Splanchnic Sympathetic Nerve Activity Research Articles

Acute Intermittent Hypoxia Induces Chemoreflex-Independent Sympathetic Plasticity and Improves Cardiovascular Function in a Rodent Model of Spinal Cord Injury

Background: Cardiovascular complications are one of the leading causes of morbidity and mortality in people with spinal cord injury (SCI). Activation of spinal sympathetic circuitry is critical to offset cardiovascular dysregulation and disease in those with high-level SCI. Acute intermittent hypoxia (AIH) elicits plasticity (i.e., a gradual increase in nerve activity post-AIH exposure) in phrenic and non-phrenic spinal motor circuitry and improves motor function — yet the cardiovascular benefits of AIH after SCI are largely unexplored. Aims: Determine mechanisms (Aim 1) and potential benefits (Aim 2) of AIH-induced sympathetic plasticity post-SCI. Hypothesis: We hypothesized that AIH elicits chemoreflex-dependent sympathetic plasticity and improves cardiovascular function in rats with SCI. Methods: For Aim 1, 40 adult (10-12 weeks) male Wistar rats were subjected to a spinal cord contusion injury (T3, 300 kdyn) and randomly assigned to 5 groups of 8 each: AIH alone, AIH after carotid body denervation (CBX, no peripheral chemoreflex), time control with no intervention (TC), simulated hemorrhage (to mimic AIH-induced hypotension), and AIH plus phenylephrine (to prevent hypotension during hypoxic episodes). Two weeks post-SCI recovery, rats were anesthetized with urethane (~2.1g.kg−1 i.v.) and underwent femoral venous and arterial catheterizations to monitor blood pressure (BP), sample arterial blood gases, and enable intravenous fluid and drug delivery. Additionally, splanchnic sympathetic nerve activity (sSNA) was recorded using a suction electrode at baseline and for up to 90-min following each experimental paradigm. AIH consisted of 10 x 1 min episodes of FiO2 = 0.1 (balanced N2) interspersed with 2 min of FiO2 = 1. Hexamethonium bromide (30 mg.kg−1) was infused to subtract noise levels from each recording, after which rats were euthanized with chloral hydrate. For Aim 2, 19 additional rats were subjected to the same injury model and time post-injury and allocated to 2 groups: AIH ( n = 11) and TC ( n = 8). These rats were instrumented with left-ventricular (LV) and arterial catheters and assessed simultaneously for cardiac and peripheral hemodynamic function at baseline and 90-min post-AIH/TC. Results: Aim 1) When expressed as % change from baseline, sSNA increased at 90-min post-treatment in AIH (93±67%, P = 0.02) and CBX (193±182%, P < 0.001), but not TC rats (50±45%, P = 0.40). Preventing BP reduction during hypoxic episodes with i.v. phenylephrine blocked the increase in sSNA 90-min post-AIH (12±66%, P > 0.99). Mimicking the AIH-induced BP reduction with simulated hemorrhage (10 x 1-min BP reductions of ~20-25 mmHg equal to those during AIH, interspersed by 2-min of euvolemia), increased sSNA 90-min post-intervention (106±86%, P = 0.01). Aim 2) The changes in maximal LV pressure (AIH = 11±8 mmHg vs. TC = 2±5 mmHg, P = 0.05) and mean arterial pressure (AIH = 8±7 mmHg vs. TC = 0±4 mmHg, P = 0.05) were greater at 90-min post-AIH treatment. Conclusions: We conclude that AIH elicits sympathetic plasticity and improves cardiovascular function post-SCI. Since AIH-induced sympathetic plasticity remains in the presence of a bilateral CBX, is replicated by episodic simulated hemorrhage, and is blocked by phenylephrine, AIH-induced sympathetic plasticity is independent of carotid (peripheral) chemoreflex activation, and is closely linked to episodic hypotension. We suggest that AIH-induced sympathetic plasticity arises from episodic reductions in spinal/medullary cardiovascular centers blood flow (and oxygen delivery) post-SCI. International Spinal Research Trust and Natural Sciences, Engineering Research Council of Canada, International Collaboration on Repair Discoveries. This is the full abstract presented at the American Physiology Summit 2024 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.

Chronic Vagotomy Blunts Cardiorespiratory Reflex Responses Through Pre- and Postsynaptic Effects in the Brainstem Nucleus Tractus Solitarii (nTS)

Sensory information from the heart, lungs, and gastrointestinal tract travels to the brain via afferents of the paired vagus nerves, where it is first integrated in the brainstem nTS that critically contributes to cardiorespiratory function. We have shown that reducing input to the nTS by unilaterally transecting the vagus nerve (vagotomy) induces astrocyte reactivity and microglial activation, and reduces sighs in hypoxia. However, the cardiorespiratory effects of vagotomy and contributing mechanisms are not well understood. We hypothesized that chronic vagotomy blunts cardiorespiratory reflex responses to vagal afferent stimulation via reduced nTS neuronal activity. Male Sprague-Dawley rats (6-wk) underwent right cervical vagotomy (or sham surgery) caudal to the nodose ganglion. After 7 days rats were anesthetized, ventilated, and instrumented to record mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA, respectively), which decreased in response to vagal afferent stimulation in sham animals. Vagotomy blunted these decreases in MAP, SSNA, and PhrNA compared to shams. In rats expressing the calcium indicator GCaMP8m in nTS neurons, vagotomy also reduced neuronal Ca2+ responses to afferent stimulation both in vivo (photometry) and in vitro (live slice imaging). We then hypothesized that these changes were due to altered signaling at the level of the tripartite synapse, where astrocytes enwrap the sensory afferent-nTS soma synapse. We found that there is likely a vagal afferent contribution to the altered reflex responses, as vagotomy reduced presynaptic Ca2+ responses in vagal afferent terminals to solitary tract stimulation in the brainstem slice. Glutamate agonists, which induce similar cardiorespiratory responses as vagal stimulation, were nanoinjected into the nTS to determine if there was a postsynaptic contribution to the blunted responses as well. Responses to glutamate injection were also blunted following vagotomy, indicating that astrocytic glutamate uptake or nTS soma glutamate receptor function may be altered. Blocking astrocytic excitatory amino acid transporters, which remove extracellular glutamate in the nTS induced similar baseline effects and did not restore cardiorespiratory responses to glutamate in vagotomy. However, nanoinjection of NMDA into the nTS to activate ionotropic glutamate receptors mimicked the blunted responses to glutamate following vagotomy, with no difference in responses to AMPA nanoinjection. Likewise, immunohistochemistry and Western blot showed that vagotomy reduced NMDA receptor subunit expression in the nTS. Together these results indicate that chronic vagotomy blunts cardiorespiratory responses to vagal afferent stimulation, likely through reduced presynaptic activation and decreased NMDA receptor expression and function. These studies will help improve our understanding of how afferent input induces neuroplasticity within the nTS, and the resulting impacts on cardiorespiratory function in health and disease. NIH HL132836, HL128454, HL98602, T32OD011126. This is the full abstract presented at the American Physiology Summit 2024 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.

N-Methyl-D-aspartate receptor (NMDAR) activity in the nucleus tractus solitarii (nTS) is required for the full expression of phrenic long-term facilitation after acute intermittent hypoxia

Exposure to acute intermittent hypoxia (AIH) induces a persistent elevation of phrenic nerve activity during the normoxic period, or phrenic long-term facilitation (pLTF). We have previously shown that nTS activity is necessary for both the development and maintenance of pLTF. However, the underlying cellular mechanisms and pathways are not yet clear. Hypoxia induces the release of glutamate from chemoafferents in the nTS, where it binds to AMPA and NMDA receptors. Previous studies have shown that activation of NMDARs increases neuronal calcium influx and triggers long-term potentiation. The present study aimed to test the role of NMDARs in pLTF and, in particular, to determine whether activation of these receptors in the nTS is required for hypoxia-induced pLTF. Mean arterial pressure (MAP) and splanchnic sympathetic and phrenic nerve activity (sSNA and PhrNA) were recorded in anesthetized, vagotomized, neuromuscularly blocked, and artificially ventilated male Sprague-Dawley rats. Animals were exposed to 10 bouts of 10% O2 (45 sec, every 5 min; AIH). Time controls (TC) were exposed to a single hypoxia bout. Parameters were measured for an additional 1 hour after the end of AIH and compared to their initial baseline (Bsl) values or TC. The NMDAR antagonist MK-801 (3 mg kg (-1), i.v.) was injected systemically, or AP5 (10 mM, 60 nl) nanoinjected bilaterally into the nTS, after the development of AIH-induced pLTF. To test whether induction of AIH or blockade of NMDARs alters neuronal function of the nTS, nTS neuronal Ca2+ responses to AIH and MK-801 were measured by fiber photometry in 3 rats previously nanoinjected with a Ca2+ indicator GCaMP-expressing virus in the nTS. Minute PhrNA (MinPhrNA, frequency x amplitude) increased 1hr after AIH (% of Bsl, 438 ± 184%; mean ± SE; p <0.01; t-test) and was significantly greater than TC (74 ± 15%Bsl p <0.01; t-test), indicating pLTF. AIH also increased nTS neuronal Ca2+. Systemic blockade of NMDARs after the development of pLTF decreased MAP, sSNA, and nTS Ca2+ and eliminated pLTF, consistent with others. Bilateral nTS nanoinjection of AP5 reduced but did not abolish pLTF (before AP5: 449 ± 109% vs. after AP5: 371 ± 107%Bsl; p <0.05; paired t-test), increased sSNA (before AP5: 192 ± 31% vs. after AP5: 219 ± 42%; p= 0.08; paired t-test) but did not alter MAP. nTS AP5 after TC had no effect on MAP, MinPhrNA (before AP5: 99 ± 46% vs. after AP5: 103 ± 43%; p = 0.44; t-test) or sSNA (before AP5: 244 ± 92% vs. after AP5: 245 ± 81%; =0.94; paired t-test). Altogether, these results show that systemic blockade of NMDARs eliminates pLTF, and our data suggest that nTS NMDARs are critical components for the maintenance of AIH-induced neuroplasticity. R01-HL-98602. This is the full abstract presented at the American Physiology Summit 2024 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.

Influence of sex and sedentary conditions on sympathetic burst characteristics in prepubertal, postpubertal, and young adult rats.

Recent evidence indicates that sex-based differences in cardiovascular disease (CVD) begin early in life, particularly when associated with risk factors such as a sedentary lifestyle. CVD is associated with elevated sympathetic nerve activity (SNA), quantified as increased SNA burst activity in humans. Whether burst characteristics are influenced by sex or sedentary conditions at younger ages is unknown. The purpose of our study is to compare SNA bursts in active and sedentary female and male rats at ages including prepuberty and young adulthood. We hypothesized that burst characteristics and blood pressure are higher under sedentary conditions and lower in female rats compared with males. We analyzed splanchnic SNA (SpSNA) recordings from Inactin-anesthetized male and female rats at 4-, 8-, and 16-wk of age. Physically active and sedentary rats were each housed in separate, environmentally controlled chambers where physically active rats had free access to an in-cage running wheel. Sympathetic bursts were obtained by rectifying and integrating the raw SpSNA signal. Burst frequency, burst height, and burst width were calculated using the Peak Parameters extension in LabChart. Our results showed that sedentary conditions produced a greater burst width in 8- and 16-wk-old rats compared with 4-wk-old rats in both males and females (P < 0.001 for both). Burst frequency and incidence were both higher in 16-wk-old males compared with 16-wk-old females (P < 0.001 for both). Our results suggest that there are sedentary lifestyle- and sex-related mechanisms that impact sympathetic regulation of blood pressure at ages that range from prepuberty into young adulthood.NEW & NOTEWORTHY The mechanisms of decreased incidence of cardiovascular disease (CVD) in reproductive-age women compared with age-matched men are unknown. The strong association between elevated sympathetic activity and CVD led us to characterize splanchnic sympathetic bursts in female and male rats. Prepubescent males and females exhibited narrower sympathetic bursts, whereas young adult males had higher resting burst frequency compared with age-matched females. Sex-based regulation of sympathetic activity suggests a need for sex-dependent therapeutic strategies to combat CVD.

Vagotomy blunts cardiorespiratory responses to vagal afferent stimulation via pre- and postsynaptic effects in the nucleus tractus solitarii.

Viscerosensory information travels to the brain via vagal afferents, where it is first integrated within the brainstem nucleus tractus solitarii (nTS), a critical contributor to cardiorespiratory function and site of neuroplasticity. We have shown that decreasing input to the nTS via unilateral vagus nerve transection (vagotomy) induces morphological changes in nTS glia and reduces sighs during hypoxia. The mechanisms behind post-vagotomy changes are not well understood. We hypothesized that chronic vagotomy alters cardiorespiratory responses to vagal afferent stimulation via blunted nTS neuronal activity. Male Sprague-Dawley rats (6weeks old) underwent right cervical vagotomy caudal to the nodose ganglion, or sham surgery. After 1week, rats were anaesthetized, ventilated and instrumented to measure mean arterial pressure (MAP), heart rate (HR), and splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA, respectively). Vagal afferent stimulation (2-50Hz) decreased cardiorespiratory parameters and increased neuronal Ca2+ measured by in vivo photometry and in vitro slice imaging of nTS GCaMP8m. Vagotomy attenuated both these reflex and neuronal Ca2+ responses compared to shams. Vagotomy also reduced presynaptic Ca2+ responses to stimulation (Cal-520 imaging) in the nTS slice. The decrease in HR, SSNA and PhrNA due to nTS nanoinjection of exogenous glutamate also was tempered following vagotomy. This effect was not restored by blocking excitatory amino acid transporters. However, the blunted responses were mimicked by NMDA, not AMPA, nanoinjection and were associated with reduced NR1 subunits in the nTS. Altogether, these results demonstrate that vagotomy induces multiple changes within the nTS tripartite synapse that influence cardiorespiratory reflex responses to afferent stimulation. KEY POINTS: Multiple mechanisms within the nucleus tractus solitarii (nTS) contribute to functional changes following vagal nerve transection. Vagotomy results in reduced cardiorespiratory reflex responses to vagal afferent stimulation and nTS glutamate nanoinjection. Blunted responses occur via reduced presynaptic Ca2+ activation and attenuated NMDA receptor expression and function, leading to a reduction in nTS neuronal activation. These results provide insight into the control of autonomic and respiratory function, as well as the plasticity that can occur in response to nerve damage and cardiorespiratory disease.

Frequency-coded patterns of sympathetic vasomotor activity are differentially evoked by the paraventricular nucleus of the hypothalamus in the Goldblatt hypertension model.

The paraventricular nucleus of the hypothalamus (PVN) contains premotor neurons involved in the control of sympathetic vasomotor activity. It is known that the stimulation of specific areas of the PVN can lead to distinct response patterns at different target territories. The underlying mechanisms, however, are still unclear. Recent evidence from sympathetic nerve recording suggests that relevant information is coded in the power distribution of the signal along the frequency range. In the present study, we addressed the hypothesis that the PVN is capable of organizing specific spectral patterns of sympathetic vasomotor activation to distinct territories in both normal and hypertensive animals. To test it, we investigated the territorially differential changes in the frequency parameters of the renal and splanchnic sympathetic nerve activity (rSNA and sSNA, respectively), before and after disinhibition of the PVN by bicuculline microinjection. Subjects were control and Goldblatt rats, a sympathetic overactivity-characterized model of neurogenic hypertension (2K1C). Additionally, considering the importance of angiotensin II type 1 receptors (AT1) in the sympathetic responses triggered by bicuculline in the PVN, we also investigated the impact of angiotensin AT1 receptors blockade in the spectral features of the rSNA and sSNA activity. The results revealed that each nerve activity (renal and splanchnic) presents its own electrophysiological pattern of frequency-coded rhythm in each group (control, 2K1C, and 2K1C treated with AT1 antagonist losartan) in basal condition and after bicuculline microinjection, but with no significant differences regarding total power comparison among groups. Additionally, the losartan 2K1C treated group showed no decrease in the hypertensive response triggered by bicuculline when compared to the non-treated 2K1C group. However, their spectral patterns of sympathetic nerve activity were different from the other two groups (control and 2K1C), suggesting that the blockade of AT1 receptors does not totally recover the basal levels of neither the autonomic responses nor the electrophysiological patterns in Goldblatt rats, but act on their spectral frequency distribution. The results suggest that the differential responses evoked by the PVN were preferentially coded in frequency, but not in the global power of the vasomotor sympathetic responses, indicating that the PVN is able to independently control the frequency and the power of sympathetic discharges to different territories.

Splanchnic sympathetic nerve denervation improves bacterial clearance and clinical recovery in established ovine Gram-negative bacteremia

BackgroundThe autonomic nervous system can modulate the innate immune responses to bacterial infections via the splanchnic sympathetic nerves. Here, we aimed to determine the effects of bilateral splanchnic sympathetic nerve denervation on blood pressure, plasma cytokines, blood bacterial counts and the clinical state in sheep with established bacteremia.MethodsConscious Merino ewes received an intravenous infusion of Escherichia coli for 30 h (1 × 109 colony forming units/mL/h) to induce bacteremia. At 24 h, sheep were randomized to have bilaterally surgically implanted snares pulled to induce splanchnic denervation (N = 10), or not pulled (sham; N = 9).ResultsSplanchnic denervation did not affect mean arterial pressure (84 ± 3 vs. 84 ± 4 mmHg, mean ± SEM; PGroup = 0.7) compared with sham treatment at 30-h of bacteremia. Splanchnic denervation increased the plasma levels of the pro-inflammatory cytokine interleukin-6 (9.2 ± 2.5 vs. 3.8 ± 0.3 ng/mL, PGroup = 0.031) at 25-h and reduced blood bacterial counts (2.31 ± 0.45 vs. 3.45 ± 0.11 log10 [CFU/mL + 1], PGroup = 0.027) at 26-h compared with sham treatment. Plasma interleukin-6 and blood bacterial counts returned to sham levels by 30-h. There were no differences in the number of bacteria present within the liver (PGroup = 0.3). However, there was a sustained improvement in clinical status, characterized by reduced respiratory rate (PGroup = 0.024) and increased cumulative water consumption (PGroup = 0.008) in splanchnic denervation compared with sham treatment.ConclusionIn experimental Gram-negative bacteremia, interrupting splanchnic sympathetic nerve activity increased plasma interleukin-6, accelerated bacterial clearance, and improved clinical state without inducing hypotension. These findings suggest that splanchnic neural manipulation is a potential target for pharmacological or non-pharmacological interventions.

Cervical spinal hemisection effects on spinal tissue oxygenation and long-term facilitation of phrenic, renal and splanchnic sympathetic nerve activity

HypothesesModerate acute intermittent hypoxia (mAIH) elicits plasticity in both respiratory (phrenic long-term facilitation; pLTF) and sympathetic nerve activity (sympLTF) in rats. Although mAIH produces pLTF in normal rats, inconsistent results are reported after cervical spinal cord injury (cSCI), possibly due to greater spinal tissue hypoxia below the injury site. There are no reports concerning cSCI effects on sympLTF. Since mAIH is being explored as a therapeutic modality to restore respiratory and non-respiratory movements in humans with chronic SCI, both effects are important. To understand cSCI effects on mAIH-induced pLTF and sympLTF, partial or complete C2 spinal hemisections (C2Hx) were performed and, 2 weeks later, we assessed: 1) ipsilateral cervical spinal tissue oxygen tension; 2) ipsilateral & contralateral pLTF; and 3) ipsilateral sympLTF in splanchnic and renal sympathetic nerves. MethodsMale Sprague-Dawley rats were studied intact, or after partial (single slice) or complete C2Hx (slice with ∼1 mm aspiration). Two weeks post-C2Hx, rats were anesthetized and prepared for recordings of bilateral phrenic nerve activity and spinal tissue oxygen pressure (PtO2). Splanchnic and renal sympathetic nerve activity was recorded in intact and complete C2Hx rats. ResultsSpinal PtO2 near phrenic motor neurons was decreased after C2Hx, an effect most prominent with complete vs. partial injuries; baseline PtO2 was positively correlated with mean arterial pressure. Complete C2Hx impaired ipsilateral but not contralateral pLTF; with partial C2Hx, ipsilateral pLTF was unaffected. In intact rats, mAIH elicited splanchnic and renal sympLTF. Complete C2Hx had minimal impact on baseline ipsilateral splanchnic or renal sympathetic nerve activity and renal, but not splanchnic, sympLTF remained intact. ConclusionGreater tissue hypoxia likely impairs pLTF and splanchnic sympLTF post-C2Hx, although renal sympLTF remains intact. Increased sympathetic nerve activity post-mAIH may have therapeutic benefits in individuals living with chronic SCI since anticipated elevations in systemic blood pressure may mitigate hypotension characteristic of people living with SCI.

Chronic Vagotomy Alters Glutamate Transmission in the Nucleus Tractus Solitarii (nTS)

The nTS is the first site of integration and processing of sensory afferent information and is one of the crucial brainstem nuclei involved in cardiorespiratory reflexes. This nucleus receives afferent information from many visceral structures in the thorax and abdomen via the peripheral branches of the vagus nerve. Vagal afferents release glutamate (Glu) which initiates signaling processes that leads to chemo- and baroreflex-mediated changes in sympathetic and phrenic nerve activity. Previous studies by us have shown that decreasing afferent input via unilateral vagotomy augmented nTS astrocyte complexity. Astrocytes contribute to regulation of Glu concentration by removing Glu from the synaptic cleft via excitatory amino acid transporters (EAATs). We hypothesized that vagotomy attenuates Glu signaling in the nTS due to increased Glu uptake by greater EAAT expression or function. Surgical transection or sham surgery of the right cervical vagus nerve caudal to the nodose ganglion was performed in male Sprague-Dawley rats. One week after surgery, mean arterial pressure (MAP), heart rate (HR), and splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA) were recorded in anesthetized and ventilated rats. Intact vagus nerves and the right phrenic nerve were acutely transected. Nanoinjections of Glu (30 nl, 1-10 mM) into the nTS decreased MAP, HR, SSNA and PhNA in both sham and vagotomy groups. Vagotomy blunted the HR, SSNA and PhrNA responses, indicating that Glu signaling was attenuated. To determine if increased EAAT function contributed to blunted Glu responses, the EAAT blocker TFB-TBOA (10nM, 60nl) was nanoinjected into the nTS. EAAT blockade mimicked the effect of Glu, producing similar depressor, bradycardic, and apneic responses in both groups. TBOA also enhanced the cardiorespiratory responses to Glu injection in both groups. EAAT blockade did not restore Glu responses in the vagotomy group compared to sham. Taken together, these data suggest that increased EAAT function did not account for the attenuated Glu signaling in the nTS. Further studies are needed to uncover the mechanisms by which vagotomy affects Glu signaling in the nTS. NIH R01 HL098602 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.

Experimental high-thoracic spinal cord injury reduces splanchnic sympathetic nerve activity and is associated with persistent hypotension

Objective: High-thoracic spinal cord injury (SCI) is associated with blood pressure (BP) dysregulation that consists of persistent hypotension, orthostatic intolerance and autonomic dysreflexia. Whilst several mechanisms have been suggested to underlie blood pressure dysregulation post-SCI, the most common inference made is that the sub-lesional sympathetic circuitry is hypoactive due to a loss of medullary input to the sympathetic preganglionic neurons. Yet, a remarkable paucity of data exists from electrophysiological studies that have assessed sympathetic nerve activity (SNA) in the setting of SCI. Aim: Assess the changes in SNA and resting BP in rats with and without high-thoracic SCI. Methods: 24 male Wistar rats (10wks old) were randomized and counterbalanced to receive a severe contusion SCI at the T3 spinal level (n=12) or remain as naïve controls (CON). Two weeks post-SCI, rats underwent our neurovascular terminal preparation. Animals were ventilated, anesthetized with inhalant isoflurane, and underwent catheterization of the left-femoral artery (BP), as well as the right femoral artery and vein for arterial blood gas sampling and i.v. fluid/drugs, respectively. Rats were then transitioned to urethane anesthesia (~2.1g.kg-1 i.v). Next, the splanchnic nerve was isolated from a retroperitoneal approach, cut, and the proximal end was recorded using a suction electrode for the measurement of splanchnic sympathetic nerve activity (sSNA). Following 30 minutes of hemodynamic stabilization, SNA and cardiovascular hemodynamics were sampled for 10 minutes, from which a 1 min recording was selected for analyses. Animals were subsequently administered a hexamethonium bolus (30 mg.kg-1) to block ganglionic transmission and the euthanized with chloral hydrate. Results: SCI animals exhibited lower systolic, diastolic and mean arterial BP (all p&lt;0.007), but heart rate was not different between groups. SCI animals also exhibited lower total sSNA (1.9 ± 0.7 vs. 3.2 ± 1.9 mv.s-1, p=0.038) as well reduced burst height (2.2 ± 1.5 vs. 3.7 ± 0.8 v.s-1, p=0.033), but the number of sSNA spikes.s-1 was higher in SCI vs. CON (182 ± 65 vs. 155 ± 33, p=0.043). Blood gases were stable and similar between groups. Conclusions: Our findings show that persistent hypotension in SCI animals is associated with lower total sub-lesional sSNA. While SCI resulted in more spontaneous sSNA spikes, these spikes fail to coordinate into equally-sized sSNA bursts. International Spinal Research Trust and Natural Sciences and Engineering Research Council of Canada (NSERC). 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.

Altered Neuronal Discharge in the Organum Vasculosum of the Lamina Terminalis Contributes to Dahl Salt-Sensitive Hypertension.

Salt-sensitive hypertension in humans and experimental models is associated with higher plasma and cerebrospinal fluid sodium chloride (NaCl) concentrations. Changes in extracellular NaCl concentrations are sensed by specialized neurons in the organum vasculosum of the lamina terminalis (OVLT). Stimulation of OVLT neurons increases sympathetic nerve activity (SNA) and arterial blood pressure (ABP), whereas chronic activation produces hypertension. Therefore, the present study tested whether OVLT neuronal activity was elevated and contributed to SNA and ABP in salt-sensitive hypertension. Male Dahl salt-sensitive (Dahl S) and Dahl salt-resistant (Dahl R) rats were fed 0.1% or 4.0% NaCl diets for 3 to 4 weeks and used for single-unit recordings of OVLT neurons or simultaneous recording of multiple sympathetic nerves during pharmacological inhibition of the OVLT. Plasma and cerebrospinal fluid Na+ and Cl- concentrations were higher in Dahl S rats fed 4% versus 0.1% or Dahl R rats fed either diet. In vivo single-unit recordings revealed a significantly higher discharge of NaCl-responsive OVLT neurons in Dahl S rats fed 4% versus 0.1% or Dahl R rats. Interestingly, intracarotid infusion of hypertonic NaCl evoked greater increases in OVLT neuronal discharge of Dahl S versus Dahl R rats regardless of NaCl diet. The activity of non-NaCl-responsive OVLT neurons was not different across strain or diets. Finally, inhibition of OVLT neurons by local injection of the gamma-aminobutyric acid agonist muscimol produced a greater decrease in renal SNA, splanchnic SNA, and ABP of Dahl S rats fed 4% versus 0.1% or Dahl R rats. A high salt diet activates NaCl-responsive OVLT neurons to increase SNA and ABP in salt-sensitive hypertension.

Revisiting differential control of sympathetic outflow by the rostral ventrolateral medulla.

The rostral ventrolateral medulla (RVLM) is an important brain region involved in both resting and reflex regulation of the sympathetic nervous system. Anatomical evidence suggests that as a bilateral structure, each RVLM innervates sympathetic preganglionic neurons on both sides of the spinal cord. However, the functional importance of ipsilateral versus contralateral projections from the RVLM is lacking. Similarly, during hypotension, the RVLM is believed to rely primarily on withdrawal of tonic gamma aminobutyric acid (GABA) inhibition to increase sympathetic outflow but whether GABA withdrawal mediates increased activity of functionally different sympathetic nerves is unknown. We sought to test the hypothesis that activation of the ipsilateral versus contralateral RVLM produces differential increases in splanchnic versus adrenal sympathetic nerve activities, as representative examples of functionally different sympathetic nerves. We also tested whether GABA withdrawal is responsible for hypotension-induced increases in splanchnic and adrenal sympathetic nerve activity. To test our hypothesis, we measured splanchnic and adrenal sympathetic nerve activity simultaneously in Inactin-anesthetized, male Sprague-Dawley rats during ipsilateral or contralateral glutamatergic activation of the RVLM. We also produced hypotension (sodium nitroprusside, i.v.) before and after bilateral blockade of GABAA receptors in the RVLM (bicuculline, 5mM 90nL). Glutamate (100mM, 30nL) injected into the ipsilateral or contralateral RVLM produced equivalent increases in splanchnic sympathetic nerve activity, but increased adrenal sympathetic nerve activity by more than double with ipsilateral injections versus contralateral injections (p < 0.05; n = 6). In response to hypotension, increases in adrenal sympathetic nerve activity were similar after bicuculline (p > 0.05), but splanchnic sympathetic nerve activity responses were eliminated (p < 0.05; n = 5). These results provide the first functional evidence that the RVLM has predominantly ipsilateral innervation of adrenal nerves. In addition, baroreflex-mediated increases in splanchnic but not adrenal sympathetic nerve activity are mediated by GABAA receptors in the RVLM. Our studies provide a deeper understanding of neural control of sympathetic regulation and insight towards novel treatments for cardiovascular disease involving sympathetic nervous system dysregulation.

Sympathoexcitatory responses to renal chemosensitive stimuli are exaggerated at nighttime in rats.

The circadian cycle impacts sympathetic nerve activity (SNA), cardiovascular hemodynamics, and renal function. Activation of renal sensory nerves by chemosensory and mechanosensory stimuli reflexively changes efferent SNA and arterial blood pressure (ABP) to maintain homeostasis. However, it is unclear to what extent circadian cycle influences reflex SNA and ABP responses to renal sensory stimuli. Renal, splanchnic, and lumbar SNA and ABP responses to intrarenal arterial infusion of bradykinin or capsaicin and elevated renal pelvic pressure were measured in male and female Sprague-Dawley rats during nighttime (wakeful/active phase) and daytime (inactive phase). Intrarenal arterial bradykinin infusion significantly increased efferent renal SNA, splanchnic SNA, and ABP but not lumbar SNA. Responses were greater during nighttime versus daytime. Similarly, intrarenal arterial capsaicin infusion significantly increased renal SNA and splanchnic SNA, and responses were again greater during nighttime. Elevated renal pelvic pressure increased renal SNA and splanchnic SNA; however, responses did not differ between daytime and nighttime. Finally, afferent renal nerve activity responses to bradykinin were not different between daytime and nighttime. Thus, renal chemokines elicit greater sympathoexcitatory responses at nighttime that cannot be attributed to differences in afferent renal nerve activity. Collectively, these data suggest that the circadian cycle alters the excitability of central autonomic networks to alter baseline SNA and ABP as well as the magnitude of visceral reflexes.NEW & NOTEWORTHY The current study discovers that the circadian cycle influences sympathetic and hemodynamic responses to activation of renal chemosensitive sensory fibers. Sympathetic responses to intrarenal bradykinin or capsaicin infusion were exaggerated during nighttime (active period), but mechanosensitive responses to elevated renal pelvic pressure were not. Importantly, renal afferent nerve responses were not different between nighttime and daytime. These data suggest that the circadian cycle modulates sympathetic responses to visceral afferent activation.

SK Channel Dysfunction in the Hypothalamic Paraventricular Nucleus Contributes to Sympathoexcitation in Dahl Salt‐Sensitive Rats

Salt‐sensitive hypertension is characterized by marked activation of the sympathetic nervous system. Previous studies have consistently demonstrated that increased excitability or activity of pre‐sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) contribute to the increased blood pressure and sympathetic nerve activity (SNA) in salt‐sensitive hypertension; however, the underlying mechanisms have not been fully elucidated. Small conductance calcium‐activated potassium (SK) channels act as negative feedback regulators of neuronal excitability in PVN neurons, and inhibition of SK channel function in the PVN is known to augment SNA and arterial blood pressure. In the present study, we tested the hypothesis that reduced SK channel function in the PVN contributes to sympathoexcitation in Dahl salt‐sensitive rats fed a high salt (HS, 4%) diet compared to a normal salt (NS, 0.4%) diet. Sympathoexcitatory responses to bilateral microinjection of the SK channel blocker apamin into the PVN were assessed in anesthetized 12–14‐week‐old male Dahl salt‐sensitive rats fed a high salt (n=7) or a normal salt (n=6) diet for 4 weeks. Baseline mean arterial pressure (MAP) was significantly elevated in rats fed a HS diet compared to NS (HS 129±4 vs. NS 114±4 mmHg, p&lt;0.05), however baseline heart rate (HR) was not different between groups (HS 411±10 vs. NS 385±13 beats/min, p=0.07). Bilateral microinjection of apamin (12.5pmol, 50nL) into the PVN significantly increased splanchnic SNA in both HS and NS rats compared to baseline, however responses were significantly attenuated in HS rats compared to NS (HS 97±22% vs. NS 249±57%, p&lt;0.01). In contrast, increases in MAP in response to PVN microinjection of apamin were similar between HS and NS rats (HS 24±4 vs. NS 29±4 mmHg, p=0.25), whereas HR responses were variable but not significantly different between groups (HS ‐8.5±9 vs. NS 7.5±7 beats/min, p=0.11). Next, we utilized qPCR to assess mRNA expression of SK1, SK2, and SK3 channels in PVN tissue from HS (n=5) and NS (n=6) rats. Interestingly, there was no significant difference in mRNA levels of either SK1, SK2, or SK3 channels between HS and NS fed rats. In summary, increases in splanchnic SNA due to PVN SK channel blockade with apamin were significantly attenuated in Dahl salt‐sensitive rats fed a HS diet, whereas there was no difference in SK channel expression between rats fed a HS vs. NS diet. These results suggest that dysfunction of SK channels in the PVN, but not decreased expression, contributes to sympathoexcitation Dahl salt‐sensitive rats fed a HS diet.

Unilateral Vagotomy Blunts Cardiorespiratory Responses to Vagal Afferent Stimulation and Nucleus Tractus Solitarii (NTS) Glutamate Microinjection

The nTS of the brainstem is the initial integration site of sensory information from the cardiorespiratory system, including afferents from the vagus nerve carrying input from the heart and lungs. The nTS is an important site of neuroplasticity, which depends on factors including neuronal and astrocytic activity, with the latter regulating glutamate (Glu) signaling. We have shown that unilateral transection of the vagus nerve to decrease afferent input results in morphologic changes in nTS glia. Vagal nerve transection also decreases nTS synaptic transmission studied in vitro. The influence of vagotomy on in vivo cardiorespiratory function via afferent‐driven Glu signaling is unknown. We hypothesized that chronic vagotomy blunts cardiorespiratory responses to vagal afferent stimulation via decreased Glu signaling. Male Sprague‐Dawley rats (6‐wk, N=19) were randomly assigned to vagotomy or sham groups. Right cervical vagus nerve transection caudal to the nodose ganglion or sham surgery was performed. One week after surgery, rats were anesthetized, ventilated, and instrumented to measure mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA, respectively). Intact vagus nerves were transected acutely to prevent efferent stimulation. Left and right vagus nerves were stimulated (20 sec, 0.2mA, 2‐10Hz, 0.5ms pulse width) and responses recorded for 3 minutes. Chronic vagotomy had no effect on baseline cardiorespiratory parameters compared to shams. Vagal nerve stimulation on either side increased MAP, HR, and SSNA and decreased PhrNA in all groups. There were no consistent group differences in MAP, HR, and SSNA responses. However, chronic vagotomy blunted the stimulation‐induced decrease in PhrNA frequency and amplitude and decreased apnea duration on the ipsilateral side compared to contralateral stimulation and to shams. To evaluate the mechanism of altered respiratory responses, Glu was nanoinjected (30nL, 1‐10mM) into the left and right nTS of chronic vagotomy and sham rats. Glu decreased MAP, HR, SSNA, and PhrNA in all groups and on both sides. Chronic vagotomy had minimal effects on MAP and SSNA responses, but blunted the decrease in HR and PhrNA frequency and amplitude during ipsilateral Glu injection compared to contralateral. The blunted responses to vagal afferent stimulation and nTS glutamate suggest that chronic vagotomy alters Glu signaling in part via postsynaptic mechanisms. Additional experiments are needed to determine the mechanisms (including the role of astrocytes) of altered nTS Glu signaling to changes in afferent input in cardiorespiratory health and disease.

Divergent Adaptations in Autonomic Nerve Activity and Neuroimmune Signaling Associated With the Severity of Inflammation in Chronic Colitis.

The autonomic nervous system (ANS) is thought to play a critical role in the anti-inflammatory reflex pathway in acute colitis via its interaction with the spleen and colon. Inflammation in the intestine is associated with a blunting of vagal signaling and increased sympathetic activity. As a corollary, methods to restore sympatho-vagal balance are being investigated as therapeutic strategies for the treatment of intestinal inflammation. Nevertheless, it is indefinite whether these autonomic signaling adaptations in colitis are detrimental or beneficial to controlling intestinal inflammation. In this study, models of moderate and severe chronic colitis are utilized to resolve the correlations between sympatho-vagal signaling and the severity of intestinal inflammation. Spleens and colons were collected from Winnie (moderate colitis), Winnie-Prolapse (severe colitis), and control C57BL/6 mice. Changes to the size and histomorphology of spleens were evaluated. Flow cytometry was used to determine the expression of adrenergic and cholinergic signaling proteins in splenic B and T lymphocytes. The inflammatory profile of the spleen and colon was determined using a RT-PCR gene array. Blood pressure, heart rate, splanchnic sympathetic nerve and vagus nerve activity were recorded. Spleens and colons from Winnie and Winnie-Prolapse mice exhibited gross abnormalities by histopathology. Genes associated with a pro-inflammatory response were upregulated in the colons from Winnie and further augmented in colons from Winnie-Prolapse mice. Conversely, many pro-inflammatory markers were downregulated in the spleens from Winnie-Prolapse mice. Heightened activity of the splanchnic nerve was observed in Winnie but not Winnie-Prolapse mice. Conversely, vagal nerve activity was greater in Winnie-Prolapse mice compared with Winnie mice. Splenic lymphocytes expressing α1 and β2 adrenoreceptors were reduced, but those expressing α7 nAChR and producing acetylcholine were increased in Winnie and Winnie-Prolapse mice. Sympathetic activity may correlate with an adaptive mechanism to reduce the severity of chronic colitis. The Winnie and Winnie-Prolapse mouse models of moderate and severe chronic colitis are well suited to examine the pathophysiology of progressive chronic intestinal inflammation.

The carotid body detects circulating tumor necrosis factor-alpha to activate a sympathetic anti-inflammatory reflex

Recent evidence has suggested that the carotid bodies might act as immunological sensors, detecting pro-inflammatory mediators and signalling to the central nervous system, which, in turn, orchestrates autonomic responses. Here, we confirmed that the TNF-α receptor type I is expressed in the carotid bodies of rats. The systemic administration of TNF-α increased carotid body afferent discharge and activated glutamatergic neurons in the nucleus tractus solitarius (NTS) that project to the rostral ventrolateral medulla (RVLM), where many pre-sympathetic neurons reside. The activation of these neurons was accompanied by an increase in splanchnic sympathetic nerve activity. Carotid body ablation blunted the TNF-α-induced activation of RVLM-projecting NTS neurons and the increase in splanchnic sympathetic nerve activity. Finally, plasma and spleen levels of cytokines after TNF-α administration were higher in rats subjected to either carotid body ablation or splanchnic sympathetic denervation. Collectively, our findings indicate that the carotid body detects circulating TNF-α to activate a counteracting sympathetic anti-inflammatory mechanism.

Patterns of renal and splanchnic sympathetic vasomotor activity in an animal model of survival to experimental sepsis.

Sepsis causes long-term disability, such as immune dysfunction, neuropsychological disorders, persistent inflammation, catabolism, and immunosuppression, leading to a high risk of death in survivors, although the contributing factors of mortality are unknown. The purpose of this experimental study in rats was to examine renal (rSNA) and splanchnic (sSNA) sympathetic nerve activity, as well as baroreflex sensitivity, in acute and chronic post-sepsis periods. The rats were divided into two groups: control group with naïve Wistar rats and sepsis group with 2-mL intravenous inoculation of Escherichia coli at 108 CFU/mL. Basal mean arterial pressure, heart rate, rSNA, sSNA, and baroreflex sensitivity were evaluated in all groups at the acute (6 h) and chronic periods (1 and 3 months). Basal rSNA and sSNA were significantly reduced in the surviving rats, as was their baroreflex sensitivity, for both pressor and hypotensive responses, and this effect lasted for up to 3 months. A single episode of sepsis in rats was enough to induce long-term alterations in renal and splanchnic sympathetic vasomotor nerve activity, representing a possible systemic event that needs to be elucidated. These findings showed that post-sepsis impairment of sympathetic vasomotor response may be one of the critical components in the inability of sepsis survivors to respond effectively to new etiological illness factors, thereby increasing their risk of post-sepsis morbidity.

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.