Neurons In Ventrolateral Medulla Research Articles

Cortical-brainstem circuitry attenuates physiological stress reactivity.

Exposure to stressful stimuli promotes multi-system biological responses to restore homeostasis. Catecholaminergic neurons in the rostral ventrolateral medulla (RVLM) facilitate sympathetic activity and promote physiological adaptations, including glycaemic mobilization and corticosterone release. While it is unclear how brain regions involved in the cognitive appraisal of stress regulate RVLM neural activity, recent studies found that the rodent ventromedial prefrontal cortex (vmPFC) mediates stress appraisal and physiological stress responses. Thus, a vmPFC-RVLM connection could represent a circuit mechanism linking stress appraisal and physiological reactivity. The current study investigated a direct vmPFC-RVLM circuit utilizing genetically encoded anterograde and retrograde tract tracers. Together, these studies found that stress-activated vmPFC neurons project to catecholaminergic neurons throughout the ventrolateral medulla in male and female rats. Next, we utilized optogenetic terminal stimulation to evoke vmPFC synaptic glutamate release in the RVLM. Photostimulating the vmPFC-RVLM circuit during restraint stress suppressed glycaemic stress responses in males, without altering the female response. However, circuit stimulation decreased corticosterone responses to stress in both sexes. Circuit stimulation did not modulate affective behaviour in either sex. Further analysis indicated that circuit stimulation preferentially activated non-catecholaminergic medullary neurons in both sexes. Additionally, vmPFC terminals targeted medullary inhibitory neurons. Thus, both male and female rats have a direct vmPFC projection to the RVLM that reduces endocrine stress responses, likely by recruiting local RVLM inhibitory neurons. Ultimately, the excitatory/inhibitory balance of vmPFC synapses in the RVLM may regulate stress reactivity and stress-related health outcomes. KEY POINTS: Glutamatergic efferents from the ventromedial prefrontal cortex target catecholaminergic neurons throughout the ventrolateral medulla. Partially segregated, stress-activated ventromedial prefrontal cortex populations innervate the rostral and caudal ventrolateral medulla. Stimulating ventromedial prefrontal cortex synapses in the rostral ventrolateral medulla decreases stress-induced glucocorticoid release in males and females. Stimulating ventromedial prefrontal cortex terminals in the rostral ventrolateral medulla preferentially activates non-catecholaminergic neurons. Ventromedial prefrontal cortex terminals target medullary inhibitory neurons.

Fasting-activated ventrolateral medulla neurons regulate T cell homing and suppress autoimmune disease in mice.

Dietary fasting markedly influences the distribution and function of immune cells and exerts potent immunosuppressive effects. However, the mechanisms through which fasting regulates immunity remain obscure. Here we report that catecholaminergic (CA) neurons in the ventrolateral medulla (VLM) are activated during fasting in mice, and we demonstrate that the activity of these CA neurons impacts the distribution of T cells and the development of autoimmune disease in an experimental autoimmune encephalomyelitis (EAE) model. Ablation of VLM CA neurons largely reversed fasting-mediated T cell redistribution. Activation of these neurons drove T cell homing to bone marrow in a CXCR4/CXCL12 axis-dependent manner, which may be mediated by a neural circuit that stimulates corticosterone secretion. Similar to fasting, the continuous activation of VLM CA neurons suppressed T cell activation, proliferation, differentiation and cytokine production in autoimmune mouse models and substantially alleviated disease symptoms. Collectively, our study demonstrates neuronal control of inflammation and T cell distribution, suggesting a neural mechanism underlying fasting-mediated immune regulation.

Exercise-induced neuroplasticity in autonomic nuclei restores the cardiac vagal tone and baroreflex dysfunction in aged hypertensive rats.

Aging is accompanied by considerable deterioration of homeostatic systems, such as autonomic imbalance characterized by heightened sympathetic activity, lower parasympathetic tone, and depressed heart rate (HR) variability, which are aggravated by hypertension. Here, we hypothesized that these age-related deficits in aged hypertensive rats can be ameliorated by exercise training, with benefits to the cardiovascular system. Therefore, male 22-mo-old spontaneously hypertensive rats (SHRs) and age-matched Wistar Kyoto (WKY) submitted to moderate-intensity exercise training (T) or kept sedentary (S) for 8 wk were evaluated for hemodynamic/autonomic parameters, baroreflex sensitivity, cardiac sympathetic/parasympathetic tone and analysis of dopamine β-hydroxylase (DBH+) and oxytocin (OT+) pathways of autonomic brain nuclei. Aged SHR-S versus WKY-S exhibited elevated mean arterial pressure (MAP: +51%) and HR (+20%), augmented pressure/HR variability, no cardiac vagal tone, and depressed reflex control of the heart (HR range, -28%; gain, -49%). SHR-T exhibited a lower resting HR, a partial reduction in the MAP (-14%), in the pressure/HR variabilities, and restored parasympathetic modulation, with improvement of baroreceptor reflex control when compared with SHR-S. Exercise training increased the ascending DBH+ projections conveying peripheral information to the paraventricular nucleus of hypothalamus (PVN), augmented the expression of OT+ neurons, and reduced the density of DBH+ neurons in the rostral ventrolateral medulla (RVLM) of SHR-T. Data indicate that exercise training induces beneficial neuroplasticity in brain autonomic circuitry, and it is highly effective to restore the parasympathetic tone, and attenuation of age-related autonomic imbalance and baroreflex dysfunction, thus conferring long-term benefits for cardiovascular control in aged hypertensive individuals.NEW & NOTEWORTHY Exercise training reduces high blood pressure and cardiovascular autonomic modulation in aged hypertensive rats. The dysfunction in the baroreflex sensitivity and impaired parasympathetic tone to the heart of aged hypertensive rats are restored by exercise training. Exercise induces beneficial neuroplasticity in the brain nuclei involved with autonomic control of cardiovascular function of aged hypertensive rats.

SGLT2 and SGLT1 inhibitors suppress the activities of the RVLM neurons in newborn Wistar rats.

Hypertension is well-known to often coexist with diabetes mellitus (DM) in humans. Treatment with sodium-glucose cotransporter 2 (SGLT2) inhibitors has been shown to decrease both the blood glucose and the blood pressure (BP) in such patients. Some reports show that SGLT2 inhibitors improve the BP by decreasing the activities of the sympathetic nervous system. Therefore, we hypothesized that SGLT2 inhibitors might alleviate hypertension via attenuating sympathetic nervous activity. Combined SGLT2/SGLT1 inhibitor therapy is also reported as being rather effective for decreasing the BP. In this study, we examined the effects of SGLT2 and SGLT1 inhibitors on the bulbospinal neurons of the rostral ventrolateral medulla (RVLM). To investigate whether bulbospinal RVLM neurons are sensitive to SGLT2 and SGLT1 inhibitors, we examined the changes in the neuronal membrane potentials (MPs) of these neurons using the whole-cell patch-clamp technique during superfusion of the cells with the SGLT2 and SGLT1 inhibitors. A brainstem-spinal cord preparation was used for the experiments. Our results showed that superfusion of the RVLM neurons with SGLT2 and SGLT1 inhibitor solutions induced hyperpolarization of the neurons. Histological examination revealed the presence of SGLT2s and SGLT1s in the RVLM neurons, and also colocalization of SGLT2s with SGLT1s. These results suggest the involvement of SGLT2s and SGLT1s in regulating the activities of the RVLM neurons, so that SGLT2 and SGLT1 inhibitors may inactivate the RVLM neurons hyperpolarized by empagliflozin. SGLT2 and SGLT1 inhibitors suppressed the activities of the bulbospinal RVLM neurons in the brainstem-spinal preparations, suggesting the possibilities of lowering BP by decreasing the sympathetic nerve activities. RVLM, rostral ventrolateral medulla. IML, intralateral cell column. aCSF, artificial cerebrospinal fluid.

Hypothalamic Corticotropin-Releasing Hormone Contributes to Hypertension in Spontaneously Hypertensive Rats.

Corticotropin-releasing hormone (CRH) is a neuropeptide regulating neuroendocrine and autonomic function. CRH mRNA and protein levels in the hypothalamic paraventricular nucleus (PVN) are increased in primary hypertension. However, the role of CRH in elevated sympathetic outflow in primary hypertension remains unclear. CRHR1 proteins were distributed in retrogradely labeled PVN presympathetic neurons with an increased level in the PVN tissue in adult spontaneously hypertensive rats (SHRs) compared with age-matched male Wistar-Kyoto (WKY) rats. CRH induced a more significant increase in the firing rate of PVN-rostral ventrolateral medulla (RVLM) neurons and sympathoexcitatory response in SHRs than in WKY rats, an effect that was blocked by preapplication of NMDA receptors (NMDARs) antagonist AP5 and PSD-95 inhibitor, Tat-N-dimer. Blocking CRHRs with astressin or CRHR1 with NBI35965 significantly decreased the firing rate of PVN-RVLM output neurons and reduced arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) in SHRs but not in WKY, whereas blocking CRHR2 with antisauvagine-30 did not. Furthermore, Immunocytochemistry staining revealed that CRHR1 colocalized with NMDARs in PVN presympathetic neurons. Blocking CRHRs significantly decreased the NMDA currents in labeled PVN neurons. PSD-95-bound CRHR1 and PSD-95-bound GluN2A in the PVN were increased in SHRs. These data suggested that the upregulation of CRHR1 in the PVN is critically involved in the hyperactivity of PVN presympathetic neurons and elevated sympathetic outflow in primary hypertension.SIGNIFICANCE STATEMENT Our study found that corticotropin-releasing hormone receptor (CRHR)1 protein levels were increased in the paraventricular nucleus (PVN), and CRHR1 interacts with NMDA receptors (NMDARs) through postsynaptic density protein (PSD)-95 in the PVN neurons in primary hypertension. The increased CRHR1 and CRHR1-NMDAR-PSD-95 complex in the PVN contribute to the hyperactivity of the PVN presympathetic neurons and elevated sympathetic vasomotor tone in hypertension in SHRs. Thus, the antagonism of CRHR1 decreases sympathetic outflow and blood pressure in hypertension. These findings determine a novel role of CRHR1 in elevated sympathetic vasomotor tone in hypertension, which is useful for developing novel therapeutics targeting CRHR1 to treat elevated sympathetic outflow in primary hypertension. The CRHR1 receptor antagonists, which are used to treat health consequences resulting from chronic stress, are candidates to treat primary hypertension.

Skeletal muscle-derived extracellular vesicles contribute to post-exercise hypotension

Post-exercise hypotension (PEH) is an immediate reduction in blood pressure (BP) after a single bout of exercise that forms the long-term hypertension amelioration by chronic exercise. The present study aims to explore a novel mechanism of PEH, with a focus of skeletal muscle-derived extracellular vesicles (SkM-EVs). We hypothesize that repetitively contracting-relaxing muscle releases antioxidant-enriched EVs that induce hypotension. Experiments were carried out in 48 male Sprague-Dawley rats. BP was recorded by a Millar catheter inserted into right carotid artery. EVs were isolated from SkM and plasma using ultracentrifugation, and then qualified and quantified using transmission electron microscopy and the ZetaView nanoparticle tracking analyzer. Results: First, in rats undergoing 1-hour of treadmill exercise (15/30/15 m/min for 10/40/10 min), we found an elevated BP during exercise (34.7 ± 5.2 mmHg, p < 0.001; n = 8), followed by a decrease in BP below baseline during the post exercise period (-7.3 ± 1.1 mmHg, p < 0.01). Pretreatment with the EV inhibitor GW4869 (1.5 mg/kg) had no effects on hypertension during exercise, but completely abolished the post-exercise hypotension (1.93 ± 0.64 mmHg, p < 0.001), suggesting a role for SkM-EVs in this response. Bolus i.v. injection of SkM-EVs from exercised rats (400 μg EV protein) induced a dramatic decrease in BP (-16.7 ± 4.4 mmHg; p < 0.01, n = 6/group) in non-exercised rats, which lasted for up to 40 min. Second, 1-hour sciatic nerve electrical stimulation (2.5V, 50Hz, 0.3s/3s)-evoked hindlimb muscle contraction in isoflurane-anesthetized rats induced a biphasic change in BP, with hypertension during the initial 20 min followed by hypotension in the last 40 min. Transection of the sciatic nerve or pretreatment with GW4869 abolished the stimulation-evoked hypertension and hypotension. Bolus i.v. injection of EVs from contracting-muscle significantly reduced BP with a maximal change of -14 ± 3.2 mmHg (p = 0.003; n = 5). Third, mass spectroscopic and western blotting analyses of these EVs identified 827 proteins, including Catalase, GSTMs, PRDXs, EPHX1, HSP90, and many others, which are involved in redox homeostasis and mitochondrial bioenergetics. Finally, using muscle-specific GFP reporter mice (MS-mG), we found increased SkM-EVs in both muscle and plasma following exercise by using flow cytometry. Confocal imaging identified GFP+-particles in the neurons of the rostral ventrolateral medulla (RVLM) of C57BL/6 mice that received an injection of SkM-EVs isolated from the exercised MS-mG mice, suggesting their active uptake across the blood-brain barrier. In summary, the major findings of this study include (1) SkM-EVs are essential for PEH; (2) i.v. administration of SkM-EVs reduced BP; (3) SkM-EVs contain enriched antioxidant enzymes and other cytoprotective proteins. These results suggest a novel mechanism underpinning PEH and provides a potential therapeutic strategy for hypertension. Supported by NIH grant R01HL160820. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neurons in the caudal ventrolateral medulla mediate descending pain control.

Supraspinal brain regions modify nociceptive signals in response to various stressors including stimuli that elevate pain thresholds. The medulla oblongata has previously been implicated in this type of pain control, but the neurons and molecular circuits involved have remained elusive. Here we identify catecholaminergic neurons in the caudal ventrolateral medulla that are activated by noxious stimuli in mice. Upon activation, these neurons produce bilateral feed-forward inhibition that attenuates nociceptive responses through a pathway involving the locus coeruleus and norepinephrine in the spinal cord. This pathway is sufficient to attenuate injury-induced heat allodynia and is required for counter-stimulus induced analgesia to noxious heat. Our findings define a component of the pain modulatory system that regulates nociceptive responses.

The interleukin-enhanced binding factor 3-mediated inhibition of nitric oxide production via phosphoinositide 3-kinase/protein kinase B pathway contributes to central cardiovascular regulation in the rostral ventrolateral medulla in hypertension.

Hypertension is characterized by sympathetic hyperactivity, which is related to the overexcitation of the presympathetic neurons in the rostral ventrolateral medulla (RVLM). Nitric oxide (NO) has been reported to be a vital neuromodulator involved in central cardiovascular regulation. However, the mechanism of interleukin-enhanced binding factor 3 (ILF3) participating in blood pressure (BP) regulation is still unclear. Therefore, this study aims to clarify the role of ILF3 within the rostral ventrolateral medulla (RVLM) in regulating NO in hypertension. It was found that the expression level of ILF3 was significantly increased in the RVLM of spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto (WKY) rats through microarray gene expression analysis, Western blot, and immunofluorescence. Overexpression of ILF3 by injecting constructed adenovirus into the RVLM increased the BP and renal sympathetic nerve activity (RSNA) of the WKY rats, significantly decreasing NO production and neuronal nitric oxide synthase (nNOS) expression. Knockdown of ILF3 in the RVLM of SHR significantly reduced BP but increased NO production and the neuronal nitric oxide synthase (nNOS) expression. Furthermore, it was found that the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway was activated via Western blotting in the RVLM after overexpression of ILF3, whereas it was attenuated after knockdown of ILF3 in SHR. In addition, inhibition of PI3K by intracisternal infusion of the PI-103 attenuated the increase in Akt phosphorylation and decrease in nNOS expression and NO production caused by overexpressing ILF3, which ultimately blunted high BP induced by overexpressing ILF3. Taken together, this current study suggests that ILF3 participates in high BP via reducing NO production in the RVLM through PI3K/Akt pathway.

In this Issue

HomeCirculation ResearchVol. 131, No. 8In this Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn this Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published29 Sep 2022https://doi.org/10.1161/RES.0000000000000573Circulation Research. 2022;131:651is related toExtracellular Vesicles Regulate Sympatho-Excitation by Nrf2 in Heart FailureImpaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2is related toTranscriptional and Immune Landscape of Cardiac SarcoidosisCardiac Sarcoidosis Immune Landscape (p 654)Download figureDownload PowerPointLiu et al examine the transcriptional and cellular composition of cardiac sarcoidosis.Sarcoidosis is a rare inflammatory disease in which granulomas—small, localized sites of inflammation—afflict various parts of the body, most commonly the lungs, skin and lymph nodes. While sarcoidosis can be managed, there is no cure for the condition and, if it manifests in the heart, it can be deadly. Because little is known about the specific pathology of cardiac sarcoidosis (CS), and because it can resemble other forms of heart inflammation, Liu and colleagues set out to map the specific transcriptional and cellular landscape of CS lesions. Using a combination of spatial transcriptomics, single-nucleus RNA sequencing and immunofluorescence, the team compared tissue samples from CS patients with those from patients with other types of myocarditis. CS granulomas had a particular cellular arrangement, the team found, with HLA-DR+ macrophages immediately adjacent to and surrounding multinucleated giant cells. The giant cells expressed transmembrane glycoprotein NMB—a novel marker of these cells—while activation of mTOR genes was a feature of the surrounding macrophages. These analyses were carried out on a small number of patient samples but, if further studies validate the findings, they could form the basis of future diagnostic tests for CS, say the team.Impaired Dynamic SR Ca Buffering Causes AD-CPVT2 (p 673)Download figureDownload PowerPointWleklinski et al reveal the molecular effects of a dominant CPVT-causing mutation.Exercise or emotional stress can prompt the release of catecholamine hormones, which induce a fast heart rate, increased blood pressure and other features of the fight-or-flight response. For people with catecholaminergic polymorphic ventricular tachycardia (CPVT), however, physical activity or stress can cause potentially lethal arrythmias. Mutation of calsequestrin-2 (CASQ2)—a sarcoplasmic reticulum (SR) calcium binding protein—is a major cause of CPVT and can be recessive or dominant in nature. For many recessive mutations, disease occurs due to loss of CASQ2 protein. Wleklenski and colleagues have investigated a dominant lysine-to-arginine mutation (K180R) and find, by contrast, protein levels remain normal. In mice carrying the mutation, not only was the level of CASQ2 comparable to that in control animals, but so too was the protein’s subcellular localization. The mutation instead interfered with CASQ2’s calcium binding, or buffering, capability within the SR. The result was that, upon catecholamine injection or exercise, the unbound calcium released prematurely from the SR triggering spontaneous cell contractions. In uncovering this novel molecular etiology of CPVT, the work provides a basis for studying the consequences of other dominant CASQ2 mutations, say the team.EV-Mediated Heart Brain Communication in CHF (p 687)Download figureDownload PowerPointTian et al discover signals from the failing heart to the brain that may further exacerbate cardiac dysfunction.After a myocardial infarction, increased oxidative stress in the heart can contribute to adverse cardiac remodeling and ultimately heart failure. Nrf2 is a master activator of antioxidant genes, suggesting a protective role, but studies in rats have shown its expression to be suppressed after MI—likely due to upregulation of Nrf2-targeting microRNAs. These miRNAs can also be packaged into vesicles and released from stressed heart cells, and now Tian and colleagues show that rats and humans with chronic heart failure (CHF) have an abundance of these miRNA-containing EVs in their blood. In the rats with CHF, these EVs were found to be taken up by neurons of the rostral ventrolateral medulla (RVLM) wherein the miRNAs suppressed Nrf2 expression. The RVLM is a brain region that controls the sympathetic nervous system and, in the presence of the EVs, it ramped up sympathetic excitation. Because such elevated sympathetic activity can induce the fight-or-flight response—including increased heart rate and blood pressure—this would likely worsen heart failure progression. The team found, however, that inhibiting the miRNAs in the EVs prevented Nrf2 suppression in the RVLM and sympathetic activation, suggesting the pathway could be targeted therapeutically. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesExtracellular Vesicles Regulate Sympatho-Excitation by Nrf2 in Heart FailureChanghai Tian, et al. Circulation Research. 2022;131:687-700Impaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2Matthew J. Wleklinski, et al. Circulation Research. 2022;131:673-686Transcriptional and Immune Landscape of Cardiac SarcoidosisJing Liu, et al. Circulation Research. 2022;131:654-669 September 30, 2022Vol 131, Issue 8 Advertisement Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000573 Originally publishedSeptember 29, 2022 PDF download Advertisement

Cardiovascular baroreflex circuit moonlights in sleep control

Sleep disturbances are strongly associated with cardiovascular diseases. Baroreflex, a basic cardiovascular regulation mechanism, is modulated by sleep-wake states. Here, we show that neurons at key stages of baroreflex pathways also promote sleep. Using activity-dependent genetic labeling, we tagged neurons in the nucleus of the solitary tract (NST) activated by blood pressure elevation and confirmed their barosensitivity with optrode recording and calcium imaging. Chemogenetic or optogenetic activation of these neurons promoted non-REM sleep in addition to decreasing blood pressure and heart rate. GABAergic neurons in the caudal ventrolateral medulla (CVLM)-a downstream target of the NST for vasomotor baroreflex-also promote non-REM sleep, partly by inhibiting the sympathoexcitatory and wake-promoting adrenergic neurons in the rostral ventrolateral medulla (RVLM). Cholinergic neurons in the nucleus ambiguous-a target of the NST for cardiac baroreflex-promoted non-REM sleep as well. Thus, key components of the cardiovascular baroreflex circuit are also integral to sleep-wake brain-state regulation.

Extracellular Vesicles Regulate Sympatho-Excitation by Nrf2 in Heart Failure.

Chronic heart failure (CHF) is associated with redox imbalance. Downregulation of Nrf2 (nuclear factor [erythroid-derived 2]-like 2) plays important roles in disrupting myocardial redox homeostasis and mediating sympathetic nerve activity in the setting of CHF. However, it is unclear if circulating extracellular vesicles (EVs) elicit sympathetic excitation in CHF by disrupting central redox homeostasis. We tested the hypothesis that cardiac-derived EVs circulate to the presympathetic rostral ventrolateral medulla and contribute to oxidative stress and sympathetic excitation via EV-enriched microRNA-mediated Nrf2 downregulation. Data were collected on rats with CHF post-myocardial infarction (MI) and on human subjects with ischemic CHF. EVs were isolated from tissue and plasma, and we determined the miRNAs cargo that related to targeting Nrf2 translation. We tracked the distribution of cardiac-derived EVs using in vitro labeled circulating EVs and cardiac-specific membrane GFP+ transgenic mice. Finally, we tested the impact of exogenously loading of antagomirs to specific Nrf2-related miRNAs on CHF-EV-induced pathophysiological phenotypes in normal rats (eg, sympathetic and cardiac function). Nrf2 downregulation in CHF rats was associated with an upregulation of Nrf2-targeting miRNAs, which were abundant in cardiac-derived and circulating EVs from rats and humans. EVs isolated from the brain of CHF rats were also enriched with Nrf2-targeting miRNAs and cardiac-specific miRNAs. Cardiac-derived EVs were taken up by neurons in the rostral ventrolateral medulla. The administration of cardiac-derived and circulating EVs from CHF rats into the rostral ventrolateral medulla of normal rats evoked an increase in renal sympathetic nerve activity and plasma norepinephrine compared with Sham-operated rats, which were attenuated by exogenously preloading CHF-EVs with antagomirs to Nrf2-targeting miRNAs. Cardiac microRNA-enriched EVs from animals with CHF can mediate crosstalk between the heart and the brain in the regulation of sympathetic outflow by targeting the Nrf2/antioxidant signaling pathway. This new endocrine signaling pathway regulating sympathetic outflow in CHF may be exploited for novel therapeutics.

Effects of ANP and BNP on the generation of respiratory rhythms in brainstem-spinal cord preparation isolated from newborn rats.

Natriuretic peptides (NPs) are a family of peptide hormones produced in cardiac muscle cells and consist mainly of three types: atrial NP (ANP), B-type (or brain) NP (BNP), and C-type NP. We herein report the effects of ANP and BNP on central respiratory activity in brainstem-spinal cord preparation isolated from newborn rats. Bath application of these peptides (100 nM) induced a weak transient depression of the respiratory rhythm followed by recovery. Respiratory-related neurons in the rostral ventrolateral medulla showed a tendency for transient hyperpolarization followed by recovery during the application of ANP or BNP. The application of a membrane-permeable cGMP, 8-Br-cGMP (10 or 20 μM), did not induce significant effects on respiratory rhythm, suggesting no involvement of guanylyl cyclase in effects of ANP or BNP. We also examined effects of BNP on respiratory depression induced by the sedative dexmedetomidine, which exerts an inhibitory influence on respiratory rhythm. When pretreated with 50 nM BNP, the inhibitory effect of 100 nM dexmedetomidine was significantly reduced. Our findings suggest that ANP and BNP act as mild excitatory agents with sustained effects on respiratory rhythm after an initial transient depression.

Control of non-REM sleep by ventrolateral medulla glutamatergic neurons projecting to the preoptic area

Understanding the neural mechanisms underlying sleep state transitions is a fundamental goal of neurobiology and important for the development of new treatments for insomnia and other sleep disorders. Yet, brain circuits controlling this process remain poorly understood. Here we identify a population of sleep-active glutamatergic neurons in the ventrolateral medulla (VLM) that project to the preoptic area (POA), a prominent sleep-promoting region, in mice. Microendoscopic calcium imaging demonstrate that these VLM glutamatergic neurons display increased activity during the transitions from wakefulness to Non-Rapid Eye Movement (NREM) sleep. Chemogenetic silencing of POA-projecting VLM neurons suppresses NREM sleep, whereas chemogenetic activation of these neurons promotes NREM sleep. Moreover, we show that optogenetic activation of VLM glutamatergic neurons or their projections in the POA initiates NREM sleep in awake mice. Together, our findings uncover an excitatory brainstem-hypothalamic circuit that controls the wake-sleep transitions.

Hypoglycemia: Control of counter‐regulatory hormone secretion by the brain

Hypoglycaemia is a common side‐effect of the treatment of type 1 diabetes with insulin. The Diabetes Control and Complications Trial indicated that, while intensive insulin therapy reduced the incidence and severity of diabetic complications such as retinopathy, neuropathy and nephropathy, the incidence of severe hypoglycaemia was increased 2‐3 fold. In non‐diabetic individuals, hypoglycaemia triggers the glucose counter‐regulatory response. This response consists of inhibition of insulin secretion and activation of glucagon secretion. Prolonged hypoglycaemia also evokes adrenaline secretion and less importantly, growth hormone and cortisol secretion. However, in individuals that have type 1 diabetes, the glucagon response to hypoglycaemia is lost. In these patients, the pancreatic α‐cells continue to synthesise glucagon since glucagon secretagogues such as arginine produce a robust glucagon secretory response. Our research has examined the hypothalamic and medullary neurocircuits that mediate adrenaline and glucagon secretion under conditions of neuroglucoprivation or hypoglycaemia. These studies have shown that the response to neuroglucoprivation involves specialised sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM) that mediate adrenaline secretion. These neurons are readily distinguishable from sympathetic vasomotorneurons also located in the RVLM. In addition, we have described the properties of perifornical hypothalamic (PeH) orexinergic (Ox) neurons that are activated by insulin‐induced hypoglycaemia (IHH) or neuroglucoprivation. PeH neurons project to many different targets including the RVLM and the dorsal motor nucleus of the vagus (DMV). Ox2‐R in the RVLM mediate the adrenaline secretory response. PeH neurons also mediate the glucagon response to IHH since the release of glucagon in response to IHH is diminished in Ataxin3 rats which have an age‐related loss of PeH orexin neurons.

The activation of C1 neurons during REM sleep is dependent on the arterial baroreflex

IntroductionNatural sleep‐wake patterns are associated with changes in sympathetic nerve activity (SNA) controlling cardiovascular function. The mechanisms underlying the effects of sleep‐wake state on SNA are not well understood. C1 neurons in the rostral ventrolateral medulla regulate cardiovascular function through SNA and mediate the sympathetic arterial baroreflex. In this study we determine the effect of sleep‐wake transitions on the activity of C1 neurons and investigate the contribution of the arterial baroreflex to these effects.MethodsThe activity of C1 neurons was measured using fiber photometry (calcium imaging) in freely behaving adult rats while monitoring the vigilance states. C1 neurons were targeted with GCaMP7s by microinjection of AAV1‐syn‐FLEX‐jGCaMP7s in the RVLM of Tyrosine Hydroxylase‐Cre transgenic rats, an optical fiber was implanted above the injection site, rats were fitted with a EEG/EMG head stage to monitor sleep‐wake state, and a radio telemetry probe to record BP. One month after the surgery, C1 neuron activity was recorded before and 7 days after sino‐aortic denervation to eliminate inputs from arterial baroreflex. Normalized fluorescence changes are expressed as Z‐score (mean ± SD) and t‐test was used to determine statistically significant differences.ResultsC1 activity was increased by acute hypotension induced by sodium nitroprusside (2.1 ± 0.5 vs. ‐0.92 ± 0.4, p<0.0001, n=5), indicating the barosensitive nature of the recorded cells. C1 activity increased during transitions from NREM to wake (‐0.60 ± 0.1 vs. 0.59 ± 0.2, p<0.0001, n=6) and decreased during transitions from wake to NREM (0.34 ± 0.2 vs. ‐0.32 ± 0.2, p<0.0001, n=6). C1 activity increased during transitions from NREM to REM sleep (‐0.74 ± 0.1 vs. 0.77 ± 0.1, p<0.0001, n=6) and decreased in transitions from REM to wake (0.58 ± 0.2 vs. ‐0.59 ± 0.2, p<0.0001, n=5). Baroreceptor denervation attenuates the increase in C1 neuron activity during REM sleep (0.4 ± 0.7 vs1.8 ± 0.4 Δ Z‐score, p=0.0054, n=4) and during wakefulness (0.59 ±0.6 vs 1.6 ± 0.4, p=0.0210, n=4) when compared to the increase before sino‐aortic denervation.ConclusionC1 neuron activity during sleep‐wake transitions mirrors the pattern of SNA. This observation indicates that C1 neurons contribute to changes in SNA associated with natural sleep‐wake patterns. Baroreceptor denervation attenuates the change in C1 activity observed during transitions to REM, indicating that the feedback from the arterial baroreflex contributes to the elevation in C1 activity in this sleep state.

Prefrontal‐medullary circuit activation attenuates stress reactivity

Organismal survival and adaptation to stress rely on brainstem catecholaminergic neurons. In particular, catecholaminergic neurons in the rostral ventrolateral medulla (RVLM) drive sympathetic activity and enable physiological adaptations, including vasoconstriction, corticosterone release, and glycemic mobilization. However, it is unclear how brain regions involved in the cognitive appraisal of stress regulate the activity of RVLM neurons. Our previous studies found that the rodent infralimbic prefrontal cortex (IL) integrates behavioral and physiological responses to stress. Thus, a potential IL‐to‐RVLM connection would represent a crucial link between stress appraisal and sympathetic reactivity. In the current study, we investigated a direct IL‐to‐RVLM circuit by targeting a genetically‐encoded anterograde tracer under the control of the CaMKIIα promoter to the IL of adult male and female rats. Analysis revealed that IL terminals apposed RVLM neurons expressing the catecholamine‐synthesizing enzyme dopamine beta hydroxylase. Further, IL input to catecholamine cells was widespread throughout the rostral to caudal extent of the VLM in both male and female rodents. Quantification of innervation density revealed that males had a larger proportion of VLM catecholamine neurons receiving IL inputs relative to female rats. Additionally, IL appositions were identified on GABAergic and glycinergic neurons in both sexes. Accordingly, we hypothesized that IL projections may activate local RVLM inhibitory cells to limit sympathetic output. To test this hypothesis, we injected a viral vector coding for channelrhodopsin‐2 (ChR2) in the IL of males and females. Next, a fiber optic cannula was implanted dorsal to the RVLM to evoke IL synaptic glutamate release. Animals then received photostimulation during restraint stress with blood sampled to determine stress reactivity. Compared to controls, male rats expressing ChR2 on IL terminals had suppressed glycemic stress responses (p < 0.05). In contrast, stimulation of the IL‐RVLM circuit in females did not affect glucose mobilization (p < 0.05). However, ChR2 decreased corticosterone responses to stress relative to control rats in both sexes (males, p < 0.01; females, p < 0.05). Thus, both male and female rats have a direct circuit from the IL portion of the mPFC to catecholamine‐synthesizing cells of the RVLM that limits glucocorticoid stress responses, likely through the activation of local inhibitory neurons. However, the density of this circuit is greater in males, potentially accounting for reduced glycemic responses. Ultimately, excitatory/inhibitory balance at IL synapses in the RVLM may be critical for the health consequences of stress.

Adrenergic C1 neurons monitor arterial blood pressure and determine the sympathetic response to hemorrhage.

Hemorrhage initially triggers a rise in sympathetic nerve activity (SNA) that maintains blood pressure (BP); however, SNA is suppressed following severe blood loss causing hypotension. We hypothesized that adrenergic C1 neurons in the rostral ventrolateral medulla (C1RVLM) drive the increase in SNA during compensated hemorrhage, and a reduction in C1RVLM contributes to hypotension during decompensated hemorrhage. Using fiber photometry, we demonstrate that C1RVLM activity increases during compensated hemorrhage and falls at the onset of decompensated hemorrhage. Using optogenetics combined with direct recordings of SNA, we show that C1RVLM activation mediates the rise in SNA and contributes to BP stability during compensated hemorrhage, whereas a suppression of C1RVLM activity is associated with cardiovascular collapse during decompensated hemorrhage. Notably, re-activating C1RVLM during decompensated hemorrhage restores BP to normal levels. In conclusion, C1 neurons are a nodal point for the sympathetic response to blood loss.

Impaired visceral pain-related functions of the midbrain periaqueductal gray in rats with colitis

The midbrain periaqueductal gray (PAG) is a key structure involved in the supraspinal modulation of pain. Previous studies have reported the association of gut inflammation-triggered chronic abdominal pain with structural and neuronal alterations within the PAG. However, whether PAG-executed visceral nociception processing and descending modulation are altered in gut pathology is not known. We used c-Fos immunohistochemistry and extracellular microelectrode recording in urethane-anesthetized male Wistar rats to evaluate the colitis-induced changes in visceral pain-related neuronal properties of the PAG and its descending outflow to visceral nociceptive neurons of the caudal ventrolateral medulla (CVLM). Analysis of c-Fos protein expression in inflamed animals has shown diminished activation of the lateral and ventrolateral PAG columns by noxious colorectal distension (CRD), although the nonstimulated c-Fos labeling in these PAG subdivisions was enhanced compared with that in controls. Microelectrode recording in the ventrolateral PAG revealed a colitis-elicited decrease in the proportion of CRD-excited neurons accompanied by an increase in the number of unresponsive cells and weakened reactions to the stimulation of CRD-inhibited PAG units. Colonic inflammation has also been found to cause a shift in the effects of ventrolateral PAG electrostimulation on CRD-excited CVLM neurons from being mostly inhibitory under normal conditions to excitatory in colitis. These findings identify impaired PAG functioning in ascending and descending visceral nociception control that may contribute to gut injury-associated visceral hyperalgesia. The data obtained can benefit a better understanding of the supraspinal mechanisms involved in the pathogenesis of postinflammatory chronic abdominal pain.

Keep in touch: Nodal connectivity in the control of breathing and blood pressure.

Brainstem and suprapontine structures controlling cardiorespiratory function are reasonably well defined, but many subtleties of the intricate neural circuitry are yet to be revealed. Contemporary experimental approaches allow for elegant delineation of nodal connectivity underpinning integrative behaviours. The highly co-ordinated contemporaneous activation of respiratory motor and autonomic outputs at rest and in response to stress is crucial to survival. In this issue of Experimental Physiology, Malheiros-Lima et al. (2022) extend our understanding of pontomedullary communication in the control of breathing and blood pressure. Medullary catecholaminergic C1 neurons and ventrolateral pontine adrenergic A5 neurons are two key nodes in the brainstem network controlling cardiorespiratory function. Both are strongly implicated in chemoreflex control of respiratory and sympathetic outflows, but the nature of the interaction between the nodes is not fully appreciated. In a sophisticated experimental series, Malheiros-Lima et al. (2022) set out to establish: (1) whether C1 neurons projecting directly to A5 neurons are glutamatergic (excitatory); (2) whether retrograde ablation of C1 neurons via the A5 region reduces hypoxia-dependent activation of C1 neurons; and (3) whether ionotropic glutamatergic receptor blockade in A5 neurons decreases the cardiorespiratory effects of selective activation of C1 neurons. The experimental findings demonstrate a crucial link between C1 and A5 neurons in the cardiorespiratory control network. In adult male rats, lentiviral transfection of catecholaminergic (tyrosine hydroxylase-positive) neurons in the rostral ventrolateral medulla (C1 neurons) with a fluorescent protein provided confirmation that transduced C1 terminals, which are predominantly glutamatergic, make direct contact with ipsilateral A5 neurons of the pons, amongst other sites, such as the A6 locus coeruleus. Retrograde destruction of C1 neurons was achieved by microinjection of a selective poison unilaterally into the A5 region, which decreased ipsilateral C1 catecholaminergic neurons by 25% compared with the contralateral neuronal count. Exposure to hypoxia for 3 h elicited the expected increases in c-Fos expression in C1 neurons; however, after selective poisoning, the density of c-Fos-positive non-catecholaminergic C1 neurons was unchanged, whereas the total number of c-Fos-positive catecholaminergic neurons was reduced compared with the contralateral population. Bilateral injections of kynurenic acid into the A5 region abrogated, in part, the increase in blood pressure, splanchnic sympathetic nerve activity and phrenic nerve motor activity elicited by selective optogenetic activation of C1 catecholaminergic neurons (transfected with channelrhodopsin2 and activated by laser). The study reveals that medullary C1 neurons activated by hypoxia project directly to pontine A5 neurons. Moreover, glutamatergic receptor blockade in the A5 node suppressed sympathetic and respiratory responses to selective C1 neuronal activation. Although C1 neurons are traditionally considered catecholaminergic, possessing the enzymatic portfolio to produce various catecholamines, Malheiros-Lima et al. (2022) found that > 90% of the varicosities in the A5 region are glutamatergic. The physiological effects of selective pharmacological blockade of glutamate neurotransmission corroborate the neuroanatomical observations suggesting that the C1–A5 connection is mediated via glutamate and accounts for an appreciable amount of C1-dependent cardiorespiratory activation. A5 neurons might also have capacity for a direct independent contribution owing to their intrinsic chemosensitivity (Kanbar et al., 2011), which appears to be a ubiquitous feature of brainstem neural circuits. There is growing evidence for a contribution by C1 neurons to breathing (Malheiros et al., 2018), extending to the respiratory response to hypoxia (Malheiros et al., 2020). The present study extends this work, revealing a contribution by the C1–A5 pathway to respiratory motor drive, shown by retardation in phrenic motor amplitude in response to hypoxia after glutamatergic inhibition in the A5 region. The precise link between A5 neurons and other pontine and medullary sites implicated in the chemoreflex-dependent drive to breathe remains to be determined, but the lack of effect of A5 inhibition on hypoxia-dependent tachypnoea might exclude connectivity with rhythmogenic sites, such as the pre-Bötzinger complex. Confusingly, however, previous work reported that A5 lesions do not affect ventilatory responses to hypoxia in conscious rats nor the phrenic nerve response in the in situ working heart–brainstem preparation (Taxini et al., 2017). This suggests that anaesthesia affects the relevant circuitry behaviour and, as such, is a confounder in the present study. Thus, it appears that the C1–A5 pathway forms an intrinsic component of the chemoreflex circuitry mediating sympathoexcitation and the pressor response to stress, with a potential contribution to the accompanying respiratory excitation. Pontine adrenergic neurons project to most structures of the brain in a highly organized manner. Therefore, it might be that specific elements of the fight or flight response, beyond cardiorespiratory adjustments, are also manifest through the C1–A5 axis. We can anticipate continued elegant work from this research group striving to unravel these complex connections. Let's keep in touch! None declared.

Activation of the Organum Vasculosum of the Lamina Terminalis Produces a Sympathetically Mediated Hypertension.

Neurons in the organum vasculosum of the lamina terminalis (OVLT) sense extracellular NaCl and angiotensin II concentrations to regulate body fluid homeostasis and arterial blood pressure. Lesion of the anteroventral third ventricular region or OVLT attenuates multiple forms of neurogenic hypertension. However, the extent by which OVLT neurons directly regulate sympathetic nerve activity to produce hypertension is not known. Therefore, the present study tested this hypothesis by using a multi-faceted approach including optogenetics, single-unit and multifiber nerve recordings, and chemogenetics. First, optogenetic activation of OVLT neurons in conscious Sprague-Dawley rats (250-400 g) produced frequency-dependent increases in arterial blood pressure and heart rate. These responses were not altered by the vasopressin receptor antagonist (β-mercapto-β,β-cyclopentamethylenepropionyl1,O-me-Tyr2,Arg8)-vasopressin but eliminated by the ganglionic blocker chlorisondamine. Second, optogenetic activation of OVLT neurons significantly elevated renal, splanchnic, and lumbar sympathetic nerve activity. Third, single-unit recordings revealed optogenetic activation of the OVLT significantly increased the discharge of bulbospinal, sympathetic neurons in the rostral ventrolateral medulla. Lastly, chronic chemogenetic activation of OVLT neurons for 7 days significantly increased 24-hour fluid intake and mean arterial blood pressure. When the 24-hour fluid intake was clamped at baseline intakes, chemogenetic activation of OVLT neurons still produced a similar increase in arterial blood pressure. Neurogenic pressor activity assessed by the ganglionic blocker chlorisondamine was greater at 7 days of OVLT activation versus baseline. Collectively, these findings indicate that acute or chronic activation of OVLT neurons produces a sympathetically mediated hypertension.