Daniela M. Sartor

Chemical stimulation of vagal afferent neurons and sympathetic vasomotor tone

Vagal afferents innervate a diverse range of structures of the thoracic and abdominal viscera. While a proportion of these afferents function as mechanoreceptors and respond to changes in intramural tension within the structures that they innervate, many also sense a broad range of chemical substances ranging from peptides, sugars and lipids present in the intraluminal contents of the gastrointestinal tract, as well as tissue prostanoids, cytokines and monoamines in the cardiopulmonary circulation. This review examines the effects of chemical stimulation of vagal afferents on circulatory and sympathetic vasomotor function. Notably, the von Bezold-Jarisch reflex is a cardiorespiratory reflex produced by chemical activation of cardiopulmonary vagal afferents. Classical stimulants of the von Bezold-Jarisch reflex include the Veratrum alkaloids and 5-HT(3) receptor agonists. Atrial natriuretic peptides are agents which also produce a von Bezold-Jarisch reflex-like response or a sensitisation of this reflex via an action on vagal afferents. Cholecystokinin (CCK) activates abdominal visceral vagal afferents, which apart from a clear role in mediation of satiety, also produces selective sympathetic vasomotor inhibition probably by inhibition of sub-groups of presympathetic vasomotor neurons of the rostral ventrolateral medulla. These actions of CCK may constitute a novel gastrointestinal-cardiovascular reflex. The afferent vagus transmits a diverse array of signals to the central nervous system, influencing sympathetic vasomotor and cardiomotor function, gastrointestinal function, neuroimmune function and endocrine function.

Cocaine‐ and amphetamine‐regulated transcript in catecholamine and noncatecholamine presympathetic vasomotor neurons of rat rostral ventrolateral medulla

Presympathetic vasomotor adrenergic (C1) and nonadrenergic (non‐C1) neurons in the rostral ventrolateral medulla (RVLM) provide the main excitatory drive to cardiovascular sympathetic preganglionic neurons in the spinal cord. C1 and non‐C1 neurons contain cocaine‐ and amphetamine‐regulated transcript (CART), suggesting that CART may be a common marker for RVLM presympathetic neurons. To test this hypothesis, we first used double‐immunofluorescence staining for CART and tyrosine hydroxylase (TH) to quantify CART‐immunoreactive (‐IR) catecholamine and noncatecholamine neurons in the C1 region. Next, we quantified the proportion of CART‐IR RVLM neurons that expressed Fos in response to a hypotensive stimulus, using peroxidase immunohistochemistry for Fos and dual immunofluorescence for CART and TH. Finally, we fluorescently detected CART immunoreactivity in electrophysiologically identified, juxtacellularly labeled RVLM presympathetic neurons. In the RVLM, 97% of TH‐IR neurons were CART‐IR, and 74% of CART‐IR neurons were TH‐IR. Nitroprusside infusion significantly increased the number of Fos‐IR RVLM neurons compared with saline controls. In nitroprusside‐treated rats, virtually all Fos/TH neurons in the RVLM were immunoreactive for CART (98% ± 1.3%, SD; n = 7), whereas 29% ± 8.3% of CART‐positive, TH‐negative neurons showed Fos immunoreactivity. Six fast (2.8–5.8 m/second, noncatecholamine)‐, two intermediate (2.1 and 2.2 m/second)‐, and five slow (<1 m/second, catecholamine)‐conducting RVLM presympathetic vasomotor neurons were juxtacellularly labeled. After fluorescent detection of CART and biotinamide, all 13 neurons were found to be CART‐IR. These results suggest that, in rat RVLM, all catecholamine and noncatecholamine presympathetic vasomotor neurons contain CART. J. Comp. Neurol. 476:19–31, 2004.

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.

Midline medullary depressor responses are mediated by inhibition of RVLM sympathoexcitatory neurons in rats

Mechanisms underlying the depressor and sympathoinhibitory responses evoked from the caudal medullary raphe (MR) region were investigated in pentobarbital sodium-anesthetized, paralyzed rats. Intermittent electrical stimulation (0.5 Hz, 0.5-ms pulses, 200 μA) of the MR elicited a mixed sympathetic response that consisted of a long-latency sympathoexcitatory (SE) peak (onset = 146 ± 7 ms) superimposed on an inhibitory phase (onset = 59 ± 10 ms). Chemical stimulation of the MR (glutamate; Glu) most frequently elicited depressor responses accompanied by inhibition of sympathetic nerve discharge. Occasionally, these responses were preceded by transient pressor and SE responses. We examined the influence of intermittent electrical stimulation (0.5 Hz, 0.5-ms pulses, 25-200 μA) and Glu stimulation of the MR on the discharge of rostral ventrolateral medulla (RVLM) premotor SE neurons. Peristimulus-time histograms of RVLM unit discharge featured a prominent inhibitory phase in response to MR stimulation (onset = 20 ± 2 ms; duration = 42 ± 4 ms; n = 12 units). Glu stimulation of the MR reduced blood pressure (-37 ± 2 mmHg, n = 19) and inhibited the discharge of RVLM SE neurons (15 of 19 neurons). Depressor and sympathoinhibitory responses elicited by chemical and electrical stimulation of the MR region are mediated by inhibition of RVLM premotor SE neurons and withdrawal of sympathetic vasomotor discharge.

Sites of action of ghrelin receptor ligands in cardiovascular control.

Circulating ghrelin reduces blood pressure, but the mechanism for this action is unknown. This study investigated whether ghrelin has direct vasodilator effects mediated through the growth hormone secretagogue receptor 1a (GHSR1a) and whether ghrelin reduces sympathetic nerve activity. Mice expressing enhanced green fluorescent protein under control of the promoter for growth hormone secretagogue receptor (GHSR) and RT-PCR were used to locate sites of receptor expression. Effects of ghrelin and the nonpeptide GHSR1a agonist capromorelin on rat arteries and on transmission in sympathetic ganglia were measured in vitro. In addition, rat blood pressure and sympathetic nerve activity responses to ghrelin were determined in vivo. In reporter mice, expression of GHSR was revealed at sites where it has been previously demonstrated (hypothalamic neurons, renal tubules, sympathetic preganglionic neurons) but not in any artery studied, including mesenteric, cerebral, and coronary arteries. In rat, RT-PCR detected GHSR1a mRNA expression in spinal cord and kidney but not in the aorta or in mesenteric arteries. Moreover, the aorta and mesenteric arteries from rats were not dilated by ghrelin or capromorelin at concentrations >100 times their EC(50) determined in cells transfected with human or rat GHSR1a. These agonists did not affect transmission from preganglionic sympathetic neurons that express GHSR1a. Intravenous application of ghrelin lowered blood pressure and decreased splanchnic nerve activity. It is concluded that the blood pressure reduction to ghrelin occurs concomitantly with a decrease in sympathetic nerve activity and is not caused by direct actions on blood vessels or by inhibition of transmission in sympathetic ganglia.

Abdominal vagal signalling: A novel role for cholecystokinin in circulatory control?

It is generally accepted that the gastrointestinal circulation is primarily under the control of the enteric nervous system. However, recent studies have demonstrated that the sympathetic nervous system may play a greater role in postprandial gastrointestinal circulatory function than was thought previously. Cholecystokinin (CCK) is a gastrointestinal hormone released from enteroendocrine cells lining the intestinal mucosa in response to feeding. Systemic administration of CCK induces gastrointestinal vasodilation mediated by withdrawal of sympathetic vasomotor drive. CCK differentially influences the discharge rate of presympathetic vasomotor neurons in the rostral ventrolateral medulla and this response is mirrored by differential responses in the gastrointestinal and skeletal muscle sympathetic vasomotor outflows. CCK1 receptors located on abdominal vagal afferent neurons are activated by CCK which, in turn, activates an intramedullary circuit in a manner analogous to that of other sympathetic cardiovascular reflexes. Evidently, abdominal vagal afferent neurons influence sympathetic vasomotor discharge in a fashion that contrasts markedly with changes in sympathetic vasomotor outflow and regional circulatory function produced by activation of vagal cardiopulmonary reflexes. The clinical implications of this mechanism may extend to the treatment of disorders such as postprandial hypotension and gastrointestinal diseases that are contingent on local blood flow.

SUPRAMEDULLARY MODULATION OF SYMPATHETIC VASOMOTOR FUNCTION

1. Supramedullary structures including the ventral medial prefrontal cortex (MPFC) and the midbrain cuneiform nucleus (CnF) project directly and indirectly to premotor sympatho‐excitatory neurons of the rostral ventrolateral medulla (RVLM) that are critically involved in the generation of sympathetic vasomotor tone.

Phenotypic identification of rat rostroventrolateral medullary presympathetic vasomotor neurons inhibited by exogenous cholecystokinin.

Systemic administration of the gastrointestinal hormone cholecystokinin (CCK) selectively inhibits splanchnic sympathetic vasomotor discharge and differentially affects presympathetic vasomotor neurons of the rostroventrolateral medulla (RVLM). Stimulation of the sympathoexcitatory region of the periaqueductal grey (PAG) produces profound mesenteric vasoconstriction. In this study, our aim was to identify phenotypically different populations of RVLM presympathetic vasomotor neurons using juxtacellular neuronal labelling and immunohistochemical detection of the adrenergic neuronal marker phenylethanolamine‐N‐methyl transferase (PNMT) and to determine whether the PAG provides functional excitatory input to these neurons. Fifty‐eight percent (36/62) of RVLM presympathetic neurons were inhibited by systemic administration of CCK. These cells had conduction velocities (3.6 ± 0.2 m/sec) in the non‐C‐fiber range consistent with neurons possessing lightly myelinated spinal axons. Of these, 79% (22/28) were excited by PAG stimulation, and 59% (10/17) were not immunoreactive for PNMT. Conversely, 42% (26/62) of RVLM presympathetic neurons were either unaffected or activated by CCK administration and had slower conduction velocities (1.4 ± 0.3 m/sec) than cells inhibited by CCK. Fifty percent (11/22) of these cells were driven by PAG stimulation, and most (11/14 or 79%) were PNMT‐positive. These results suggest that cardiovascular responses elicited by PAG stimulation occur via activation of non‐C1 and C1 RVLM presympathetic neurons. RVLM neurons inhibited by CCK were more likely to be driven by PAG stimulation and may be a subset of neurons responsible for driving gastrointestinal sympathetic vasomotor tone. CCK‐induced inhibition of a subpopulation of RVLM presympathetic neurons may be implicated in postprandial hyperemia and postprandial hypotension. J. Comp. Neurol. 465:467–479, 2003.

Chemical stimulation of visceral afferents activates medullary neurones projecting to the central amygdala and periaqueductal grey.

Cholecystokinin (CCK) stimulates gastrointestinal vagal afferent neurones that signal visceral sensations. We wished to determine whether neurones of the nucleus of the solitary tract (NTS) or ventrolateral medulla (VLM) convey visceral afferent information to the central nucleus of the amygdala (CeA) or periaqueductal grey region (PAG), structures that play a key role in adaptive autonomic responses triggered by stress or fear. Male Sprague-Dawley rats received a unilateral microinjection of the tracer cholera toxin subunit B (CTB, 1%) into the CeA or PAG followed, 7 days later, by an injection of CCK (100 microg/kg, i.p.) or saline. Brains were processed for detection of Fos protein (Fos-IR) and CTB. CCK induced increased expression of Fos-IR in the NTS and the VLM, relative to control. When CTB was injected into the CeA, CTB-immunoreactive (CTB-IR) neurones were more numerous in the rostral NTS ipsilateral to the injection site, whereas they were homogeneously distributed throughout the VLM. Double-labelled neurones (Fos-IR+CTB-IR) were most numerous in the ipsilateral NTS and caudal VLM. The NTS contained the higher percentage of CTB-IR neurones activated by CCK. When CTB was injected into the PAG, CTB-IR neurones were more numerous in the ipsilateral NTS whereas they were distributed relatively evenly bilaterally in the rostral VLM. Double-labelled neurones were not differentially distributed along the rostrocaudal axis of the NTS but were more numerous in this structure when compared with the VLM. NTS and VLM neurones may convey visceral afferent information to the CeA and the PAG.

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.