Physiology & Behavior, Vol. 49, pp. 653--656. ©PergamonPress plc, 1991. Printedin the U.S.A.
0031-9384/91 $3.00 + .00
BRIEF COMMUNICATION
Plasma Catecholamine Responses to Acute Motion Stress in Laboratory Rats R I C H A R D M c C A R T Y , 1 G L E N E I S E N A N D C Y N T H I A L. B A R T H O L O W
Department o f Psychology, University o f Virginia, Charlottesville, VA 22903-2477 Received 29 A u g u s t 1990
McCARTY, R., G. EISEN AND C. L. BARTHOLOW. Plasma catecholamine responses to acute motion stress in laboratory rats. PHYSIOL BEHAV 49(3) 653--656, 1991.--Plasma levels of norepinephrine (NE) and epinephrine (EPI) were measured in adult male Fischer 344 (F-344) laboratory rats exposed to acute motion stress. Two days prior to testing, rats were prepared with chronic tail artery catheters to permit remote sampling of blood from conscious, freely behaving animals. Animals remained in their home cages during the entire testing protocol. After collection of basal blood samples, cages were rotated at 45 revolutions per minute for a 10-minute period each hour for 5 consecutive hours. Additional blood samples were collected immediately after each of the 10minute rotation stress sessions. Plasma levels of NE remained unchanged from baseline immediately following each of the rotation stress sessions. In contrast, plasma EPI increased significantly above baseline levels following each of the 5 rotation stress sessions. These data indicate that rotation stress provides a selective activation of epinephrine-containing adrenal chromaffin cells as reflected in an increase in plasma EPI but not NE. This stress model could prove valuable in examining the physiological and behavioral consequences of adrenal EPI release in freely behaving animals without the confounding effects of increases in circulating norepinephrine. Sympathetic nervous system
Adrenal medulla
Plasma
Catecholamines
IN their pioneering work on stress and immune function, Riley and his colleagues (18) developed a novel form of stressful stimulation for use in studies of laboratory mice. Using plasma corticosterone as an endocrine index of the response to stress, these investigators exposed mice in their home cages to rotation stress at speeds of 16 to 78 revolutions per minute. With the levels of stressful stimulation employed in their studies, animals developed mild spatial disorientation and possibly vertigo and dizziness. Their standard protocol involved exposure of laboratory mice to 10 minutes of motion stress followed by 50 minutes of recovery. This cycle was then repeated for any of several periodicities. Based upon their extensive studies, these investigators suggested that motion stress resulted in an activation of the adrenal cortex but not other stress-responsive hormonal systems. In addition, they reported that plasma levels of corticosterone increased monotonically with increasing intensity of motion stress (18). The hypothalamic-pituitary-adrenal cortical axis represents only one of several stress-responsive endocrine systems (1, 7, 12, 19, 21). In particular, the sympathetic-adrenal medullary system is especially sensitive to acutely stressful stimuli and responds in a graded fashion to stimuli of increasing intensity (10,16). In the present study with laboratory rats, we have employed a motion
Motion stress
Fischer 344 rat
stress paradigm similar to the one developed originally by Riley. Our intent was to examine more closely the effects of repeated bouts of motion stress on plasma levels of norepinephrine and epinephrine. Our findings demonstrate that plasma epinephrine but not plasma norepinephrine increases significantly above basal values in laboratory rats with each exposure to motion stress. METHOD Ninety-day-old male Fischer 344 (F-344) rats were purchased from Charles River Breeding Laboratories, Wilmington, MA. Upon arrival in our laboratory, rats were housed in groups of 3--4 in Wahmann suspended metal cages for at least 1 week prior to use. At all times during this study, laboratory chow and tap water were freely available. The vivarium room was maintained on a 12-hour light-dark photoperiod (lights on at 0700 hours) at a temperature of 20-22°C. At approximately 100 days of age, rats were anesthetized with pentobarbital (35--40 mg/kg, IP) and a PE-50 catheter was placed into the ventral caudal artery as described by Chiueh and Kopin (2). After exteriorizing the catheter in the mid-scapula region, a 30-cm length of spring wire was placed over the tubing for pro-
~Requests for reprints should be addressed to Richard McCarty, Ph.D., Department of Psychology, 102 Gilmer Hall, University of Virginia, Charlottesville, VA 22903-2477.
653
654
McCARTY, EISEN AND BARTH()I.O~'
tection and was secured to the rat with an adhesive tape collar. The tubing was filled with saline containing heparin (300 IU/ml) and the end of the tubing was occluded with a 23-ga needle and 1-ml tuberculin syringe. Following recovery from surgery, rats were placed singly into plastic cages (25 x 25 x 15 cm) that contained a layer of bedding material, laboratory chow and tap water. The spring wire and syringe were led out the top-center of the cage and positioned such that each rat could move freely within the cage. Patency of catheters was maintained with twice daily infusions of 0.5 ml of the heparinized saline in the early morning and the late afternoon. Two days after surgery, basal blood samples (0.5 ml) were collected from each rat with a minimum of disturbance between 0800-0930 hours. The volume of blood collected was replaced immediately with a slow infusion of heparinized saline (100 IU/ ml). After collection of all basal blood samples, rats were divided into 3 groups of 3 rats each. Beginning at 1000 hours, 3 home cages containing individual rats were taken to an adjacent room and secured onto each of 3 wooden platforms that rotated at 45 revolutions per minute. Platforms were chain driven by an electric motor with a variable speed control. After 10 minutes of motion stress, a 0.5-ml blood sample was collected from each rat and the volume was replaced with heparinized saline. This group of rats was then returned to the housing area for 50 minutes and the procedure was repeated for the two additional groups of 3 rats each. Each rat was exposed to 5 bouts of 10 minutes of motion stress followed by 50 minutes of rest over a 5-hour period. Additional baseline blood samples were collected from each rat immediately prior to the third and fifth bouts of motion stress. Blood samples were collected into 10 × 75-mm glass culture tubes that were kept in an ice bath. Samples were centrifuged at 4000 x g for 10 minutes at 4°C and the plasma was removed and stored at - 70°C for no longer than 3 weeks. Aliquots of plasma was deproteinated and levels of norepinephrine and epinephrine were determined by a radioenzymatic-thin layer chromatographic procedure (3,17). The sensitivity of the assay was less than 6 pg for each amine and intra- and interassay coefficients of variations were less than 7%. For purposes of analysis of these data, the 3 basal values of plasma norepinephrine and epinephrine were averaged for each rat. Then, significance of differences between baseline and each of 5 motion stress values were determined by paired t-tests. Two animals were excluded from the study as a result of catheter failure during the motion stress protocol. RESULTS
F-344 rats displayed a minimum of disturbance during the 10minute periods of motion stress. Each rat was positioned in the center of the cage and remained inactive during rotation of the cage. Within 5-10 minutes after being returned to the housing area, rats were resting in their home cages. Plasma levels of both catecholamines returned to prestress basal levels in each animal following the second and fourth bouts of motion stress. Plasma levels of norepinephrine did not change significantly from basal levels immediately following the 5 bouts of motion stress (ps>0.30). Indeed, plasma levels of norepinephrine were slightly reduced below basal levels following the 2nd4th bouts of motion stress and were only slightly higher than basal values following the 1st and 5th bouts of motion stress (Fig. 1). In contrast, plasma epinephrine increased significantly above basal values following each of the 5 bouts of motion stress (ps
0031-9384/91 $3.00 + .00
BRIEF COMMUNICATION
Plasma Catecholamine Responses to Acute Motion Stress in Laboratory Rats R I C H A R D M c C A R T Y , 1 G L E N E I S E N A N D C Y N T H I A L. B A R T H O L O W
Department o f Psychology, University o f Virginia, Charlottesville, VA 22903-2477 Received 29 A u g u s t 1990
McCARTY, R., G. EISEN AND C. L. BARTHOLOW. Plasma catecholamine responses to acute motion stress in laboratory rats. PHYSIOL BEHAV 49(3) 653--656, 1991.--Plasma levels of norepinephrine (NE) and epinephrine (EPI) were measured in adult male Fischer 344 (F-344) laboratory rats exposed to acute motion stress. Two days prior to testing, rats were prepared with chronic tail artery catheters to permit remote sampling of blood from conscious, freely behaving animals. Animals remained in their home cages during the entire testing protocol. After collection of basal blood samples, cages were rotated at 45 revolutions per minute for a 10-minute period each hour for 5 consecutive hours. Additional blood samples were collected immediately after each of the 10minute rotation stress sessions. Plasma levels of NE remained unchanged from baseline immediately following each of the rotation stress sessions. In contrast, plasma EPI increased significantly above baseline levels following each of the 5 rotation stress sessions. These data indicate that rotation stress provides a selective activation of epinephrine-containing adrenal chromaffin cells as reflected in an increase in plasma EPI but not NE. This stress model could prove valuable in examining the physiological and behavioral consequences of adrenal EPI release in freely behaving animals without the confounding effects of increases in circulating norepinephrine. Sympathetic nervous system
Adrenal medulla
Plasma
Catecholamines
IN their pioneering work on stress and immune function, Riley and his colleagues (18) developed a novel form of stressful stimulation for use in studies of laboratory mice. Using plasma corticosterone as an endocrine index of the response to stress, these investigators exposed mice in their home cages to rotation stress at speeds of 16 to 78 revolutions per minute. With the levels of stressful stimulation employed in their studies, animals developed mild spatial disorientation and possibly vertigo and dizziness. Their standard protocol involved exposure of laboratory mice to 10 minutes of motion stress followed by 50 minutes of recovery. This cycle was then repeated for any of several periodicities. Based upon their extensive studies, these investigators suggested that motion stress resulted in an activation of the adrenal cortex but not other stress-responsive hormonal systems. In addition, they reported that plasma levels of corticosterone increased monotonically with increasing intensity of motion stress (18). The hypothalamic-pituitary-adrenal cortical axis represents only one of several stress-responsive endocrine systems (1, 7, 12, 19, 21). In particular, the sympathetic-adrenal medullary system is especially sensitive to acutely stressful stimuli and responds in a graded fashion to stimuli of increasing intensity (10,16). In the present study with laboratory rats, we have employed a motion
Motion stress
Fischer 344 rat
stress paradigm similar to the one developed originally by Riley. Our intent was to examine more closely the effects of repeated bouts of motion stress on plasma levels of norepinephrine and epinephrine. Our findings demonstrate that plasma epinephrine but not plasma norepinephrine increases significantly above basal values in laboratory rats with each exposure to motion stress. METHOD Ninety-day-old male Fischer 344 (F-344) rats were purchased from Charles River Breeding Laboratories, Wilmington, MA. Upon arrival in our laboratory, rats were housed in groups of 3--4 in Wahmann suspended metal cages for at least 1 week prior to use. At all times during this study, laboratory chow and tap water were freely available. The vivarium room was maintained on a 12-hour light-dark photoperiod (lights on at 0700 hours) at a temperature of 20-22°C. At approximately 100 days of age, rats were anesthetized with pentobarbital (35--40 mg/kg, IP) and a PE-50 catheter was placed into the ventral caudal artery as described by Chiueh and Kopin (2). After exteriorizing the catheter in the mid-scapula region, a 30-cm length of spring wire was placed over the tubing for pro-
~Requests for reprints should be addressed to Richard McCarty, Ph.D., Department of Psychology, 102 Gilmer Hall, University of Virginia, Charlottesville, VA 22903-2477.
653
654
McCARTY, EISEN AND BARTH()I.O~'
tection and was secured to the rat with an adhesive tape collar. The tubing was filled with saline containing heparin (300 IU/ml) and the end of the tubing was occluded with a 23-ga needle and 1-ml tuberculin syringe. Following recovery from surgery, rats were placed singly into plastic cages (25 x 25 x 15 cm) that contained a layer of bedding material, laboratory chow and tap water. The spring wire and syringe were led out the top-center of the cage and positioned such that each rat could move freely within the cage. Patency of catheters was maintained with twice daily infusions of 0.5 ml of the heparinized saline in the early morning and the late afternoon. Two days after surgery, basal blood samples (0.5 ml) were collected from each rat with a minimum of disturbance between 0800-0930 hours. The volume of blood collected was replaced immediately with a slow infusion of heparinized saline (100 IU/ ml). After collection of all basal blood samples, rats were divided into 3 groups of 3 rats each. Beginning at 1000 hours, 3 home cages containing individual rats were taken to an adjacent room and secured onto each of 3 wooden platforms that rotated at 45 revolutions per minute. Platforms were chain driven by an electric motor with a variable speed control. After 10 minutes of motion stress, a 0.5-ml blood sample was collected from each rat and the volume was replaced with heparinized saline. This group of rats was then returned to the housing area for 50 minutes and the procedure was repeated for the two additional groups of 3 rats each. Each rat was exposed to 5 bouts of 10 minutes of motion stress followed by 50 minutes of rest over a 5-hour period. Additional baseline blood samples were collected from each rat immediately prior to the third and fifth bouts of motion stress. Blood samples were collected into 10 × 75-mm glass culture tubes that were kept in an ice bath. Samples were centrifuged at 4000 x g for 10 minutes at 4°C and the plasma was removed and stored at - 70°C for no longer than 3 weeks. Aliquots of plasma was deproteinated and levels of norepinephrine and epinephrine were determined by a radioenzymatic-thin layer chromatographic procedure (3,17). The sensitivity of the assay was less than 6 pg for each amine and intra- and interassay coefficients of variations were less than 7%. For purposes of analysis of these data, the 3 basal values of plasma norepinephrine and epinephrine were averaged for each rat. Then, significance of differences between baseline and each of 5 motion stress values were determined by paired t-tests. Two animals were excluded from the study as a result of catheter failure during the motion stress protocol. RESULTS
F-344 rats displayed a minimum of disturbance during the 10minute periods of motion stress. Each rat was positioned in the center of the cage and remained inactive during rotation of the cage. Within 5-10 minutes after being returned to the housing area, rats were resting in their home cages. Plasma levels of both catecholamines returned to prestress basal levels in each animal following the second and fourth bouts of motion stress. Plasma levels of norepinephrine did not change significantly from basal levels immediately following the 5 bouts of motion stress (ps>0.30). Indeed, plasma levels of norepinephrine were slightly reduced below basal levels following the 2nd4th bouts of motion stress and were only slightly higher than basal values following the 1st and 5th bouts of motion stress (Fig. 1). In contrast, plasma epinephrine increased significantly above basal values following each of the 5 bouts of motion stress (ps
