Psychoneuroendocrinology.Vol. 17, No. 6. pp. 691-699, 1992
0306-4530192 $5.00+0.00 ©1993 Pergamon Press Ltd.
Printed in Great Britain
EFFECTS OF CHRONIC CORTICOSTERONE TREATMENT ON ELECTROPHYSIOLOGICAL A N D BEHAVIORAL MEASURES IN THE RAT C. L. EHLERS, R. I. CHAPLIN, and W. M. KANEKO Department of Neuropharmacology,The Scripps Research Institute, La Jolla, California, U.S.A. (Received 17 September 1991; in final form 13 November 1991)
SUMMARY The electrophysiological and behavioral effects of daily oral exposure to a corticosterone or vehicle solution was evaluated in 20 male Wistar rats over a 10-wk period. Evaluation of the rats' behavior in an open field apparatus, as well as in automated locomotor cages, revealed no significant differences between steroid-treated and control animals following 5-6 wk of exposure. No differences in mean EEG power, as estimated by spectral analysis of cortical and dorsal hippocampal recordings, were observed between the two groups following 8 wk of exposure. However, some increases in EEG "stability" were noted in the corticosterone-treated rats. At 9 wk, responses to auditory stimuli, as assessed by evoked responses, in cortex and dorsal hippocampus were also found to be unaltered by corticosterone exposure. These studies suggest that exposure to daily oral corticosterone, in the doses used, over a period of 2-3 mo is not associated with gross electrophysiological or spontaneous behavioral effects in the brain areas assessed.
INTRODUCTION SEVERAL CLINICALSYNDROMESare associated with prolonged elevations in circulating levels of glucocorticoids. In addition to Cushing's syndrome, marked increases in cortisol secretion rates have been observed in endogenous depressives (Linkowski et al., 1985), as well as in chronic alcoholics (Van Thiel, 1983). In both of these disorders, failure of plasma cortisol to suppress in response to administration of the synthetic glucocorticoid dexamethasone has been reported (Carroll, 1982; Schwartz & Dunner, 1982). In addition, in certain chronic alcoholics, hypercortisolemia can be so severe that several features of "pseudo-Cushing's syndrome" are present (Rees et al., 1977). Over the last 5 yr, evidence from animal studies has been accumulating which suggests that prolonged exposure to supraphysiological levels of adrenal glucocorticoids may produce changes in neuronal morphology, particularly in the hippocampus. For example, chronic injections of glucocorticoids can result in depletion of corticosteroid receptors in hippocampus and reduction in hippocampal cell numbers (Sapolsky, 1985; Sapolsky et al., 1985; Packan & Sapolsky, 1990). Other investigators have not found hippocampal pyramidal cell loss following chronic steroid administration but have observed a reduction in the number of acetylAddress correspondence and reprint requests to: Dr. C. L. Ehlers, The Scripps Research Institute, BCR-1, 10666 North Torrey Pines Road, La Jolla CA 92037, USA. 691
692
c.L. EHLER5et al.
cholinesterase-stained neurons in the region of the medial septal area (Tizabi et al., 1989). Other evidence has led to the proposal that glucocorticoids themselves may not kill hippocampal neurons outright, but rather may make them more susceptible to damage from neurological insults (Armanini et al., 1990). The results of these animal studies have led to the speculation that sustained glucocorticoid exposure in humans, whether due to endocrine disorders, exogenous administration, or perhaps depression (Holsboer, 1988) and/or alcoholism, might, lead to changes in brain function. If chronic elevations of corticosteroids, in fact, do lead to changes in brain and behavioral functioning, then such changes should be observable in animal models. Significant differences in b e h a v i o r a l and e l e c t r o p h y s i o l o g i c a l measures have b e e n observed in a n i m a l models of depression (Ehlers et al., 1989) and in a n i m a l s e x p o s e d c h r o n i c a l l y to e t h a n o l (Ehlers & Chaplin 1987, 1991). However, fewer studies have focused on the effects of chronic glucocorticoid administration on in vivo functioning, as assessed by behavioral and/or electrophysiological m e a n s . T h u s , the aims o f the p r e s e n t s t u d y were to e x p o s e rats to d a i l y e x o g e n o u s glucocorticoids over a period of 2 - 3 mo and assess their functioning by both behavioral and electrophysiological methods. MATERIALS AND METHODS The experimental animals were 20 male Wistar rats initially weighing 380-450 g. Body weights for all rats were monitored weekly. They were housed in pairs and maintained in a temperature- and light-controlled room (12 hr:12 hr light:dark cycle; lights on 0600h-1800h). Water and rat chow were given ad libitum throughout the study. At least 2 wk prior to the experimental procedures, the rats were surgically prepared with recording electrodes. They were anesthetized (Nembutal, 50 mg/kg IP), and stainless steel bipolar electrodes were aimed at the dorsal hippocampus (AP 3.0, ML 3.0, DV 3.1). Screw electrodes were placed in the calvarium overlying the frontal and posterior cortices. A "reference" electrode, which was grounded, was placed in the thick bony area of the calvarium 3 mm posterior to lambda, which lies parallel to the cerebellum. In all animals, electrode connections were made to a multipin (Amphenol) connector, and the entire assembly was anchored to the skull with acrylic dental cement. The study was approved by the institutional animal research committee. The rats were exposed to corticosterone (Steraloids, Whilton, NH) or vehicle once daily, over a 10-wk period. Every day between 0600h and 0800h, for this 10-wk period, the rats were placed in individual cages for I hr. For the following 2 hr, half the rats were given a bottle containing 50 ml of a corticosterone solution (20 mg corticosterone, 1.204 ml 95% ETOH, and 48.796 ml 5% sucrose). The control rats received the identical ethanol and sucrose solution without the steroid. At the end of the 2-hr session, the amount of solution remaining in each of the bottles was measured, and the rats were returned to their home cages. One control and one steroid-treated rat died during the study. Two behavioral measures were ascertained on these animals. After 5 - 6 wk of treatment, each rat was individually tested in an open field apparatus between ll00h and 1200b. This was accomplished by placing the rat in a topless, black, opaque plexiglass box (115 x 115 x 46 cm) in a dark room, with a white-noise level of 70 dB. A 150-watt bulb was centered over the open field to produce a light intensity of 100-150 ft-candles. The rats were tested by placing them in the center of the open field; over a 5-rain session, the latency for the rat to reach an outer square was recorded, as was the number of inner squares and outer squares entered, number of free rearings and wall rearings, number of grooming bouts, and number of defecations and urinations. At 6 wk, the locomotor behavior of the rats was quantified. For measurements of locomotion, the rats were individually placed from 1800h-0600h in wire mesh cages equipped with two infrared photocell beams aimed across the long axis of the floor. Activity was quantified by totaling photocell beam interruptions over 10-min intervals for 180 rain, at the beginning of the session, just after lights-off, and for 180 min at the end of the session, just before lights-on. Electrophysiological recordings were obtained on the test day between 0930h and 1300h, after 8-9 wk of daily steroid- or control-solution exposure. EEG recordings were obtained in freely moving rats. Twenty min of EEG recordings of bipolar signals were obtained from frontal cortex and dorsal hippocampus, referenced to
CHRONIC CORTICOSTERONEEFFEc-rs ON EEG AND BEHAVIOR
693
ground. EEG signals were recorded on a Grass polygraph, with a bandpass of 1-70 Hz, and transfered to a Vetter Model D recorder for off-line analysis. For quantification of the EEG, 20 min of EEG were digitized (128 Hz), and the power spectra of continuous 4-sec epochs were determined for a 0.25-64 Hz range. The Fourier-transformed data were further compressed into seven frequency bands (1-2, 2 - 4 , 4 - 6 , 6-8, 8-16, 16-32, and 32-64 Hz). Mean power density was calculated for each band, as well as EEG "stability", as described previously (Ehlers & Havstad, 1982; Ehlers, 1986). Evoked responses (EPs) to auditory stimuli also were assessed by placing an individual rat in a Naugahyde sling, which comfortably supported the animal in an awake state but prevented movement-induced artifact. The sling was placed in an electrically shielded, light-, sound-, and temperature-controlled BRS/LVE recording chamber. All animals were adapted to the chamber prior to recordings. On a test day, the rats were placed individually into the chamber, and a connector attached to a microdot cable was used to transfer the monopolar (referred to the lambda ground screw) EEG signals to a polygraph. The bandpass for recordings was set at 0.3-75 Hz with a 60 Hz notch filter in. The signals were amplified (50% gain), and the EEG and calibration signals, were transferred from the polygraph on-line to a DEC (LSI 11-2) computer, which also controlled the presentation of the auditory stimuli. Free-field auditory stimuli were presented through a small speaker centered approximately 20 cm above the rat's head. EPs were elicited with two paradigms: an acoustic oddball, and an oddball+noise paradigm. The tones were generated by a programmable, multiple-tone generator, the characteristics of which have been described previously (Polich et al., 1983). The acoustic parameters for this paradigm were two square-wave tones (rise/fall time < 1 msec): a standard tone (20 msec, 1 kHz, 70 dB SPL) presented in 84% of the trials, a rare tone (20 msec, 2 kHz, 80 dB SPL) presented in 16% of the trials in the 2-tone paradigm and 10% of the trials in the 3-tone paradigm, and a noise burst (20 msec, noise, 95 dB SPL) presented in 6% of the trials in the 3-tone paradigm. Rare tones were interspersed with standards such that no two rare tones occurred successively, with a noise burst substituted for a rare tone every 12 trials in the 3-tone paradigm. The digitizing epoch for each trial was 1 sec, and a 0.5-1.0 sec intertrial interval was used to reduce habituation. The total number of trials in a recording session was 312 in the 3-tone paradigm and 150 in the 2-tone paradigm. At 10 wk post-steroid treatment, the rats were no longer given access to the steroid or vehicle solutions. Four to five days later, the rats were sacrificed by decapitation, and their adrenals were dissected and weighed. For statistical analysis, analysis of variance (ANOVA) techniques were utilized, with simple main-effects post hoc tests for repeated measures. RESULTS Rats w e r e f o u n d to r e a d i l y c o n s u m e the s u c r o s e / s t e r o i d a n d s u c r o s e / v e h i c l e solutions. While there was no overall difference b e t w e e n the groups in the amount consumed, there were some time effects. As shown in Fig. 1, the rats drank an average o f 19 m l solution a day for the first wk, b u t w e r e d r i n k i n g 24 m l b y the 10 th w k (ANOVA for t i m e : F = 5 . 6 1 ; d f = 9 , 1 4 4 ; p < 0 . 0 1 ) . These data suggest that the steroid group was consuming an average o f 7 . 6 - 9 . 6 m g of corticosterone daily. The result o f this c o n s u m p t i o n o f steroid solution was evident in the group difference in body weight over the 10 w k period (ANOVA for group: F = 7.74; d f = 1,16; p < 0.01). As shown in Fig. 2, while no difference in b o d y weight was o b s e r v e d b e t w e e n the groups for the first 4 wk, the steroid-consuming group failed to gain as much weight as did their controls between weeks 4 - 1 0 (ANOVA for group × time: F = 2 4 . 5 8 ; d f = 9 , 1 4 4 ; p < 0 . 0 0 1 ) . A t 10 w k the steroidtreated rats weighed 84% o f the weight o f their controls. In spite o f significant differences in b o d y weight, the two groups of rats did not display significant differences in the behaviors assessed. As shown in Fig. 3, no differences were noted between the two groups in any o f the behaviors exhibited in the open field, such as rearings, e x p l o r i n g i n n e r or outer squares, or signs o f " a n x i e t y " such as u r i n a t i o n or defecation. In a d d i t i o n , no s i g n i f i c a n t d i f f e r e n c e in the a m o u n t o f l o c o m o t i o n q u a n t i f i e d b y p h o t o b e a m
694
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0306-4530192 $5.00+0.00 ©1993 Pergamon Press Ltd.
Printed in Great Britain
EFFECTS OF CHRONIC CORTICOSTERONE TREATMENT ON ELECTROPHYSIOLOGICAL A N D BEHAVIORAL MEASURES IN THE RAT C. L. EHLERS, R. I. CHAPLIN, and W. M. KANEKO Department of Neuropharmacology,The Scripps Research Institute, La Jolla, California, U.S.A. (Received 17 September 1991; in final form 13 November 1991)
SUMMARY The electrophysiological and behavioral effects of daily oral exposure to a corticosterone or vehicle solution was evaluated in 20 male Wistar rats over a 10-wk period. Evaluation of the rats' behavior in an open field apparatus, as well as in automated locomotor cages, revealed no significant differences between steroid-treated and control animals following 5-6 wk of exposure. No differences in mean EEG power, as estimated by spectral analysis of cortical and dorsal hippocampal recordings, were observed between the two groups following 8 wk of exposure. However, some increases in EEG "stability" were noted in the corticosterone-treated rats. At 9 wk, responses to auditory stimuli, as assessed by evoked responses, in cortex and dorsal hippocampus were also found to be unaltered by corticosterone exposure. These studies suggest that exposure to daily oral corticosterone, in the doses used, over a period of 2-3 mo is not associated with gross electrophysiological or spontaneous behavioral effects in the brain areas assessed.
INTRODUCTION SEVERAL CLINICALSYNDROMESare associated with prolonged elevations in circulating levels of glucocorticoids. In addition to Cushing's syndrome, marked increases in cortisol secretion rates have been observed in endogenous depressives (Linkowski et al., 1985), as well as in chronic alcoholics (Van Thiel, 1983). In both of these disorders, failure of plasma cortisol to suppress in response to administration of the synthetic glucocorticoid dexamethasone has been reported (Carroll, 1982; Schwartz & Dunner, 1982). In addition, in certain chronic alcoholics, hypercortisolemia can be so severe that several features of "pseudo-Cushing's syndrome" are present (Rees et al., 1977). Over the last 5 yr, evidence from animal studies has been accumulating which suggests that prolonged exposure to supraphysiological levels of adrenal glucocorticoids may produce changes in neuronal morphology, particularly in the hippocampus. For example, chronic injections of glucocorticoids can result in depletion of corticosteroid receptors in hippocampus and reduction in hippocampal cell numbers (Sapolsky, 1985; Sapolsky et al., 1985; Packan & Sapolsky, 1990). Other investigators have not found hippocampal pyramidal cell loss following chronic steroid administration but have observed a reduction in the number of acetylAddress correspondence and reprint requests to: Dr. C. L. Ehlers, The Scripps Research Institute, BCR-1, 10666 North Torrey Pines Road, La Jolla CA 92037, USA. 691
692
c.L. EHLER5et al.
cholinesterase-stained neurons in the region of the medial septal area (Tizabi et al., 1989). Other evidence has led to the proposal that glucocorticoids themselves may not kill hippocampal neurons outright, but rather may make them more susceptible to damage from neurological insults (Armanini et al., 1990). The results of these animal studies have led to the speculation that sustained glucocorticoid exposure in humans, whether due to endocrine disorders, exogenous administration, or perhaps depression (Holsboer, 1988) and/or alcoholism, might, lead to changes in brain function. If chronic elevations of corticosteroids, in fact, do lead to changes in brain and behavioral functioning, then such changes should be observable in animal models. Significant differences in b e h a v i o r a l and e l e c t r o p h y s i o l o g i c a l measures have b e e n observed in a n i m a l models of depression (Ehlers et al., 1989) and in a n i m a l s e x p o s e d c h r o n i c a l l y to e t h a n o l (Ehlers & Chaplin 1987, 1991). However, fewer studies have focused on the effects of chronic glucocorticoid administration on in vivo functioning, as assessed by behavioral and/or electrophysiological m e a n s . T h u s , the aims o f the p r e s e n t s t u d y were to e x p o s e rats to d a i l y e x o g e n o u s glucocorticoids over a period of 2 - 3 mo and assess their functioning by both behavioral and electrophysiological methods. MATERIALS AND METHODS The experimental animals were 20 male Wistar rats initially weighing 380-450 g. Body weights for all rats were monitored weekly. They were housed in pairs and maintained in a temperature- and light-controlled room (12 hr:12 hr light:dark cycle; lights on 0600h-1800h). Water and rat chow were given ad libitum throughout the study. At least 2 wk prior to the experimental procedures, the rats were surgically prepared with recording electrodes. They were anesthetized (Nembutal, 50 mg/kg IP), and stainless steel bipolar electrodes were aimed at the dorsal hippocampus (AP 3.0, ML 3.0, DV 3.1). Screw electrodes were placed in the calvarium overlying the frontal and posterior cortices. A "reference" electrode, which was grounded, was placed in the thick bony area of the calvarium 3 mm posterior to lambda, which lies parallel to the cerebellum. In all animals, electrode connections were made to a multipin (Amphenol) connector, and the entire assembly was anchored to the skull with acrylic dental cement. The study was approved by the institutional animal research committee. The rats were exposed to corticosterone (Steraloids, Whilton, NH) or vehicle once daily, over a 10-wk period. Every day between 0600h and 0800h, for this 10-wk period, the rats were placed in individual cages for I hr. For the following 2 hr, half the rats were given a bottle containing 50 ml of a corticosterone solution (20 mg corticosterone, 1.204 ml 95% ETOH, and 48.796 ml 5% sucrose). The control rats received the identical ethanol and sucrose solution without the steroid. At the end of the 2-hr session, the amount of solution remaining in each of the bottles was measured, and the rats were returned to their home cages. One control and one steroid-treated rat died during the study. Two behavioral measures were ascertained on these animals. After 5 - 6 wk of treatment, each rat was individually tested in an open field apparatus between ll00h and 1200b. This was accomplished by placing the rat in a topless, black, opaque plexiglass box (115 x 115 x 46 cm) in a dark room, with a white-noise level of 70 dB. A 150-watt bulb was centered over the open field to produce a light intensity of 100-150 ft-candles. The rats were tested by placing them in the center of the open field; over a 5-rain session, the latency for the rat to reach an outer square was recorded, as was the number of inner squares and outer squares entered, number of free rearings and wall rearings, number of grooming bouts, and number of defecations and urinations. At 6 wk, the locomotor behavior of the rats was quantified. For measurements of locomotion, the rats were individually placed from 1800h-0600h in wire mesh cages equipped with two infrared photocell beams aimed across the long axis of the floor. Activity was quantified by totaling photocell beam interruptions over 10-min intervals for 180 rain, at the beginning of the session, just after lights-off, and for 180 min at the end of the session, just before lights-on. Electrophysiological recordings were obtained on the test day between 0930h and 1300h, after 8-9 wk of daily steroid- or control-solution exposure. EEG recordings were obtained in freely moving rats. Twenty min of EEG recordings of bipolar signals were obtained from frontal cortex and dorsal hippocampus, referenced to
CHRONIC CORTICOSTERONEEFFEc-rs ON EEG AND BEHAVIOR
693
ground. EEG signals were recorded on a Grass polygraph, with a bandpass of 1-70 Hz, and transfered to a Vetter Model D recorder for off-line analysis. For quantification of the EEG, 20 min of EEG were digitized (128 Hz), and the power spectra of continuous 4-sec epochs were determined for a 0.25-64 Hz range. The Fourier-transformed data were further compressed into seven frequency bands (1-2, 2 - 4 , 4 - 6 , 6-8, 8-16, 16-32, and 32-64 Hz). Mean power density was calculated for each band, as well as EEG "stability", as described previously (Ehlers & Havstad, 1982; Ehlers, 1986). Evoked responses (EPs) to auditory stimuli also were assessed by placing an individual rat in a Naugahyde sling, which comfortably supported the animal in an awake state but prevented movement-induced artifact. The sling was placed in an electrically shielded, light-, sound-, and temperature-controlled BRS/LVE recording chamber. All animals were adapted to the chamber prior to recordings. On a test day, the rats were placed individually into the chamber, and a connector attached to a microdot cable was used to transfer the monopolar (referred to the lambda ground screw) EEG signals to a polygraph. The bandpass for recordings was set at 0.3-75 Hz with a 60 Hz notch filter in. The signals were amplified (50% gain), and the EEG and calibration signals, were transferred from the polygraph on-line to a DEC (LSI 11-2) computer, which also controlled the presentation of the auditory stimuli. Free-field auditory stimuli were presented through a small speaker centered approximately 20 cm above the rat's head. EPs were elicited with two paradigms: an acoustic oddball, and an oddball+noise paradigm. The tones were generated by a programmable, multiple-tone generator, the characteristics of which have been described previously (Polich et al., 1983). The acoustic parameters for this paradigm were two square-wave tones (rise/fall time < 1 msec): a standard tone (20 msec, 1 kHz, 70 dB SPL) presented in 84% of the trials, a rare tone (20 msec, 2 kHz, 80 dB SPL) presented in 16% of the trials in the 2-tone paradigm and 10% of the trials in the 3-tone paradigm, and a noise burst (20 msec, noise, 95 dB SPL) presented in 6% of the trials in the 3-tone paradigm. Rare tones were interspersed with standards such that no two rare tones occurred successively, with a noise burst substituted for a rare tone every 12 trials in the 3-tone paradigm. The digitizing epoch for each trial was 1 sec, and a 0.5-1.0 sec intertrial interval was used to reduce habituation. The total number of trials in a recording session was 312 in the 3-tone paradigm and 150 in the 2-tone paradigm. At 10 wk post-steroid treatment, the rats were no longer given access to the steroid or vehicle solutions. Four to five days later, the rats were sacrificed by decapitation, and their adrenals were dissected and weighed. For statistical analysis, analysis of variance (ANOVA) techniques were utilized, with simple main-effects post hoc tests for repeated measures. RESULTS Rats w e r e f o u n d to r e a d i l y c o n s u m e the s u c r o s e / s t e r o i d a n d s u c r o s e / v e h i c l e solutions. While there was no overall difference b e t w e e n the groups in the amount consumed, there were some time effects. As shown in Fig. 1, the rats drank an average o f 19 m l solution a day for the first wk, b u t w e r e d r i n k i n g 24 m l b y the 10 th w k (ANOVA for t i m e : F = 5 . 6 1 ; d f = 9 , 1 4 4 ; p < 0 . 0 1 ) . These data suggest that the steroid group was consuming an average o f 7 . 6 - 9 . 6 m g of corticosterone daily. The result o f this c o n s u m p t i o n o f steroid solution was evident in the group difference in body weight over the 10 w k period (ANOVA for group: F = 7.74; d f = 1,16; p < 0.01). As shown in Fig. 2, while no difference in b o d y weight was o b s e r v e d b e t w e e n the groups for the first 4 wk, the steroid-consuming group failed to gain as much weight as did their controls between weeks 4 - 1 0 (ANOVA for group × time: F = 2 4 . 5 8 ; d f = 9 , 1 4 4 ; p < 0 . 0 0 1 ) . A t 10 w k the steroidtreated rats weighed 84% o f the weight o f their controls. In spite o f significant differences in b o d y weight, the two groups of rats did not display significant differences in the behaviors assessed. As shown in Fig. 3, no differences were noted between the two groups in any o f the behaviors exhibited in the open field, such as rearings, e x p l o r i n g i n n e r or outer squares, or signs o f " a n x i e t y " such as u r i n a t i o n or defecation. In a d d i t i o n , no s i g n i f i c a n t d i f f e r e n c e in the a m o u n t o f l o c o m o t i o n q u a n t i f i e d b y p h o t o b e a m
694
C. L. EHLER5 et al. 5O Control Steroid
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