Physiology& Behavior,Vol. 50, pp. 1175-1179. © Pergamon Press plc, 1991. Printed in the U.S.A.
0031-9384/91 $3.00 + .00
Sensitization of the Plasma Corticosterone Response to Novel Environments M I C H A E L B. H E N N E S S Y
Department of Psychology, Wright State University, Dayton, OH 45435 R e c e i v e d 24 February 1987 HENNESSY, M. B. Sensitization of the plasma corticosterone response to novel environments. PHYSIOL BEHAV 50(6) 11751179, 1991.--In Experiment l, ICR Swiss mice were exposed to one of two novel cages for 1, 6, or l0 30-min trials. Plasma corticosterone elevations were greater in the more novel than in the less novel cage and following the sixth as compared to the first and tenth exposures. In Experiment 2, BALB/c and C57BL/6J mice and Sprague-Dawley rats also exhibited significant, though modest, increases in corticosterone levels from their first to their sixth 30-min exposures to the more novel cage. These findings demonstrate generalization of an apparent sensitization of the plasma corticosterone response across the two levels of novelty and four strains of laboratory rodents tested here. The transience of the effect may explain some earlier inconsistencies in the literature. In Experiment 3, five daily 30-rain exposures to the more novel environment minimally raised resting corticosterone levels of ICR Swiss mice. Further, corticosterone levels during a first exposure were no greater at 15 or 45 rain than at 30 min. These results suggest that the increase in corticosterone with repeated exposures reflects a true sensitization of the corticosterone response rather than an increase in resting levels with repeated exposures or a shift in the time course of the corticosterone response. Corticosterone
Novelty
Sensitization
Stress
IN the study of stress, the most widely investigated physiological system is undoubtedly that of the hypothalamic-pituitary-adrenal (HPA) axis. Despite the wealth of information obtained regarding the response of this system to numerous aversive and challenging stimuli, basic issues remain unresolved. Prominent among these is the effect of repeated stimulation. In studies using aversive stimulation, such as electric shock and restraint, the circulating corticosterone levels of rats and mice have been found to decrease, to increase, and to show no change over the course of a series of stimulus presentations (2, 4, 7, 9, 14, 21). The reasons for these inconsistent findings are unclear, yet a grasp of the factors determining whether a stressor's impact grows or diminishes with experience clearly is fundamental to our understanding of stress physiology and adaptability under aversive conditions. One of the most interesting "stressors" used in such research is exposure to a novel environment. This is a potent stimulus for HPA activation; rats and mice reliably exhibit elevated levels of circulating corticosterone following their first exposure to a novel environment (1,13), and this response cannot be accounted for by the handling required to place the animal into its new surroundings (11, 13, 15, 17, 18). When one considers the simple novel environments used in such studies (e.g., a small clean cage), it would seem that the plasma corticosterone response would invariably habituate within a few trials. That is, the simple novel environment presumably would quickly become familiar, so that the effective stimulus for HPA activation (the novelty) would be diminished substantially upon subsequent exposures. However, the empirical findings have not been this straightforward. As has been found in studies using aversive
stimulation discussed above, rodents exposed to novel environments have been observed to show decreased (5, 8, 18, 19), increased (10,12), and unchanged (9, 12, 17) plasma corticosterone levels with repeated exposures. The most curious of these three experimental outcomes is the increase in corticosterone levels with increasing familiarity of the environment. This apparent sensitization of the corticosterone response to a novel environment was the focus of the present study. The inconsistent results of earlier studies using similar procedures suggest that subtle differences in protocol may determine whether sensitization occurs, and if it occurs, whether it is detected in a particular experiment. Therefore, Experiments 1 and 2 examined the effect of several variables (degree of novelty, number of exposures, and subject genotype) hypothesized to influence the outcome of studies in this area. Experiment 3 asked whether the previously observed increase in corticosterone levels following repeated exposure to a novel environment did, in fact, represent sensitization of the corticosterone response or if this increase could instead be accounted for by an increase in resting corticosterone levels or a shift in the time course of the corticosterone response. EXPERIMENT 1 Two variables were of interest in Experiment 1. The first was the intensity of stimulation, i.e., degree of novelty of the test environment, Studies utilizing electric shock and restraint indicate that higher intensifies of a particular type of stimulation are more likely to result in sensitization and less likely to result in habituation of the plasma corticosterone response than are lower intensities (16, 19, 20). In an earlier experiment from this labo-
1175
1176
HENNESS~
ratory, Hennessy and Foy (10) found that mice exposed to a less novel cage (clean cage with clean wood shavings) exhibited a smaller plasma corticosterone elevation than did mice exposed to a more novel cage (clean cage with no bedding), but that exposure to either environment produced apparent sensitization-corticosterone concentrations were greater following the sixth 30-min exposure than they were following the first. However, this latter finding was based on a significant main effect embedded in a larger factorial design. Although relevant interactions involving the degree of novelty were not significant, visual inspection of the data suggested that the increase with repeated trials was a function of exposure to the more novel environment, and that this effect of the degree of novelty was obscured by the results of other groups included in the design that had an additional treatment imposed during novelty exposure. Therefore, Experiment 1 examined corticosterone levels of mice following one and six exposures to low- and high-novelty environments using the procedures of our earlier study (10). but in a design that did not include other treatments. The second variable examined was the number of exposures to the environment. When sensitization occurs, whether or not it is detected is likely to depend on the trial chosen to test for the effect. In particular, an initial period of increased responsiveness may be transitory. Thus, in studies in which plasma corticosterone levels were found to be of the same magnitude following 8 or 10 exposures as they were following the first (9,17), sensitization may have occurred during earlier trials, but waned by the trial on which sensitization was tested. To examine this issue, Experiment 1 included groups of mice exposed to the novel environments on 10, as well as on 1 and 6, occasions. METHOD
Animals
Seventy male mice of the ICR Swiss strain were obtained at 7-10 weeks of age from Harlan Laboratories in Indianapolis, IN. They were individually housed in 33.3 x 20.0 x 12.7-cm polycarbonate mouse cages with ad lib food and water, and wood shavings for bedding. The colony room, which contained only the animals for this study, was maintained at 74 + 2°F on an 11-h light/13-h dark cycle (lights on at 0630). All animals were allowed at least 2 weeks to adapt to these housing conditions prior to testing. Procedure
Sixty mice were divided into 6 groups of 10 each, based on the two-way factorial combination of two degrees of novelty (low or high) and three numbers of exposures (1, 6, or 10). The remaining 10 mice were assigned to a base condition used to estimate resting corticosterone levels. One mouse in the lownovelty, 10-exposure group was excluded due to experimenter error. The test environments were 45.7 x 23.8 x 20.0-cm clear polycarbonate rodent cages. For the low-novelty groups, the floor of the cage was covered with clean wood shavings of the type used in the home cage, whereas for the high-novelty groups, the cage was bare. The test cages were cleaned in a commercial cage washer prior to each use. An exposure consisted of gently lifting the mouse from its home cage by its tail and placing it individually into a test cage for 30 rain. Mice were returned to their home cages in the same manner immediately following exposure for all but the last designated exposure period. Those mice given multiple exposures were tested once on each of six or ten consecutive days at the same time of day.
hnmediately following each mouse's last exposure, it was carried in its test cage to an adjacent room where it was placed in a glass jar containing ether-soaked cotton. After the mouse was lightly anesthetized, it was decapitated and trunk blood was collected in heparinized tubes. Animals in the base condition were carried to the blood-sampling room in their home cages prior to any disturbance. Blood was collected within 2.5 rain of the time the cages were removed, which is rapid enough to ensure that the disturbance and anesthesia involved in the bloodsampling procedure did not affect corticosterone levels in the samples obtained (6,22). All blood samples were collected between 0830 and 1030 h. The experiment was scheduled such that blood was always sampled on weekdays to ensure that corticosterone levels were not influenced by any change of routine in the animal care facilities during weekends. Samples were centrifuged to separate plasma which was then frozen until analyzed for corticosterone using standard radioimmunoassay procedures. Duplicate aliquots were assayed using antisera, tritiated corticosterone and general protocol provided by Radioassay Systems Laboratories, Carson. CA. RESULTS AND DISCUSSION
As is clear in Fig. 1, the plasma corticosterone levels of all groups of mice exposed to the test cages were significantly elevated over levels in the base condition (Mann-Whitney U-test, U s = 0 to 4, all ps
0031-9384/91 $3.00 + .00
Sensitization of the Plasma Corticosterone Response to Novel Environments M I C H A E L B. H E N N E S S Y
Department of Psychology, Wright State University, Dayton, OH 45435 R e c e i v e d 24 February 1987 HENNESSY, M. B. Sensitization of the plasma corticosterone response to novel environments. PHYSIOL BEHAV 50(6) 11751179, 1991.--In Experiment l, ICR Swiss mice were exposed to one of two novel cages for 1, 6, or l0 30-min trials. Plasma corticosterone elevations were greater in the more novel than in the less novel cage and following the sixth as compared to the first and tenth exposures. In Experiment 2, BALB/c and C57BL/6J mice and Sprague-Dawley rats also exhibited significant, though modest, increases in corticosterone levels from their first to their sixth 30-min exposures to the more novel cage. These findings demonstrate generalization of an apparent sensitization of the plasma corticosterone response across the two levels of novelty and four strains of laboratory rodents tested here. The transience of the effect may explain some earlier inconsistencies in the literature. In Experiment 3, five daily 30-rain exposures to the more novel environment minimally raised resting corticosterone levels of ICR Swiss mice. Further, corticosterone levels during a first exposure were no greater at 15 or 45 rain than at 30 min. These results suggest that the increase in corticosterone with repeated exposures reflects a true sensitization of the corticosterone response rather than an increase in resting levels with repeated exposures or a shift in the time course of the corticosterone response. Corticosterone
Novelty
Sensitization
Stress
IN the study of stress, the most widely investigated physiological system is undoubtedly that of the hypothalamic-pituitary-adrenal (HPA) axis. Despite the wealth of information obtained regarding the response of this system to numerous aversive and challenging stimuli, basic issues remain unresolved. Prominent among these is the effect of repeated stimulation. In studies using aversive stimulation, such as electric shock and restraint, the circulating corticosterone levels of rats and mice have been found to decrease, to increase, and to show no change over the course of a series of stimulus presentations (2, 4, 7, 9, 14, 21). The reasons for these inconsistent findings are unclear, yet a grasp of the factors determining whether a stressor's impact grows or diminishes with experience clearly is fundamental to our understanding of stress physiology and adaptability under aversive conditions. One of the most interesting "stressors" used in such research is exposure to a novel environment. This is a potent stimulus for HPA activation; rats and mice reliably exhibit elevated levels of circulating corticosterone following their first exposure to a novel environment (1,13), and this response cannot be accounted for by the handling required to place the animal into its new surroundings (11, 13, 15, 17, 18). When one considers the simple novel environments used in such studies (e.g., a small clean cage), it would seem that the plasma corticosterone response would invariably habituate within a few trials. That is, the simple novel environment presumably would quickly become familiar, so that the effective stimulus for HPA activation (the novelty) would be diminished substantially upon subsequent exposures. However, the empirical findings have not been this straightforward. As has been found in studies using aversive
stimulation discussed above, rodents exposed to novel environments have been observed to show decreased (5, 8, 18, 19), increased (10,12), and unchanged (9, 12, 17) plasma corticosterone levels with repeated exposures. The most curious of these three experimental outcomes is the increase in corticosterone levels with increasing familiarity of the environment. This apparent sensitization of the corticosterone response to a novel environment was the focus of the present study. The inconsistent results of earlier studies using similar procedures suggest that subtle differences in protocol may determine whether sensitization occurs, and if it occurs, whether it is detected in a particular experiment. Therefore, Experiments 1 and 2 examined the effect of several variables (degree of novelty, number of exposures, and subject genotype) hypothesized to influence the outcome of studies in this area. Experiment 3 asked whether the previously observed increase in corticosterone levels following repeated exposure to a novel environment did, in fact, represent sensitization of the corticosterone response or if this increase could instead be accounted for by an increase in resting corticosterone levels or a shift in the time course of the corticosterone response. EXPERIMENT 1 Two variables were of interest in Experiment 1. The first was the intensity of stimulation, i.e., degree of novelty of the test environment, Studies utilizing electric shock and restraint indicate that higher intensifies of a particular type of stimulation are more likely to result in sensitization and less likely to result in habituation of the plasma corticosterone response than are lower intensities (16, 19, 20). In an earlier experiment from this labo-
1175
1176
HENNESS~
ratory, Hennessy and Foy (10) found that mice exposed to a less novel cage (clean cage with clean wood shavings) exhibited a smaller plasma corticosterone elevation than did mice exposed to a more novel cage (clean cage with no bedding), but that exposure to either environment produced apparent sensitization-corticosterone concentrations were greater following the sixth 30-min exposure than they were following the first. However, this latter finding was based on a significant main effect embedded in a larger factorial design. Although relevant interactions involving the degree of novelty were not significant, visual inspection of the data suggested that the increase with repeated trials was a function of exposure to the more novel environment, and that this effect of the degree of novelty was obscured by the results of other groups included in the design that had an additional treatment imposed during novelty exposure. Therefore, Experiment 1 examined corticosterone levels of mice following one and six exposures to low- and high-novelty environments using the procedures of our earlier study (10). but in a design that did not include other treatments. The second variable examined was the number of exposures to the environment. When sensitization occurs, whether or not it is detected is likely to depend on the trial chosen to test for the effect. In particular, an initial period of increased responsiveness may be transitory. Thus, in studies in which plasma corticosterone levels were found to be of the same magnitude following 8 or 10 exposures as they were following the first (9,17), sensitization may have occurred during earlier trials, but waned by the trial on which sensitization was tested. To examine this issue, Experiment 1 included groups of mice exposed to the novel environments on 10, as well as on 1 and 6, occasions. METHOD
Animals
Seventy male mice of the ICR Swiss strain were obtained at 7-10 weeks of age from Harlan Laboratories in Indianapolis, IN. They were individually housed in 33.3 x 20.0 x 12.7-cm polycarbonate mouse cages with ad lib food and water, and wood shavings for bedding. The colony room, which contained only the animals for this study, was maintained at 74 + 2°F on an 11-h light/13-h dark cycle (lights on at 0630). All animals were allowed at least 2 weeks to adapt to these housing conditions prior to testing. Procedure
Sixty mice were divided into 6 groups of 10 each, based on the two-way factorial combination of two degrees of novelty (low or high) and three numbers of exposures (1, 6, or 10). The remaining 10 mice were assigned to a base condition used to estimate resting corticosterone levels. One mouse in the lownovelty, 10-exposure group was excluded due to experimenter error. The test environments were 45.7 x 23.8 x 20.0-cm clear polycarbonate rodent cages. For the low-novelty groups, the floor of the cage was covered with clean wood shavings of the type used in the home cage, whereas for the high-novelty groups, the cage was bare. The test cages were cleaned in a commercial cage washer prior to each use. An exposure consisted of gently lifting the mouse from its home cage by its tail and placing it individually into a test cage for 30 rain. Mice were returned to their home cages in the same manner immediately following exposure for all but the last designated exposure period. Those mice given multiple exposures were tested once on each of six or ten consecutive days at the same time of day.
hnmediately following each mouse's last exposure, it was carried in its test cage to an adjacent room where it was placed in a glass jar containing ether-soaked cotton. After the mouse was lightly anesthetized, it was decapitated and trunk blood was collected in heparinized tubes. Animals in the base condition were carried to the blood-sampling room in their home cages prior to any disturbance. Blood was collected within 2.5 rain of the time the cages were removed, which is rapid enough to ensure that the disturbance and anesthesia involved in the bloodsampling procedure did not affect corticosterone levels in the samples obtained (6,22). All blood samples were collected between 0830 and 1030 h. The experiment was scheduled such that blood was always sampled on weekdays to ensure that corticosterone levels were not influenced by any change of routine in the animal care facilities during weekends. Samples were centrifuged to separate plasma which was then frozen until analyzed for corticosterone using standard radioimmunoassay procedures. Duplicate aliquots were assayed using antisera, tritiated corticosterone and general protocol provided by Radioassay Systems Laboratories, Carson. CA. RESULTS AND DISCUSSION
As is clear in Fig. 1, the plasma corticosterone levels of all groups of mice exposed to the test cages were significantly elevated over levels in the base condition (Mann-Whitney U-test, U s = 0 to 4, all ps
