Role of the Sympathetic Nervous System in Schlager Genetically Hypertensive Mice
Early studies indicate that the hypertension observed in the Schlager inbred mouse strain may be attributed to a neurogenic mechanism. In this study, we examined the contribution of the sympathetic nervous system in maintaining hypertension in the BPH/2J mouse and used c-Fos immunohistochemistry to elucidate whether neuronal activation in specific brain regions was associated with waking blood pressure. Male hypertensive (BPH/2J; n=14), normotensive (BPN/3J; n=18), and C57/Bl6 (n=5) mice were implanted with telemetry devices, and after 10 days of recovery, recordings of blood pressure, heart rate, and locomotor activity were measured to determine circadian variation. Mean arterial pressure was higher in BPH/2J than in BPN/3J or C57/Bl6 mice (P<0.001), and BPH/2J animals showed exaggerated day-night differences (17±2 versus 6±1 mm Hg in BPN/3J or +8±2 mm Hg in C57/Bl6 mice; P<0.001). Acute sympathetic blockade with pentolinium (7.5 mg/kg IP) during the active and inactive phases reduced blood pressure to comparable levels in BPH/2J and BPN/3J mice. The number of c-Fos–labeled cells was greater in the amygdala (+180%; P<0.01), paraventricular nucleus (+110%; P<0.001), and dorsomedial hypothalamus (+48%; P<0.001) in the active (hypertensive) phase in BPH/2J compared with BPN/3J mice. The level of neuronal activation was mostly similar in these regions in the inactive phase. Of all of the regions studied, neuronal activation in the medial amygdala, as detected by c-Fos, was highly correlated to mean arterial pressure (r=0.98). These findings indicate that the hypertension is largely attributable to sympathetic nervous system activity, possibly generated through greater levels of arousal regulated by neurons located in the medial amygdala.
- BPH/2J mice
- sympathetic nervous system
- cardiovascular responses
- baroreceptor reflex
- c-Fos immunohistochemistry
Conventional wisdom suggests that the sympathetic nervous system (SNS) plays a small role in the long-term control of arterial pressure and is confined to regulating blood pressure (BP) in the short term. However, there is increasing evidence suggesting the importance of sympathetic hyperactivity in a number of disease states, such as hypertension, congestive heart failure, and renal failure.1 Studies from Esler2 have demonstrated that noradrenaline release from renal nerves is elevated in young, borderline hypertensive patients. The morning surge in BP is markedly greater in hypertensive patients and can be reduced by drugs that specifically inhibit the SNS, such as clonidine and guanabenz.3 These and other studies suggest that the SNS makes an important contribution to human hypertension, but whether the SNS can itself generate a long-term hypertensive state has been difficult to demonstrate in experimental animal models.4–7⇓⇓⇓
One hypertensive model that has been largely overlooked is the spontaneously hypertensive mouse. In the late 1970s, Schlager and Sides8 developed 3 inbred lines of mice with low (BPL/1J), high (BPH/2J), and normal (BPN/3J) BPs. The strain was started from a mixture of an 8-way cross of unrelated, inbred normotensive mice. Previous reports of BP in these mice have been obtained mainly by the tail-cuff method, and only recently has the hypertension been confirmed using radiotelemetry.9 Early studies focused on catecholamines and found reduced brain noradrenaline content compared with BPN/3J and BPL/1J mice.10,11⇓ The hypothalamus, amygdala, and cerebellum were the main areas exhibiting a reduction in noradrenaline content in young hypertensive mice.10 A later analysis by Denoroy et al12 showed that the noradrenaline content of the preoptic area was higher in the BPH/2J mouse but lower in the paraventricular nucleus (PVN). The lower levels in the PVN may reflect increased turnover of noradrenaline that would result in increased sympathetic activity. In support of this, BPH/2J mice have 41% greater noradrenaline content in the superior cervical ganglion but normal levels of catecholamines and metabolites in the adrenal medulla compared with random-bred mice.12
On the basis of these findings, there is a distinct possibility that the high BP identified in BPH/2J mice involves a neurogenic mechanism, but there has been no systematic investigation of the contribution of the SNS to the hypertension in these animals. Therefore, in the current study, we examined the effect of the SNS on BP with ganglionic blockade and determined whether, during the waking arousal period (lights off), we could observe greater increases in BP in the hypertensive animals. This, for a nocturnal animal, parallels the morning surge in BP observed in humans. We also used c-Fos immunohistochemistry to determine whether this surge in BP was associated with greater neuronal activation in specific brain regions.
Cardiovascular experiments were carried out on 15- and 23-week–old male normotensive (BPN/3J, n=18) and hypertensive (BPH/2J, n=14) mice. For comparison 5 normotensive C57/Bl6 mice were also included. Animals were kept on a 12:12 hour light-dark cycle (6:00 am to 6:00 pm light). All of the mice were allowed access ad libitum to water and mouse chow (Specialty Feeds, Glen Forrest; 19.0% protein, 5.0% fat, 5.0% fiber, and 0.2% sodium). Under isoflurane open-circuit anesthesia (5.0% induction and 1.5% to 2.0% maintenance), mice were implanted with radiotelemetry transmitters, with the catheter inserted into the carotid artery and the transmitter body implanted along the right flank.13 After a 10-day recovery period, mice were housed individually for the remaining duration of the study at the Baker IDI Heart and Diabetes Institute. The experiments were approved previously by the Alfred Medical Research Education Precinct Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for Scientific Use of Animals.
Measurement of BP, Heart Rate, and Locomotor Activity in Freely Moving Mice in Home Cages
At the end of the recovery period, 48-hour continuous recordings of systolic arterial pressure (SAP), diastolic arterial pressure (DAP), calculated mean arterial pressure (MAP), heart rate (HR), and locomotor activity were measured continuously and sampled at 1000 Hz using an analog-to-digital data acquisition card (National Instruments 6024E), as described previously.14
Relationship Between Locomotor Activity and MAP
To calculate the reactivity index, average MAP, HR, and locomotor activity values expressed as arbitrary units were calculated during 1-minute intervals over 3.4 days, and the sensitivity score was logarithmically transformed to correct for positive skew.15
Pharmacological Test: Sympathetic Blockade
During the night period when the animals were active or during the day when the animals were inactive, BP and HR were determined before and after IP administration of the sympathetic blocker, pentolinium (7.5 mg/kg; Sigma) in 27-week–old BPN/3J (n=8) and BPH/2J (n=5) mice.
Cardiovascular and Baroreflex Assessments
Evaluation of cardiovascular function, cardiovascular variability, and the baroreceptor HR reflex were by spectral analysis (please see the online Data Supplement for more details at http://hyper.ahajournals.org).
Details on c-Fos immunohistochemical analysis are also available in the online Data Supplement.
Cardiovascular data are expressed as mean or mean change±SEM. The acceptable level of type II error was set at 5%; therefore, data were deemed significant when P<0.05. The data were analyzed by multifactor, nested split-plot ANOVA, which allowed for within-animal and between-animal contrasts.16 Statistical evaluation of c-Fos counts expressed as mean±SEM was performed by 2-way ANOVA with a Greenhouse-Geisser correction for repeated measures.
Cardiovascular Measurements and Locomotor Activity in BPH/2J and BPN/3J Mice
At 15 and 23 weeks of age, average 24-hour values of SAP, DAP, MAP, HR, and locomotor activity were greater in hypertensive BPH/2J mice when compared with normotensive BPN/3J mice (P<0.01; Table 1). MAP was 23% higher in BPH/2J than in BPN/3J mice at 15 weeks and 15% greater at 23 weeks. BP increased with age in both groups but to a lesser extent in BPH/2J mice (Table 1, effect of age; P<0.001). There was no difference in HR at both ages; however, HR was 27% higher in BPH/2J mice (Table 1). Both groups of mice increased body weight with age, but BPH/2J mice were consistently lighter than BPN/3J mice (−17% average of both ages; Table 1; P<0.001). Locomotor activity was 93% greater in BPH/2J mice compared with BPN/3J mice (P<0.002; Table 1), and no effect of age was observed in either group.
Analysis of the hourly levels of cardiovascular variables in BPN/3J and BPH/2J mice showed a similar pattern with higher values throughout the active (night) phase and lower values throughout the inactive (day) phase (Figure 1, average of 15 and 23 weeks). BPH/2J mice were hypertensive and tachycardic during both periods. Although the hypertension was particularly evident during the active period, it was also present during the daytime period when activity levels were very low and similar in both groups (Figure 1). Thus, day-night differences in MAP were greatly exaggerated in BPH/2J animals in comparison with BPN/3J animals (+20±1 versus 7±1 mm Hg at 15 weeks and +17±3 versus 7±1 mm Hg at 23 weeks; P<0.001; Table 1). Likewise, day-night differences in HR were 69% greater and locomotion 10-fold greater in BPH/2J compared with BPN/3J mice (P<0.001; Table 1). There were no significant effects of age on these differences between groups (Table 1).
Comparisons With 23-Week–Old C57/Bl6 Mice
There were no differences in average 24-hour SAP, DAP, MAP, or body weight between BPN/3J mice and normotensive C57/Bl6 mice (24-hour MAP was 114±1 mm Hg in C57/Bl6 and 111±1 mm Hg in BPN/3J). However, HR and activity levels were greater in C57/Bl6 mice compared with BPN/3J mice (571±9 versus 468±10 bpm and 1.7±0.4 versus 0.8±0.1 units, respectively; P<0.001). BPH/2J mice had higher BP than C57/Bl6 mice but similar levels of HR and activity (please see the online Data Supplement).
Similar day-night differences in cardiovascular variables, including SAP, DAP, MAP, and HR, were observed in BPN/3J and C57/Bl6 mice (+8±2 mm Hg, +7±2 mm Hg, +8±2 mm Hg, and +50±13 bpm, respectively, in C57/Bl6) but were less than those observed in BPH/2J mice (please see the online Data Supplement). Greater levels of day-night differences in activity were apparent in C57/Bl6 mice compared with BPN/3J mice (P<0.001) but not with BPH/2J mice (1.76±0.80, 0.23±0.10, and 1.56±0.60 U, respectively).
Relationship Between Day-Night Differences in MAP and “Active-Period” MAP
There was a very strong correlation between the day-night difference in MAP and the active- (night) phase MAP across all 3 of the strains (n=37; r=0.7; P<0.001). There was no correlation when only the normotensive BPN/3J and C57/Bl6 mice were included (n=23; r=0.07; P value not significant; please see the online Data Supplement).
Relationship Between Locomotor Activity and MAP
Plotting the relationship between BP and locomotor activity for each minute over an average of 3.4 days (minimum: 1.8 days; maximum: 4.9 days) using a method that we developed recently15 identified a linear relationship between log activity and BP for 23-week–old BPN/3J and BPH/2J mice. The slope of the line was similar (BPN/3J 10.3±1.1 versus BPH/2J 10.4±1.9 mm Hg per log unit activity; P>0.05), but the line was elevated in BPH/2J mice (intercept for BPN/3J, 117±2 versus BPH/2J, 135±1 mm Hg; P<0.001; Figure 2). The correlation coefficient for the regression was similar, at 0.46 (P<0.001; degrees of freedom: 11) and 0.51 (P<0.001; degrees of freedom: 11) for BPN/3J and BPH/2J mice, respectively (effect of strain; P>0.05).
Cardiovascular and Locomotor Activity Response to Sympathetic Blockade
During the active (night) phase, sympathetic blockade by pentolinium reduced MAP in the 2 groups to comparable levels (102±1 in BPN/3J compared with 101±3 mm Hg in BPH/2J mice; P=0.771; Figure 3). The change in MAP between 10 and 20 minutes was greater in BPH/2J compared with BPN/3J mice (−55±5 and −34±3, respectively; P<0.001; Figure 3). When expressed as a percentage of change, the MAP responses were larger in BPH/2J compared with BPN/3J mice (−39±2% and −28±2%; P<0.01; Figure 3). During the inactive (day) phase, there was no difference in the hypotensive effect of pentolinium in BPH/2J compared with BPN/3J mice when expressed as a change in MAP or as a percentage of change (please see the online Data Supplement).
Cardiovascular Variability and Baroreceptor Reflex Using Power Spectral Analysis
The gain of the baroreceptor HR reflex was calculated from the cross-spectral analysis of the transfer function between MAP and HR in the midfrequency range (0.3 to 0.5 Hz). During the inactive (day) phase, there were no differences in MAP or HR midfrequency powers between BPH/2J and BPN/3J mice or in baroreflex sensitivity (Figure 4). However, baroreflex gain was slightly lower in C57/Bl6 mice (−23%; P<0.05) because of a 70% lower HR power and a 42% lower MAP power in this frequency band (Figure 4).
During the active (night) phase there was a 4.6-fold increase in the MAP power in BPH/2J mice and a doubling of the MAP power in BPN/3J and C57/Bl6 mice compared with the inactive period. HR power was reduced in BPN/3J and BPH/2J mice and similar in C57/Bl6 mice in the active compared with the inactive period. Thus, the baroreflex gain was well maintained in the normotensive strains during the active period (compare top and bottom panels, Figure 4) such that BPN/3J and C57/Bl6 mice had similar baroreflex values. We observed a markedly lower baroreflex gain in the hypertensive mice compared with both normotensive strains during the active period (Figure 4; P<0.001). This was because of a greater MAP power and reduced HR power in BPH/2J compared with BPN/3J mice. The coherence between MAP and HR was similar in the 3 strains and during the 2 phases.
Effect of Pentolinium on Cardiovascular Variability Using Power Spectral Analysis
To assess whether spectral MAP power in the midfrequency range reflected the level of sympathetic nerve activity, we compared values before and after sympathetic blockade with pentolinium. During the active (night) phase, pentolinium reduced midfrequency MAP power in the 23-week–old BPH/2J mice by 65% and 68% in BPN/3J mice but had no effect during the inactive phase when MAP power was lower (please see the online Data Supplement). Only the high-frequency power of BPH/2J mice during the active period was reduced by pentolinium (−53%; P<0.001; please see the online Data Supplement).
Circadian differences were observed within both of the Schlager strains in the same nuclei with greater c-Fos expression identified in the medial amygdala (MeAm), PVN, dorsomedial hypothalamus (DMH), and rostral ventrolateral medulla (RVLM) in the active (night) compared with the inactive (day) phase. By contrast, no circadian difference in c-Fos counts was observed in the central amygdala (CeAm), suprachiasmatic nucleus, caudal ventrolateral medulla, and nucleus of the solitary tract (Table 2).
Two hours after lights out in the active (night) phase of the 24-hour cycle, greater neuronal activation, as detected by c-Fos immunohistochemistry, was observed in the MeAm (+161%), CeAm (+198%), PVN (+110%), and DMH (+48%) of hypertensive BPH/2J mice compared with normotensive BPN/3J mice (P<0.01; Table 2). Examples of the distribution of c-Fos in brain regions from BPN/3J and BPH/2J mice in the dark phase are shown in Figure 5. Conversely, there were no differences in these brain regions between groups 2 hours before lights out in the inactive (day) phase of the 24-hour cycle with the exception of the MeAm, which showed 73% greater activation in BPH/2J mice (Table 2).
An analysis of the correlation between the degree of activation in the various brain regions compared with the level of MAP during the day and night showed the highest linkage (correlation coefficient) with the MeAm (r=0.98), followed by PVN (r=0.95) and DMH (r=0.92), with lesser correlation with RVLM (r=0.79), nucleus of the solitary tract (r=0.77), and caudal ventrolateral medulla (r=0.73) and lowest correlation with the CeAm (r=0.68; please see the online Data Supplement). In addition, the relationship between the fall in MAP attributed to pentolinium (indicating SNS contribution) was again most related to the MeAm (r=0.95), followed by the nucleus of the solitary tract (r=0.90) and caudal ventrolateral medulla (r=0.91), PVN (r=0.87), CeAm (r=0.80), and DMH (r=0.79), with lesser correlation with the RVLM (r=0.63; please see the online Data Supplement).
The major novel findings from the present study demonstrated that hypertensive BPH/2J mice have a markedly amplified day-night difference in BP compared with normotensive BPN/3J or C57/Bl6 mice, as revealed by radiotelemetry. Furthermore, acute sympathetic inhibition with ganglionic blockade in BPN/3J and BPH/2J mice completely abolished the hypertension in BPH/2J mice reducing BP between the 2 groups to comparable levels. Although the surge in BP after onset of the active (night) phase may be accentuated by the increase in locomotor activity, the hypertension was sustained in BPH/2J compared with C57/Bl6 mice who had the same night-induced activity increase. This shows that the hypertension is independent of locomotor activity in BPH/2J mice. Importantly, the hypertension is associated with greater activation of neurons in hypothalamic regions (PVN and DMH) that are critical for the regulation of cardiovascular autonomic function. Greater levels of c-Fos expression were also detected in the amygdala, suggesting that limbic pathways associated with arousal and stress are also hyperactive in the BPH/2J mouse. Indeed, the strongest association (r=0.95) between the level of MAP and the effect of pentolinium and CNS regional activation occurred with the MeAm. Together these findings suggest that the SNS is a major determinant of the hypertension in the BPH/2J mouse driven by heightened nighttime arousal. Thus, the BPH/2J mouse model could be considered mainly as a neurogenic form of hypertension.
Early studies in Schlager hypertensive and normotensive mice focused on catecholamine levels in the brain and showed reduced brain noradrenaline content in the hypothalamus, amygdala, and cerebellum of young hypertensive BPH/2J mice compared with normotensive BPN/3J mice.10,11⇓ An additional study identified lower levels of noradrenaline in the PVN that may reflect increased turnover of noradrenaline, indicating greater sympathetic nerve activity in BPH/2J mice.12 These early findings of changes in catecholamine levels in the amygdala and hypothalamus, together with greater activation of neurons in these brain regions in BPH/2J mice, strongly suggest that the cause of the hypertension is likely an overactive sympathetic nervous system. Cardiovascular studies of these mice, and mice in general, have been hampered for technical reasons associated with measuring BP in such small animals. This has only been addressed in the last few years with the development of miniature radiotelemetry probes for mice and an appropriate implantation method.13 There has been only 1 other telemetry study of the BPH/2J mouse, by McGuire et al,9 who studied 20-week–old mice (n=6), and although they observed a similar degree of hypertension as we did, their assessment of differences in the day-night rhythm between BPH/2J and BPN/3J mice did not reach statistical significance (−14±1.5 versus −10.7±1.3 mm Hg, respectively; P=0.09).
We suggest that the hypertension in the BPH/2J mouse is strongly influenced by circadian patterns, which drive the SNS during the active hours and inhibit the SNS during normal sleep. This mechanism appears to be markedly exaggerated in BPH/2J mice. Indeed, there is a strong correlation between MAP observed in the active (night) phase in each mouse and the day-night differences in MAP (r=0.7; P<0.001), suggesting that much of the hypertension is circadian in origin. Our strongest evidence of a neurogenic mechanism was the effectiveness of ganglionic blockade to abolish the hypertension when it is most evident in the night period, bringing the BP of both groups to the same level. Pentolinium also had a marked effect in reducing the high levels of midfrequency (autonomic) power in BP, which is consistent with an activated SNS. Furthermore, the effects of pentolinium during the inactive (day) phase were similar in the 2 groups, suggesting that the SNS contribution to the hypertension occurs mainly during the active period. Because there is considerable evidence that the SNS is a major contributor to the day-night changes in BP in both humans17 and experimental animals,18 it is likely that the SNS is also a major contributor to the hypertension in the Schlager mouse model. Circadian rhythms are endogenously generated, with the circadian rhythmicity being closely timed by neurons located within the suprachiasmatic nucleus.19 We observed no difference in the number of activated neurons in the suprachiasmatic nucleus between BPN/3J and BPH/2J mice during either the active or inactive phases, suggesting that the circadian clock mechanisms do not differ in these groups. Our suggestion of an “overactive SNS” is consistent with previous studies in BPH/2J mice that showed, using a microarray analysis of the adrenal gland, that a number of catecholamine and sympathetic activity–related loci were altered, with genes coding for catecholamine biosynthetic enzymes being overexpressed in BPH/2J mice, whereas those coding for catecholamine degradation enzymes were underexpressed compared with BPL/1J mice.20
An important question relates to the underlying mechanism for the sympathetic overactivity. Our results indicate that neurons located in the amygdala are likely prime candidates for mediating the greater BP surge identified in hypertensive BPH/2J mice. The MeAm showed greater activation in BPH/2J mice during the active and inactive phases, suggesting a circadian pattern that closely follows the BP. Indeed, the activity of the MeAm showed the highest correlation of any brain region studied when compared with the level of BP in both groups of Schlager mice during the day and night periods (r=0.98), as well as when compared with the reduced BP produced by SNS inhibition by ganglionic blockade (r=0.95). This suggests that the MeAm reflects the level of SNS activation contributing to BP and, as such, is likely to be involved in generating this activity rather than simply responding to it. It has been shown that the MeAm, rather than the CeAm, is an important integrative site for processing emotions such as anxiety and fear by relaying the appropriate neuroendocrine, cardiovascular, and behavioral responses.21 Stimulation of the MeAm increases BP through SNS activation,22 whereas inhibition blocks the pressor effect of restraint stress in rats.23 Furthermore, the amygdala provides the major inputs to the hypothalamic regions integrating cardiovascular responses to acute stress, such as the PVN and DMH.24 For example, there is electrophysiological evidence for an angiotensinergic projection from the MeAm to the hypothalamic area, which involves angiotensin II type 1 receptors25 and which may be related to particular behaviors.26 Indeed, we identified clear neuronal activation, as detected by c-Fos immunohistochemistry, in the DMH and PVN, as well as in the RVLM of both groups of Schlager mice associated with day-night differences. Although there was no observable difference in these brain regions between groups during the inactive phase, there was markedly greater neuronal activation in the DMH and PVN of BPH/2J mice in the active phase. Thus, it appears that these brain regions do not act to regulate cardiovascular responses in BPH/2J mice during extended periods of rest. Instead, the present data are consistent with putative projections arising from the amygdala innervating both the DMH and the PVN, likely acting concurrently to modulate the output of presympathetic neurons in hypertensive BPH/2J mice during the waking arousal period. Therefore, our findings are supportive of other studies that suggest that projections arising from the MeAm are intimately involved in the integration of moment-to-moment information required to adjust HR and BP and associated behavioral responses.27
BPH/2J mice were markedly more active than BPN/3J mice during a 24-hour period, with a clear increase in behavior transitioning from the inactive phase to the active phase. These results confirm our general findings in mice that activity is associated with an increase in BP.14 However, activity, per se, did not explain the hypertension in this strain of mouse nor the greater day-night difference. We observed that C57/Bl6 mice have similar activity to BPH/2J mice but were normotensive and had a day-night difference in MAP that was similar to BPN/3J mice. Furthermore, an analysis of the relationship between BP and activity for each minute over several days, using a method that we developed recently,15 showed that, whereas the relationship between activity and BP is positive for both BPH/2J and BPN/3J mice, the regression lines were parallel, with the line for the hypertensive mice elevated (set at a higher BP) compared with the normotensive mice.
In addition to hypertension in BPH/2J mice, we also observed that these mice had considerably higher HRs than BPN/3J mice during both the active and inactive periods. However, the HRs in BPH/2J mice were similar to normotensive C57/Bl6 mice and, as such, do not appear to be a major factor in the hypertension, per se. The tachycardia persisted during periods of inactivity but to a lesser extent, and therefore may be only partly related to the greater locomotor activity observed in both the BPH/2J and C57/Bl6 strains. Importantly, tachycardia in BPN/3J mice was abolished by pentolinium, suggesting that it is mainly sympathetically mediated. Cross-spectral analysis identified less baroreceptor HR reflex gain in BPH/2J mice because of a much higher MAP power in the autonomic (mid) frequency band compared with the normotensive strains, but only during the active period. This is consistent with most forms of established hypertensive animal models, as well as human hypertension.28 Only small differences were noted during the inactive period, with lower baroreflex gain observed in C57/Bl6 mice, which is, therefore, not related directly to the hypertension.
The present study provides evidence for functional changes to the integration of sympathetic activity in genetically hypertensive mice, such that the Schlager BPH/2J mouse can be considered a neurogenic form of hypertension, driven by an overactive SNS. The BPH/2J mouse has markedly greater neuronal activation in specific regions of the amygdala and hypothalamus, possibly related to greater levels of arousal during the night period, causing an exaggerated circadian BP rhythm. Therefore, this mouse model may be most relevant to human hypertensive patients who have a centrally mediated SNS hyperresponsivity associated with circadian rhythms29,30⇓ or perhaps to white coat hypertensives. The focus now is to determine the genetic influences that are responsible for this phenomenon. Thus, systematic examination of this mouse strain, particularly regarding the regulation of the MeAm, may well contribute to unraveling the central mechanisms associated with human neurogenic hypertension.
Sources of Funding
This work was funded by a project grant from the National Health and Medical Research Council, Australia (526662).
- Received May 12, 2009.
- Revision received June 5, 2009.
- Accepted July 9, 2009.
- ↵Hashimoto J, Chonan K, Aoki Y, Ugajin T, Yamaguchi J, Nishimura T, Kikuya M, Michimata M, Matsubara M, Araki T, Hozawa A, Ohkubo T, Imai Y. Therapeutic effects of evening administration of guanabenz and clonidine on morning hypertension: evaluation using home-based blood pressure measurements. J Hypertens. 2003; 21: 805–811.
- ↵Burke SL, Head GA, Lambert GW, Evans RG. Renal sympathetic neuroeffector function in renovascular and angiotensin II-dependent hypertension in rabbits. Hypertension. 2007; 49: 932–938.
- ↵Evans RG, Burke SL, Lambert GW, Head GA. Renal responses to acute reflex activation of renal sympathetic nerve activity and renal denervation in secondary hypertension. Am J Physiol Regul Integr Comp Physiol. 2007; 293: R1247–R1256.
- ↵Head GA, Burke SL. Sympathetic responses to stress and rilmenidine in 2K1C hypertensive rabbits: evidence of enhanced non-vascular neuroeffector mechanism. Hypertension. 2004; 43: 636–642.
- ↵Burke SL, Evans RG, Moretti J-L, Head GA. Levels of renal and extrarenal sympathetic drive in Ang II-induced hypertension. Hypertension. 2008; 51: 878–883.
- ↵Schlager G, Freeman R, El Seoudy AA. Genetic study of norepinephrine in brains of mice selected for differences in blood pressure. J Hered. 1983; 74: 97–100.
- ↵Butz GM, Davisson RL. Long-term telemetric measurement of cardiovascular parameters in awake mice: a physiological genomics tool. Physiol Genomics. 2001; 5: 89–97.
- ↵Jackson K, Head GA, Morris BJ, Chin-Dusting J, Jones E, La Greca L, Mayorov DN. Reduced cardiovascular reactivity to stress but not feeding in renin enhancer knockout mice. Am J Hypertens. 2007; 20: 893–899.
- ↵Adams DJ, Head GA, Markus MA, Lovicu FJ, van der Weyden L, Köntgen F, Arends MJ, Thiru S, Mayorov DN, Morris BJ. Renin enhancer is critical for regulation of renin gene expression and control of cardiovascular function. J Biol Chem. 2006; 281: 31753–31761.
- ↵Snedecor GW, Cochran WG: Statistical Methods. Ames, IA: Iowa State University Press; 1980.
- ↵Carvalho MJ, van Den Meiracker AH, Boomsma F, Lima M, Freitas J, Veld AJ, Falcao De Freitas A. Diurnal blood pressure variation in progressive autonomic failure. Hypertension. 2000; 35: 892–897.
- ↵Fries RS, Mahboubi P, Mahapatra NR, Mahata SK, Schork NJ, Schmid-Schoenbein GW, O'Connor DT. Neuroendocrine transcriptome in genetic hypertension: multiple changes in diverse adrenal physiological systems. Hypertension. 2004; 43: 1301–1311.