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Serotonergic modulation of the hamster wheelrunning rhythm: response to lighting conditions and food deprivation
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.
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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
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184 - 5b 154 - 4
2.56.4- 0 . 0 7 b 1.97 ± 0.06
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DHT animals. There were no effects of treatment, repeated measure evaluation or interaction according to an analysis of variance of daily wheel revolutions. As in Expt. 1, the maximum running rate was calculated for each animal before treatment, after treatment and after 15 days of DD exposure. The results (Fig. 3B) are similar to those from Expt. 1. Analysis of variance revealed a significant between groups effect (dr = 1,29, F = 6.06, P < 0.02), a significant within groups effect (dr = 2,58, F ffi 29.8, e < 0.001) and an interaction effect ((if = 2,58, F = 15,25, P < 0.001). As in Expt. 1,
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Fig. 3. Maximum running rate before and after treatment 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 different, P < 0.05.
there was no pre-surgical difference in maximum running rate (231 -- 7 vs 245 ± 9 for CON and DHT, respectively), but post-surgical maximum running rate in LD 14:10 was significantly greater for the CON group (maximum per 5 min = 243 -- 10 revs) than the DHT group (maximum per 5 min = 191 ± 10 revs; P < 0.05, Newman-Keuls test). Unlike the results under LL, CON animals maintained a higher maximum rate during DD (maximum per 5 min = 210 ± 8) than did DHT animals (maximum per 5 min = 163 ± 8 revs; P < 0.05, Newman-Keuls test). The average daily wheel revolutions after 56 days in DD did not differ between D I E and CON groups (4866 ± 535 vs 5751 ± 721). Food deprivation acutely boosted the average daily revolutions for 18119 DHT animals and 11/11 CON animals. Nevertheless, the magnitude of the average increase was much less for DHT animals than for CON animals (mean increase: DHT = 3192 -- 646; CON = 9144 ± 1357; t ffi 4.52, df = 28, P < 0.001). Body weight and foot measurements. Each animal was weighed before perfusion and its 4 feet were weighed afterward. The body weights of DHT animals were significantly greater than those of CON animals (184 ± 5 vs 154 ± 4 g; t = 4.27, df = 28, P < 0.001). Similarly, total foot weight of DHT animals was greater than that for CON animals (2.57 .4- 0.07 vs 1.97 ± 0.05 g, df 28, P < 0.001). In general, foot weight was positively correlated with body weigh, acro3s all animals (r 0.55). However, such a correlation existed only for CON animals (r = 0.69, P < 0.02) and not for the DHT group (r = 0.07, ns). When the contribution of body weight to the foot weight measure was removed, relative foot weights of the two groups did not differ. Neither was there a significant correlation between the mean daily wheel revolutions during the last 5 days before perfu~ion and the DHT animals' foot weight (r = -0.04, ns). DISCUSSION Wheelrunning activity of hamsters is o n e of the most widely studied variables in the field of circadian rhythms.
190 Interest has focused on environmental, anatomical and humorai factors that modulate the frequency of the wheelrunning rhythm and facilitate its entrainment to a time-giving stimulus. Relatively little attention has been paid to the mechanisms regulating wheelrunning itself. Several investigators have shown that the circadian period of hamsters with wheel access differs from that of hamsters without wheel access 1'~2'~9 or that access to a wheel at a particular circadian phase can modify circadian rhythm expression ~3. In rats, the number of wheel revolutions per day appears to be inversely related to the circadian period, but directly related to the amount of serotonin in the SCN ~. Previously, we showed that general depletion of brain serotonin would cause marked alterations in the phase angle of entrainment and the duration of the daily activity phase. These changes occurred and remained stable without modification of the amount of daily wheelrunning ~°'~s. The present results support the previous observations: lesions of the serotonergic system do not modify the number of daily wheel revolutions under entrained conditions. The pattern of the running rhythm has been altered in DHT animals with the pre-lesion number of wheel revolutions becoming spread, after the lesion, across a nocturnal activity phase that is about 1.8 h longer 1°'is. The changed pattern is reflected by the fact that maximum running rate of lesioned animals under entrained conditions is lower than for CON animals. Under DD conditions, a similar phenomenon is observed in the DHT group which has a prolonged activity phase 10 combined with no change in average daily running, producing a decline in maximum running rate compared to that for CON animals (Fig, 3B). In contrast, the large drop in daily running by CON animals in LL reduces the maximum running rate (Fig. 3A) to a level comparable to that for DHT animals which are relatively unresponsive to the effects of LL on daily wheel revolutions. The circadian rhythm systems of LL-housed animals with DHT lesions appear to act 'hypernormar, as if they are extra-sensitive to the phasic effects of light t'. In contrast to this putative increase in sensitivity to light that is discerned from circadian perio~ and phase changes, analysis of the amount of running suggests exactly the opposite conclusion. CON hamste;s exposed to LL show an acute 58% decline in daily wheel revolutions compared to only 19% for DHT animals. At the same time, CON animals have shorter circadian periods in LL than serotonin depleted animals IU. The two sets of data again demonstrate the relative independence of the central control of wheelrunning and the control of circadian rhythmicity (cfrefs. 14 and 16). The fact that the longer circadian periods by serotonin depleted hamsters in LL are associated with relatively more wheelrunning could
also be construed as demonstrating a direct relationship between rhythm period and the magnitude of the running rhythm. If true, then the result is opposite to what is observed in rats 17. It is unlikely that there is a relationship between circadian period and amount of running by hamsters because acute food deprivation greatly increases daily wheel revolutions by DD-housed CON animals compared to DHT animals, but the r suiting changes in circadian period of the two groups are equal. In DD, the amount of daily running by both treatment groups decreased by a percentage roughly equal to that shown by the DHT group exposed to LL. Thus, it is not simply the presence of constant environmental conditions that causes the diminished running in CON animals. Rather, it is the 24 h presence of light. Prolonged exposure to either LL or DD is apparently associated with a general decrease in wheelrunning. DHT treated animals do not differ from CON animals after 56 days exposure to LL, and the number of wheel revolutions at that time is not substantially different from what is found after a similar number of days in DD. The present approach to understanding the role of serotonergic neurons as modulators of 'motivated' behavior suggests that novel research strategies are wa~anted. Loss of serotonin per se is not sufficient to alter properties of the hamster locomotor regulatory system. However, modest environmental challenges facilitate a clear distinction between lesioned and unlesioned animals, The two challenges evaluated in these studies were LL exposure and acute food deprivation, LL generally depresses wheelrunning, while food deprivation has the opposite effect, In each case, animals sustaining massive damage to the serotonergic system did not respond with nearly the same magnitude as CON animals. Food deprivation, a stimulus condition that is known to increase wheelrunning in both ratss and hamsters3, greatly elevated the number of daily wheel revolutions by CON animals (159% increase). There was a similar, but attenuated (66%), increase by the DHT group. This result is consistent with the hypothesis that depletion of serotonin reduces the behavioral responsiveness of the wheelrunning system to various environmental stimuli. Obese, hypoactive hamsters are restored to normal running levels following serotonin depletion4. That observation, and the present results from food deprived animals, may derive from a common feature. Hamsters lacking serotonin may be relatively unable to detect an internal signal related to management of metabolic fuels. The foregoing discussion must be tempered by the knowledge of prior work showing that food deprivation of DHT treated rats is associated with large increases in wheelrunningzz. Rats also show large increases in both nocturnal and diurnal wheelrunning following DHT
191 treatment 2°, although no information is available concerning shifts in phase angle of entrainment. It appears certain, however, ~hat total daily revolutions by rats are increased by DHT lesions. A major difficulty inherent in both the present study and those with rats 2°'22 concerns the location specificity of the lesions. The present experiments intentionally and effectively produced damage to the serotonergic system throughout the brain. In contrast, Waldbillig and colleagues aimed the bilateral DHT infusions at the ventrolateral hypothalamic area 2° or the anterior hypothalamic area 2L22 limiting damage to hippocampus, septum and, to a lesser extent, hypothalamus. The apparent contradiction between the present results and those for rats may be attributable to species differences or to differences between the several studies in extent, or location, of serotonergic system destruction. The observation that serotonin depleted animals had larger feet initially raised the question of whether the rhythm changes observed in LL-housed animals were possibly related to foot problems. The fact that animals housed in DD also had large feet indicates that such an interpretation is not correct. Food intake, body weight and adiposity of rats are known to increase after depletion of forebrain serotonin by DHT2t; hamsters also gain weight after generalized serotonin depletion TM. Foot measurements across the combined groups of animals in Expt, 1 were correlated with body weight. In Expt. 2, a good correlation between body weight and foot weight was also obtained. However, when the CON and DHT groups in Expt, 2 were examined separately, there was a strong correlation between foot weight and body weight for the CON animals, but not for the DHT group. An increase in body size predicts, in a normal animal, an appropriate increase in the size of the platform on which the body stands, While this prediction appears true for
CON animals, it is not true for animals sustaining DHTinduced depletions of brain serotonin. The data suggest that factors controlling foot size in DHT treated animals may be effective independent of body weight. At this time, it is not possible to ascertain a reason for the increased foot size of the experimental animals. We have previously suggested that neurons modulating phase of the circadian running rhythm project through the medial forebrain bundle 6'~° and that these projections are not involved in the regulation of the amount of wheelrunning. Proximity to the SCN appears to be a major factor concerning whether or not a lesion will reduce the amount of daily wheelrunnings. Lesions causing the greatest decrease in wheelrunning actually destroy parts of the SCN itself. The major loss of 5-HT-IR cells from the dorsal raphe apparently has no impact on the amount of running. Therefore, it is unlikely that projections to the SCN from this nucleus regulate the running reductions seen after the peri-SCN damage. Numerous reports have demonstrated that lesions of the median raphe induce hyperactivity in rats (see ref. 2 for references). However, this phenomenon may be more related to destruction of non-serotonergic cells and fibers of passage than to loss of serotonergic transmitter systems2. In summary, the present results show stimulus-specific changes (either increases or decreases) in the amount of daily wheelrunning by CONtreated, but not by DHT-lesioned, hamsters. This effect appears to be the result of central serotonin depletion, but further research is necessary to identify the specific projections mediating stimulus-specific modulation of wheelrunning activity.
REFERENCES
6 Goodless-Sanchez, N., Moore, R.Y. and Morin, L.P., Lateral hypothalamic regulation of hamster circadian rhythm phase, Physiol. Behav., 49 (1991) 533-537. 7 Hsu, S.-M., Raine, L. and Fanger, H., Use of avidin-biotin-perioxidase com?lex (ABC) in immunoperioxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem., 29 (1981) 577-580. 8 Johnson, R.E, Moore, R.Y. and Morin, L.P., Running wheel activity in hamsters with hypothalamic damage, Physiol. Be. hay., 43 (1988) 755-763. 9 McLean, I.W. and Nakane, P.K., Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy, J. Histochem. Cytochem., 22 (1974) 1077-1083. I0 Morin, L.P. and Bianchard, J.H., Depletion of brain serotonin by 5,7-DHT modifies hamster circadian rhythm response to light, Brain Research, 566 (1991) 173-185. 11 Pittendrigh, C.S. and Daan, S., A functional analysis of circadian pacemakels in nocturnal rodents. I. The stability and lability of spontaneous frequency, J. Comp. Physiol., 106 (1976) 223-252.
1 Aschoff, J., Figala, J. and Poppel, E., Circadian rhythms of locomotor activity in the golden hamster (Mesocricetus auratus) measured with two different techniques, J. Comp. Physiol. Psy. choL, 85 (1973) 20-28. 2 Asin, K.E. and Fibiger, H.C., An analysisof neuronal elements within the median nucleus of the raphe that mediate lesion-induced increases in locomotor activity, Brain Research, 268 (1983) 211-223. 3 Borer, K.T., The nonhomeostatic motivation to run. In A.R. Morrison ~nclP L. Strick (Eds.), Changing Concepts of the Ner. vous System, Academic Press, New York, 1982, pp. 539-567. 4 Borer, K.T., Bonna, R. and Kielb, M., Hippocampal serotonin mediates hypoactivity in dietarily obese hamsters: a possible manifestation of aging?, Pharmacol. Biochem. Behav., 31 (1988) 885-892. 5 Collier, G. and Hirsch, E., Reinforcingproperties of spontaneous activity in the rat, J. Comp. Physiol. PsychoL, 77 (1971) 155-160.
Acknowledgements. Supported by research Grant NS22168. We are grateful to A. Borel;a for excellent technical assistance.
192 12 Pratt, B.L. and Goldman, B.D., Environmental influences upon circadian periodicity of Syrian hamsters, Physiol. Behav., 36 (1986) 91-.95. 13 Reebs, S.G. and Mrosovsky, N., Effects of induced wheel running on the circadian activity rhythms of Syrian hamsters: entrainment and phase response curve, J. Biol. Rhythms, 4 (1989) 39-48. 14 Richter, C.P., Sleep and activity: their relation to the 24-h clock, Proc. Assoc. Res. Nerv. Meat. Dis., 45 (1967) 8-27. 15 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 16 Schwartz, W.J., Gross, R.A. and Morton, M.T., The suprachiasmatic nuclei contain a tetrodotoxin-resistant circadian pacemaker, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 1694-1698. 17 Shioiri, T., Takahashi, K., Yamada, N. and Takahashi, S., Motor activity correlates negatively with free-running period, while positively with serotonin contents in SCN in free-running rats, Physiol. Behav., 49 (1991) 779-786. 18 Smale. L.. Micheis, K.M., Moore, R.Y. and Morin, L.P., Destruction of the hamster serotonergic system by 5,7-DHT: ef-
fects on circadian rhythm phase, entrainment and response to triazolam, Brain Research, 515 (1990) 9-19. 19 Turek, F.W., Effects of stimulated physical activity on the circadian pacemaker of vertebrates, l. Biol. Rhythms, 4 (1989) 135-147. 20 Waldbillig, R.J., Bartness, T.J. and Stanley, B.G., Disproportionate increases in locomotor activity in response to hormonal and photic stimuli following regional neurochemical depletions of serotonin, Brain Research, 217 (1981) 79-91. 21 Waldbillig, R.J., Bartness, T.J. and Stanley, B.G., Increased food intake, body weight, and adiposity in rats after regional neurochemical depletion of serotonin, J. Comp. Physiol. Psychol., 95 (1981) 391-405. 22 Waldbillig, R.J., Welder, G.E., Clemmons, R.M. and Bartness, T.J., Rostral hypothalamic microinfusions of 5,7-dihydroxytryptamine produce anatomically and neurochemically selective depletions of hippocampal serotonin and increase the influence of estrogen and food deprivation on locomotor activity, Brain Re. search, 347 (1985) 149-153.
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.
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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
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DHT animals. There were no effects of treatment, repeated measure evaluation or interaction according to an analysis of variance of daily wheel revolutions. As in Expt. 1, the maximum running rate was calculated for each animal before treatment, after treatment and after 15 days of DD exposure. The results (Fig. 3B) are similar to those from Expt. 1. Analysis of variance revealed a significant between groups effect (dr = 1,29, F = 6.06, P < 0.02), a significant within groups effect (dr = 2,58, F ffi 29.8, e < 0.001) and an interaction effect ((if = 2,58, F = 15,25, P < 0.001). As in Expt. 1,
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there was no pre-surgical difference in maximum running rate (231 -- 7 vs 245 ± 9 for CON and DHT, respectively), but post-surgical maximum running rate in LD 14:10 was significantly greater for the CON group (maximum per 5 min = 243 -- 10 revs) than the DHT group (maximum per 5 min = 191 ± 10 revs; P < 0.05, Newman-Keuls test). Unlike the results under LL, CON animals maintained a higher maximum rate during DD (maximum per 5 min = 210 ± 8) than did DHT animals (maximum per 5 min = 163 ± 8 revs; P < 0.05, Newman-Keuls test). The average daily wheel revolutions after 56 days in DD did not differ between D I E and CON groups (4866 ± 535 vs 5751 ± 721). Food deprivation acutely boosted the average daily revolutions for 18119 DHT animals and 11/11 CON animals. Nevertheless, the magnitude of the average increase was much less for DHT animals than for CON animals (mean increase: DHT = 3192 -- 646; CON = 9144 ± 1357; t ffi 4.52, df = 28, P < 0.001). Body weight and foot measurements. Each animal was weighed before perfusion and its 4 feet were weighed afterward. The body weights of DHT animals were significantly greater than those of CON animals (184 ± 5 vs 154 ± 4 g; t = 4.27, df = 28, P < 0.001). Similarly, total foot weight of DHT animals was greater than that for CON animals (2.57 .4- 0.07 vs 1.97 ± 0.05 g, df 28, P < 0.001). In general, foot weight was positively correlated with body weigh, acro3s all animals (r 0.55). However, such a correlation existed only for CON animals (r = 0.69, P < 0.02) and not for the DHT group (r = 0.07, ns). When the contribution of body weight to the foot weight measure was removed, relative foot weights of the two groups did not differ. Neither was there a significant correlation between the mean daily wheel revolutions during the last 5 days before perfu~ion and the DHT animals' foot weight (r = -0.04, ns). DISCUSSION Wheelrunning activity of hamsters is o n e of the most widely studied variables in the field of circadian rhythms.
190 Interest has focused on environmental, anatomical and humorai factors that modulate the frequency of the wheelrunning rhythm and facilitate its entrainment to a time-giving stimulus. Relatively little attention has been paid to the mechanisms regulating wheelrunning itself. Several investigators have shown that the circadian period of hamsters with wheel access differs from that of hamsters without wheel access 1'~2'~9 or that access to a wheel at a particular circadian phase can modify circadian rhythm expression ~3. In rats, the number of wheel revolutions per day appears to be inversely related to the circadian period, but directly related to the amount of serotonin in the SCN ~. Previously, we showed that general depletion of brain serotonin would cause marked alterations in the phase angle of entrainment and the duration of the daily activity phase. These changes occurred and remained stable without modification of the amount of daily wheelrunning ~°'~s. The present results support the previous observations: lesions of the serotonergic system do not modify the number of daily wheel revolutions under entrained conditions. The pattern of the running rhythm has been altered in DHT animals with the pre-lesion number of wheel revolutions becoming spread, after the lesion, across a nocturnal activity phase that is about 1.8 h longer 1°'is. The changed pattern is reflected by the fact that maximum running rate of lesioned animals under entrained conditions is lower than for CON animals. Under DD conditions, a similar phenomenon is observed in the DHT group which has a prolonged activity phase 10 combined with no change in average daily running, producing a decline in maximum running rate compared to that for CON animals (Fig, 3B). In contrast, the large drop in daily running by CON animals in LL reduces the maximum running rate (Fig. 3A) to a level comparable to that for DHT animals which are relatively unresponsive to the effects of LL on daily wheel revolutions. The circadian rhythm systems of LL-housed animals with DHT lesions appear to act 'hypernormar, as if they are extra-sensitive to the phasic effects of light t'. In contrast to this putative increase in sensitivity to light that is discerned from circadian perio~ and phase changes, analysis of the amount of running suggests exactly the opposite conclusion. CON hamste;s exposed to LL show an acute 58% decline in daily wheel revolutions compared to only 19% for DHT animals. At the same time, CON animals have shorter circadian periods in LL than serotonin depleted animals IU. The two sets of data again demonstrate the relative independence of the central control of wheelrunning and the control of circadian rhythmicity (cfrefs. 14 and 16). The fact that the longer circadian periods by serotonin depleted hamsters in LL are associated with relatively more wheelrunning could
also be construed as demonstrating a direct relationship between rhythm period and the magnitude of the running rhythm. If true, then the result is opposite to what is observed in rats 17. It is unlikely that there is a relationship between circadian period and amount of running by hamsters because acute food deprivation greatly increases daily wheel revolutions by DD-housed CON animals compared to DHT animals, but the r suiting changes in circadian period of the two groups are equal. In DD, the amount of daily running by both treatment groups decreased by a percentage roughly equal to that shown by the DHT group exposed to LL. Thus, it is not simply the presence of constant environmental conditions that causes the diminished running in CON animals. Rather, it is the 24 h presence of light. Prolonged exposure to either LL or DD is apparently associated with a general decrease in wheelrunning. DHT treated animals do not differ from CON animals after 56 days exposure to LL, and the number of wheel revolutions at that time is not substantially different from what is found after a similar number of days in DD. The present approach to understanding the role of serotonergic neurons as modulators of 'motivated' behavior suggests that novel research strategies are wa~anted. Loss of serotonin per se is not sufficient to alter properties of the hamster locomotor regulatory system. However, modest environmental challenges facilitate a clear distinction between lesioned and unlesioned animals, The two challenges evaluated in these studies were LL exposure and acute food deprivation, LL generally depresses wheelrunning, while food deprivation has the opposite effect, In each case, animals sustaining massive damage to the serotonergic system did not respond with nearly the same magnitude as CON animals. Food deprivation, a stimulus condition that is known to increase wheelrunning in both ratss and hamsters3, greatly elevated the number of daily wheel revolutions by CON animals (159% increase). There was a similar, but attenuated (66%), increase by the DHT group. This result is consistent with the hypothesis that depletion of serotonin reduces the behavioral responsiveness of the wheelrunning system to various environmental stimuli. Obese, hypoactive hamsters are restored to normal running levels following serotonin depletion4. That observation, and the present results from food deprived animals, may derive from a common feature. Hamsters lacking serotonin may be relatively unable to detect an internal signal related to management of metabolic fuels. The foregoing discussion must be tempered by the knowledge of prior work showing that food deprivation of DHT treated rats is associated with large increases in wheelrunningzz. Rats also show large increases in both nocturnal and diurnal wheelrunning following DHT
191 treatment 2°, although no information is available concerning shifts in phase angle of entrainment. It appears certain, however, ~hat total daily revolutions by rats are increased by DHT lesions. A major difficulty inherent in both the present study and those with rats 2°'22 concerns the location specificity of the lesions. The present experiments intentionally and effectively produced damage to the serotonergic system throughout the brain. In contrast, Waldbillig and colleagues aimed the bilateral DHT infusions at the ventrolateral hypothalamic area 2° or the anterior hypothalamic area 2L22 limiting damage to hippocampus, septum and, to a lesser extent, hypothalamus. The apparent contradiction between the present results and those for rats may be attributable to species differences or to differences between the several studies in extent, or location, of serotonergic system destruction. The observation that serotonin depleted animals had larger feet initially raised the question of whether the rhythm changes observed in LL-housed animals were possibly related to foot problems. The fact that animals housed in DD also had large feet indicates that such an interpretation is not correct. Food intake, body weight and adiposity of rats are known to increase after depletion of forebrain serotonin by DHT2t; hamsters also gain weight after generalized serotonin depletion TM. Foot measurements across the combined groups of animals in Expt, 1 were correlated with body weight. In Expt. 2, a good correlation between body weight and foot weight was also obtained. However, when the CON and DHT groups in Expt, 2 were examined separately, there was a strong correlation between foot weight and body weight for the CON animals, but not for the DHT group. An increase in body size predicts, in a normal animal, an appropriate increase in the size of the platform on which the body stands, While this prediction appears true for
CON animals, it is not true for animals sustaining DHTinduced depletions of brain serotonin. The data suggest that factors controlling foot size in DHT treated animals may be effective independent of body weight. At this time, it is not possible to ascertain a reason for the increased foot size of the experimental animals. We have previously suggested that neurons modulating phase of the circadian running rhythm project through the medial forebrain bundle 6'~° and that these projections are not involved in the regulation of the amount of wheelrunning. Proximity to the SCN appears to be a major factor concerning whether or not a lesion will reduce the amount of daily wheelrunnings. Lesions causing the greatest decrease in wheelrunning actually destroy parts of the SCN itself. The major loss of 5-HT-IR cells from the dorsal raphe apparently has no impact on the amount of running. Therefore, it is unlikely that projections to the SCN from this nucleus regulate the running reductions seen after the peri-SCN damage. Numerous reports have demonstrated that lesions of the median raphe induce hyperactivity in rats (see ref. 2 for references). However, this phenomenon may be more related to destruction of non-serotonergic cells and fibers of passage than to loss of serotonergic transmitter systems2. In summary, the present results show stimulus-specific changes (either increases or decreases) in the amount of daily wheelrunning by CONtreated, but not by DHT-lesioned, hamsters. This effect appears to be the result of central serotonin depletion, but further research is necessary to identify the specific projections mediating stimulus-specific modulation of wheelrunning activity.
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Acknowledgements. Supported by research Grant NS22168. We are grateful to A. Borel;a for excellent technical assistance.
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