Cardiovascular Responses to Aversive and Nonaversive Stressors in

original contributions

nature publishing group

Cardiovascular Responses to Aversive and Nonaversive Stressors in Schlager Genetically Hypertensive Mice Pamela J. Davern1, Kristy L. Jackson1, Thu-Phuc Nguyen-Huu1, Luisa La Greca1 and Geoffrey A. Head1 Background Schlager inbred hypertensive mice (BPH/2J) have been suggested to have high blood pressure (BP) due to an overactive sympathetic nervous system (SNS). The brain nuclei associated with the hypertension are also those involved in the integration of the cardiovascular responses to stress. Therefore, in the present study, we hypothesize that an increased contribution of the SNS in BPH/2J mice may culminate in a greater pressor response to stressful stimuli in these hypertensive mice than normotensive (BPN/3J) mice. Methods Male hypertensive BPH/2J and normotensive BPN/3J mice were implanted with telemetry devices and exposed to a series of behavioral “stress” tests including aversive stress (shaker, clean cage‑switch, and restraint) and nonaversive stress (feeding).

Results Aversive stress caused a 67–88% greater pressor response in BPH/2J compared with BPN/3J mice. By contrast, the feedinginduced pressor response was not different between groups. All stressors induced tachycardia that was less in BPH/2J mice (feeding and restraint) and others were not different between groups (clean cage‑switch and shaker). Conclusions These findings indicate that hypertension in BPH/2J mice is associated with greater pressor responsiveness to aversive stress but not to appetitive arousal. Thus, BPH/2J hypertensive mice may be a particularly relevant model for human hypertensive patients that overrespond to daily stressors. Keywords: blood pressure; heart rate; hypertension; mice; stress Am J Hypertens 2010; 23:838-844 © 2010 American Journal of Hypertension, Ltd.

There is a growing realization that the sympathetic nervous system (SNS) may participate in the long-term regulation of blood pressure (BP) and importantly contribute to the development of hypertension. Esler and colleagues have established that young hypertensives have higher levels of activity of the SNS in key organs such as the kidney1 that is related to a high release rate of subcortical noradrenaline that is likely to arise from the hypothalamus.2 The SNS may also contribute to a greater early morning surge in BP that is markedly greater in hypertensive patients3 because it can be effectively reduced by drugs that specifically inhibit the SNS such as clonidine and guanabenz.4 Thus, although it is clear that the SNS makes an important contribution to human hypertension, to explore the central mechanisms that contribute to the overactivity requires the development of appropriate experimental animal models and techniques. Schlager and colleagues developed an inbred strain 1Neuropharmacology Laboratory, Baker IDI Heart and Diabetes Research Institute, Melbourne, Australia. Correspondence: Pamela J. Davern ([email protected])

Received 3 January 2010; first decision 2 February 2010; accepted 4 March 2010; advance online publication 8 April 2010. doi:10.1038/ajh.2010.69 © 2010 American Journal of Hypertension, Ltd. 838

of hypertensive mice (BPH/2J) in the 1970s that have a systolic BP 35 mm Hg greater than a normotensive control group of mice (BPN/3J) bred in parallel. But this strain has received only relatively modest attention in the field of hypertension research.5 Early studies of this strain compared brain catecholamine levels between hypertensive BPH/2J mice and low BP mice and found no difference in the adult,6,7 but lower levels of noradrenaline in the midbrain and cerebellum of young hypertensive mice.6 Another study showed greater noradrenaline content in the preoptic area, but reduced levels in the paraventricular nucleus of the hypothalamus.8 At the time, reduced levels of noradrenaline in the hypothalamus were suggested as a possible hypertension mechanism, but further studies have been hampered by the difficulty of measuring BP in conscious mice. Recently, we studied these mice using radiotelemetry and found that BPH/2J mice display profound hypertension during the active period and lesser hypertension during the inactive period. This exaggerated day–night BP difference was not associated with locomotor activity or differences in heart rate (HR), but was completely blocked by the ganglion blocker pentolinium. We also found that regions of the limbic system (medial amygdala (MeAm)) and the hypothalamus (dorsomedial and paraventricular nucleus) were more activated after

august 2010 | VOLUME 23 NUMBER 8 | 838-844 | AMERICAN JOURNAL OF HYPERTENSION

Stress Responses in Genetically Hypertensive Mice


the onset of darkness than control normotensive mice.9 These findings not only suggest that the hypertension observed is likely due to an overactive SNS, the pattern of night-time arousal activation is highly suggestive of the pattern associated with stress similar to that observed in human early morning BP surge.3 Thus, we hypothesize that Schlager BPH/2J mice may respond considerably more in terms of BP rise than BPN/3J mice to a stressful stimulus. In the present study, we therefore investigated cardiovascular reactivity in normotensive BPN/3J and hypertensive BPH/2J Schlager mice before, during, and after exposure to aversive stress and compared this to a nonaversive stimulus. We induced a range of high arousal states by exposing mice to a series of behavioral “stress” tests that have previously been shown to affect cardiovascular reactivity in mice,10 including an unstable rotating table (shaker stress), clean cage-switch (new environment), and eating an almond (appetitive stimulus). We also included a restraint stress and utilized a tele metry BP monitoring system to enable measures of activity to account for the confounding factor of locomotion. As exaggerated activity of the SNS in response to stress has been linked to elevated cardiovascular disease risk and hypertension,11 we propose that this model may be crucial to our understanding of how the SNS might contribute to chronic hypertension.

cardiovascular parameters of all mice to baseline values. Feeding was initiated by placing a piece of almond through the drinking bottle hole in the cage lid (BPN/3J, n = 8 and BPH/2J, n = 7). The mouse was observed for when it commenced eating until when it ceased, and recordings were only included from mice that were seen feeding on the almond for a continuous period of not less than 5 min. If the mice ceased eating before 5 min, the experiment was repeated on an alternate day. Shaker stress involved moving the home cage containing the mouse onto an orbital mixer machine at a speed of 90 rotations/min (BPN/3J, n = 8 and BPH/2J, n = 7). After 5 min, the orbital mixer was switched off and the home cage containing the mouse was returned to its original position. In restraint stress, the mouse cage lid was removed, and the mouse was then guided into a cylindrical plexiglass restrainer with a sliding back plate to confine the mouse, without applying physical pressure to the animal (BPN/3J, n = 8 and BPH/2J, n = 7). The restrained mouse was left in the restrainer for 5 min before being released back into its cage and the cage lid replaced. After a 1-week recovery, clean cage-switch was performed that involved removing the mouse from its home cage and placing it in a clean, previously unoccupied cage (BPN/3J, n = 8 and BPH/2J, n = 6). After 60 min, the mouse was returned to its original cage.

Methods

Assessment of sympathetic blockade. Cardiovascular and locomotor responses were determined before and after intraperitoneal injection of the ganglionic blocker, pentolinium (7.5 mg/kg; Sigma , Sydney, Australia) in BPN/3J (n = 11) and BPH/2J (n = 7) mice, and 50 min following drug administration, mice were restrained for a period of 5 min.

Animals. Experiments were carried out in 18 ± 1 week-old conscious male normotensive (BPN/3J, n = 11) and hypertensive (BPH/2J, n = 7) mice. Animals were kept on a 12:12-h light–dark cycle (6 AM–6 PM light). All mice were allowed access ad libitum to water and mouse chow (19% protein, 5% fat, 5% fiber, 0.2% sodium; Specialty Feeds, Glen Forrest, Australia). The experiments were previously approved 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, HR, and locomotor activity in freely moving mice. Under isoflurane open-circuit anesthesia, mice were implanted with radiotelemetry transmitters, with the catheter inserted into the carotid artery and transmitter body implanted along the right flank.12 Following a 10-day recovery period, mice were housed individually for the remaining duration of the study. During the recording session, pulsatile arterial pressure and gross locomotor activity were monitored continuously, and were sampled at 1,000 Hz using an analog-to-digital data acquisition card, and the beat-to-beat mean arterial pressure, and HR were detected online and analyzed as described previously.10 Assessment of cardiovascular reactivity in response to behavioral “stress” tests. Induction of a high arousal state was induced by carrying out a series of behavioral “stress” tests as described previously.10,13 Each of the stressors were randomized on the first day with a period of 60–90 min in between to allow full recovery. This time period was sufficient to return AMERICAN JOURNAL OF HYPERTENSION | VOLUME 23 NUMBER 8 | august 2010

Statistical analysis. Cardiovascular data were analyzed by a split-plot repeated measure analysis of variance to determine the effects of genetic hypertension (strain) and stress on cardiovascular parameters and locomotion. The autocorrelation influence of repeated measures was adjusted by the Greenhouse–Geisser coefficient.14 The sum of squares calculated as the effect of stress between groups was compared using a between and within-animal combined residual mean square as described previously.15 Values were expressed as mean ± standard error of the mean or mean difference standard error of the difference. Values were considered significant when P < 0.05. Results Resting cardiovascular parameters

The average basal mean arterial pressure prior to stress in BPH/2J mice was 122 ± 2 mm Hg (n = 7) compared with 105 ± 1 mm Hg in BPN/3J (n = 11) (P < 0.0001), whereas HR was also higher in BPH/2J (549 ± 13 vs. 432 ± 7 beats/min, P < 0.001). Cardiovascular response to appetitive behavioral tests

Almond feeding. Feeding on a piece of almond caused a rapid and sustained pressor and tachycardic response in both 839



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Figure 1 | Line graphs represent the MAP, HR, and locomotor activity responses between BPN/3J mice (white circles; n = 8) and BPH/2J mice (black circles; n = 7) before, during, and after feeding. Each dot represents mean value, averaged across a 30-s period. Bar graphs represent average absolute changes (left) and percentage changes (right) in MAP and HR, and absolute change in locomotor activity in BPN/3J (white bar) and BPH/2J (black bar) in response to stress. The average responses were calculated over 5 min of stress exposure and a 5-min control period in each animal. Values are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 vs. BPN/3J mice. bpm, beats/min; BPH, hypertensive; BPN, normotensive; HR, heart rate; MAP, mean arterial pressure.

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Figure 2 | Line graphs represent the MAP, HR, and locomotor activity responses between BPN/3J mice (white circles; n = 8) and BPH/2J mice (black circles; n = 7) before, during, and after restraint stress. Each dot represents mean value; averaged across a 30-s period. Bar graphs represent average absolute changes (left) and percentage changes (right) in MAP and HR, and absolute change in locomotor activity in BPN/3J mice (white bar) and BPH/2J mice (black bar) in response to stress. These average responses were calculated over 5 min of stress exposure and a 5-min control period in each animal. Values are mean ± s.e.m. ***P < 0.001 vs. BPN/3J mice. bpm, beats/min; BPH, hypertensive; BPN, normotensive; HR, heart rate; MAP, mean arterial pressure.

groups of mice (Figure 1). The pressor response to feeding was greater in BPH/2J mice compared with BPN/3J mice (+27.4 ± 1.5 and +22.6 ± 1.1 mm Hg; F1,280 = 5.2; P = 0.02; Figure 1). However, when expressed as percentage change, there was no difference between groups. The tachycardic 840

response to feeding of +107 ± 9.5 beats/min in BPH/2J mice was smaller than that observed in BPN/3J (+149 ± 9.6 beats/ min; F1,280 = 9; P = 0.0025 for delta difference; P < 0.001 for % difference; Figure 1). No difference in activity was observed between groups (Figure 1). August 2010 | VOLUME 23 NUMBER 8 | AMERICAN JOURNAL OF HYPERTENSION



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Figure 3 | Line graphs represent the MAP, HR, and locomotor activity responses between BPN/3J mice (white circles; n = 11) and BPH/2J mice (black circles; n = 7) during restraint stress 50 min following pentolinium. Each dot represents mean value; averaged every 30 s across a 15-min period after pentolinium treatment. Bar graphs represent average absolute changes (left) and percentage changes (right) in MAP and HR, and absolute change in locomotor activity in BPN/3J mice (white bar) and BPH/2J mice (black bar) in response to stress. These average responses were calculated over 5 min of stress exposure and a 5-min control period in each animal. Values are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 vs. BPN/3J mice. bpm, beats/min; BPH, hypertensive; BPN, normotensive; HR, heart rate; MAP, mean arterial pressure.

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Figure 4 | Line graphs represent the MAP, HR, and locomotor activity responses between BPN/3J mice (white circles; n = 8) and BPH/2J mice (black circles; n = 7) before, during, and after shaker stress. Each dot represents mean value; averaged across a 30-s period. Bar graphs represent average absolute changes (left) and percentage changes (right) in MAP and HR, and absolute change in locomotor activity in BPN/3J mice (white bar) and BPH/2J mice (black bar) in response to stress. These average responses were calculated over 5 min of stress exposure and a 5-min control period in each animal. Values are mean ± s.e.m. **P < 0.01, ***P < 0.001 vs. BPN/3J mice. bpm, beats/min; BPH, hypertensive; BPN, normotensive; HR, heart rate; MAP, mean arterial pressure.

Cardiovascular response to aversive behavioral tests

Restraint. A 5-min restraint stress elicited rapid and sustained pressor and tachycardic responses in both groups of mice (Figure 2). The pressor response to restraint was 17 mm Hg AMERICAN JOURNAL OF HYPERTENSION | VOLUME 23 NUMBER 8 | august 2010

greater in BPH/2J than BPN/3J mice (+38 ± 3 mm Hg compared with +21 ± 2 mm Hg, respectively; F1,280 between strains = 23.8; P < 0.001; Figure 2). Expressed as a percent of baseline, the response was 77% greater in BPH/2J mice (P < 0.001). 841



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Figure 5 | Line graphs represent the MAP, HR, and locomotor activity responses between BPN/3J mice (white circles; n = 8) and BPH/2J mice (white circles; n = 6) before and during clean cage-switch stress. Each dot represents mean value, averaged across a 10-min period. Bar graphs represent average absolute changes (left) and percentage changes (right) in MAP and HR, and absolute change in locomotor activity in BPN/3J mice (white bar) and BPH/2J mice (black bar) in response to stress. These average responses were calculated over 60 min of stress exposure and a 60-min control period in each animal. Values are mean ± s.e.m. **P < 0.01, ***P < 0.001. BPH, hypertensive; BPN, normotensive.

Conversely, the tachycardic response to restraint was less in BPH/2J compared with BPN/3J mice (+161 ± 11 compared with +233 ± 10 beats/min; F1,280 = 19.4; P < 0.001; Figure 2). A small but similar increase in activity was observed (F1,280 between strains = 0.1; P > 0.05; Figure 2). Restraint following sympathetic blockade. Administration of pentolinium in hypertensive BPH/2J and normotensive BPN/3J mice abolished the pressor responses to restraint stress. Mean arterial pressure actually fell below control levels in BPH/2J mice (−13 ± 4 mm Hg), whereas there was a small increase in mean arterial pressure in BPN/3J (+5 ± 2 mm Hg). There was therefore a difference between strains (F1,100 = 21; P < 0.001). HR responses increased following sympathetic blockade and stress, but to a much lesser extent than in nontreated animals and also to a lesser extent in BPH/2J mice (F1,100 = 8.6; P = 0.005). Activity levels did not change during restraint stress in either group, but increased markedly after cession of the restraint (Figure 3). Shaker stress. A 5-min period on an orbital platform caused rapid pressor and tachycardic responses in both groups of mice. The pressor and tachycardic response was sustained in BPH/2J mice, but not in BPN/3J mice, where the BP and HR response declined after 3 min. Overall, the pressor response to shaker stress was 67% greater in BPH/2J mice when compared with BPN/3J mice (+29 ± 2 and +17 ± 2 mm Hg, respectively; F1,280 = 28; P < 0.001; Figure 4). The onset of shaker stress induced an increase in tachycardia in both groups of mice that remained elevated in BPH/2J mice, but returned to resting levels in BPN/3J mice before the shaker was switched off. Even so, 842

the average absolute changes and percentage changes (+26%) from resting HR to the stress-induced HR were not different between groups (F1,280 < 3.5; P > 0.05). Average responses in activity in both groups, during and after shaker stress, appear to be almost opposite in pattern with the BPN/2J more active during shaker than BPH/2J (F1,280 = 7.4; P = 0.01) and less active during recovery (F1,280 = 34; P < 0.001; Figure 4). Clean cage-switch. Pressor response to clean cage-switch was 85% greater in BPH/2J mice when compared with BPN/3J mice (+23 ± 1 and +12 ± 1 mm Hg, respectively; F1,190 = 54; P < 0.001). There was a slightly greater tachycardic response to clean cage-switch in BPH/2J mice (+162 ± 6 beats/min) compared with +144 ± 7 beats/min in BPN/3J mice (F1,72 = 7; P = 0.007). The change in activity levels was greater in BPH/2J mice (F1,72 = 18; P < 0.001; Figure 5). Discussion

The major findings from the present study indicate that hypertensive BPH/2J mice exhibit greater pressor responses to all aversive stressors (+67–88%) than normotensive BPN/3J mice. However, no difference in the pressor response was observed following nonaversive feeding (when expressed as a percentage of baseline). In absolute terms, there was a small difference in pressor response due to eating with the BPH/2J mice showing a 5 mm Hg greater response compared with BPN/3J mice. This might be expected due to the higher BP levels and probably reflects the impact of changes to mechanotransduction mechanisms associated with greater levels of vasoconstriction and chronic hypertension.16 However, scaling as percentage and hence greater vascular August 2010 | VOLUME 23 NUMBER 8 | AMERICAN JOURNAL OF HYPERTENSION


r eactivity change does not eliminate the marked disparity between pressor responses to the aversive stimuli between strains. The ability of pentolinium to completely abolish the pressor response to restraint stress strongly supports the conclusion that an aberrant response to aversive stress is leading to a greater sympathetic activation and therefore a greater increase in BP. This suggests that pathways regulating sympathetic vasomotor tone in response to aversive stress are the key to understanding the neural mechanism responsible for hypertension in BPH/2J mice. The very high levels of BP in BPH/2J mice are most evident during the active period, but the arousal associated with nocturnal behavior would not typically be considered “aversive.” We have previously shown that the hypertension is directly correlated to the activity of the MeAm,9 and this region is known to integrate the emotional response to stress.17 It is known to be necessary for the manifestation of the pressor response to stress.18 Taken together, these findings suggest that hypertension in the BPH/2J mice may be due to an exaggerated emotional response to aversive stimuli at the limbic or possibly higher cortical levels resulting in neurogenic hypertension. To explain night-time hypertension in the BPH/2J mice, the sympathetic over-reactivity must be related to the arousal stimuli associated with the onset of darkness. It remains to be determined whether the sounds and smells of other male mice are actually inducing a relative state of fear in these mice. The finding that the mechanism appears to be related to the integration of aversive but not appetitive arousal associated with food is consistent with a defect in the MeAm that is not thought to affect appetite19 apart from mineralocorticoidinduced sodium appetite.20 However, even at the level of the hypothalamus, these mechanisms are still functionally separate but colocated in the dorsomedial hypothalamus that projects to the rostral ventrolateral medulla. In rabbits, we found that aversive stress could be attenuated by blocking angiotensin AT1 receptors and through increased reactive oxygen species within the dorsomedial hypothalamus21 and also within the rostral ventrolateral medulla.22 By contrast, the response to food presentation was not affected. In the present study, we included a range of stimuli because not all the aversive stimuli result in the same hemodynamic patterns. Differences in tachycardia may induce a somewhat different contribution from cardiac output and resistance to the rise in BP during stress.23 Shaker stress has been noted previously as increasing BP but not HR or cardiac output in rabbits, and was referred to as a passive “freezing” type of stress.24 Apart from the initial disturbance of moving the mouse’s cage that increases HR for a few minutes, HR rapidly returned to control values during the latter part of shaker stress suggesting the passive “freezing” pattern that has been observed in rabbits was also observed in BPN/3J mice. By contrast, BPH/2J mice responded with a gradually increasing tachycardia suggesting that they respond inappropriately with a defense rather than a freezing pattern. One can also see this in the BP pattern suggesting some influence of the HR on the BP response to stress. The inappropriate response to shaker stress fits well with the known role of MeAm that is to recognize, AMERICAN JOURNAL OF HYPERTENSION | VOLUME 23 NUMBER 8 | august 2010

original contributions analyze, and categorize incoming chemosensory information and to determine appropriate emotional response whether it be reproductive, territorial/competitive, or predator passive.25 Thus, BPH/2J mice manifest an aberrant pattern of responses possibly by being unable to discriminate correctly the aversive stimulus but have a typical arousal response to food. In addition to differences in HR, one would expect some contribution of locomotor activity to BP responses in freely moving mice.10 Regardless of the type of stress, both groups of mice were immediately more active following stress exposure in a pattern that closely paralleled cardiovascular responses. We have previously described this in more detail looking at the relationship between locomotor activity and BP by correlation in the various forms of stress.26 This comparison supports our earlier findings in mice indicating that activity is associated with an increase in BP. Even so, the difference in the change in locomotor levels from rest to stress in BPH/2J compared with BPN/3J mice was inconsistent. Activity was greater in hypertensive mice during clean cage-switch as the mice explored the new environment more vigorously and for longer compared to normotensive mice. By contrast, activity was less during oscillation stress, and there was very little activity recorded during restraint as would be expected. Nevertheless, in all three cases, the pressor response was greater in BPH/2J mice. Thus, the disparate changes in locomotion induced by these different stressors suggest that altered activity in BPH/2J mice cannot explain the greater pressor responses to stress in this strain of mouse. In conclusion, the present study further implicates the SNS in the cause of hypertension in BPH/2J mice that appears to be driven particularly by a heightened state of arousal to aversive, but not to appetitive arousal stimuli. This predisposition appears to carry over into the daily pattern of activity resulting in a marked degree of hypertension. Given that these mice were originally selected as hypertensive using a tailcuff restrainer,27 which is identical to our method of giving restraint stress, this may not appear surprising in hindsight. Thus, these mice represent a type of white coat hypertensive subject who reacts more to stressful stimuli to the extent of becoming hypertensive. Given our previous analysis showing that the greater reactivity can be measured within the MeAm, the cause may lie at the “emotional” limbic level rather than the autonomic levels in the hypothalamus or brainstem. Disclosure: The authors declared no conflict of interest. 1. Esler M. Sympathetic nervous system: contribution to human hypertension and related cardiovascular diseases. J Cardiovasc Pharmacol 1995; 26(Suppl 2):S24–S28. 2. Ferrier C, Jennings GL, Eisenhofer G, Lambert G, Cox HS, Kalff V, Kelly M, Esler MD. Evidence for increased noradrenaline release from subcortical brain regions in essential hypertension. J Hypertens 1993; 11:1217–1227. 3. Head GA, Lukoshkova EV. Understanding the morning rise in blood pressure. Clin Exp Pharmacol Physiol 2008; 35:516–521. 4. 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. 5. Schlager G, Sides J. Characterization of hypertensive and hypotensive inbred strains of mice. Lab Anim Sci 1997; 47:288–292. 6. 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. 843

original contributions 7. Schlager G, Freeman R. Norepinephrine level in the hypothalamus of the genetically hypertensive mouse. Experientia 1983; 39:793–794. 8. Denoroy L, Sautel M, Schlager G, Sacquet J, Sassard J. Catecholamine concentrations in discrete brain nuclei and sympathetic tissues of genetically hypertensive mice. Brain Res 1985; 340:148–150. 9. Davern PJ, Nguyen-Huu TP, La Greca L, Abdelkader A, Head GA. Role of the sympathetic nervous system in Schlager genetically hypertensive mice. Hypertension 2009; 54:852–859. 10. 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. 11. Esler M, Rumantir M, Kaye D, Jennings G, Hastings J, Socratous F, Lambert G. Sympathetic nerve biology in essential hypertension. Clin Exp Pharmacol Physiol 2001; 28:986–989. 12. Butz GM, Davisson RL. Long-term telemetric measurement of cardiovascular parameters in awake mice: a physiological genomics tool. Physiol Genomics 2001; 5:89–97. 13. Davern PJ, Chen D, Head GA, Chavez CA, Walther T, Mayorov DN. Role of angiotensin II Type 1A receptors in cardiovascular reactivity and neuronal activation after aversive stress in mice. Hypertension 2009; 54:1262–1268. 14. Ludbrook J. Repeated measurements and multiple comparisons in cardiovascular research. Cardiovasc Res 1994; 28:303–311. 15. Korner PI, Badoer E, Head GA. Cardiovascular role of the major noradrenergic cell groups in the rabbit: analysis based on 6-hydroxydopamine-induced transmitter release. Brain Res 1987; 435:258–272. 16. Folkow B, Grimby G, Thulesius O. Adaptive structural changes of the vascular walls in hypertension and their relation to the control of the peripheral resistance. Acta Physiol Scand 1958; 44:255–272. 17. Singewald N, Chicchi GG, Thurner CC, Tsao KL, Spetea M, Schmidhammer H, Sreepathi HK, Ferraguti F, Singewald GM, Ebner K. Modulation of basal and

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August 2010 | VOLUME 23 NUMBER 8 | AMERICAN JOURNAL OF HYPERTENSION