Blood pressure is maintained during dehydration by hypothalamic

J Physiol 592.17 (2014) pp 3783–3799 3783

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Blood pressure is maintained during dehydration byhypothalamic paraventricular nucleus-driven tonicsympathetic nerve activity

Walter W. Holbein1, Megan E. Bardgett1 and Glenn M. Toney1,2

1Department of Physiology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA2Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA

Key points

� At normal resting mean arterial pressure (MAP), sympathetic nerve activity (SNA) mostlyconsists of respiratory and cardiac rhythmic bursts of action potentials.

� In animal models of sympathetic hyperactivity, elevated SNA and MAP become reliant onactivity of neurones in the hypothalamic paraventricular nucleus (PVN).

� Dehydrated (DH) rats (48 h water deprived) were used as a model of sympathetic hyperactivity.As expected, acute PVN inhibition reduced MAP and integrated splanchnic SNA (sSNA) inDH rats, but had no effect in euhydrated controls. Unexpectedly, the fall of sSNA in DH ratswas due to a reduction of irregular, tonic activity as neither respiratory nor cardiac rhythmicbursting was significantly affected.

� We conclude that MAP is largely maintained during dehydration by PVN-driven tonic SNAand speculate that a normally quiescent tonic component of SNA might also be recruited inchronic diseases (hypertension, heart failure, obesity) where PVN activation drives sympathetichyperactivity.

Abstract Resting sympathetic nerve activity (SNA) consists primarily of respiratory and cardiacrhythmic bursts of action potentials. During homeostatic challenges such as dehydration, thehypothalamic paraventricular nucleus (PVN) is activated and drives SNA in support of arterialpressure (AP). Given that PVN neurones project to brainstem cardio-respiratory regions thatgenerate bursting patterns of SNA, we sought to determine the contribution of PVN to supportof rhythmic bursting of SNA during dehydration and to elucidate which bursts dominantlycontribute to maintenance of AP. Euhydrated (EH) and dehydrated (DH) (48 h water deprived)rats were anaesthetized, bilaterally vagotomized and underwent acute PVN inhibition by bilateralinjection of the GABA-A receptor agonist muscimol (0.1 nmol in 50 nl). Consistent with previousstudies, muscimol had no effect in EH rats (n = 6), but reduced mean AP (MAP; P < 0.001) andintegrated splanchnic SNA (sSNA; P < 0.001) in DH rats (n = 6). Arterial pulse pressure wasunaffected in both groups. Muscimol reduced burst frequency of phrenic nerve activity (P < 0.05)equally in both groups without affecting the burst amplitude–duration integral (i.e. area underthe curve). PVN inhibition did not affect the amplitude of the inspiratory peak, expiratory troughor expiratory peak of sSNA in either group, but reduced cardiac rhythmic sSNA in DH rats only(P < 0.001). The latter was largely reversed by inflating an aortic cuff to restore MAP (n = 5),suggesting that the muscimol-induced reduction of cardiac rhythmic sSNA in DH rats was anindirect effect of reducing MAP and thus arterial baroreceptor input. We conclude that MAP is

C© 2014 The Authors. The Journal of Physiology C© 2014 The Physiological Society DOI: 10.1113/jphysiol.2014.276261

3784 W. W. Holbein and others J Physiol 592.17

largely maintained in anaesthetized DH rats by a PVN-driven component of sSNA that is neitherrespiratory nor cardiac rhythmic.

(Resubmitted 18 April 2014; accepted after revision 16 June 2014; first published online 27 June 2014)Corresponding author G. M. Toney: Department of Physiology, University of Texas Health Science Center at SanAntonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. Email: [email protected]

Abbreviations AP, arterial pressure; AUC, area under the curve; CIH, chronic intermittent hypoxia; DH, dehydrated;ECG, electrocardiogram; EH, euhydrated; EP, expiratory peak; ET, expiratory trough; IP, inspiratory peak; MAP,mean arterial pressure; PNA, phrenic nerve activity; PVN, hypothalamic paraventricular nucleus; Posm, plasmaosmolality; Pprotein, plasma protein; RVLM, rostral ventrolateral medulla; rVRG, rostral ventral respiratory group;SHR, spontaneously hypertensive rats; SNA, sympathetic nerve activity; sSNA, splanchnic sympathetic nerve activity.

Introduction

Ongoing sympathetic nerve activity (SNA) is thedominant source of resting vasomotor tone and istherefore a major determinant of resting arterial pressure(AP). Sympathetic nerve traffic consists primarily ofdiscrete respiratory and cardiac rhythmic bursts of actionpotentials (Adrian et al. 1932; Numao et al. 1987;Darnall & Guyenet, 1990; Dampney, 1994) generated byconvergent respiratory network and arterial baroreceptorinputs to reticulospinal neurones in the rostral ventro-lateral medulla (RVLM) (Sun & Guyenet, 1985; Haselton& Guyenet, 1989; Guyenet et al. 1990; Dampney, 1994;Miyawaki et al. 1996, 2002).

Rhythmic excitation and inhibition of RVLM neuronesby respiratory network inputs generates a multi-modalpattern of rhythmic SNA (Haselton & Guyenet, 1989;Dampney, 1994; Miyawaki et al. 1996, 2002; Paton, 1996).Early work by Traube and Hering (Killip, 1962; Simms et al.2010) suggested that respiratory rhythmic bursting of SNAcontributed to the regulation of AP. This concept has beenfortified recently by studies indicating that enhancementof respiratory rhythmic SNA contributes to developmentand maintenance of elevated AP in models of neurogenichypertension (Czyzyk-Krzeska & Trzebski, 1990; Zoccalet al. 2008; Simms et al. 2009; Toney et al. 2010; Zoccal &Machado, 2010; Moraes et al. 2012).

Brain regions and mechanisms that actively modulaterespiratory rhythmic bursting of SNA are under activeinvestigation (Dick et al. 2009; Simms et al. 2010),but remain incompletely understood. The hypothalamicparaventricular nucleus (PVN) is a brain region wellknown for its capacity to regulate SNA. Another importantfunction of the PVN is to modulate respiratory activity.Indeed, acute activation of the PVN drives inspiratoryand expiratory activity and strengthens respiratoryactivity/coupling (Mack et al. 2002, 2007; Kc et al. 2010).This dual function of PVN is also subject to adaptivechange. For example, PVN activation in rats exposed tochronic intermittent hypoxia (CIH) elicits exaggeratedincreases of sympathetically driven AP and respiratoryactivity compared to normoxic controls (Prabha et al.

2011). Interestingly, pressor and respiratory responsesare prevented by blockade of vasopressin V1a receptorsin the region of RVLM (Kc et al. 2010; Prabha et al.2011). These findings are consistent with anatomicalevidence that descending axons of PVN neurones projectto sympathetic control neurons of the RVLM and to keyrespiratory-related regions of the lateral pons, dorsal andventral respiratory groups in the medulla, and to spinalphrenic motor nuclei (Kc & Dick, 2010). Collectively,functional and anatomical evidence indicates that PVNneurones are probably capable of dynamically modulatingrespiratory rhythmic bursting of SNA.

Pulse rhythmic discharge of arterial baroreceptorsactively buffers transient fluctuations of AP by gradingthe strength of rhythmic GABAergic inhibition ofsympathoexcitatory RVLM neurones (Mifflin, 2001).Attenuated baroreflex buffering is considered a potentiallyimportant contributor to the elevation of SNA thatdrives neurogenic forms of arterial hypertension (Mifflin,2001; Barrett & Malpas, 2005). Although most studiesdocumenting reduced baroreflex function have notdirectly demonstrated how this impacts cardiac rhythmicbursting of SNA, activation of CNS inputs to the centralbaroreflex arc that attenuate synaptic transmission arelikely to weaken pulse rhythmic inhibition of RVLMneurones, thereby reducing the oscillation amplitude ofcardiac rhythmic SNA. In the latter regard, activation of thePVN evokes a net increase of integrated multi-fibre SNAwhile concurrently attenuating arterial baroreflex function(Patel & Schmid, 1988; Page et al. 2011). Thus, availableliterature evidence indicates that PVN activation operatesin both a feed-forward sympathoexcitatory fashionto support/raise AP while also attenuating baroreflexactivity that would otherwise interfere with heightenedsympathoexcitatory drive. Together these actions of PVNhave the potential to increase SNA while also reducingbaroreflex-driven cardiac rhythmic bursting of SNA.

In sum, evidence indicates that PVN activation drivesSNA while also increasing respiration and attenuatingthe arterial baroreflex. Physiological or disease conditionsthat activate the PVN therefore might not only drive a

C© 2014 The Authors. The Journal of Physiology C© 2014 The Physiological Society

J Physiol 592.17 Arterial pressure during dehydration 3785

net increase of SNA, but do so by increasing respiratoryrhythmic bursts while decreasing cardiac rhythmic bursts.

Sympathetic regulatory PVN neurones are activated bynumerous homeostatic challenges but perhaps the mostwell studied is dehydration (Brooks et al. 2004b; Stockeret al. 2004a, 2005), which drives SNA largely by recruitinga monosynaptic glutamatergic projection from PVN to theRVLM (Brooks et al. 2004a; Stocker et al. 2006). Given theevidence discussed above, one might reasonably expect fordehydration to strongly modulate respiratory and cardiacrhythmic bursting of SNA. However, we recently reportedthat this is not the case. Instead, dehydration increased atonic component of splanchnic SNA without changingrespiratory or cardiac rhythmic bursting (Holbein &Toney, 2013). Given this unexpected finding, the pre-sent study sought to establish the role of PVN neuro-nal activity in the patterning of SNA during dehydrationby testing the hypothesis that acute inhibition of PVNneuronal activity in dehydrated rats will reduce AP byselectively reducing a tonic component of SNA. Stateddifferently, despite the capacity of PVN to modulatecardio-respiratory control, we hypothesized that PVNneuronal activity in the specific setting of dehydration doesnot strongly modulate respiratory or cardiac rhythmicbursting of SNA.

Methods

Ethical approval

Experimental and surgical procedures complied withguidelines set forth by the National Institutes of Healthand were approved by the Institutional Animal Care andUse Committee of the University of Texas Health ScienceCenter at San Antonio.

Animals

Adult male Sprague–Dawley rats (n = 23, 250–400 g)(Charles River Laboratories, Wilmington, MA, USA) werehoused in a temperature-controlled room (22–23°C) witha 14:10 h light/dark cycle (lights on at 0700 h). All rats hadcontinuous access to food (Harlan Teklad LM-485, 0.3%NaCl), but water was withheld from dehydrated (DH) ratsfor 48 h prior to initiating experimental protocols.

Experimental procedures

On the day of each experiment, rats were anaesthetizedby intraperitoneal injection of a mixture of α-chloralose(80 mg kg−1) and urethane (800 mg kg−1) (Sigma-Aldrich,St Louis, MO, USA). Catheters (PE-50 tubing) wereimplanted in the left femoral artery and both femoralveins for recording AP and administration of drugs,

respectively. Cervical Vagus nerves were transected toeliminate transmission of pulmonary stretch receptorinputs to the respiratory network and thereby pre-vent network entrainment to the frequency of artificialventilation. Aortic depressor nerves were left intactto retain full transmission of arterial baroreceptorinputs. Rats were instrumented to record a lead 1electrocardiogram. After tracheal cannulation, rats weresubjected to neuromuscular blockade with gallaminetriethiodide (25 mg kg−1 h−1, IV) and artificially ventilatedwith oxygen-enriched room air. End-tidal CO2 wasmaintained between 5.0 and 5.5% by adjusting ventilationrate (80–100 breaths min−1) and/or tidal volume (2–3 ml).Rats were placed in a stereotaxic device and bodytemperature was maintained at 37 ± 1°C with a ventrallylocated water-circulating pad. An adequate depth ofanaesthesia was assessed by absence of a withdrawal reflexbefore neuromuscular blockade and lack of a pressorresponse to foot pinch, thereafter. Supplemental doses ofanaesthetic (10% of initial dose) were given as necessary.

Phrenic and sympathetic nerve recording

To record phrenic nerve activity (PNA), tissue over-lying the left scapula was incised and retracted. Thephrenic nerve was isolated near the brachial plexus,transected and its proximal end placed on a bipolarsilver wire electrode (A-M systems, Sequim, WA, USA;0.005-inch outer diameter). To record splanchnic SNA(sSNA), the left greater splanchnic nerve was exposedthrough a retroperitoneal incision, isolated proximal tothe adrenal gland and placed on a bipolar stainlesssteel wire electrode (A-M systems, 0.005-inch outerdiameter). To insulate recordings from body fluid,each nerve–electrode interface was covered with asilicon-based impression material (Super-Dent Light,Carlisle Laboratories, Bridgetown, Barbados). Signalswere obtained through high-impedance probes connectedto AC amplifiers that were equipped with half-amplitudefrequency filters (band pass: 30–1000 Hz) and a60 Hz notch filter. Nerve signals were amplified(20 000–50 000x), full-wave rectified, RC integrated(τ =10 ms) and digitized at 1.5 kHz using a Micro 1401MKII analog-to-digital converter and Spike2 software (v7.1,Cambridge Electronic Design, Cambridge, UK). Noise insSNA recordings was determined as a 3 min average ofintegrated voltage recorded 5 min after bolus injection ofthe ganglionic blocker hexamethonium (30 mg kg−1, IV).

Chemical inhibition of the PVN

To inhibit neuronal activity in the PVN, the long-actingGABA-A receptor agonist muscimol (Sigma) wasnanoinjected (0.1 nmol in 50 nl) bilaterally into the



PVN as previously described (Stocker et al. 2004a, 2005;Bardgett et al. 2014). Briefly, with the skull levelled betweenbregma and lambda a craniotomy was performed anda glass micropipette was lowered into the PVN at thefollowing coordinates (in mm): AP, 1.8–2.1 caudal tobregma; ML, 0.2–0.4 from midline; DV, 7.5 ventral todura. Prior to injections, recorded variables were allowedto stabilize for 30 min to establish baseline values. Avenous blood sample (0.5 ml) was collected duringthis period for determination of haematocrit, plasmaosmolality and protein concentration. Each blood samplewas replaced with an equal volume of isotonic saline.Muscimol or vehicle (artificial cerebrospinal fluid) wasnanoinjected bilaterally into the PVN using a pneumaticpump (World Precision Instruments, Sarasota, FL, USA)connected to a single-barrelled glass micropipette (tip:�50 μm outer diameter). Injections into PVN were eachmade over 20–30 s, first on one side then the other.Injections were separated by 2–3 min and sites weremarked by including 0.2% rhodamine beads in the injectedsolution.

To determine if any change in cardiac rhythmic burstingof SNA observed after PVN muscimol in DH rats wasa direct result of inhibiting neuronal activity or arosesecondary to the accompanying fall of mean AP (MAP),an additional group of DH rats (n = 5) was instrumentedwith an inflatable cuff around the abdominal aorta torestore MAP (recorded from the forearm brachial artery)during the period when PVN muscimol had producedits full effect (�30 min after injection). Based on evidencethat arterial baroreceptors are responsive to both static andpulsatile changes in AP (Franz, 1969; Thoren et al. 1977;Chapleau & Abboud, 1987; Seagard et al. 1990; Mahdiet al. 2013), we reasoned that pulse (cardiac) rhythmicbaroreceptor afferent input to the sympathetic networkwould be restored by raising MAP to baseline duringPVN inhibition. We further reasoned that this would holdas long as arterial pulse pressure was not significantlychanged during aortic cuff inflation.

Histology

At the end of experiments, rats received an over-dose of α-chloralose/urethane cocktail. Brains wereremoved, post-fixed in 4% paraformaldehyde for 24–48 h,cryoprotected in 30% sucrose in PBS, and then sectionedat 50 μm with a freezing microtome. Injection sites wereidentified by mapping the outermost distribution of beadsonto standard plates from the atlas of Paxinos & Watson(1998). Images from similar rostral–caudal levels of PVNfrom all subjects within each treatment group were over-laid. Hence, the outermost distribution of beads in theoverlaid image revealed the full range of injection siteswithin each group.

Haematocrit, plasma osmolality and protein

Haematocrit was determined from duplicate capillarytubes measured with a Lancer microhaematocrit tubereader (Sigma). Plasma osmolality (Posm) was determinedfrom the average of duplicate plasma samples usinga freezing-point depression osmometer (model 3320,Advanced Instruments, Norwood, MA, USA). Plasmaprotein (Pprotein) was determined by refractometry(VWR International, Buffalo Grove, IL, USA).

Data analysis

Values of sSNA, MAP and PNA were determined from5 min segments of stable data just prior to and 30 minutesafter performing PVN injections. Changes in sSNA (μV)were calculated by subtracting the voltage due to noiseafter administration of hexamethonium (3 min average).MAP was calculated as Pdiastolic + (Psystolic–Pdiastolic)/3.

To quantify respiratory rhythmic bursting of SNA,triggered averages of sSNA were constructed before and30 min after muscimol injections. Each average used theonset of 150 consecutive phrenic nerve bursts as the triggerevents. Averages consisted of a 0.3 s pre-trigger and a1.6 s post-trigger period. The latter was equal to theaverage respiratory cycle length. The amplitude of eachrespiratory rhythmic sSNA oscillation was determinedas the difference between the mean voltage of thepost-triggered period and the voltage of each post-triggerpeak or trough (Fig. 2A, left). Amplitudes were expressedin microvolts. The area under the curve (AUC) of eachrhythmic oscillation was also determined and expressedin units of μV.s. Changes in central respiratory drive inresponse to PVN muscimol were quantified from PNAburst-triggered averages of integrated PNA constructedfrom the same PNA bursts used to trigger averages ofsSNA. Neural inspiration and expiration were defined asburst duration and inter-burst interval, respectively. PNAburst amplitudes were quantified as the difference betweenthe average expiratory phase voltage and peak inspiratoryphase voltage. PNA burst amplitude was expressed inmicrovolts. PNA burst AUC was also calculated andexpressed in units of μV.s. PNA burst frequency wasderived from the mean frequency of phrenic onset eventsat baseline and after muscimol. Frequency was expressedas bursts per minute (BPM).

To quantify cardiac rhythmic bursting of sSNA,triggered averages were constructed from �1600 electro-cardiogram (ECG) R-waves concurrently recorded withsegments of sSNA used for construction of PNAburst-triggered averages. Each R-wave-triggered averageconsisted of a 0.15 s post-trigger period (�one cardiaccycle). The amplitude of cardiac rhythmic sSNA wascalculated as the voltage difference between the peakand trough of the oscillation (Fig. 3A, left). Amplitudes



Table 1. Baseline values of recorded variables

Group n MAP (mmHg) HR (beats min−1) sSNA (mV) Posm (mosmol kg−1) Protein (g dl−1) Hct (%)

Euhydrated (EH) 10 106 ± 4 396 ± 14 4.3 ± 0.8 (2.7 ± 0.7) 332 ± 4† 4.6 ± 0.2 47 ± 1Dehydrated (DH) 13 98 ± 6 382 ± 5 7.0 ± 1.0∗ (2.4 ± 0.3) 352 ± 4∗† 5.6 ± 0.1∗ 54 ± 2∗

Values are the mean ± SEM; n, no. of rats; MAP, mean arterial pressure; HR, heart rate; sSNA, splanchnic sympathetic nerve activity;Posm, plasma osmolality; Pprotein, plasma protein concentration; Hct, haematocrit. †Values elevated (�30 mosmol kg−1) in bothgroups due to urethane anaesthesia. ∗P < 0.01 vs. EH. Note that sSNA voltages are values with noise subtracted. Noise was determinedas the signal remaining after ganglionic blockade. Voltages in parentheses are average noise levels in sSNA recordings of each group.

were expressed in microvolts. AUC was calculated for onecardiac cycle and expressed in units of μV.s.

Values of MAP and arterial pulse pressure weredetermined over data segments used for constructingtriggered averages by constructing R-wave-triggeredaverages of AP using the same �1600 ECG R-waves usedto construct averages of sSNA. From triggered averagesof AP, pulse pressure was determined as the differencebetween the signal minimum (diastolic pressure) andmaximum (systolic pressure). MAP was calculated asdescribed above.

Statistics

Baseline integrated sSNA, MAP, PNA burst amplitude/frequency, ETCO2 , haematocrit, Posm and Pprotein wereeach compared across euhydrated (EH) and DH groupswith unpaired Student’s t tests. Effects of PVN muscimolacross groups on integrated sSNA, MAP, heart rate (HR),PNA bursting (amplitude, AUC and frequency), neuralinspiratory and expiratory duration, and rhythmic sSNAburst amplitudes were analysed by two-way analysisof variance (ANOVA). When significant F-values wereobtained, independent t tests with layered Bonferronicorrections were performed for pair-wise comparisonsbetween groups. Statistical tests were performed usingPrism software (v5.0, GraphPad, La Jolla, CA, USA). In allcases, a critical value of P<0.05 was considered statisticallysignificant.

Results

Effects of water deprivation on sSNA,haemodynamics and haematology

Table 1 shows baseline values of recorded variables. Aspreviously reported (Stocker et al. 2004a, 2005; Holbein& Toney, 2013), EH and DH rats had similar baselinevalues of MAP and HR whereas haematocrit, plasmaprotein concentration and plasma osmolality were eachsignificantly elevated in the DH group (P < 0.01).These haematology values indicate that DH rats wereboth hyperosmotic and hypovolaemic. Consistent with

literature evidence (Colombari et al. 2011; Holbein &Toney, 2013), the mean voltage of integrated sSNA wassignificantly greater in DH than EH rats (P < 0.05). Thisdifference is probably not attributable to differences inthe size of nerve bundles selected or to variations in thequality of nerve recordings as electrical noise (values inparentheses), quantified as the signal voltage remainingafter ganglionic blockade, was similar in both groups.Because noise was subtracted from the mean voltage ofintegrated sSNA, the magnitude of noise was not a factorin these between-group differences.

Effects of water deprivation on baseline respiratoryparameters

As recently reported (Holbein & Toney, 2013), for similarvalues of ETCO2 across groups (EH: 5.6 ± 0.2%; DH:5.5 ± 0.1%), neural inspiratory time (EH: 0.50 ± 0.05 s;DH: 0.51 ± 0.03 s), neural expiratory time (EH:1.16 ± 0.08 s; DH: 1.05 ± 0.05 s), PNA burst amplitude(EH: 11.9 ± 2.0 μV; DH: 10.8 ± 2.0 μV) and PNA burstAUC (EH: 3.2 ± 0.5 μV.s; DH, 3.6 ± 0.7 μV.s) were allsimilar in EH and DH rats (n = 6 per group), indicatingthat dehydration had no effect on the strength of centralrespiratory drive. Note that ventilation parameters (depthand frequency) were similar in EH and DH rats and werenot affected by PVN inhibition in either group.

PVN support of integrated sSNA, MAP and PNA

Figure 1A shows the response of an EH (left) and DH(right) rat to inhibition of PVN by nanoinjection ofmuscimol. As previously reported for lumbar and renalSNA (Stocker et al. 2004b, 2005), integrated sSNA waslargely unaltered by muscimol in the EH rat but wasreduced in the DH rat. Likewise, AP was unchanged inthe EH rat but promptly fell in the DH rat. Muscimolhad little effect on PNA burst amplitude in either sub-ject, though a slight increase can be seen in the EH rat.Summary data (n = 6 per group) in Fig. 1B show that PVNmuscimol was without effect on integrated sSNA (top, left)or MAP (top, right) in EH rats (sSNA: −0.4 ± 0.2 μV;



MAP: −2 ± 3 mmHg), but significantly (P < 0.001)reduced values in DH rats (sSNA: −3.8 ± 0.8 μV; MAP:−28 ± 4 mmHg). Bonferroni post hoc tests revealedthat muscimol-induced reductions of sSNA and MAPwere significantly greater in DH rats than EH controls(P < 0.001). Interestingly, ANOVA testing revealed thatvoltages of integrated sSNA after PVN muscimol were nolonger different in DH compared to EH rats. Figure 1Balso shows that PVN muscimol had no effect on PNAburst amplitude (bottom, left) in EH (+6.2 ± 2.3 μV)or DH (+4.5 ± 2.1 μV) rats, and the average AUC of

PNA bursts (bottom, middle) was likewise unaffected(EH: +0.8 ± 0.5 μV.s; DH: −0.3 ± 0.2 μV.s). Bycontrast, PNA burst frequency (bottom, right) wassignificantly (P < 0.001) reduced after PVN muscimolin both groups (EH: −5.2 ± 0.8 BPM; DH: −7.8 ± 0.6BPM). The reduction of PNA frequency was mainlydue to significant lengthening of expiratory duration(P < 0.05) in both groups (EH: +0.44 ± 0.07 s; DH:+0.51 ± 0.07 s) as inspiratory durations were unchanged(EH: −0.07 ± 0.04 s; DH: −0.06 ± 0.04 s). Injection ofvehicle into PVN did not affect recorded variables (n = 5;

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Figure 1. Effects of chemical inhibition of PVN on sSNA, AP and PNAA, nanoinjection of muscimol (0.1 nmol in 50 nl per side) into PVN had little effect on sSNA, AP or PNA in aEH rat (left), but caused a prompt fall of sSNA and AP without affecting PNA in a dehydrated (DH) rat (right).Arrows indicate times of muscimol injection. B, summary data (n = 6 per group) showing the effects of PVNnanoinjection of muscimol on sSNA (top, left) and MAP (top, right). In EH rats, muscimol had no effect on eithervariable (compare open and light grey bars). In DH rats, muscimol significantly decreased sSNA and MAP (compareblack and dark grey bars). Note that after PVN muscimol the mean voltage of sSNA in DH rats fell to a value notdifferent from that of EH rats. PVN muscimol had no effect on the amplitude or area under the curve of PNA burst,but decreased (P < 0.05) PNA burst frequency in both groups (bottom left). Collectively, the data indicate thatPVN neuronal activation in DH rats supports sSNA and MAP but does not modulate respiratory activity. ∗P < 0.01vs. EH; †P < 0.01 vs. baseline. Summary data are the mean ± SEM.



data not shown). Collectively, the data in Fig. 1 indicatethat PVN neuronal activity during dehydration supportsongoing sSNA and MAP, but not the strength of neuralinspiration.

Effects of PVN inhibition on respiratory and cardiacrhythmic sSNA and pulse pressure

Summary PNA burst-triggered averages of sSNA fromEH (left) and DH (right) rats (n = 6 per group) areshown in Fig. 2A. Note that PVN muscimol had noeffect on the duration, onset latency or amplitude ofthe inspiratory peak (IP), expiratory trough (ET) orexpiratory peak (EP) of respiratory rhythmic sSNA (inset:left) in either EH (left) or DH (right) rats. In DH ratsonly, the mean level of sSNA fell, consistent with effectson integrated sSNA shown in Fig. 1B (top, left). Figure 2Bshows R-wave-triggered averages of sSNA from EH (left)and DH (right) rats (n = 6 per group). Whereas PVNmuscimol had no effect on the oscillation amplitudeor mean voltage (inset: left) of cardiac rhythmic sSNA

in EH rats, it reduced the oscillation amplitude in DHrats (right). Consistent with effects on integrated sSNA(Fig. 1B; top, left), PVN muscimol reduced mean voltagein R-wave-triggered averages from DH rats. SummaryR-wave-triggered averages of AP from EH (left) and DH(right) rats (n = 6 per group) are shown in Fig. 2C.Whereas PVN muscimol had no effect on pulse pressurein EH or DH rats, it reduced MAP in R-wave-triggeredaverages from DH rats. The latter is consistent with effectsof muscimol on MAP shown in Fig. 1B (top, right).

Analysis of the above data is summarized inFig. 3. PVN muscimol significantly reduced the meanvoltage of PNA-triggered sSNA averages from DH rats(−3.7 ± 0.5 μV; P < 0.005), but had no effect onaverages from EH controls (−0.4 ± 0.4 μV) (Fig. 3A).PVN muscimol did not affect the amplitude (top, right)or AUC (bottom, left) of respiratory rhythmic bursts ofsSNA in either group. Figure 3A (bottom, right) also showsthat the average value of sSNA supported by PVN neuro-nal activity (i.e. PVN-dependent sSNA) was significantlygreater in DH rats compared to EH controls (P < 0.01).Similarly, PVN-dependent sSNA within each rhythmic

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Figure 2. Effect of PVN injection ofmuscimol on rhythms of sSNAA, nanoinjection of muscimol into the PVN didnot change the mean voltage or amplitudes ofrespiratory rhythmic peaks of PNAburst-triggered averages of sSNA (inset) in EHcontrol rats (n = 6, left). In DH rats (n = 6,right), PVN muscimol reduced mean sSNAwithout affecting amplitudes of respiratoryrhythmic peaks. B, similarly, injection ofmuscimol into the PVN did not change themean voltage or the amplitude of the cardiacrhythmic oscillation of R-wave-triggeredaverages of sSNA (inset) in EH rats, butreduced mean sSNA and the cardiac oscillationamplitude in WD rats (right). C, data fromR-wave-triggered AP averages confirm thatPVN muscimol did not change MAP or pulsepressure (inset) in EH rats (left), but reducedMAP in DH rats (right) without changing pulsepressure (right). Data are the mean ± SEM.



component (IP, ET, EP) was also significantly greaterin DH rats. Interestingly, PVN neuronal activity in DHrats contributed similarly to mean sSNA voltage and toeach respiratory rhythmic component of sSNA, suggestingthat PVN activation causes a uniform upward shift ofsSNA voltage such that the magnitude of PVN-drivensSNA is similar across all phases of the respiratory cycle.Collectively, the data in Figs 2A and 3A indicate that PVNneuronal activity during dehydration supports a greater

level of tonic sSNA and does not modulate respiratoryrhythmic bursting.

The summary data in Fig. 3B indicate that dehydrationincreased resting sSNA and PVN muscimol causeda significantly greater (P < 0.01) reduction of meansSNA voltage (top, left) in R-wave-triggered averagesfrom DH rats (−3.9 ± 0.6 μV) compared to EHcontrols (−0.4 ± 0.2 μV). The oscillation amplitude ofcardiac rhythmic sSNA (top, right) was unaffected in

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Figure 3. Summary data of effects of PVNmuscimol on sSNA rhythms and APA, bar graphs showing that PVN muscimolreduced (P < 0.01) the mean voltage (top,left) of PNA-triggered averages of sSNA (seeFig. 2A) in DH rats (n = 6) (compare black anddark grey bars), but not in EH controls(n = 6). By contrast, PVN muscimol had noeffect on either the amplitude (top, right) orthe area under the curve (bottom, left) of theinspiratory peak (IP), expiratory trough (ET) orexpiratory peak (EP) of sSNA. SubtractingsSNA values after PVN muscimol from thoseat baseline revealed that PVN-dependentmean sSNA (bottom, right) was significantlygreater (P < 0.01) in DH than EH rats.Analysis further revealed that PVN-dependentsSNA during IP, ET and EP were not differentfrom the mean in either group. Thus, PVNinhibition caused equivalent reductions ofsSNA across all phases of the respiratorycycle. B, summary data of R-wave-triggeredaverages of sSNA (see Fig. 2B) showing thatPVN muscimol in EH rats (compare open andlight grey bars) had no effect on mean sSNA(top, left) and no effect on either theamplitude (top, right) or the area under thecurve (AUC; bottom, left) of cardiac rhythmicsSNA. By contrast, PVN inhibition in DH rats(compare black and dark grey bars) reduced(P < 0.01) mean sSNA and cardiac rhythmicsSNA oscillation amplitude and AUC.Subtracting values of R-wave-triggered sSNAafter PVN muscimol from those at baselinerevealed that PVN-dependent mean sSNA(bottom, right) was again significantly greater(P < 0.01) in DH rats than EH controls. C,although PVN muscimol reduced (P < 0.01)MAP in DH rats (left), it did not affect pulsepressure (right) in either group (see Fig. 2C).These data indicate that PVN neuronal activityin DH rats contributes to mean sSNA and itscardiac rhythmicity but not respiratoryrhythmic sSNA. ∗P < 0.01 vs. control;†P < 0.01 vs. baseline; NS, not significant.Summary data are mean ± SEM.



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Figure 4. Effects of restoring MAP on PVN inhibition induced lowering of cardiac rhythmic sSNA in DHratsA, a representative experiment showing the fall of MAP and integrated sSNA caused by PVN muscimol in a DHrat. During the nadir of MAP, inflation of an aortic cuff restored MAP toward baseline. Expanded traces (�threecardiac cycles) below show the ECG (grey) and simultaneously recorded sSNA at baseline (left), during the PVNmuscimol-induced nadir of sSNA (centre) and during restoration of MAP by cuff inflation (right). Note that cardiacrhythmic bursts at baseline and during cuff inflation are similar, and larger than during PVN inhibition. B, analysis ofcardiac rhythmic sSNA (top) revealed that PVN muscimol reduced the amplitude of the cardiac rhythmic oscillation(grey line) compared to baseline (black line). Amplitude was largely restored when MAP was returned towardbaseline (grey dashed line). Note that oscillation amplitudes in R-wave-triggered sSNA averages are somewhatless than in example traces (A, bottom) due to effects of averaging across many (�1600) cardiac cycles (seeMethods for details). R-wave-triggered averages of AP revealed that neither PVN muscimol nor aortic cuff inflationaffected arterial pulse pressure (bottom), but each graded the level of MAP. C, group data (n = 5) indicate thatPVN muscimol significantly reduced the mean voltage of ongoing sSNA. Restoring MAP did not affect eithermean sSNA voltage (top left) or PVN-dependent sSNA (top right). However, restoring MAP (grey bars) significantlyincreased the amplitude (bottom left) and AUC (bottom right) of cardiac rhythmic sSNA as compared to valuesbefore restoration of MAP (black bars). Note that the cardiac oscillation of sSNA determined at baseline is not



EH rats (+0.1 ± 0.2 μV), but was significantly reduced(P < 0.001) in DH rats (−3.8 ± 0.3 μV). Differentialeffects of PVN muscimol on cardiac rhythmic sSNA wasalso evident as a significant reduction (P < 0.01) inthe AUC of the cardiac rhythmic oscillation (bottom,left) in DH rats (−0.13 ± 0.1 μV.s) but not EHcontrols (+0.01 ± 0.01 μV.s). Figure 3B also shows thatPVN-dependent sSNA (bottom, right) was significantlygreater (P < 0.01) in DH (3.9 ± 0.6 μV) than EH(0.4 ± 0.2 μV) rats and was quantitatively similar to valuesdetermined from analysis of PNA burst-triggered averagesof sSNA (see Fig. 3A, bottom, right). Consistent withresults shown in Figs 1B (right) and 2C, Fig. 3C shows thatwhereas MAP determined from R-wave-triggered averages(left) was unaffected by PVN muscimol in EH rats, it wassignificantly (P < 0.001) reduced in DH rats. Arterial pulsepressure was unaffected by PVN muscimol in either group(right).

Effects of restoring MAP on PVN inhibition-inducedlowering of cardiac rhythmic sSNA

To determine the extent to which the muscimol-inducedreduction of cardiac rhythmic sSNA in DH rats wasa direct effect of inhibiting PVN neuronal activity orwas secondary to the accompanying fall of MAP, aseparate group of DH rats (n = 5) was instrumentedwith a cuff surrounding the abdominal aorta. Figure 4Ashows an example cuff inflation experiment in a DH rat.Note that PVN muscimol elicited the expected fall ofsSNA and MAP (compared to Fig. 1A, right). When thecuff was inflated to restore MAP, ongoing sSNA under-went little change, but expanded ECG (grey trace) andsSNA (black trace) waveforms (bottom) show that themuscimol-induced reduction of cardiac rhythmic burstingwas largely restored by cuff inflation. Figure 4B (top) showsR-wave-triggered sSNA averages constructed from rawdata in Fig. 4A at baseline (black line), after PVN muscimol(grey line), and after muscimol and cuff inflation (darkgrey dashed line). Again note that muscimol reduced themean voltage and cardiac rhythmic oscillation amplitudeof sSNA. Figure 4B (bottom) shows R-wave-triggered APaverages from the raw data in Fig. 4A. Note that althoughPVN muscimol reduced MAP and cuff inflation restoredit toward baseline, neither had an obvious effect on pulsepressure.

Summary data in Fig. 4C show that PVN muscimolsignificantly reduced mean sSNA voltage (−1.8 ± 0.5 μV;

P < 0.05; top, left), cardiac oscillation amplitude(−1.0 ± 0.2 μV; P < 0.05; bottom, left) and cardiacoscillation AUC (−0.04 ± 0.01 μV.s; P < 0.05; bottom,right). Inflating the cuff had no effect on meansSNA voltage, but restored cardiac oscillation amplitude(+0.4 ± 0.4 μV) and AUC (+0.05 ± 0.02 μV.s) to base-line. PVN-dependent sSNA (top, right) was similar beforeand after restoration of MAP. Figure 4D shows summarydata from R-wave-triggered AP averages. MAP (left) wassignificantly reduced by PVN muscimol (P < 0.001) andwas restored by cuff inflation (P < 0.001). Neither PVNinhibition nor cuff inflation significantly changed arterialpulse pressure (right). Collectively, the data in Fig. 4indicate that PVN neuronal activity in DH rats supportsa greater level of tonic sSNA without directly altering itscardiac rhythmic bursting.

Histology

Figure 5A is a representative photomicrograph of a brainsection through the PVN and shows the distributionof fluorescent microspheres co-injected with muscimol.Figure 5B shows a schematic drawing of PVN fromrostral (top) to caudal (bottom). Grey regions representthe overall distribution of injected microspheres. Notethat muscimol injections were made bilaterally and beaddistributions were generally symmetrical on the right andleft sides. Therefore, injection sites are shown unilaterally– EH on the left, DH on the right. In both groups,injections targeted the dorsal and lateral parvocellularregions throughout the rostral caudal extent of PVN. Thecentral magnocellular region was unavoidably targeted aswell.

Discussion

Studies in anaesthetized rats indicate that activity of hypo-thalamic PVN neurones does not normally contribute tosupport of ongoing SNA or MAP (Stocker et al. 2004b,2005). The situation is quite different in DH rats whereacute PVN inhibition causes a significant fall of MAPthat studies have previously shown to be accompanied byreductions of both renal and lumbar SNA (Stocker et al.2004b, 2005). Here we showed that the fall of MAP was alsoaccompanied by a reduction of sSNA. The PVN activationduring dehydration appears to drive sympathetic activityto multiple end organs. This is probably neededto raise systemic vascular resistance sufficiently to

different from that determined after restoration of MAP. D, PVN muscimol reduced MAP and cuff inflation restoredit to baseline (left). Neither muscimol nor cuff inflation affected arterial pulse pressure (right). These data indicatethat reduced cardiac rhythmic sSNA during PVN inhibition in DH rats is likely to occur secondary to the fall ofMAP. sSNA was integrated with τ = 10 ms. ∗P < 0.05 compared to before cuff inflation; †P < 0.05 compared tobaseline. Summary data are the mean ± SEM.



maintain AP in the face of dehydration-inducedhypovolaemia.

It is notable that PVN inhibition in DH rats causedsSNA to fall to a level that was no longer differentfrom that of EH rats. This indicates that increasedsSNA during dehydration is almost entirely driven bymuscimol-inhibitable PVN neuronal activity. The latterinterpretation is based on comparing sSNA voltages(i.e. microvolts) before and after PVN inhibition acrossgroups of EH and DH rats. Although comparing SNAvoltages is an often used analytical approach (Mandel &Schreihofer, 2009; Simms et al. 2009; Huber & Schreihofer,2010; Mischel & Mueller, 2011; Mueller & Mischel, 2012;Holbein & Toney, 2013), it should be emphasized that

A

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Figure 5. Histological verification of PVN injection sitesA, a representative photomicrograph showing the location offluorescent microspheres following bilateral injections into PVN. B,schematic drawings of rostral middle and caudal planes through thePVN. Grey regions indicate the overlapping distributions of injectedfluorescent microspheres for EH (n = 2–6; left) and DH (n = 2–6;right) rats. Values to the right of each schematic are distances caudalto bregma.

group differences in resting SNA are more frequentlycompared by expressing SNA values as a percentage ofthe ‘maximum’ SNA obtained in each group (Guildet al. 2010). Maximum SNA is typically determinedby evoking one or another sympathoexcitatory reflex(i.e. arterial chemoreflex, somatosympathetic reflex,nasopharyngeal reflex, etc.). Here, we chose not totake this approach due to lack of evidence regardingeffects of dehydration to modulate sympathoexcitatoryreflexes. If dehydration were to differentially modulatesympathoexcitation evoked by various stimuli, then thevalue of maximum SNA used for normalization purposescould differ depending on which reflex was evoked to elicit‘maximum’ SNA.

We acknowledge that comparing SNA voltages is alsonot without potential confounds. Indeed, voltage of SNAcan vary across experiments even when action potentialtraffic is identical. This can arise, for example, if differentsized nerve bundles, each with a different number ofhealthy/active axons, were selected from one experimentto the next. Under such conditions, differences in voltageper se would not necessarily reflect different levels ofCNS activity driving sympathetic outflow. Voltages canalso vary due to differences in the thickness of nervesheaths, variable nerve–electrode contact or changes inamplifier settings from across experiments (Guild et al.2010). Therefore, reporting SNA values in voltage unitsrequires that care be taken to minimize possible sourcesof voltage variation across recordings. In the presentstudy, the supra-adrenal branch of the splanchnic nervewas recorded, which in Sprague-Dawley rats is almostinvariably a large single bundle. Thus, inter-experimentvariability arising from selecting different sized nervebundles was minimized. Because the splanchnic bundleis large, voltage variability due to dissection injury is alsominimal, especially when a single investigator performsall the dissections and recordings – as was the case in thepresent study. Likewise, variability due to differences inelectrode material, nerve–electrode interface and amplifierperformance were largely avoided because all experimentsused the same electrode/insulating material and identicalamplifier settings. In addition, rats of similar age and sizewere used. Therefore, sources of possible variability werecontrolled for and largely avoided in the present study.Support for the latter conclusion comes from the fact thatvoltage due to noise in recordings of sSNA (i.e. the signalremaining after ganglionic blockade) was nearly identicalin EH and DH rats (see Table 1). We acknowledge thatalthough increased extracellular sodium concentration inDH rats might be capable of altering electrophysiologicalproperties of sympathetic fibres and/or soma, suchbiophysical changes seem unlikely to have influencedvoltages of recorded nerves since these would probablycause a generalized voltage increase in recordings from



DH compared to EH rats. However, this was not observedas PNA burst amplitudes were not different across groups.

Literature evidence indicates that acutepharmacological activation of the PVN increasesSNA (Porter & Brody, 1985; Martin et al. 1991; Kenneyet al. 2003) while also stimulating respiration (Patel &Schmid, 1988; Mack et al. 2002, 2007; Kenney et al. 2003;Reddy et al. 2005; Kc & Dick, 2010) and modulatingthe arterial baroreflex (Patel & Schmid, 1988; Dampney,1994; Page et al. 2011). From this it might reasonably beexpected that physiological challenges that activate thePVN would modulate respiratory and cardiac rhythmicbursting of SNA. In the present study, however, weobserved that acute PVN inhibition selectively reduceda tonic component of sSNA in DH but not EH rats.This suggests that PVN activation during dehydrationsupports arterial pressure by increasing a componentof SNA that is neither respiratory nor strongly cardiacrhythmic. As with our comparison of resting SNA acrossgroups (see above), our interpretation that amplitudesof respiratory and cardiac rhythmic sSNA bursting wereunaffected by dehydration or by acute PVN inhibitionis based on comparing burst amplitudes expressed involtage units, not as percentages of baseline (or percentagechange from baseline). Our rationale for reporting burstamplitudes in voltage units is based on the fact thatvoltages obtained through AC-coupled bipolar electrodesare proportional to the density of action potentials thatpass first across one electrode then the other (Guild et al.2010). Like most modern amplifiers, ours is linear over awide range of input voltages and so the voltage amplitudeof a post-trigger burst is not sensitive to the baseline(pre-trigger) level of voltage. On this basis, it would beinaccurate to report bursts of equal voltage amplitude(equivalent action potential density) as different simplybecause baseline activity in one group was larger orsmaller than in the other.

It should be acknowledged that reduced respiratoryand/or cardiac rhythmic bursting among non-splanchnicsympathetic nerves might have contributed to the fallof MAP caused by PVN inhibition in DH rats. Thisbeing the case, the reduction of tonic sSNA in thepresent study might have merely been correlated withthe fall of MAP. Although we cannot rule out thispossibility at the present time, it seems likely that thefall in sSNA contributed to the fall of MAP becausethe mesenteric circulation is a major contributor tototal systemic resistance. Additional experiments will beneeded to determine if PVN inhibition during dehydrationselectively reduces a tonic component of activity recordedfrom multiple sympathetic nerves that regulate vasomotortone.

Although studies indicate that acute PVNactivation increases the frequency and depth ofrespiration and cardio-respiratory coupling through a

vasopressin-dependent mechanism in the RVLM/rostralventral respiratory group (rVRG) (Yeh et al. 1997; Macket al. 2002, 2007; Kc et al. 2010; Kc & Dick, 2010), weobserved that acute PVN inhibition in EH and DH ratscaused only a small reduction of respiratory frequencyand no effect on the depth of inspiration. It thereforeappears that dehydration does not actively recruit thevasopressinergic PVN–RVLM/rVRG pathway (Kc et al.2010) that others have shown to be activated in ratsexposed to chronic intermittent hypoxia (Prabha et al.2011). We did not directly measure the strength ofexpiration, but on the whole it would appear from thepresent findings that ongoing PVN neuronal activityunder normal conditions or during dehydration does notplay a major role in driving respiration, at least not inanaesthetized rats. That respiratory drive was not stronglymodified in DH rats is consistent with our observationthat PVN inhibition also failed to change respiratoryrhythmic bursting of sSNA. It should be emphasized thatfailure of PVN inhibition to change the strength of neuralinspiration or respiratory rhythmic bursting of sSNA inDH rats does not exclude a contribution of respiratoryrhythmic SNA to maintenance of MAP. Indeed, PVNinhibition might have caused an even greater reduction ofMAP in DH rats if not for the persistence of respiratoryrhythmic SNA.

In the present study, we initially observed that PVNinhibition in DH rats concurrently reduced integratedsSNA and MAP. As noted above, this suggests thatongoing sSNA contributes to support of MAP in DHrats, which is consistent with literature evidence (Scroginet al. 1999, 2002; Brooks et al. 2004a,b, 2005; Stockeret al. 2004a; Stocker et al. 2005, 2006; Antunes et al.2006; Holbein & Toney, 2013). It is worth noting thatthe reduction of integrated sSNA by PVN inhibition inDH rats was associated with reduced cardiac rhythmicsSNA bursting. This observation raises the possibility thatPVN neuronal activity during dehydration might activelyfacilitate baroreflex-mediated cardiac rhythmic sSNAinhibition. Literature evidence is consistent with such apossibility (Page et al. 2011). However, if PVN activationduring dehydration were to significantly facilitate thebaroreflex then the amplitude of the cardiac rhythmicsSNA oscillation might be expected to be greater at base-line (i.e. prior to PVN inhibition) in DH rats compared toEH rats. This would be expected because greater cardiacrhythmic inhibition in DH rats would produce peri-ods of greater synaptic inhibition of RVLM (and other)sympathoexcitatory neurons compared to EH rats. Asnoted, however, this was not observed. It therefore seemslikely that reduced cardiac rhythmic bursting of sSNAthat occurred during PVN inhibition in DH rats wasprobably due to reduced pulse rhythmic baroreceptorinhibition that occurred secondary to the accompanyingfall of arterial pressure.



Consistent with the latter interpretation, we observedthat restoring MAP by inflation of an aortic cuff duringPVN inhibition largely normalized the cardiac rhythmicoscillation of sSNA in DH rats. Somewhat unexpectedly,cuff inflation restored MAP without increasing arterialpulse pressure. However, arterial baroreceptors areresponsive to pulsatile and non-pulsatile changes inpressure (Franz, 1969; Thoren et al. 1977; Chapleau& Abboud, 1987; Seagard et al. 1990; Mahdi et al.2013). Consequently, for a given arterial pulse pressure,rhythmic baroreceptor discharge increases in proportionto the mean level of pressure (Franz, 1969; Thorenet al. 1977; Chapleau & Abboud, 1987). On this basis,aortic cuff inflation could have increased baroreceptorinput and restored cardiac rhythmic inhibition of sSNAby raising MAP even without an increase of pulsepressure.

Another observation from the aortic cuff experimentin DH rats is that inflation caused only a small netreduction of integrated sSNA that was not statisticallysignificant (see Fig. 4). The most likely explanation isthat the cardiac rhythmic component of sSNA comprisesonly about 10% of total integrated voltage in dehydratedrats after PVN muscimol (Holbein & Toney, 2013).Consequently, even though cuff inflation largely restoredthe cardiac rhythmic inhibitory oscillation of sSNA, theabsolute voltage loss attributable to these periods ofmore effective pulse rhythmic inhibition was insufficientto produce a statistically significant reduction of totalintegrated sSNA. We cannot entirely exclude the possibilitythat indirect sympathoexcitatory effects of cuff inflationmight have masked the expected magnitude of baroreflexsympathoinhibition. For example, cuff inflation couldhave stimulated SNA by causing lower body ischaemia(Fujii et al. 2003; Mizuno et al. 2011) or by reducing venousreturn to unload cardiopulmonary baroreceptors (Vissinget al. 1989). These are perhaps unlikely explanationsbecause arterial pulse pressure was maintained and itappears that venous return/cardiac output was alsolargely preserved during cuff inflation given that MAPincreased in the face of greater aortic resistance. Thishaemodynamic profile also suggests that it is unlikely thata reduction of carotid body blood flow would have causedsympathoexcitation by activating the arterial chemoreflex.With regard to the latter, arterial chemoreflex activationhas been linked to increased expiratory bursting of SNA(Zoccal et al. 2008; Zoccal & Machado, 2010; Moraes et al.2012) and elevated circulating angiotensin II, which ischaracteristic of dehydration, has been shown to ‘sensitize’the arterial chemoreflex (Peng et al. 2011). The lack ofan increase of expiratory bursting of sSNA in the pre-sent study lends further credence to the argument againstincreased arterial chemoreflex activity contributing toSNA regulation in DH rats before or during cuff inflation.Cuff inflation could have acutely activated the peripheral

renin–angiotensin system (Blaine & Davis, 1971) andthereby offset baroreflex sympathoinhibition by increasingcentral sympathoexcitatory drive (Fink et al. 1980). This,too, seems unlikely because central angiotensin II-inducedsympathoexcitation depends on activation of the PVN(Ferguson, 1988; Ferguson & Kasting, 1988), which wasinhibited with muscimol during cuff inflation. A finalfactor that might have masked a net reduction of sSNAduring cuff inflation is that stimulation of angiotensinII could have facilitated synaptic transmission throughsympathetic ganglia (Aiken & Reit, 1968; Dendorferet al. 2002), thereby amplifying transmission of cardiacrhythmic inputs from sympathetic preganglionic neurons.Whichever factors contributed to the observed effects ofcuff inflation in DH rats, the present findings overallappear consistent with the conclusion that PVN neuronalactivation during dehydration supports heightened sSNAwithout having a prominent direct effect on its cardiacrhythmicity.

As noted above, the most unexpected finding of thepresent study was that the fall of MAP produced byinhibition of PVN in DH rats was accompanied by aselective reduction of a tonic component of sSNA. It isacknowledged that anaesthesia might have differentiallyaffected network behaviour in DH compared to EHrats, thereby activating a tonic component of SNA thatmight not exist while rats are conscious. To the extentthat PVN activation during dehydration does increasea tonic component of SNA and does so to multipleend organs, our findings suggest that this tonic SNAcontributes significantly to neurogenic vasomotor toneand support of AP in the dehydrated state. This ideais not without precedent. Koshiya & Guyenet (1995),for example, used PNA-triggered averaging of sSNA todemonstrate that low-dose intravenous administration ofclonidine lowers MAP and, as in the present study, causesa selective reduction of the pre-trigger level of sSNA. Astriking similarity between their study and ours is thatboth recorded sSNA to monitor patterning of sympatheticactivity. This raises the question of whether tonic SNAis a particularly prominent or perhaps unique featureof splanchnic sympathetic outflow. Consistent with thispossibility, a recent study, again recording sSNA, reportedthat exposure of rats to acute intermittent hypoxia inducedlong-term facilitation of sympathetic activity. Of note isthat the increase of sSNA was characterized by a selectiveincrease of tonic activity without a change of respiratoryrhythmic bursting (Xing & Pilowsky, 2010). Anotherstudy reported that acute activation of peripheral chemo-receptors after elimination of baroreflex transmission andcentral respiratory network activity increased a toniccomponent of both sSNA and lumbar SNA (Koshiya& Guyenet, 1996). The latter observation indicates thattonic activity is not an exclusive feature of splanchnicsympathetic activity.



The source of enhanced PVN-driven tonic SNA duringwater deprivation is currently unknown, but availableevidence suggests that plasma hyperosmolality could playa significant role. Not only does acute internal carotidartery infusion of hypertonic saline increase SNA viaa forebrain- and PVN-dependent mechanism (Chen &Toney, 2001; Brooks et al. 2005; Antunes et al. 2006;Shi et al. 2007), acute carotid infusions of hypotonicsaline to reduce forebrain osmolality reduce MAP indehydrated rats (Brooks et al. 2004b, 2005). Given that theneurohumoral environment of dehydration is complex,additional studies are needed to determine the relativecontribution of hypertonicity versus multiple hormonesand viscero-sensory inputs to PVN-driven tonic SNA inDH rats.

A major question that emerges from the present studyis whether activation of tonic SNA occurs mainly orexclusively in response to acute homeostatic challengessuch as dehydration or if tonic SNA is driven in diseasestates where sympathetic outflow is chronically increased?The answer to this question is not presently known.Of interest, however, are studies in normal animals thathave identified tonic and rhythmic patterns of dischargeamong identified PVN (Chen & Toney, 2003, 2010),RVLM (Tseng et al. 2009), and sympathetic pre- andpost-ganglionic neurones (Darnall & Guyenet, 1990).Thus, neuronal substrates capable of generating bothtonic and rhythmic patterns of SNA clearly exist, butstudies to date have principally focused on modulation ofrhythmic patterns. For example, studies in spontaneouslyhypertensive rats suggest that the IP of SNA plays anexaggerated role in the development (Simms et al. 2009)and maintenance of hypertension (Czyzyk-Krzeska &Trzebski, 1990; Simms et al. 2009). In rats made hyper-tensive by exposure to CIH (Zoccal et al. 2008; Zoccal &Machado, 2010; Moraes et al. 2012) and by treatmentwith angiotensin II and a high salt diet (Toney et al.2010), the late-expiratory peak of SNA is exaggerated andhas been implicated in the accompanying hypertension.Mechanisms driving sympathetic outflow in these chronicmodels are almost certainly not identical to those ofDH rats and experimental differences further confounddirect comparison. Additional studies are needed todetermine how acute versus chronic challenges activatedistinct or common circuit elements and mechanisms togenerate and modulate tonic versus rhythmic patterns ofSNA.

In summary, the present findings indicate that PVNneuronal discharge in anaesthetized dehydrated ratsprimarily supports a tonic component of sSNA –one that is neither respiratory nor strongly cardiacrhythmic. Importantly, this reduction of tonic sSNA byPVN inhibition was linked to the fall of MAP, raisingthe possibility that it subserves vasomotor function.Additional studies are needed to determine if PVN

activation drives tonic SNA to non-splanchnic vascularbeds and to determine the overall role of PVN-driven tonicSNA in maintaining vasomotor tone and arterial pressureduring homeostatic challenges other than dehydrationand in chronic cardiovascular disease models. Studiesare also needed to determine if tonic SNA resultsfrom an irregular pattern of discharge among individualsympathetic–regulatory PVN neurones or if tonic SNA isan emergent property of an extended sympathetic networkin which the PVN participates. A full understanding ofneural mechanisms that generate and modulate tonic SNAcould provide novel avenues for improved treatment ofneurogenic cardiovascular diseases.

References

Adrian ED, Bronk DW & Phillips G (1932). Discharges inmammalian sympathetic nerves. J Physiol 74,115–133.

Aiken JW & Reit E (1968). Stimulation of the cat stellateganglion by angiotensin. J Pharmacol Exp Ther 159, 107–114.

Antunes VR, Yao ST, Pickering AE, Murphy D & Paton JF(2006). A spinal vasopressinergic mechanism mediateshyperosmolality-induced sympathoexcitation. J Physiol 576,569–583.

Bardgett ME, Holbein WW, Herrera-Rosales M & Toney GM(2014). Ang II-salt hypertension depends on neuronalactivity in the hypothalamic paraventricular nucleus but noton local actions of tumor necrosis factor-alpha. Hypertension63, 527–534.

Barrett CJ & Malpas SC (2005). Problems, possibilities, andpitfalls in studying the arterial baroreflexes’ influence overlong-term control of blood pressure. Am J Physiol RegulIntegr Comp Physiol 288, R837–R845.

Blaine EH & Davis JO (1971). Evidence for a renal vascularmechanism in renin release: new observations with gradedstimulation by aortic constriction. Circ Res 28, Suppl 2,118–126.

Brooks VL, Freeman KL & Clow KA (2004a). Excitatory aminoacids in rostral ventrolateral medulla support blood pressureduring water deprivation in rats. Am J Physiol Heart CircPhysiol 286, H1642–H1648.

Brooks VL, Freeman KL & O’Donaughy TL (2004b). Acute andchronic increases in osmolality increase excitatory aminoacid drive of the rostral ventrolateral medulla in rats. Am JPhysiol Regul Integr Comp Physiol 287, R1359–R1368.

Brooks VL, Qi Y & O’Donaughy TL (2005). Increasedosmolality of conscious water-deprived rats supports arterialpressure and sympathetic activity via a brain action. Am JPhysiol Regul Integr Comp Physiol 288, R1248–R1255.

Chapleau MW & Abboud FM (1987). Contrasting effects ofstatic and pulsatile pressure on carotid baroreceptor activityin dogs. Circ Res 61, 648–658.

Chen QH & Toney GM (2001). AT1-receptor blockade in thehypothalamic PVN reduces central hyperosmolality-inducedrenal sympathoexcitation. Am J Physiol Regul Integr CompPhysiol 281, R1844–1853.



Chen QH & Toney GM (2003). Identification andcharacterization of two functionally distinct groups of spinalcord-projecting paraventricular nucleus neurons withsympathetic-related activity. Neuroscience 118, 797–807.

Chen QH & Toney GM (2010). In vivo discharge propertiesof hypothalamic paraventricular nucleus neurons withaxonal projections to the rostral ventrolateral medulla. JNeurophysiol 103, 4–15.

Colombari DS, Colombari E, Freiria-Oliveira AH, Antunes VR,Yao ST, Hindmarch C, Ferguson AV, Fry M, Murphy D &Paton JF (2011). Switching control of sympathetic activityfrom forebrain to hindbrain in chronic dehydration.J Physiol 589, 4457–4471.

Czyzyk-Krzeska MF & Trzebski A (1990). Respiratory-relateddischarge pattern of sympathetic nerve activity in thespontaneously hypertensive rat. J Physiol 426, 355–368.

Dampney RA (1994). Functional organization of centralpathways regulating the cardiovascular system. Physiol Rev74, 323–364.

Darnall RA & Guyenet P (1990). Respiratory modulation ofpre- and postganglionic lumbar vasomotor sympatheticneurons in the rat. Neurosci Lett 119, 148–152.

Dendorfer A, Thornagel A, Raasch W, Grisk O, Tempel K &Dominiak P (2002). Angiotensin II induces catecholaminerelease by direct ganglionic excitation. Hypertension 40,348–354.

Dick TE, Baekey DM, Paton JF, Lindsey BG & Morris KF(2009). Cardio-respiratory coupling depends on the pons.Respir Physiol Neurobiol 168, 76–85.

Ferguson AV (1988). Systemic angiotensin acts at thesubfornical organ to control the activity of paraventricularnucleus neurons with identified projections to the medianeminence. Neuroendocrinology 47, 489–497.

Ferguson AV & Kasting NW (1988). Angiotensin acts at thesubfornical organ to increase plasma oxytocinconcentrations in the rat. Regul Pept 23, 343–352.

Fink GD, Haywood JR, Bryan WJ, Packwood W & Brody MJ(1980). Central site for pressor action of blood-borneangiotensin in rat. Am J Physiol 239, R358–R361.

Franz GN (1969). Nonlinear rate sensitivity of the carotid sinusreflex as a consequence of static and dynamic nonlinearitiesin baroreceptor behaviour. Ann N Y Acad Sci 156, 811–824.

Fujii T, Kurata H, Takaoka M, Muraoka T, Fujisawa Y, ShokojiT, Nishiyama A, Abe Y & Matsumura Y (2003). The role ofrenal sympathetic nervous system in the pathogenesis ofischemic acute renal failure. Eur J Pharmacol 481, 241–248.

Guild SJ, Barrett CJ, McBryde FD, Van Vliet BN, Head GA,Burke SL & Malpas SC (2010). Quantifying sympatheticnerve activity: problems, pitfalls and the need forstandardization. Exp Physiol 95, 41–50.

Guyenet PG, Darnall RA & Riley TA (1990). Rostralventrolateral medulla and sympathorespiratory integrationin rats. Am J Physiol 259, R1063–R1074.

Haselton JR & Guyenet PG (1989). Electrophysiologicalcharacterization of putative C1 adrenergic neurons in therat. Neuroscience 30, 199–214.

Holbein WW & Toney GM (2013). Sympathetic network driveduring water deprivation does not increase respiratory orcardiac rhythmic sympathetic nerve activity. J Appl Physiol(1985) 114, 1689–1696.

Huber DA & Schreihofer AM (2010). Attenuated baroreflexcontrol of sympathetic nerve activity in obese Zucker rats bycentral mechanisms. J Physiol 588, 1515–1525.

Kc P, Balan KV, Tjoe SS, Martin RJ, Lamanna JC, Haxhiu MA& Dick TE (2010). Increased vasopressin transmission fromthe paraventricular nucleus to the rostral medulla augmentscardiorespiratory outflow in chronic intermittenthypoxia-conditioned rats. J Physiol 588, 725–740.

Kc P & Dick TE (2010). Modulation of cardiorespiratoryfunction mediated by the paraventricular nucleus. RespirPhysiol Neurobiol 174, 55–64.

Kenney MJ, Weiss ML, Mendes T, Wang Y & Fels RJ (2003).Role of paraventricular nucleus in regulation of sympatheticnerve frequency components. Am J Physiol Heart Circ Physiol284, H1710–H1720.

Killip T, 3rd (1962). Oscillation of blood flow and vascularresistance during Mayer waves. Circ Res 11, 987–993.

Koshiya N & Guyenet PG (1995). Sympatholytic effect ofclonidine depends on the respiratory phase in rat splanchnicnerve. J Auton Nerv Syst 53, 82–86.

Koshiya N & Guyenet PG (1996). Tonic sympatheticchemoreflex after blockade of respiratory rhythmogenesis inthe rat. J Physiol 491, 859–869.

Mack SO, Kc P, Wu M, Coleman BR, Tolentino-Silva FP &Haxhiu MA (2002). Paraventricular oxytocin neurons areinvolved in neural modulation of breathing. J Appl Physiol92, 826–834.

Mack SO, Wu M, Kc P & Haxhiu MA (2007). Stimulation ofthe hypothalamic paraventricular nucleus modulatescardiorespiratory responses via oxytocinergic innervation ofneurons in pre-Botzinger complex. J Appl Physiol 102,189–199.

Mahdi A, Sturdy J, Ottesen JT & Olufsen MS (2013). Theafferent dynamics of the baroreflex control system. PLoSComput Biol 9, e1003384.

Mandel DA & Schreihofer AM (2009). Modulation of thesympathetic response to acute hypoxia by the caudalventrolateral medulla in rats. J Physiol 587,461–475.

Martin DS, Segura T & Haywood JR (1991). Cardiovascularresponses to bicuculline in the paraventricular nucleus of therat. Hypertension 18, 48–55.

Mifflin SW (2001). What does the brain know about bloodpressure? News Physiol Sci 16, 266–271.

Mischel NA & Mueller PJ (2011) (In)activity-dependentalterations in resting and reflex control of splanchnicsympathetic nerve activity. J Appl Physiol (1985) 111,1854–1862.

Miyawaki T, Goodchild AK & Pilowsky PM (2002). Evidencefor a tonic GABA-ergic inhibition of excitatoryrespiratory-related afferents to presympathetic neurons inthe rostral ventrolateral medulla. Brain Res 924, 56–62.

Miyawaki T, Minson J, Arnolda L, Chalmers J, Llewellyn-SmithI & Pilowsky P (1996). Role of excitatory amino acidreceptors in cardiorespiratory coupling in ventrolateralmedulla. Am J Physiol 271, R1221–R1230.

Mizuno M, Murphy MN, Mitchell JH & Smith SA (2011).Skeletal muscle reflex-mediated changes in sympatheticnerve activity are abnormal in spontaneously hypertensiverats. Am J Physiol Heart Circ Physiol 300, H968–H977.



Moraes DJ, Zoccal DB & Machado BH (2012). Medullaryrespiratory network drives sympathetic overactivity andhypertension in rats submitted to chronic intermittenthypoxia. Hypertension 60, 1374–1380.

Mueller PJ & Mischel NA (2012). Selective enhancement ofglutamate-mediated pressor responses after GABAA receptorblockade in the RVLM of sedentary versus spontaneouswheel running rats. Front Physiol 3, 447.

Numao Y, Koshiya N, Gilbey MP & Spyer KM (1987). Centralrespiratory drive-related activity in sympathetic nerves ofthe rat: the regional differences. Neurosci Lett 81, 279–284.

Page MC, Cassaglia PA & Brooks VL (2011). GABA in theparaventricular nucleus tonically suppresses baroreflexfunction: alterations during pregnancy. Am J Physiol RegulIntegr Comp Physiol 300, R1452–R1458.

Patel KP & Schmid PG (1988). Role of paraventricular nucleus(PVH) in baroreflex-mediated changes in lumbarsympathetic nerve activity and heart rate. J Auton Nerv Syst22, 211–219.

Paton JF (1996). The ventral medullary respiratory network ofthe mature mouse studied in a working heart-brainstempreparation. J Physiol 493, 819–831.

Paxinos GW (1998). (Ed.) The rat brain in stereotaxiccoordinates. Academic Press, San Diego, CA.

Peng YJ, Raghuraman G, Khan SA, Kumar GK & Prabhakar NR(2011). Angiotensin II evokes sensory long-term facilitationof the carotid body via NADPH oxidase. J Appl Physiol(1985) 111, 964–970.

Porter JP & Brody MJ (1985). Neural projections fromparaventricular nucleus that subserve vasomotor functions.Am J Physiol 248, R271–R281.

Prabha K, Balan KV, Martin RJ, Lamanna JC, Haxhiu MA &Dick TE (2011). Chronic intermittent hypoxia-inducedaugmented cardiorespiratory outflow mediated byvasopressin-V1A receptor signalling in the medulla. Adv ExpMed Biol 701, 319–325.

Reddy MK, Patel KP & Schultz HD (2005). Differential role ofthe paraventricular nucleus of the hypothalamus inmodulating the sympathoexcitatory component ofperipheral and central chemoreflexes. Am J Physiol RegulIntegr Comp Physiol 289, R789–R797.

Scrogin KE, Grygielko ET & Brooks VL (1999). Osmolality: aphysiological long-term regulator of lumbar sympatheticnerve activity and arterial pressure. Am J Physiol 276,R1579–R1586.

Scrogin KE, McKeogh DF & Brooks VL (2002). Is osmolality along-term regulator of renal sympathetic nerve activity inconscious water-deprived rats? Am J Physiol Regul IntegrComp Physiol 282, R560–R568.

Seagard JL, van Brederode JF, Dean C, Hopp FA, GallenbergLA & Kampine JP (1990). Firing characteristics ofsingle-fibre carotid sinus baroreceptors. Circ Res 66,1499–1509.

Shi P, Stocker SD & Toney GM (2007). Organum vasculosumlaminae terminalis contributes to increased sympatheticnerve activity induced by central hyperosmolality.Am J Physiol Regul Integr Comp Physiol 293,R2279–R2289.

Simms AE, Paton JF, Allen AM & Pickering AE (2010). Isaugmented central respiratory-sympathetic couplinginvolved in the generation of hypertension? Respir PhysiolNeurobiol 174, 89–97.

Simms AE, Paton JF, Pickering AE & Allen AM (2009).Amplified respiratory–sympathetic coupling in thespontaneously hypertensive rat: does it contribute tohypertension? J Physiol 587, 597–610.

Stocker SD, Cunningham JT & Toney GM (2004a). Waterdeprivation increases Fos immunoreactivity in PVNautonomic neurons with projections to the spinal cord androstral ventrolateral medulla. Am J Physiol Regul Integr CompPhysiol 287, R1172–R1183.

Stocker SD, Hunwick KJ & Toney GM (2005). Hypothalamicparaventricular nucleus differentially supports lumbar andrenal sympathetic outflow in water-deprived rats. J Physiol563, 249–263.

Stocker SD, Keith KJ & Toney GM (2004b). Acute inhibition ofthe hypothalamic paraventricular nucleus decreases renalsympathetic nerve activity and arterial blood pressure inwater-deprived rats. Am J Physiol Regul Integr Comp Physiol286, R719–R725.

Stocker SD, Simmons JR, Stornetta RL, Toney GM & GuyenetPG (2006). Water deprivation activates a glutamatergicprojection from the hypothalamic paraventricular nucleus tothe rostral ventrolateral medulla. J Comp Neurol 494,673–685.

Sun MK & Guyenet PG (1985). GABA-mediated baroreceptorinhibition of reticulospinal neurons. Am J Physiol 249,R672–R680.

Thoren P, Saum WR & Brown AM (1977). Characteristics ofrat aortic baroreceptors with nonmedullated afferent nervefibers. Circ Res 40, 231–237.

Toney GM, Pedrino GR, Fink GD & Osborn JW (2010). Doesenhanced respiratory-sympathetic coupling contribute toperipheral neural mechanisms of angiotensin II-salthypertension? Exp Physiol 95, 587–594.

Tseng WT, Chen RF, Tsai ML & Yen CT (2009). Correlation ofdischarges of rostral ventrolateral medullary neurons withthe low-frequency sympathetic rhythm in rats. Neurosci Lett454, 22–27.

Vissing SF, Scherrer U & Victor RG (1989). Relation betweensympathetic outflow and vascular resistance in the calfduring perturbations in central venous pressure. Evidencefor cardiopulmonary afferent regulation of calf vascularresistance in humans. Circ Res 65, 1710–1717.

Xing T & Pilowsky PM (2010). Acute intermittent hypoxia inrat in vivo elicits a robust increase in tonic sympathetic nerveactivity that is independent of respiratory drive. J Physiol588, 3075–3088.

Yeh ER, Erokwu B, LaManna JC & Haxhiu MA (1997). Theparaventricular nucleus of the hypothalamus influencesrespiratory timing and activity in the rat. Neurosci Lett 232,63–66.

Zoccal DB & Machado BH (2010). Sympathetic overactivitycoupled with active expiration in rats submitted to chronicintermittent hypoxia. Respir Physiol Neurobiol 174,98–101.



Zoccal DB, Simms AE, Bonagamba LG, Braga VA, PickeringAE, Paton JF & Machado BH (2008). Increased sympatheticoutflow in juvenile rats submitted to chronic intermittenthypoxia correlates with enhanced expiratory activity.J Physiol 586, 3253–3265.

Additional Information

Competing Interests

The authors have no competing or conflicting interests relatedto the proposed work.

Author contributions

W.W.H., M.E.B. and G.M.T. developed the hypothesis anddesigned experiments. W.W.H. performed experiments and

collected data. W.W.H. and G.M.T. analysed data and preparedthe manuscript text and figures. W.W.H., M.E.B. and G.M.T.revised the manuscript and all are qualified authors. The finalversion of the manuscript was approved by all authors.

Funding

This research was supported by NIH grants HL102310 andHL088052 (G.M.T.). M.E.B. was supported by NIH traininggrant T32 HL07446.

Acknowledgements

We gratefully acknowledge Mary Ann Andrade, Alfredo S.Calderon and Myrna Herrera-Rosales for excellent technicalassistance.


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