Physiology & Behavior, Vol. 47, pp. 647-652. ¢ PergamonPress plc, 1990. Printed in the U.S.A.

0031-9384/90 $3.00 + .00

Habituation and Sensitization of Plasma Catecholamine Responses to Chronic Intermittent Stress: Effects of Stressor Intensity MARIA KONARSKA,

R O B E R T E. S T E W A R T A N D R I C H A R D M c C A R T Y 1

Department o f Psychology, University o f Virginia, Charlottesville, VA 22903 R e c e i v e d 11 S e p t e m b e r 1989

KONARSKA, M., R. E. STEWART AND R. McCARTY. Habituation and sensitization of plasma catecholamine responses to chronic intermittent stress: Effects of stressor intensity. PHYSIOL BEHAV 47(4) 647-652, 1990.--Adult male Sprague-Dawley rats were exposed acutely (1 time for 30 minutes) or chronically (30 minutes per day for 27 days) to swim stress in water maintained at either 18°C, 24°C or 34°C. Each rat was prepared with an indwelling tail artery catheter to allow for direct measures of mean arterial pressure (MAP, mmHg) and heart rate (HR, beats per minute) and for remote collection of blood samples before, during and after the 1st or 27th swim stress session. Blood samples were later analyzed for plasma levels of norepinephrine and epinephrine to serve as an assessment of sympathetic-adrenal medullary activity. Compared to handled controls, body weight gain was reduced significantly in rats exposed chronically to swim stress at any of the 3 temperatures. However, baseline values of MAP and HR and plasma levels of norepinephrine and epinephrine were similar in chronically stressed rats compared to their handled controls. The plasma norepinephrine response of rats exposed chronically to either 18°C or 24°C swim stress was significantly greater than that of matched controls stressed for the fast time. In contrast, the plasma epinephrine response of chronically stressed rats from these two groups was slightly but not significantly reduced compared to matched controls. For swim stress at 34°C, the plasma norepinephrine and epinephrine responses of chronically stressed rats were reduced significantly compared to controls stressed for the first time. These findings demonstrate that stressor intensity affects sensitization and habituation of plasma catecholamine responses in laboratory rats exposed to chronic intermittent stress. In addition, the results of these experiments suggest that the sympathetic nervous system and the adrenal medulla respond differentially to manipulations of stressor intensity. Plasma catecholamines Norepinephrine Habituation Stressor intensity


Acute stress

THE study of psychological influences on activity of the sympathetic nerves and the adrenal medulla began with the pioneering studies by Walter B. Cannon and his colleagues during the early years of this century (3-6). As noted by Brooks (2), some of Cannon's major findings related to the role of the autonomic nervous system in emotional expression, defense reactions, stressful stimulation, and maintenance of internal homeostasis. The discovery of norepinephrine by von Euler (33) in 1946 marked the advent of systematic studies on the role of norepinephrine released from sympathetic nerve terminals and epinephrine secreted from the adrenal medulla in physiological and behavioral adjustments to stressful stimulation in laboratory animals and humans (26). Mason and coworkers were among the first to examine plasma catecholamine responses to stressful stimulation in animals under controlled laboratory conditions. In their studies of chair-restrained rhesus monkeys, they observed moderate elevations in

Chronic stress


plasma norepinephrine when subjects were exposed to a variety of acute stressors. In contrast, elevations in plasma epinephrine occurred less frequently and were often associated with conditions of novelty or unpredictability (27). Unfortunately, these groundbreaking experiments were hampered by the use of relatively insensitive methods for the assay of catecholamines in plasma. In addition, the focus of many early experiments was an examination of plasma catecholamine responses following acute exposure to one of several time-limited stressors. Few experiments were concerned with plasma catecholamine responses of animals following exposure to chronic intermittent stress. In the mid-1970s, several biochemical methods were developed which, for the first time, permitted the assay of catecholamines in very small volumes of plasma with sensitivities in the femtomole range (9). A number of these studies revealed that plasma levels of norepinephrine are derived primarily from sym-

IRequests for reprints should be addressed to Richard McCarty, Ph.D., Department of Psychology, 102 Gilmer Hall, University of Virginia, Charlottesville, VA 22903-2477.




pathetic nerve terminals, although some norepinephrine in plasma is secreted from the adrenal medulla during exposure of animals to stressful stimulation. In contrast, virtually all circulating epinephrine is secreted directly from the adrenal medulla (20, 24, 34). Several important principles related to regulation of the sympathetic-adrenal medullary system have been formulated based upon studies of plasma catecholamines in laboratory animals and humans conducted over the past 15 years (1, 12, 17). First, plasma levels of both catecholamines are elevated above basal values in animals following even minor disturbances. In addition, great care must be taken to obtain blood samples from animals using remote sampling from chronic arterial or venous catheters (19,22). Second, plasma catecholamines are cleared from blood with a half-life of approximately 1 minute in laboratory rats and 2 minutes in humans. Thus, blood sampling time points must be chosen with this factor in mind (22,34). Third, elevations in plasma catecholamines are positively correlated with the intensity of stressful stimulation (18,28). Fourth, elevations in plasma catecholamines are dampened in animals exposed repeatedly to the same stressor. In contrast, elevations in plasma catecholamines are exaggerated in chronically stressed animals following acute exposure to a novel stressor (21, 23, 25). Finally, the sympathetic nerves and the adrenal medulla are regulated independently and under some experimental conditions plasma levels of one catecholamine may increase substantially, while plasma levels of the other may remain at basal levels (11, 17, 18, 22). In the present experiment, we have expanded upon our recent studies by examining the effects of stressor intensity on the plasma catecholamine responses of laboratory rats exposed to chronic intermittent swim stress. We were particularly interested in determining if the plasma norepinephrine and epinephrine responses of chronically stressed animals were matched over a range of stressor intensities. That is, in the course of adaptation to chronic intermittent exposure to swim stress, would changes in responses of plasma norepinephrine occur in parallel with changes in plasma epinephrine? METHOD

Animals Adult male Sprague-Dawley rats weighing 250-300 g were purchased from Dominion Laboratories, Dublin, VA. Upon arrival in our vivarium, rats were housed individually in suspended metal cages and were allowed at least one week to recover from the disruption of shipment and to acclimate to our laboratory. At all times, tap water and Purina rat chow were provided in ad lib quantities. The vivarium and testing rooms were maintained on a 12 hour light-dark cycle (lights on 0700-1900 hours) at an ambient temperature of 22 -+ I°C.

Stress Procedures In each of 3 separate experiments, animals were assigned at random to one of two groups (N = 9-12 rats per group at the start of the experiment). For each experiment, one group was weighed and then exposed to swim stress (water temperature maintained at either 18°C, 24°C, or 34°C) for 30 minutes per day for each of 26 consecutive days. Matched controls for each experiment were handled, weighed and returned to their home cages each day. All manipulations occurred between 0900-1300 hours.

Surgery At least 1 hour after handling or swim stress on day 26, rats were anesthetized with sodium brevital (60 mg/kg, IP) and

prepared with tail artery catheters as described originally by Chiueh and Kopin (7). Briefly, a 2 cm midline incision was made through the ventral tail sheath near the base of the tail. The ventral tail artery was dissected free and elevated and a PE50 catheter was inserted up into the artery and secured with sutures. The tubing was led under the tail sheath and skin to exit in the midscapula region and was covered with a stainless steel extension spring (Dieco Company, Macedonia, OH) which was secured to the rat with an adhesive tape collar. The tubing was filled with heparinized saline (300 I.U. per ml) and the end of the tubing was occluded with a 23 ga. needle and sterile tuberculin syringe. After complete recovery from anesthesia (usually 30--45 minutes), rats were placed individually into plastic cages (35 x 35 x 22 cm), which contained bedding material and laboratory chow and tap water. The spring wire and catheter were led out of the top-center of the cage and secured such that each rat had complete freedom of movement about the cage. Catheters were flushed in the early morning and late afternoon with 0.5 ml of the heparinized saline to maintain catheter patency.

Sampling Protocol Between 0900-1100 hours on the day following surgery, baseline values of mean arterial pressure (MAP, mmHg) and heart rate (HR, beats per minute) were measured, while rats were resting and undisturbed in their home cages. The end of each catheter was connected to a Statham pressure transducer and MAP was recorded on a Grass muttichannel polygraph. HR was recorded by a cardiotachometer triggered by the arterial pulse. Two days after surgery, baseline blood samples (0.6 ml) were obtained from the tail artery catheters of each rat between 0900-1100 hours with a minimum of disturbance. After this and each subsequent blood sample, an equal volume of heparinized saline (100 I.U. per ml) was infused slowly back into the catheter to maintain blood volume. Chronically stressed and control rats were then exposed to swim stress at the appropriate temperature (18°C, 24°C or 34°C) for a 30-minute period between 1100-1500 hours. Additional blood samples were collected at 5, 15 and 30 minutes of the stress session and 15 and 60 minutes following stress when animals had been returned to their home cages. As many as 6 consecutive 0.5-ml blood samples may be collected at 3-minute intervals from adult male laboratory rats without affecting basal plasma levels of norepinephrine (NE) or epinephrine (EPI) (18).

Assay of Plasma Catecholamines All blood samples were collected into iced 10 x 75 glass culture tubes and were placed into an ice water bath immediately. Samples were later centrifuged at 4000 x g for 10 minutes at 4°C. Aliquots of plasma were removed and stored at - 7 0 ° C until assayed for content of NE and EPI within 6 weeks (9,29). The sensitivity of the radioenzymatic-thin layer chromatographic assay was less than 5 pg for each amine and the coefficient of variation within and between assays was less than 6%.

Data Reduction and Analysis The results for each experiment are presented as means - SEM for the indicated numbers of animals. Comparisons between groups were made using two-tailed t-tests for unpaired data. Data for plasma catecholamines were analyzed by computing an area under the curve (AUC, pg x minutes) for each animal. To obtain this measure, the plasma NE and EPI responses above baseline for swim stress were plotted on graph paper for each animal and the






Treatment Group (N)

Body Weight (Day 1)

Body Weight (Day 26)

Body Gain (Day 26-Day 1)

Control (12) SS-18°C (9)

303 +- 2 299 - 4

400 +_ 5 378 _ 11

96 -+ 5 78 +__ 6*

Control (10) SS-24°C (12)

322 -+ 2 323 - 6

409 +-- 3 365 _ 8:~

86 _ 3 42 +_ 12t

Control (12) SS-34°C (12)

273 - 3 272 - 3

398 373 _

4 61

126 101 _

50004000v Ld Z < :S 0"3 .< 0_





Control (8) SS-18°C (9)

111 _ 1 107 - 3

335 ~- 8 329 ± 3

Control (9) SS-24°C (9)

99 +_ 2 107 -+ 2

340 +_ 7 348 -4- 5

Control (11) SS-34°C (12)

107 _+ 2 106 _ 4

345 _ 4 353 +- 7










TIME (minutes) 6000-

As summarized in Table 1, starting body weights of control and stressed rats were quite similar. On day 26, however, body weights of chronically stressed rats were significantly less than matched controls for the 24°C and the 34°C groups (p

Habituation and sensitization of plasma catecholamine responses to chronic intermittent stress: effects of stressor intensity.

Adult male Sprague-Dawley rats were exposed acutely (1 time for 30 minutes) or chronically (30 minutes per day for 27 days) to swim stress in water ma...
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