI in two PVN subnuclei: posterior magnocellular neurons and medial parvocellular (mp) neurons, as well as in the SON. These findings support the hypothesis that increasing neural activity plays a role in chronic DOCA‐salt hypertension, and that benzamil attenuates this increase. Benzamil seems to specifically target neurons that play a role in neuroendocrine responses, as it attenuated CFI in the magnocellular vasopressinergic neurons of the PVN and SON, as well as the mp (corticotropin‐releasing hormone positive) neurons of the PVN. (HL064178)

Activation of nucleus tractus solitarius 5‐HT2Abut not other 5‐HT2receptor subtypes inhibits the sympathetic activity in rats

Our first aim was to elucidate the mechanisms underlying the hypotensive response elicited by 5-HT(2) receptor activation in the nucleus tractus solitarius (NTS). In pentobarbitone-anaesthetized rats, intra-NTS administration of 2,5-dimethoxy-4-iodoamphetamine (DOI), a wide spectrum 5-HT(2) receptor agonist, but not an antagonist of selective 5-HT(2B) and 5-HT(2C) receptors, produced a decrease in blood pressure and heart rate. The maximal cardiovascular changes obtained by DOI (0.5 pmol) could be almost completely abolished by prior intra-NTS microinjection (10 pmol) of MDL-100907, a selective 5-HT(2A) receptor antagonist, but not by 5-HT(2B) or 5-HT(2C) receptor antagonists. In addition, using extracellular recordings we found that the large majority of identified cardiovascular rostroventrolateral medulla (RVLM) neurons were almost totally inhibited by NTS 5-HT(2A) receptor stimulation. We then investigated whether intra-NTS administration of a subthreshold dose (0.05 pmol) of DOI, known to facilitate the cardiovagal component of the baroreflex, could also modulate the sympathoinhibitory component of this reflex. These experiments showed that neither the decrease in the activity of the cardiovascular RVLM neurons and lumbar sympathetic nerve activities produced by aortic occlusion (gain of the baroreflex), nor the hypotensive response elicited by aortic nerve stimulation, were potentiated by the microinjection of DOI under such conditions. These data show that activation of 5-HT(2A), but not 5-HT(2B) or 5-HT(2C), receptors, located on NTS neurons, elicits depressor and bradycardic responses, and that this 5-HT(2A)-mediated hypotension is produced via the inhibition of RVLM cardiovascular neurons. In addition, NTS 5-HT(2A) receptor activation facilitates the cardiac but not the sympathetic baroreflex response.

Efferent projections of rat rostroventrolateral medulla C1 catecholamine neurons: Implications for the central control of cardiovascular regulation

A replication-defective lentivirus vector that expresses enhanced green fluorescent protein (EGFP) under the control of a synthetic dopamine-beta-hydroxylase (DbetaH) promoter was used to define efferent projections of C1 catecholamine neurons in rat rostral ventrolateral medulla (RVLM). EGFP expression was restricted to C1 neurons and filled their somatodendritic compartments and efferent axons 7-28 days after vector injection. This included the descending projections to thoracic spinal cord and a network in brainstem, midbrain, and diencephalon. In caudal brainstem, restricted terminal fields were present in the dorsal motor vagal complex, A1, raphe pallidus and obscurus, and marginal layer of ventrolateral medulla. Innervation of raphe nuclei was most dense at the level of RVLM, but rostral levels of pallidus were devoid of innervation. A sparse commissural projection to contralateral RVLM was observed, and pericellular arbors were present in the dorsal reticular formation among the projection pathway of catecholamine axons. Rostral brainstem contained a dense innervation of locus coeruleus and the nucleus subcoeruleus. A restricted innervation of the ventrolateral column of the periaqueductal gray distinguished the midbrain. Forebrain labeling was restricted to the diencephalon, where distinctive terminal fields were observed in the paraventricular thalamic nucleus; the lateral hypothalamic area; and the paraventricular, dorsomedial, supraoptic, and median preoptic nuclei of hypothalamus. Projection fibers also coursed through the tuberal hypothalamus into the median eminence. Collectively, these data demonstrate that RVLM C1 neurons modulate the activity of other central cell groups known to participate in the regulation of cardiovascular and autonomic function.

The sympathoinhibitory effects of systemic cholecystokinin are dependent on neurons in the caudal ventrolateral medulla in the rat

The gastrointestinal hormone CCK inhibits a subset of presympathetic neurons in the rostroventrolateral medulla (RVLM) that may be responsible for driving the sympathetic vasomotor outflow to the gastrointestinal circulation. We tested the hypothesis that the central neurocircuitry of this novel sympathoinhibitory reflex involves a relay in the caudal ventrolateral medullary (CVLM) depressor area. Blood pressure and greater splanchnic sympathetic nerve discharge (SSND) or lumbar sympathetic nerve discharge (LSND) were monitored in anesthetised, paralyzed male Sprague-Dawley rats. The effects of phenylephrine (PE, 10 microg/kg iv; baroreflex activation), phenylbiguanide (PBG, 10 microg/kg iv; von Bezold-Jarisch reflex) and CCK (4 or 8 microg/kg iv) on SSND or LSND, were tested before and after bilateral injection of 50-100 nl of the GABAA agonist muscimol (1.75 mM; n=6, SSND; n=7, LSND) or the excitatory amino acid antagonist kynurenate (55 mM; n=7, SSND) into the CVLM. PE and PBG elicited splanchnic and lumbar sympathoinhibitory responses that were abolished by bilateral muscimol or kynurenate injection into the CVLM. Similarly, the inhibitory effect of CCK on SSND was abolished after neuronal inhibition within the CVLM. In contrast, CCK-evoked lumbar sympathoexcitation was accentuated following bilateral CVLM inhibition. In control experiments (n=7), these agents were injected outside the CVLM and had no effect on splanchnic sympathoinhibitory responses to PE, PBG, and CCK. In conclusion, neurons in the CVLM are necessary for the splanchnic but not lumbar sympathetic vasomotor reflex response to CCK. This strengthens the view that subpopulations of RVLM neurons supply sympathetic vasomotor outflow to specific vascular territories.