Blood pressure is maintained during dehydration by hypothalamic paraventricular nucleus-driven tonic sympathetic nerve activity

Abstract

Resting sympathetic nerve activity (SNA) consists primarily of respiratory and cardiac rhythmic bursts of action potentials. During homeostatic challenges such as dehydration, the hypothalamic paraventricular nucleus (PVN) is activated and drives SNA in support of arterial pressure (AP). Given that PVN neurones project to brainstem cardio-respiratory regions that generate bursting patterns of SNA, we sought to determine the contribution of PVN to support of rhythmic bursting of SNA during dehydration and to elucidate which bursts dominantly contribute to maintenance of AP. Euhydrated (EH) and dehydrated (DH) (48 h water deprived) rats were anaesthetized, bilaterally vagotomized and underwent acute PVN inhibition by bilateral injection of the GABA-A receptor agonist muscimol (0.1 nmol in 50 nl). Consistent with previous studies, muscimol had no effect in EH rats (n = 6), but reduced mean AP (MAP; P < 0.001) and integrated splanchnic SNA (sSNA; P < 0.001) in DH rats (n = 6). Arterial pulse pressure was unaffected 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 under the curve). PVN inhibition did not affect the amplitude of the inspiratory peak, expiratory trough or 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 an indirect effect of reducing MAP and thus arterial baroreceptor input. We conclude that MAP is largely maintained in anaesthetized DH rats by a PVN-driven component of sSNA that is neither respiratory nor cardiac rhythmic.

Key points

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

• In animal models of sympathetic hyperactivity, elevated SNA and MAP become reliant on activity 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) in DH rats, but had no effect in euhydrated controls. Unexpectedly, the fall of sSNA in DH rats was due to a reduction of irregular, tonic activity as neither respiratory nor cardiac rhythmic bursting was significantly affected.

• We conclude that MAP is largely maintained during dehydration by PVN-driven tonic SNA and speculate that a normally quiescent tonic component of SNA might also be recruited in chronic diseases (hypertension, heart failure, obesity) where PVN activation drives sympathetic hyperactivity.

Introduction

Ongoing sympathetic nerve activity (SNA) is the dominant source of resting vasomotor tone and is therefore a major determinant of resting arterial pressure (AP). Sympathetic nerve traffic consists primarily of discrete respiratory and cardiac rhythmic bursts of action potentials (Adrian et al. 1932; Numao et al. 1987; Darnall & Guyenet, 1990; Dampney, 1994) generated by convergent respiratory network and arterial baroreceptor inputs to reticulospinal neurones in the rostral ventrolateral 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 neurones by respiratory network inputs generates a multi-modal pattern 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 SNA contributed to the regulation of AP. This concept has been fortified recently by studies indicating that enhancement of respiratory rhythmic SNA contributes to development and maintenance of elevated AP in models of neurogenic hypertension (Czyzyk-Krzeska & Trzebski, 1990; Zoccal et al. 2008; Simms et al. 2009; Toney et al. 2010; Zoccal & Machado, 2010; Moraes et al. 2012).

Brain regions and mechanisms that actively modulate respiratory rhythmic bursting of SNA are under active investigation (Dick et al. 2009; Simms et al. 2010), but remain incompletely understood. The hypothalamic paraventricular nucleus (PVN) is a brain region well known for its capacity to regulate SNA. Another important function of the PVN is to modulate respiratory activity. Indeed, acute activation of the PVN drives inspiratory and expiratory activity and strengthens respiratory activity/coupling (Mack et al. 2002, 2007; Kc et al. 2010). This dual function of PVN is also subject to adaptive change. For example, PVN activation in rats exposed to chronic intermittent hypoxia (CIH) elicits exaggerated increases of sympathetically driven AP and respiratory activity compared to normoxic controls (Prabha et al. 2011). Interestingly, pressor and respiratory responses are prevented by blockade of vasopressin V1a receptors in the region of RVLM (Kc et al. 2010; Prabha et al. 2011). These findings are consistent with anatomical evidence that descending axons of PVN neurones project to sympathetic control neurons of the RVLM and to key respiratory-related regions of the lateral pons, dorsal and ventral respiratory groups in the medulla, and to spinal phrenic motor nuclei (Kc & Dick, 2010). Collectively, functional and anatomical evidence indicates that PVN neurones are probably capable of dynamically modulating respiratory rhythmic bursting of SNA.

Pulse rhythmic discharge of arterial baroreceptors actively buffers transient fluctuations of AP by grading the strength of rhythmic GABAergic inhibition of sympathoexcitatory RVLM neurones (Mifflin, 2001). Attenuated baroreflex buffering is considered a potentially important contributor to the elevation of SNA that drives neurogenic forms of arterial hypertension (Mifflin, 2001; Barrett & Malpas, 2005). Although most studies documenting reduced baroreflex function have not directly demonstrated how this impacts cardiac rhythmic bursting of SNA, activation of CNS inputs to the central baroreflex arc that attenuate synaptic transmission are likely to weaken pulse rhythmic inhibition of RVLM neurones, thereby reducing the oscillation amplitude of cardiac rhythmic SNA. In the latter regard, activation of the PVN evokes a net increase of integrated multi-fibre SNA while concurrently attenuating arterial baroreflex function (Patel & Schmid, 1988; Page et al. 2011). Thus, available literature evidence indicates that PVN activation operates in both a feed-forward sympathoexcitatory fashion to support/raise AP while also attenuating baroreflex activity that would otherwise interfere with heightened sympathoexcitatory drive. Together these actions of PVN have the potential to increase SNA while also reducing baroreflex-driven cardiac rhythmic bursting of SNA.

In sum, evidence indicates that PVN activation drives SNA while also increasing respiration and attenuating the arterial baroreflex. Physiological or disease conditions that activate the PVN therefore might not only drive a net increase of SNA, but do so by increasing respiratory rhythmic bursts while decreasing cardiac rhythmic bursts.

Sympathetic regulatory PVN neurones are activated by numerous homeostatic challenges but perhaps the most well studied is dehydration (Brooks et al. 2004b; Stocker et al. 2004a, 2005), which drives SNA largely by recruiting a monosynaptic glutamatergic projection from PVN to the RVLM (Brooks et al. 2004a; Stocker et al. 2006). Given the evidence discussed above, one might reasonably expect for dehydration to strongly modulate respiratory and cardiac rhythmic bursting of SNA. However, we recently reported that this is not the case. Instead, dehydration increased a tonic component of splanchnic SNA without changing respiratory or cardiac rhythmic bursting (Holbein & Toney, 2013). Given this unexpected finding, the present study sought to establish the role of PVN neuronal activity in the patterning of SNA during dehydration by testing the hypothesis that acute inhibition of PVN neuronal activity in dehydrated rats will reduce AP by selectively reducing a tonic component of SNA. Stated differently, despite the capacity of PVN to modulate cardio-respiratory control, we hypothesized that PVN neuronal activity in the specific setting of dehydration does not strongly modulate respiratory or cardiac rhythmic bursting of SNA.

Methods

Ethical approval

Experimental and surgical procedures complied with guidelines set forth by the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio.

Animals

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

Experimental procedures

On the day of each experiment, rats were anaesthetized by 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) were implanted in the left femoral artery and both femoral veins for recording AP and administration of drugs, respectively. Cervical Vagus nerves were transected to eliminate transmission of pulmonary stretch receptor inputs to the respiratory network and thereby prevent network entrainment to the frequency of artificial ventilation. Aortic depressor nerves were left intact to retain full transmission of arterial baroreceptor inputs. Rats were instrumented to record a lead 1 electrocardiogram. After tracheal cannulation, rats were subjected to neuromuscular blockade with gallamine triethiodide (25 mg kg⁻¹ h⁻¹, iv) and artificially ventilated with oxygen-enriched room air. End-tidal CO₂ was maintained between 5.0 and 5.5% by adjusting ventilation rate (80–100 breaths min⁻¹) and/or tidal volume (2–3 ml). Rats were placed in a stereotaxic device and body temperature was maintained at 37 ± 1°C with a ventrally located water-circulating pad. An adequate depth of anaesthesia was assessed by absence of a withdrawal reflex before neuromuscular blockade and lack of a pressor response to foot pinch, thereafter. Supplemental doses of anaesthetic (10% of initial dose) were given as necessary.

Phrenic and sympathetic nerve recording

To record phrenic nerve activity (PNA), tissue overlying the left scapula was incised and retracted. The phrenic nerve was isolated near the brachial plexus, transected and its proximal end placed on a bipolar silver wire electrode (A-M systems, Sequim, WA, USA; 0.005-inch outer diameter). To record splanchnic SNA (sSNA), the left greater splanchnic nerve was exposed through a retroperitoneal incision, isolated proximal to the adrenal gland and placed on a bipolar stainless steel wire electrode (A-M systems, 0.005-inch outer diameter). To insulate recordings from body fluid, each nerve–electrode interface was covered with a silicon-based impression material (Super-Dent Light, Carlisle Laboratories, Bridgetown, Barbados). Signals were obtained through high-impedance probes connected to AC amplifiers that were equipped with half-amplitude frequency filters (band pass: 30–1000 Hz) and a 60 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 1401MK II analog-to-digital converter and Spike2 software (v7.1, Cambridge Electronic Design, Cambridge, UK). Noise in sSNA recordings was determined as a 3 min average of integrated voltage recorded 5 min after bolus injection of the ganglionic blocker hexamethonium (30 mg kg⁻¹, iv).

Chemical inhibition of the PVN

To inhibit neuronal activity in the PVN, the long-acting GABA-A receptor agonist muscimol (Sigma) was nanoinjected (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 between bregma and lambda a craniotomy was performed and a glass micropipette was lowered into the PVN at the following coordinates (in mm): AP, 1.8–2.1 caudal to bregma; ML, 0.2–0.4 from midline; DV, 7.5 ventral to dura. Prior to injections, recorded variables were allowed to stabilize for 30 min to establish baseline values. A venous blood sample (0.5 ml) was collected during this period for determination of haematocrit, plasma osmolality and protein concentration. Each blood sample was replaced with an equal volume of isotonic saline. Muscimol or vehicle (artificial cerebrospinal fluid) was nanoinjected bilaterally into the PVN using a pneumatic pump (World Precision Instruments, Sarasota, FL, USA) connected to a single-barrelled glass micropipette (tip: ∼50 μm outer diameter). Injections into PVN were each made over 20–30 s, first on one side then the other. Injections were separated by 2–3 min and sites were marked by including 0.2% rhodamine beads in the injected solution.

To determine if any change in cardiac rhythmic bursting of SNA observed after PVN muscimol in DH rats was a direct result of inhibiting neuronal activity or arose secondary to the accompanying fall of mean AP (MAP), an additional group of DH rats (n = 5) was instrumented with an inflatable cuff around the abdominal aorta to restore MAP (recorded from the forearm brachial artery) during the period when PVN muscimol had produced its full effect (∼30 min after injection). Based on evidence that arterial baroreceptors are responsive to both static and pulsatile changes in AP (Franz, 1969; Thoren et al. 1977; Chapleau & Abboud, 1987; Seagard et al. 1990; Mahdi et al. 2013), we reasoned that pulse (cardiac) rhythmic baroreceptor afferent input to the sympathetic network would be restored by raising MAP to baseline during PVN inhibition. We further reasoned that this would hold as long as arterial pulse pressure was not significantly changed during aortic cuff inflation.

Histology

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

Haematocrit, plasma osmolality and protein

Haematocrit was determined from duplicate capillary tubes measured with a Lancer microhaematocrit tube reader (Sigma). Plasma osmolality (Posm) was determined from the average of duplicate plasma samples using a freezing-point depression osmometer (model 3320, Advanced Instruments, Norwood, MA, USA). Plasma protein (Pprotein) was determined by refractometry (VWR International, Buffalo Grove, IL, USA).

Data analysis

Values of sSNA, MAP and PNA were determined from 5 min segments of stable data just prior to and 30 minutes after performing PVN injections. Changes in sSNA (μV) were calculated by subtracting the voltage due to noise after administration of hexamethonium (3 min average). MAP was calculated as P_diastolic + (P_systolic–P_diastolic)/3.

To quantify respiratory rhythmic bursting of SNA, triggered averages of sSNA were constructed before and 30 min after muscimol injections. Each average used the onset of 150 consecutive phrenic nerve bursts as the trigger events. Averages consisted of a 0.3 s pre-trigger and a 1.6 s post-trigger period. The latter was equal to the average respiratory cycle length. The amplitude of each respiratory rhythmic sSNA oscillation was determined as the difference between the mean voltage of the post-triggered period and the voltage of each post-trigger peak or trough (Fig. 2A, left). Amplitudes were expressed in microvolts. The area under the curve (AUC) of each rhythmic oscillation was also determined and expressed in units of μV.s. Changes in central respiratory drive in response to PVN muscimol were quantified from PNA burst-triggered averages of integrated PNA constructed from the same PNA bursts used to trigger averages of sSNA. Neural inspiration and expiration were defined as burst duration and inter-burst interval, respectively. PNA burst amplitudes were quantified as the difference between the average expiratory phase voltage and peak inspiratory phase voltage. PNA burst amplitude was expressed in microvolts. PNA burst AUC was also calculated and expressed in units of μV.s. PNA burst frequency was derived from the mean frequency of phrenic onset events at baseline and after muscimol. Frequency was expressed as bursts per minute (BPM).

Figure 2. Effect of PVN injection of muscimol on rhythms of sSNA.

A, nanoinjection of muscimol into the PVN did not change the mean voltage or amplitudes of respiratory rhythmic peaks of PNA burst-triggered averages of sSNA (inset) in EH control rats (n = 6, left). In DH rats (n = 6, right), PVN muscimol reduced mean sSNA without affecting amplitudes of respiratory rhythmic peaks. B, similarly, injection of muscimol into the PVN did not change the mean voltage or the amplitude of the cardiac rhythmic oscillation of R-wave-triggered averages of sSNA (inset) in EH rats, but reduced mean sSNA and the cardiac oscillation amplitude in WD rats (right). C, data from R-wave-triggered AP averages confirm that PVN muscimol did not change MAP or pulse pressure (inset) in EH rats (left), but reduced MAP in DH rats (right) without changing pulse pressure (right). Data are the mean ± SEM.

To quantify cardiac rhythmic bursting of sSNA, triggered averages were constructed from ∼1600 electrocardiogram (ECG) R-waves concurrently recorded with segments of sSNA used for construction of PNA burst-triggered averages. Each R-wave-triggered average consisted of a 0.15 s post-trigger period (∼one cardiac cycle). The amplitude of cardiac rhythmic sSNA was calculated as the voltage difference between the peak and trough of the oscillation (Fig. 3A, left). Amplitudes were expressed in microvolts. AUC was calculated for one cardiac cycle and expressed in units of μV.s.

Figure 3. Summary data of effects of PVN muscimol on sSNA rhythms and AP.

A, bar graphs showing that PVN muscimol reduced (P < 0.01) the mean voltage (top, left) of PNA-triggered averages of sSNA (see Fig. 2A) in DH rats (n = 6) (compare black and dark grey bars), but not in EH controls (n = 6). By contrast, PVN muscimol had no effect on either the amplitude (top, right) or the area under the curve (bottom, left) of the inspiratory peak (IP), expiratory trough (ET) or expiratory peak (EP) of sSNA. Subtracting sSNA values after PVN muscimol from those at baseline revealed that PVN-dependent mean sSNA (bottom, right) was significantly greater (P < 0.01) in DH than EH rats. Analysis further revealed that PVN-dependent sSNA during IP, ET and EP were not different from the mean in either group. Thus, PVN inhibition caused equivalent reductions of sSNA across all phases of the respiratory cycle. B, summary data of R-wave-triggered averages of sSNA (see Fig. 2B) showing that PVN muscimol in EH rats (compare open and light grey bars) had no effect on mean sSNA (top, left) and no effect on either the amplitude (top, right) or the area under the curve (AUC; bottom, left) of cardiac rhythmic sSNA. By contrast, PVN inhibition in DH rats (compare black and dark grey bars) reduced (P < 0.01) mean sSNA and cardiac rhythmic sSNA oscillation amplitude and AUC. Subtracting values of R-wave-triggered sSNA after PVN muscimol from those at baseline revealed 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 pulse pressure (right) in either group (see Fig. 2C). These data indicate that PVN neuronal activity in DH rats contributes to mean sSNA and its cardiac rhythmicity but not respiratory rhythmic sSNA. *P < 0.01 vs. control; †P < 0.01 vs. baseline; NS, not significant. Summary data are mean ± SEM.

Values of MAP and arterial pulse pressure were determined over data segments used for constructing triggered averages by constructing R-wave-triggered averages of AP using the same ∼1600 ECG R-waves used to construct averages of sSNA. From triggered averages of AP, pulse pressure was determined as the difference between the signal minimum (diastolic pressure) and maximum (systolic pressure). MAP was calculated as described above.

Statistics

Baseline integrated sSNA, MAP, PNA burst amplitude/frequency, , haematocrit, Posm and Pprotein were each compared across euhydrated (EH) and DH groups with unpaired Student's t tests. Effects of PVN muscimol across groups on integrated sSNA, MAP, heart rate (HR), PNA bursting (amplitude, AUC and frequency), neural inspiratory and expiratory duration, and rhythmic sSNA burst amplitudes were analysed by two-way analysis of variance (ANOVA). When significant F-values were obtained, independent t tests with layered Bonferroni corrections were performed for pair-wise comparisons between groups. Statistical tests were performed using Prism software (v5.0, GraphPad, La Jolla, CA, USA). In all cases, a critical value of P < 0.05 was considered statistically significant.

Results

Effects of water deprivation on sSNA, haemodynamics and haematology

Table 1 shows baseline values of recorded variables. As previously reported (Stocker et al. 2004a, 2005; Holbein & Toney, 2013), EH and DH rats had similar baseline values of MAP and HR whereas haematocrit, plasma protein concentration and plasma osmolality were each significantly elevated in the DH group (P < 0.01). These haematology values indicate that DH rats were both hyperosmotic and hypovolaemic. Consistent with literature evidence (Colombari et al. 2011; Holbein & Toney, 2013), the mean voltage of integrated sSNA was significantly greater in DH than EH rats (P < 0.05). This difference is probably not attributable to differences in the size of nerve bundles selected or to variations in the quality of nerve recordings as electrical noise (values in parentheses), quantified as the signal voltage remaining after ganglionic blockade, was similar in both groups. Because noise was subtracted from the mean voltage of integrated sSNA, the magnitude of noise was not a factor in these between-group differences.

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 ± 1
Dehydrated (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 both groups due to urethane anaesthesia.

^*P < 0.01 vs. EH. Note that sSNA voltages are values with noise subtracted. Noise was determined as the signal remaining after ganglionic blockade. Voltages in parentheses are average noise levels in sSNA recordings of each group.

Effects of water deprivation on baseline respiratory parameters

As recently reported (Holbein & Toney, 2013), for similar values of 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 burst AUC (EH: 3.2 ± 0.5 μV.s; DH, 3.6 ± 0.7 μV.s) were all similar in EH and DH rats (n = 6 per group), indicating that dehydration had no effect on the strength of central respiratory drive. Note that ventilation parameters (depth and frequency) were similar in EH and DH rats and were not 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 of muscimol. As previously reported for lumbar and renal SNA (Stocker et al. 2004b, 2005), integrated sSNA was largely unaltered by muscimol in the EH rat but was reduced in the DH rat. Likewise, AP was unchanged in the EH rat but promptly fell in the DH rat. Muscimol had little effect on PNA burst amplitude in either subject, though a slight increase can be seen in the EH rat. Summary data (n = 6 per group) in Fig. 1B show that PVN muscimol 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 revealed that muscimol-induced reductions of sSNA and MAP were significantly greater in DH rats than EH controls (P < 0.001). Interestingly, ANOVA testing revealed that voltages of integrated sSNA after PVN muscimol were no longer different in DH compared to EH rats. Figure 1B also shows that PVN muscimol had no effect on PNA burst 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). By contrast, PNA burst frequency (bottom, right) was significantly (P < 0.001) reduced after PVN muscimol in both groups (EH: −5.2 ± 0.8 BPM; DH: −7.8 ± 0.6 BPM). The reduction of PNA frequency was mainly due 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 of vehicle into PVN did not affect recorded variables (n = 5; data not shown). Collectively, the data in Fig. 1 indicate that PVN neuronal activity during dehydration supports ongoing sSNA and MAP, but not the strength of neural inspiration.

Figure 1. Effects of chemical inhibition of PVN on sSNA, AP and PNA.

A, nanoinjection of muscimol (0.1 nmol in 50 nl per side) into PVN had little effect on sSNA, AP or PNA in a EH 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 PVN nanoinjection of muscimol on sSNA (top, left) and MAP (top, right). In EH rats, muscimol had no effect on either variable (compare open and light grey bars). In DH rats, muscimol significantly decreased sSNA and MAP (compare black and dark grey bars). Note that after PVN muscimol the mean voltage of sSNA in DH rats fell to a value not different 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 that PVN neuronal activation in DH rats supports sSNA and MAP but does not modulate respiratory activity. *P < 0.01 vs. EH; †P < 0.01 vs. baseline. Summary data are the mean ± SEM.

Effects of PVN inhibition on respiratory and cardiac rhythmic sSNA and pulse pressure

Summary PNA burst-triggered averages of sSNA from EH (left) and DH (right) rats (n = 6 per group) are shown in Fig. 2A. Note that PVN muscimol had no effect on the duration, onset latency or amplitude of the inspiratory peak (IP), expiratory trough (ET) or expiratory peak (EP) of respiratory rhythmic sSNA (inset: left) in either EH (left) or DH (right) rats. In DH rats only, the mean level of sSNA fell, consistent with effects on integrated sSNA shown in Fig. 1B (top, left). Figure 2B shows R-wave-triggered averages of sSNA from EH (left) and DH (right) rats (n = 6 per group). Whereas PVN muscimol had no effect on the oscillation amplitude or mean voltage (inset: left) of cardiac rhythmic sSNA in EH rats, it reduced the oscillation amplitude in DH rats (right). Consistent with effects on integrated sSNA (Fig. 1B; top, left), PVN muscimol reduced mean voltage in R-wave-triggered averages from DH rats. Summary R-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 pressure in EH or DH rats, it reduced MAP in R-wave-triggered averages from DH rats. The latter is consistent with effects of muscimol on MAP shown in Fig. 1B (top, right).

Analysis of the above data is summarized in Fig. 3. PVN muscimol significantly reduced the mean voltage of PNA-triggered sSNA averages from DH rats (−3.7 ± 0.5 μV; P < 0.005), but had no effect on averages 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 of sSNA in either group. Figure 3A (bottom, right) also shows that the average value of sSNA supported by PVN neuronal activity (i.e. PVN-dependent sSNA) was significantly greater in DH rats compared to EH controls (P < 0.01). Similarly, PVN-dependent sSNA within each rhythmic component (IP, ET, EP) was also significantly greater in DH rats. Interestingly, PVN neuronal activity in DH rats contributed similarly to mean sSNA voltage and to each respiratory rhythmic component of sSNA, suggesting that PVN activation causes a uniform upward shift of sSNA voltage such that the magnitude of PVN-driven sSNA is similar across all phases of the respiratory cycle. Collectively, the data in Figs 2A and 3A indicate that PVN neuronal activity during dehydration supports a greater level of tonic sSNA and does not modulate respiratory rhythmic bursting.

The summary data in Fig. 3B indicate that dehydration increased resting sSNA and PVN muscimol caused a significantly greater (P < 0.01) reduction of mean sSNA voltage (top, left) in R-wave-triggered averages from DH rats (−3.9 ± 0.6 μV) compared to EH controls (−0.4 ± 0.2 μV). The oscillation amplitude of cardiac rhythmic sSNA (top, right) was unaffected in EH rats (+0.1 ± 0.2 μV), but was significantly reduced (P < 0.001) in DH rats (−3.8 ± 0.3 μV). Differential effects of PVN muscimol on cardiac rhythmic sSNA was also evident as a significant reduction (P < 0.01) in the AUC of the cardiac rhythmic oscillation (bottom, left) in DH rats (−0.13 ± 0.1 μV.s) but not EH controls (+0.01 ± 0.01 μV.s). Figure 3B also shows that PVN-dependent sSNA (bottom, right) was significantly greater (P < 0.01) in DH (3.9 ± 0.6 μV) than EH (0.4 ± 0.2 μV) rats and was quantitatively similar to values determined from analysis of PNA burst-triggered averages of sSNA (see Fig. 3A, bottom, right). Consistent with results shown in Figs 1B (right) and 2C, Fig. 3C shows that whereas MAP determined from R-wave-triggered averages (left) was unaffected by PVN muscimol in EH rats, it was significantly (P < 0.001) reduced in DH rats. Arterial pulse pressure was unaffected by PVN muscimol in either group (right).

Effects of restoring MAP on PVN inhibition-induced lowering of cardiac rhythmic sSNA

To determine the extent to which the muscimol-induced reduction of cardiac rhythmic sSNA in DH rats was a direct effect of inhibiting PVN neuronal activity or was secondary to the accompanying fall of MAP, a separate group of DH rats (n = 5) was instrumented with a cuff surrounding the abdominal aorta. Figure 4A shows an example cuff inflation experiment in a DH rat. Note that PVN muscimol elicited the expected fall of sSNA and MAP (compared to Fig. 1A, right). When the cuff was inflated to restore MAP, ongoing sSNA underwent little change, but expanded ECG (grey trace) and sSNA (black trace) waveforms (bottom) show that the muscimol-induced reduction of cardiac rhythmic bursting was largely restored by cuff inflation. Figure 4B (top) shows R-wave-triggered sSNA averages constructed from raw data in Fig. 4A at baseline (black line), after PVN muscimol (grey line), and after muscimol and cuff inflation (dark grey dashed line). Again note that muscimol reduced the mean voltage and cardiac rhythmic oscillation amplitude of sSNA. Figure 4B (bottom) shows R-wave-triggered AP averages from the raw data in Fig. 4A. Note that although PVN muscimol reduced MAP and cuff inflation restored it toward baseline, neither had an obvious effect on pulse pressure.

Figure 4. Effects of restoring MAP on PVN inhibition induced lowering of cardiac rhythmic sSNA in DH rats.

A, a representative experiment showing the fall of MAP and integrated sSNA caused by PVN muscimol in a DH rat. During the nadir of MAP, inflation of an aortic cuff restored MAP toward baseline. Expanded traces (∼three cardiac cycles) below show the ECG (grey) and simultaneously recorded sSNA at baseline (left), during the PVN muscimol-induced nadir of sSNA (centre) and during restoration of MAP by cuff inflation (right). Note that cardiac rhythmic bursts at baseline and during cuff inflation are similar, and larger than during PVN inhibition. B, analysis of cardiac 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 toward baseline (grey dashed line). Note that oscillation amplitudes in R-wave-triggered sSNA averages are somewhat less than in example traces (A, bottom) due to effects of averaging across many (∼1600) cardiac cycles (see Methods for details). R-wave-triggered averages of AP revealed that neither PVN muscimol nor aortic cuff inflation affected arterial pulse pressure (bottom), but each graded the level of MAP. C, group data (n = 5) indicate that PVN muscimol significantly reduced the mean voltage of ongoing sSNA. Restoring MAP did not affect either mean sSNA voltage (top left) or PVN-dependent sSNA (top right). However, restoring MAP (grey bars) significantly increased the amplitude (bottom left) and AUC (bottom right) of cardiac rhythmic sSNA as compared to values before restoration of MAP (black bars). Note that the cardiac oscillation of sSNA determined at baseline is not different from that determined after restoration of MAP. D, PVN muscimol reduced MAP and cuff inflation restored it to baseline (left). Neither muscimol nor cuff inflation affected arterial pulse pressure (right). These data indicate that reduced cardiac rhythmic sSNA during PVN inhibition in DH rats is likely to occur secondary to the fall of MAP. sSNA was integrated with τ = 10 ms. *P < 0.05 compared to before cuff inflation; †P < 0.05 compared to baseline. Summary data are the mean ± SEM.

Summary data in Fig. 4C show that PVN muscimol significantly 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 cardiac oscillation AUC (−0.04 ± 0.01 μV.s; P < 0.05; bottom, right). Inflating the cuff had no effect on mean sSNA voltage, but restored cardiac oscillation amplitude (+0.4 ± 0.4 μV) and AUC (+0.05 ± 0.02 μV.s) to baseline. PVN-dependent sSNA (top, right) was similar before and after restoration of MAP. Figure 4D shows summary data from R-wave-triggered AP averages. MAP (left) was significantly reduced by PVN muscimol (P < 0.001) and was restored by cuff inflation (P < 0.001). Neither PVN inhibition nor cuff inflation significantly changed arterial pulse pressure (right). Collectively, the data in Fig. 4 indicate that PVN neuronal activity in DH rats supports a greater level of tonic sSNA without directly altering its cardiac rhythmic bursting.

Histology

Figure 5A is a representative photomicrograph of a brain section through the PVN and shows the distribution of fluorescent microspheres co-injected with muscimol. Figure 5B shows a schematic drawing of PVN from rostral (top) to caudal (bottom). Grey regions represent the overall distribution of injected microspheres. Note that muscimol injections were made bilaterally and bead distributions were generally symmetrical on the right and left sides. Therefore, injection sites are shown unilaterally – EH on the left, DH on the right. In both groups, injections targeted the dorsal and lateral parvocellular regions throughout the rostral caudal extent of PVN. The central magnocellular region was unavoidably targeted as well.

Figure 5. Histological verification of PVN injection sites.

A, a representative photomicrograph showing the location of fluorescent microspheres following bilateral injections into PVN. B, schematic drawings of rostral middle and caudal planes through the PVN. Grey regions indicate the overlapping distributions of injected fluorescent microspheres for EH (n = 2–6; left) and DH (n = 2–6; right) rats. Values to the right of each schematic are distances caudal to bregma.

Discussion

Studies in anaesthetized rats indicate that activity of hypothalamic PVN neurones does not normally contribute to support of ongoing SNA or MAP (Stocker et al. 2004b, 2005). The situation is quite different in DH rats where acute PVN inhibition causes a significant fall of MAP that studies have previously shown to be accompanied by reductions of both renal and lumbar SNA (Stocker et al. 2004b, 2005). Here we showed that the fall of MAP was also accompanied by a reduction of sSNA. The PVN activation during dehydration appears to drive sympathetic activity to multiple end organs. This is probably needed to raise systemic vascular resistance sufficiently to maintain AP in the face of dehydration-induced hypovolaemia.

It is notable that PVN inhibition in DH rats caused sSNA to fall to a level that was no longer different from that of EH rats. This indicates that increased sSNA during dehydration is almost entirely driven by muscimol-inhibitable PVN neuronal activity. The latter interpretation is based on comparing sSNA voltages (i.e. microvolts) before and after PVN inhibition across groups of EH and DH rats. Although comparing SNA voltages 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 group differences in resting SNA are more frequently compared by expressing SNA values as a percentage of the ‘maximum’ SNA obtained in each group (Guild et al. 2010). Maximum SNA is typically determined by evoking one or another sympathoexcitatory reflex (i.e. arterial chemoreflex, somatosympathetic reflex, nasopharyngeal reflex, etc.). Here, we chose not to take this approach due to lack of evidence regarding effects of dehydration to modulate sympathoexcitatory reflexes. If dehydration were to differentially modulate sympathoexcitation evoked by various stimuli, then the value of maximum SNA used for normalization purposes could differ depending on which reflex was evoked to elicit ‘maximum’ SNA.

We acknowledge that comparing SNA voltages is also not without potential confounds. Indeed, voltage of SNA can vary across experiments even when action potential traffic is identical. This can arise, for example, if different sized nerve bundles, each with a different number of healthy/active axons, were selected from one experiment to the next. Under such conditions, differences in voltage per se would not necessarily reflect different levels of CNS activity driving sympathetic outflow. Voltages can also vary due to differences in the thickness of nerve sheaths, variable nerve–electrode contact or changes in amplifier settings from across experiments (Guild et al. 2010). Therefore, reporting SNA values in voltage units requires that care be taken to minimize possible sources of voltage variation across recordings. In the present study, the supra-adrenal branch of the splanchnic nerve was recorded, which in Sprague-Dawley rats is almost invariably a large single bundle. Thus, inter-experiment variability arising from selecting different sized nerve bundles was minimized. Because the splanchnic bundle is large, voltage variability due to dissection injury is also minimal, especially when a single investigator performs all the dissections and recordings – as was the case in the present study. Likewise, variability due to differences in electrode material, nerve–electrode interface and amplifier performance were largely avoided because all experiments used the same electrode/insulating material and identical amplifier settings. In addition, rats of similar age and size were used. Therefore, sources of possible variability were controlled for and largely avoided in the present study. Support for the latter conclusion comes from the fact that voltage due to noise in recordings of sSNA (i.e. the signal remaining after ganglionic blockade) was nearly identical in EH and DH rats (see Table 1). We acknowledge that although increased extracellular sodium concentration in DH rats might be capable of altering electrophysiological properties of sympathetic fibres and/or soma, such biophysical changes seem unlikely to have influenced voltages of recorded nerves since these would probably cause a generalized voltage increase in recordings from DH compared to EH rats. However, this was not observed as PNA burst amplitudes were not different across groups.

Literature evidence indicates that acute pharmacological activation of the PVN increases SNA (Porter & Brody, 1985; Martin et al. 1991; Kenney et 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 modulating the arterial baroreflex (Patel & Schmid, 1988; Dampney, 1994; Page et al. 2011). From this it might reasonably be expected that physiological challenges that activate the PVN would modulate respiratory and cardiac rhythmic bursting of SNA. In the present study, however, we observed that acute PVN inhibition selectively reduced a tonic component of sSNA in DH but not EH rats. This suggests that PVN activation during dehydration supports arterial pressure by increasing a component of SNA that is neither respiratory nor strongly cardiac rhythmic. As with our comparison of resting SNA across groups (see above), our interpretation that amplitudes of respiratory and cardiac rhythmic sSNA bursting were unaffected by dehydration or by acute PVN inhibition is based on comparing burst amplitudes expressed in voltage units, not as percentages of baseline (or percentage change from baseline). Our rationale for reporting burst amplitudes in voltage units is based on the fact that voltages obtained through AC-coupled bipolar electrodes are proportional to the density of action potentials that pass first across one electrode then the other (Guild et al. 2010). Like most modern amplifiers, ours is linear over a wide range of input voltages and so the voltage amplitude of a post-trigger burst is not sensitive to the baseline (pre-trigger) level of voltage. On this basis, it would be inaccurate to report bursts of equal voltage amplitude (equivalent action potential density) as different simply because baseline activity in one group was larger or smaller than in the other.

It should be acknowledged that reduced respiratory and/or cardiac rhythmic bursting among non-splanchnic sympathetic nerves might have contributed to the fall of MAP caused by PVN inhibition in DH rats. This being the case, the reduction of tonic sSNA in the present study might have merely been correlated with the fall of MAP. Although we cannot rule out this possibility at the present time, it seems likely that the fall in sSNA contributed to the fall of MAP because the mesenteric circulation is a major contributor to total systemic resistance. Additional experiments will be needed to determine if PVN inhibition during dehydration selectively reduces a tonic component of activity recorded from multiple sympathetic nerves that regulate vasomotor tone.

Although studies indicate that acute PVN activation increases the frequency and depth of respiration and cardio-respiratory coupling through a vasopressin-dependent mechanism in the RVLM/rostral ventral respiratory group (rVRG) (Yeh et al. 1997; Mack et al. 2002, 2007; Kc et al. 2010; Kc & Dick, 2010), we observed that acute PVN inhibition in EH and DH rats caused only a small reduction of respiratory frequency and no effect on the depth of inspiration. It therefore appears that dehydration does not actively recruit the vasopressinergic PVN–RVLM/rVRG pathway (Kc et al. 2010) that others have shown to be activated in rats exposed to chronic intermittent hypoxia (Prabha et al. 2011). We did not directly measure the strength of expiration, but on the whole it would appear from the present findings that ongoing PVN neuronal activity under normal conditions or during dehydration does not play a major role in driving respiration, at least not in anaesthetized rats. That respiratory drive was not strongly modified in DH rats is consistent with our observation that PVN inhibition also failed to change respiratory rhythmic bursting of sSNA. It should be emphasized that failure of PVN inhibition to change the strength of neural inspiration or respiratory rhythmic bursting of sSNA in DH rats does not exclude a contribution of respiratory rhythmic SNA to maintenance of MAP. Indeed, PVN inhibition might have caused an even greater reduction of MAP in DH rats if not for the persistence of respiratory rhythmic SNA.

In the present study, we initially observed that PVN inhibition in DH rats concurrently reduced integrated sSNA and MAP. As noted above, this suggests that ongoing sSNA contributes to support of MAP in DH rats, which is consistent with literature evidence (Scrogin et al. 1999, 2002; Brooks et al. 2004a,b2004b, 2005; Stocker et al. 2004a; Stocker et al. 2005, 2006; Antunes et al. 2006; Holbein & Toney, 2013). It is worth noting that the reduction of integrated sSNA by PVN inhibition in DH rats was associated with reduced cardiac rhythmic sSNA bursting. This observation raises the possibility that PVN neuronal activity during dehydration might actively facilitate baroreflex-mediated cardiac rhythmic sSNA inhibition. Literature evidence is consistent with such a possibility (Page et al. 2011). However, if PVN activation during dehydration were to significantly facilitate the baroreflex then the amplitude of the cardiac rhythmic sSNA oscillation might be expected to be greater at baseline (i.e. prior to PVN inhibition) in DH rats compared to EH rats. This would be expected because greater cardiac rhythmic inhibition in DH rats would produce periods of greater synaptic inhibition of RVLM (and other) sympathoexcitatory neurons compared to EH rats. As noted, however, this was not observed. It therefore seems likely that reduced cardiac rhythmic bursting of sSNA that occurred during PVN inhibition in DH rats was probably due to reduced pulse rhythmic baroreceptor inhibition that occurred secondary to the accompanying fall of arterial pressure.

Consistent with the latter interpretation, we observed that restoring MAP by inflation of an aortic cuff during PVN inhibition largely normalized the cardiac rhythmic oscillation of sSNA in DH rats. Somewhat unexpectedly, cuff inflation restored MAP without increasing arterial pulse pressure. However, arterial baroreceptors are responsive to pulsatile and non-pulsatile changes in pressure (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 proportion to the mean level of pressure (Franz, 1969; Thoren et al. 1977; Chapleau & Abboud, 1987). On this basis, aortic cuff inflation could have increased baroreceptor input and restored cardiac rhythmic inhibition of sSNA by raising MAP even without an increase of pulse pressure.

Another observation from the aortic cuff experiment in DH rats is that inflation caused only a small net reduction of integrated sSNA that was not statistically significant (see Fig. 4). The most likely explanation is that the cardiac rhythmic component of sSNA comprises only about 10% of total integrated voltage in dehydrated rats after PVN muscimol (Holbein & Toney, 2013). Consequently, even though cuff inflation largely restored the cardiac rhythmic inhibitory oscillation of sSNA, the absolute voltage loss attributable to these periods of more effective pulse rhythmic inhibition was insufficient to produce a statistically significant reduction of total integrated sSNA. We cannot entirely exclude the possibility that indirect sympathoexcitatory effects of cuff inflation might have masked the expected magnitude of baroreflex sympathoinhibition. For example, cuff inflation could have stimulated SNA by causing lower body ischaemia (Fujii et al. 2003; Mizuno et al. 2011) or by reducing venous return to unload cardiopulmonary baroreceptors (Vissing et al. 1989). These are perhaps unlikely explanations because arterial pulse pressure was maintained and it appears that venous return/cardiac output was also largely preserved during cuff inflation given that MAP increased in the face of greater aortic resistance. This haemodynamic profile also suggests that it is unlikely that a reduction of carotid body blood flow would have caused sympathoexcitation by activating the arterial chemoreflex. With regard to the latter, arterial chemoreflex activation has 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 is characteristic of dehydration, has been shown to ‘sensitize’ the arterial chemoreflex (Peng et al. 2011). The lack of an increase of expiratory bursting of sSNA in the present study lends further credence to the argument against increased arterial chemoreflex activity contributing to SNA regulation in DH rats before or during cuff inflation. Cuff inflation could have acutely activated the peripheral renin–angiotensin system (Blaine & Davis, 1971) and thereby offset baroreflex sympathoinhibition by increasing central sympathoexcitatory drive (Fink et al. 1980). This, too, seems unlikely because central angiotensin II-induced sympathoexcitation depends on activation of the PVN (Ferguson, 1988; Ferguson & Kasting, 1988), which was inhibited with muscimol during cuff inflation. A final factor that might have masked a net reduction of sSNA during cuff inflation is that stimulation of angiotensin II could have facilitated synaptic transmission through sympathetic ganglia (Aiken & Reit, 1968; Dendorfer et al. 2002), thereby amplifying transmission of cardiac rhythmic inputs from sympathetic preganglionic neurons. Whichever factors contributed to the observed effects of cuff inflation in DH rats, the present findings overall appear consistent with the conclusion that PVN neuronal activation during dehydration supports heightened sSNA without having a prominent direct effect on its cardiac rhythmicity.

As noted above, the most unexpected finding of the present study was that the fall of MAP produced by inhibition of PVN in DH rats was accompanied by a selective reduction of a tonic component of sSNA. It is acknowledged that anaesthesia might have differentially affected network behaviour in DH compared to EH rats, thereby activating a tonic component of SNA that might not exist while rats are conscious. To the extent that PVN activation during dehydration does increase a tonic component of SNA and does so to multiple end organs, our findings suggest that this tonic SNA contributes significantly to neurogenic vasomotor tone and support of AP in the dehydrated state. This idea is not without precedent. Koshiya & Guyenet (1995), for example, used PNA-triggered averaging of sSNA to demonstrate that low-dose intravenous administration of clonidine lowers MAP and, as in the present study, causes a selective reduction of the pre-trigger level of sSNA. A striking similarity between their study and ours is that both recorded sSNA to monitor patterning of sympathetic activity. This raises the question of whether tonic SNA is a particularly prominent or perhaps unique feature of splanchnic sympathetic outflow. Consistent with this possibility, a recent study, again recording sSNA, reported that exposure of rats to acute intermittent hypoxia induced long-term facilitation of sympathetic activity. Of note is that the increase of sSNA was characterized by a selective increase of tonic activity without a change of respiratory rhythmic bursting (Xing & Pilowsky, 2010). Another study reported that acute activation of peripheral chemoreceptors after elimination of baroreflex transmission and central respiratory network activity increased a tonic component of both sSNA and lumbar SNA (Koshiya & Guyenet, 1996). The latter observation indicates that tonic activity is not an exclusive feature of splanchnic sympathetic activity.

The source of enhanced PVN-driven tonic SNA during water deprivation is currently unknown, but available evidence suggests that plasma hyperosmolality could play a significant role. Not only does acute internal carotid artery infusion of hypertonic saline increase SNA via a forebrain- and PVN-dependent mechanism (Chen & Toney, 2001; Brooks et al. 2005; Antunes et al. 2006; Shi et al. 2007), acute carotid infusions of hypotonic saline to reduce forebrain osmolality reduce MAP in dehydrated rats (Brooks et al. 2004b, 2005). Given that the neurohumoral environment of dehydration is complex, additional studies are needed to determine the relative contribution of hypertonicity versus multiple hormones and viscero-sensory inputs to PVN-driven tonic SNA in DH rats.

A major question that emerges from the present study is whether activation of tonic SNA occurs mainly or exclusively in response to acute homeostatic challenges such as dehydration or if tonic SNA is driven in disease states where sympathetic outflow is chronically increased? The answer to this question is not presently known. Of interest, however, are studies in normal animals that have identified tonic and rhythmic patterns of discharge among identified PVN (Chen & Toney, 2003, 2010), RVLM (Tseng et al. 2009), and sympathetic pre- and post-ganglionic neurones (Darnall & Guyenet, 1990). Thus, neuronal substrates capable of generating both tonic and rhythmic patterns of SNA clearly exist, but studies to date have principally focused on modulation of rhythmic patterns. For example, studies in spontaneously hypertensive rats suggest that the IP of SNA plays an exaggerated role in the development (Simms et al. 2009) and maintenance of hypertension (Czyzyk-Krzeska & Trzebski, 1990; Simms et al. 2009). In rats made hypertensive by exposure to CIH (Zoccal et al. 2008; Zoccal & Machado, 2010; Moraes et al. 2012) and by treatment with angiotensin II and a high salt diet (Toney et al. 2010), the late-expiratory peak of SNA is exaggerated and has been implicated in the accompanying hypertension. Mechanisms driving sympathetic outflow in these chronic models are almost certainly not identical to those of DH rats and experimental differences further confound direct comparison. Additional studies are needed to determine how acute versus chronic challenges activate distinct or common circuit elements and mechanisms to generate and modulate tonic versus rhythmic patterns of SNA.

In summary, the present findings indicate that PVN neuronal discharge in anaesthetized dehydrated rats primarily supports a tonic component of sSNA – one that is neither respiratory nor strongly cardiac rhythmic. Importantly, this reduction of tonic sSNA by PVN inhibition was linked to the fall of MAP, raising the possibility that it subserves vasomotor function. Additional studies are needed to determine if PVN activation drives tonic SNA to non-splanchnic vascular beds and to determine the overall role of PVN-driven tonic SNA in maintaining vasomotor tone and arterial pressure during homeostatic challenges other than dehydration and in chronic cardiovascular disease models. Studies are also needed to determine if tonic SNA results from an irregular pattern of discharge among individual sympathetic–regulatory PVN neurones or if tonic SNA is an emergent property of an extended sympathetic network in which the PVN participates. A full understanding of neural mechanisms that generate and modulate tonic SNA could provide novel avenues for improved treatment of neurogenic cardiovascular diseases.

Glossary

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

plasma osmolality

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

Additional Information

Competing Interests

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

Author contributions

W.W.H., M.E.B. and G.M.T. developed the hypothesis and designed experiments. W.W.H. performed experiments and collected data. W.W.H. and G.M.T. analysed data and prepared the manuscript text and figures. W.W.H., M.E.B. and G.M.T. revised the manuscript and all are qualified authors. The final version of the manuscript was approved by all authors.

Funding

This research was supported by NIH grants HL102310 and HL088052 (G.M.T.). M.E.B. was supported by NIH training grant T32 HL07446.