Brain Research, 566 (1991) 186-192 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/911503.50
186 BRES 17221
Serotonergic modulation of the hamster wheelrunning rhythm: response to lighting conditions and food deprivation L.P. M o r i n a n d J. B l a n c h a r d Department of Psychiatry, State Universin.' of New York, Stony Brook, r~~. 11794 (U.S.A.) (Accepte-i i6 July i991)
Key words: 5,7-Dihydroxytryptamine; Body weight; Hamster; $erotonin; Light; Food deprivation; Wheelrunning; Environmental stimulus
Depletion of brain serotonin by 5,7-d~hydroxytryptamine (DHT) produces large changes in photic regulation of the hamster circadian running rhythm. This study documents th, changes in daily wheelrunning caused by DHT lesions and their relationship to changes in photic conditions o:" food avadabiiity. Hamsters were given bilateral infusions of the selective neurotoxin during entrainment to a light-dark cycle (LD) of 14:10 h. At a later time, anim; ~; were transferred to constant light (LL) or dark (DD) for a prolonged period. Animals in DD were also st,Jject to 3 days of f~od .ieprivati~n. Destruction of the serotonergic system does not change the amount of daily running in LD 14:10, but does alter the "~s¢ of runni~ ~ rol animals respond to LL by greatly decreasing running compared to those with lesions. Food deprivation, a condi~.,on that greatly elev ~,:s running in control animals, is not nearly as effective in lesioned animals. The results suggest tha~ serotonin-depletea hamsters have diminished responsiveness to environmental stimuli.
INTRODUCTION
The suprachiasmatic nuclei ($CN) contain one or more circadian ~locks controlling a large number of physiological and behavioral rhythms is. The hamster locomotor (wheelrunning) rhythm may be the most thoroughly studied ~irc~dian rhythm. Each such rhythm is characterized by its period, amplitude and phase. Amplitude has been le;s well studied than either rhythm phase or period contcol. Phase and period are timing properties of a rhythm and are thought to directly reflect correspondJ~ig rharacteristics of a central pacemaker (i.e., clock mecaanism) it. In contrast, the magnitude of any measured rhythm is not necessarily related to the amplit~de of the pacemaker. With a mechanical timepiece, a clock mechanism generates the period and controls the phase of the clock hands. Amplitude of the mechanical clock is purely a function of the length of the hands. Magnitude of the running rhythm is a secondary characteristic coupled to the circadian clock and adjustable through amplification (positively or negatively) of the circadian signal indicating rhythm phase. There is presently little understanding of the mammalian pacemaker mechanism and it is impossible to watch the hands of the clock change position or to measure their ;ength. Instead, investigators are required to mea-
sure repetitive changes in magnitude of an overt rhythm and assume that the timing of these changes reflect the underlyi~Ig pacemaker functions of period and phase. A classic example of this was provided by Richter t4 who anesthetized freerunning rats for several days. The magnitude of the circadian wheelrunning rhythm was reduced to zero for th~ duration of the anesthesia, but reappeared with the appropriate period, phase and magnitude as the anesthesia wore off. In a similar experiment, Schwartz et al. t6 infused tetrodotoxin onto the rat SCN. This also reduced magnitude of the running rhythm to zero, although the animals were awake and ran substantially throughout the duration of drug, infusion. Afterward, the rhythm resumed with the proper period, phase and magnitude. In each experiment, the animals became arrhythmic, but for quite different reasons. In the first case, all locomotor activity was abolished. In the second, circadian pacemaker mechanisms were dissociated from motor control systems. Thus, the magnitude of a measured rhythm is potentially modifiable at or near the central pacemaker as well as at sites along the path of motor output regulation. In contrast, period and phase must always be controlled through an action involving the pacemaker. We have previously shown that several lesion types modify the daily amount of hamster wheelrunning activ-
Correspondence: L.P. Morin, Department of Psychiatry, Health Sciences Center, SUNY, Stony Brook, NY 11794, U.S.A.
187 ity and that the most effective sites also disturb the circadian clock s. Hypothalamic lesions that damage the SNC will reduce running about 90%. Lesions elsewhere in the hypothalamus or thalamus can alter phase angle of entrainment or block phasic actions of light on the circadian period with smaller, or no, alterations in the amount of daily running 6.s'~°. The present data were collected during studies of the role played by serotonin in the regulation of the hamster circadian wheelrunning rhythm. They offcJ" an opportunity to indirectly evaluate the relationship between circadian rhythm regulation and magnitude of the running rhythm. MATERIALS AND METHODS Detailed methods are described in the companion paperm. Briefly, adult male golden hamsters (90-100 g) from Charle~ River/ Lakeview were individually caged under 14 h light/10 h dark (LD 14:10) conditions in a temperature controlled room (21 -+ 2 °C). After 3 weeks, each animal was transferred to a plastic cage containing a running wheel. Wheel revolutions were recorded per 5 rain interval (288 data 'bins' per day) by computer. Water and food (except when explicitly removed in Expt. 2) were continuously available. Light intensity under LD 14:10 conditions averaged 31 lux.
Experiment 1 Animals remained undisturbed for 3 weeks in LD 14:10 before receiving surgical treatment. All animals then remained in LD 14:10 for 4 additional weeks after which the lights in the animal room were continuously on (LL) for 85 days. The lights were then turned off continuously (DD) for 50 days and this was followed by a return to LD 14:10 for 14 days at which time each animal was anesthetized and peffused.
Experiment 2 The initial conditions were similar to those in Expt, 1 except that after LD 14:10, all animals were placed into DD that lasted abou~ 170 days. After 55 days in DD, all food was removed from each animal's cage. Deprivation began at 12,00 h and lasted 72 h. AboL~t 35 days later, all animals entered a paradigm for generating a phase response curve (PRC) to light. This procedure is described ~n detail elsewherem.
Surgery In both experiments, each animal was pre-treated with an intraperitoneai injection of desipramine hydrochloride (Sigma, 25 mg/kg in saline) about 30 min before being anesthetized with an intraperitoneal injection of sodium pentobarbital (10.6 m[~kg) during the light phase of the photoperiod and placed in a stercotaxic instrument. Animals received 5,7-dihydroxytryptamine creatinine sulfate (DHT; Sigma; 75/~g of free base in 2.5 t~l 0.5% ascorbic acid per lateral ventricle) or vehicle (CON group).
Histology At the conclusion of the experiments, each animal was deeply anesthetized and perfused9. Brains were removed, postfixed, cryoprotected in sucrose, then frozen and serially sectioned (30 #m) in the coronal plane. The avidin-biotin complex technique7 was used for serotonin immunohistochemistry with a 1:50 dilution of a monoclonal antibody to serotonin (Accurate). The sections were reacted using an ABC kit (Vector) with diaminobenzidine as the chromogen. Sections through the entire SCN or raphe nuclei of each animal were microscopically examined and evaluated. A digitizing imaging system (MCID) provided an index of relative optical density through the SCN and IGL. Cells in the dorsal and median ra-
phe nuclei were also counted using the digitizing ~ystem.
Statistics The CSS statistical package (StatSoft) was used. All probability values reported are twr~-tailed unless specifically stated otherwise. RESULTS
Histology Experiments 1 and 2. Optical density of serotonin immuncre.activity (5-HT-IR) in the SCN was significantly reduced by DHT treatment. Optical density of 5-HT-IR in the intergeniculate leaflet (IGL) was also significantly reduced by DHT treatment. In the dorsal raphe, cells with 5-HT-IR were reduced by about 90% in DHTtreated animals (Fig. 1). A more thorough presentation of the histological data is presented elsewhere t°.
Behavior--Experiment 1 Daily wheel revolutions during entrainment and LL. The daily activity levels of the treatment groups were evaluated before surge~, after surgery and during days 15-20 of LL (Fig. 2A). One I3AIT animal was not included in the analysis because it only averaged 6 revolutions/day (>8.5 S.D.'s from the mean) during the 5 days of LL. Analysis of variance revealed a significant within groups effect (F = 13.35, ,ff = 2.44, P < 0.001) and a group x treatment interaction (F = 3.42, P = 0.04). Under LD 14:10 conditions, the DHT and control (CON) groups did not differ either before or after ~,~ur~ery with respect to average wheel revolutions per 24 h. However, upon exposure to LL, CON animals responded with ar~ average 57.9 -+ 10,0% decline in daily revolutions compared to a 19.2 -. 11.5% drop for the D H T animals (t ffi 2.32, df ffi 22, P < 0.03). After 56 days in LL, CON animals ran an average 5626 - 1564 daily revolutions which did not differ significantly from the 3933 *'- 515 revolutions by the DHT group. A second measure of wheelrunning rate was also examined for each animal and the 5 min data bin containing the largest average number of revolutions was recorded as the maximum running rate. The results are shown in Fig. 3A. Analysis of variance revealed a significant between groups effect (df = 1,23, F = 6.12, P < 0.02) and a significant within groups effect (df = 2,44, F = 12.99, P < 0.001), but no interaction effect. Postsurgical maximum running rate in LD 14:10 was significantly greater for the CON group than the DHT group (P < 0.05, Newman-Keuls test; there were no other between group differences). Whereas only the DHT group showed a significant drop in maximum running rate after surgery (P < 0.03), maximum running rate decreased significantly in LL for both D H T and CON groups (P < 0.01 and P < 0.02, respectively; paired t-tests). The two
188 groups did not differ with respect to maximum running rate in LL.
Body weight and foot measurements. During the process of perfusing the animals for histology, it was noted that the feet of several individuals had the appearance of being bigger than normal. Therefore, after perfusion, the front and back feet of the remaining animals (DHT 5; CON = 4) were measured and weighed (Table I). Feet of DHT animals tended to be both thicker and wider than those of CON animals. Combined foot
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weight, a measure that presumably reflects total foot size and density, was significantly greater for DHT animals despite the small sample sizes (t = 2.93, df = 7, P < 0.025). Body weight also tended to be greater for the 5 DHT animals, but was not significant (however, termir,a| body weights were available for 14/17 DHT and 7/8 CON ~nimals; the groups differed significantly: 178 +- 5 vs 155 -+- 7, t = 2.52, df = 19, P < 0.025). Footweight was highly correlated with body weight, r = 0.85.
groups were evaluated before surgery, after surgery and during days 15-20 of D D (Fig. 2B). Analysis of variance revealed a significant within groups effect (F = 24,95, df = 2,58, P < 0.001). As in Expt. 1, the DHT and CON groups did not differ either before or after surgery with respect to average wheel revolutions per 24 h (Fig. 2B). In contrast to the effects of LL, DD did not differentially suppress daily wheel revolutions. CON animals responded with an average 28.8 +-- 14.0% decline in daily revolutions compared to a 29.5 - 19.5% drop for the
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Fig. 1. Camera lucida drawings of representative dorsal raphe nuclei from the brains of (A) a control (CON)-treated hamster and (B) a 5,7-dihydroxytryptamine (DHT)-Iesioned hamster. Aq, cerebral aqueduct: DR, dorsal raphe; MLF, medial longitudinal fasciculus.
AFTER
DD
Fig, 2, Average daily wheel revolutions before and after surgical treatment under LD 14:10, and under constant lighting conditions. A: experiment 1; B: experiment 2, Open bars represent CON data; hatched bars represent DHT data, *groups differ with respect to percent change from the post surgical state, P < 0.03.
189
TABLE I
Body weight (SWt) and foot weight (FtWt), width (Wid) and thickness (Thn)) measures at the termination of Experiments I and 2 BWt
FtWt
FrontWid
FrontThk
HindWid
HindThk
180 --+ 6
2.11 +- 0.10 •
8.3 _ O.T
162.4- 10
1.76.4- 0.07
7.8 +- 0.1
5.2 - 0.2 c 4.9 + 0.1
7.7 -+ 0.2 '74 +_ 0.1
5.1 - 0.1 4.9 - 0.1
184 - 5b 154 - 4
2.56.4- 0 . 0 7 b 1.97 ± 0.06
Experiment 1 D l - r r (~, = 5)
CON (n = 4) Experiment 2 DHT (n = 19) CON (n = 11) ap < 0.025 and
bp
186 BRES 17221
Serotonergic modulation of the hamster wheelrunning rhythm: response to lighting conditions and food deprivation L.P. M o r i n a n d J. B l a n c h a r d Department of Psychiatry, State Universin.' of New York, Stony Brook, r~~. 11794 (U.S.A.) (Accepte-i i6 July i991)
Key words: 5,7-Dihydroxytryptamine; Body weight; Hamster; $erotonin; Light; Food deprivation; Wheelrunning; Environmental stimulus
Depletion of brain serotonin by 5,7-d~hydroxytryptamine (DHT) produces large changes in photic regulation of the hamster circadian running rhythm. This study documents th, changes in daily wheelrunning caused by DHT lesions and their relationship to changes in photic conditions o:" food avadabiiity. Hamsters were given bilateral infusions of the selective neurotoxin during entrainment to a light-dark cycle (LD) of 14:10 h. At a later time, anim; ~; were transferred to constant light (LL) or dark (DD) for a prolonged period. Animals in DD were also st,Jject to 3 days of f~od .ieprivati~n. Destruction of the serotonergic system does not change the amount of daily running in LD 14:10, but does alter the "~s¢ of runni~ ~ rol animals respond to LL by greatly decreasing running compared to those with lesions. Food deprivation, a condi~.,on that greatly elev ~,:s running in control animals, is not nearly as effective in lesioned animals. The results suggest tha~ serotonin-depletea hamsters have diminished responsiveness to environmental stimuli.
INTRODUCTION
The suprachiasmatic nuclei ($CN) contain one or more circadian ~locks controlling a large number of physiological and behavioral rhythms is. The hamster locomotor (wheelrunning) rhythm may be the most thoroughly studied ~irc~dian rhythm. Each such rhythm is characterized by its period, amplitude and phase. Amplitude has been le;s well studied than either rhythm phase or period contcol. Phase and period are timing properties of a rhythm and are thought to directly reflect correspondJ~ig rharacteristics of a central pacemaker (i.e., clock mecaanism) it. In contrast, the magnitude of any measured rhythm is not necessarily related to the amplit~de of the pacemaker. With a mechanical timepiece, a clock mechanism generates the period and controls the phase of the clock hands. Amplitude of the mechanical clock is purely a function of the length of the hands. Magnitude of the running rhythm is a secondary characteristic coupled to the circadian clock and adjustable through amplification (positively or negatively) of the circadian signal indicating rhythm phase. There is presently little understanding of the mammalian pacemaker mechanism and it is impossible to watch the hands of the clock change position or to measure their ;ength. Instead, investigators are required to mea-
sure repetitive changes in magnitude of an overt rhythm and assume that the timing of these changes reflect the underlyi~Ig pacemaker functions of period and phase. A classic example of this was provided by Richter t4 who anesthetized freerunning rats for several days. The magnitude of the circadian wheelrunning rhythm was reduced to zero for th~ duration of the anesthesia, but reappeared with the appropriate period, phase and magnitude as the anesthesia wore off. In a similar experiment, Schwartz et al. t6 infused tetrodotoxin onto the rat SCN. This also reduced magnitude of the running rhythm to zero, although the animals were awake and ran substantially throughout the duration of drug, infusion. Afterward, the rhythm resumed with the proper period, phase and magnitude. In each experiment, the animals became arrhythmic, but for quite different reasons. In the first case, all locomotor activity was abolished. In the second, circadian pacemaker mechanisms were dissociated from motor control systems. Thus, the magnitude of a measured rhythm is potentially modifiable at or near the central pacemaker as well as at sites along the path of motor output regulation. In contrast, period and phase must always be controlled through an action involving the pacemaker. We have previously shown that several lesion types modify the daily amount of hamster wheelrunning activ-
Correspondence: L.P. Morin, Department of Psychiatry, Health Sciences Center, SUNY, Stony Brook, NY 11794, U.S.A.
187 ity and that the most effective sites also disturb the circadian clock s. Hypothalamic lesions that damage the SNC will reduce running about 90%. Lesions elsewhere in the hypothalamus or thalamus can alter phase angle of entrainment or block phasic actions of light on the circadian period with smaller, or no, alterations in the amount of daily running 6.s'~°. The present data were collected during studies of the role played by serotonin in the regulation of the hamster circadian wheelrunning rhythm. They offcJ" an opportunity to indirectly evaluate the relationship between circadian rhythm regulation and magnitude of the running rhythm. MATERIALS AND METHODS Detailed methods are described in the companion paperm. Briefly, adult male golden hamsters (90-100 g) from Charle~ River/ Lakeview were individually caged under 14 h light/10 h dark (LD 14:10) conditions in a temperature controlled room (21 -+ 2 °C). After 3 weeks, each animal was transferred to a plastic cage containing a running wheel. Wheel revolutions were recorded per 5 rain interval (288 data 'bins' per day) by computer. Water and food (except when explicitly removed in Expt. 2) were continuously available. Light intensity under LD 14:10 conditions averaged 31 lux.
Experiment 1 Animals remained undisturbed for 3 weeks in LD 14:10 before receiving surgical treatment. All animals then remained in LD 14:10 for 4 additional weeks after which the lights in the animal room were continuously on (LL) for 85 days. The lights were then turned off continuously (DD) for 50 days and this was followed by a return to LD 14:10 for 14 days at which time each animal was anesthetized and peffused.
Experiment 2 The initial conditions were similar to those in Expt, 1 except that after LD 14:10, all animals were placed into DD that lasted abou~ 170 days. After 55 days in DD, all food was removed from each animal's cage. Deprivation began at 12,00 h and lasted 72 h. AboL~t 35 days later, all animals entered a paradigm for generating a phase response curve (PRC) to light. This procedure is described ~n detail elsewherem.
Surgery In both experiments, each animal was pre-treated with an intraperitoneai injection of desipramine hydrochloride (Sigma, 25 mg/kg in saline) about 30 min before being anesthetized with an intraperitoneal injection of sodium pentobarbital (10.6 m[~kg) during the light phase of the photoperiod and placed in a stercotaxic instrument. Animals received 5,7-dihydroxytryptamine creatinine sulfate (DHT; Sigma; 75/~g of free base in 2.5 t~l 0.5% ascorbic acid per lateral ventricle) or vehicle (CON group).
Histology At the conclusion of the experiments, each animal was deeply anesthetized and perfused9. Brains were removed, postfixed, cryoprotected in sucrose, then frozen and serially sectioned (30 #m) in the coronal plane. The avidin-biotin complex technique7 was used for serotonin immunohistochemistry with a 1:50 dilution of a monoclonal antibody to serotonin (Accurate). The sections were reacted using an ABC kit (Vector) with diaminobenzidine as the chromogen. Sections through the entire SCN or raphe nuclei of each animal were microscopically examined and evaluated. A digitizing imaging system (MCID) provided an index of relative optical density through the SCN and IGL. Cells in the dorsal and median ra-
phe nuclei were also counted using the digitizing ~ystem.
Statistics The CSS statistical package (StatSoft) was used. All probability values reported are twr~-tailed unless specifically stated otherwise. RESULTS
Histology Experiments 1 and 2. Optical density of serotonin immuncre.activity (5-HT-IR) in the SCN was significantly reduced by DHT treatment. Optical density of 5-HT-IR in the intergeniculate leaflet (IGL) was also significantly reduced by DHT treatment. In the dorsal raphe, cells with 5-HT-IR were reduced by about 90% in DHTtreated animals (Fig. 1). A more thorough presentation of the histological data is presented elsewhere t°.
Behavior--Experiment 1 Daily wheel revolutions during entrainment and LL. The daily activity levels of the treatment groups were evaluated before surge~, after surgery and during days 15-20 of LL (Fig. 2A). One I3AIT animal was not included in the analysis because it only averaged 6 revolutions/day (>8.5 S.D.'s from the mean) during the 5 days of LL. Analysis of variance revealed a significant within groups effect (F = 13.35, ,ff = 2.44, P < 0.001) and a group x treatment interaction (F = 3.42, P = 0.04). Under LD 14:10 conditions, the DHT and control (CON) groups did not differ either before or after ~,~ur~ery with respect to average wheel revolutions per 24 h. However, upon exposure to LL, CON animals responded with ar~ average 57.9 -+ 10,0% decline in daily revolutions compared to a 19.2 -. 11.5% drop for the D H T animals (t ffi 2.32, df ffi 22, P < 0.03). After 56 days in LL, CON animals ran an average 5626 - 1564 daily revolutions which did not differ significantly from the 3933 *'- 515 revolutions by the DHT group. A second measure of wheelrunning rate was also examined for each animal and the 5 min data bin containing the largest average number of revolutions was recorded as the maximum running rate. The results are shown in Fig. 3A. Analysis of variance revealed a significant between groups effect (df = 1,23, F = 6.12, P < 0.02) and a significant within groups effect (df = 2,44, F = 12.99, P < 0.001), but no interaction effect. Postsurgical maximum running rate in LD 14:10 was significantly greater for the CON group than the DHT group (P < 0.05, Newman-Keuls test; there were no other between group differences). Whereas only the DHT group showed a significant drop in maximum running rate after surgery (P < 0.03), maximum running rate decreased significantly in LL for both D H T and CON groups (P < 0.01 and P < 0.02, respectively; paired t-tests). The two
188 groups did not differ with respect to maximum running rate in LL.
Body weight and foot measurements. During the process of perfusing the animals for histology, it was noted that the feet of several individuals had the appearance of being bigger than normal. Therefore, after perfusion, the front and back feet of the remaining animals (DHT 5; CON = 4) were measured and weighed (Table I). Feet of DHT animals tended to be both thicker and wider than those of CON animals. Combined foot
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Behavior-- Experiment 2 Daily wheel revolutions in LD, DD and effect of food deprivation. The daily activity levels of the treatment
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weight, a measure that presumably reflects total foot size and density, was significantly greater for DHT animals despite the small sample sizes (t = 2.93, df = 7, P < 0.025). Body weight also tended to be greater for the 5 DHT animals, but was not significant (however, termir,a| body weights were available for 14/17 DHT and 7/8 CON ~nimals; the groups differed significantly: 178 +- 5 vs 155 -+- 7, t = 2.52, df = 19, P < 0.025). Footweight was highly correlated with body weight, r = 0.85.
groups were evaluated before surgery, after surgery and during days 15-20 of D D (Fig. 2B). Analysis of variance revealed a significant within groups effect (F = 24,95, df = 2,58, P < 0.001). As in Expt. 1, the DHT and CON groups did not differ either before or after surgery with respect to average wheel revolutions per 24 h (Fig. 2B). In contrast to the effects of LL, DD did not differentially suppress daily wheel revolutions. CON animals responded with an average 28.8 +-- 14.0% decline in daily revolutions compared to a 29.5 - 19.5% drop for the
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Fig. 1. Camera lucida drawings of representative dorsal raphe nuclei from the brains of (A) a control (CON)-treated hamster and (B) a 5,7-dihydroxytryptamine (DHT)-Iesioned hamster. Aq, cerebral aqueduct: DR, dorsal raphe; MLF, medial longitudinal fasciculus.
AFTER
DD
Fig, 2, Average daily wheel revolutions before and after surgical treatment under LD 14:10, and under constant lighting conditions. A: experiment 1; B: experiment 2, Open bars represent CON data; hatched bars represent DHT data, *groups differ with respect to percent change from the post surgical state, P < 0.03.
189
TABLE I
Body weight (SWt) and foot weight (FtWt), width (Wid) and thickness (Thn)) measures at the termination of Experiments I and 2 BWt
FtWt
FrontWid
FrontThk
HindWid
HindThk
180 --+ 6
2.11 +- 0.10 •
8.3 _ O.T
162.4- 10
1.76.4- 0.07
7.8 +- 0.1
5.2 - 0.2 c 4.9 + 0.1
7.7 -+ 0.2 '74 +_ 0.1
5.1 - 0.1 4.9 - 0.1
184 - 5b 154 - 4
2.56.4- 0 . 0 7 b 1.97 ± 0.06
Experiment 1 D l - r r (~, = 5)
CON (n = 4) Experiment 2 DHT (n = 19) CON (n = 11) ap < 0.025 and
bp
