Psychophysiology, 38 ~2001!, 490–499. Cambridge University Press. Printed in the USA. Copyright © 2001 Society for Psychophysiological Research
Blood pressure, heart rate, and behavioral responses to psychological “novelty” stress in freely moving rats
MAARTEN VAN DEN BUUSE,a SASKIA A.B.E. VAN ACKER,b MARC FLUTTERT,b and E. RONALD DE KLOET b a b
Neuropharmacology Laboratory, Baker Medical Research Institute, Melbourne, Australia Leiden0Amsterdam Center for Drug Research, Leiden University, Sylvius Laboratories, Leiden, The Netherlands
Abstract We developed a new model of psychological “open-field” stress in freely moving rats. Blood pressure and heart rate of the rats were measured by radiotelemetry and behavior analyzed by video tracking software. Open-field exposure induced marked increases in blood pressure and heart rate. Repeated daily exposure induced pressor responses that were slightly higher on Day 4 when compared to Day 1. Pretreatment with the b1-adrenoceptor antagonist atenolol inhibited the tachycardia whereas the ganglion blocker pentolinium inhibited the pressor response, indicating involvement of the sympathetic nervous system. Pretreatment with diazepam prevented the novelty stress-induced pressor response and reduced the tachycardia. These results show that the psychological stress of exposing rats to an open field induces marked cardiovascular effects that are mediated by sympathetic hyperactivity. This model is unique in that it focuses on psychological stress and allows concomitant measurement of blood pressure, heart rate, and behavior in freely moving rats. Descriptors: Stress, Blood pressure, Heart rate, Sympathetic, Rats, Behavior
Little information is available on the central nervous system mechanisms in stress-induced cardiovascular responses in humans ~Soufer et al., 1998; Wilkinson et al., 1998!. By contrast, several experimental animal studies have addressed the involvement of a variety of brain mechanisms in such responses but have done so using a wide variety of methods ~Bohus, Koolhaas, Korte, Roozendaal, & Wiersma, 1996; Davis, Falls, Campeau, & Kim, 1993; Smith, De Vito, & Astley, 1984!. While several of these studies have used physical stressors such as restraint, footshock, and air-jet stress ~Burke, Malpas, & Head, 1998; DiBona & Jones, 1995!, it is particularly the more psychological stressors such as novelty, conditioned fear ~Carrive, Leung, Harris, & Paxinos, 1997; Davis et al., 1993! and dominant0submissive behavior ~Blanchard, Sakai, McEwen, Weiss, & Blanchard, 1993! that are relevant for human mental and psychological stress. Importantly, physical stressors such as the widely used immobilization stress have been shown to involve different CNS pathways compared with psychological stressors such as novelty stress or conditioned fear ~Iványi, Wiegant, & De Wied, 1991; Petty, Kramer, & Larrison, 1996; Van de Kar, Piechowski, Rittenhouse, & Gray, 1991!, and, in addition, show a different extent and pattern of cardiovascular responses and habituation ~Lawler, Naylor, & Abel, 1993; McCarty & Gold, 1996; Roozendaal, Koolhaas, & Bohus, 1997!. Exposure of an animal to a novel environment elicits a number of central and hormonal responses ~Papa, Pellicano, Welzl, & Sadile, 1993; Petty et al., 1996!. The “large open field” has been used for several years as a simple novelty stimulus ~Denenberg, 1969; Ramos & Mormede, 1998; Van den Buuse & De Jong, 1988, 1989!. No obviously noxious stimulus, such as footshock, re-
There is growing epidemiological evidence that mental stress is a risk factor increasing cardiovascular morbidity and mortality ~Rosengren, Tibblin, & Wilhelmsen, 1991; Ruberman, Weinblatt, Goldberg, & Chaudhary, 1984!. For example, subjects who showed the presence of mental stress-induced cardiac ischemia had significantly higher rates of subsequent fatal and nonfatal cardiac events in a long-term follow-up study when compared to subjects who did not show stress-induced ischemia ~Jiang et al., 1996!. Prolonged stress in the workplace, caused by high work load and low rewards, predicted a higher incidence of cardiac ischemia and fatal cardiovascular events when compared to control subjects who experienced low levels of stress ~Bosma, Peter, Siegrist, & Marmot, 1998; Lynch, Krause, Kaplan, Tuomilehto, & Salonen, 1997!. The acute effects of stress in humans include marked and regionally specific increases in sympathetic vasomotor and cardiac tone ~Benschop et al., 1994; Esler et al., 1995; Thompson & Moss, 1994!, leading to vasoconstriction and increased heart rate. In addition, psychological stress induces a variety of hemodynamic, renal, and hormonal responses ~Herd, 1991!.
This study was supported by a grant from the Dutch Heart Foundation ~Grant Number 94-122! and a block-institute grant of the National Health and Medical Research Council of Australia to the Baker Medical Research Institute. The authors gratefully acknowledge the helpful suggestions of Dr. Melly S. Oitzl. Address reprint requests to: M. van den Buuse, Ph.D., Mental Health Research Institute, Locked Bag 11, Parkville, Victoria 3052, Australia. E-mail:
[email protected].
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Novelty stress and blood pressure straint, or noise, needs to be applied, making the “open field” test an attractive model in terms of animal experimentation ethics. Moreover, the open-field method allows for easy quantification of different aspects of exploratory and locomotor behavior. Behavioral analysis can be done by a trained observer or by using specialized analysis software, the latter being more objective and considerably less labor intensive. In the open field, rats experience acute novelty-induced fear, unpredictability, and unfamiliarity, but they also display exploratory behavior, curiosity, and coping ~Dulawa, Grandy, Low, Paulus, & Geyer, 1999!. Quantified components of behavioral responses relate to different aspects of these characteristics ~Denenberg, 1969; Ramos & Mormede, 1998!. With repeated exposure to the open field, behavioral responses change, which is usually referred to as behavioral habituation. However, little information is available about cardiovascular responses to repeated exposure to stress such as the open field. While trying to delineate central mechanisms involved in cardiovascular stress responses ~or test new drugs!, surprisingly few studies have attempted to measure behavioral as well as cardiovascular responses simultaneously. This is important in order to separate anxiolytic, sedative, and motor effects of any particular treatment applied and control for nonspecificity. One problem might have been the use of catheters, harnesses, and tethers, which by themselves can greatly influence behavioral responses. To record behavioral and cardiovascular responses without the use of indwelling cannulae, in the present study we used radiotelemetry and small implantable radiotransmitters allowing the rats to be truly freely moving and unrestrained ~Brockway, Mills, & Azar, 1991; Guiol, Ledoussal, & Surgé, 1992; Van den Buuse, 1994!. In the present study, we thus set out to characterize the open field test in rats as a stimulus model to investigate cardiovascular responses to psychological stress. We used automated methodology, including radiotelemetric assessment of blood pressure and heart rate, and video-tracking analysis of exploratory locomotor behavior. The specific objects of the study were ~a! to assess the feasibility of this model by quantifying and comparing changes in blood pressure, heart rate, and behavioral parameters in response to open-field exposure; ~b! to verify the possible role of the sympathetic nervous system in this new model by using pretreatment with the ganglion blocker pentolinium to block sympathetic vasomotor activity or the b1-adrenoceptor antagonist atenolol to block cardiac sympathetic activity; ~c! to assess the effect of a classical anxiolytic drug, diazepam; and ~d! to compare cardiovascular and behavioral responses over repeated exposure of the rats to the open field. Methods Experiments were conducted on three separate groups of 8 male wistar rats ~Wagrij line, Charles River, Germany!, which were obtained from the Broekman instituut ~Amsterdam, The Netherlands! and were 230 6 3 g body weight. The animals were anesthetized with a mixture of N2O0O 2 ~2:1! and isoflurane and instrumented with TA11PA-C40 telemetry transmitters ~Data Sciences, USA! as previously described ~Brockway et al., 1991; Van den Buuse, 1994!. Briefly, through a midline abdominal incision, the abdominal aorta was exposed and clamped off. A small hole was punctured in the aortic wall and the flexible tip of the transmitter cannula inserted and fixed in place with a drop of tissue glue ~Loctite, Australia!. The aortic clamps were then removed and blood flow to the hindquarters could resume, always within 2 min of the initial occlusion. The body of the transmitter was sutured to the inside abdominal wall and all incisions were suture closed.
491 Animals were given a subcutaneous injection of Temgesic ~Buprenorphin! and allowed 10 days to recover from the surgery. The rats were housed in individual cages with rat chow and tap water available ad libitum. In the animal house, lights were on at 7 a.m. and off at 7 p.m. and experiments were performed between 9 a.m. and 1 p.m. Baseline data for blood pressure, heart rate, and behavioral activity were obtained by telemetry while the animals were in the home cage either the day before the first open field test ~Experiment 1! or during 10 min before treatments and 10 min before subsequent transfer to the open field ~Experiments 2 and 3!. In Experiment 1, baseline values were the average of four 1-min readings, taken 15 min apart, overlapping the time of day the rats were going to be tested. Blood pressure values were obtained as absolute pressures, using calibration values obtained from Data Science ~USA!. Ambient pressure was obtained with a Data Sciences ambient pressure monitor and substracted by the system from the telemetry pressure values to give blood pressure. Activity counts were obtained by the system by monitoring changes in the signal strength that occurred as a result of whole body movements. Changes in signal strength of more than a predetermined amount generate a digital pulse that was counted by the system. Open Field Telemetry and Behavioral Analysis The open field consisted of a 90-cm circular arena with a wall approximately 30 cm high ~Oitzl, Fluttert, & De Kloet, 1994; Van den Buuse & De Jong, 1988!. An “object” ~small centrifuge bucket! was placed in the center of the open field to arouse the rat’s curiosity and draw it away from the perimeter of the open field, which is where it would normally spend most of the time ~Denenberg, 1969; Oitzl et al., 1994; Ramos & Mormede, 1998!. Lighting was provided by a 60-W bulb approximately 1 m above the floor of the open field; otherwise the room was only dimly lit. A commercial video camera was mounted about 150 cm above the open field and connected to a commercial video recorder for recording the rat’s behavior. Eight RA1010 receivers ~Data Sciences, USA! were placed under the floor of the open field covering as much as possible of the open-field area. The eight receivers were connected to a receiver multiplexer ~RMX10, Data Sciences, USA! that was connected to one channel on the system’s consolidation matrix ~BCM100, Data Sciences, USA!. We used the Dataquest IV data acquisition program ~Data Sciences, USA! to obtain data for systolic, mean, and diastolic blood pressure, heart rate, and gross behavioral activity every 12 s while each rat was in the open field. Behavior was analyzed from the video recordings by the Ethovision videotracking system ~version 3.0, Noldus Information Technology, Wageningen, The Netherlands!. In the analysis program, the open field was divided into outer circle and inner circle ~of approximately 40-cm diameter! and analysis distinguished data for “total arena” ~outer circle plus inner circle! from those obtained in the “inner zone.” Parameters calculated were distance moved in total arena ~in centimeters per minute!, distance moved in inner zone ~in centimeters per minute!, velocity of movement in total arena ~in centimeters per second! and percentage time spent in the inner zone. It should be noted that the Noldus analysis package determines velocity only when the animal is actually in motion and ignores times at which no movement occurs. Experiment 1: Cardiovascular and Behavioral Effects of Exposure to the Open Field Blood pressure, heart rate, and gross behavioral activity were recorded the day before the first open-field exposure during the
492 same time as the animals would be transferred to the open field. On the day of the experiments, the rats were taken in their home cage from the animal holding room to the experiment room and transferred into the open field, close to the wall but facing the center, within 5 min. After 15 min, the rat was gently removed from the open field, transferred to its home cage and carried back to the animal holding room. This procedure was repeated at the same time of day on 4 subsequent days. After the last open field exposure, the rats were killed and the telemetry probes retrieved. Experiment 2: Effect of Pretreatment with Atenolol or Pentolinium Because Experiment 1 showed a marked difference in behavioral activity between Day 1 and Day 2 of exposure to the open field with only small changes in cardiovascular responses, in subsequent experiments the rats were first put in the open field on one day ~no recordings taken! and subsequently treated and tested on the following day. On this experiment day, baseline blood pressure, heart rate, and behavioral activity were recorded 5 min before and 20 min after treatment with saline ~1 ml0kg s.c.!, the b1-adrenoceptor antagonist atenolol ~1 mg0kg s.c.! or the ganglion blocker pentolinium ~5 mg0kg s.c.! ~Keeton & Biediger, 1991!. The injections were given 25 min before the rats were placed into the open field. As in the first experiment, the rats were brought to the experiment room in their home cages, then gently transferred into the open field, and 15 min later gently transferred back into their home cages and brought back to the animal holding room. The design of this experiment was a randomized “crossover” design where the rats were treated randomly with different drugs on different experiment days. Thus, three sessions, each consisting of one initial exposure to the open field, without drug treatment, and one subsequent experimental day, were done with at least 4-day intervals. At the end of the three experiments, all rats had thus received all treatments and none of the treatments were repeated in one and the same rat. After the last open-field exposure, the rats were killed and the telemetry probes retrieved. Experiment 3: Effect of Pretreatment with Diazepam or Clonidine The protocol for this experiment was similar to that of Experiment 2, except that the rats were treated randomly with either saline ~1 ml0kg s.c.!, the benzodiazepine anxiolytic diazepam ~2 mg0kg s.c.!, or the centrally acting a2-adrenoceptor agonist clonidine ~5 mg0kg s.c.!. Data Analysis Data obtained by the telemetry system or the behavioral tracking analysis when the rats were in the open field were averaged over 1-min periods. Baseline data were either the average of four readings, each of 1 min, 15 min apart ~Experiment 1!, or of 10 min between 5 and 15 min before treatment ~before open field0before treatment values, Experiments 2 and 3! and 10 min between 10 and 20 min after treatment ~before open field0after treatment values, Experiments 2 and 3!. In Experiment 1, the time courses of blood pressure, heart rate, and behavioral parameters were analyzed with a two-way analysis of variance ~ANOVA! for repeated measures using Time ~15 subsequent min! and Day ~4 subsequent days! as factors. A Bonferroni-corrected t test was used to determine significant differences between different days. In Experiments 2 and 3, the effect of treatments on baseline blood pressure and heart rate
M. van den Buuse et al. was analyzed with a two-way ANOVA for repeated measures with Treatment ~saline, atenolol, pentolinium or saline, diazepam, clonidine, respectively! and Time ~before versus after treatment! as factors. Between-group comparisons were again done with a Bonferroni-corrected t test. The time course of changes in changes in blood pressure and heart rate during the 15-min open field exposure were analyzed with a two-way ANOVA for repeated measures with Treatment and Time ~15-min observation period! as factors. Average 15-min values for behavioral parameters were analyzed with a one-way ANOVA for repeated measures, followed by a Bonferroni-corrected t test for between-group comparisons. Statistical analysis was carried out using commercially available statistics software ~Sigmastat, version 1.0, Jandel Scientific!. In all cases a p of less than 5% was considered as indicating a significant difference. Data are presented as mean 6 standard error of the mean ~SEM!. Results Experiment 1: Cardiovascular and Behavioral Effects of Exposure to the Open Field While in the open field, rats displayed blood pressure and heart rate values markedly higher than those obtained in the home cage on previous days ~Figure 1!. During the 15-min of their first open-field exposure, systolic, diastolic, and mean blood pressure of the rats initially rose and then tended to fall again, whereas heart rate tended to increase gradually ~Figure 1!. One-way ANOVA with repeated measures of blood pressure data confirmed these changes over the 15 min, F~7,14! 5 7.0, p , .001, F~7,14! 5 8.8, p , .001, and F~7,14! 5 9.1, p , .001, for systolic, diastolic, and mean blood pressure, respectively, and F~7,14! 5 3.4, p 5 .001, for heart rate. Significant changes in gross activity, F~7,14! 5 2.4, p 5 .005, reflected the high initial activity followed by a gradual decrease. Total distance moved showed a significant decline during the 15 min, F~7,14! 5 4.8, p , .001, whereas values for distance moved in the inner circle were variable and did not change significantly. The percentage time spent in the inner circle increased significantly, F~7,15! 5 2.4, p 5 .005. Velocity of movement, as measured in the total field, significantly declined during the 15-min observation period, F~7,14! 5 4.2, p , .001 ~Figure 1!. Analysis of cardiovascular and behavioral data over repeated exposures revealed significant changes ~Figure 2 and Table 1!. Overall values of mean blood pressure on Day 4, but not Days 2 and 3, were higher than on Day 1, whereas the pattern of changes within the 15-min open field exposure was similar between all 4 days ~significant effect only of Time, F~14,479! 5 22.7, p , .001, and of Day, F~3,479! 5 3.5, p 5 .034; Figure 2!. On all 4 days, heart rate gradually rose above the initial increased values during the 15-min open-field exposure, although the overall levels were similar ~significant effect only of Time, F~14,479! 5 3.9, p , .001!. Systolic and diastolic blood pressures showed a similar pattern to mean blood pressure with the values on Day 4, but not Day 2 or Day 3, being significantly higher than those on the first day of open field exposure ~Table 1!. Behavioral activity, as measured by telemetry, showed a marked decrease in activity counts from the first to the second day of open field exposure, but no further changes on Days 3 and 4 ~significant effect only of Day, F~3,479! 5 14.8, p , .001, and of Time, F~14,479! 5 3.97, p , .001!. Behavioral parameters obtained from the automated tracking analysis generally showed a similar trend as the behavioral counts obtained by telemetry. Thus, counts
Novelty stress and blood pressure
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Figure 2. Mean blood pressure ~top panel! and heart rate ~bottom panel! of 8 freely moving rats during 15 min in the open field ~curves!. The rats were exposed to the open field on 4 consecutive days; data from Day 1 and Day 4 are shown ~see Table 1 for average values of all 4 days!. Baseline values obtained on previous days at similar times as the open-field exposure are shown for comparison ~hatched bars!. Data were obtained with radiotelemetry and expressed as mean values 6 the average within-animal SEM. Blood pressure on Day 4 was significantly higher than on Day 1.
Figure 1. Parallel changes of systolic, diastolic, and mean blood pressure, heart rate, and behavioral parameters of rats in the open field as measured by radiotelemetry and video-tracking analysis. Data are mean 6 SEM of 8 rats during 15 min. The rats were exposed to the open field on 4 consecutive days; data from Day 1 is shown ~see Table 1 for values of all 4 days!. Baseline blood pressure, heart rate, and locomotor counts were obtained in the home cage.
were significantly reduced upon repeated exposure to the open field compared to the first day for total distance moved ~effect of Day, F~3,479! 5 13.2, p , .001!; distance moved in the inner zone of the open field, F~3,479! 5 5.0, p 5 .009!; and velocity of movement, F~3,479! 5 13.2, p , .001!. The time-related trends over the 15-min open-field exposure were similar on the 4 consecutive days ~Table 1!. Because of the large decrease in behavioral activity from the first to the second day of exposure to the open field with relative maintenance of the cardiovascular response, in the subsequent series of experiments rats were always exposed to the open field on one day and treated and tested on the next day.
Experiment 2: Effects of Pretreatment with Atenolol or Pentolinium Baseline values. A significant decrease in resting blood pressure was observed after treatment with pentolinium but not atenolol ~significant overall effect of Treatment, F~2,41! 5 9.2, p 5 .004; of Time, F~1,41! 5 10.7, p 5 .017; and a significant Treatment 3 Time interaction, F~2,41! 5 24.5, p , .001; Table 2!. Neither pentolinium or atenolol pretreatment significantly affected resting heart rate ~Table 2!, although there was a significant Treatment 3 Time interaction, F~2,41! 5 6.2, p 5 .014, probably reflecting the tendency for heart rate values to increase in saline-treated rats but to decrease after atenolol or pentolinium pretreatment. Open field. In marked contrast to rats that had been pretreated with saline or atenolol, in pentolinium-pretreated rats, the pressor response during exposure to the open field was markedly reduced ~Figures 3 and 4!. Although the overall effect of Pretreatment was of borderline significance, F~2,314! 5 3.6, p 5 .059, there was a significant effect of Time, F~14,314! 5 2.1, p 5 .018, and a significant Treatment 3 Time interaction, F~28,314! 5 3.1, p , .001 ~Figure 3!, reflecting the marked difference in time course between the groups. The 15-min average values for mean blood pressure confirmed this difference ~Figure 4! and a similar effect was observed for systolic and diastolic blood pressure ~not shown!. Rats pretreated with atenolol showed a significantly smaller tachycardia response in the open field than saline- or pentoliniumpretreated rats ~Figure 3!. Statistical analysis showed a significant overall effect of Treatment, F~2,314! 5 8.1, p 5 .006, and of Time, F~14,314! 5 3.4, p , .001, and a Treatment 3 Time interaction, F~28,314! 5 2.1, p 5 .002. Analysis of the average 15-min values confirmed the effect of atenolol ~Figure 4!.
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M. van den Buuse et al. Table 1. Average Values of Cardiovascular and Behavioral Parameters Obtained from 8 Conscious, Freely Moving Rats During 15-Min Exposure to the Open Field on 4 Subsequent Days
Systolic blood pressure ~mm Hg! Mean blood pressure ~mm Hg! Diastolic blood pressure ~mm Hg! Heart rate ~b0min! Behavioral activity ~counts! Distance moved in total open field ~cm0min! Distance moved in inner circle ~cm0min! Velocity of movement ~cm0s! Percentage time in inner circle
Day 0
Day 1
Day 2
Day 3
Day 4
113 6 2 97 6 1 82 6 1 383 6 6 461
135 6 1 117 6 1 99 6 1 500 6 2 805 6 1 355 6 11 81 6 4 6.0 6 0.2 29.3 6 2.0
137 6 1 119 6 1 101 6 1 503 6 2 501 6 2* 228 6 12* 50 6 5 3.9 6 0.2* 21.7 6 2.3
138 6 1 119 6 1 101 6 1 501 6 2 469 6 2* 202 6 13* 46 6 5* 3.4 6 0.2* 21.7 6 2.3
139 6 1* 121 6 1* 103 6 1* 507 6 3 439 6 2* 190 6 11* 46 6 5* 3.2 6 0.2* 23.2 6 2.6
Note. Basal cardiovascular values were obtained one day before the first open-field test ~Day 0!. “Behavioral activity” was obtained by the telemetry system; other behavioral parameters were obtained from automated tracking analysis. Data are mean 6 SEM. *p , .05 for difference with Day 1 ~two-way ANOVA for repeated measures and Bonferroni-corrected t test!.
Behavioral activity, as measured by telemetry, was significantly reduced in rats that had been pretreated with pentolinium, but was unaffected by atenolol pretreatment ~Figure 4!. Similar findings were obtained from the automated tracking analysis. Thus, distance moved in the total open field or the inner circle, velocity of movement, and percentage time spent in the inner circle were all significantly reduced in pentolinium-pretreated rats but not in atenolol-pretreated rats ~Figure 4!. Experiment 3: Effects of Pretreatment with Diazepam or Clonidine Baseline values. Pretreatment with diazepam produced a small, but significant increase in resting blood pressure ~Table 2!. There was a significant overall effect of Treatment, F~2,41! 5 4.5, p 5
.035, and of Time, F~1,41! 5 10.0, p 5 .02. Resting heart rate was also slightly, but significantly increased in rats treated with saline or diazepam, but not clonidine ~significant overall effect of Treatment, F @2,41# 5 8.8, p 5 .0045, and of Time, F @1,41# 5 46.3, p , .001; Table 2!. Open field. In marked contrast to rats pretreated with saline or clonidine, in diazepam-pretreated rats, the pressor response was completely absent ~Figures 5 and 6!. There was a significant effect of Treatment, F~2,314! 5 14.4, p , .001!; of Time, F~14,314! 5 15.8, p , .001; and a significant interaction between these factors, F~28,314! 5 4.0, p , .001. The 15-min average values for mean blood pressure confirmed this difference ~Figure 6! and a similar effect was observed for systolic and diastolic blood pressure ~not
Table 2. The Effect of Pretreatments on Mean Blood Pressure and Heart Rate as Measured by Telemetry in Conscious, Freely Moving Rats in the Home Cage
Experiment 2: saline Mean blood pressure ~mm Heart rate ~b0min! Atenolol ~1 mg0kg s.c.! Mean blood pressure ~mm Heart rate ~b0min! Pentolinium ~5 mg0kg s.c.! Mean blood pressure ~mm Heart rate ~b0min! Experiment 3: saline Mean blood pressure ~mm Heart rate ~b0min! Diazepam ~2 mg0kg s.c.! Mean blood pressure ~mm Heart rate ~b0min! Clonidine ~5 mg0kg s.c.! Mean blood pressure ~mm Heart rate ~b0min!
Before open field0 before treatment
Before open field0 after treatment
Change
Hg!
100 6 3 341 6 3
103 6 4 388 6 15
n.s. n.s.
Hg!
99 6 5 365 6 17
99 6 4 344 6 5
n.s. n.s.
Hg!
104 6 4 370 6 15
81 6 3* 347 6 11
222.4 6 2.9 n.s.
Hg!
99 6 1 354 6 6
106 6 3 412 6 17*
n.s. 58 6 18
Hg!
99 6 1 357 6 10
109 6 4* 437 6 13*
9.9 6 3.5 80 6 14
Hg!
99 6 2 355 6 8
99 6 3 381 6 13
Note. Data are mean 6 SEM of 7 rats in each experiment. *p , .05 for difference with baseline values ~two-way ANOVA for repeated measures and Bonferroni-corrected t test!.
n.s. n.s.
Novelty stress and blood pressure
Figure 3. Change in mean blood pressure ~top panel! and heart rate ~bottom panel! in 7 conscious, freely moving rats during a 15-min exposure to the open field. Rats were exposed to the open field on 3 different days with at least 4-day intervals after random pretreatment with saline ~open circles!, 1 mg0kg of atenolol ~closed squares!, or 5 mg0kg of pentolinium ~closed triangles!. Pretreatment with pentolinium blocked the stressinduced rise in blood pressure whereas pretreatment with atenolol inhibited the tachycardia. Data are expressed as mean 6 the average within-animal SEM. For average 15-min values, see Figure 4.
shown!. Rats that had been treated with diazepam showed only a reduced and short-lasting tachycardia response in the open field. The tachycardia was also smaller in rats that had been treated with clonidine, resulting in values in between those of the saline- and diazepam-treated rats ~Figure 5!. Statistical analysis showed a significant overall effect only of Treatment, F~2,314! 5 4.6, p 5 .033, and of Time, F~14,314! 5 30.3, p , .001. Analysis of the average 15-min values showed a significant reduction of the tachycardia after both diazepam and clonidine treatment ~Figure 6!. Behavioral activity, as measured by the telemetry system, was significantly reduced in rats that had been pretreated with diazepam, but was not significantly affected by clonidine treatment ~Figure 6!. The automated tracking analysis showed a significantly reduced total distance moved in the open field, whereas none of the other parameters was significantly altered ~Figure 6!. Discussion We developed a new model of psychological stress in rats combining exposure to a novel environment and measurement of cardiovascular and behavioral parameters by radiotelemetry and automated tracking analysis, respectively. We observed that rats, when exposed to the novelty stress of being placed in a large open field, displayed marked pressor responses and tachycardia in addition to a range of behavioral manifestations. The pressor response and tachycardia could be blocked to a large extent by pretreatment with a ganglion blocker and b1-adrenoceptor antag-
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Figure 4. The effect of pretreatment with atenolol ~1 mg0kg s.c.! or pentolinium ~5 mg0kg s.c.! on the change in blood pressure and heart rate, and on behavioral parameters during a 15-min exposure to the open field in conscious, freely moving rats ~n 5 7!. Pretreatment with pentolinium blocked the stress-induced rise in blood pressure whereas pretreatment with atenolol inhibited the tachycardia. Pretreatment with pentolinium also caused a reduction in behavioral activity. Data are expressed as mean 6 SEM. *p , .05 for difference with values obtained after saline-pretreatment. IZ 5 inner zone of the open field. The double bar for distance indicates total values or values for inner circle only.
onist, respectively, suggesting these responses were mediated by increased sympathetic vasomotor tone and cardiac sympathetic activity, respectively. Interestingly, although behavioral hyperactivity in the open field was reduced upon repeated exposure, the cardiovascular responses did not show similar habituation. Indeed, there was a small, but significant trend for the blood pressure response to become greater after repeated exposure. Pretreatment with the benzodiazepine anxiolytic diazepam blocked both the pressor response and virtually all of the tachycardia while selectively inhibiting aspects of behavior. Pretreatment with a low dose of the a2-adrenoceptor agonist clonidine inhibited the tachycardia. In general, changes in behavior or baseline cardiovascular values were unrelated to the differential changes in open-field cardiovascular stress responses. Repeated Exposure to Stress It is widely accepted that repeated exposure to a stressful situation leads to a gradual reduction in behavioral, hormonal, and cardiovascular responses, a process usually referred to as habituation ~Chen & Herbert, 1995; McCarty & Gold, 1996!. In our experiments, behavioral responses were reduced upon repeated exposure to the open field, in accordance with this principle. The contrasting
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Figure 5. Change in mean blood pressure ~top panel! and heart rate ~bottom panel! in 7 conscious, freely moving rats during a 15-min exposure to the open field. Rats were exposed to the open field on 3 different days with at least 4-day intervals after random pretreatment with saline ~open circles!, 2 mg0kg of diazepam ~closed squares!, or 5 mg0kg of clonidine ~closed triangles!. Pretreatment with diazepam prevented the stressinduced rise in blood pressure whereas pretreatment with clonidine attenuated the tachycardia. Data are expressed as mean 6 the average withinanimal SEM. For average 15-min values, see Figure 6.
finding, that cardiovascular responses were not reduced upon repeated exposure, could be explained in two ways. Habituation will occur when stressors are predictable in the type of stimulus, intensity, and duration, allowing the animal to reduce its behavioral and neurohormonal responses to the minimum required to maintain homeostasis ~De Boer, Slangen, & Van der Gugten, 1989; McCarty & Gold, 1996!. Our results could then be explained by suggesting that exposure of a rat to an open-field situation, as used in the present experiments, confronts the animal with a high degree of unpredictability that persists over consecutive sessions and prevents habituation of the cardiovascular responses. This does not, however, explain why behavioral responses were reduced, unless we assume that the reduced scores for distance moved and velocity reflect an increased immobility or “freezing” behavior. Immobility or freezing is a widely described effect of exposure to severe stressors such as footshock ~Carrive et al., 1997; Davis et al., 1993; Ramos & Mormede, 1998! and the possible increase in immobility in our experiments could reflect sensitization of the animal to repeated unpredictability, similar to the blood pressure responses. Another, contrasting, explanation of the results could be that repeated exposure to the open field confronts the rat with a familiar and predictable environment allowing the animal to learn from its previous experience and adjust its behavioral and neurohormonal responses accordingly. Upon repeated exposure to the open field, the animal may well recognize the environment and display reduced exploration. On the other hand, because there is no place to
M. van den Buuse et al.
Figure 6. The effect of pretreatment with diazepam ~2 mg0kg s.c.! or clonidine ~5 mg0kg s.c.! on the change in blood pressure and heart rate, and on behavioral parameters during a 15-min exposure to the open field in conscious, freely moving rats ~n 5 7!. Pretreatment with diazepam completely prevented the stress-induced rise in blood pressure whereas pretreatment with clonidine attenuated the tachycardia. Pretreatment with diazepam also caused a reduction in behavioral activity and distance moved. Data are expressed as mean 6 SEM. *p , .05 for difference with values obtained after saline pretreatment. IZ 5 inner zone of the open field. The double bar for distance indicates total values or values for inner circle only.
hide or escape, the cardiovascular system ~and, presumably, hormonal release! remains activated in readiness of possibly necessary flight or defensive responses. A dissociation between exploration and stress levels has been suggested by other studies as well. Ramos and Mormede ~1998! recently reviewed this literature on multidimensional aspects of stress responses in animals. Although not specifically dealing with cardiovascular responses or habituation mechanisms, their review discussed several studies showing that “locomotion in novel stressful environments and measures of anxiety represent two different dimensions of emotionality”. The authors furthermore argued that neuroendocrine and autonomic parameters are “adequate indicators of stress levels,” whereas locomotor activity can be influenced independently ~Ramos & Mormede, 1998!. If these theoretical concepts apply to our experimental model, then it could be argued that repeated exposure to the open field does not result in reduced levels of stress in the animals. By comparing the pattern of changes of behavioral activity over the 4 consecutive days with that of the pressor response and tachycardia, it can at least be concluded that the cardiovascular effects are not merely a consequence of the increased exploratory locomotor activity of the rats in the open field. If this were the
Novelty stress and blood pressure case, both the pressor response and tachycardia would be reduced over repeated exposure. Few previous studies have dealt with the effect of repeated psychological stress on cardiovascular parameters in rats. Exposing intruder rats to four subsequent social defeats has been shown to result in progressively smaller tachycardia responses ~Tornatzky & Miczek, 1994!. On the other hand, Nijsen et al. ~1998! observed that over five consecutive exposures to a novel cage, behavioral, but not sympathetic heart rate responses were reduced, similar to our results. Involvement of the Sympathetic Nervous System Our findings on the effect of pentolinium and atenolol treatment suggest that the stress-induced pressor response and tachycardia are mediated to a large extent by increased sympathetic vasomotor tone and increased cardiac sympathetic activity, respectively. It was somewhat surprising that pentolinium pretreatment did not block the tachycardia, but this may be due to pharmacokinetic and pharmacodynamic characteristics of this particular ganglion blocker. For example, it has been shown that the pressor action of cocaine could be prevented by pretreatment with the noncompetitive ganglion blocker chlorisondamine, but not by the competitive agents pentolinium and hexamethonium ~Tella, Schindler, & Goldberg, 1993!. Alternatively, the stress-induced tachycardia may consist of two mechanisms, one via the cardiac sympathetic and the other possibly a nonsympathetic or vagal mechanism. This would explain why pretreatment with atenolol did not completely block the tachycardia, but left a significant stress-induced increase in heart rate. The nonsympathetic mechanism could become particularly prominent in situations of sympatho-inhibition, such as after ganglion blockade with pentolinium, whereupon it would manifest itself in an apparently unaffected tachycardia response. There is good evidence for vagal withdrawal as an additional mechanism regulating heart rate in some forms of stress ~Nijsen et al., 1998!. Our results are in agreement with several other studies that showed marked increases in sympathetic nerve firing ~DiBona & Jones, 1995; Randall, Brown, Brown, & Kilgore, 1994! and plasma levels of noradrenaline and adrenaline in rats after psychological stress ~De Boer et al., 1989; Kvetnansky, McCarty, Thoa, Lake, & Kopin, 1979; McCarty & Kopin, 1978!. Diazepam and Clonidine Pretreatment with the benzodiazepine anxiolytic diazepam completely blocked the open-field stress-induced pressor response and markedly inhibited the tachycardia. This action can most likely be attributed to the well-known behavioral effect of diazepam, reducing the “stressfulness” of the open field to the rat. Diazepam treatment induced selective changes in behavior, including a reduction in distance moved but not velocity of movement, suggesting a specific effect on behavior rather than sedation. Previous studies have also shown differential effects of diazepam on psychological stress responses ~Beck & Fibiger, 1995!. Interestingly, in our experiments, diazepam treatment was more effective in preventing the pressor response than the tachycardia, as was also found for cardiovascular responses to restraint stress ~Conahan & Vogel, 1986!. A number of studies have suggested direct cardiovascular effects of diazepam on cardiovascular regulation. As in our studies, it has been shown by others that diazepam treatment causes an increase in resting heart rate, most likely through vagal withdrawal ~Conahan & Vogel, 1986!. The site of action of diazepam could be
497 the nucleus tractus solitarius, where diazepam binding sites have been shown to be present and local administration of this drug modulates cardiovascular function ~Barron, Pavelka, & Garrett, 1997!. However, the importance of these effects in the cardiovascular effects of open-field stress remains unclear. Clonidine is widely known for its central action, reducing sympathetic vasomotor tone and cardiac sympathetic activity. In our experiments, pretreatment with clonidine caused a modest reduction in the stress-induced tachycardia, but had little effect on the blood pressure response. We chose to use a low dose of clonidine, as higher doses are known to induce sedation ~Van den Buuse & De Jong, 1989!, which could have influenced cardiovascular responses nonspecifically. Previously it has been shown that clonidine treatment dose-dependently reduced anticipatory tachycardia and hyperthermia in a resident0intruder model of social stress in rats ~Tornatzky & Miczek, 1994!. Clonidine pretreatment, at a dose only slightly higher than that used in our study, reduced the pressor response and the increased heart rate variability in response to air-jet stress in rats ~Blanc, Grichois, & Elghozi, 1991!. This opens the possibility that the effect of clonidine on stress-induced cardiovascular responses depends on the nature of the stressor, that is, the central nervous system pathways involved. Methodological Considerations In this study, the novel combination of radiotelemetry, videotracking analysis, and open-field stress yielded consistent blood pressure, heart rate, and behavioral activity data, allowing detailed analysis of cardiovascular responses versus behavioral responses within each 15-min stress test and over several consecutive days. The detailed behavioral analysis allows detection of subtle changes in behavior, distinguishing specific psychopharmacological effects of particular drugs used from nonspecific sedative effects. Such a distinction would be impossible with more widely used stress models, such as restraint or air-jet stress. It should also be emphasized that all of the observations were obtained without the use of an apparently noxious stimulus, such as pain, noise, or air-jet, as has been used widely in previous experiments ~Bhatnagar, Dallman, Roderick, Basbaum, & Taylor, 1998; Burke et al., 1988; Chen & Herbert, 1995; Conahan & Vogel, 1986; Mansi & Drolet, 1997!. We recently completed experiments showing markedly greater increases in blood pressure and heart rate in spontaneously hypertensive rats ~SHR! compared to two control normotensive rat strains. As in the present study, differences in cardiovascular stress responses between the strains were unrelated to differences in behavior ~Van den Buuse, Lambert, Fluttert, & Eikelis, 2001!. Conclusions Novelty stress, by exposure of rats to an open-field situation, induces marked pressor responses and tachycardia, associated with a variety of behavioral responses. Repeated exposure of the animals to the stress does not lead to reduced cardiovascular responses ~habituation! but, instead, tends to induce progressively greater pressor responses. Both the pressor response and tachycardia are to a large extent caused by increased sympathetic nervous system activity. Pretreatment with the anxiolytic diazepam blocks the pressor response and most of the tachycardia. This novel radiotelemetry method, combined with video analysis of behavior, allows simple and effective stress testing, which may prove useful to elucidate central nervous system mechanisms in the cardiovascular responses to psychological stress.
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~Received April 28, 1999; Accepted September 7, 2000!
