Neurons In Rostral Ventrolateral Medulla Research Articles

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.

PDZD8-mediated endoplasmic reticulum–mitochondria associations regulate sympathetic drive and blood pressure through the intervention of neuronal mitochondrial homeostasis in stress-induced hypertension

Neuronal hyperexcitation in the rostral ventrolateral medulla (RVLM) drives heightened sympathetic nerve activity and contributes to the etiology of stress-induced hypertension (SIH). Maintenance of mitochondrial functions is central to neuronal homeostasis. PDZD8, an endoplasmic reticulum (ER) transmembrane protein, tethers ER to mitochondria. However, the mechanisms of PDZD8-mediated ER–mitochondria associations regulating neuronal mitochondrial functions and thereby mediating blood pressure (BP) in the RVLM of SIH were largely unknown. SIH rats were subjected to intermittent electric foot shocks plus noise for 2 h twice daily for 15 consecutive days. The underlying mechanisms of PDZD8 were investigated through in vitro experiments by using small interfering RNA and through in vivo experiments, such as intra-RVLM microinjection and Western blot analysis. The function of PDZD8 on BP regulation in the RVLM was determined in vivo via the intra-RVLM microinjection of adeno-associated virus (AAV)2-r-Pdzd8. We found that the c-Fos-positive RVLM tyrosine hydroxylase (TH) neurons, renal sympathetic nerve activity (RSNA), plasma norepinephrine (NE) level, BP, and heart rate (HR) were elevated in SIH rats. ER–mitochondria associations in RVLM neurons were significantly reduced in SIH rats. PDZD8 was mainly expressed in RVLM neurons, and mRNA and protein levels were markedly decreased in SIH rats. In N2a cells, PDZD8 knockdown disrupted ER–mitochondria associations and mitochondrial structure, decreased mitochondrial membrane potential (MMP) and respiratory metabolism, enhanced ROS levels, and reduced catalase (CAT) activity. These effects suggested that PDZD8 dysregulation induced mitochondrial malfunction. By contrast, PDZD8 upregulation in the RVLM of SIH rats could rescue neuronal mitochondrial function, thereby suppressing c-Fos expression in TH neurons and decreasing RSNA, plasma NE, BP, and HR. Our results indicated that the dysregulation of PDZD8-mediated ER–mitochondria associations led to the loss of the activity homeostasis of RVLM neurons by disrupting mitochondrial functions, thereby participating in the regulation of SIH pathology.

Ovariectomy Exacerbates Acute Ethanol-Induced Tachycardia: Role of Nitric Oxide and NMDA Receptors in the Rostral Ventrolateral Medulla

Ethanol consumption influences cardiovascular functions. In humans, acute consumption of ethanol causes dose-dependent tachycardia. Our previous study showed that ethanol-induced tachycardia might involve decreased nitric oxide (NO) signaling in the brain's medulla. NMDA receptors, another important target of ethanol, are one of the upstream signals of nitric oxide. Reports showed the modulation of NMDA receptor function by estrogen or estrogen receptors. The present study aims to examine the hypothesis that depletion of estrogen by ovariectomy (OVX) might modulate ethanol-induced tachycardia by regulating NMDA receptor function and NO signaling in the cardiovascular regulatory nucleus of the brain. Ethanol (3.2 g/kg, 40% v/v, 10 mL/kg) or saline (10 mL/kg) was administered by oral gavage in sham or OVX female Sprague-Dawley (SD) rats. The blood pressure (BP) and heart rate (HR) were measured using the tail-cuff method. The levels of phosphoserine 896 of the GluN1 subunit (pGluN1-serine 896) and NMDA GluN1 subunits (GluN1) were determined by immunohistochemistry. The expressions of nitric oxide synthase (NOS) and estrogen receptors in the tissue were measured by Western blotting. Nitric oxide contents were measured as total nitrate-nitrite by colorimetric assay kit. In a 2-h observation, there was no significant change in BP between the saline and ethanol groups. However, compared with saline, ethanol caused an increase in HR (tachycardia) in sham control or OVX rats. Interestingly, ethanol produced more significant tachycardia in the OVX group than in the sham control group. Nitric oxide levels were lower in the area of the rostral ventrolateral medulla (RVLM) 60 min following ethanol administration in OVX compared with sham control, without significant changes in the expression of NOS and estrogen receptors (ERα and ERβ). In addition, a decrease in the immunoreactivity of pGluN1-serine 896, without significant changes in GluN1, was found in neurons of RVLM 40 min following ethanol administration in OVX compared with sham control. Our results suggest that depletion of estradiol (E2) by OVX might exacerbate the tachycardia following ethanol administration, the underlying mechanism of which might be associated with decreased NMDA receptor function and NO level in the RVLM.

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.

Increased neuronal activation in sympathoregulatory regions of the brain and spinal cord in type 2 diabetic rats.

Increased cardiac sympathetic nerve activity in type 2 diabetes mellitus (DM) suggests impaired autonomic control of the heart. However, the central regions that contribute to the autonomic cardiac pathologies in type 2 DM are unknown. Therefore, we tested the hypothesis that neuronal activation would be increased in central sympathoregulatory areas in a pre-clinical type 2 DM animal model. Immunohistochemistry in 20-week-old male Zucker diabetic fatty (ZDF) rats revealed an increased number of neurones expressing ΔFosB (a marker of chronic neuronal activation) in the intermediolateral column (IML) of the spinal cord in DM compared to non-diabetic (non-DM) rats (P<0.05). Rostral ventrolateral medulla (RVLM) neurones activate IML neurones and receive inputs from the hypothalamic paraventricular nucleus (PVN), as well as the nucleus tractus solitarius (NTS) and area postrema (AP), in the brainstem. We observed more ΔFosB-positive noradrenergic RVLM neurones (P<0.001) and corticotrophin-releasing hormone PVN neurones (P<0.05) in DM compared to non-DM rats. More ΔFosB-positive neurones were also observed in the NTS (P<0.05) and AP (P<0.01) of DM rats compared to non-DM rats. Finally, because DM ZDF rats are obese, we also expected increased activation of pro-opiomelanocortin (POMC) arcuate nucleus (ARC) neurones in DM rats; however, fewer ΔFosB-positive POMC ARC neurones were observed in DM compared to non-DM rats (P<0.01). In conclusion, increased neuronal activation in the IML of type 2 DM ZDF rats might be driven by RVLM neurones that are possibly activated by PVN, NTS and AP inputs. Elucidating the contribution of central sympathoexcitatory drive in type 2 DM might improve the effectiveness of pharmacotherapies for diabetic heart disease.

Reduction of oxidative stress and inflammatory signaling in the commissural nucleus of the solitary tract (commNTS) and rostral ventrolateral medulla (RVLM) in treadmill trained rats

Inflammation has been associated with cardiovascular diseases and the key point is the generation of reactive oxygen species (ROS). Exercise modulates medullary neurons involved in cardiovascular control. We investigated the effect of chronic exercise training (Tr) in treadmill running on gene expression (GE) of ROS and inflammation in commNTS and RVLM neurons. Male Wistar rats (N = 7/group) were submitted to training in a treadmill running (1 h/day, 5 days/wk/10 wks) or maintained sedentary (Sed). Superoxide dismutase (SOD), catalase (CAT), neuroglobin (Ngb), Cytoglobin (Ctb), NADPH oxidase (Nox), cicloxigenase-2 (Cox-2), and neuronal nitric oxide synthase (NOS1) gene expression were evaluated in commNTS and RVLM neurons by qPCR. In RVLM, Tr rats increased Ngb (1.285 ± 0.03 vs. 0.995 ± 0.06), Cygb (1.18 ± 0.02 vs.0.99 ± 0.06), SOD (1.426 ± 0.108 vs. 1.00 ± 0.08), CAT (1.34 ± 0.09 vs. 1.00 ± 0.08); and decreased Nox (0.55 ± 0.146 vs. 1.001 ± 0.08), Cox-2 (0.335 ± 0.05 vs. 1.245 ± 0.02), NOS1 (0.51 ± 0.08 vs. 1.08 ± 0.209) GE compared to Sed. In commNTS, Tr rats increased SOD (1.384 ± 0.13 vs. 0.897 ± 0.101), CAT GE (1.312 ± 0.126 vs. 0.891 ± 0.106) and decreased Cox-2 (0.052 ± 0.011 vs. 1.06 ± 0.207) and NOS1 (0.1550 ± 0.03559 vs. 1.122 ± 0.26) GE compared to Sed. Therefore, GE of proteins of the inflammatory process reduced while GE of antioxidant proteins increased in the commNTS and RVLM after training, suggesting a decrease in oxidative stress of downstream pathways mediated by nitric oxide.

Activation of the Rostral Ventrolateral Medulla Reveals Specialized Control of Sympathetic Nerves Involved in Regulation of Blood Glucose and Blood Pressure

BACKGROUND The sympathetic nervous system mediates the “fight-or-flight” response, including stimulation of the adrenal glands to secrete epinephrine. Epinephrine increases heart rate and contractility and increases blood glucose. Sympathetic nerve activity to functionally distinct targets is regulated differentially by the rostral ventrolateral medulla (RVLM). Therefore, it is important to recognize the numerous roles of the RVLM in control of both blood pressure and blood glucose. Anatomical evidence suggests that innervation of the adrenal gland is from ipsilateral spinal sympathetic neurons and, in turn by RVLM neurons primarily from the ipsilateral side; however, the functional implications of lateralized innervation are lacking. Functional evidence also suggests that withdrawal of tonic GABAergic inhibition in the RVLM during decreases in blood pressure is an important regulatory mechanism in the excitation of sympathetic vasoconstrictor nerves (e.g., splanchnic SNA). We were interested in whether similar neurotransmitter mechanisms mediate hypotension-induced increases in adrenal SNA (AdSNA). Thus, the goals of the present study were to 1) compare effects of contralateral vs. ipsilateral RVLM activation on sSNA and AdSNA, 2) determine the role of RVLM GABAA receptors in hypotension-induced increases in sSNA and AdSNA and 3) investigate functional outcomes in a physiologically relevant model of altered glucose regulation. We hypothesized that activation of the RVLM produces differential increases in sSNA versus AdSNA, given the unique functional role of AdSNA in glucose regulation. METHODS We measured AdSNA and sSNA in male Sprague-Dawley rats during ipsilateral or contralateral glutamatergic activation of the RVLM. We produced decreases in blood pressure (sodium nitroprusside, i.v.) before and after bilateral GABAA receptor blockade (bicuculline, 5mM 90nL). In related experiments, blood glucose was also recorded before and after 2-deoxyglucose (2-DG, i.v.). Lastly, adrenal glands from active (12 wk running wheels) and sedentary animals were analyzed to measure adrenal medulla size. RESULTS Our results indicated that glutamate (100 mM, 30nL) injected into the left RVLM produced increases in ipsilateral AdSNA that were more than twice that of increases following right RVLM (contralateral) injections (p<0.05; n=6). In contrast, sSNA responses were equivalent to glutamate injections on either side of the RVLM. In response to hypotension, both sSNA and AdSNA increased; however, increases in sSNA but not AdSNA were eliminated by bicuculline (p<0.05; n=4). In preliminary experiments, sedentary animals demonstrated potentially greater increases in glucose in response to 2-DG stimulation (p=0.21, n=4) and possibly smaller adrenal medulla size (p=0.142, n=3). CONCLUSION The results confirm previous anatomical findings that the RVLM has predominantly ipsilateral innervation of adrenal nerves. Baroreflex-mediated increases in sSNA but not AdSNA are mediated by GABAA receptors in the RVLM. A deeper understanding of neural control of adrenal function provides insight into comorbidities between cardiovascular and metabolic disease pathologies.

Androgenic modulation of arterial baroreceptor dysfunction and neuroinflammation in endotoxic male rats

Autonomic neuropathy contributes to cardiovascular derangements induced by endotoxemia. In this communication, we tested the hypothesis that androgenic hormones improve arterial baroreflex dysfunction and predisposing neuroinflammatory response caused by endotoxemia in male rats. Baroreflex curves relating changes in heart rate to increases or decreases in blood pressure evoked by phenylephrine (PE) and sodium nitroprusside (SNP), respectively, were constructed in conscious sham-operated, castrated, and testosterone-replaced castrated rats treated with or without lipopolysaccharide (LPS, 10 mg/kg i.v.). Slopes of baroreflex curves were taken as measures of baroreflex sensitivity (BRS). In sham rats, LPS significantly reduced reflex bradycardia (BRSPE) and tachycardia (BRSSNP) and increased immunohistochemical expression of nuclear factor kappa B (NFκB) in heart and brainstem neurons of nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM). The baroreflex depressant effect of LPS was maintained in castrated rats despite the remarkably attenuated inflammatory response. Testosterone replacement of castrated rats counteracted LPS-evoked BRSPE, but not BRSSNP, depression and increased cardiac, but not neuronal, NFκB expression. We also evaluated whether LPS responses could be affected following pharmacologic inhibition of androgenic biosynthetic pathways. Whereas none of LPS effects were altered in rats pretreated with formestane (aromatase inhibitor) or finasteride (5α-reductase inhibitor), the LPS-evoked BRSPE, but not BRSSNP, depression and cardiac and neuronal inflammation disappeared in rats pretreated with degarelix (gonadotropin-releasing hormone receptor blocker). Overall, despite the seemingly provocative role for the hypothalamic-pituitary–gonadal axis in the neuroinflammatory and baroreflex depressant effects of LPS, testosterone appears to distinctly modulate the two LPS effects.

Impaired Kv7 channel activity in the central amygdala contributes to elevated sympathetic outflow in hypertension.

Elevated sympathetic outflow is associated with primary hypertension. However, the mechanisms involved in heightened sympathetic outflow in hypertension are unclear. The central amygdala (CeA) regulates autonomic components of emotions through projections to the brainstem. The neuronal Kv7 channel is a non-inactivating voltage-dependent K+ channel encoded by KCNQ2/3 genes involved in stabilizing the neuronal membrane potential and regulating neuronal excitability. In this study, we investigated if altered Kv7 channel activity in the CeA contributes to heightened sympathetic outflow in hypertension. The mRNA and protein expression levels of Kv7.2/Kv7.3 in the CeA were significantly reduced in spontaneously hypertensive rats (SHRs) compared with Wistar-Kyoto (WKY) rats. Lowering blood pressure with coeliac ganglionectomy in SHRs did not alter Kv7.2 and Kv7.3 channel expression levels in the CeA. Fluospheres were injected into the rostral ventrolateral medulla (RVLM) to retrogradely label CeA neurons projecting to the RVLM (CeA-RVLM neurons). Kv7 channel currents recorded from CeA-RVLM neurons in brain slices were much smaller in SHRs than in WKY rats. Furthermore, the basal firing activity of CeA-RVLM neurons was significantly greater in SHRs than in WKY rats. Bath application of specific Kv7 channel blocker 10, 10-bis (4-pyridinylmethyl)-9(10H)-anthracnose (XE-991) increased the excitability of CeA-RVLM neurons in WKY rats, but not in SHRs. Microinjection of XE-991 into the CeA increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA), while microinjection of Kv7 channel opener QO-58 decreased ABP and RSNA, in anaesthetized WKY rats but not SHRs. Our findings suggest that diminished Kv7 channel activity in the CeA contributes to elevated sympathetic outflow in primary hypertension. This novel information provides new mechanistic insight into the pathogenesis of neurogenic hypertension.

Responses of Neurons in the Medullary Lateral Tegmental Field and Nucleus Tractus Solitarius to Vestibular Stimuli in Conscious Felines.

Considerable evidence shows that the vestibular system contributes to adjusting sympathetic nervous system activity to maintain adequate blood pressure during movement and changes in posture. However, only a few prior experiments entailed recordings in conscious animals from brainstem neurons presumed to convey baroreceptor and vestibular inputs to neurons in the rostral ventrolateral medulla (RVLM) that provide inputs to sympathetic preganglionic neurons in the spinal cord. In this study, recordings were made in conscious felines from neurons in the medullary lateral tegmental field (LTF) and nucleus tractus solitarius (NTS) identified as regulating sympathetic nervous system activity by exhibiting changes in firing rate related to the cardiac cycle, or cardiac-related activity (CRA). Approximately 38% of LTF and NTS neurons responded to static 40° head up tilts with a change in firing rate (increase for 60% of the neurons, decrease for 40%) of ~50%. However, few of these neurons responded to 10° sinusoidal rotations in the pitch plane, in contrast to prior findings in decerebrate animals that the firing rates of both NTS and LTF neurons are modulated by small-amplitude body rotations. Thus, as previously demonstrated for RVLM neurons, in conscious animals NTS and LTF neurons only respond to large rotations that lead to changes in sympathetic nervous system activity. The similar responses to head-up rotations of LTF and NTS neurons with those documented for RVLM neurons suggest that LTF and NTS neurons are components of the vestibulo-sympathetic reflex pathway. However, a difference between NTS/LTF and RVLM neurons was variability in CRA over time. This variability was significantly greater for RVLM neurons, raising the hypothesis that the responsiveness of these neurons to baroreceptor input is adjusted based on the animal's vigilance and alertness.

Neural pathway between the nucleus accumbens and the rostral ventrolateral medulla in a rat model of anorexia nervosa

To investigate the potential neural pathway connecting the nucleus accumbens (NAc) and the rostral ventrolateral medulla (RVLM), and whether the pathway participates in the regulation of cardiovascular function in a model rat of anorexia nervosa (AN). Rat models of AN were established by allowing voluntary activity in a running wheel with restricted feeding, with the rats having free access to normal chow without exercise as the control group. FluoroGold (FG) retrograde tracing method and multi-channel simultaneous recording technique were used to explore the possible pathway between the NAc and the RVLM. The rats in AN group exhibited significantly reduced systolic blood pressure (SBP), mean arterial pressure (MAP) and heart rate (HR) with significantly increased discharge frequency of RVLM neurons in comparison with the control rats. After the injection of FG into the RVLM, retrograde labeled neurons were observed in the NAc of the rats in both the normal control and AN groups. In both groups, SBP and HR were significantly decreased in response to 400 μA electrical stimulation of the NAc accompanied by an obvious increase in the discharge frequency of the RVLM neurons; the diastolic blood pressure (DBP) and MAP were significantly lower in AN model rats than in the normal rats in response to the stimulation. We successfully established a rat model of AN via hyperactivity and restricted feeding and confirm the presence of a neural pathway connecting the NAc and the RVLM. This pathway might participate in the regulation of cardiovascular function in AN model rats.

Co‐release of inhibitory neurotransmitters in the RVLM

Sympathetic nerve activity (SNA) and blood pressure (BP) are largely determined by the level of excitability of presympathetic neurons (C1 neurons) in the rostral ventrolateral medulla (RVLM). GABAergic inhibition in the RVLM is the primary mechanism for depressor and sympathoinhibitory responses; however, the role of glycinergic inhibition in the control of RVLM excitability is unknown. There is evidence that both, GABA and glycine efficiently inhibit RVLM neurons, but functional evidence for their co‐release or the mechanism of co‐inhibition is lacking. In our study we used both male and female glycine transporter 2 Cre (GlyT2‐Cre) mice. Glycinergic neurons sending projections to the RVLM were identified with viral labeling. Targeted expression of channelrhodopsin 2 (ChR2) in GlyT2‐expressing neurons and whole‐cell recordings from presympathetic RVLM neurons were used to study the mechanism of glycine release. We found that under steady‐state conditions spontaneous inhibitory postsynaptic current (sIPSC) are primarily mediated by GABA. Photostimulation of glycinergic fibers expressing GlyT2 in the RVLM resulted in evoked IPSCs with both glycinergic and GABAergic components. The time course of synaptic signaling were identical for the two transmitters. Bath application of GABAA receptors antagonist bicuculline (10 μM) or glycine receptor antagonist strychnine (1 μM) revealed that ~40% of the evoked inhibitory currents are GABAergic. High frequency photostimulation (20 Hz) of co‐releasing inhibitory fibers produced a short‐term synaptic depression. Blockade of GlyT2 prevented the glycinergic component of the light evoked IPSCs. In this study we showed that GABA and glycine are co‐released in the RVLM. Our findings suggest that the two neurotransmitters may be functionally interchangeable or complement each other, and thus provide diverse mechanisms for maintaining inhibitory homeostasis.

Prorenin Induces Intracellular Signaling And Reactive Oxygen Species In The Brainstem

The importance of the brain renin angiotensin system (RAS) in blood pressure (BP) regulation and water homeostasis is well accepted. However, it remains unclear whether renin and/or its precursor, prorenin, are expressed in the brain and whether they trigger signaling pathways in specific brain regions. We previously used double transgenic mice expressing enhanced green fluorescent protein controlled by renin promoter and beta‐galactosidase controlled by the human angiotensinogen (AGT) promoter to localize their expression in the brain. We reported that neurons expressing renin are localized in close proximity to AGT‐expressing cells in the rostral ventrolateral medulla (RVLM), a brainstem nucleus involved in sympathetic activity and BP control. Here, we hypothesized that prorenin induces phosphorylation of intracellular cascades such as extracellular signal‐regulated kinases (ERK1/2) and activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPHox) in the brainstem. Neonatal brainstem cells were cultured from C57BL/6J mice at postnatal day 1–3 and maintained for 7–10 days in B27‐supplemented neurobasal medium. Immunofluorescence experiments demonstrated the cultures contain both MAP2+ neurons and GFAP+ astrocytes. Expression of type 1 angiotensin receptor (AT1R), renin, and prorenin receptor (PRR) was confirmed by qPCR. To quantify ERK1/2 phosphorylation, cells were stimulated with recombinant prorenin (rProrenin) at 100 nM for 0, 30, and 60 min. Total and phosphorylated ERK1/2 were detected by Western Blot. The effect of rProrenin on NADPHox activity was measured by lucigenin chemiluminescence assay. Specific activity of NADPHox was expressed as chemiluminescence units per min per microgram of protein. Vehicle and phorbol myristate acetate were used as negative and positive controls, respectively. Incubation with rProrenin increased the ratio of phosphorylated‐to‐total ERK1/2 from 0.65±0.07 A.U. at baseline to 1.01±0.13 A.U. at 30 min (54% increase; p=0.0035; n=7) and 1.29±0.09 A.U. at 60 min (102% increase; p&lt;0.0001; n=7). We generated preliminary data showing that both PRO20, a PRR blocker, and Ro‐31, a protein kinase C inhibitor, abrogated prorenin‐induced ERK1/2 phosphorylation. In an additional experiment, we observed that Angiotensin II (Ang II) at 100 nM induced ERK1/2 phosphorylation from 0.98 A.U. to 1.52 A.U. at 15 min (55 % increase), 1.61 A.U. at 30 min (65% increase), and 1.35 A.U. at 60 min (38% increase). Incubation of cells with 100 nM rProrenin for 6 hours resulted in 47.2±9.5 % induction of NADPHox activity compared to vehicle control (p&lt;0.05, n=5). Finally, fluorescent in situ hybridization (RNAscope) data suggests that PRR is expressed in the RVLM neurons and RVLM‐targeted stereotactic microinjection of glutamate, rProrenin, or Ang II, but not vehicle, induced an acute BP elevation in isoflurane‐anesthetized mice. We conclude that prorenin in the brainstem induces downstream signaling involving ERK/2 phosphorylation and generation of ROS, which might contribute to BP elevation and sympathoexcitation. Future studies will determine whether prorenin‐elicited signaling in the brainstem requires binding to PRR, generation of Ang II, and/or AT1R.Support or Funding InformationP01 HL084207 to Sigmund and 5T32HL134643 to Nakagawa

SPARC‐Network‐Scale Interactions Among Simultaneously Recorded Brainstem Neurons In The Dorsal Medulla, Ventrolateral Medulla, And Pons: A Substrate For Central Autonomic Control And Processing Of Cardiovascular Afferent Feedback

The central autonomic control system provides parasympathetic and sympathetic drive to peripheral ganglia that regulate a host of visceral functions. Detailed knowledge of the network scale organization of this central circuit is incomplete. We hypothesized that functional synaptic interactions between many simultaneously recorded neurons in and around the nucleus of the tractus solitarius (NTS), the retrotrapezoid nucleus (RTN), ventral respiratory column (VRC) and pons would yield information about the network‐scale strategy that is employed by this system to regulate autonomic drive. Experiments were conducted in 73 decerebrated, paralyzed, and artificially ventilated cats. Multi‐electrode arrays including up to 100 channels were placed in the areas of the NTS, RTN, and VRC. A total of 2,643 neurons were challenged with alterations in blood pressure (BP) and cardiac modulation was assessed in 3,531 neurons. The spike activity patterns of many simultaneously recorded single neurons were evaluated during breathing, the cardiac cycle, increased activation of baroreceptors by inflation of a balloon in the abdominal aorta and unloading of baroreceptors by inflation of a balloon in the vena cava. There was no apparent anatomical segregation of neurons in these areas with regard to cardiac or BP modulation based on stereotaxic coordinates. Cross correlation analysis revealed robust excitatory and inhibitory functional interactions between neurons in the areas of the NTS, VRC, RTN, and pons. Plots customized to reveal patterns of information transfer between neurons in different areas provided evidence that units in the RTN and VRC were predominantly exciting those in the NTS area, while these same NTS neurons were exciting and inhibiting different neurons in the RTN and VRC. Specific evidence was found for putative intermediate ventrolateral medulla (iVLM) neurons that were excited by BP and involved in inhibitory interactions with putative rostral ventrolateral medulla (RVLM) neurons that were inhibited by increased BP. However, this mechanism was a low frequency feature of the dataset. Evidence for synaptic inhibition between neurons that responded to increased BP was a predominant finding. Among neurons that responded to decreased BP, evidence for synaptic excitation was more common. These results support differential organization of brainstem networks that mediate autonomic responses to increased BP relative to those that mediate decreased BP. The brainstem network that mediates sympatho‐inhibition in response to increased blood pressure is sparsely excitatory and may depend on abundant inhibitory mechanisms to enhance signal/noise ratios in support of information transmission from baroreceptors.Support or Funding InformationSupported by 3OT2OD023854

Brainstem catecholaminergic neurons mediate cognitive impairment in heart failure rats through regulation of hippocampal synaptic‐related gene expression.

Heart failure (HF) is common in elder population and it is highly associated with cognitive impairment (CI). Several studies suggest that autonomic imbalance contribute to age‐related CI. We previously showed that catecholaminergic neurons (C1) from the rostral ventrolateral medulla (RVLM) displayed chronic activation in HF. In addition, we also shown that HF rats showed CI. Whether RVLM‐C1 neurons contribute to CI in the setting of HF is completely unknown. Therefore, we aimed to study the contribution of RVLM‐C1 neurons on cognitive function in HF. Adult male Sprague‐Dawley rats underwent volume overload to induce HF. Anti‐dopamine β‐hydroxylase‐saporin (DβH–SAP: 5ng/150nl) was used to selectively destroy RVLM‐C1 neurons. Morris water maze (MWM) test was used to assess learning and memory performance. Micropunches containing the hippocampus were dissected to measure the expression of several synaptic‐related genes using a custom 12‐gene array by qRT‐PCR. HF rats displayed learning and memory impairment compared to control animals. Indeed, HF showed an increased traveled distance to the target platform along 5 trial/days during MWM test (15.1±0.5 vs. 30.7±2.1 m; Control vs. HF, respectively; p&lt;.05). In addition, when the platform was changed daily to a different quadrant (i.e. to evaluate reference memory) HF rats showed an increase in the number of trials required to reach the platform compared to control rats (4.2±0.1 ± vs. 8.2±1.2 trials; Control vs. HF, respectively; p&lt;.05). Partial ablation of RVLM‐C1 neurons in HF rats decreased traveled distance to reach the target platform compared to HF rats without treatment (18.1±1 vs 30.7±2.1 m; HFDβH–SAP vs. HF, respectively; p&lt;.05). Also, during memory testing HF untreated rats needed almost twice the number of trials to accomplish the task when compared to HF+DβH–SAP rats (4.4±0.4 vs. 8.2±1.2 trials; HFDβH–SAP vs. HF, respectively; p&lt;.05). Finally, compared to control rats, HF rats display (Ctrl vs. HF, in AU) decreased levels of PSD‐95 (1.0±0.2 vs. 0.4±0.3), Hommer1 (1.0±0.2 vs. 0.6±0.3), CaMKIV (1.0±0.2 vs. 0.5±0.3), Piccolo (1.0±0.3 vs. 0.4±0.2), Erc2 (1.0±0.3 vs. 0.2±0.1), RIM (1.0±0.5 vs. 0.3±0.2), Neuroligin 3 (1.0±0.4 vs. 0.5±0.3), Neuroxin 1 (1.0±0.3 vs. 0.6±0.4) and Synaptotagmin‐1 (1.0±0.3 vs. 0.6±0.1). Importantly, DβH–SAP treatment in HF significantly restored the expression levels of Hommer1, Piccolo, Erc2, RIM and Synaptotagmin‐1 to levels observed in control rats. Taken together, our results show that catecholaminergic RVLM neurons contribute to CI in HF rats. In addition, our results suggest that RVLM‐C1 may regulate hippocampal synaptic transmission in the setting of HF through the regulation of gene expression in the hippocampus.Support or Funding InformationSupported by FONDECYT 1180172 and 3190659 and the basal Center of Excellence in Aging and Regeneration (AFB 170005).

Sex‐ and Time‐Dependent Differences in Glutamate Receptor Subunit (GLUN1) in the Rat Rostral Ventrolateral Medulla

The cardioprotective effects of ovarian hormones are evident by the fact that women have a lower risk of cardiovascular disease compared to men, at least until menopause, at which time the relative risks reverse. Several studies have also demonstrated the importance of ovarian hormones in the regulation of sympathetic outflow, potentially via actions in the rostral ventrolateral medulla (RVLM), a brain region critical to sympathetic outflow and blood pressure. However, the time course and mechanisms of sex‐dependent changes in excitation of RVLM neurons have not been fully characterized. Therefore, the purpose of this study was to compare glutamate receptor (GluN1) subunit expression in the RVLM of male and female rats, focusing specifically on ages of development in female rats which precede the onset of the estrous cycle (4 wks old) versus when the estrous cycle is fully established (8 wks old). We hypothesized that eight week‐old female rats would exhibit reduced GluN1 compared to four week‐old females. In contrast, we also predicted that eight week‐old male rats would have higher GluN1 compared to four week‐old males. To test our hypothesis, we obtained brain punches from RVLM subregions of male and female Sprague‐Dawley rats (n=3 ea; 4 and 8 wks old) based on their anatomical location relative to the facial nucleus (FN). Punches were processed for western blotting of GluN1 subunit in four subregions: FN‐480; FN‐240; FN+240 and FN+480. At FN‐480, GluN1 was lower in eight week‐compared to four week‐old female rats (0.67 ± 0.11 vs. 1.11 ± 0.08 protein/GAPDH, respectively). In contrast, at FN‐480, eight week‐old male rats exhibited increased NR1 expression compared to four week old males (1.05 ± 0.06 vs. 0.65 ± 0.10; protein/GAPDH, respectively p&lt;0.05). These results suggest that lower levels of ovarian hormones in prepubescent females may increase the excitability of RVLM neurons and lead to greater sympathoexcitation, which then decrease with the onset of the reproductive cycle. The absence of ovarian hormones in males (perhaps coupled with a lack of regular exercise) may exacerbate RVLM excitability, leading to greater sympathoexcitation in sedentary males. Collectively, these data suggest that ovarian hormones exert cardioprotective effects via actions in the RVLM, reducing sympathetic outflow and contributing to a lower incidence of cardiovascular disease in women of reproductive age.Support or Funding InformationHL096787‐08; AHA25810010

Rostral ventrolateral medulla neuron activity is suppressed by Klotho and stimulated by FGF23 in newborn Wistar rats.

Hypertension often occurs in patients with chronic kidney disease (CKD). Considering the decrease in serum Klotho and increase in serum FGF23 levels in such patients, decreased Klotho and increased FGF23 levels were thought to be associated with hypertension. Presympathetic neurons at the rostral ventrolateral medulla (RVLM) contribute to sympathetic activity and regulation of blood pressure. Therefore, we hypothesized that Klotho would reduce the activities of RVLM neurons and FGF23 would stimulate them. Accordingly, this study examined the effects of Klotho and FGF23 on bulbospinal neurons in the RVLM. We used a brainstem-spinal cord preparation to record from RVLM presympathetic neurons and to evaluate the effects of Klotho and FGF23 on firing rate and membrane potentials of these neurons. Our results showed that Klotho-induced RVLM neuron hyperpolarization, while ouabain, a Na+/K+-ATPase inhibitor, suppressed the effects of Klotho on such neurons. Moreover, FGF23 induced RVLM neuron depolarization, while SU5402, an FGF23 receptor (FGFR1) antagonist, induced RVLM neuron hyperpolarization. Histological examinations revealed that Klotho, Na+/K+-ATPase, FGF23, and FGFR1 were present in RVLM neurons and that Klotho was localized in the same neurons as FGFR1. These results suggest that Klotho and FG23 regulate the activity of RVLM neurons. Klotho may reduce the activity of RVLM neurons via stimulating Na+/K+-ATPase on those neurons while FGF23 may activate those neurons via FGFR1.

Responses of neurons in the rostral ventrolateral medulla of conscious cats to anticipated and passive movements.

The vestibular system contributes to regulating sympathetic nerve activity and blood pressure. Initial studies in decerebrate animals showed that neurons in the rostral ventrolateral medulla (RVLM) respond to small-amplitude (<10°) rotations of the body, as in other brain areas that process vestibular signals, although such movements do not affect blood distribution in the body. However, a subsequent experiment in conscious animals showed that few RVLM neurons respond to small-amplitude movements. This study tested the hypothesis that RVLM neurons in conscious animals respond to signals from the vestibular otolith organs elicited by large-amplitude static tilts. The activity of approximately one-third of RVLM neurons whose firing rate was related to the cardiac cycle, and thus likely received baroreceptor inputs, was modulated by vestibular inputs elicited by 40° head-up tilts in conscious cats, but not during 10° sinusoidal rotations in the pitch plane that affected the activity of neurons in brain regions providing inputs to the RVLM. These data suggest the existence of brain circuitry that suppresses vestibular influences on the activity of RVLM neurons and the sympathetic nervous system unless these inputs are physiologically warranted. We also determined that RVLM neurons failed to respond to a light cue signaling the movement, suggesting that feedforward cardiovascular responses do not occur before passive movements that require cardiovascular adjustments.

Subregional differences in GABAA receptor subunit expression in the rostral ventrolateral medulla of sedentary versus physically active rats.

Neurons in the rostral ventrolateral medulla (RVLM) regulate blood pressure through direct projections to spinal sympathetic preganglionic neurons. Only some RVLM neurons are active under resting conditions due to significant, tonic inhibition by gamma-aminobutyric acid (GABA). Withdrawal of GABAA receptor-mediated inhibition of the RVLM increases sympathetic outflow and blood pressure substantially, providing a mechanism by which the RVLM could contribute chronically to cardiovascular disease (CVD). Here, we tested the hypothesis that sedentary conditions, a major risk factor for CVD, increase GABAA receptors in RVLM, including its rostral extension (RVLMRE ), both of which contain bulbospinal catecholamine (C1) and non-C1 neurons. We examined GABAA receptor subunits GABAAα1 and GABAAα2 in the RVLM/RVLMRE of sedentary or physically active (10-12 weeks of wheel running) rats. Western blot analyses indicated that sedentary rats had lower expression of GABAAα1 and GABAAα2 subunits in RVLM but only GABAAα2 was lower in the RVLMRE of sedentary rats. Sedentary rats had significantly reduced expression of the chloride transporter, KCC2, suggesting less effective GABA-mediated inhibition compared to active rats. Retrograde tracing plus triple-label immunofluorescence identified fewer bulbospinal non-C1 neurons immunoreactive for GABAAα1 but a higher percentage of bulbospinal C1 neurons immunoreactive for GABAAα1 in sedentary animals. Sedentary conditions did not significantly affect the number of bulbospinal C1 or non-C1 neurons immunoreactive for GABAAα2 . These results suggest a complex interplay between GABAA receptor expression by spinally projecting C1 and non-C1 neurons and sedentary versus physically active conditions. They also provide plausible mechanisms for both enhanced sympathoexcitatory and sympathoinhibitory responses following sedentary conditions.

Effects of age on feeding response: Focus on the rostral C1 neuron and its glucoregulatory proteins.

Older people are likely to develop anorexia of aging. Rostral C1 (rC1) catecholaminergic neurons in rostral ventrolateral medulla (RVLM) are recently discovered its role in food intake control. It is well established that these neurons regulate cardiovascular function. This study aims to determine the effect of age on the function of rostral C1 (rC1) neurons in mediating feeding response. Male Sprague Dawley rats at 3-months (n = 22) and 24-months (n = 22) old were used and further divided into two subgroups; 1) treatment group with 2-deoxy-d-glucose (2DG) and 2) vehicle group. Feeding hormones such as cholecystokinin (CCK), ghrelin and leptin were analysed using enzyme-linked immunosorbent assay (ELISA). Rat brain was carefully dissected to obtain the brainstem RVLM region. Further analysis was carried out to determine the level of proteins and genes in RVLM that were associated with feeding pathway. Protein expression of tyrosine hydroxylase (TH), phosphorylated TH at Serine40 (pSer40TH), AMP-activated protein kinase (AMPK), phosphorylated AMPK (phospho AMPK) and neuropeptide Y Y5 receptor (NPY5R) were determined by western blot. Expression of TH, AMPK and NPY genes were determined by real-time PCR. This study showed that blood glucose level was elevated in young and old rats following 2DG administration. Plasma CCK-8 concentration was higher in the aged rats at basal and increased with 2DG administration in young rats, but the leptin and ghrelin showed no changes. Old rats showed higher TH and lower AMPK mRNA levels. Glucoprivation decreased AMPK mRNA level in young rats and decreased TH mRNA in old rats. Aged rC1 neurons showed higher NPY5R protein level. Following glucoprivation, rC1 neurons produced distinct molecular changes across age in which, in young rats, AMPK phosphorylation level was increased and in old rats, TH phosphorylation level was increased. These findings suggest that glucose-counterregulatory responses by rC1 neurons at least, contribute to the ability of young and old rats in coping glucoprivation. Age-induced molecular changes within rC1 neurons may attenuate the glucoprivic responses. This situation may explain the impairment of feeding response in the elderly.