Physiology & Behavior, Vol. 22, pp. 653-656. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Corticosterone "Basal Levels" and Response to Ether Anesthesia in Rats on a Water Deprivation Regimen I G A R Y D. C O O V E R , J O H N P. H E Y B A C H ,
JENNIFER
LENZ AND JOHN F. MILLER
Department of Psychology, Northern Illinois University, DeKalb, IL 60115 ( R e c e i v e d 2 A u g u s t 1978) COOVER, G. D., J. P. HEYBACH, J. LENZ AND J. F. MILLER. Corticosterone "basal levels" and response to ether anesthesia in rats on a water deprivation regimen. PHYSIOL. BEHAV. 22(4) 653-656, 1979.--Male hooded rats were
subjected to a 23 2/3-hr water deprivation schedule for 8 weeks. The corticosterone levels of these rats increased between 0800 and I000 hr if tbey had always been watered at 1200 hr, but increased between 0600 and 0800 if the daily watering time had been randomized between 0800 and 1200 hr the previous 8 weeks. The corticosterone levels remained high until 1200 hr for both treatments. The results indicate temporal entrainment and a stable, elevated baseline corticosterone concentration. Eight weeks on the deprivation schedule resulted in accelerated corticosterone responses to acute stressors, but the acceleration was minimized when the rats were maintained under continuous ether anesthesia. The procedure of blood sampling under ether anesthesia is valid for water-deprived rats if the sample is drawn within 1.5 min, but it is not valid for up to 3 min as in ad lib rats. Corticosterone
Water deprivation
Temporal entrainment
FOOD and/or water deprivation schedules that restrict the nocturnal rat to a short period of consumption in the morning alter the circadian rhythm of plasma corticosterone concentration [8, 9, 12]. Peak basal levels of corticosterone occur in the morning, before feeding or watering, instead of, or as well as, at the normal lights-off time in the evening. The altered rhythm seems due, in part, to an altered sleep/wake cycle, since the rats also exhibit alterations in the time of the daily peak in body temperature, running activity and hippocampal norepinephrine and serotonin [9]. The high prewatering morning corticosterone levels of rats maintained on a 23 2/3-hr water deprivation schedule have also been interpreted as due to a chronic stress effect of the deprivation regimen [15]. Sakellaris and Vernikos-Danellis [15] favored this interpretation for two reasons: (1) the high morning corticosterone levels were found to diminish over 1, 4 and 8 weeks on the deprivation regimen, and (2) after 8 weeks on the water deprivation schedule the corticosterone response to the acute stress o f an intraperitoneal injection of saline was accelerated, such that a large corticosterone increase occurred within 2.5 min. These findings appear to show (1) the adaptation of high corticosterone levels that occurs under chronic stress and (2) the increase in adrenal response to additional, acute stressors in chronically stressed rats [4, 14, 16]. These findings also raise practical issues for research on the behavioral effects of appetitively motivated behavior on corticosterone responses. Is the high corticosterone baseline stable, and can blood samples be obtained under
Stress response
Blood sampling
ether anesthesia as in previous behavioral studies [1, 2, 7, 111? The first experiment of the present study was designed to determine whether the decrease in morning corticosterone levels during the 8 weeks on water deprivation reported by Sakellaxis and Vernikos-Danellis [15] was due to temporal entrainment to the watering time. Sakellaris and VernikosDaneUis had provided water to the rats at 1200 hr each day but took blood samples at 0800 hr, so it is possible that the pre-watering rise became delayed beyond 0800 with extended training. We thus examined the corticosterone levels at 0800, 1000 and 1200 hr after 8 weeks, and in rats that had been watered at random times between 0800 and 1200 hr as well as in rats always watered at 1200 hr. In the second experiment we replicated the rapid corticosterone increase to a saline injection produced by 8 weeks on the water deprivation schedule, but also examined the time course of the corticosterone response to ether anesthesia.
METHOD
The animals were male hooded rats from the colony of the Psychology Department, Northern Illinois University. They were 110 -+ 10 days of age at the beginning of the experiments, when they were individually housed in plastic cages with wire lids (Plastic Products) and wood chip bedding
1This study was supported in part by the Graduate School Fund, Northern Illinois University. The authors wish to thank, for their aid in collecting blood samples, Leonard Nenja, Mark Offerman, Allan Shnerson~ Gregory Urban and Stephen Welle.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/040653-04502.00/0
COOVER ET AL.
654 (Ab-Sorb-Dri) in a room maintained at 21-23°C with light onset at 0700 and offset at 1900 hr. All animals were provided ad lib access to food (Wayne Lab Blox) throughout the experiment. After 48 hr of adaptation to the new quarters the majority of animals were restricted to 20 min access to water/day for 56-59 days. Background noise and human presence in the animal quarters was structured during the course of the experiments by having one of the experimenters (JPH) utilize the room as a study room. An experimenter was thus almost continuously present in the room from 0730 to 1600 hr on weekdays, and from 0900 to 1230 on weekends. Removal of animals for blood sampling after 8 weeks occured only on the days Tuesday through Friday. Blood samples were collected in heparinized tubes or syringes, centrifuged and the plasma separated and stored frozen for subsequent assay. Corticosterone assay was by a modification [3] of the fluorometric micromethod of Glick, von Redlich and Levine [6].
Experiment 1: Morning Basal Levels The first experiment used 72 water-deprived and 12 ad lib rats. Half of the water-deprived rats were always provided their 20 min access to water at 1200 hr each day (Fixed Watering Time), while the other half were provided water at different times each day: twice each week at 0800, once each week at 0900, 1000 and 1100, and twice each week at 1200 hr (Random Watering Time). The Random animals all received their water at 1200 hr on the next to final day of the experiment, in order that they would be deprived for equivalent times compared to Fixed animals when the terminal blood sample was taken. On the 56th, 57th and 58th days of deprivation 7 or 8 rats were decapitated at each of the times 0800, 1000 and 1200 hr. A total of 9 Fixed and 9 Random animals, and 4 ad lib rats, were decapitated at each time, taking one-third of the animals of each subgroup each day (except that there was an odd, additional ad lib rat at each time point on the second day). One experimenter entered the rat quarters and handed rat cages to each of 7 or 8 experimenters acting as runners, and then watered any rats scheduled to receive water. The runners carded the rats to a guillotine attendant 15 m away from the rat quarters, and bled the trunks of the decapitated rats. All 7 or 8 rats were decapitated within 1.2 min of disturbing the first rat. At 1201 on the third day o f decapitation 6 Fixed and 6 Random animals were provided water bottles at an even pace over a 9-min period, while the remaining 3 Fixed and 3 Random rats were left unwatered. The 6 unwatered rats were then decapitated at 1210, to determine whether the cues of watering the other rats over the prior 10 min would produce the conditioned decrease in corticosterone that has been reported previously [3,10]. On the morning of the fourth day, the last remaining 6 Fixed and 6 Random animals were decapitated between 0600 and 0602, 1 hr before light onset, to provide supplemental data regarding the corticosterone level in the early morning before watering.
Experiment 2: Corticosterone Response The animals were 35 water-deprived and 25 ad lib rats, run in two waves of equal size staggered by 1 week. All water-deprived rats were provided access to water for 20 min/day at 1200 hr for 8 weeks. Blood samples were obtained
between 0730 and 0800 on eight mornings (Tuesday through Friday for 2 weeks). Time 0 blood samples were obtained from 12 deprived and 12 ad lib rats by decapitation within 15 sec of disturbing the rat in its quarters. Another 10 deprived rats were removed to the decapitation room at Time 0, injected with 1.0 ml saline intraperitoneally, and placed in their cage on the floor. These rats were decapitated at Time 2.5 min, to verify the rapid rise of corticosterone in deprived rats reported by Sakellaris and Vernikos-Danellis [15]. The remaining 13 deprived and 13 ad lib rats were also removed from their quarters at Time 0, placed in a jar containing ether-soaked cotton for 1.1 min, and then taped supine on a table top. A pad of ether-soaked cotton was placed over their snouts to maintain the anesthetized state, and their jugular vein was exposed on one side. A blood sample (0.6 ml) was drawn at Time 1.5 - 0.1 min, and another at 2.6 +- 0.1 min. Five each of these deprived and ad lib rats were then quickly decapitated and a blood sample obtained in order to compare the corticosterone concentrations of jugular and trunk blood samples. The other 8 rats each of the deprived and ad lib groups were maintained anesthetized and jugular vein samples were again drawn at Times 5.0 -+ 0.1 and 10.0 - 0.1 min. In order to obtain 26 jugular-vein animals in eight mornings, sampling from only 2 animals at a time, 10 of the animals were tested beginning (Time 0) at 0750. Their corticosterone values were not different from those of the 16 animals tested first, at 0730. Animals of each treatment condition were tested each of the eight mornings, and corticosterone values did not differ as a function of the morning on which the sample was taken. RESULTS AND DISCUSSION
Experiment 1: Morning Basal Levels Analysis of the corticosterone concentrations of Fixed and Random animals at 0800, 1000 and 1200 hr (Fig. 1) yielded a significant Watering Time Condition by Sampling Time interaction, F(2,48)= 10.58, p
Corticosterone "Basal Levels" and Response to Ether Anesthesia in Rats on a Water Deprivation Regimen I G A R Y D. C O O V E R , J O H N P. H E Y B A C H ,
JENNIFER
LENZ AND JOHN F. MILLER
Department of Psychology, Northern Illinois University, DeKalb, IL 60115 ( R e c e i v e d 2 A u g u s t 1978) COOVER, G. D., J. P. HEYBACH, J. LENZ AND J. F. MILLER. Corticosterone "basal levels" and response to ether anesthesia in rats on a water deprivation regimen. PHYSIOL. BEHAV. 22(4) 653-656, 1979.--Male hooded rats were
subjected to a 23 2/3-hr water deprivation schedule for 8 weeks. The corticosterone levels of these rats increased between 0800 and I000 hr if tbey had always been watered at 1200 hr, but increased between 0600 and 0800 if the daily watering time had been randomized between 0800 and 1200 hr the previous 8 weeks. The corticosterone levels remained high until 1200 hr for both treatments. The results indicate temporal entrainment and a stable, elevated baseline corticosterone concentration. Eight weeks on the deprivation schedule resulted in accelerated corticosterone responses to acute stressors, but the acceleration was minimized when the rats were maintained under continuous ether anesthesia. The procedure of blood sampling under ether anesthesia is valid for water-deprived rats if the sample is drawn within 1.5 min, but it is not valid for up to 3 min as in ad lib rats. Corticosterone
Water deprivation
Temporal entrainment
FOOD and/or water deprivation schedules that restrict the nocturnal rat to a short period of consumption in the morning alter the circadian rhythm of plasma corticosterone concentration [8, 9, 12]. Peak basal levels of corticosterone occur in the morning, before feeding or watering, instead of, or as well as, at the normal lights-off time in the evening. The altered rhythm seems due, in part, to an altered sleep/wake cycle, since the rats also exhibit alterations in the time of the daily peak in body temperature, running activity and hippocampal norepinephrine and serotonin [9]. The high prewatering morning corticosterone levels of rats maintained on a 23 2/3-hr water deprivation schedule have also been interpreted as due to a chronic stress effect of the deprivation regimen [15]. Sakellaris and Vernikos-Danellis [15] favored this interpretation for two reasons: (1) the high morning corticosterone levels were found to diminish over 1, 4 and 8 weeks on the deprivation regimen, and (2) after 8 weeks on the water deprivation schedule the corticosterone response to the acute stress o f an intraperitoneal injection of saline was accelerated, such that a large corticosterone increase occurred within 2.5 min. These findings appear to show (1) the adaptation of high corticosterone levels that occurs under chronic stress and (2) the increase in adrenal response to additional, acute stressors in chronically stressed rats [4, 14, 16]. These findings also raise practical issues for research on the behavioral effects of appetitively motivated behavior on corticosterone responses. Is the high corticosterone baseline stable, and can blood samples be obtained under
Stress response
Blood sampling
ether anesthesia as in previous behavioral studies [1, 2, 7, 111? The first experiment of the present study was designed to determine whether the decrease in morning corticosterone levels during the 8 weeks on water deprivation reported by Sakellaxis and Vernikos-Danellis [15] was due to temporal entrainment to the watering time. Sakellaris and VernikosDaneUis had provided water to the rats at 1200 hr each day but took blood samples at 0800 hr, so it is possible that the pre-watering rise became delayed beyond 0800 with extended training. We thus examined the corticosterone levels at 0800, 1000 and 1200 hr after 8 weeks, and in rats that had been watered at random times between 0800 and 1200 hr as well as in rats always watered at 1200 hr. In the second experiment we replicated the rapid corticosterone increase to a saline injection produced by 8 weeks on the water deprivation schedule, but also examined the time course of the corticosterone response to ether anesthesia.
METHOD
The animals were male hooded rats from the colony of the Psychology Department, Northern Illinois University. They were 110 -+ 10 days of age at the beginning of the experiments, when they were individually housed in plastic cages with wire lids (Plastic Products) and wood chip bedding
1This study was supported in part by the Graduate School Fund, Northern Illinois University. The authors wish to thank, for their aid in collecting blood samples, Leonard Nenja, Mark Offerman, Allan Shnerson~ Gregory Urban and Stephen Welle.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/040653-04502.00/0
COOVER ET AL.
654 (Ab-Sorb-Dri) in a room maintained at 21-23°C with light onset at 0700 and offset at 1900 hr. All animals were provided ad lib access to food (Wayne Lab Blox) throughout the experiment. After 48 hr of adaptation to the new quarters the majority of animals were restricted to 20 min access to water/day for 56-59 days. Background noise and human presence in the animal quarters was structured during the course of the experiments by having one of the experimenters (JPH) utilize the room as a study room. An experimenter was thus almost continuously present in the room from 0730 to 1600 hr on weekdays, and from 0900 to 1230 on weekends. Removal of animals for blood sampling after 8 weeks occured only on the days Tuesday through Friday. Blood samples were collected in heparinized tubes or syringes, centrifuged and the plasma separated and stored frozen for subsequent assay. Corticosterone assay was by a modification [3] of the fluorometric micromethod of Glick, von Redlich and Levine [6].
Experiment 1: Morning Basal Levels The first experiment used 72 water-deprived and 12 ad lib rats. Half of the water-deprived rats were always provided their 20 min access to water at 1200 hr each day (Fixed Watering Time), while the other half were provided water at different times each day: twice each week at 0800, once each week at 0900, 1000 and 1100, and twice each week at 1200 hr (Random Watering Time). The Random animals all received their water at 1200 hr on the next to final day of the experiment, in order that they would be deprived for equivalent times compared to Fixed animals when the terminal blood sample was taken. On the 56th, 57th and 58th days of deprivation 7 or 8 rats were decapitated at each of the times 0800, 1000 and 1200 hr. A total of 9 Fixed and 9 Random animals, and 4 ad lib rats, were decapitated at each time, taking one-third of the animals of each subgroup each day (except that there was an odd, additional ad lib rat at each time point on the second day). One experimenter entered the rat quarters and handed rat cages to each of 7 or 8 experimenters acting as runners, and then watered any rats scheduled to receive water. The runners carded the rats to a guillotine attendant 15 m away from the rat quarters, and bled the trunks of the decapitated rats. All 7 or 8 rats were decapitated within 1.2 min of disturbing the first rat. At 1201 on the third day o f decapitation 6 Fixed and 6 Random animals were provided water bottles at an even pace over a 9-min period, while the remaining 3 Fixed and 3 Random rats were left unwatered. The 6 unwatered rats were then decapitated at 1210, to determine whether the cues of watering the other rats over the prior 10 min would produce the conditioned decrease in corticosterone that has been reported previously [3,10]. On the morning of the fourth day, the last remaining 6 Fixed and 6 Random animals were decapitated between 0600 and 0602, 1 hr before light onset, to provide supplemental data regarding the corticosterone level in the early morning before watering.
Experiment 2: Corticosterone Response The animals were 35 water-deprived and 25 ad lib rats, run in two waves of equal size staggered by 1 week. All water-deprived rats were provided access to water for 20 min/day at 1200 hr for 8 weeks. Blood samples were obtained
between 0730 and 0800 on eight mornings (Tuesday through Friday for 2 weeks). Time 0 blood samples were obtained from 12 deprived and 12 ad lib rats by decapitation within 15 sec of disturbing the rat in its quarters. Another 10 deprived rats were removed to the decapitation room at Time 0, injected with 1.0 ml saline intraperitoneally, and placed in their cage on the floor. These rats were decapitated at Time 2.5 min, to verify the rapid rise of corticosterone in deprived rats reported by Sakellaris and Vernikos-Danellis [15]. The remaining 13 deprived and 13 ad lib rats were also removed from their quarters at Time 0, placed in a jar containing ether-soaked cotton for 1.1 min, and then taped supine on a table top. A pad of ether-soaked cotton was placed over their snouts to maintain the anesthetized state, and their jugular vein was exposed on one side. A blood sample (0.6 ml) was drawn at Time 1.5 - 0.1 min, and another at 2.6 +- 0.1 min. Five each of these deprived and ad lib rats were then quickly decapitated and a blood sample obtained in order to compare the corticosterone concentrations of jugular and trunk blood samples. The other 8 rats each of the deprived and ad lib groups were maintained anesthetized and jugular vein samples were again drawn at Times 5.0 -+ 0.1 and 10.0 - 0.1 min. In order to obtain 26 jugular-vein animals in eight mornings, sampling from only 2 animals at a time, 10 of the animals were tested beginning (Time 0) at 0750. Their corticosterone values were not different from those of the 16 animals tested first, at 0730. Animals of each treatment condition were tested each of the eight mornings, and corticosterone values did not differ as a function of the morning on which the sample was taken. RESULTS AND DISCUSSION
Experiment 1: Morning Basal Levels Analysis of the corticosterone concentrations of Fixed and Random animals at 0800, 1000 and 1200 hr (Fig. 1) yielded a significant Watering Time Condition by Sampling Time interaction, F(2,48)= 10.58, p
