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Clorgycline delays the phase-position of circadian neurotransmitter receptor rhythms
Brain Research, 241 (1982) 115-122
115
Elsevier Biomedical Press
Clorgycline Delays the Phase-position of Circadian Neurotransmitter Receptor Rhythms ANNA WIRZ-JUSTICE*, MARIAN S. KAFKA, DIETER NABER**, IAIN C. CAMPBELL, PAUL J. MARANGOS, LAWRENCE TAMARKIN and THOMAS A. WEHR Clinical Psychobiology Branch, Biological Psychiatry Branch, and Clinical Neuropharmacology Branch, National Institute of Mental Health and National Institute of Child Health and Human Development, Bethesda, MD 20205 (U.S.A.)
(Accepted November 3rd, 1981) Key words: circadian rhythm - - monoamine oxidase inhibitor - - adrenergic, cholinergic, opiate, dopamine, benzodiazepine
receptors - - melatonin
The number of a- and fl-adrenergic, muscarinic cholinergic, opiate, and benzodiazepine receptors in rat forebrain, and dopamine and benzodiazepine receptors in striatum, change throughout the day. The diurnal rhythms of these receptors were altered by treatment with the monoamine-oxidase inhibitor clorgyline: following treatment some or all rhythm characteristics of wave form, amplitude, 24-h mean, and phase, were affected. One common effect of treatment was a delay in phase-position of binding to a- and fl-adrenergic, opiate and benzodiazepine receptors. Additionally, the nocturnal elevation in pineal melatonin which normally returns to baseline at light onset, persisted 3 h into the light period after clorgyline administration. These biochemical observations extend behavioural findings that clorgyline can delay the phase-position of rodent nocturnal activity onset, and does so by slowing the central circadian pacemaker. INTRODUCTION Neurotransmitter systems in the C N S undergo daily changes in most aspects o f their metabolism - - f r o m uptake of precursor amino acids to enzyme activity and neurotransmitter turnover. Endogenous circadian rhythms have also been f o u n d in neurotransmitter receptor numbed0,11, 20-z~-. As studies of neurotransmitter receptors are an important p a r t o f current biochemical strategies in n e u r o p h a r m a c o l o gy, the design of such studies and the interpretation o f their results need to take into account short-term changes in receptor n u m b e r due to time o f day. Furthermore, the existence o f daily fluctuations in receptor numbers raises fundamental issues related to the mode of action o f drugs which have been reported to change receptor numbers: such changes could conceivably arise f r o m a phase shift in the time o f m a x i m u m and m i n i m u m receptor n u m b e r without
any change in the total n u m b e r o f receptors available over the 24-h period. We present evidence that clorgyline, a m o n o amine-oxidase type A inhibitor ( M A O I ) antidepressant drug, that has previously been shown to decrease the n u m b e r of a- and fl-adrenergic receptors measured at a single time point 2,a, has, in addition, p r o f o u n d effects on the circadian rhythms o f these and o f other receptors. MATERIALS AND METHODS Drug
Clorgyline hydrochloride in distilled deionized water was administered to rats at a constant rate o f 4 mg/kg/day/14 days using subcutaneously implanted Alzet minipumps (Model 1701) 29. The minipumps were implanted during the light phase under shortlasting halothane anaesthesia. Clorgyline was a gift
* Fellow of the Swiss Foundation for Biomedical Research. To whom correspondence should be addressed at: Psychiatrische Universit~itsklinik, Wilhelm Kleinstrasse 27, CH-4025 Basel, Switzerland. ** Fellow of ther German Academic Exchange Service. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
116 from May and Baker Pharmaceuticals, Dagenham, U.K. ; Alzet minipumps were purchased from the Alza Corporation, Palo Alto, CA. Animals Male Sprague-Dawley rats (Taconic Farms, MD), 100 g weight at the beginning of the experiment, were kept 7 to a cage under a controlled L :D schedule (12:12, light on from 07.00 to 19.00 h). After one week of adaptation to the LD schedule, 42 animals were implanted with minipumps containing clorgyline; 42 control animals received the minor surgical procedure used for the implantation. The animals were kept on the same LD schedule, and on day 15, paired groups of 7 controls and 7 treated rats were decapitated at 4-h intervals throughout 24 h (beginning at 10.00 h). The controls and clorgyline-treated animals did not differ significantly in weight (243 ~ 9 g; 220 ± 10 g). Pineal glands were rapidly removed and frozen until assayed for melatonin; forebrains (anterior to the cerebellum) were sagitally divided and frozen after striata had been dissected out. The experiment was carried out from October 3 to 26, 1979. Tissue preparation For a given time point, alternate left and right forebrains of the 7 animals/group were homogenized in Tris buffer (pH 7.7, 0.05 M), washed and centrifuged 3 times, and a final suspension (1 g tissue wet weight/10 ml) used directly in binding assays of a- and /3-adrenergic and benzodiazepine receptors. Frozen aliquots of this suspension were subsequently used in binding assays of muscarinic cholinergic and opiate receptors. The striata were homogenized as for forebrain, but in a final dilution of 0.1 g tissue wet weight/10 ml, and used directly in binding assays of dopamine and benzodiazepine receptors. Melatonin was measured in pineal glands by radioimmunoassay28. Receptor assays Receptors was assayed by the specific binding of tritiated ligands (New England Nuclear) as previously described: the a-adrenergic receptor using [aH]WB4101 (24.0 Ci/mmol, 0.39 nM), with and without phentolamine (10-5 M) as antagonisp0; the fl-adrenergic receptor using [3H]dihydroalprenolol
(DHA, 40 Ci/mmol, 0.95 nM), with and without D,L-propranolol (2 × 10-5 M)10; the acetylcholine muscarinic receptor using [3H]quinuclidinyl benzylate (QNB, 29.4 Ci/mmol, 0.16 nM) with and without atropine (2 × 10-6 M)11; the dopamine receptor using [3H]spiroperidol (21 Ci/mmol, 0.38 nM), with and without (+)-butaclamol (10 -6 M)e0; the opiate receptor using [aH]naloxone (25.4 Ci/mmol, 2.8 nM), with and without naloxone (10 -6 M)~-2; the benzodiazepine receptor using [3H]diazepam (80 Ci/mmol, 3 nM) with and without diazepam (2 x 10-6 M) 17. In addition, Scatchard analysis25 of saturation experiments were done for each receptor on homogenates from the time points of maximal and minimal binding. All time points were assayed in random order on a single day; at each time point individual brain homogenates from 7 animals were assayed in triplicate. The reliability of the assays was estimated by the coefficient of variation (S.D. x 100/mean). The intra- and inter-individual coefficient of variation for each receptor is summarized in Table III. RESULTS Circadian rhythms in receptor number were found in all receptors investigated. Chronic clorgyline had marked effects on some or all rhythm characteristics : wave-form (shape), phase (timing of peak binding), amplitude (peak: trough ~), and 24h mean number of each receptor. a-Adrenergic receptor binding (Fig. 1A) showed two main peaks at the end of the light and beginning of the dark phase. After chronic clorgyline treatment the rhythm was clearly sinusoidal with a single peak at the beginning of the light phase. Control fladrenergic receptor binding (Fig. 1B) showed a peak in the mid-dark phase. After clorgyline treatment, peak binding occurred later in the dark phase, with an apparent second peak in the middle of the light phase. A bimodal rhythm of binding to the cholinergic receptor in the forebrains from control rats was reduced in amplitude and modified by clorgyline treatment to a single broader peak occurring at the beginning of the light phase (Fig. 2A). Binding to the benzodiazepine receptor in controls also showed two peaks, at the beginning and end of the dark
117 5.5
A.
production with an abrupt fall at light onset, was observed in pineals from controls (Fig. 4). After clorgyline treatment, this abrupt fall did not occur, and melatonin remained high 3 h into the light phase and was higher than normal throughout the light
~ 5.C .o1 O
co 3~ 3 rn o
TE
4.E
phase. Scatchard analyses of saturation curves from control animals (n = 6) and from clorgyline-treated animals at times of maximal and minimal binding (n = 2) indicated that the changes in binding throughout the day were not due to changes in the apparent affinity for the ligand but changes in the number of binding sites: Kes for control and clorgyline-treated animals, respectively (mean 4- S.E.M.): a-adrenergic receptor (2.6 4- 0.3 nM and 2.7 4- 0.4 nM);/3adrenergic receptor (1.5 4- 0.2 nM and 1.4 ~ 0.3
0
40
[5.
~:
20
o~ ,~ ~
1.5
, 5 1£
2
6
10
14
18
22
2
6
10
T I M E O F DAY ( h r )
Fig. 1. Specific binding of [sH]WB4101 to the ct-adrenergic receptor (A) and [3H]DHA to the fl-adrenergic receptor (B) measured at 6 time points throughout the 24-h day. Each point represents the mean 4: S.E.M. of 5-7 animals: 3 points are repeated without S.E.M. to illustrate the rhythm more clearly without an arbitrary cut-off time. The shaded areas represent the dark phase of the LD cycle. The rhythm in control animals is indicated by the solid line; in clorgylinetreated animals by the dotted line. Details are in the Materials and Methods section, and statistics in Tables I-III. phase. Clorgyline treatment shifted the second peak to the middle of the light phase (Fig. 2B). Opiate receptor binding in controls was maximal at the beginning of the dark phase: clorgyline treatment had the effect of inducing a second major peak at the beginning of the light phase (Fig. 2C). Binding to receptors in striatal homogenates showed similar unimodal rhythms for dopamine (peak binding in the dark phase) and benzodiazepine receptors (peak binding at the end of the light and beginning of the dark phase (Fig. 3A, B). Clorgyline treatment markedly decreased the 24-h mean binding to both receptors, although as a relative measure, the amplitude did not decrease. While the rhythm in binding to the dopamine receptor after clorgyline treatment was, as a whole, no longer statistically significant, there were significant differences between time points suggesting the persistence of time-dependent processes (Table I). The well-established nocturnal peak of melatonin
0
a
B
~3 o~ 11
,o
•
<~
24
C
ZO O 3
2O
o~
10 2
6
10 14 18 22 T I M E O F DAY (hr)
10
Fig. 2. Specificbinding of [ZH]QNB to the muscarinic cholinergic receptor (A), [ZH]diazepamto the benzodiazepine receptor (B) and [3H]naloxone to the opiate receptor (C) in controls (solid line) and clorgyline-treated animals (dotted line). Details as in legend to Fig. 1, Materials and Methods, section and in Tables I-III.
118 TABLE I Statistical analyses of" circadian rhythms." one-way analysis of variance Controls F
Clorgyline P
Least-squares-difference* P < 0.01
Forebrain receptor binding Alpha-adrenergic 6.53
< 0.0003 2 > 14, 6 18 > 6
F
P
P < 0.05
P < 0.01
2 > 10, 22 11.34 18 > 2 2 , 1 4 10, 22, > 6 22, 2 > 6, 14,18 4.26
< 0.0001 6, 10, 14 > 18, 22, 2
< 0.001
Beta-adrenergic
3.53
< 0.013
22, 2 > 18 22>6
Muscarinic cholinergic
27.23
< 0.0001
10, 2, > all
14 > 18, 22
5.17
Benzodiazepine
13.8
< 0.0001
14 > 2
9.1
Opiate
6.5
< 0.001
10, 14, 22, 6 > 18 10, 22, 6 > 2 6>14 22 > 6, 10, 14, 18 2>6
Striatal receptor binding Dopamine 2.94
< 0.025
Benzodiazepine
3.47
< 0.014
Concentration Pineal melatonin
7,19
< 0.0001 2, 6 > 14, 18, 22 6>10
18, 22, > 6
Least squares-difference*
22, 2, 6 > 10, 14 all > 6 18 > 14
< 0.004
P < 0.05
2,6,14>18 6>22
2>22 6 > 10
2, 6, 10, 14 > 18 < 0.0001 10, 14, 22 >2,6
10 > 22 > 18
23.3
< 0.0001 2, 6, 10 > 14, 18, 2
10 > 22
2.15
< 0.09
22>6
14>6
4.98
< 0.002
18, 22 > 10 1 8 > 14, 22
2 > 14, 22 18>6
4.46
< 0.003
2, 6, 1 0 > 2 2
6>14
18 > 6
* Procedure determining which time points (in hours) are significantly different from each other ~6. n M ) ; a c e t y l c h o l i n e r e c e p t o r (0.021 ~: 0.002 n M a n d 0.032 :~ 0.004 n M ) ; d o p a m i n e r e c e p t o r (1.7 zk 0.08
A
n M a n d 1.5 ~: 1.1 n M ) ; o p i a t e r e c e p t o r (1.2 ~ 0.05 n M a n d 1.2 ± 0.02 n M ) ; a n d b e n z o d i a z e p i n e r e c e p t o r (3.1 Jz 0.3 n M a n d 3.5 ~: 0.5 n M ) .
~ 2
A c h a n g e i n n u m b e r , n o t affinity, h a d p r e v i o u s l y b e e n o b s e r v e d i n studies o f c i r c a d i a n c h a n g e in, a n d the effects o f p s y c h o t r o p i c d r u g s o n , b i n d i n g to different receptors.
The significance of changes over time were established b y o n e - w a y analysis o f v a r i a n c e ( A N O V A ) : all receptors showed highly significant rhythms both
11 ~o
i~ ~ -~ ~-2
B.
8
i n c o n t r o l s a n d c l o r g y l i n e - t r e a t e d g r o u p s , w i t h the single e x c e p t i o n o f the d o p a m i n e r e c e p t o r after
zO
7
Fig. 3. Specific binding of [SH]spiroperidol to the dopamine receptor (A), [SH]diazepam to the benzodiazepine receptor (B) in striatal membrane preparations. The rhythm in control animals is indicated by the solid line; in clorgyline-treated animals by the dotted line. Details as in legend to Fig. 1, Materials and Methods, section and in Tables I-III.
<~ o~ 4 3
2
6
10
14
18
22
T I M E OF DAY ( h r )
2
6
10
119 800
melatonin (Tables II, III). The time-dependent process (two-way interaction of the drug with time) was significant for all receptors (Table II).
!
DISCUSSION z
400
-
200
-
Z
0 _J uJ
2
N N
6
I0
14
18
22
2
6
I0
TIME OF DAY (hr)
Fig. 4. Melatonin concentrations in pineal glands measured at 6 times points throughout the 24-h day, in controls (solid line) and clorgyline-treated animals (dotted line). Melatonin concentrations in the light phase are significantly higher after clorgyIine treatment (see Tables I-III). clorgyline treatment (P < 0.09) (Table I). For each group, a further analysis by the least-squares-difference procedure indicated which time points were different from each other and at which level of significance za (Table I). The effect of drug treatment was analyzed by two-way ANOVA, i.e. the effect of clorgyline on time-independent (e.g. 24-h mean) and time-dependent (e.g. phase) processes. Analyzed as a time-independent process, clorgyline treatment significantly affected a-adrenergic, cholinergic, striatal dopamine and benzodiazepine binding, and pineal
There are two levels of analysis in this study: first, the effect of clorgyline on individual neurotransmitter systems, and second, the possible effect of this drug on a central circadian oscillatory system. The limitations in determining the shape of a circadian rhythm imposed by the sampling intervals (4 h) need to be noted, and the lack of precise temporal resolution might account for the apparent irregularity in many of the receptor rhythms. Seven similar 24-h studies of receptor rhythms in untreated rats have documented that these receptors change with time of day l°,n,13,2°-22,z7,a°. In our studies we have observed that there are apparent phase shifts in the timing of peak binding to a given receptor. However a circadian variation is always present, with changes throughout the day of an order of magnitude commonly reported for differences between groups studied at a single time point (e.g. after lesions). The effects of drugs on these rhythms are profoundll,12,20,34,35,ag. Clorgyline changes the temporal pattern of binding to each receptor. The changes are in part consistent with the MAOI action of clorgyline 1,z. Clorgyline is a specific inhibitor of MAO-A, and would preferentially block the deamination of noradrenaline and serotonin. It is likely that the dose schedule
TABLE II Statistical attalysis of clorgyline effects: two-way analysis of variance Drug treatment (time independent process)
Two-way interaction (time-dependent process)
F
P
F
5.94 2.99 199.23 0.21 0.62
< 0.02 n.s. < 0.0001 n.s. n.s.
12.84 4.10 5.14 8.93 11.25
952.82 211.80
< 0.0001 < 0.0001
2.57 2.76
< 0.035 < 0.03
< 0.013
0.97
n.s.
P
Forebrain receptor binding
Alpha-adrenergic Beta-adrenergic Muscarinic cholinergic Benzodiazepine Opiate
< < < < <
0.0001 0.003 0.001 0.0001 0.0001
Striatal receptor binding
Dopamine Benzodiazepine Concentration
Pineal melatonin
6.46
120 TABLE HI
Methodology, 24-h mea~ binding, and rhythm amplitude Coefficient of var24-h mean binding ( ± S.E.M.) iation in controls* (%) (n) pmol/g wet weight
Amplitude Peak: Trough (%)
bttraInterbtdividual
Controls
Clorgyline
P**
Controls
Clorgyline
Alpha-adrenergic
5.4
11.3
45
38
5.4
11.2
n,s.
26
26
Muscarinic cholinergic
4.6
6.8
< 0.001
56
30
Benzodiazepine
3.1
3.8
n.s.
25
20
Opiate
7.9
39.0
4.23 ~ 0.10 (41) 1.50 =E 0.04 (39) 18.40 zb 0.34 (38) 11.60 zb 0.20 (41) 1.42 ~ 0.11 (31)
< 0.055
Beta-adrenergie
4.53 ± 0.12 (38) 1.56 ~- 0.04 (35) 14.77 ± 0.44 (37) 11.60 ~ 0.20 (41) 1.39 ± 0.07 (27)
n.s.
78
161
Doparnine
6.5
21.8
21
35
3.0
10.8
2.53 % 0.08 (36) 3.76 ~ 0.14 (38)
< 0.001
Benzodiazepine
9.36 2~ 0.22 (41) 6.32 ± 0.15 (35)
< 0.001
32
47
274 ± 46 (42)
410 ~ 48 (42)
< 0.04
520
355
Forebrain receptor binding
Striatal receptor binding
Concentratiott Pineal rnelatonin (pg/gland)
* Mean coefficient of variation calculated as S.D. × 100/mean for each assay (n = 42 for intra; n ~ 6 for inter). ** Student's two-tailed t-test. used in this study may cause some inhibition of MAO-B. By inhibiting monoamine metabolism and inducing prolonged increases in noradrenaline and possibly dopamine, one effect would be a subsensitivity of the adrenergic and dopamine receptors. The results suggest that two weeks of clorgyline treatment tended to decrease adrenergic receptor binding; previous results had shown that 3 weeks of clorgyline treatment induced a significant decrease (when measured at a single time point)2,4. The finding of a large decrease in mean binding to the dopamine receptor is consistent with this hypothesis, although it does not support other studies whose results suggest suggest an agonist-induced supersensitivityS,14. There are no single time point studies of the effects of clorgyline on cholinergic, opiate, or benzodiazepine receptors, as these systems have not been considered related to the drug's use as an MAOI. In terms of up and down regulation of receptor number over 24 h, there are but 3 significant changes: marked decreases in binding to striatal dopamine and benzodiazepine receptors at all times of day,
and increased binding to forebrain cholinergic receptors at all times of day. The 24-h mean number of forebrain benzodiazepine and opiate receptors is unchanged. In general, the results are interesting in that they demonstrate that both increased and decreased binding to a given receptor can coexist, separated only in the temporal dimension. This may explain some of the discrepancies in the literature, since results may vary depending on the time of day the experiment was carried out : for example, after clorgyline treatment there is increased a-adrenergic recep tot binding in the early morning (27 % increase, t = 2.51, P < 0.03), no significant differences in the afternoon, and decreased binding in the evening (32 % decrease, t = 6.32, P < 0.0002) (Fig. IA). The effect of clorgyline treatment on pineal melatonin is particularly interesting. Light at any time suppresses metatonin secretion, and on a normal light-dark cycle the onset of light suppresses the last hours of the nocturnal secretory phase, i.e. it truncates the rhythm. The most parsimonious explanation of the drug effect is that it does not change the
121 phase-position of the rhythm, but blocks the suppressing effect of light, so that melatonin secretion persists during the early light phase. Other experiments support this interpretation 5. Two further effects of clorgyline on rhythm parameters are (a) there is often a change in the shape of the rhythm with drug, and (b) there is a shift in the timing of peak binding to later in the day. Interpretation of the phase-shifting effects of clorgyline on circadian receptor rhythms can be made with respect to the phase-shifting effects oF clorgyline on the circadian rest-activity cycle. Chronic clorgyline delays the onset of nocturnal running activity in hamsters entrained to a light-dark cycle, and does so in a dose-dependent mannerL The phase-delay under entrained conditions is related to the lengthening of the period of the rest-activity cycle by clorgyline under free-running conditions of constant darkness35,36,d8, 39. Furthermore, preliminary studies in vivo show that clorgyline lengthens the period of free-running rhythms when administered directly to the postulated central pacemaker, the suprachiasmatic nuclei (SCN) of the anterior hypothalamus 19, but not when administered in other brain regions (Groos, unpublished observations) 3s. Thus the effect of clorgyline to slow the frequency of a central circadian pacemaker may be reflected in the delayed phase-position of many neurotransmitter receptor rhythms. Very few drugs have been found that can modify the frequency of circadian rhythmsZ3, 24. It may be
REFERENCES
important that among these few are 3 antidepressant drugs: lithium 6, imipramine 35,zs,~9, and clorgyline35,3s, 39. Lithium has been the most extensively studied, and found to lengthen the free-running circadian period in many species 6-9,16,z7, as well as delay phase position under entrained conditions 12 15,18. Imipramine disrupts circadian rhythmicity when applied to the SCN 38, and we have previously shown that imipramine delays the phase position of many neurotransmitter receptor rhythms in a manner analogous to that reported here for clorgyline TM z0,34,zg. These findings support the hypothesis that imipramine and clorgyline modify circadian rhythm parameters by pharmacological modulation of a pacemaker in the SCN rather than by diffuse action on a number of brain structures 3s,39. Even though the antidepressants may be relatively selective in their action of inhibiting monoamine uptake or deamination, this effect on frequency may explain why they change the rhythms even of those neurotransmitters whose metabolism they do not affect directly. If, as has been suggested 3°-z3, abnormally phaseadvanced circadian rhythms play an important role in depressive pathophysiology, the ability of clinically effective antidepressants to phase-delay circadian rhythms may be important in their therapeutic mechanisms of action. Thus the up- or down-regulation of receptor number may be less relevant to antidepressant drug action than effects on the temporal organisation of neurotransmitter systems.
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5 Craig, C., Tamarkin, L., Garrick, N. and Wehr, T. A., Long-term and short-term effects of clorgyline (a monoamine-oxidase type A inhibitor) on locomotor activity and on pineal melatonin in the hamster, Nearosci. Abstr., 1981. 6 Engelmann, W., A slowing down of circadian rhythms by lithium ions, Z. Naturforsch., 28c (1973) 733-736. 7 Hofmann, K., Gfinderoth-Palmowski, M., Wiedenmann, G. and Engelmann, W., Further evidence for period lengthening effect of Li+ on circadian rhythms, Z. Naturforsch., 32c (1978) 231-234. 8 Johnsson, A., Pflug, B., Engelmann, W. and Klemke, W., Effect of lithium carbonate on circadian periodicity in humans, Pharmakopsychiat. Neuro. PsychopharmacoL, 12 (1979) 423-425. 9 Johnsson, A., Engelmann, W., Pflug, B. and Klemke, W., Influence of lithium ions on human circadian rhythms, Z. Naturforsch., 35c (1980) 503-507.
122 10 Kafka, M. S., Wirz-Justice, A. and Naber, D., Circadian and seasonal rhythms in a- and fl-adrenergic receptors in the rat brain, Brain Research, 207 (1981) 409-419. 11 Kafka, M. S., Wirz-Justice, A., Naber, D. and Wehr, T. A., Circadian acetylcholine receptor rhythm in rat brain and its modification by imipramine, Neuropharmacology, 20 (1981) 421-425. 12 Kafka, M. S., Wirz-Justice, A., Naber, D., Marangos, P. J., O'Donahue, T. and Wehr, T. A., Effect of lithium on circadian neurotransmitter receptor rhythms, Neuropsychobiology, 8 (1982) 41-50. 13 Kafka, M. S., Moore, R. Y. and Marangos, P. J., Suprachiasmatic nucleus ablation: effects on rat brain neurotransmitter receptor rhythms. Neurosci. Abstr., 1981. 14 Klawans, H. L., Goetz, C., Nausieda, P. A. and Weiner, W. J., Levo-DOPA induced dopamine receptor supersensitivity, Trans. Amer. neurol. Assoc., 102 (1977) 80-83. 15 Kripke, D. F., Judd, L. L., Hubbard, B., Janowsky, D. S. and Huey, L. Y., The effect of lithium carbonate on the circadian rhythm of sleep in normal human subjects, Biol. Psychiat., 14 (1979) 545-548. 16 Kripke, D. F. and Wyborney, V. G., Lithium slows rat circadian activity rhythms, Life Sci., 26 (1980) 1319-1321. 17 Marangos, P. J., Paul, S. M., Parma, A. M., Goodwin, F. K., Syapin, P. and Skolnick, P., Purinergic inhibition of diazepam binding to rat brain (in vitro), Life Sci., 24 (1979) 851-858. 18 McEachron, D. L., Kripke, D. F., Hawkins, R., Haus, E., Pavlinac, D. and Deftos, L., Lithium delays biochemical circadian rhythms in rats, Neuropsychobiology, 8 (1982) 12-29. 19 Moore, R. Y., Central control of circadian rhythms. In W. F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinology, Vol. 5, Raven Press, New York, NY, 1978, pp. 185-206. 20 Naber, D., Wirz Justice, A., Kafka, M. S. and Wehr, T. A., Dopamine receptor binding in rat striatum: ultradian rhythm and its modification by chronic imipramine, Psychopharmacology, 68 (1980) 1-5. 21 Naber, D., Wirz-Justice, A., Kafka, M. S., ToNer, I. and Borb61y, A. A., Seasonal variations in the endogenous rhythm of dopamine receptor binding in rat striatum, BiologicalPsychiat., 16 (1981) 831-835. 22 Naber, D., Wirz-Justice, A. and Kafka, M. S., Circadian rhythm in rat brain opiate receptor, Neurosci. Lett., 21 (1981) 45-50. 23 Pittendrigh, C. S. and Caldarola, P. C., General homeostasis of the frequency of circadian oscillations, Proc. nat. Acad. Sci. U.S.A., 70 (1973) 2697-2701. 24 Richter, C. P., Biological Clocks in Medicine and Psychiatry, Charles C. Thomas, Springfield, IL., 1965. 25 Scatchard, G., The attractions of proteins for small molecules and ions, Ann. N.Y. Acad. Sci., 5l (1949) 660-672. 26 Snedecor, G. W. and Cochran, W. G., Statistical Methods (6th edn.), Iowa State University Press, Ames, IA, 1967, p. 271.
27 Strumwasser, F. and Viele, D. P., Lithium increases the period of a neuronal circadian oscillator, Neurosei. Abstr., 241 (1980) 5. 28 Tamarkin, L., Reppert, S. M. and Klein, D. C., Regulation of pineal melatonin in the syrian hamster, Endocrinology, 104 (1979) 385-389. 29 Theewes, F. and Yum, S. I., Principles of the design and operation of generic osmotic pumps for the delivery of semisolid or liquid drug formulations, Ann. biomed. Eng., 4 (1976) 343-353. 30 Wehr, T. A., Wirz-Justice, A., Goodwin, F. K., Duncan, W. and Gillin, J. C., Phase advance of the sleep-wake cycle as an antidepressant, Science, 206 (1979) 710-713. 31 Wehr, T. A. and Wirz-Justice, A., Circadian rhythm mechanisms in affective illness and in antidepressant drug action, Pharmacopsychiatria, 15 (1982) 31-39. 32 Wehr, T. A. and Goodwin, F. K., Biological rhythms and psychiatry. In S. Arieti and H. K. H. Brodie (Eds.), American Handbook of Psychiatry, Vol. VI, Basic Books, New York, NY, 1981. 33 Wehr, T. A., Goodwin, F. K., Wirz-Justice, A., Craig, C. and Breitmaier, J., 48-Hour sleep-wake cycles in manicdepressive illness: naturalistic observations and sleep deprivation experiments, Arch. gen. Psychiat., in press. 34 Wirz-Justice, A., Kafka, M. S., Naber, D. and Wehr, T. A., Circadian rhythms in rat brain a- and fl-adrenergic receptors are modified by chronic imipramine, Life Sci., 27 (1980) 341-347. 35 Wirz-Justice, A., Wehr, T. A., Goodwin, F. K., Kafka, M. S., Naber, D., Marangos, P. J. and Campbell, I. C., Antidepressant drugs slow circadian rhythms in behavior and brain neurotransmitter receptors, PsychopharmacoL Bldl., 16 (1980) 45M7. 36 Wirz-Justice, A. and Wehr, T. A., Uncoupling of circadian rhythms in hamsters and man. In W. P. Koella (Ed.), Sleep 1980, Proceedings of the 5th European Congress of Sleep Research, Amsterdam, 1980, Karger, Basel, pp. 64-72. 37 Wirz-Justice, A., ToNer, I., Kafka, M. S., Naber, D., Marangos, P. J., Borb61y, A. A. and Wehr, T. A., Sleep deprivation: effects on circadian rhythms of rat brain neurotransmitter receptors, Psychiat. Res., 5 (1981) 67-76. 38 Wirz-Justice, A., Groos, G. A. and Wehr, T. A., The neuropharmacology of circadian timekeeping in mammals. In J. Aschoff, S. Daan and G. A. Groos (Eds.), Vertebrate Circadian Systems: Structure and Physiology, Springer, Berlin, 1982. 39 Wirz-Justice, A., Wehr, T. A. and Goodwin, F. K., Antidepressant drugs: effects on the circadian system. In T. A. Wehr and F. K. Goodwin (Eds.), Circadian Rhythms in Psychiatry, Boxwood Press, Los Angeles, CA, 1981. 40 Wirz-Justice, A., Krguchi, K., Campbell, l.C.and Feer, H., Altered circadian rhythms in neurotransmitter receptors and plasma hormones in spontaneous hypertensive rats, Experientia, in press.
115
Elsevier Biomedical Press
Clorgycline Delays the Phase-position of Circadian Neurotransmitter Receptor Rhythms ANNA WIRZ-JUSTICE*, MARIAN S. KAFKA, DIETER NABER**, IAIN C. CAMPBELL, PAUL J. MARANGOS, LAWRENCE TAMARKIN and THOMAS A. WEHR Clinical Psychobiology Branch, Biological Psychiatry Branch, and Clinical Neuropharmacology Branch, National Institute of Mental Health and National Institute of Child Health and Human Development, Bethesda, MD 20205 (U.S.A.)
(Accepted November 3rd, 1981) Key words: circadian rhythm - - monoamine oxidase inhibitor - - adrenergic, cholinergic, opiate, dopamine, benzodiazepine
receptors - - melatonin
The number of a- and fl-adrenergic, muscarinic cholinergic, opiate, and benzodiazepine receptors in rat forebrain, and dopamine and benzodiazepine receptors in striatum, change throughout the day. The diurnal rhythms of these receptors were altered by treatment with the monoamine-oxidase inhibitor clorgyline: following treatment some or all rhythm characteristics of wave form, amplitude, 24-h mean, and phase, were affected. One common effect of treatment was a delay in phase-position of binding to a- and fl-adrenergic, opiate and benzodiazepine receptors. Additionally, the nocturnal elevation in pineal melatonin which normally returns to baseline at light onset, persisted 3 h into the light period after clorgyline administration. These biochemical observations extend behavioural findings that clorgyline can delay the phase-position of rodent nocturnal activity onset, and does so by slowing the central circadian pacemaker. INTRODUCTION Neurotransmitter systems in the C N S undergo daily changes in most aspects o f their metabolism - - f r o m uptake of precursor amino acids to enzyme activity and neurotransmitter turnover. Endogenous circadian rhythms have also been f o u n d in neurotransmitter receptor numbed0,11, 20-z~-. As studies of neurotransmitter receptors are an important p a r t o f current biochemical strategies in n e u r o p h a r m a c o l o gy, the design of such studies and the interpretation o f their results need to take into account short-term changes in receptor n u m b e r due to time o f day. Furthermore, the existence o f daily fluctuations in receptor numbers raises fundamental issues related to the mode of action o f drugs which have been reported to change receptor numbers: such changes could conceivably arise f r o m a phase shift in the time o f m a x i m u m and m i n i m u m receptor n u m b e r without
any change in the total n u m b e r o f receptors available over the 24-h period. We present evidence that clorgyline, a m o n o amine-oxidase type A inhibitor ( M A O I ) antidepressant drug, that has previously been shown to decrease the n u m b e r of a- and fl-adrenergic receptors measured at a single time point 2,a, has, in addition, p r o f o u n d effects on the circadian rhythms o f these and o f other receptors. MATERIALS AND METHODS Drug
Clorgyline hydrochloride in distilled deionized water was administered to rats at a constant rate o f 4 mg/kg/day/14 days using subcutaneously implanted Alzet minipumps (Model 1701) 29. The minipumps were implanted during the light phase under shortlasting halothane anaesthesia. Clorgyline was a gift
* Fellow of the Swiss Foundation for Biomedical Research. To whom correspondence should be addressed at: Psychiatrische Universit~itsklinik, Wilhelm Kleinstrasse 27, CH-4025 Basel, Switzerland. ** Fellow of ther German Academic Exchange Service. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
116 from May and Baker Pharmaceuticals, Dagenham, U.K. ; Alzet minipumps were purchased from the Alza Corporation, Palo Alto, CA. Animals Male Sprague-Dawley rats (Taconic Farms, MD), 100 g weight at the beginning of the experiment, were kept 7 to a cage under a controlled L :D schedule (12:12, light on from 07.00 to 19.00 h). After one week of adaptation to the LD schedule, 42 animals were implanted with minipumps containing clorgyline; 42 control animals received the minor surgical procedure used for the implantation. The animals were kept on the same LD schedule, and on day 15, paired groups of 7 controls and 7 treated rats were decapitated at 4-h intervals throughout 24 h (beginning at 10.00 h). The controls and clorgyline-treated animals did not differ significantly in weight (243 ~ 9 g; 220 ± 10 g). Pineal glands were rapidly removed and frozen until assayed for melatonin; forebrains (anterior to the cerebellum) were sagitally divided and frozen after striata had been dissected out. The experiment was carried out from October 3 to 26, 1979. Tissue preparation For a given time point, alternate left and right forebrains of the 7 animals/group were homogenized in Tris buffer (pH 7.7, 0.05 M), washed and centrifuged 3 times, and a final suspension (1 g tissue wet weight/10 ml) used directly in binding assays of a- and /3-adrenergic and benzodiazepine receptors. Frozen aliquots of this suspension were subsequently used in binding assays of muscarinic cholinergic and opiate receptors. The striata were homogenized as for forebrain, but in a final dilution of 0.1 g tissue wet weight/10 ml, and used directly in binding assays of dopamine and benzodiazepine receptors. Melatonin was measured in pineal glands by radioimmunoassay28. Receptor assays Receptors was assayed by the specific binding of tritiated ligands (New England Nuclear) as previously described: the a-adrenergic receptor using [aH]WB4101 (24.0 Ci/mmol, 0.39 nM), with and without phentolamine (10-5 M) as antagonisp0; the fl-adrenergic receptor using [3H]dihydroalprenolol
(DHA, 40 Ci/mmol, 0.95 nM), with and without D,L-propranolol (2 × 10-5 M)10; the acetylcholine muscarinic receptor using [3H]quinuclidinyl benzylate (QNB, 29.4 Ci/mmol, 0.16 nM) with and without atropine (2 × 10-6 M)11; the dopamine receptor using [3H]spiroperidol (21 Ci/mmol, 0.38 nM), with and without (+)-butaclamol (10 -6 M)e0; the opiate receptor using [aH]naloxone (25.4 Ci/mmol, 2.8 nM), with and without naloxone (10 -6 M)~-2; the benzodiazepine receptor using [3H]diazepam (80 Ci/mmol, 3 nM) with and without diazepam (2 x 10-6 M) 17. In addition, Scatchard analysis25 of saturation experiments were done for each receptor on homogenates from the time points of maximal and minimal binding. All time points were assayed in random order on a single day; at each time point individual brain homogenates from 7 animals were assayed in triplicate. The reliability of the assays was estimated by the coefficient of variation (S.D. x 100/mean). The intra- and inter-individual coefficient of variation for each receptor is summarized in Table III. RESULTS Circadian rhythms in receptor number were found in all receptors investigated. Chronic clorgyline had marked effects on some or all rhythm characteristics : wave-form (shape), phase (timing of peak binding), amplitude (peak: trough ~), and 24h mean number of each receptor. a-Adrenergic receptor binding (Fig. 1A) showed two main peaks at the end of the light and beginning of the dark phase. After chronic clorgyline treatment the rhythm was clearly sinusoidal with a single peak at the beginning of the light phase. Control fladrenergic receptor binding (Fig. 1B) showed a peak in the mid-dark phase. After clorgyline treatment, peak binding occurred later in the dark phase, with an apparent second peak in the middle of the light phase. A bimodal rhythm of binding to the cholinergic receptor in the forebrains from control rats was reduced in amplitude and modified by clorgyline treatment to a single broader peak occurring at the beginning of the light phase (Fig. 2A). Binding to the benzodiazepine receptor in controls also showed two peaks, at the beginning and end of the dark
117 5.5
A.
production with an abrupt fall at light onset, was observed in pineals from controls (Fig. 4). After clorgyline treatment, this abrupt fall did not occur, and melatonin remained high 3 h into the light phase and was higher than normal throughout the light
~ 5.C .o1 O
co 3~ 3 rn o
TE
4.E
phase. Scatchard analyses of saturation curves from control animals (n = 6) and from clorgyline-treated animals at times of maximal and minimal binding (n = 2) indicated that the changes in binding throughout the day were not due to changes in the apparent affinity for the ligand but changes in the number of binding sites: Kes for control and clorgyline-treated animals, respectively (mean 4- S.E.M.): a-adrenergic receptor (2.6 4- 0.3 nM and 2.7 4- 0.4 nM);/3adrenergic receptor (1.5 4- 0.2 nM and 1.4 ~ 0.3
0
40
[5.
~:
20
o~ ,~ ~
1.5
, 5 1£
2
6
10
14
18
22
2
6
10
T I M E O F DAY ( h r )
Fig. 1. Specific binding of [sH]WB4101 to the ct-adrenergic receptor (A) and [3H]DHA to the fl-adrenergic receptor (B) measured at 6 time points throughout the 24-h day. Each point represents the mean 4: S.E.M. of 5-7 animals: 3 points are repeated without S.E.M. to illustrate the rhythm more clearly without an arbitrary cut-off time. The shaded areas represent the dark phase of the LD cycle. The rhythm in control animals is indicated by the solid line; in clorgylinetreated animals by the dotted line. Details are in the Materials and Methods section, and statistics in Tables I-III. phase. Clorgyline treatment shifted the second peak to the middle of the light phase (Fig. 2B). Opiate receptor binding in controls was maximal at the beginning of the dark phase: clorgyline treatment had the effect of inducing a second major peak at the beginning of the light phase (Fig. 2C). Binding to receptors in striatal homogenates showed similar unimodal rhythms for dopamine (peak binding in the dark phase) and benzodiazepine receptors (peak binding at the end of the light and beginning of the dark phase (Fig. 3A, B). Clorgyline treatment markedly decreased the 24-h mean binding to both receptors, although as a relative measure, the amplitude did not decrease. While the rhythm in binding to the dopamine receptor after clorgyline treatment was, as a whole, no longer statistically significant, there were significant differences between time points suggesting the persistence of time-dependent processes (Table I). The well-established nocturnal peak of melatonin
0
a
B
~3 o~ 11
,o
•
<~
24
C
ZO O 3
2O
o~
10 2
6
10 14 18 22 T I M E O F DAY (hr)
10
Fig. 2. Specificbinding of [ZH]QNB to the muscarinic cholinergic receptor (A), [ZH]diazepamto the benzodiazepine receptor (B) and [3H]naloxone to the opiate receptor (C) in controls (solid line) and clorgyline-treated animals (dotted line). Details as in legend to Fig. 1, Materials and Methods, section and in Tables I-III.
118 TABLE I Statistical analyses of" circadian rhythms." one-way analysis of variance Controls F
Clorgyline P
Least-squares-difference* P < 0.01
Forebrain receptor binding Alpha-adrenergic 6.53
< 0.0003 2 > 14, 6 18 > 6
F
P
P < 0.05
P < 0.01
2 > 10, 22 11.34 18 > 2 2 , 1 4 10, 22, > 6 22, 2 > 6, 14,18 4.26
< 0.0001 6, 10, 14 > 18, 22, 2
< 0.001
Beta-adrenergic
3.53
< 0.013
22, 2 > 18 22>6
Muscarinic cholinergic
27.23
< 0.0001
10, 2, > all
14 > 18, 22
5.17
Benzodiazepine
13.8
< 0.0001
14 > 2
9.1
Opiate
6.5
< 0.001
10, 14, 22, 6 > 18 10, 22, 6 > 2 6>14 22 > 6, 10, 14, 18 2>6
Striatal receptor binding Dopamine 2.94
< 0.025
Benzodiazepine
3.47
< 0.014
Concentration Pineal melatonin
7,19
< 0.0001 2, 6 > 14, 18, 22 6>10
18, 22, > 6
Least squares-difference*
22, 2, 6 > 10, 14 all > 6 18 > 14
< 0.004
P < 0.05
2,6,14>18 6>22
2>22 6 > 10
2, 6, 10, 14 > 18 < 0.0001 10, 14, 22 >2,6
10 > 22 > 18
23.3
< 0.0001 2, 6, 10 > 14, 18, 2
10 > 22
2.15
< 0.09
22>6
14>6
4.98
< 0.002
18, 22 > 10 1 8 > 14, 22
2 > 14, 22 18>6
4.46
< 0.003
2, 6, 1 0 > 2 2
6>14
18 > 6
* Procedure determining which time points (in hours) are significantly different from each other ~6. n M ) ; a c e t y l c h o l i n e r e c e p t o r (0.021 ~: 0.002 n M a n d 0.032 :~ 0.004 n M ) ; d o p a m i n e r e c e p t o r (1.7 zk 0.08
A
n M a n d 1.5 ~: 1.1 n M ) ; o p i a t e r e c e p t o r (1.2 ~ 0.05 n M a n d 1.2 ± 0.02 n M ) ; a n d b e n z o d i a z e p i n e r e c e p t o r (3.1 Jz 0.3 n M a n d 3.5 ~: 0.5 n M ) .
~ 2
A c h a n g e i n n u m b e r , n o t affinity, h a d p r e v i o u s l y b e e n o b s e r v e d i n studies o f c i r c a d i a n c h a n g e in, a n d the effects o f p s y c h o t r o p i c d r u g s o n , b i n d i n g to different receptors.
The significance of changes over time were established b y o n e - w a y analysis o f v a r i a n c e ( A N O V A ) : all receptors showed highly significant rhythms both
11 ~o
i~ ~ -~ ~-2
B.
8
i n c o n t r o l s a n d c l o r g y l i n e - t r e a t e d g r o u p s , w i t h the single e x c e p t i o n o f the d o p a m i n e r e c e p t o r after
zO
7
Fig. 3. Specific binding of [SH]spiroperidol to the dopamine receptor (A), [SH]diazepam to the benzodiazepine receptor (B) in striatal membrane preparations. The rhythm in control animals is indicated by the solid line; in clorgyline-treated animals by the dotted line. Details as in legend to Fig. 1, Materials and Methods, section and in Tables I-III.
<~ o~ 4 3
2
6
10
14
18
22
T I M E OF DAY ( h r )
2
6
10
119 800
melatonin (Tables II, III). The time-dependent process (two-way interaction of the drug with time) was significant for all receptors (Table II).
!
DISCUSSION z
400
-
200
-
Z
0 _J uJ
2
N N
6
I0
14
18
22
2
6
I0
TIME OF DAY (hr)
Fig. 4. Melatonin concentrations in pineal glands measured at 6 times points throughout the 24-h day, in controls (solid line) and clorgyline-treated animals (dotted line). Melatonin concentrations in the light phase are significantly higher after clorgyIine treatment (see Tables I-III). clorgyline treatment (P < 0.09) (Table I). For each group, a further analysis by the least-squares-difference procedure indicated which time points were different from each other and at which level of significance za (Table I). The effect of drug treatment was analyzed by two-way ANOVA, i.e. the effect of clorgyline on time-independent (e.g. 24-h mean) and time-dependent (e.g. phase) processes. Analyzed as a time-independent process, clorgyline treatment significantly affected a-adrenergic, cholinergic, striatal dopamine and benzodiazepine binding, and pineal
There are two levels of analysis in this study: first, the effect of clorgyline on individual neurotransmitter systems, and second, the possible effect of this drug on a central circadian oscillatory system. The limitations in determining the shape of a circadian rhythm imposed by the sampling intervals (4 h) need to be noted, and the lack of precise temporal resolution might account for the apparent irregularity in many of the receptor rhythms. Seven similar 24-h studies of receptor rhythms in untreated rats have documented that these receptors change with time of day l°,n,13,2°-22,z7,a°. In our studies we have observed that there are apparent phase shifts in the timing of peak binding to a given receptor. However a circadian variation is always present, with changes throughout the day of an order of magnitude commonly reported for differences between groups studied at a single time point (e.g. after lesions). The effects of drugs on these rhythms are profoundll,12,20,34,35,ag. Clorgyline changes the temporal pattern of binding to each receptor. The changes are in part consistent with the MAOI action of clorgyline 1,z. Clorgyline is a specific inhibitor of MAO-A, and would preferentially block the deamination of noradrenaline and serotonin. It is likely that the dose schedule
TABLE II Statistical attalysis of clorgyline effects: two-way analysis of variance Drug treatment (time independent process)
Two-way interaction (time-dependent process)
F
P
F
5.94 2.99 199.23 0.21 0.62
< 0.02 n.s. < 0.0001 n.s. n.s.
12.84 4.10 5.14 8.93 11.25
952.82 211.80
< 0.0001 < 0.0001
2.57 2.76
< 0.035 < 0.03
< 0.013
0.97
n.s.
P
Forebrain receptor binding
Alpha-adrenergic Beta-adrenergic Muscarinic cholinergic Benzodiazepine Opiate
< < < < <
0.0001 0.003 0.001 0.0001 0.0001
Striatal receptor binding
Dopamine Benzodiazepine Concentration
Pineal melatonin
6.46
120 TABLE HI
Methodology, 24-h mea~ binding, and rhythm amplitude Coefficient of var24-h mean binding ( ± S.E.M.) iation in controls* (%) (n) pmol/g wet weight
Amplitude Peak: Trough (%)
bttraInterbtdividual
Controls
Clorgyline
P**
Controls
Clorgyline
Alpha-adrenergic
5.4
11.3
45
38
5.4
11.2
n,s.
26
26
Muscarinic cholinergic
4.6
6.8
< 0.001
56
30
Benzodiazepine
3.1
3.8
n.s.
25
20
Opiate
7.9
39.0
4.23 ~ 0.10 (41) 1.50 =E 0.04 (39) 18.40 zb 0.34 (38) 11.60 zb 0.20 (41) 1.42 ~ 0.11 (31)
< 0.055
Beta-adrenergie
4.53 ± 0.12 (38) 1.56 ~- 0.04 (35) 14.77 ± 0.44 (37) 11.60 ~ 0.20 (41) 1.39 ± 0.07 (27)
n.s.
78
161
Doparnine
6.5
21.8
21
35
3.0
10.8
2.53 % 0.08 (36) 3.76 ~ 0.14 (38)
< 0.001
Benzodiazepine
9.36 2~ 0.22 (41) 6.32 ± 0.15 (35)
< 0.001
32
47
274 ± 46 (42)
410 ~ 48 (42)
< 0.04
520
355
Forebrain receptor binding
Striatal receptor binding
Concentratiott Pineal rnelatonin (pg/gland)
* Mean coefficient of variation calculated as S.D. × 100/mean for each assay (n = 42 for intra; n ~ 6 for inter). ** Student's two-tailed t-test. used in this study may cause some inhibition of MAO-B. By inhibiting monoamine metabolism and inducing prolonged increases in noradrenaline and possibly dopamine, one effect would be a subsensitivity of the adrenergic and dopamine receptors. The results suggest that two weeks of clorgyline treatment tended to decrease adrenergic receptor binding; previous results had shown that 3 weeks of clorgyline treatment induced a significant decrease (when measured at a single time point)2,4. The finding of a large decrease in mean binding to the dopamine receptor is consistent with this hypothesis, although it does not support other studies whose results suggest suggest an agonist-induced supersensitivityS,14. There are no single time point studies of the effects of clorgyline on cholinergic, opiate, or benzodiazepine receptors, as these systems have not been considered related to the drug's use as an MAOI. In terms of up and down regulation of receptor number over 24 h, there are but 3 significant changes: marked decreases in binding to striatal dopamine and benzodiazepine receptors at all times of day,
and increased binding to forebrain cholinergic receptors at all times of day. The 24-h mean number of forebrain benzodiazepine and opiate receptors is unchanged. In general, the results are interesting in that they demonstrate that both increased and decreased binding to a given receptor can coexist, separated only in the temporal dimension. This may explain some of the discrepancies in the literature, since results may vary depending on the time of day the experiment was carried out : for example, after clorgyline treatment there is increased a-adrenergic recep tot binding in the early morning (27 % increase, t = 2.51, P < 0.03), no significant differences in the afternoon, and decreased binding in the evening (32 % decrease, t = 6.32, P < 0.0002) (Fig. IA). The effect of clorgyline treatment on pineal melatonin is particularly interesting. Light at any time suppresses metatonin secretion, and on a normal light-dark cycle the onset of light suppresses the last hours of the nocturnal secretory phase, i.e. it truncates the rhythm. The most parsimonious explanation of the drug effect is that it does not change the
121 phase-position of the rhythm, but blocks the suppressing effect of light, so that melatonin secretion persists during the early light phase. Other experiments support this interpretation 5. Two further effects of clorgyline on rhythm parameters are (a) there is often a change in the shape of the rhythm with drug, and (b) there is a shift in the timing of peak binding to later in the day. Interpretation of the phase-shifting effects of clorgyline on circadian receptor rhythms can be made with respect to the phase-shifting effects oF clorgyline on the circadian rest-activity cycle. Chronic clorgyline delays the onset of nocturnal running activity in hamsters entrained to a light-dark cycle, and does so in a dose-dependent mannerL The phase-delay under entrained conditions is related to the lengthening of the period of the rest-activity cycle by clorgyline under free-running conditions of constant darkness35,36,d8, 39. Furthermore, preliminary studies in vivo show that clorgyline lengthens the period of free-running rhythms when administered directly to the postulated central pacemaker, the suprachiasmatic nuclei (SCN) of the anterior hypothalamus 19, but not when administered in other brain regions (Groos, unpublished observations) 3s. Thus the effect of clorgyline to slow the frequency of a central circadian pacemaker may be reflected in the delayed phase-position of many neurotransmitter receptor rhythms. Very few drugs have been found that can modify the frequency of circadian rhythmsZ3, 24. It may be
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