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Circadian rhythm of drinking and running-wheel activity in rats with 6-hydroxydopamine lesions of the ventral tegmental area
Brain Research 899 (2001) 187–192 www.elsevier.com / locate / bres
Research report
Circadian rhythm of drinking and running-wheel activity in rats with 6-hydroxydopamine lesions of the ventral tegmental area Yoshiaki Isobe*, Hitoo Nishino Department of Physiology, Nagoya City University Medical School, Mizuho-ku, Nagoya 467 -8601, Japan Accepted 30 January 2001
Abstract Circadian rhythms in drinking and running-wheel (locomotor) activity of rats with 6-hydroxydopamine (6-OHDA, 4 mg / 2 ml per rat)-induced lesions in the ventral tegmental area (VTA) were examined under a light–dark (LD) cycle and constant dim light (5 lux). Under the LD cycle, the length of the locomotor activity period was decreased during the dark, and increased during the light period in the lesioned rats. Under the constant dim light conditions, the free-running circadian period (t) of drinking and activity rhythm was longer in lesioned rats than in sham-operated controls. The elongation of the circadian period was accompanied by decrements in activity. These observations suggest that the mesolimbic dopaminergic system modulates rhythms in circadian drinking and locomotor activity. 2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Circadian rhythm; Locomotor activity; Drinking; Ventral tegmental area; 6-Hydroxydopamine
1. Introduction The suprachiasmatic nucleus (SCN) is important for control of circadian rhythm in mammals. The neuronal network of a putative SCN pacemaker with the nucleus accumbens (N. Acc) might modify the circadian locomotor activity rhythms in rats [2]. The N. Acc receives dopaminergic afferent input from the ventral tegmental area (VTA) [17]. Mesolimbic neuronal alterations contribute to behavioral motivation [23]. Experiments using bright light lesioning of the retina have suggested that the VTA is connected to retinal input in Sprague–Dawley rats [11]. The integration of circadian and visual signals modulates hypothalamic dopamine neurons whose activity is also affected by the VTA [10]. The lesions of ventral mesencephalic tegmentum caused disturbances of spontaneous activity, while modifications of the circadian rhythms was *Corresponding author. Tel.: 181-52-853-8136; fax: 181-52-8423069. E-mail address: [email protected] (Y. Isobe).
not induced [6]. However, the role played by midbrain VTA in the generation and entrainment of the circadian rhythm has not been defined. VTA lesions induced by high doses (4 mg / rat) of 6-hydroxydopamine (6-OHDA) decreased the activity [3,12,17], and lower doses (2 mg / rat) of 6-OHDA [17,21] and high-frequency lesions [28] increased the activity levels. A short-term increase in locomotor activity occurs when the circadian activity rhythm of hamsters was phase-shifted by light pulse [1]. After a triazolam injection, the increase of locomotor activity accompanies a phase shift [29]. Behavioral events trigger a phase advance during the subjective day, when the ‘clock’ is insensitive to light [19]. Non-photic resetting has the opposite effect to light-pulse induced phase shift [19,24]. These findings indicate that the phase shift of the circadian activity rhythm is coupled with the activity level. The free-running circadian period is negatively correlated with motor activity level [27], although the correlation between the circadian rhythm period and locomotor activity levels is not always evident in hamsters [20].
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02223-5
Y. Isobe, H. Nishino / Brain Research 899 (2001) 187 – 192
188
The present study was undertaken to determine whether VTA chemical lesioning (6-OHDA) would affect wheelactivity levels and modulate circadian drinking and wheelactivity rhythms.
2. Materials and methods
2.1. Animals Thirty-one male Wistar rats, bred in our animal room, were used. Rats were housed in plexiglass cages, maintained under a 12:12-h light–dark cycle (LD cycle, lights on at 07:00 h) at a room temperature of 25618C. Illumination intensity at the light period was 150 lux produced by a fluorescent lamp and 5 lux in the dark produced by an incandescent bulb. The rats were provided with food and water ad libitum.
2.2. Rhythms of running-wheel activity and drinking activity Each rat was maintained in a cage with an attached side running wheel (Natume, Japan). Each revolution of the running wheel was recorded by an event recorder (Fuji Electric, Japan) with a digital recorder simultaneously (Shimadzu, Japan). The wheel cage was set in a temperature-controlled room at 25618C with the same lighting conditions as in the animal room. Drinking was monitored with a drop counter attached to the water bottle, which was connected to the event recorder with digital recorder (same apparatus used for recording running-wheel activity). The measurements of wheel activity and drinking rhythm were analyzed over 0.5- or 1-h intervals, and these rhythms are represented as actograms by the double plot method, in which the actograms of succeeding 2 days were arranged side by side [9,13,14]. In comparing the 24-h variations of wheel activity under the LD cycle, the eight lesioned rats and eight age-matched sham-operated control rats were measured at the fifth week after the operation. After 3 days setting to the wheel in each rat, we started to record the running-wheel activity. Amounts of wheel revolutions during periods of 30 min were counted using a cumulative recorder. These rats were sighted. To compare the wheel-activity level and circadian rhythmicity in lesioned and sham-operated animals, drink-
ing and running-wheel activity rhythms were simultaneously measured under conditions of constant dim light in intact (sighted, n59) and blinded (n56) rats (see Table 1). When stable free-running rhythms of drinking and wheel activity were evident, the rats received a microinjection of 6-OHDA in bilateral VTA. Six sighted and three blinded rats were lesioned, and three rats from each group received the sham injection (saline only) around the eighth week of age. The operation was performed during the circadian time (CT; CT 0 corresponds to lights on) of CT 4 to CT 6 h. These time points correspond to the dead zone (no phase shift) of the phase response curve for light pulse. The circadian period (t) was estimated by autocorrelation analysis and periodogram analysis with the slope on eyefitted line of onset of activity inactogram. Uncertainty of the estimated t was less than 60.02 h. Blinding was performed by bilateral ocular enucleation on the day of birth. Closed eyelids were opened with a razor and the eyeballs were removed under hypothermic anesthesia [8].
2.3. 6 -OHDA lesion 6-OHDA (Sigma, Japan) was dissolved in sterile isotonic saline, containing 0.1% ascorbic acid, at a concentration of 2 mg / ml. 6-OHDA (2 mg / 1 ml) was injected stereotaxically into the bilateral VTA at around 8 weeks of age, taking 1 min under Na-pentobarbital (5 mg / 100 g) anesthesia, through a 30-gauge stainless-steel needle. The injection needle was lowered according to the following coordinates in rats (body weight 260 g): 5.2 mm posterior to bregma, 0.4 mm (both side) laterally from the midline and 8 mm from the cerebral surface. The incisor bar was set 2.5 mm below the interaural line. Sham-operated controls received bilateral injections of 1 ml of saline.
2.4. Histology Tyrosine hydroxylase (TH) immunohistochemical staining was performed after the experiments to establish the extent of the lesion in the VTA. Briefly, anesthetized animals were perfused through the heart, firstly with saline then with modified Zamboni’s fixative solution (0.2% picric acid, 0.05% glutaraldehyde, 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.5). Brains were removed and stored in phosphate buffer containing 10% sucrose for 1 h, then 20% sucrose overnight at 48C. Sections (50 mm thick) were cut from relevant areas for immunohistochemi-
Table 1 Lengthening of circadian period in running-wheel activity and drinking rhythms after the ventral tegmental area 6-OHDA lesion a 6-OHDA
Sighted Wheel activity
Drinking
Wheel activity
Drinking
Pre Post
24.4960.10 (4) 24.6460.09 (4)*
24.5260.12 (4) 24.7160.06 (4)*
23.5760.04 (3) 23.7060.10 (3)*
23.5660.06 (3) 23.6560.07 (3)
a
Blinded
The number of rats is indicated in parentheses. *Two-way ANOVA, P,0.05.
Y. Isobe, H. Nishino / Brain Research 899 (2001) 187 – 192
cal staining using anti-TH antibody and the PAP (ICN Biochemicals, USA) method [12].
189
creased in lesioned animals. 6-OHDA injection into the VTA produced a complete loss of TH-positive cell bodies in the A10 area in seven cases in eight animals.
2.5. Statistics To evaluate the statistical differences in groups, analysis of variance (ANOVA) was used. To compare the circadian period of pre- and post-lesion, two-way ANOVA was used. The differences were considered to be significant at P, 0.05.
3. Results
3.1. Wheel activity changes under the LD cycle Twenty-four-hour variations of wheel activity measured under the LD cycle are shown in Fig. 1. The wheel activities of lesioned and sham-operated rats were 2186104 (Mean6S.D.) and 3596172 turns per 24 h, respectively. Significant hypoactivity was thus observed in the lesioned rats (P,0.05). The amount of wheel activity during the dark period in the lesioned rats was reduced to 61% that of sham-operated rats. The wheel activity during the light period just after lights on was increased in the lesioned rats. Synchronization to the LD cycle was de-
3.2. Wheel activity and drinking rhythms during free running The results of simultaneously measured free-running rhythms of drinking and running-wheel activity in the sighted and blinded rats are shown in Fig. 2. In the sham-operated rats, no marked changes in the patterns of drinking or wheel-activity rhythms, nor in their daily amounts of intake or wheel activity were recognized after the sham operation. In the lesioned rats, however, the circadian periods (t) of both drinking and wheel-activity rhythms were elongated after the VTA lesion compared with that before injection of 6-OHDA (Table 1). The average circadian period (t) of sighted rats during the running-wheel activity rhythm determined by periodogram analysis was 24.4960.10 h (Mean6S.D.) and 24.6460.09 h before and after the lesion, respectively (n54) (Table 1). The t of the drinking rhythm was 24.5260.12 h and 24.7160.06 h prior to and post-lesion, respectively (n54). In the blinded rats, free-running periods of drinking and wheel-activity rhythm were calculated in three lesioned and three sham-operated rats (Table 1). The pre- and post-lesion in circadian period (t) of wheel-activity rhythm were 23.5760.04 h (Mean6S.D.) and 23.7060.10 h, respectively (Table 1). The period was significantly longer after the lesion when compared with before the operation using the Spearman’s rank correlation test (P,0.05). The circadian period of drinking rhythm was elongated after the VTA lesion, although it was not significant. Blinding caused shortening (,24 h) of the periods of both drinking and wheel-activity rhythms, as is well known. Circadian rhythms in rats kept under the constant dim light and blinded rats did not show a similar circadian period, as shown in our previous report [14]. In the sham-operated rats, shortening of the period after the operation was not significant (Fig. 2, Sight-Cont. lower one, and BlindCont.), reflected by the large inter-individual variations. The circadian drinking rhythm was similar in shape to the wheel-activity rhythm. The VTA lesion did not induce characteristic changes in the amounts of drinking (Fig. 2). Although the drinking and wheel-activity rhythms were different in phase, these rhythms were well synchronized (Fig. 2).
4. Discussion Fig. 1. Circadian running-wheel activity rhythms of sham-operated (upper frame) and 6-OHDA lesioned (lower frame) rats at the fifth week after the operation. In both groups (n58), the mean6S.D. of the amounts of activity measured during each 30-min period are shown. The horizontal dark bar indicates the dark period in the LD cycle.
Free-running circadian periods (ts) of drinking and wheel-activity rhythms were lengthened by the VTA 6OHDA lesion both in blinded and sighted groups. No phase shift of the circadian rhythm by 6-OHDA or saline administration into the VTA under pentobarbital anesthesia
190
Y. Isobe, H. Nishino / Brain Research 899 (2001) 187 – 192
Fig. 2. Actograms of drinking and running-wheel activity rhythms measured under constant dim light (5 lux) on the sham-operated (Cont.) and VTA-lesioned (Lesion) rats that was sighted (Sight) or blinded (Blind); e.g. Blind-Cont. indicates the sham-operated blinded rat. In each figure, the upper and lower frames show the drinking and running-wheel activity rhythms, respectively. The operation was performed on the day marked by the arrow. The daily amounts of wheel activity are shown in each actogram. Time of day and the amounts of wheel activity or drinking were similar to those shown in the schematic inset in the lower left corner.
was observed in either lesioned or sham-operated rats. These findings indicate that neither 6-OHDA nor saline, administered into the VTA during the circadian time 4–6 h, induces the phase shift at this time zone (dead zone). However, we have not excluded the possibility of a phasedependent variation of locomotor activity by 6-OHDA, since synergistic behavioral effects of dopamine D1 and D2 agonists were reported to be different at subjective day and night [18]. Hypoactivity caused by the 6-OHDA for a dose of 4 mg in each rat was found (Figs. 1 and 2). The present findings confirm those of previous studies where larger doses of 6-OHDA (.4 mg per rat)-induced hypoactivity [3,17]. The decrease in locomotor activity following the VTA lesion is suggested to be related to the dopamine release from the N. Acc. which is a center of motivation of movement [17,23]. An animal’s ‘level of excitement’ and ‘spontaneous
circadian period’ are related [1]. The period of the wheelactivity rhythm tended to be longer in hamsters housed with a running wheel than in those without a wheel [29]. The free-running circadian period is correlated negatively with motor activity level [27]. These phenomena indicate that decrements in the amount of activity are related to the elongation of the circadian period. The circadian rhythm of drinking showed similar rhythm with wheel activity in shape. It was reported that bilateral 6-OHDA lesions of substantia nigra caused aphasia and adipsia [25]. Compared with the substantia nigra, the VTA lesion did not induce characteristic changes in the amounts of drinking (Fig. 2). Although the mechanism is not clear, the onset of drinking rhythm was delayed 1 h with that of running-wheel activity rhythm in both blinded and sighted rats. Studies of midbrain dopamine neurons in vivo and in
Y. Isobe, H. Nishino / Brain Research 899 (2001) 187 – 192
vitro have demonstrated that A10 cells (VTA) fire regularly (self-oscillation), and also fire in the absence of synaptic input [7,26]. This self-oscillation is an important factor for a pacemaker. Although the firing rate is different from that of SCN, the VTA might be one of the candidates for extra-SCN pacemaker modulating systems in circadian rhythm generation. Methamphetamine induces an increase in dopamine release accompanied by increments in locomotor activity [9,12]. Daily administration of methamphetamine activates an endogenous oscillator(s) and evokes free-running circadian rhythms of activity, corticosterone and body temperature in SCN-lesioned rats [9]. The body temperature rhythms measured under the LD cycle did not show marked changes after the VTA-6-OHDA lesions (data not shown). Tyrosine hydroxylase-positive cells (catecholaminergic neurons) innervate the SCN and these neurons mainly lack vasoactive intestinal peptide [16]. Vasoactive intestinal peptide, which is contained in neurons localized in the ventrolateral area of the SCN that receive retinal input, have an important role in the entrainment of circadian rhythm [5]. It could be suggested that catecholaminergic input to the SCN, including the mesolimbic dopaminergic system, functions independently of the retinal input to the SCN. Corticosterone receptor was recognized in VTA [22]. Corticosterone treatment decreased levels of glucocorticoid receptor immunoreactivity in VTA of rat [22]. The dopamine-lesioned rats exhibited a lower basal corticosterone secretion than sham-lesioned animals [4]. Intraventricular injection of 6-OHDA induced a loss of circadian corticosterone rhythm under constant light conditions [8]. These results indicate that the dopaminergic system (VTA) may have a stimulatory influence on the hypothalamopituitary-adrenal axis. The attenuated functions of corticosterone might influence the circadian rhythm [15]. In conclusion, the mesolimbic dopaminergic system arising from VTA may modulate the circadian rhythms of wheel activity and drinking in SCN intact rats.
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[5] S. Daikoku, S. Hisano, Y. Kagotani, R. Yokote, H. Kawano, Peptidergic neurons in the suprachiasmatic nucleus in rats. Development and functional properties, in: H. Nakagawa, Y. Oomura, K. Nagai (Eds.), New Functional Aspects of the Suprachiasmatic Nucleus of the Hypothalamus, John Libbey, London, 1993, pp. 53–60. [6] O. Gaffori, M. Le Moal, L. Stinus, Locomotor hyperactivity and hypoexploration after lesion of the dopaminergic-A10 area in the ventral mesencephalic tegmentum (VMT) of rats, Behav. Brain Res. 1 (1980) 313–329. [7] J. Grenhoff, L. Ugedo, T.H. Svensson, Firing patterns of midbrain dopamine neurons: differences between A9 and A10 cells, Acta Physiol. Scand. 134 (1988) 127–132. [8] K. Honma, T. Hiroshige, Participation of brain catecholaminergic neurons in a self-sustained circadian oscillation of plasma corticosterone in the rat, Brain Res. 169 (1979) 519–529. [9] S. Honma, K. Honma, T. Shirakawa, T. Hiroshige, Rhythms in behaviors, body temperature and plasma corticosterone in SCN lesioned rats given methamphetamine, Physiol. Behav. 44 (1988) 247–255. [10] T.L. Horvath, An alternate pathway for visual signal integration into the hypothalamo-pituitary axis: retinorecipient intergeniculate neurons project to various regions of the hypothalamus and innervate neuroendocrine cells including those producing dopamine, J. Neurosci. 18 (1998) 1546–1558. [11] C. Humpel, A. Saria, Retinal lesion by bright artificial light increases vasoactive intestinal polypeptide in the rat ventral tegmental area / substantia nigra, Neurosci. Lett. 130 (1991) 92–94. [12] Y. Ishida, T. Hashitani, M. Kumazaki, T. Ikeda, H. Nishino, Behavioral and biochemical effects of intra-accumbens dopaminergic grafts, Brain Res. Bull. 24 (1990) 487–492. [13] Y. Isobe, K. Aoki, F. Furuyama, T. Hashitani, Effects of continuous light on the circadian rhythms of running-wheel activity and body temperature in spontaneously hypertensive rats, Med. Principles Pract. 2 (1991) 138–147. [14] Y. Isobe, Development of body temperature rhythms in blinded and intact rats nursed under a light–dark cycle or in constant darkness, Biol. Rhythm Res. 28 (Suppl.) (1997) 5–18. [15] Y. Isobe, M. Isobe, Circadian rhythm of Arg-vasopressin contents in the suprachiasmatic nucleus in relation to corticosterone, Brain Res. 800 (1998) 78–85. [16] H. Jacomy, O. Bosler, Catecholaminergic innervation of the suprachiasmatic nucleus in the adult rat: ultrastructural relationships with neurons containing vasoactive intestinal peptide or vasopressin, Cell Tissue Res. 280 (1995) 87–96. [17] G.F. Koob, L. Stinus, M. Le Moal, Hyperactivity and hypoactivity produced by lesions to the mesolimbic dopamine system, Behav. Brain Res. 3 (1981) 341–359. [18] M.T. Martin-Iverson, N. Yamada, Synergistic behavioral effects of dopamine D1 and D2 receptor agonists are determined by circadian rhythms, Eur. J. Pharmacol. 215 (1992) 119–125. [19] E.S. Maywood, N. Mrosovsky, M.D. Field, M.H. Hastings, Rapid down-regulation of mammalian period genes during behavioral resetting of the circadian clock, Proc. Natl. Acad. Sci. USA 96 (1999) 15211–15216. [20] N. Mrosovsky, Further experiments on the relationship between the period of circadian rhythms and locomotor activity levels in hamsters, Physiol. Behav. 66 (1999) 797–801. [21] R.D. Oades, K. Taghzouti, J.-M. Rivet, H. Simon, M. Le Moal, Locomotor activity in relation to dopamine and noradrenaline in the nucleus accumbens, septal and frontal areas: a 6-hydroxydopamine study, Neuropsychobiology 16 (1986) 37–42. [22] J. Oritz, J.L. DeCaprio, T.A. Kosten, E.J. Nestler, Strain-selective effects of corticosterone on locomotor sensitization to cocaine and on levels of tyrosine hydroxylase and glucocorticoid receptor in the ventral tegmental area, Neuroscience 67 (1995) 383–397. [23] R.C. Pierce, P.W. Kalivas, A circuitry model of the expression of
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behavioral to amphetamine-like psychostimulants, Brain Res. Rev. 25 (1997) 192–216. [24] C.S. Pittendrigh, S. Daan, A functional analysis of circadian pacemakers in nocturnal rodents. I. The stability and lability of spontaneous frequency, J. Comp. Physiol. 106 (1976) 223–252. [25] K. Sakai, D.M. Gash, Effect of bilateral 6-OHDA lesions of the substantia nigra on locomotor activity in the rat, Brain Res. 633 (1994) 144–150. [26] M. K Sanghera, M.E. Trulson, D.C. German, Electrophysiological properties of mouse dopamine neurons: in vivo and in vitro studies, Neuroscience 12 (1984) 793–801.
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Research report
Circadian rhythm of drinking and running-wheel activity in rats with 6-hydroxydopamine lesions of the ventral tegmental area Yoshiaki Isobe*, Hitoo Nishino Department of Physiology, Nagoya City University Medical School, Mizuho-ku, Nagoya 467 -8601, Japan Accepted 30 January 2001
Abstract Circadian rhythms in drinking and running-wheel (locomotor) activity of rats with 6-hydroxydopamine (6-OHDA, 4 mg / 2 ml per rat)-induced lesions in the ventral tegmental area (VTA) were examined under a light–dark (LD) cycle and constant dim light (5 lux). Under the LD cycle, the length of the locomotor activity period was decreased during the dark, and increased during the light period in the lesioned rats. Under the constant dim light conditions, the free-running circadian period (t) of drinking and activity rhythm was longer in lesioned rats than in sham-operated controls. The elongation of the circadian period was accompanied by decrements in activity. These observations suggest that the mesolimbic dopaminergic system modulates rhythms in circadian drinking and locomotor activity. 2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Circadian rhythm; Locomotor activity; Drinking; Ventral tegmental area; 6-Hydroxydopamine
1. Introduction The suprachiasmatic nucleus (SCN) is important for control of circadian rhythm in mammals. The neuronal network of a putative SCN pacemaker with the nucleus accumbens (N. Acc) might modify the circadian locomotor activity rhythms in rats [2]. The N. Acc receives dopaminergic afferent input from the ventral tegmental area (VTA) [17]. Mesolimbic neuronal alterations contribute to behavioral motivation [23]. Experiments using bright light lesioning of the retina have suggested that the VTA is connected to retinal input in Sprague–Dawley rats [11]. The integration of circadian and visual signals modulates hypothalamic dopamine neurons whose activity is also affected by the VTA [10]. The lesions of ventral mesencephalic tegmentum caused disturbances of spontaneous activity, while modifications of the circadian rhythms was *Corresponding author. Tel.: 181-52-853-8136; fax: 181-52-8423069. E-mail address: [email protected] (Y. Isobe).
not induced [6]. However, the role played by midbrain VTA in the generation and entrainment of the circadian rhythm has not been defined. VTA lesions induced by high doses (4 mg / rat) of 6-hydroxydopamine (6-OHDA) decreased the activity [3,12,17], and lower doses (2 mg / rat) of 6-OHDA [17,21] and high-frequency lesions [28] increased the activity levels. A short-term increase in locomotor activity occurs when the circadian activity rhythm of hamsters was phase-shifted by light pulse [1]. After a triazolam injection, the increase of locomotor activity accompanies a phase shift [29]. Behavioral events trigger a phase advance during the subjective day, when the ‘clock’ is insensitive to light [19]. Non-photic resetting has the opposite effect to light-pulse induced phase shift [19,24]. These findings indicate that the phase shift of the circadian activity rhythm is coupled with the activity level. The free-running circadian period is negatively correlated with motor activity level [27], although the correlation between the circadian rhythm period and locomotor activity levels is not always evident in hamsters [20].
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02223-5
Y. Isobe, H. Nishino / Brain Research 899 (2001) 187 – 192
188
The present study was undertaken to determine whether VTA chemical lesioning (6-OHDA) would affect wheelactivity levels and modulate circadian drinking and wheelactivity rhythms.
2. Materials and methods
2.1. Animals Thirty-one male Wistar rats, bred in our animal room, were used. Rats were housed in plexiglass cages, maintained under a 12:12-h light–dark cycle (LD cycle, lights on at 07:00 h) at a room temperature of 25618C. Illumination intensity at the light period was 150 lux produced by a fluorescent lamp and 5 lux in the dark produced by an incandescent bulb. The rats were provided with food and water ad libitum.
2.2. Rhythms of running-wheel activity and drinking activity Each rat was maintained in a cage with an attached side running wheel (Natume, Japan). Each revolution of the running wheel was recorded by an event recorder (Fuji Electric, Japan) with a digital recorder simultaneously (Shimadzu, Japan). The wheel cage was set in a temperature-controlled room at 25618C with the same lighting conditions as in the animal room. Drinking was monitored with a drop counter attached to the water bottle, which was connected to the event recorder with digital recorder (same apparatus used for recording running-wheel activity). The measurements of wheel activity and drinking rhythm were analyzed over 0.5- or 1-h intervals, and these rhythms are represented as actograms by the double plot method, in which the actograms of succeeding 2 days were arranged side by side [9,13,14]. In comparing the 24-h variations of wheel activity under the LD cycle, the eight lesioned rats and eight age-matched sham-operated control rats were measured at the fifth week after the operation. After 3 days setting to the wheel in each rat, we started to record the running-wheel activity. Amounts of wheel revolutions during periods of 30 min were counted using a cumulative recorder. These rats were sighted. To compare the wheel-activity level and circadian rhythmicity in lesioned and sham-operated animals, drink-
ing and running-wheel activity rhythms were simultaneously measured under conditions of constant dim light in intact (sighted, n59) and blinded (n56) rats (see Table 1). When stable free-running rhythms of drinking and wheel activity were evident, the rats received a microinjection of 6-OHDA in bilateral VTA. Six sighted and three blinded rats were lesioned, and three rats from each group received the sham injection (saline only) around the eighth week of age. The operation was performed during the circadian time (CT; CT 0 corresponds to lights on) of CT 4 to CT 6 h. These time points correspond to the dead zone (no phase shift) of the phase response curve for light pulse. The circadian period (t) was estimated by autocorrelation analysis and periodogram analysis with the slope on eyefitted line of onset of activity inactogram. Uncertainty of the estimated t was less than 60.02 h. Blinding was performed by bilateral ocular enucleation on the day of birth. Closed eyelids were opened with a razor and the eyeballs were removed under hypothermic anesthesia [8].
2.3. 6 -OHDA lesion 6-OHDA (Sigma, Japan) was dissolved in sterile isotonic saline, containing 0.1% ascorbic acid, at a concentration of 2 mg / ml. 6-OHDA (2 mg / 1 ml) was injected stereotaxically into the bilateral VTA at around 8 weeks of age, taking 1 min under Na-pentobarbital (5 mg / 100 g) anesthesia, through a 30-gauge stainless-steel needle. The injection needle was lowered according to the following coordinates in rats (body weight 260 g): 5.2 mm posterior to bregma, 0.4 mm (both side) laterally from the midline and 8 mm from the cerebral surface. The incisor bar was set 2.5 mm below the interaural line. Sham-operated controls received bilateral injections of 1 ml of saline.
2.4. Histology Tyrosine hydroxylase (TH) immunohistochemical staining was performed after the experiments to establish the extent of the lesion in the VTA. Briefly, anesthetized animals were perfused through the heart, firstly with saline then with modified Zamboni’s fixative solution (0.2% picric acid, 0.05% glutaraldehyde, 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.5). Brains were removed and stored in phosphate buffer containing 10% sucrose for 1 h, then 20% sucrose overnight at 48C. Sections (50 mm thick) were cut from relevant areas for immunohistochemi-
Table 1 Lengthening of circadian period in running-wheel activity and drinking rhythms after the ventral tegmental area 6-OHDA lesion a 6-OHDA
Sighted Wheel activity
Drinking
Wheel activity
Drinking
Pre Post
24.4960.10 (4) 24.6460.09 (4)*
24.5260.12 (4) 24.7160.06 (4)*
23.5760.04 (3) 23.7060.10 (3)*
23.5660.06 (3) 23.6560.07 (3)
a
Blinded
The number of rats is indicated in parentheses. *Two-way ANOVA, P,0.05.
Y. Isobe, H. Nishino / Brain Research 899 (2001) 187 – 192
cal staining using anti-TH antibody and the PAP (ICN Biochemicals, USA) method [12].
189
creased in lesioned animals. 6-OHDA injection into the VTA produced a complete loss of TH-positive cell bodies in the A10 area in seven cases in eight animals.
2.5. Statistics To evaluate the statistical differences in groups, analysis of variance (ANOVA) was used. To compare the circadian period of pre- and post-lesion, two-way ANOVA was used. The differences were considered to be significant at P, 0.05.
3. Results
3.1. Wheel activity changes under the LD cycle Twenty-four-hour variations of wheel activity measured under the LD cycle are shown in Fig. 1. The wheel activities of lesioned and sham-operated rats were 2186104 (Mean6S.D.) and 3596172 turns per 24 h, respectively. Significant hypoactivity was thus observed in the lesioned rats (P,0.05). The amount of wheel activity during the dark period in the lesioned rats was reduced to 61% that of sham-operated rats. The wheel activity during the light period just after lights on was increased in the lesioned rats. Synchronization to the LD cycle was de-
3.2. Wheel activity and drinking rhythms during free running The results of simultaneously measured free-running rhythms of drinking and running-wheel activity in the sighted and blinded rats are shown in Fig. 2. In the sham-operated rats, no marked changes in the patterns of drinking or wheel-activity rhythms, nor in their daily amounts of intake or wheel activity were recognized after the sham operation. In the lesioned rats, however, the circadian periods (t) of both drinking and wheel-activity rhythms were elongated after the VTA lesion compared with that before injection of 6-OHDA (Table 1). The average circadian period (t) of sighted rats during the running-wheel activity rhythm determined by periodogram analysis was 24.4960.10 h (Mean6S.D.) and 24.6460.09 h before and after the lesion, respectively (n54) (Table 1). The t of the drinking rhythm was 24.5260.12 h and 24.7160.06 h prior to and post-lesion, respectively (n54). In the blinded rats, free-running periods of drinking and wheel-activity rhythm were calculated in three lesioned and three sham-operated rats (Table 1). The pre- and post-lesion in circadian period (t) of wheel-activity rhythm were 23.5760.04 h (Mean6S.D.) and 23.7060.10 h, respectively (Table 1). The period was significantly longer after the lesion when compared with before the operation using the Spearman’s rank correlation test (P,0.05). The circadian period of drinking rhythm was elongated after the VTA lesion, although it was not significant. Blinding caused shortening (,24 h) of the periods of both drinking and wheel-activity rhythms, as is well known. Circadian rhythms in rats kept under the constant dim light and blinded rats did not show a similar circadian period, as shown in our previous report [14]. In the sham-operated rats, shortening of the period after the operation was not significant (Fig. 2, Sight-Cont. lower one, and BlindCont.), reflected by the large inter-individual variations. The circadian drinking rhythm was similar in shape to the wheel-activity rhythm. The VTA lesion did not induce characteristic changes in the amounts of drinking (Fig. 2). Although the drinking and wheel-activity rhythms were different in phase, these rhythms were well synchronized (Fig. 2).
4. Discussion Fig. 1. Circadian running-wheel activity rhythms of sham-operated (upper frame) and 6-OHDA lesioned (lower frame) rats at the fifth week after the operation. In both groups (n58), the mean6S.D. of the amounts of activity measured during each 30-min period are shown. The horizontal dark bar indicates the dark period in the LD cycle.
Free-running circadian periods (ts) of drinking and wheel-activity rhythms were lengthened by the VTA 6OHDA lesion both in blinded and sighted groups. No phase shift of the circadian rhythm by 6-OHDA or saline administration into the VTA under pentobarbital anesthesia
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Fig. 2. Actograms of drinking and running-wheel activity rhythms measured under constant dim light (5 lux) on the sham-operated (Cont.) and VTA-lesioned (Lesion) rats that was sighted (Sight) or blinded (Blind); e.g. Blind-Cont. indicates the sham-operated blinded rat. In each figure, the upper and lower frames show the drinking and running-wheel activity rhythms, respectively. The operation was performed on the day marked by the arrow. The daily amounts of wheel activity are shown in each actogram. Time of day and the amounts of wheel activity or drinking were similar to those shown in the schematic inset in the lower left corner.
was observed in either lesioned or sham-operated rats. These findings indicate that neither 6-OHDA nor saline, administered into the VTA during the circadian time 4–6 h, induces the phase shift at this time zone (dead zone). However, we have not excluded the possibility of a phasedependent variation of locomotor activity by 6-OHDA, since synergistic behavioral effects of dopamine D1 and D2 agonists were reported to be different at subjective day and night [18]. Hypoactivity caused by the 6-OHDA for a dose of 4 mg in each rat was found (Figs. 1 and 2). The present findings confirm those of previous studies where larger doses of 6-OHDA (.4 mg per rat)-induced hypoactivity [3,17]. The decrease in locomotor activity following the VTA lesion is suggested to be related to the dopamine release from the N. Acc. which is a center of motivation of movement [17,23]. An animal’s ‘level of excitement’ and ‘spontaneous
circadian period’ are related [1]. The period of the wheelactivity rhythm tended to be longer in hamsters housed with a running wheel than in those without a wheel [29]. The free-running circadian period is correlated negatively with motor activity level [27]. These phenomena indicate that decrements in the amount of activity are related to the elongation of the circadian period. The circadian rhythm of drinking showed similar rhythm with wheel activity in shape. It was reported that bilateral 6-OHDA lesions of substantia nigra caused aphasia and adipsia [25]. Compared with the substantia nigra, the VTA lesion did not induce characteristic changes in the amounts of drinking (Fig. 2). Although the mechanism is not clear, the onset of drinking rhythm was delayed 1 h with that of running-wheel activity rhythm in both blinded and sighted rats. Studies of midbrain dopamine neurons in vivo and in
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vitro have demonstrated that A10 cells (VTA) fire regularly (self-oscillation), and also fire in the absence of synaptic input [7,26]. This self-oscillation is an important factor for a pacemaker. Although the firing rate is different from that of SCN, the VTA might be one of the candidates for extra-SCN pacemaker modulating systems in circadian rhythm generation. Methamphetamine induces an increase in dopamine release accompanied by increments in locomotor activity [9,12]. Daily administration of methamphetamine activates an endogenous oscillator(s) and evokes free-running circadian rhythms of activity, corticosterone and body temperature in SCN-lesioned rats [9]. The body temperature rhythms measured under the LD cycle did not show marked changes after the VTA-6-OHDA lesions (data not shown). Tyrosine hydroxylase-positive cells (catecholaminergic neurons) innervate the SCN and these neurons mainly lack vasoactive intestinal peptide [16]. Vasoactive intestinal peptide, which is contained in neurons localized in the ventrolateral area of the SCN that receive retinal input, have an important role in the entrainment of circadian rhythm [5]. It could be suggested that catecholaminergic input to the SCN, including the mesolimbic dopaminergic system, functions independently of the retinal input to the SCN. Corticosterone receptor was recognized in VTA [22]. Corticosterone treatment decreased levels of glucocorticoid receptor immunoreactivity in VTA of rat [22]. The dopamine-lesioned rats exhibited a lower basal corticosterone secretion than sham-lesioned animals [4]. Intraventricular injection of 6-OHDA induced a loss of circadian corticosterone rhythm under constant light conditions [8]. These results indicate that the dopaminergic system (VTA) may have a stimulatory influence on the hypothalamopituitary-adrenal axis. The attenuated functions of corticosterone might influence the circadian rhythm [15]. In conclusion, the mesolimbic dopaminergic system arising from VTA may modulate the circadian rhythms of wheel activity and drinking in SCN intact rats.
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