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Circadian rhythms in mammalian neurotransmitter receptors
Progress in Neurobiology Vol. 29, pp. 219 to 259, 1987 Printed in Great Britain. All rights reserved
0301-0082/87/$0.00 + 0.50 Copyright © 1987 Pergamon Journals Ltd
CIRCADIAN RHYTHMS IN MAMMALIAN NEUROTRANSMITTER RECEPTORS ANNA WIRZ-JUSTICE
Psychiatric University Clinic, Basel Switzerland
(Received 10 February 1986)
Contents 1. 2. 3. 4. 5.
6.
7. 8. 9. 10. 11. 12.
Summary Introduction Methodological issues The beta-adrenergic receptor of the pineal gland The mammalian CNS 5.1. Changes in receptor binding over 24 hours: first CNS studies 5.2. Replication of receptor binding rhythms and possible seasonal modulation 5.3. Persistance of circadian rhythms under constant conditions but not after SCN lesions 5.4. Regional differences in receptor rhythms 5.4.1. Frontal cortex and hypothalamus 5.4.2. Discrete brain regions 5.4.3. Negative results 5.4.4. Regional differences in rhythmicity: relationship to regional function? 5.5. Strain differences in receptor rhythms 5.6. Age and gender 5.7. Receptor rhythms: correlation with neurotransmitter turnover or second messenger 5.7.1. Presynaptic events 5.7.2. Postsynaptic events Receptor rhythms and receptor-mediated function 6.1. Opiate receptors and pain sensitivity 6.2. Serotonin receptor stimulation and behavioural response 6.3. Alpha2-adrenergic receptor stimulated feeding 6.4. Alphas- and beta-adrenergic stimulated behaviours in the open field Retinal input to the circadian system Antidepressant drugs and receptor rhythms Human receptor rhythms Hormone receptors Mechanisms of rapid receptor change Conclusions Acknowledgements References
219 220 221 223 223 223 226 227 229 229 232 232 232 238 240 241 242 244 245 245 246 246 247 248 248 252 253 254 254 255 255
1. Summary At the present time, the following summary statements can be made as to 24-hour changes in receptor binding. (1) In all receptors studied in homogenates from whole rat forebrain (~t~, ~t2, fl-adrenergic, muscarinic cholinergic, dopaminergic, 5HT-I, 5HT-2, adenosine, opiate, benzodiazepine, GABA, imipramine), significant variations over 24 hours have been documented. (2) The receptor rhythms measured change in wave form, amplitude, and phase throughout the year, even though the animals have been kept on a defined and constant LD cycle. Whether these rhythms are truly seasonal requires further investigation. (3) The rhythms are circadian: i.e. they persist in the absence of time cues, and the unimodal rhythms do not persist after lesion of the putative circadian pacemaker in the suprachiasmatic nuclei. 219
220
A. WIRZ-JUSTICE
(4) The rhythms can be uni- or bimodal, and each brain region shows a particular pattern. The pattern can be different for the same ligand in different nuclei of a given brain region (e.g. hypothalamus). (5) Nearly all studies of receptor rhythms have been carried out in rats; the results vary according to strain and even within the same strain from different breeding lines. (6) Receptor rhythm characteristics are modified by age: e.g. the amplitude, phase, as well as the 24-hour mean of binding to a given ligand in a defined brain region. (7) The changes in number of binding sites over 24 hours can be correlated with amine turnover, second messenger, or function of that brain region; however these relationships, although consistent within a region, do not hold for all regions. (8) If gradual changes in CNS neurotransmitter receptor function are considered important in the pathogenesis of schizophrenia and affective disorders and the mode of action of psychopharmacological agents, then consideration of the short term rapid change over 24 hours is equally necessary. Chronic treatment with a number of psychoactive drugs known to induce up- or down-regulation of receptor number, also induces marked changes in circadian rhythm parameters of wave form, amplitude, phase and 24-hour mean. This is of methodological importance for single time-point studies, since the interpretation of the results will depend on time of day. (9) Preliminary evidence supports the assumption that the significant variation in receptor binding throughout the day may underlie the well-known circadian rhythms of susceptibility to many CNS drugs. (10) New findings of circadian rhythms in receptors on blood cells indicate the relevance of these changes also in human physiology. 2. Introduction
The existence of non-decaying, repetitive phenomena in biology was long considered a consequence of specific vital forces in living organisms. However, spontaneous selfsustaining periodicity is found in chemical reactions and physical systems; all major biological processes are periodic (Garfinkel, 1983). Oscillations are observed at all levels of organisation from single organisms, collections of cells, or in populations of subcellular organelles. The period length ranges from less than a second to days to a year. Periodicity can be considered to represent a basic phenomenon in biological systems that confers positive functional advantages. Rapp has concisely defined the evolutionary advantages of periodic biological control: the mechanisms of entrainment and synchronisation are a natural property of biological oscillators; oscillators that structure time can also be used to order physically separated events; endogenous oscillators can predict repetitive events; periodic signalling is metabolically efficient; and finally, frequency dependent control systems can succeed in environments in which amplitude dependent control fails (Rapp, 1979; 1987). Circadian rhythms (circa die = about a day) are the subject of this review, rhythms of ca. 24 hours that are intrinsic to the organism, not the consequence of periodic stimulation, as they persist in the absence of external time cues. Endogenous circadian rhythms can be found not only in the entire organism but also in organs, tissues or even isolated cells. In the absence of time cues or "zeitgebers", the natural frequency usually differs slightly from 24 hours. Light entrains the circadian system to the 24 hour day. Circadian rhythms have been shown to be genetically determined--at the molecular level or within species or strains. This intrinsic temporal order is an adaptation to the temporal structure of the environment. The selective advantages are that the peak of each rhythm occurs at a particular phase of the light: dark cycle, and the sequential order of metabolic and physiologic rhythms coordinate the optimal timing of interdependent functions, while separating incompatible ones. Thus the organism can respond appropriately in its behaviour to the demands of the changing environment. The formal properties of the circadian system have been elucidated over the past twenty years, in particular through the work of Pittendrigh, Aschoff and Wever. With the
CIRCADIAN RHYTHMS
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discovery of a neuroanatomy of mammalian temporal organisation, the accent of much research has moved towards neurobiology. Some of the questions now being asked are: Is one central circadian pacemaker--the suprachiasmatic nuclei (SCN) of the anterior hypothalamus--sufficient to explain the diversity of rhythmic phenomena, or is a multi-oscillator system necessary? What zeitgebers synchronise circadian rhythms and through what receptive organs is this information mediated? What is the neurochemistry of rhythm generation itself and of the pathways for entrainment? A large body of neurobiological literature attests to the phenomenology of circadian change. The pulsatile secretion of hormones shows a circadian profile and a monthly or seasonal modification of rhythm parameters--total secretion over 24 hours, wave form, phase and amplitude. The sleep-wake cycle manifests a rhythm of REM-NREM sleep with period of ca. 90' as well as marked dependence of sleep architecture on circadian phase. Since such physiological and endocrinological parameters are regulated by neurotransmitters and neuropeptides in discrete brain regions, and temporally organised by the SCN, it is not surprising that circadian variations in neurotransmitters and their precursors, their synthetic and metabolic enzymes have been well documented. In addition, the effect of drugs is modified by time of day (for review see Reinberg and Smolensky, 1983). First, by the kinetic parameters of absorption, liver metabolism, renal elimination, binding to plasma proteins, peak plasma concentration or the time required to attain this peak, distribution volume or clearance. Second, and independently, by the circadian variation in susceptibility of a system to a given drug. Since it is the interaction of a medicament with a receptor that permits pharmacologic activity to be expressed, it may be that receptors undergo circadian variations in their number or in their availability. It is from this field of chronopharmacology that the concept of possible CNS receptor change throughout 24 hours was developed. The concept was met with scepticism from "classical" pharmacologists, although less from neurobiologists acquainted with the characteristics of circadian rhythms. The reasons for this scepticism centred around two important issues: the difficulties of using receptor binding techniques to study the functional significance of receptor populations; the general assumption that in vivo the process of receptor change is slow, possibly requiring days to weeks to occur. Studies of the mechanisms of membrane-receptor regulation, especially those concerning rapid changes in brain receptor binding, may provide a conceptual and experimental approach to understanding mechanisms of circadian rhythms in CNS receptors.
3. Methodological Issues The development of the remarkably simple technique of radioligand binding has been instrumental in detailed study of how biologically active substances interact with their target tissues. This review deals with receptor binding techniques that are in common use. Radioligands used for such experiments have been defined pharmacologically as showing different specificities for a given receptor population. A CNS receptor binding site is defined as showing stereospecific, high affinity for a radioligand with Kd in the piconanomolar range; a saturation curve with a maximal number of binding sites (Bmax); and selective displacement with a series of related compounds. In spite of the apparent simplicity and straightforward technique, there are many subtle details of a binding assay that may account for discrepancies in the apparent number of binding sites measured in different laboratories.
222
A. WIRZ-JUSTICE
In this review, attention will be paid to factors that are also important in single time point studies of receptor binding: age and gender, species, strain and line, light: dark cycle and its intensity, time of year, correlation with amine release or receptor stimulated post-synaptic events. Outside the scope of this review, but recognised as a real source of variance between different assays, are: membrane preparation (centrifugation speed, number of washings); differences in timing of the assay after collection (storage effect); buffer systems and their concentrations; addition of the antioxidant ascorbate or a monoamineoxidase inhibitor; binding of radioligand to glass filters or plastic tubes; filtration set-up (multiple ports with vacuum gradient); differences in protein content used in the assay or methods used to calculate protein concentration (Leslie et al., 1980; Kraeuchi et al., 1981; Weiner et al., 1982; Muscettola et al., 1986). Such methodological factors at the level of the binding assay itself are not trivial, as pedantic neurochemists who try to standardise and optimise their assay well know. Much time needs to be invested to attain the level of reproducibility that is necessary for measuring relatively small differences in receptor binding. Most pharmacological and lesion studies of receptor populations deal with changes of the order of magnitude of 15-40% , rarely more. Further methodological issues in 24-hour studies of receptor binding are found in Section 5.2. Circadian rhythms are defined as having a period of ca. 24 hours, ultradian rhythms a period of less than 20 hours, and infradian, long-term rhythms a period of greater than 28 hours (usually days, months, years). There are practical limitations to the experimental measurement of circadian rhythms in vitro. Receptor binding studies are of necessity invasive, cross-sectional measures of values from individual animals, and therefore subject to greater variance than if intraindividual receptor sensitivity could be measured over time. The requirement that the number of animals per study be kept to a reasonable minimum has led to various strategies to increase information yield. Too few time points can skew the shape of a rhythm and the conclusions drawn: wave form is defined by the interval between time points. Studies using four or less time points are most likely to be confounded by type II errors, when no temporal variation is found. A single 24-hour period may also not be representative: more convincing (but more tiring to do) are studies repeating the first time point (Brennan et al., 1985; Di Lauro et al., 1986), or the entire 24-hour span (Kafka et al., 1986a; Campbell et al., 1986) and finding similar patterns. A further methodological issue involves the assay itself: if information is to be attained about whether circadian changes are in receptor number or affinity, it is necessary to carry out saturation or displacement curves. However the more precise the dissection, the less tissue is available for complete characterisation of binding. Additionally, if each membrane preparation is used for triplicate assays of total and nonspecific binding, the daunting aspect of such circadian studies--where all time points should be measured in random order in a single assay--becomes clear: an average day of around 600 samples. There is not only the qualitative aspect of changes in number or affinity. Quantitatively, for statistical analysis, an adequate number of individual animals needs to be measured at a given time point: a compromise has been to measure binding at one concentration of radioligand, followed by saturation or displacement curves in pooled homogenates at the times of maxima or minima. A few heroic studies have done both, thus establishing simultaneously the 24-hour time course of the number of binding sites together with appropriate statistics (Lee et al., 1984; Brennan et al., 1985; Di Lauro et al., 1986) (see Tables 3-6; Figs 8, 11). Receptor binding is followed by alterations of cellular metabolic events, such as enzyme activities or ion fluxes, that are ultimately expressed as characteristic physiologic or pharmacologic effects. In general, receptor binding studies are currently moving from phenomenology to this more difficult but more relevant aspect of receptor occupancy and its functional correlates. Circadian studies of receptors are still, however, largely at the descriptive level.
CIRCADIAN RHYTHMS
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4. The Beta-Adrenergic Receptor of the Pineal Gland The pineal gland has proved to be a useful model for studying the interaction of environmental and neural influences. The large amplitude rhythm in nocturnal secretion of the neurohormone melatonin is generated by a large rhythm in serotonin N-acetyltransferase activity. This in turn is regulated by a neural circuit from the retina via the SCN that stimulates rhythmic noradrenaline release from pineal sympathetic nerve endings: binding to fl-adrenergic receptors, cyclic-AMP and isoproterenol-stimulated increase of cyclic-AMP (Romero et al., 1975; Mikuni et al., 1981) all undergo circadian rhythms. In contrast, there is no large change in pineal ~tl-adrenergic receptor binding with time of day (Sudgen and Klein, 1985) (Fig. 1). In the pineal, fl-adrenergic receptor binding rhythms, as measured by conventional techniques, are correlated with physiological and pharmacologic response. A rapid change in the number of binding sites occurs within a few hours. It is surprising that the study of pineal fl-adrenergic receptors, one of the very first applications of the new technique of using highly labelled antagonist drugs to measure high affinity specific binding to membranes, did not lead to any further investigation of circadian variation in other receptor populations, nor was the pineal used as a model for studying the mechanisms of rapid and large amplitude changes (ca. 100%) in fl-adrenergic receptor binding. PINEAL ADRENERGIC RECEPTOR BINDING 120(]
111X
E lO :
i".
p
TiME OF DAY (hr) FIG. I. A comparison of the pattern of specific binding to cq- and fl-adrenergic receptors of rat pineal gland homogenates. The former is redrawn from Sudgen and Klein (rats kept on a 14:l0 LD cycle; radioligand: [t25I]-HEAT; N = 5-7 pineals; ANOVA n.s.); the latter, unpublished data, Wirz-Justice e t a / . (rats kept on a 12:12 LD cycle and then released into DD; radioligand: [3H]-DHA; N = 4 pools of 3 pineals; ANOVA p < 0.01).
5. The Mammalian CNS 5.1. CHANGESIN RECEPTOR BINDING OVER 24 HOURS: FIRST CNS STUDIES Although circadian and ultradian rhythms in all aspects of CNS neurotransmitter metabolism, from enzyme activity to release, have been repeatedly demonstrated, the possibility that neurotransmitter receptors also show circadian rhythmicity was considered seriously only for the fl-adrenergic receptor on the pineal gland. The classical concepts of long-term change in receptor number, as found after denervation or chronic treatment with drugs, prevailed. Thus the first CNS receptor rhythm studies emphasised the use of straightforward, established binding techniques (Table l), wholebrain or forebrain homogenates from rats that had been entrained to the usual laboratory 12:12 LD cycle,
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A. WIRZ-JUSTICE
TABLE 1. LIGANDS USED FOR MEASUREMENT OF SPECIFIC BINDING TO RECEPTORS IN TISSUE HOMOGENATES
Receptor fl-adrenergic
3H-Ligand DHA
~q-adrenergic
WB-4101 prazosin clonidine QNB spiroperidol
ct2-adrenergic muscarinic cholinergic dopaminergic
serotonergic opiate GABA benzodiazepine adenosine
5HT spiroperidol ketanserin naloxone morphine GABA diazepam flunitrazepam Ro 15-1788 1-PIA
Displacing drug propranolol alprenolol phentolamine phentolamine atropine ( + )butaclamol spiroperidol ( - )sulpiride haloperidol LSD, 5HT ketanserin methysergide naloxone levorphanol morphine muscimol diazepam clonazepam clonazepam 1 -PIA
and binding studies carried out in a single run (using randomised sampling) to minimise systematic errors, variance across days and across assays. The strategy was to have groups of animals similar to those that compare different treatment paradigms, the treatment being simply time of day. Statistical analysis in the former case is usually that of Student's t-test for independent variables; in the latter, multiple t-tests across the different time points cannot be used (although permitted for comparing peak to trough values). Furthermore, it is important not to make assumptions as to the wave form of the rhythm (as done in cosinor analysis), since wave form is modified by the interval between time points taken. It is more important to choose a conservative test of significance likely to be biologically relevant, than obtaining significance itself (e.g. analysis of variance, ANOVA). As a first step in defining methodology, a summary of the variation coefficients (SD/mean x 100) at three levels of experimentation can be made. First, for a given ligand, the assay variance of triplicate samples; second, for each time point, the interindividual variance; and third, the variance in ligand binding across the 24-hour time span, which reflects the amplitude of rhythmic change (Table 2). Table 3 clearly demonstrates that the variation coefficients of receptor binding over 24 hours exceed the single time point interindividual variance by a factor of 3-10. Thus an
TABLE 2. COEFFICIENT OF VARIATION* OVER A SERIES OF BINDING STUDIES
Inter-individual Amplitudeof Receptor Method (N = 7 rats/ rhythm binding (triplicates) timepoint) peak : trough % fl-adrenergic 5.2% 9.5% 35% cq-adrenergic 3.9% 10% 27% ~t2-adrenergic~ 7.0% 9% 39% muscarinic cholinergic 6.5% 7.9% 28% dopaminergic 6.7% 15.6% 75% opiate 10.4% 23. 1% 87% benzodiazepine 3. 1% 3.8% 40% imipramine:~ 5.7% 10% 35% * SD x 100/mean. Analysed from data summarised in Kafka et al., 1983; tKrauchi et al., 1984 (N = 3 pools of 2/time point); :~Wirz-Justiceet aL, 1983b(N = 4 pools of 9/time point).
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TABLE 3. 24-HouR RECEPTOR BINDING IN WHOLE FOREBRAIN HOMOGENATESFROM MALE RATS (IN A 12:12 LD CYCLE UNLESS OTHERWISE INDICATED)
Ligand/assay*/strain/ age/conditions
Rhythm (wave form)t/ amplitude (peak : trough %)/ N (time points) [hi (animals)/ significance (ANOVA unless otherwise noted)
Reference
DHA/I Sprague-Dawley 250 g/LD DD
C/26%/6 [7]/p < 0.01 C/28%/6 [7]/p < 0.001
Kafka et al., 1981a Kafka et al., 1981a
DHA/I Sprague-Dawley 200 g/DD SCN lesion
U/73%/6 [8]/p < 0.05 U/100%/6 [8]/,0 < 0.05
Kafka el al., 1985 Kafka et al., 1985
U/21%/8/n.s.
Lemmer et al., 1985
C/65%/8/p
< 0.001 C/120%/8/p < 0.0001 U / 2 2 % / 8 / p < 0.01
Lemmer et al., 1985 Lemmer et al., 1985 Lemmer et al., 1985
WB-4101/I Spragne-Dawley 250 g/LD DD
C/45%/6 [7]/p < 0.001 C/22%/6 [7]/p < 0.01
Kafka et al., 1981a Kafka et al., 1981a
WB-4101/I Sprague-Dawley 200 g/DD SCN lesion
C/28%/6 [8]/p < 0.01 U/14%/6 [8]/p < 0.05
Kafka et al., 1985 Kafka et al., 1985
QNB/I Sprague-Dawley 250 g/LD DD
U/56%/6 [7]/p < 0.001 C/71%/6 [7]/p < 0.001
Kafka et al., 1981b Kafka et al., 1981b
QNB/1 Sprague-Dawley 200 g/DD SCN lesion
U/20%/6 [8]/p < 0.05 U/28%/6 [8]/p < 0.05
Kafka et al., 1985 Kafka et al., 1985
QNB/Bm.~ Sprague-Dawley 200 g (receptor occupation)/LD
C/100%/4 [6]/p < 0.001
Mash et al., 1985
5HT/2 Wistar-Wu 200 g/LD
C/215%/12 [3]/p < 0.001
Wesemann et al., 1986a
DHA/Bm, x Wistar-TNO, 150 g (adenylate cyclase, c-AMP, phosphodiesterase)
Spiroperidol/l Sprague-Dawley LD 14:10 Ontogeny: Postnatal day 15 prepubertal day 31 adult day 90
- / - / 8 [3-10]/n.s. t-test U/28%/10 [3-17]/p < 0.01 C/93%/13 [3-13]/p < 0.01
Morphine/Bronx Sprague-Dawley 200g/LD DD
C/106%/6 [6]/p < 0.05 C/314%/6 [6]/p < 0.01
Lee et al., 1984 Lee et al., 1984
Naloxone/l Sprague-Dawley 250 g/LD DD
C/78%/6 [7]/p < 0.001 C/89%/6 [7]/p < 0.001
Naber et al., 1981 Naber et al., 1981
- / 2 0 8 % / 2 [5]/p < 0.001 t-test
Hendrie et al., 1983
U/25%/6 [7]/p < 0.001 C/31%/6 [7]/p < 0.001
Kafka et aL, 1983 Kafka et aL, 1983
Naloxone/B~x Hooded-Lister 300 g/LD Diazepam/1 Sprague-Dawley 250 g/LD DD
Bruinink et al., 1983
Continued
A. WIRZ-JUSTICE
226
TABLE 3---continued
Rhythm (wave fo~'m)t/ amplitude (peak : trough %)/ N (time points) [n] (animals)/ significance (ANOVA unless otherwise noted)
Ligand/assay*/strain/ age/conditions Diazepam/1 Sprague-Dawley 200 g/DD SCN lesion N6-(l-phenyl-isopropyl) adenosine/B=ax Sprague-Dawley/LD
Reference
C/33%/6 [8]/p < 0.01 U/34%/6 [8]/p < 0.01
Kafka et al., 1985 Kafka et al., 1985
C/74%/6 [6]/p < 0.02
Virus et al., 1984
* Number of ligand concentrations = 1, 2, etc; or Scatchard Plot = B ~ . * Circadian (unimodal) = C; Ultradian (bimodal) = U. LD = light:dark cycle; DD = continuous darkness.
explanation of these daily changes as mere "biological noise" is, at least in our studies, insufficient. Furthermore, the argument that a 20-40% change over 24 hours is unlikely to be relevant to receptor function should also be as exigently applied to non-circadian studies: this range is precisely that consistently demonstrated with most drug or lesion protocols. The first studies of binding to forebrain homogenates from rats on a 12:12 LD cycle showed significant changes throughout 24 hours. Although single concentrations of radioligand were used to measure specific binding, in further experiments pooled homogenates were subjected to saturation curves (at each time point, or at the times of maximum and minimum). In none of the experiments was a change in Kd observed (e.g. Fig. 2), indicating that the changes in specific binding throughout the day were changes in the apparent number of receptors (Kafka et al., 1983). 5.2. REPLICATION OF RECEPTOR BINDING RHYTHMS AND POSSIBLE SEASONAL MODULATION
Similar experiments in the same laboratory using the same rat strain and assays were repeated four times: each time, significant rhythms were found in st and fl-adrenergic, muscarinic cholinergic, opiate and benzodiazepine receptor binding in forebrain and in dopaminergic receptor binding in striatum (summarised in Kafka et al., 1983). One problem was immediately made clear: a given rhythm was not as reproducible in its wave-form and phase characteristics as was the pineal fl-adrenergic receptor rhythm. Since the animals were strictly entrained to the same LD cycle, the data suggested that either the changes were due to random variance, or due to an effect of season that was not one of photoperiod. The latter explanation would require finding the same pattern during the same month of consecutive years--a somewhat mammoth undertaking. The former was considered unlikely for reasons of methodology described above. A similar variability of circadian patterns had been found in repeated measurement of noradrenaline concentration in rat brain (Davies et al., 1972). In this context, some observations of apparent seasonal changes in number of receptor binding sites had already been made in single time point studies (in the light phase). For example, the density of naloxone binding sites in mouse brain homogenates was 73% higher in winter than in summer (Codd and Byrne, 1981); similarly, the number of muscarinic cholinergic receptors was 54% higher in spring than in summer (Kastrup and Christensen, 1984). In the seasonally reproductive ewe, pineal fl-adrenergic receptor density and affinity peak in May and November (Foldes et al., 1985). A phase-shift could explain such seasonal changes just as could a change in the 24-hour mean. In our studies, QNB binding in the early light phase could be high or low, depending on the rhythm at that time of year; additionally, changes in the 24-hour mean across
CIRCADIAN RHYTHMS
227
SCATCHARD PLOT OF OPIATE RECEPTORBINDING
10 hr
0.015 0.010i
? ~ !
0.015 0.010
0.006
14 hr • t
•
0.005 0.05 0.10 0.15
B/F
,
i
r• 0.015~
18 hr
0.010' • •
0.05 0.10 0.15 B/F L z\ 0.015g 0.010i
0.06 0.10 0.15
B/F 0.015
2 hr
0.05 0.10 0.15
6 hr
0.015
•
0.010
0.005
0,006 0.10 0.15
•e'X~
B/F
0.010
0.06
22 hr
"
0.06 0.10 0.15
[ IH]-NALOXONE SPECIFICALLYBOUND (pmol / 10 mO wet weigkt) FIG. 2. Scatchard plot of opiate receptor binding at 6 different times of day. Male Sprague-Dawley rats were kept on an LD cycle, and the entire forebrain assayed for specific naloxone binding (pool of N = 6 at each time point, experiment carried out in April) (from Naber et al., 1981).
seasons were found. This meant that although the lowest 24-hour mean QNB binding occurred in October, the value in the early light phase was higher in October than in April (Fig. 3) (Kafka et al., 1981b). In those receptors where binding showed both uni- and bimodal patterns [such as muscarinic cholinergic, Fig. 3, or ~l- and ~-adrenergic, benzodiazepine, or striatal dopamine receptors (Kafka et al., 1983)] no straightforward statement as to at which time maximum and minimum binding occurs could be made with respect to whole for•brain. However a unimodal rhythm in opiate receptor binding did show replicable characteristics of peak binding occurring in the dark phase (Naber et al., 198!; Wirz-Justice et al., 1981; Lee et al., 1984) (Figs 2, 18, 22). All data available for whole for•brain receptor rhythms are summarised in Table 3. 5.3. PERSISTENCEOF CIRCADIAN RHYTHMS UNDER CONSTANT CONDITIONS BUT NOT AFTER SCN LESIONS
The previous studies had been carried out in animals kept on an LD cycle. It is well known that light can directly modify behaviour and physiology by "masking" (Aschoff et al., 1981). By studying animals that had been kept in constant darkness (DD) during the last day(s) of the experiment, possible masking effects of light itself on receptor binding could be eliminated. In addition, a rhythm persisting under conditions without time cues satisfies an important criterion for an endogenous circadian rhythm generated within the brain itself.
228
A. WIRZ-JUSTICE pmol3H-QNB/ gramwetweight 24 forebrain January
i %
2O FEBRUARY
18
t
March 12 APRIL
10
I
I
22
/
May
S
J
JUNE
i 1/
July
August
18
*~
,
**
i
September 14] OCTOBER
November December
12:
* *p<0.01
I
* p<0.05 ,,e,,,i,A, 10
14
18
22
2
10
14
18
Timeof Day (hr) F I G . 3. 24-Hour patterns of [3H]-QNB specific binding to muscarinic cholinergic receptors in rat forebrain in four separate experiments (mean + s . e . m , o f N = 7 animals at 6 time points, except for April, where the 7 animals were pooled for Scatchard analyses). The shaded area indicates the dark phase to which the animals were exposed, the dotted lines the changing external photoperiod (from Kafka et al., 198 l b ) . A fifth experiment (data not shown) carried out in December-January in Switzerland with the Sprague-Dawley Ivanovas rat strain showed a bimodal pattern with peaks in the mid-dark and mid-light phase intermediate between the experiments in October and February ( W i r z - J u s t i c e et al., 1981).
Indeed, it was found that all binding rhythms remained significant in DD, and their wave form was greatly simplified to a unimodal pattern in most cases (Table 3, Fig. 4). Thus the persistence of significant changes over 24 hours in the absence of time cues indicates their endogenous nature (see also Naber et al., 1981; Lee et al., 1984 for similar patterns of opiate receptor binding in LD and DD). To address the even more crucial question of whether these circadian rhythms in receptor binding are driven by the putative circadian pacemaker in the SCN, a difficult study with SCN lesioned animals was undertaken by the Bethesda group (Kafka et al., 1985). The endogenous circadian rhythm in ~-adrenergic and benzodiazepine receptor binding found in controls was no longer present after SCN lesions: the patterns became ultradian with two peaks in 24 hours (Fig. 5). The/~-adrenergic and muscarinic cholinergic receptor rhythms were ultradian in control animals and this pattern persisted after SCN lesions. Thus SCN ablation appeared to cause a selective loss of circadian function in those receptors that showed unimodal rhythmicity: ultradian rhythms were not affected. This experiment indicates that the SCN is necessary for the maintenance of circadian expression of receptor binding, as it is for most other circadian functions that have been investigated.
229
CIRCADIAN RHYTHMS
RECEPTOR BINDING IN DO 12
BZP
Z L--J D .c
I~
1
•-~ ~
|,.~
Z FIG. 4. Circadian rhythm of binding to e~- and ~-adrenergic and benzodiazepine receptors in forebrain homogenates from rats kept under LD 12:12 for three weeks (as indicated by the vertical lines at 7 hr for lights on, at 19 hr for lights off), and then maintained for three days in DD. They were sacrificed under red light (N = 6 rats at 6 time points). All rhythms were significant by ANOVA; the peak values indicated are significantly different from the troughs (redrawn from Kafka et al., 1983). 5.4. REGIONAL DIFFERENCES IN RECEPTOR RHYTHMS M o n o a m i n e s , their enzymes and metabolites undergo circadian variations in the C N S that vary f r o m region to region and even a m o n g the nuclei o f a given region (e.g. N A and 5 H T metabolism in different brainstem nuclei ( K a n et al., 1977; Daiguji et al., 1978; Natali et al., 1980a, b; Chevillard et al., 1981; D a s z u t a et al., 1982; Semba et al., 1984; K o u l u et al., 1985; Rosenwasser et al., 1985). Thus the rhythms measured in whole forebrain are the sum o f all nuclei included in the dissection. F o r this reason, the focus shifted to studies o f regional differences in receptor rhythms. 5.4.1. Frontal cortex and hypothalamus Frontal cortex is the region most often used for studies o f drug effects on receptor binding: for this reason a n u m b e r o f groups have begun their investigations o f 24-hour rhythms in a brain area not noted for high amplitude rhythmicity. These results are summarised in Table 4. cc c A D R E N O C E P T O R B I N D I N G mCONTROLS ....SCN LESION
a O
,
o
L+
I 3
i 7
I | 11 D + 3
I 7
| 11
TIME OF DAY (hr)
FIG. 5. Circadian rhythm in ~q-adrenergic receptor binding in controls and ultradian rhythm in SCN lesioned animals (redrawn from Kafka et aL, 1985).
230 TABLE 4.
A. WmZ-JUSTtCE 24-Hour RECEPTORBINDINGIN CORTEX(OR REGIONSTrmREOF)FROMMALERATS(IN 12:12 LD CYCLE UNLESSOTHERWISEINDICATED)
Ligand/Assay/Strain/ Age/Conditions DHA/Bmu Sprague-Dawley 200 g/DD (fl-stimulated c-AMP) DHA/I Sprague-Dawley 200 g/DD
Rhythm (wave form)t/ amplitude (peak:trough %)/ N (time points) In] (animals)/ significance (ANOVA unless otherwise noted)
Reference
U/40%/6 [6]/p < 0.01 U/80%/6 [6]/p < 0.05 C/18-47%/6 [8]/p < 0.05 in 5/9 discrete cortical regions
Benedito and Kafka, 1982 Benedito and Kafka, 1982
DHA/B~x Wistar-King 250 g/LD
- / - / 4 [?]/n.s.
Matsubara et al., 1982
DHA/Bmx /
-/20%/7 [4]/n.s.
Di Lauro et al., 1986
- / 13%/4 [3-5]/n.s.
Fowler et al., 1985
DHA/l Wistar-Kyoto SHR 300 g/DD
- / - / 8 [6]/n.s. - / - / 8 [6]/n.s.
Wirz-Justice et al., 1983a
DHA/1 Wistar-Fuellinsdor f/LD 3 months 12 months 24 months
-/33%/6 [4]/n.s. -/23%/6 [4]/n.s. -/29%/6 [4]/n.s.
Jenni-Eiermann et al., 1985
- / 7 % / 6 [3]/n.s.
Wirz-Justice et al., unpubl, data
-/28%/2 [8]/p < 0.05 t-test
Mogilnicka et al., 1985
C/26%/6 [6]/p < 0.05 C/150%/6 [6]/p < 0.05
Benedito and Kafka, 1982 Benedito and Kafka, 1982
-/17%/7 [4]/n.s.
Di Lauro et al., 1986
-/14%/6 [4]/n.s. -/18%/6 [4]/n.s. -/22%/6 [4]/n.s.
Jenni-Eiermann et al., 1985
Kafka et al., 1986a
Wistar-Morini 300 g/DD DHA/Bmx Sprague-Dawley/LD
DHA/1 Wistar-Fuellinsdorf 350 g/LD DHA/Bmax Wistar 200 g/LD WB-4101/Bm,x Sprague-Dawley 200 g/DD or-stimulated c-AMP WB-4101/B~x Wistar-Morini 300 g/DD WB-4101/l Wistar-Fuellinsdorf/LD 3 months 12 months 24 months Prazosin/Bmax Wistar 200 g/LD Prazosin/l Sprague-Dawley 200 g/DD
-/19%/2 [8]/p < 0.05 t-test
Mogilnicka et al., 1985
C/44-117%/6 [8]/p < 0.01 in 7/7 discrete cortical regions
Kafka et al., 1986a
p-aminoclonidine/l Sprague-Dawley 200 g/DD
C/49-92%/6 [8]/,o < 0.05 in 3/6 discrete cortical regions
Kafka et al., 1986a
Clonidine/l Wistar-Fuellinsdorf 350 g/DD QNB/1 Fischer 10 weeks/LD
C/61%/8 [4]/,o < 0.05
Kraeuchi et al., unpubl, data
- / 10%/6 [3-4]/n.s.
Por and Bondy, 1981 Contmued
CIRCADIAN RHYTHMS
231
TABLE 4---continued
Ligand/Assay/Strain/ Age/Conditions
Rhythm (wave form)t/ amplitude (peak:trough %)/ N (time points) In] (animals)/ significance (ANOVA unless otherwise noted)
Reference
C/25-85%/6 [8]/p < 0.01 in 2/4 discrete cortical regions
Kafka et aL, 1986a
-/29%/6 [4]/n.s. -/19%/6 [4]/n.s. -/32%/6 [4l/n.s. -/14%/8 [4]/n.s. occipital cortex
Jenni-Eiermann et al., 1985
- / 9 % / 6 [3]/n.s. occipital cortex
Wirz-Justice et al., unpubl, data
25% increase in temporal 65% in visual cortex of dark-reared rats after 3 hr light/p < 0.01 t-test
Murphy et al., 1980
LSD/I Wistar-Fuellinsdorf/LD 3 months 12 months 24 months
U/29%/6 [41/p < 0.05 - / 1 ! %/6 [4]/n.s. U/24%/6 [4]/p < 0.05
Jenni-Eiermann et al., 1985
Spiroperidol/1 Wistar-Fuellinsdorf/LD 3 months 12 months 24 months
--/41%/6 [4]/n.s. -/17%/6 [4]/n.s. --/31%/6 [4]/n.s.
Jenni-Eiermann et al., 1985
Spiroperidol/1 Wistar-Fuellinsdorf 350 g/DD
-/20%/8 [4]/n.s. occipital cortex
Kraeuchi et al., unpubl, data
Spiroperidol/1 Wistar-Fuellinsdorf 350 g/LD
C/36%/6 [3l/p < 0.05 occipital cortex
Wirz-Justice et al., unpubl, data
Spiroperidol/Bmax Wistar-Morini 300 g/DD
-/10%/7 [4]/n.s.
Di Lauro et al., 1986
Ketanscrin/B~,ax Wistar (UK) 300 g/LD
-/50%/6 [6]/n.s.
Campbell et al., 1986
Imipramine/B~x Wistar-Morini 300 g/DD
-/16%/7 [4]/n.s.
Di Lauro et aL, 1986
Imipramine/1 Wistar-Fuellinsdorf 350 g/DD
U/36%/8 [4]/p < 0.05 occipital cortex
Kraeuchi et al., 1986
Imipramine/1 Wistar-Fuellinsdorf 350 g/LD
U/31%/6 [3]/p < 0.01 occipital cortex
Kraeuchi et al., 1986
Naloxone/l Sprague-Dawley/ 9, 15, 26, 30 days Female/Male
- / - / 4 / n . s . t-test
Jacobson and Wilkinson, 1985
Flunitrazepam Sprague-Dawley 200 g/DD Flunitrazepam/Bmx Sprague-Dawley 350 g/LD 14:10
C/45-125%/6 [81/p < 0.05 in 3/5 discrete cortical regions
Kafka et al., 1986a
C/50%/6 [3]/p < 0.001 t-test frontal cortex
Brennan et al., 1985
QNB/1 Sprague-Dawley 200 g/DD QNB/I Wistar-Fuellinsdorf/LD 3 months 12 months 24 months QNB/1 Wistar-Fuellinsdorf 350 g/DD QNB/I Wistar-Fuellinsdorf 350 g/LD 5HT/1 CFHB 55 days
Kraeuchi et aL, unpubl, data
Continued
232
A. WIRZ-JUSTICE TABLE 4 - - c o n t i n u e d
Ligand/Assay/Strain/ Age/Conditions Human Autopsy Material Scopolamine/1 5HT, Spiperone/Bmax
Rhythm (wave form)t/ amplitude (peak:trough %)/ N (time points) [n] (animals)/ significance(ANOVA unless otherwise noted) C/137%/8 [345]/p < 0.02 t-test - / - / 4 [18-20]/n.s. t-test
Reference Perry et al., 1977 Wester et al., 1984
Whereas in whole forebrain significant 24-hour variations were found in all studies (Table 3), only about 40% of measurements in cortex (or defined areas thereof) evidenced significant rhythmicity (Table 4). The hypothalamus more often (60%) showed large amplitude rhythms (Table 5).
5.4.2. Discrete brain regions The diversity of results with respect to the existence or not of circadian rhythms in neurotransmitter receptors predicates caution. More detailed dissection has revealed circadian or ultradian receptor rhythms in some, but not all regions, even within a given area such as the cortex (e.g. Fig. 6). This complexity is entirely analogous to that found in the numerous studies on circadian rhythms in neurotransmitters themselves. We have been able to replicate receptor patterns when precisely defined punched-out brain areas have been used. For example, the SCN itself shows a robust circadian rhythm in imipramine binding with a minimum at dusk and a maximum at dawn (Fig. 7). A smaller amplitude, ultradian rhythm in imipramine binding is found in the occipital cortex, whereas the caudate putamen shows no significant changes over 24 hours (Wirz-Justice et al., 1983b; Kraeuchi et al., 1986).
5.4.3. Negative results Some groups have carefully executed studies that show no rhythm in maximal number of binding sites (Fowler et al., 1985; D'i Lauro et al., 1986; Campbell et al., 1986) (e.g. Fig. 8). These negative findings need to be included in any discussion of receptor rhythms, but cannot be used to refute categorically their existence; there are a number of experimental factors apart from brain regions that can account for such differences, such as strain, LD or DD conditions, the number of time points and the number of animals per time point.
5.4.4. Regional differences in rhythmicity: relationship to regional function? It is only when repeated studies under adequate conditions consistently show no change over 24 hours, that a negative finding could be as functionally relevant for the given receptor system as a positive finding in another region. A detailed example illustrating this concept is therefore presented: ct2-adrenergic receptor binding in the locus coeruleus (LC) and in the medial hypothalamus (MH). The LC contains the largest clusters of noradrenaline-containing neurons in the CNS. There is evidence for ~tE-adrenergic receptors in the LC mediating inhibitory responses (Aghajanian and VanDer Maelen, 1982), and being involved in regulation of the sleep-wake cycle (Drew et al., 1979). There is no circadian modification in binding to ct2-adrenergic receptors in the LC of control Wistar rats nor in animals whose sleep-wake cycle is phase shifted by chronic methamphetamine treatment, nor after one or four weeks drug withdrawal (Wirz-Justice et al., 1985a and unpublished data) (Fig. 9). Noradrenaline
CIRCADIANRHYTHMS
233
TABLE 5. 24-HouR RECEPTORBINDINGIN HYPOTHALAMUS(OR REGIONS* THEREOF) FROMMALE RATS(IN 12:12 LD CYCLE UNLESSOTHERWISEINDICATED)
Ligand/Assay/Strain/ Age/Conditions
Rhythm (wave form)f/ amplitude (peak : trough %)/ N (time points) [hi (animals)/ significance (ANOVA unless otherwise noted)
Reference
DHA/1 Sprague-Dawley 200 g/DD
C/100%/6 [81/p < 0.05
Kafka et al., 1986a
DHA/1 Wistar-Fuellinsdorf 350 g/LD
U/40%/6 [3]/LH, p < 0.05
Kraeuchi et al., 1984
DHA/Bm~/
-/29%/7 [41/n.s.
Di Lauro et al., 1986
Matsubara et al., 1982
Wistar-Morini 300 g/DD DHA/Bm,~
- / -/4
Prazosin/1 Sprague-Dawley 200 g/DD
C/70%/6 [8]/p < 0.05
Kafka et al., 1986a
WB-4101/Bma~ Wistar-Morini 300 g/DD
-/27%/7 [4]/n.s.
Di Lauro et al., 1986
U/21%/8 [1/pool of 6 ] / C/39%/8 [l/pool of 6 ] / -
Wirz-Justice et al., 1983a
p-Amino-clonidine/1 Sprague-Dawley 200 g/DD
C/50%/6 [8]/n.s.
Kafka et al., 1986a
p-Amino-clonidine/1, B~x Sprague--Dawley 300 g/LD
C in SCN, PVN, SON, LH (of 8 discrete nuclei)
Jhanwar-Uniyal et al., 1986
Clonidine/1 Wistar-Fuellinsdorf 350 g/LD
C/39%/6 [31/p < 0.05
Kraeuchi et al., 1984
Clonidine/1 Wistar-Fuellinsdorf 350 g/DD
U/18%/8 [4I/VMH, p < 0.05 C/46%/8 [4]/PVN, p < 0.05
Kraeuchi et al., unpubl, data
U/20%/8 [pool of 6]/ C/31%/8 [pool of 6]/
Wirz-Justice et al., 1983a
QNB/I Fischer 10 weeks
C/20%/6 [3-4]/p < 0.06
Por and Bondy, 1981
QNB/1 Wistar-Fuellinsdorf 350 g/DD
- / 11%/8 [4I/POM, n.s.
Kraeuchi et al., unpubl, data
Spiperone/Bmax Wistar-Morini 300 g/DD
-/45%/7 [4l/n.s.
Di Lauro et al., 1986
Spiperone/1 Wistar-Fuellinsdorf 350 g/LD
C/23%/6 [3]/LH, p < 0.05
Wirz-Justice et al., unpubl, data
Imipramine/Bm,x Wistar-Morini 300 g/DD Imipramine/l Wistar-Fuellinsdorf 350 g/DD
-/15%/7 [4]/n.s.
Di Lauro et al., 1986
C/35%/8 [4I/SCN, p < 0.02 C/27%/8 [4]/POM, p < 0.05 C / 4 1 % / 8 [4]/AH, p < 0.05
Wirz-Justice et al., 1983b
C/30%/6 [3]/SCN, p < 0.05
Kraeuchi et al., 1986
WB-4101/I Wistar-Kyoto SHR 300 g/DD
•
Clonidine/l Wistar-Kyoto SHR 300 g/DD
lmipramine/1 Wistar-Fuellinsdorf 350 g/LD
[?]/n.s.
Continued J,PN 293--B
234
A. WIRZ-JUSTICE TABLE 5--continued
Rhythm (wave form)t/ amplitude (peak:trough %)/ N (time points) [n] (animals)/ significance (ANOVA unless otherwise noted)
Ligand/Assay/Strain/ Age/Conditions Naloxone/l Sprague-Dawley 9, 15, 26, 30 days
C/
Flunitrazepam/Bma x Sprague-Dawley
- / - / 6 [3]/n.s. t-test
Reference Jacobson and Wilkinson, 1985
/ 4 / V M H / p < 0.01
in females only Brennan et al., 1985
350 g/LD 14:10
* Abbreviations for hypothalamic regions: SCN, suprachiasmatic nucleus; PVN, paraventricular nucleus; SON, supraoptic nucleus; POM medial preoptic area; VMH, ventral medial hypothalamus; LH, lateral hypothalamus; AH, anterior hypothalamus.
]H-DIHYOROALPRENOLOL ($H-OHA) BOUNO
*=p<005 "* = o , ~ 0 , 0 1
50 OLFACTORY BUL8
ANOVA
40 30 SO
FRONTAL CORTEX
50
ENTORHINAL CORTEX
40 30
CINGULATE CORTEX
SO
.
.
.
.
.
.
4
7O
SO PIRIFORM CORTEX
SO 70 SO
INSULAR CORTEX
PARIETAL CORTEX
SO 5O
70 SO 8O
TEMPORAL 70 CORTEX SO 5O SO OCCIPITAL CORTEX HYPOTHALAMUS
50 40 20 50
HIPPOCAMPUS
40 3O
CEREBELLUM
30 22
PONS-MEDULLA
20 12
CAUOATE-PUTAMEN
50
THALAMUS SEPTUM
40 3O 20 10
14
18 22 TIME (hi)
2
6
FIG. 6. [3H]-DHA (0.8 riM) specific binding to fl-adrenergic receptors at 6 time-points throughout 24 hours in discrete brain regions. Sprague-Dawley rats were maintained on a 12:12 LD cycle as indicated (dark phase from 19-7 hr), and enucleated 2-3 days before the experiment (functionally without time cues). Each time point is the mean from 6-8 pools of regions from 3 rats. Significance rhythms were found in 7 of the 15 regions investigated (from Kafka et al., 1986a).
CIRCADIAN RHYTHMS
235
I,a~ Exp.1 e~Q Exp2 800
750
f .g
7OO
/ i
550 500=
550. 5o0
i
I
SCN
t
350. 300=
~
250=
30C | ~
25C
'
%
OC
200 |
*2
i
*6
|
|
*8 D *2
|
•
*6
*8
FIG. 7. A comparison between 24-hour rhythms and overall means of specific [3H]-imipramine binding in three different brain regions in two separate experiments. Exp. 1 was carried out in DD, with 6 month-old male Fuellinsdorf-Wistar rats in October; mean + SEM of 3--4 pools of 9 animals; N = 36 for the 24-hour mean. Exp. 2 was carried out in LD, with 3 month-old rats in March; mean + SEM of 3 pools of 2 animals (and a larger punch for the SCN area); N = 18 for the 24-hour mean. The black line represents the dark phase of the entraining LD cycle (from Kraeuchi et al., 1986).
in the LC also shows no circadian rhythmicity in one study of Wistar rats (Semba et al., 1984), which is consistent with these findings, but a significant rhythm in another study using Sprague-Dawley rats (Koulu et al., 1985). It is surprising that a drug that increases wake time by 4 hours does not even change the 24-hour mean binding to ~2-adrenergic receptors in the LC. This overall stability indicates that these noradrenergic receptors are involved neither in the rhythmicity of the sleep-wake cycle, nor in the homeostasis of sleep regulation. A different pattern is shown in another region studied in the same experiment. The medial hypothalamus, in particular the paraventricular nucleus, is involved in the regulation of feeding and corticosterone rhythms, both via an ~2-adrenergic receptor mechanism (Marino et al., 1983; Jhanwar-Uniyal et al., 1986). In this region we found a significant rhythm in [3H]-clonidine binding (Kraeuchi et al., 1984). After chronic methamphetamine treatment, ~2-adrenergic receptor binding increased at dawn together with increased feeding at this time of day. After drug withdrawal, the rhythm gradually returned to the control pattern (Fig. 10). Moreover, at one week's withdrawal--a time characterised by rebound eating and weight gain--an increase in the 24 hour mean of • 2-adrenergic receptor binding was found. Thus both the circadian and homeostatic aspects of feeding are reflected in parallel changes in medial hypothalamic ~-receptors (Kraeuchi et al., 1984).
236
A. WIRZ-JUSTICE
CORTEX
HYPOTHALAMUS
BRAINSTEM
200. 150, E I0050Oz
30( It ~1 2 5 0 -
- -L
_ -11
_ __T
200 E 150
X
100 -
--
" ....
,
...............
, .....
,.
•
0
300, 250
200 .~
150.
E
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o
00. 50.
X
/_ . . . . . .
"Z .......
.r_" . . . . . . .t" . . . . . . . .
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/-
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c
~5ooi ,150.
400. 350
_~
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T. ...... ....
. I . . . . Z . ......... ....... I ....... .T
T I M E OF DAY
FIG. 8. N o significant differences in Bmax over 7 time points in cq- and fl-adrenergic, spiroperidol and imipramine receptor binding in cortex, hypothalamus, or brain stem homogenates. Rats were kept on a 12:12 LD cycle as indicated, and then released into D D the day before the experiment. N = 4 pools of 6 rats at each time point (from Di Lauro et al., 1986).
Recently, Leibowitz' group has observed decreased ~(2-adrenergic receptor binding sites in the medial hypothalamus after 48 hours food deprivation; furthermore receptor change was circadian phase dependent and unusually rapid (Jhanwar-Uniyal et al., 1985). Three hours food deprivation at the beginning of the dark phase reduced binding by 65%, and six hours food deprivation at the end of the dark phase increased binding by 54%. These studies support our contention that it should be possible to correlate rapid changes in receptor number in specific regions with the function of the neurotransmitter in that region. A similar circadian rhythm of ~2-adrenergic receptor binding has been found in the paraventricular nucleus itself (Jhanwar-Uniyal et al., 1986). This study in punched-out brain areas is the most detailed up until now and beautifully illustrates the phenomenon: in only three out of fourteen brain areas--and these all in the hypothalamus--could a circadian rhythm in ct2-adrenergic receptor binding be demonstrated. However these three areas are precisely those with an important role in rhythmic function: the SCN, showing a rhythm with the same pattern as in the paraventricular nucleus, and the supraoptic nucleus, with a rhythm 180 ° out of phase with the other two regions (Jhanwar-Uniyal et al., 1986).
CIRCADIAN RHYTHMS
237
locus coeruleus [3HI - clonidine binding
~"*dp( 80. 6O 40. 20-
L+2
f.f~¢
+~ +10 D~2 +6 +10
A
80 60
20. ,
L+2
~(.
)
i
,
+6 +10 D+2 +6 +10
A-1
80_ 60.
20.
L+2
+6 +10 D•2
+6 ÷10
fmol/ mg pr. A - 4 80.
60. 40.
• & .5 ,lb D,2 ,6 .lb FIG. 9. Lack of circadian rhythm in [3H]-clonidine binding (3 riM) to ~2-adrenergic receptors in the locus coeruleus from 4 groups of rats kept on an LD cycle, as indicated by the shading. Group C = controls; A = methamphetamine for 4 weeks (100 mg/l drinking water; mean brain tissue concentration, 238 ng/ml); A - 1 = 1 week withdrawal from drug; A - 4 = 4 weeks withdrawal. Mean -t- SEM from 3 pools of two rats at each time point (Wirz-Justice et al.).
Thus an adequate description of receptor rhythms still remains to be established. Neurotransmitter receptors in discrete brain areas with known functional correlates are most likely to show replicable rhythmicity, or a lack thereof; in addition, parallel measurement of amine turnover or second messenger rhythms may be necessary (see Section 5.7).
238
A. WIRZ-JUSTICE medial hypothalamus
aH-Clonidine binding ~¢,.-r c 1001
'[
80
;'~S):" : '
L+2
+6 *tO D+2
+6
+10
r
r
T
L+2
+6 +10 D~2
+6
+6 ÷10 D~2
+6 +K)
fmolet mgw A 100'
+10
frrmbj
80
T
L+2
A-4 100,
no k+2
....... +6
+100+2
÷6
~i *10
FIG. 10. Patterns of [3H]-clonidine binding to ct2-adrenergic receptors in the medial hypothalamus. Details as in Fig. 9. All rhythms except after chronic methamphetamine treatment are significant (from Kraeuchi et aL, 1984).
5.5. STRAIN DIFFERENCES IN RECEPTOR RHYTHMS
The initial studies on receptor rhythms were carried out in the same line of SpragueDawley rats (Kafka et al., 1983). Naturally, replication studies in different laboratories have used different strains and/or lines of animals. Differences in sleep-wake or restactivity rhythms have been shown between strains of mice and rats (Oliverio and Malorni, 1979; Valatx, 1984; Lemmer et al., 1981), and differences in neurotransmitter rhythms found in the nuclei involved in sleep regulation (Natali et al., 1980a, b). Thus it is not surprising that such differences also exist for neurotransmitter receptors. Single time point studies have shown differences in catecholamine receptors between rat strains (Wolf et al., 1980; Helmeste et al., 1982; Chiu et al., 1982; Helmeste, 1983); another study found no differences in cholinergic receptors (Blaker et al., 1983). The circadian approach has been applied to the Wistar-Kyoto strain of spontaneous hypertensive rats (SHR) and their normotensive controls (WKY). Although blood pressure is higher in SHRs at all times of day, there are marked differences in many circadian rhythms: the rest-activity cycle shows earlier onset of nocturnal locomotion; plasma prolactin and stress-induced corticosterone secretion begins earlier in the light phase in SHR than in WKY rats; and adrenergic receptors show circadian phase dependent differences (Kraeuchi et al., 1983; Wirz-Justice et al., 1983a).
CIRCADIAN RHYTHMS
239
The mean 24-hour binding to ~-, ~2- and fl-adrenergic receptors was higher in SHR than WKY in all regions investigated, but greatest differences were found at the end of the dark phase. Again, there were pronounced regional differences• In the cerebellum, fl-adrenergic receptor binding was higher in SHR rats at all times of day, with an identical rhythm to that in WKY rats. Displacement curves indicated no difference in affinity either between time points or between strains (Fig. 11) (Wirz-Justice et al., 1983a). These detailed studies in WKY and SHR rats indicate that the differences between strains in most brain regions are small, and very dependent on time of day, the increase in adrenergic receptor binding being most consistently seen at the end of the dark phase. Furthermore, with respect to most efficacious therapy, it has been observed in rats that the anti-hypertensive drug, ct-methyl-DOPA, produces the greatest and most long-lasting decrease in blood pressure when administered in the dark phase (Post and Nair, 1975). A less obvious source of differences in receptor rhythms is not the strain of rats but the separate lines of a given strain that are used. There is as yet only one systematic investigation of this issue (Jenni-Eiermann et al., 1986). Large differences in a number of receptor rhythms were found in many brain regions from two rat lines of Wistar origin (Kfm: WlST and Fuellinsdorf Albino)• The 24-hour mean of QNB and spiroperidol binding in striatum, fl-adrenergic receptor binding in hippocampus, and GABA-receptor binding in cerebellum was more than double in Kfm compared with Fuellinsdorf rats. In seven out of eleven assays the Kfm line had rhythm amplitudes greater than 50%, whereas this was the case in only one out eleven assays for the Fuellinsdorf line. Finally, marked differences in phase position were observed between the two lines in six out of eleven assays• Since most of the studies of receptor rhythms in Basel have used the FueUinsdorf line, our increased number of negative findings compared with earlier studies (Kafka et al., 1983) may be related to this chance choice of one of the less rhythmic lines in the available sources of animals. An example of this difference between Wistar lines is shown in Fig. 12A. Such major differences in two closely related rat lines underline the difficulties in determining the patterns and timing of receptor rhythms. This problem is not unique to receptors: diurnal fluctuations of acetylcholine (Saito et al., 1975) and choline acetyl-
~-ADRENERGIC RECEPTOR BINDING IN CEREBELLUM - W K Y .....
SHR
~.0
•
,.
""" ••'•' i
"''/' ' '
.~\~(~ .'rm 32i o~
i
2-'L. ,~ ~ TIME OF DAY (hr)
~'° ~' ,o'° ~-o DrL~9 Concentration (M)
,~',
,o;
,~°
'
,°4
20
i
,co
23
,o-,
,~'
s
,~°
,~"
,e'
,
,~'
,
,°4
FIG. 11. [aH]-DHAbinding (1 riM)to fl-adrenergicreceptors in cerebellum.At each time point, N = 2 poolsof tissue from3 rats wereused for 6-pointdisplacementcurveswith L-propranol(from Wirz-Justice et al., 1983a).
A. WIRZ-JUST1CE
240
(A)
NB St r i a t u m
3000
e-
$ 0 e~
E \
1000 0
E
07
(B)
11
15
19
23
03
07
hr
19
23
03
07 hr
striatum
.E 5 0 e~
E ¢-
~) 3o 0 0 >,
'\+/
0 <
~
10
E
07
11
15
FIG. 12. QNB Binding (1 nM) (A) and choline acetyltransferase (CAT) activity (B) in striatum from Wistar-Fuellinsdorf (open circles, QNB: p < 0.05; CAT: n.s.) and Wistar-Kfm rats (closed circles, QNB: p < 0.001; CAT: p < 0.002) throughout the LD cycle (from Jenni-Eiermann et al., 1986).
transferase activity varied across region and between two strains of Sprague-Dawley rats (Nordberg and Wahlstroem, 1980) as well as Wistars (Figure 12b) (Jenni-Eiermann et al., 1986). 5.6. AGE AND GENDER
Most studies of receptor binding are carried out in adult male rats of ca. 200-300 g body weight. Similarly, there are as yet few studies investigating the modification of receptor rhythms by gender (in particular possible differential effects of puberty), or ontogeny and senescence. The only study comparing male and female rats at different ages found no opiate receptor rhythms in cortical slices (Jacobson and Wilkinson, 1985). However, in mediobasal hypothalamic slices, a complex developmental pattern was seen in both sexes. Female hypothalamus by 26 and 30 days of age showed a clear diurnal decrease in opiate receptor binding. These binding changes in prepubertal female rats have a functional correlate: the diurnal variation in LH response to opiate receptor blockade is maximal in the morning and minimal in the late afternoon.
CIRCADIAN RHYTHMS
241
A detailed study of the ontogeny of high affinity spiperone binding sites in forebrain (Bruinink, 1983) found no marked diurnal variations at postnatal day 15. At 30 days a sharp peak in the mid-dark phase was found for both dopaminergic and serotonergic binding sites. This pattern inversely reflected the bimodal activity-rest cycle at this age, and was similar to earlier findings of a bimodal rhythm of spiroperidol binding in striatum from another line of Sprague-Dawley rats (Naber et al., 1980). In adult rats (90 days) a different rhythm of spiperone binding was observed, with a decrease in the dark phase again inverse to the unimodal peak of nocturnal activity. A similar pattern was found in striatal spiroperidol binding in adult rats of the same strain (Wirz-Justice et al., 1981). Thus during ontogeny, certain receptor rhythms increase in 24-hour mean and amplitude, and change in wave form from ultradian to circadian. It is therefore of interest to note that ageing can induce the reverse: a decrease in 24-hour mean and amplitude, and dispersal of phase. Yet there is no linear trend with ageing that could be used as a generalisation (Jenni-Eiermann et al., 1985). Many ligands were used to measure regional receptor binding in 3, 12 and 24 month old male Fuellinsdorf Albino rats. In general, but not always, 24-hour mean binding was lowest in old rats. The amplitude of receptor rhythms was considerably less pronounced in adult than in young rats; however aged rats again had many rhythms of high amplitude. The phase position of peak binding of a given ligand in a given region also varied with age, with respect to each other and to the LD cycle. Noticeable was the increased dispersion of phase with age. An example is shown in Fig. 13. 5.7. RECEPTOR RHYTHMS: CORRELATIONWITH NEUROTRANSMITTERTURNOVER OR SECOND MESSENGER With this list of problematic issues related to replicability of receptor rhythms, a more general problem is introduced. For all studies involving receptor binding, there is no functional correlate of changes in apparent number. Thus the importance of a 30% change in binding can be assessed only with respect to statistical, but not biological significance. This is a fundamental drawback of the elegant and deceptively easy technique of radioligand binding. It is only in the pineal gland that the chain of metabolic events triggered by noradrenaline release from the superior cervical ganglion has been extensively studied (see Section 4). The rhythms in the pineal are of large amplitude. Since rapid changes in apparent number of fl-adrenergic receptors have been replicably found in the pineal, and linked to the sensitivity of adenylate cyclase stimulation, parallel phenomena should be measurable in CNS tissue.
CIRCADIAN RHYTHMS OF GABA-RECEPTOR BINDING WITH AGE
[3M
[tam
[ F M O L ] CEREBELLUHt 12S0.9
124
3H-GRBR/MU$CIHOL L. . . . . .
1000.0 750.~ 500.0
.
.
,
111~19230307 P=O,O03
111~19230307 P=O~O!
II1519230382 p= o,o13
FIG. 13. [3H]-GABAbinding(25 riM)in cerebellumfrom three age groups of malerats. N = 4/time point. All three rhythms are significant:however the 24 hour mean decreases with age, and a circadian rhythmbecomesan ultradianrhythmin 24 monthold animals(fromJenni-Eiermannet al., 1985, and Ph.D. thesis, Universityof Basel).
242
A. WIRZ-JUSTICE 5.7.1. Presynaptic events
The relationship between neurotransmitter turnover and receptor occupancy is not at all clear. Initially the concept prevailed that increased release of a neurotransmitter would lead to gradual development of post-synaptic receptor down-regulation. However this direct relationship is in reality complicated by differential changes in pre- and post-synaptic receptor populations to transmitter release. The circadian rhythms in the pineal gland do show this relationship. But already here, in well-defined systems, receptor differences are seen. Noradrenaline release over 24 hours and fl-adrenergic receptor binding show an inverse relationship, with only small changes in ~-adrenergic receptor binding throughout the day (Fig. 1). We have found CNS adrenergic receptor rhythms that follow a parallel, not inverse, time course. For example, the functional importance of noradrenaline in feeding regulation has been established. Feeding is regulated in the lateral hypothalamus via a fl-adrenergic receptor mechanism, and in the medial hypothalamus via an ~2-adrenergic receptor mechanism (Leibowitz, 1980) (Figs 10, 14 and Sections 5.4.4; 6.3). In the lateral hypothalamus, the patterns of presynaptic noradrenaline uptake (Van der Gutgen and Slangen, 1975), rate of neuronal firing (Schmitt, 1973), and fl-adrenergic receptor binding (Kraeuchi et al., 1984) are all bimodal. More important, the functional response of feeding induced by local injection of noradrenaline in the lateral hypothalamus also shows a bimodal rhythm (Margules et al., 1972): thus maximal fl-adrenergic receptor binding occurs at dusk and dawn when the feeding response is also highest (Fig. 14). Here begins the difficulty of interpretation: whereas in the pineal, high noradrenaline release led to decline in the number of fl-adrenergic binding sites, in the lateral hypothalamus, high I,J~T[RAL HYPOTHALAI'~ RHYThRS
3.-NA
VA~ DER GUGTEN
UPTAKE
1975
RATE OF DISCHARGE
so~mmtt 1973
I~AN RILl( INT~E AptIR 25vl NA znJ.
MaeGULtS 1972
3.-CHA BI~DIN6
KRAUeH[ [T AL.,
1983
FIG. 14. Lateral hypothalamic rhythms taken fromdifferentstudiesall showingbimodality:uptake of [3H]-NAand [3H]-DHAbinding together with firing rate and feedingstimulation after direct LH injection of NA.
CIRCADIAN
RHYTHMS
243
noradrenaline release (as indirectly measured by reuptake and firing rates) occurs at the same times as high fl-adrenergic receptor binding. In this same region we have also measured serotoninergic parameters (Fig. 15A, unpublished data). First, two putative serotonergic binding sites using the ligands spiroperidol (5HT-2 postsynaptic receptors) and imipramine (presynaptic 5HT-uptake sites) follow identical patterns: indeed, there is a high correlation between the amount of binding to the two ligands in individual animals (r = 0.75, p < 0.001). Second, the circadian rhythms of 5HT and its metabolite 5HIAA in the tissue extract also follow a remarkably similar pattern. In this context, it is perhaps interesting to note that a third serotonergic binding site, using 5HT as ligand (5HT-1 postsynaptic receptors) shows an inverse rhythm to that of 5HT itself (at least, when measured in forebrain) (Weseman et al., 1986a) (Fig. 16). Third, the pattern of 5HT-parameters in the LH is inverse to that found for fl-adrenergic receptors in the same region (Fig. 14). Do these patterns indicate that synthesis and release of 5HT and NA in the lateral hypothalamus are not directly responsible for receptor density, as in the classical model? Interactions between the levels of neurotransmitter regulation are still incompletely understood at the single time point level. Studies of circadian rhythms may appear more
cA,
LH
CONTROLS
(B)
LH M E T H A M P H E T A M I N E
n n
n
E
E
Iz
I-r
"6o"6=Ec~ ~E
.| E .~.
._r E i
o J Z
FIG. 15. Patterns of specific [3H]-spiroperidol and [3H]-imipramine binding, in LH homogenates, and 5HT and 5HIAA concentration in the supernatant (determined by HPLC, Dr. P. Waldmeier). A represents the group of control animals, B the group of animals treated 4 weeks with methamphetamine (both with N = 3 pools of 2 rats/time point), on a 12:12 LD cycle (unpublished data; experimental details in Kraeuchi e t al., 1984).
244
A. WIRZ-JUSTICE NORMAL
z
LIGHT/DARK
CTCLE
50' rio
30
,
\ \
20
T
/
I
~
10
~ -10 ~L x
~
Z
~ --30 ~, . . . . ,,
~ -40
m
5HT CONCENTRATION 5HT BINDING
UA if21 !
'0200
' '
0800
1,08
2000 0200 TIME OF DAY (hr)
Fla. 16. Diurnal variations of specific5HT binding (open circles)and 5HT concentrations(closed triangles) in rat forebrain homogenates, expressed as % deviation from the 24-hour mean for comparison. Animals were kept on a 12: 12 LD cycle;one way ANOVA for both rhythmsp < 0.001 (from Wesemann et al., 1986a).
complex, yet on the other hand could provide a physiological model to unravel the functional process of change unperturbed by pharmacological intervention. That pharmacological intervention itself may disrupt circadian organisation of these interrelated parameters is shown by the chronic methamphetamine study (Fig. 15B). Whereas in control animals (Fig. 15A) all 5HT parameters measured showed parallel rhythmicity in the LH, this was not the case after drug treatment. Peak 5HT and 5HIAA were both shifted to the light phase. Imipramine binding was not significantly different from controls. Spiroperidol binding showed quite a different wave-form and timing from controls that had no obvious correlation with any of the other parameters. Thus maximum release of 5HT and maximum receptor binding were no longer in phase. 5.7.2. P o s t s y n a p t i c events When ~j- and /~-adrenergic receptor binding in cerebral cortical slices, measured over 24 hours, are compared with noradrenaline-stimulated cyclic-AMP production in the slices, the circadian rhythms are similar (Benedito and Kafka, 1982) (Fig. 17). Thus it appears that the number of ~1- and/~-adrenergic receptors stimulated by noradrenaline in vitro can regulate the magnitude of cyclic-AMP production dependent on time of day. Yet in vivo, if discrete brain regions are analysed, the patterns become quite complex. First, the rhythms in noradrenaline release and metabolism do not confer the rhythms measured in adrenergic receptors; and second, the rhythms of adrenergic receptors and cyclic-AMP concentration are not in phase in every region (Kafka et al., 1986a,b). In the cingulate cortex, the peak cyclic-AMP concentration parallels the peak binding to the cq-adrenergic receptor; in contrast, in occipital, parietal, and temporal cortices, the rhythms are 180 ° out of phase. A further study of such relationships has recently been carried out by Lemmer, who had characterised circadian rhythms in the /3-receptor-adenylate cyclase-eyclic-AMPphosphodiesterase system in rat heart ventricles that varied with season (Lemmer and Lang, 1983; Lang et al., 1983; Lemmer et al., 1983). In forebrain,/~-adrenergic receptor binding showed little rhythmicity. However brain adenylate cyclase activity and cyclicAMP showed highly significant circadian rhythms of large amplitude ( > 1 0 0 % peak: trough), and phosphodiesterase an ultradian rhythm (Lemmer et al., 1985). Thus it appears that in brain the existence of circadian rhythms in functional response of receptors is
245
CIRCADIAN RHYTHMS
1 4
o=
s~
1
I
i
I
,~
1
Total cAMP (a- and ~-stimulated)
B
~-receptor (WB4101)
/x,
-
/J-receptor (DHA} * p = 0.05 * * p = 0.01
E_=
~'~
I
I
I,,
'
6
10 14 18 TIME OF DAY (hr)
1
J
22
2
FIG. 17. ~q- and fl-adrenergicreceptor mediated cAMP production, and specificbinding to cq- and fl-adrenergicreceptors in rat cerebral cortical slices measured at 6 points throughout the 24-hour day. Rats were maintained under a 12:12 LD cycle (lights on from 7-19 hr) for 18 days, then without time cues (enucleation)for 2-3 days before sacrifice.Each point representsthe mean + sem of 5-6 sliced hemicortices measured in triplicate. Significance(ANOVA) for cAMP: p < 0.01; ~-adrenergie receptor: p < 0.05; fl-adrenergic receptor: p < 0.01. Peaks marked significantly different from troughs (from Benedito and Kafka, 1982; Kafka et al., 1986b).
probably far more pronounced than the rhythms in the receptors themselves. As in all binding studies, the issue remains unsolved of how much change in receptor number is necessary for a functional modification of response.
6. Receptor Rhythms and Receptor-Mediated Function 6. l. OPIATE RECEPTORS AND PAIN SENSITIVITY We have described regional dependence of receptor rhythms and related function (Section 5.4.4). An example of a more general phenomenon is the correlation of opiate receptors and behavioural pain sensitivity. There are well-defined circadian rhythms in tail-pinch or paw-lick nociceptive responses in mice and rats (summarised in Kavaliers and Hirst, 1983). In nearly all studies, peak sensitivity was found in the dark phase. Circadian variations in brain endorphin content (Simantov et aL, 1980; Kerdelhue et aL, 1983; Jung et aL, 1984) also show maximal concentrations in the dark phase. Whole brain opiate receptor binding, whether measured with [3HI-morphine (Lee et al., 1984) or [3H]-naloxone (Naber et al., 1981; Wirz-Justice et al., 1981) showed a large peak in the dark phase. Some examples of opiate receptor binding and opiate analgesia are shown in Fig. 18. Measurement in brain regions such as the pontomesencephalic region (Lopez-Colome and Drucker-Colin, 1984) or hypothalamus (Jacobson and Wilkinson, 1985) (Fig. 18A) have indicated that a bimodal rhythm can also be present, with peaks in both the dark and light phase. However opiate receptor changes in these specific brain areas may be more related to other functions such as ultradian R E M - N R E M sleep rhythms or gonadotrophic hormone release, respectively. Only one study has directly compared receptor binding and nociception: greatest pain sensitivity occurs at the end of the dark phase when the maximal number of naloxone binding sites are found (Hendrie et al., 1983).
246
A. WIRZ-JUSTICE
6.2. SEROTONIN RECEPTOR STIMULATIONAND BEHAVIOURAL RESPONSE [3H]-Serotonin is considered to bind to 5HT-1 receptor sites. A significant circadian rhythm has been found in rat brain with maximum in the dark phase (Wesemann et al., 1983, 1986a). [3H]-Spiroperidol is the ligand used to measure 5HT-2 receptor sites in frontal cortex. A bimodal pattern of binding is seen in both frontal cortex and lateral hypothalamus: these rhythms are correlated with those found for [3H]-imipramine binding--a putative presynaptic site related to 5HT uptake. None of these receptor rhythms have been directly related to function. However 5HT behavioural rhythms have been investigated separately. The head twitch response induced in mice and rats when 5HT-2 receptors are stimulated by 5-MeODMT shows a circadian rhythm (Singleton and Marsden, 1981; Moser and Redfern, 1985, 1986). In contrast, the 5HT-syndrome that has been suggested to be mediated by 5HT-1 receptors shows no circadian rhythm (Moser and Redfern, 1985, 1986). In order to provide direct correlations between 5HT-induced behaviours and receptor rhythms, parallel studies should be made of 5HT receptors in that brain region relevant for the 5HT-induced behaviour measured. 6.3. ALPHA2-ADRENERGIC RECEPTOR STIMULATED FEEDING Clonidine elevates food intake in rats after peripheral or intra-hypothalamic application through an cq-adrenergic receptor mechanism in the paraventricular nucleus (McCabe et
(A)
OPIATE
RECEPTOR BINDING : O R E B R A I N c~ qaber et al.,
01
"B E n
198~ ~laber et al..
O
1981
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-6 E
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o i,I u)
1981
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1984
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.8
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CIRCADIANRHYTHMS
OPIATE
247
ANALGESIA
(B) Oliverio et al.. O
_z
1982
~,, >. o(J
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Puglisi-Allegra et al.,
ut
z
1982
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Hendrie et al., (J m
_1
1983
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Buckett,
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o. z < aJ (.)
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1981
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*4
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4
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FxG. 18. Diurnal rhythms of specific opiate receptor binding using [3H]-naloxone in rats on an LD cycle (A), compared with a number of different studies of opiate analgesia (B). References as cited. A study using [3H]-morphine (Lee et al., 1984) as ligand also found highest opiate receptor binding in the first half of the dark phase.
al., 1984). ct2-Adrenergic receptors in the PVN (and medial hypothalamus) show a circadian rhythm with peak binding at dusk and a trough at dawn (Leibowitz et al., 1984; Kraeuchi et al., 1984) (Fig. 10). We carried out a functional test of this rhythm: low doses
of i.p. clonidine stimulated feeding only at dusk when ~2-adrenergic receptors are high and not at dawn when they are low (Kraeuchi et al., 1985).
6.4. ALPHA I- AND BETA-ADRENERGIC STIMULATED BEHAVIOURS IN THE OPEN FIELD
Many behaviours in the open field show circadian rhythmicity (Vassout et al., 1982; Gentsch et aL, 1982). ~t- and //-Adrenergic receptors together mediate exploratory behaviour: an gragonist can enhance, fl-agonists suppress ambulation at the beginning of the dark but not at the beginning of the light phase. Parallel measurements indicated that both ~ - and fl-adrenergic receptors are higher at this sensitive phase (Mogilnicka et ai., 1985).
248
A. WIRZ-JUSTICE
7. Retinal Input to the Circadian System We have considered circadian rhythms in the pineal and in the CNS areas that drive them. Largely neglected has been consideration of the sensory organ that mediates visual information synchronising these rhythms, that itself is of CNS origin: the retina. The retinal-hypothalamic tract to the SCN directly relays LD information to the circadian pacemaker (Moore, 1983). However the retina does not merely passively code for luminance (that aspect of visual information primarily required for entrainment; Groos, 1982). The mammalian retina itself may contain a self-sustaining circadian pacemaker (Terman and Terman, 1985), modulating the light signal to the SCN through its own circadian rhythm of visual sensitivity. The visual cells manifest endogenous circadian rhythms in metabolic activity on the morphological level e.g. disk-shedding from outer segments (Besharse, 1982) and organelle turnover in inner segments (Rem~ et al., 1985). The retinal neurotransmitters, dopamine and melatonin, that are considered to be involved in the regulation of these phenomena, also undergo circadian variation (Pang et al., 1980; Besharse, 1982; Dubocovich, 1983; Wirz-Justice et al., 1984). Retinal dopamine synthesis is stimulated by light; this is mimicked by GABA antagonists and blocked by benzodiazepines (Morgan, 1982). Even though these retinal neurotransmitters show circadian rhythms and respond to light, neither dopamine nor benzodiazepine receptors in the retina do so (Dubocovich et al., 1985; Wirz-Justice et al., 1985b), although both are modified by longer exposure to light or darkness. Thus a negative finding in receptor binding can occur with a positive finding of a rhythm in the neurotransmitter itself (Fig. 19). It may be particularly important in the retina for the system to be buffered against rapid changes in receptor sensitivity related to the external LD cycle (see Section 8, Table 6 for drug effects).
8. Antidepressant Drugs and Receptor Rhythms Our specific interest in the action of antidepressant drugs on receptor rhythms arose from behavioural studies of the circadian rest-activity cycle. Lithium was the first psychopharmacological agent to have been shown to lengthen period and delay phase of the rest-activity cycle in many species (for review see Wirz-Justice, 1983). Clorgyline, a monoamine oxidase inhibitor, also had similar effects (Wirz-Justice and Campbell, 1982; Duncan et al., 1986), as did amphetamine (summarised in Wirz-Justice et al., 1985a). Since a great deal of work on the mechanisms of antidepressant drug action has focussed on receptor binding, it seemed important to evaluate such drug effects across 24 hours. RETINAL
CIRCADIAN
RHYTHMS
e-me
o,,,,,,o
-80
DA
Ro 15-1788 SPECIFIC BINDING • 60 fmolJmg prot.
SYNTHESIS ng/retina/min
20-
I
LI 2.5 5
11 D
4
11
FIG. 19. Circadian rhythm of DA synthesis (measured using AMPT) in retina from rats kept in DD after entrainment to the 12:12 LD cycle shown. DA synthesis practically ceases in the dark phase, and rises rapidly at the beginning of the light phase (even without the direct stimulus of light). The diurnal pattern of specific binding of the benzodiazepine antagonist Ro 15-1788 is not statistically significant, even though the pattern bears some resemblance to the DA synthesis rhythm. N = 5 retinae/time point (redrawn from Wirz-Justice et al., 1984; 1985b).
CIRCADIANRHYTHMS
249
TABLE 6. DRUG EFFECTSON CIRCADIANRECEPTORRHYTHMS
Drug/dosage/duration
Radioligand
Modification of rhythm wave form*/ ampl./mean/phaset
Reference
Uptake lnhibitors
Imipramme I0 mg/kg/day i.p., 21 days
QNB
C--* U / - 11%/-20%/
Kafka et al., 1981b
Imipramme 10 t*mol/kg oral, 21 days
DHA
-/-/-35%/
Fowler et al., 1985
Imipramlne 10 mg/kg/day i.p., 21 days
DHA
C ~ U / + 19%/-30%/
Wirz-Justice et al., 1980
Imipramme I0 mg/kg/day i.p., 21 days
WB-4101
C / + 2 9 % / - 1 5 % / - 8 hr
Wirz-Justice et al., 1980
Imipramme 10 mg/kg p.o., 14 days
Prazosin
N = 2/ /+30%/8 hr + 10%/20 hr
Maj et al., 1985
Imipramlne 10 mg/kg/day i.p., 21 days
Spiroperidol (striatum)
U/-14%/-20%/-4 - 60%
Naber et al., 1980
Imipramlne 15 mg/kg/day water, 14 days
5HT
C/=/-54%/-2hr
Wesemann et al., 1986b
Imipramme I0 mg/kg/day i.p., 21 days
Diazepam
C/+20%/=/=
Marangos et al., unpubl. data
Imipramme 10 mg/kg/day Alzet, 14 days
Flunitrazepam Ro 1 5 - 1 7 8 8 (retina)
O ~ C/+ / - 23 %/ O~C/+/-16%/
Wirz-Justice et al., 1985b
Imipramlne 10 mg/kg/day i.p., 14 days
Opiate
C --*U / + 100%/- 7%/
Lee et al., 1984
Zimelidine 10/~mol/kg p.o., 21 days
DHA
-/-/-20%/
Fowler et al., 1985
Amitryptiline I 0 mg/kg p.o., 14 days
Prazosin
N=2/
+18% 8hr/ +3% 20hr/
Maj et al., 1985
Citalopram 10 mg/kg p.o., 14 days MAOI
Prazosin
N=2/
/+20% 8hr/ +9% 20 hr/
Maj et al., 1985
Pargyline 5 mg/kg i.p., 14 days
Opiate
C~U/+82%/=/
Lee et al., 1984
Clorgyline (irreversible MAO-A inhibitor) 4 mg/kg/day Alzet s.c. 14 days
Opiate DHA WB-4101 QNB Spiroperidol (striatum) Diazepam
C --*U / + 83 % / = / C--*/=/=/ U ~ C/= / = / U ---,C / - 26%/+ 25%/ C / + 1 4 % / - 7 3 % / + 6 hr
Wirz-Justice Wirz-Justice Wirz-Justice Wirz-Justice Wirz-Justice
U --*C / = / = /
Wirz-Justice et al., 1982
Ro 15-1788 (retina)
-/-/-/
Wirz-Justice et al., unpubl, data
Ketanserin
-/-/-17%/
Campbell et aL, 1986
DHA
-/-/-
Fowler et al., 1985
Clorgyline I mg/kg/day p.o., 25 days Amiflamine (reversible MAO-A inhibitor) 3 #mol/kg p.o., 21 days JPN
29'3~-
14%/
hr
et et et et et
al., al., al., al., al.,
1982 1982 1982 1982 1982
Continued
250
A. WIRZ-JUSTICE
TABLE6~continued
Modification of rhythm Drug/dosage/duration Lithium Lithium 0.06% food 14 days
wave form*;;' ampl./mean/phaset
Radioligand DHA WB4101 Diazepam Opiate QNB
Reference
C-*O/-13%/+28%/ U-*0/-36%/+ 13%/ C --* O / - 2 2 % / - 11%/ C/-39%/+43%/-4 hr U/-28%/+45%/-6 hr C --* U / + 1 6 % / - 31%/
Kafka Kafka Kafka Kafka Kafka Kafka
et et et et et et
al., al., al., al., al., al.,
1982 1982 1982 1982 1982 1982
C--*U/=/+55%/ U ~ C / + 2 2 % / + 17%/ U ~ C / - 2 4 % / + 37%/ C -* U / + 3 3 % / - 195%/
Naber Naber Naber Naber
et et et et
al., al., al., al.,
1982 1982 1982 1982
Opiate
C/-47%/+34%/12hr
Naber et al., 1982
Opiate
C / + 7 7 % / - 1 6 % / + 4 hr
Lee et al., 1984
Opiate
C/+23%/-16%/=
Lee et al., 1984
DHA (LH)
U -~ C/= / =/
Kraeuchi et al., 1984
Clonidine
C~O/-23%/+
Spiroperidol (striatum) Neuroleptica
Fluphenazine decanoate 2 x 10mg in 14 days
DHA WB4101 QNB Spiroperidol
(striatum) Chlorpromazine 10 mg/kg i.p., 14 days Amphetamines
Amphetamine 5 mg/kg i.p., 14 days Methamphetamine 100 mg/l water 28 days
13%/
Kraeuchi et al., 1984
(MH)
Imipramine
C -* O / - / = /
Kraeuchi et al., 1986
C or U/decreased
Wirz-Justice et al., 1981
(SCN, LH)
Non-Pharmacological Antidepressant: Sleep Deprivation 12-24 hr SD
DHA WB4101 QNB Diazepam Opiate Spiroperidol
amplitude in 5/6 rhythms/ = /=
(striatum) 24-48 hr SD
5HT
U --* C / = / + 20%/
Wesemann et al., 1986b
* C = circadian; U = ultradian; O = no significant rhythm; -~ change of wave form. t Phase: - = DELAY; + = A D V A N C E (in hours).
In a series of detailed studies using many ligands and a number of antidepressant drugs, we found that inclusion of the temporal framework precluded any simple explanation of drug effects. The number of investigators who have carried out circadian rhythm studies with antidepressants is increasing. The main points are summarised in Table 6. A replication of findings from single time points studies was made with respect to most receptors (e.g. fl-adrenergic down-regulation with the tricyclic antidepressant imipramine). An example of the methodological problem raised by such circadian studies is shown in Fig. 20. a,-Adrenergic binding showed two main peaks at the end of the light and beginning of the dark phase. Chronic imipramine treatment changed the wave form, and the main peak occurred ca. 8 hours later. However comparison of control and treated animals at each time point yields different results depending on time of day: higher 0q-adrenergic receptor binding after imipramine treatment at dawn; no difference at the beginning of the light phase; and a marked decrease at all other times of day. A similar result is found with 0q-adrenergic receptor binding after chronic clorgyline (Wirz-Justice
CIRCADIAN
RHYTHMS
251
I-ADRENOCEPTOR BINDING 4.5
~nCONTROLS ""CHRONIC IMIPRAMINE
4'01 ~
3.5
**-x-
m ~ 3.0
--
~
|
L÷ 3
!
!
|
|
7 11D+ 3 7 TIME OF DAY (hr)
|
11
FIG. 20. Effect of chronic imipramine treatment (10 mg/kg/day, i.p. for 3 weeks) on binding to the cq-adrenergic receptor in forebrain homogenates. Rats were kept on a 12:12 LD cycle; N = 7/time point; both rhythms significant (ANOVA) (Redrawn from Wirz-Justice et al., 1980).
1982). Maj has also found that chronic imipramine significantly increases Ctl-adrenergic receptor binding at dawn but has no effect at dusk (two time points only) (Maj et al., 1985). Similarly, zimeline-induced down-regulation of cortical fl-adrenergic receptors was dependent on time of day (Fowler et al., 1985). The time-dependent modification of receptor binding is not limited to brain tissue: in the retina itself, imipramine may modify light input information via down-regulation of benzodiazepine receptors (Fig. 21) (Wirz-Justice et al., 1985b). Binding to both agonist and antagonist drugs was reduced only in the dark phase. This suggests the possibility that active antidepressant drugs may affect circadian rhythms by modulating visual sensitivity to light (since the photoreceptor response to light is dependent on the DA-GABAbenzodiazepine receptor complex). At the photoreceptor level, the few drugs investigated do reduce light responses (Rem6 et al., 1984; Rem6 and Wirz-Justice, 1985); at the behavioural level, lithium changes the sensitivity of hamster rhythms to light pulses (Han, 1984), and in man, characteristics of the EOG (Emrich et al., 1984). In contrast to the marked effects on different receptor rhythm characteristics by psychopharmacological agents, sleep deprivation, a non-pharmacological antidepressant modality, only slightly decreases amplitude. Essentially, all receptor rhythms showed a convincing replication (e.g. opiate receptor binding, Fig. 22) (Wirz-Justice et al., 1981). et al.,
9. Human Receptor Rhythms Postmortem studies of monoamines in human hypothalamus have documented large amplitude rhythms (Carlsson et al., 1980). Perry et al. (1977) were the first to demonstrate circadian variation in any CNS receptor: muscarinic cholinergic receptor binding in cerebral cortex was 100% lower during the day than during the night. No variation in 5HT-1 or -2 receptor binding could be demonstrated when 6-hour intervals were analysed (Wester et al., 1984). There are now a number of studies documenting diurnal rhythms of catecholaminergic binding sites on blood cells, fl-Adrenergic receptors on intact human lymphocytes show a circadian rhythm in males and an ultradian rhythm in females (Pangerl et al., 1986). The peak number of binding sites occur at the same time as maximal blood pressure. Two other studies show higher fl-adrenergic receptor binding during the day than night (Froehler et
252
A. W1RZ-JusTICE
RETINAL BENZODIAZEPINE RECEPTOR BINDING (Scatchard plots) Effect of chronic imipramine depends on time of day , - , D+IO
L*IO 3H - F L U N I T R A Z E P A M
006
0.04
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FIG. 21. Scatchard plots from two time points (end of the dark and end of the light phase) to illustrate (a) the similarity of agonist ([3H]-flunitrazepam) and antagonist ([3H]-Ro 15-1788) binding characteristics in retinal homogenates after chronic imipramine treatment; (b) that imipramine reduces the apparent number and not affinity of benzodiazepine receptor binding and; (c) that the effect of imipramine is dependent on time of day. Rats were kept on a 12:12 LD cycle, given imipramine via Alzet osmotic minipumps (10 mg/kg/day for 2 weeks) and transferred to DD on the experimental day (from Wirz-Justice et al., 1985b).
al., 1985; Titinchi et al., 1984). There appear to be both direct effects of noradrenaline or
agonists, as well as an independently regulated circadian rhythm. For example, stress of highly active control subjects raises plasma catecholamines in the morning and lowers fl-adrenergic receptor number; however both active and inactive subjects have lower binding in the evening independent of catecholamine levels (Krawietz et al., 1985). The agonist salbutamol induces down-regulation in the morning but not in the evening (Titinchi et al., 1984). A biphasic effect of i.v. agonist (doubled fl-adrenergic receptor binding after 30 minutes; halved after 4 hours) has not been investigated at different times of day (Tomeh and Cryer, 1980). One study of ~2-adrenergic receptor binding in platelets showed a 30% amplitude rhythm (Froehler et al., 1985), another found no modulation by time of day (Jones et al., 1983). The number of "peripheral type" benzodiazepine receptor binding sites in platelets did undergo significant circadian variation (Levi et al., 1986).
C I R C A D I A N RHYTHMS
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Specific binding of [3H]-naloxone to the opiate receptor during the last 13 hours of a sleep deprivation protocol and the first 12 hours of recovery sleep, together with non-sleep-deprived control groups at the same time points in the light and dark phase. N = 6/time point; both rhythms highly significant by ANOVA (from Wirz-Justice et aL, 1981). F I G . 22. 24-hour
A well known hormonal receptor manifests both a circadian rhythm in afffinity--insulin binding to erythrocytes is 25% less at midnight than during the day--and insulin sensitivity is higher in the afternoon than at night (Schultz et al., 1983a,b). 10. Hormone Receptors The analogy with hormone receptors in the brain may also be made. The rhythm in rat brain glucocorticoid receptor binding in hippocampus and septum persists independent of the pituitary (Angelucci et al., 1980); the number of cytoplasmic estrogen receptors from rat and hamster hypothalamus also show a circadian rhythm in the absence of circulating estrogens (Roy and Wilson, 1981; Wilson et aL, 1983; O'Connor et aL, 1985). These results support the idea that there are centrally driven diurnal fluctuations in the sensitivity of target cells to their respective steroids. It has even been postulated that the diurnal variations in catecholaminergic transmission might be a factor underlying the hormonal receptor rhythm (O'Connor et al., 1985). 11. Mechanisms of Rapid Receptor Change The number of receptors on cells is very dynamically regulated. Altered receptor concentration often determines altered tissue sensitivity to drug or hormone action, since the concept of a biologic receptor includes two distinct functions: not only must a receptor bind biologically active agonist hormones or their functionally inert antagonists, but it must also activate enzymes and ion channels to lead to a characteristic physiologic response. Mechanisms of membrane receptor regulation have been extensively investigated for adrenergic receptors (Harden, 1983; Lefkowitz et al., 1984). Two possible mechanisms have been proposed for adenylat¢ cyclase coupled receptor desensitisation. Within minutes of exposure of cells to isoproterenol, the receptors become functionally uncoupled i.e.
254
A. WIRZ-JUSTICE
incapable of forming the high affinity complex and therefore of stimulating adenylate cyclase. Subsequently the uncoupled receptors are removed from the cell surface and sequestered within the cell--the receptors in this internalised compartment are removed from contact with the effector components of the system. Their functional inadequacy is due to their physical separation: when agonist is removed, the receptors recycle to the cell surface and become recoupled to the adenylate cyclase. The demonstration of such a rapid and reversible functional change, resulting in changes in the apparent number of fl-adrenergic receptor binding sites, provides a potential model for circadian rhythms in CNS receptors. The second mechanism of desensitisation is phosphorylation of fl-adrenergic receptors--this is irreversible and thus not able to be invoked for short term changes. Not only in model systems (Lefkowitz et al., 1984) are rapid changes in receptor binding found: incubation of cortical slices with isoproterenol decreases both fl-adrenergic receptors and increase 5HT-2 receptors within 30 minutes (Scott and Crews, 1985); 5HT-2 receptors are rapidly down-regulated with a combination of ~2-antagonists and antidepressant drugs (Scott and Crews, 1983). One exception to the general finding that agonists induce a decrease in receptor number is the doubling of 13-adrenergic receptors on lymphocytes during the first 30 minutes of agonist infusion, followed by large decreases after 4-6 hours further infusion (Tomeh and Cryer, 1980). An agonist-induced increase in receptor number potentially could be related to the observation that phospholipid methylation unmasks "cryptic" fl-adrenergic receptors in rat reticulocytes (Strittmatter et al., 1979). Further evidence that circadian variations in radioligand binding may be dependent on agonist occupation has been found (Mash et al., 1985). Since endogenous, high affinity agonist-receptor complexes can prevent the assay of muscarinic receptors, even with potent antagonists, assay conditions were selected to fully uncouple such complexes and yield "total" receptor levels. Binding was then constant over 24 hours. Under normal assay conditions in Krebs buffer, binding assays measure only unoccupied receptors. Free QNB binding sites varied 100% over 24 hours. This interesting study indicates that agonists in binding experiments offer a new approach to investigating mechanisms of neurotransmitter receptor rhythms. It also indicates that circadian changes in cholinergic receptors may be due to alterations in the use of proteins rather than to changes in the amount of protein, a conclusion applicable to the studies of fl-adrenergic receptors, where no new synthesis of receptors is involved (Lefkowitz et al., 1984). 12. Conclusions
Lefkowitz concludes "Important clinical ramifications of this new information about agonist-induced receptor alterations leading to desensitisation is that whenever possible, such agonist therapy should be as intermittent as possible to minimise the development of desensitisation to drug effects" (1984). Precisely analogous statements form the basis of chronopharmacology (Reinberg and Smolensky, 1983). Since there are circadian rhythms in sensitivity to drugs, clinical application should, where possible, take this into account in designing treatment schedules. Sensitivity to drugs is itself mediated by receptor function: a drug binds to a receptor and activates, or blocks, a biological process. Thus circadian rhythms in receptor number may underlie pharmacological rhythms in response; intermittent drug application could fulfil both criteria--dosage at the right time of day for maximum effect, pulse dosage to minimise development of desensitisation. However, as this review notes, research into dynamic changes of receptors with respect to circadian physiology has a short history, in spite of the demonstration of pineal fl-adrenergic receptor rhythmicity over a decade ago. The approach to molecular mechanisms of receptor desensitisation induced by agonist drugs could also be applied to mechanisms of endogenous receptor rhythms. Given the caveats delineated above (with respect to age, strain, gender, LD and housing conditions, and in particular the importance
CIRCADIANRHYTHMS
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of precise brain regions) the non-pharmacological model of circadian rhythms in CNS receptors may provide a new approach to receptor mechanisms and receptor function in neurobiology. Acknowledgements I thank all my collaborators in these studies, and in particular Kurt Kraeuchi, Marian Kafka, Thomas Wehr, Iain Campbell and Gianni Muscettola for extensive discussions over the years. A fellowship from the Swiss Foundation for Biomedical Research, and Twinning Grants from the European Science Foundation (with I.C. and G.M.) are gratefully acknowledged. Further thanks are due to the many researchers who sent preprints of their recent data on receptor rhythms. All previously published Figures are reprinted with permission.
References AGHAJANIAN,G. K. and MANNERMAELEN, C. P. (1982) ~t2-Adrenoceptor-mediated hyperpolarization of locus coeruleus neurons: intracellular studies /n vivo. Science 215, 1394-1396. ANGELUCCI,L., VALERI,P., PALMERY,M., PATACCHIOLI,F. R. and CATALANI,A. (1980) Brain glucocorticoid receptor: correlation of in vivo uptake of corticosterone with behavioral, endocrine, and neuropharmacological events. In: Receptors for Neurotransmitters and Peptide Hormones, pp. 391-406. Eds. G. PEPEU, i . J. KUHAR and S. J. ENNA. Raven Press: New York. ASCHOFF,J., DAAN,S. and HONMA,K. I. (1982) Zeitgebers, entrainment and masking: some unsettled questions. In: Vertebrate Circadian Systems, pp. 13-24. Eds. J. ASCHOFF,S. DAANand G. GROOS.Springer: Heidelberg. BENEDITO, M. A. and KAFKA, i . S. (1982) Circadian rhythms in alpha- and beta-adrenergic receptor number and cyclic-AMP production in rat cerebral cortical slices. Soc. Neurosci. Abstr. 8, 660. BESHARSE, J. C. (1982) The daily light,lark cycle and rhythmic metabolism in the photoreceptor-pigment epithelial complex. In: Progress in Retinal Research, Vol. 1, pp. 82-124. Eds. N. OSBORNEand G. CHADER. Pergamon Press: Oxford. BLAKER, W. D., CHENEY, D. L. and STOFF, D. M. (1983) Interstrain comparison of avoidance behavior and neurochemical parameters of brain cholinergic function. Pharmac. Biochem. Behav. 18, 189-193. BRENNAN,M. J. W., VOLICER,L., MOORE-EDE,M. C. and BORSOOK,D. (1985) Daily rhythms of benzodiazepine receptor numbers in frontal lobe and cerebellum of the rat. Life Sci. 36, 2333-2337. BRUININK,A., LICHTENSTEIGER,W. and SCHLUMPF,i . (1983) Ontogeny of diurnal rhythms of central dopamine, serotonin and spirodecanone binding sites and of motor activity in the rat. Life Sci. 33, 31-38. BUCKETT,W. R. (1981) Stimulation-produced analgesia is dependent upon the influence of both circadian and circannual rhythms. Br. J. Pharmac. 281P. CAMPBELL, I. C., DURCAN, M. J., DUNN, G., KOSHIKAWA,F. and KOSHIKAWA,N. (1986) Circadian studies of central neurotransmitter populations in the rat. Abstract, 2nd International Conference o f Chronopharmacology, Montreux. CARLSSON, A., SVENNERHOLM,L. and WINBLAO, B. (1980) Seasonal and circadian monoamine variations in human brains examined post mortem. Acta psychiat, scand. Suppl. 280, 75-85. CHEVILLARD, C., BARDEN,N. and SAAVEDRA,J. M. (1981) Twenty-four hour rhythm in monoamine oxidase activity in specific areas of the rat brain stem. Brain Res. 223, 205-209. CHIU, P., RAJAKUMAR,G., CHIU, S., CHIu-YIN, K. and MISHRA,R. K. (1982) Enhanced [aH]-spiroperidol binding in striatum of spontaneously hypertensive rat (SHR). Eur. d. Pharmac. 82, 243-244. COOD, E. E. and BYRNE,W. L. (1981) Seasonal variation in the apparent number of binding sites for 3H-opioid agonists and antagonists. Life Sci. 28, 2577-2583. DIAGUJI, i . , MIKUNI, M., OKADA, F. and YAMASHITA, I. (1978) The diurnal variations in dopamine-flhydroxylase activity in the hypothalamus and locus coeruleus of the rat. Brain Res. 155, 409-412. DASZUTA,A., FAUOON, M. and TERNAUX,J.-P. (1982) Uptake of [3H]-serotonin and [3H]-noradrenaline in the raphe nuclei and the locus coeruleus of C57BL/6RHOLCO and BALB/cCENCLO mice at three times of the day. Neurosei. Lett. 29, 141-146. DAVIES,J. A., ANCILL,R. J. and REDFERN,P. H. (1972) Hallucinogenic drugs and circadian rhythms. In: Progress in Brain Research, pp. 79-95. Eds. P. S. BRADLEYand R. W. BRIMBLECOMBE.Elsevier: Amsterdam. Dl LAURO, A., GIANNINI, C. P., MUSCETTOLA,G., GRECO, A. M. and DE FRANCISCIS,P. (1986) No circadian rhythms of serotoninergic, alpha-, beta-adrenergic and imipramine binding sites in rat brain regions. Chronobiol. Int. 3, 123-126. DREW, G. M., GOWER, A. J. and MARRIOT,A. S. (1979) ~t2-Adrenoceptors mediate clonidine-induced sedation in the rat. Br. J. Pharmac. 67, 133-141. DUBOCOVlCH, M. L. (1983) Melatonin is a potent modulator of dopamine release in the retina. Nature 306, 782-784. DUBOCOVICH,M. L., LUCAS,R. C. and TAKAHASHI,J. S. (1985) Light-dependent regulation ofdopamine receptors in mammalian retina. Brain Res. 335, 321-325. DUNCAN,W. C., TAMARKIN,L. and WEHR, m. A. (1986) Clorgyline increases the circadian period, the activity-rest ratio and total activity in syrian hamsters. A. Rev. Chronopharmac. 3, 17-20. EMRICH, H. M., ULLRICH, A., ADAMCZYK,J. and STEIN, R. (1984) Lithium affects EOG in humans. Psychiat. Res. 13, 355-357.
256
A. WIRZ-JUSTICE
FOLDE$, A., MAXWELL, C. A., SCARAMUZZ1,R. J., DONNELLY, J. B., HOSKINSON, R. M. and RINTOUL, A. J. (1985) Seasonal changes in the sensitivity of ovine pineal fl-adrenoceptors to steroids. Neuroendocrinol. 41, 494---498. FOWLER, C. J., HALL, H., SAAF, J., ASK, A.-L. and Ross, S. B. (1985) Pharmacological manipulation of biochemically measured rhythms in the mammalian central nervous system. In: Circadian Rhythms in the Central Nervous System, pp. lll-120. Eds. P. H. REDFERN, I. C. CAMPBELL,J. A. DAVIES and K. F. MARTIN. Macmillan: London. FROEHLER, i . , SAITO, Y., ACKENHEIL, i . , BAK, R., BONDY, B., FEISTENAUER, E., HOFSCHUSTER, E., VAKIS, A. and WELTER, n . (1985) Catecholaminergic binding sites on blood cells of healthy volunteers with special respect to circadian rhythms. Pharmacopsychiat. lg, 147-148. GARFINKEL, A. (1983) A mathematics for physiology. Am. J. Physiol. 245, R455-R466. GENTSCH, C., LICHTSTEINER, M. and FEER, H. (1982) Behavioural comparisons between individuallyand group-housed male rats: effects of novel environments and diurnal rhythm. Behav. Brain Res. 6, 93-100. GROOS, G. (1982) The neurophysiology of the mammalian suprachiasmatic nucleus and its visual afferents. In: Vertebrate Circadian Systems pp. 96-105. Eds. J. ASCHOFF, S. DAAN and G. GROOS. Springer: Heidelberg. HAN, S.-Z. (1984) Lithium chloride changes sensitivity of hamster rhythm to light pulses. J. interdiscipl. Cycle Res. 15, 139-145. HARDEN, T. K. (1983) Agonist-induced desensitization of the fl-adrenergic receptor-linked adenylate cyclase. Pharmac. Rev. 35, 5 32. HELMESTE, D. M. (1983) Spontaneous and apomorphine-induced locomotor changes parallel dopamine receptor differences in two rat strains. Pharmacol. Biochem. Behav. 19, 153-155. HELMESTE, D. M., SEEMAN, P. and COSClNA D. V. (1981) Relation between brain catecholamine receptors and dopaminergic stereotypy in rat strains. Fur. J. Pharmac. 69, 465-470. HENDRIE, C. A., NOHRIA, V. and ROOGERS, R. J. (1983) Circadian variation in pain sensitivity in rats may be related to alterations in opiate receptor density. Br. J. Pharmac. 78, 46P. JACOBSON, W. and WILKINSON, M. (1985) Diurnal changes in the opiatergic control of LH release in prepubertal rats are associated with diurnal variations in hypothalamic but not cortical [3H]-naloxone binding sites. Soc. Neurosci. Abstr. 11. JENNI-EIERMANN, S., YON HAHN, H. P. and HONEGGER, C. G. (1985) Circadian variations of neurotransmitter binding in three age groups of rats. Gerontology 31, 138-149. JENNI-EIERMANN, S., VON HAHN, H. P. and HONEGGER, C. G. (1986) Diurnal rhythms in neurotransmitter receptor binding and CAT activity: different patterns in two rat lines of Wistar origin. Brain Res. 370, 54-60. JHANWAR-UNIYAL, M., DARWlSH, M., BEITMIRZA,G. and LEIBOWITZ, S. F. (1985) Impact of food deprivation on hypothalamic and cortical cq- and ~t2-noradrenergic receptors. Soc. Neurosci. Abstr. l l, No. 21.1. JHANWAR-UNIYAL, M., ROLAND, C. R. and LEIBOWITZ,S. F. (1986) Diurnal rhythm of ct2-noradrenergic receptors in the paraventricular nucleus and other brain areas: relation to circulating corticosterone and feeding behavior. Life Sci. 38, 473-482. JONES, S. B., BYLUND, D. B., RIESER, C. A., SHEKIM, W. O., BYER, J. A. and CAgR, G. W. (1983) ~-Adrenergic receptor binding in human platelets: alterations during the menstrual cycle. Clin. Pharmac. Ther. 34, 90-96. JUNG, C.-Y., KIM, K.-W., CHO, K.-P. (1984) A study on the diurnal variation of endorphin in rat brain. Korean J. Pharmac. 20, 35-47. KAFKA, M. S., WIRZ-JUSTICE,A. and NABER, n. (1981a) Circadian and seasonal rhythms in ~t- and fl-adrenergic receptors in the rat brain. Brain Res. 207, 409-419. KAFKA, M. S., WIRZ-JUSTICE, A., NABER, D. and WEHR, T. A. (1981b) Circadian acetylcholine receptor rhythm in rat brain and its modification by imipramine. Neuropharmacol. 20, 421-425. KAFKA, M. S., WIRZ-JUST1CE,A., NABER, D., MARANGOS,P. J., O'DONOHUE, T. L. and WEHR, T. A. (1982) Effect of lithium on circadian neurotransmitter receptor rhythms. Neuropsychobiol. 8, 41--50. KAFKA, i . S., WIRZ-JUSTICE, A., NABER, D., MOORE, R. Y. and BENEDITO, M. A. (1983) Circadian rhythms in rat brain neurotransmitter receptors. Fedn Proc. 42, 2796-2801. KAFKA, m. S., MARANGOS,P. J. and MOORE, R. Y. (1985) Suprachiasmatic nucleus ablation abolishes circadian rhythms in rat brain neurotransmitter receptors. Brain Res. 327, 344-347. KAFKA, i . S., BENEDITO,M. A., BLENDY,J. A. and TOKOLA, N. S. (1986a) Circadian rhythms in neurotransmitter receptors in discrete rat brain regions. Chronobiol. Int. 3, 91-100. KAFKA, M. S., BENEDITO, M. A., ROTH, R. H., STEELE, L. K., WOLFE, W. W. and CATRAVAS,G. N. (1986b) Circadian rhythms in catecholamine metabolites and cyclic nucleotide production. Chronobiol. Int. 3, 101-115. KAN, J. P., CHOUVET, G., HI~RY, F., DEBILLY, G., MERMET, A., GLOWINSKI, J. and PUJOL, J. F. (1977) Daily variations of various parameters of serotonin metabolism in the rat brain. 1. Circadian variations of tryptophan-5-hydroxylase in the raphe nuclei and the striatum. Brain Res. 123, 125-136. KASTRUP,J. and CHRISTENSEN,J. (1984) Lack of effects of thyroid hormones on muscarine cholinergic receptors in rat brain and heart. Scand. J. clin. Lab. Invest. 44, 33 38. KAVALIERS, M. and HIRST, M. (1983) Daily rhythms of analgesia in mice: effects of age and photoperiod. Brain Res. 279, 387-393. KERDELHUE, B., KARTESZl, M., PASQUALINI, C. REINBERG, A., MEZEY, E. and PALKOVITS, M. (1983) Circadian variations in fl-endorphin concentrations in pituitary and in some brain nuclei of the adult male rat. Brain Res. 261, 243-248. KOULU, M., BJELOGRLIC, N., AGREN, H., SAAVEDRA, J. M. and L1NNOILA, M. (1985) Circadian changes in the concentrations of monoamines in discrete rat brain stem nuclei. Acts physiol, scand. 124, Suppl. 542. 268. KRAEUCHI, K., ALTHAUS, A. and FEER, H. (1981) Easier binding studies: no more washing! Experientia 37, 672.
CIRCADIANRHYTHMS
257
KRAEUCHI, K., WIRZ-JUSTICE, A., WILLE~R, R., CAMPBELL, I. C. and FEER, H. (1983) Spontaneous hypertensive rats: behavioral and corticosterone response depend on circadian phase. Physiol. Behav. 30, 35~,0. KRAEUCHI, K., WIRZ-JUSTICE, A., MORIMASA, T., WILLENER, R. and FEER, H. (1984) Hypothalamic cq- and /~-adrenoceptor rhythms are correlated with circadian feeding: evidence from chronic methamphetamine treatment and withdrawal. Brain Res. 321, 83-90. KRAEUCm, K., WIRZ-JUSTICE, A., SUETTERLIN-WILLENER, R. and FEER, H. (1985) Clonidine stimulation of food intake is circadian phase dependent. IRCS Med. Sci. 13, 561. KRAEUCHI, K., WIRZ-JUSTICE, A., MORIMASA, T., SUETTERLIN-WILLENER, R. and FEER, H. (1986) Temporal distribution of [3H]-imipramine binding in rat brain regions is not changed by chronic methamphetamine. Chronobiol. Int. 3, 127-133. KRAWIETZ, W., KLEIN, E. M., UNTERBERG, CH. and ACKENHEIL, M. (1985) Physical activity decreases the number of/~-adrenergic receptors on human lymphocytes. Klin. Wschr. 63, 73-78. LANG, P.-H., BISSINGER, H. and LEMMER, B. (1983) Circadian rhythm in cAMP content and in the activities of basal adenylate cyclase and phosphodiesterase in rat heart ventricles, d. Rev. Chronopharmae. I, 219-222. LEE, M.-S., KIM, K.-W. and CHO, K.-P. (1984) A study on the circadian rhythm of opiate receptor in rat brain. Korean J. Pharmac. 20, 23-34. LEFKOWITZ, R. J., CARGN, M. G. and STILES, G. L. (1984) Mechanisms of membrane-receptor regulation. Biochemical, physiological, and clinicalinsights derived from studies of the adrenergic receptor. New Engl. J. Med. 310, 1570-1579. " LEmOWITZ, S. F. (1980) Neurocbemical systems of the hypothalamus: control of feeding and drinking behavior and water-electrolyte excretion. In: Handbook of the Hypothalamus, Vol. I, Part A, Behavioral Studies of the Hypothalamus, pp. 299-437. F_As. P. J. MORGANE and J. PANKSEPP. Marcel Dekker: New York. LnM~R, B. and LANG,P.-H. (1983) Circadian-stage-dependency in antagonist binding of [3H]-dihydroalprenolol to rat heart ventricular membranes. A. Rev. Chronapharmac. 1, 321-324. LEMMER, B., CASPARI-IRVING, G. and WEIMER, R. (1981) Strain dependency in motor activity and in concentration and turnover of catecholamines in synchronized rats. Pharmac. Biochem. Behav. 15, 173-178. LEMMER, B., LANG, P.-H. and BISSINGER,H. (1983) Circannual peak-shift in circadian rhythm in basal cAMP content in rat heart ventricles..4. Rev. Chronopharmac. 1, 223-225. LEMMER, B., LANG, P.-H., GORKA, Z., SCHMIDT, S. and BAERME1ER,H. (1985) Circadian rhythms in the beta-receptor-adenylate-cyclase-cAMP-phosphodiesterase-system in heart ventricles and brain of the rat. J. Interdiscipl. Cycle Res. 16, 142. LESLIE, F. M., DUNLAPIII, C. E. and Cox, B. M. (1980) Ascorbate decreases ligand binding to neurotransmitter receptors. J. Neurochem. 34, 219-221. LEVI, F., BANAVIDES,J., TOUITOU,V., QUARTERRONET,D., CANTON,T. UZAN, A., GUEREMY,C., LE FUR G. and REINBERG,A. (1986) Circadian rhythm in peripheral type benzodiazepine binding sites in human platelets. Biochem. Pharmac. 35, 2623-2625. LOPEZ-COLOME,A. M. and DRUCKER-COLIN,R. (1984) Ultradian rhythm of pontomesencephalic opiate receptor: modification by chloramphenicol. Exp. Neurol. 85, 209 215. MAJ, J., KLIMEK, V. and NOWAK, G. (1985) Antidepressant drugs given repeatedly increase the binding to cq-adrenoceptors in the rat cortex. Fur. J. Pharmac. 119, 113-116. MARGULES,n. L., LEWIS, M. J., DRAGOVICH,J. A. and MARGULES,A. S. (1972) Hypothalamic norepinephrine: circadian rhythms and the control of feeding behavior. Science 178, 640~43. MARINO, L. A., DEBELLIS,M. and LEIBOWlTZ,S. F. (1983) ct2-Adrenergic receptors in the paraventricular nucleus mediate feeding induced by norepinephrine and clonidine. Soc. Neurosci. Abstr. 9, 467. MASH, n., FLYNN, D. D. KALINOSKI,L. and POTTER, L. T. (1985) Circadian variations in radioligand binding to muscarinc receptors in rat brain dependent upon endogenous agonist occupation. Brain Res. 331, 35-38. MATSUBARA,S., KAZAWA,T., OKA, I., KOYAMA,T. and YAMASHITA,[. (1982) Circadian variation in rat brain fl-adrenergic receptor. Folia psychiat, neurol, jap. 36, 478-479. MCCABE, J. T., DEBELLIS,M. and LEIBOWITZ,S. F. (1984) Clonidine-induced feeding: analysis of central sites of action and fiber projections mediating this response. Brain Res. 309, 85-104. MIKUNI, M., SAITO,Y., KOYAMA,T. and YAMASHITA,I. (1981) Circadian variation of cyclic AMP in the rat pineal gland. J. Neurochem. 36, 1295-1297. MOGILNICKA, E., WEDZONY,K., KLIMEK, V. and NOWAK, G. (1985) The importance of timing for the action of ctI- or fl-agonists: possible relationship with the density of [3H]-prazosin and [3H]-dihydroalprenolol binding sites. Eur. J. Pharmac. I13, 279-282. MOORE, R. Y. (1983) Organisation and function of a CNS circadian oscillator: the suprachiasmatic nucleus. Fedn Proc. 42, 2783-2789. MORGAN, W. W. (1982) Dopamine neurons in the retina: a new pharmacological model. In: Cell Biology o f the Eye, pp. 533-554. Ed. D. S. McDEVITT. Academic Press: New York. MOSER, P. C. and REDFERN,P. H. (1985) Circadian variation in behavioural responses to central 5HT receptor stimulation in the mouse. Psychopharmac. 86, 223-227. MOSER, P. C. and REDFERN,P. H. (I 986) Circadian rhythms in behaviours mediated by 5HT receptor stimulation in the rat. A. Rev. Chronopharmac. 3, 13-16. MURPHY, S., UZBEKOV,M. G. and ROSE,S. P. R. (1980) Changes in serotonin receptors in different brain regions after light exposure of dark-reared rats. Neurosci. Left. 17, 317-321. MUSCETTOLA,G., DI LAURO,A. and GIANNINI,C. P. (1986) Platelet 3H-imipramine binding in bipolar patients. Psychiat. Res. 18, 343-353. NABER, D., WIRZ-JUSTICE,A., KAFKA,M. S. and WEHR, T. A. (1980) Dopamine receptor binding in rat striatum: ultradian rhythm and its modification by chronic imipramine. Psychopharmac. 68, 1-5.
258
A. WIRZ-JusTICE
NABER, D., WIRZ-JUSTICE,A. and KAFKA, M. S. (1981) Circadian rhythm in rat brain opiate receptor. Neurosci. Lett. 21, 45-50. NAaER, D., WmZ-JUSTICE, A. and KAFKA, M. S. (1982) Chronic fluphenazine treatment modifies circadian rhythms of neurotransmitter receptor binding in rat brain. J. Neural Transm. 55, 277-288. NATALI, J. P., MCRAE-DEGOEURCE,A., CHOUVET, G. and PUJOL, J. F. (1980a) Genetic studies of daily variations of first-step enzymes of monoamine metabolism in the brain of inbred strains of mice and hybrids. I. Daily variations of tryptophan hydroxylase activity in the nuclei raphe dorsalis, raphe centralis and in the striatum. Brain Res. 191, 191-203. NATALI, J. P., MCRAE-DEGUEURCE, A., KEANE, P., DEBILLY, G. and PUJOL, J. F. (1980b). II. Daily variations of tyrosine hydroxylase activity in the locus coeruleus. Brain Res. 191, 205 213. NORDBERG,A. and WAHLSTROEM,G. (1980) Diurnal fluctuation in striatal choline acetyltransferase activity and strain difference in brain protein content of the rat. Acta physiol, scand. 108, 385-388. O'CONNOR, L. H., MORIN, L. P. and FEDER, H. H. (1985) A diurnal fluctuation in medial basal hypothalamicpreoptic area cytosol estrogen receptors in ovariectomized hamsters. Brain Res. 347, 376-380. OUVERIO, A. and MALORNI, W. (1979) Wheel running and sleep in two strains of mice: plasticity and rigidity in the expression of circadian rhythmicity. Brain Res. 163, 121-133. OLIVERIO,A., CASTELLANO,C. and PUGLISI-ALLEGRA,S. (1982) Opiate analgesia: evidence for circadian rhythms in mice. Brain Res. 249, 265-270. PANG, S. F., YU, H. S., SUEN, H. C. and BROWN, G. M. (1980) Melatonin in the retina of rats: a diurnal rhythm. J. Endocr. 87, 89-93. PANGERL, A., REMIEN, J. and HAEN, E. (1986) The number of fl-adrenoceptor sites on intact human lymphocytes depends on time of day, on season, and on sex. ,4. Rev. Chronopharmac. 3, 331-334. PERRY, E. K., PERRY, R. H. and TOMLINSON, B. E. (1977) Circadian variations in cholinergic enzymes and muscarinic receptor binding in human cerebral cortex. Neurosci. Lett. 4, 185-189. PILCHER, C. W. T., JONES, S. M. and BROWNE, J. (1982) Rhythmic nature of naloxone-induced aversions and nociception in rats. Life Sci. 31, 1249-1252. POR, S. B. and BONDY, S. C. (1981) Regional circadian variation of acetylcholine muscarinic receptors in the rat brain. J. Neurosci. Res. 6, 315-318. POST, J. and NAIR, V. (1975) Circadian variations in the action of antihypertensive drugs. In: Proceedings of the XII International Conference of the International Society for Chronobiology, pp. 375-386. PUGLIS1-ALLEGRA,CASTELLANO,C. and OLIVERIO,A. (1982) Circadian variations in stress-induced analgesia. Brain Res. 252, 373-376. RAPP, P. E. (1979) Bifurcation theory, control theory and metabolic regulation. In: Biological Systems, Modelling and Control, pp 1-83. Ed. D. A. LtNKENS. Peter Peregrinus Ltd: New York. RAPP, P. E. (1987) Why are so many biological systems periodic? Prog. Neurobiol. 29, 261 273. REINaERG, A. and SMOLENSKY, M. (1983) Biological rhythms and medicine. Springer Verlag: New York, Heidelberg. REML C. and WIRZ-JUST1CE,A. (1985) Circadian rhythms of the retina and their pharmacological modulation. In: Circadian Rhythms in the Central Nervous System, pp. 135-145. Eds. P. H. REDFERN, I. C. CAMPBELL, J. A. DAVIES, K. F. MARTIN. MacMillan: London. REMI~, C., WIRZ-JusTICE, A., AEBERHARD, B. and RHYNER, A. (1984) Chronic clorgyline dampens rat retinal rhythms. Brain Res. 298, 99 106. REMIt, C., AEBERHARD,B. and SCHOCH, M. (1985) Circadian rhythm of autophagy and light responses of autophagy and disk-shedding in the rat retina. J. comp. Physiol. A, 156, 669-677. ROMERO, J. A., ZATZ, M., KEBABIAN,J. W. and AXELROD,J. (1975) Circadian cycles in binding of [3H]-alprenolol to fl-adrenergic receptor sites in rat pineal. Nature 258, 435-436. ROSENWASSER, A. M., TRUBOWITSCH, G. and ADLER, N. T. (1985) Circadian rhythm in metabolic activity of suprachiasmatic, supraoptic and raphe nuclei. Neurosci. Lett. 58, 183 187. Roy, E. J. and WILSON, M. A. (1981) Diurnal rhythm of cytoplasmic estrogen receptors in the rat brain in the absence of circulating estrogens. Science 213, 1525-1527. SAITO, Y., YAMASHITA,I., YAMAZAKI, K., OKADA, F., SATOMI, R. and FUJIEDA, T. (1975) Circadian fluctuation of brain acetylcholine in rats. I. On the variations in the total brain and discrete brain areas. Life Sci. 16, 281-288. SCHMITT,M. (1973) Circadian rhythmicity in responses of cells in the lateral hypothalamus. Am. J. Physiol. 225, 1096-1101. SCHULZ, B., RATZMANN, K. P., ALBRECHT, G. and BIBERGEIL, H. (1983a) Diurnal rhythm of insulin sensitivity in subjects with normal and impaired glucose tolerance. Exp. clin. Endocr. 81, 263-272. SCHULZ, B., GREENFIELD, M. and REAVEN, G. M. (1983b) Diurnal variation in specific insulin binding to erythrocytes. Exp. clin. Endocr. 81, 273-279. SCOTT, J. A. and CREws, F. T. (1983) Rapid decrease in rat brain beta-adrenergic receptor binding during combined antidepressant alpha-2 antagonist treatment. J. Pharmac. exp. Ther. 224, 640-646. SCOTT, J. A. and CREWS, F. T. (1985) Increase in serotonin-2 receptor density in rat cerebral cortex slices by stimulation of beta-adrenergic receptors. Biochem. Pharmac. 34, 1585-1588. SEMBA,J., TORU, M. and MATAGA,N. (1984) Twenty-four hour rhythms of norepinephrine and serotonin in nucleus suprachiasmaticus, raphe nuclei, and locus coeruleus in the rat. Sleep 7, 211-218. SIMANTOV,R., ZANG,D. and YEHUDA,S. (1980) Regulation of pituitary endorphins under steroids treatment and during day-night cycle. Adv. bioehem. Psychopharmac. 22, 247-256. SINGLETON, C. and MARSOEN, C. A. (1981) Circadian variation in the head twitch response produced by 5-methoxy-N,N-dimethyltryptamine and p-chloroamphetamine in the mouse. Psychopharmac. 74, 173-176. STRITTMATTER, W. J., HIRATA, F. and AXELROD, J. (1979) Phospholipid methylation unmasks cryptic fl-adrenergic receptors in rat reticulocytes. Science 204, 1205-1207. SUDGEN, D. and KLEIN, D. C. (1985) Regulation of rat pineal cq-adrenoceptors. J. Neurochem. 44, 63-67. TERMAN, M. and TERMAN, J. (1985) A circadian pacemaker for visual sensitivity? Ann. N.Y. Acad. Sci. 453, 147-161.
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TITINCHI, S., AL SHAMMA,M., PATEL, K. R., KERR, J. W. and CLARK,B. (1984) Circadian variation in number and affinity of fl~-adrenoceptors in lymphocytes of asthmatic patients. Clin. Sci. 66, 323-328. TOMEH, J. F. and CRYER, P. E. (1980) Biphasic adrenergic modulation of beta-adrenergic receptors in man: agonist-induced early increment and late decrement in beta-adrenergic receptor number. J. clin. Invest. 65, 836-840. VALAXX,J. L. (1984) Genetics as a model for studying the sleep-wake cycle. In: Sleep mechanisms, pp. 135-145. Eds. A. BORBELYand J. L. VALATX.Springer: Berlin. VAN DER GtrrGEN, J. and SLANGEN,J. L. (1975) Norepinephrine uptake by hypothalamic tissue from the rat related to feeding. Pharmac. Biochem. Behav. 3, 855-860. VASSOUT,A., FIDALGO, D., HtmN, CH. WIRZ-JUsTICE,A. and DELINI-STULA,A. (1982) Circadian activity in socially-raised and isolated rats as assessed in an open-field device. Experientia 38, 720. VIRUS, R. M., BAOLA~WSKI, T. and RADULOVACKI,M. (1984) Circadian variation of [aH]N6-(l-phenylisopropyl)adenosine binding in rat brain. Neurosei. Lett. 46, 219-222. WEINER, N., AROLD, N. and WESEMANN,W. (1982) Possible hazards in serotonin binding assays in human and rat brain: effects of ascorbate, pargyline, and buffer concentration. J. Neurosci. Meth. 5, 41-45. WESEMANN, W., WEINER, N., ROXSCH, M. and SCHULZ, E. (1983) Serotonin binding in rat brain: circadian rhythm and effect of sleep deprivation. ]. Neural Transm. Suppl. 18, 287-294. W~SEUANN, W., ROXSCH, M., SCHULZ, E., STURM, G. and ZOEFEL,P. (1986a) Circadian rhythm of serotonir] binding in rat brain: I. Effect of the light/dark cycle. Chronobiol. Int. 3, 135-139. WESEMANN,W., ROTSCH,M., SCnULZ, E. and ZOEFEL,P. (1986b) Circadian rhythm of serotonin binding in rat brain: II. Influence of sleep deprivation and imipramine. Chronobiol. Int. 3, 141-146. WESTER, P. HARDY, J. A., MARCUSSON,J., NYBERG, P. and WINBLAD,B. (1984) Serotonin concentrations in normal aging human brains: relation to serotonin receptors. Neurobiol. Aging 5, 199-203. WILSON, M. A., CLARK,A. S., CLYDE,V. and RoY, E. J. (1983) Characterisation of a pineal-independent diurnal rhythm in neural estrogen receptors and its possible behavioral consequences. Neuroendocr. 37, 14-22. WIRZ-JUSTICE, A. (1983) Antidepressant drugs: effects on the circadian system. In: Circadian Rhythms in Psychiatry, pp. 235-264. Eds. T. A. WEHR and F. K. GOODWIN. The Boxwood Press: Pacific Grove. W1RZ-JUSTICE,A. and CAMPBELL,I. C. (1982) Antidepressant drugs can slow or dissociate carcadian rhythms. Experientia 38, 1301-1309. WIRZ-JUSTICE, A., KAFKA, M. S., NABER, D. and WEHR, T. A. (1980) Circadian rhythms in rat brain ~t- and fl-adrenergic receptors are modified by chronic imipramine. Life Sei. 27, 341-347. WIRZ-JUSTICE, A., TOBLER, I., KAFKA, U. S., NABER, D., MARANGOS,P. J., BORBELY, A. A. and WEHR, T. A. (1981) Sleep deprivation: effect on circadian rhythms of rat brain neurotransmitter receptors. Psychiat. Res. 5, 67-76. WIRZ-JUSTICE, A., KAFKA, M. S., NABER, D., CAMPBELL, I. C., MARANGOS,P. J., TAMARKIN,L. and WEHR, T. A. (1982) Clorgyline delays the phase-position of circadian neurotransmitter receptor rhythms. Brain Res. 241, 115-122. WIRZ-JUSTICE,A., KRAEUCHI,K., CAMPBELL,I. C. and FEER, H. (1983a) Adrenoceptor changes in spontaneous hypertensive rats: a circadian approach. Brain Res. 262, 233-242. WIRZ-JusxICE, A., KRAEUCHI, K., MORIMASA,T., WILLENER,R. and FEER, H. (1983b) Circadian rhythm of [3H]-imipramine binding in the rat suprachiasmatic nuclei. Eur. J. Pharmac. 87, 331-333. WIRZ-JUSTICE,A., DAPRADA,M. and REM/LC. (1984) Circadian rhythm in rat retinal dopamine. Neurosci. Left. 45, 21-25. WIRZ-JUSTICE,A., KRAEUCHI,K., MORIMASA.T. and RIETVELD,W. (1985a) Circadian feeding rhythms: central mechanisms and methamphetamine modulation. In: Circadian Rhythms in the Central Nervous System, pp. 123-133. Eds P. H. REDFERN,I. C. CAMPBELL,J. A. DAVIESand K. F. MARTIN. Macmillan: London. WIRZ-JUSTICE, A., SUETTERLIN-WILLENER,R., REMIt, C., MUSCETTOLA,G. and DI LAURO, A. (1985b) Retinal benzodiazepine receptor binding. Experientia 41, 793. WOLF, M. D., WILCOX,R. E., RIFFLE,W. H. and ABRAHAM,L. n. (1980) Strain differences in dopamine receptor function and the initiation of movement. Pharmacol. Biochem. Behav. 13, 5-7.
0301-0082/87/$0.00 + 0.50 Copyright © 1987 Pergamon Journals Ltd
CIRCADIAN RHYTHMS IN MAMMALIAN NEUROTRANSMITTER RECEPTORS ANNA WIRZ-JUSTICE
Psychiatric University Clinic, Basel Switzerland
(Received 10 February 1986)
Contents 1. 2. 3. 4. 5.
6.
7. 8. 9. 10. 11. 12.
Summary Introduction Methodological issues The beta-adrenergic receptor of the pineal gland The mammalian CNS 5.1. Changes in receptor binding over 24 hours: first CNS studies 5.2. Replication of receptor binding rhythms and possible seasonal modulation 5.3. Persistance of circadian rhythms under constant conditions but not after SCN lesions 5.4. Regional differences in receptor rhythms 5.4.1. Frontal cortex and hypothalamus 5.4.2. Discrete brain regions 5.4.3. Negative results 5.4.4. Regional differences in rhythmicity: relationship to regional function? 5.5. Strain differences in receptor rhythms 5.6. Age and gender 5.7. Receptor rhythms: correlation with neurotransmitter turnover or second messenger 5.7.1. Presynaptic events 5.7.2. Postsynaptic events Receptor rhythms and receptor-mediated function 6.1. Opiate receptors and pain sensitivity 6.2. Serotonin receptor stimulation and behavioural response 6.3. Alpha2-adrenergic receptor stimulated feeding 6.4. Alphas- and beta-adrenergic stimulated behaviours in the open field Retinal input to the circadian system Antidepressant drugs and receptor rhythms Human receptor rhythms Hormone receptors Mechanisms of rapid receptor change Conclusions Acknowledgements References
219 220 221 223 223 223 226 227 229 229 232 232 232 238 240 241 242 244 245 245 246 246 247 248 248 252 253 254 254 255 255
1. Summary At the present time, the following summary statements can be made as to 24-hour changes in receptor binding. (1) In all receptors studied in homogenates from whole rat forebrain (~t~, ~t2, fl-adrenergic, muscarinic cholinergic, dopaminergic, 5HT-I, 5HT-2, adenosine, opiate, benzodiazepine, GABA, imipramine), significant variations over 24 hours have been documented. (2) The receptor rhythms measured change in wave form, amplitude, and phase throughout the year, even though the animals have been kept on a defined and constant LD cycle. Whether these rhythms are truly seasonal requires further investigation. (3) The rhythms are circadian: i.e. they persist in the absence of time cues, and the unimodal rhythms do not persist after lesion of the putative circadian pacemaker in the suprachiasmatic nuclei. 219
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(4) The rhythms can be uni- or bimodal, and each brain region shows a particular pattern. The pattern can be different for the same ligand in different nuclei of a given brain region (e.g. hypothalamus). (5) Nearly all studies of receptor rhythms have been carried out in rats; the results vary according to strain and even within the same strain from different breeding lines. (6) Receptor rhythm characteristics are modified by age: e.g. the amplitude, phase, as well as the 24-hour mean of binding to a given ligand in a defined brain region. (7) The changes in number of binding sites over 24 hours can be correlated with amine turnover, second messenger, or function of that brain region; however these relationships, although consistent within a region, do not hold for all regions. (8) If gradual changes in CNS neurotransmitter receptor function are considered important in the pathogenesis of schizophrenia and affective disorders and the mode of action of psychopharmacological agents, then consideration of the short term rapid change over 24 hours is equally necessary. Chronic treatment with a number of psychoactive drugs known to induce up- or down-regulation of receptor number, also induces marked changes in circadian rhythm parameters of wave form, amplitude, phase and 24-hour mean. This is of methodological importance for single time-point studies, since the interpretation of the results will depend on time of day. (9) Preliminary evidence supports the assumption that the significant variation in receptor binding throughout the day may underlie the well-known circadian rhythms of susceptibility to many CNS drugs. (10) New findings of circadian rhythms in receptors on blood cells indicate the relevance of these changes also in human physiology. 2. Introduction
The existence of non-decaying, repetitive phenomena in biology was long considered a consequence of specific vital forces in living organisms. However, spontaneous selfsustaining periodicity is found in chemical reactions and physical systems; all major biological processes are periodic (Garfinkel, 1983). Oscillations are observed at all levels of organisation from single organisms, collections of cells, or in populations of subcellular organelles. The period length ranges from less than a second to days to a year. Periodicity can be considered to represent a basic phenomenon in biological systems that confers positive functional advantages. Rapp has concisely defined the evolutionary advantages of periodic biological control: the mechanisms of entrainment and synchronisation are a natural property of biological oscillators; oscillators that structure time can also be used to order physically separated events; endogenous oscillators can predict repetitive events; periodic signalling is metabolically efficient; and finally, frequency dependent control systems can succeed in environments in which amplitude dependent control fails (Rapp, 1979; 1987). Circadian rhythms (circa die = about a day) are the subject of this review, rhythms of ca. 24 hours that are intrinsic to the organism, not the consequence of periodic stimulation, as they persist in the absence of external time cues. Endogenous circadian rhythms can be found not only in the entire organism but also in organs, tissues or even isolated cells. In the absence of time cues or "zeitgebers", the natural frequency usually differs slightly from 24 hours. Light entrains the circadian system to the 24 hour day. Circadian rhythms have been shown to be genetically determined--at the molecular level or within species or strains. This intrinsic temporal order is an adaptation to the temporal structure of the environment. The selective advantages are that the peak of each rhythm occurs at a particular phase of the light: dark cycle, and the sequential order of metabolic and physiologic rhythms coordinate the optimal timing of interdependent functions, while separating incompatible ones. Thus the organism can respond appropriately in its behaviour to the demands of the changing environment. The formal properties of the circadian system have been elucidated over the past twenty years, in particular through the work of Pittendrigh, Aschoff and Wever. With the
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discovery of a neuroanatomy of mammalian temporal organisation, the accent of much research has moved towards neurobiology. Some of the questions now being asked are: Is one central circadian pacemaker--the suprachiasmatic nuclei (SCN) of the anterior hypothalamus--sufficient to explain the diversity of rhythmic phenomena, or is a multi-oscillator system necessary? What zeitgebers synchronise circadian rhythms and through what receptive organs is this information mediated? What is the neurochemistry of rhythm generation itself and of the pathways for entrainment? A large body of neurobiological literature attests to the phenomenology of circadian change. The pulsatile secretion of hormones shows a circadian profile and a monthly or seasonal modification of rhythm parameters--total secretion over 24 hours, wave form, phase and amplitude. The sleep-wake cycle manifests a rhythm of REM-NREM sleep with period of ca. 90' as well as marked dependence of sleep architecture on circadian phase. Since such physiological and endocrinological parameters are regulated by neurotransmitters and neuropeptides in discrete brain regions, and temporally organised by the SCN, it is not surprising that circadian variations in neurotransmitters and their precursors, their synthetic and metabolic enzymes have been well documented. In addition, the effect of drugs is modified by time of day (for review see Reinberg and Smolensky, 1983). First, by the kinetic parameters of absorption, liver metabolism, renal elimination, binding to plasma proteins, peak plasma concentration or the time required to attain this peak, distribution volume or clearance. Second, and independently, by the circadian variation in susceptibility of a system to a given drug. Since it is the interaction of a medicament with a receptor that permits pharmacologic activity to be expressed, it may be that receptors undergo circadian variations in their number or in their availability. It is from this field of chronopharmacology that the concept of possible CNS receptor change throughout 24 hours was developed. The concept was met with scepticism from "classical" pharmacologists, although less from neurobiologists acquainted with the characteristics of circadian rhythms. The reasons for this scepticism centred around two important issues: the difficulties of using receptor binding techniques to study the functional significance of receptor populations; the general assumption that in vivo the process of receptor change is slow, possibly requiring days to weeks to occur. Studies of the mechanisms of membrane-receptor regulation, especially those concerning rapid changes in brain receptor binding, may provide a conceptual and experimental approach to understanding mechanisms of circadian rhythms in CNS receptors.
3. Methodological Issues The development of the remarkably simple technique of radioligand binding has been instrumental in detailed study of how biologically active substances interact with their target tissues. This review deals with receptor binding techniques that are in common use. Radioligands used for such experiments have been defined pharmacologically as showing different specificities for a given receptor population. A CNS receptor binding site is defined as showing stereospecific, high affinity for a radioligand with Kd in the piconanomolar range; a saturation curve with a maximal number of binding sites (Bmax); and selective displacement with a series of related compounds. In spite of the apparent simplicity and straightforward technique, there are many subtle details of a binding assay that may account for discrepancies in the apparent number of binding sites measured in different laboratories.
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In this review, attention will be paid to factors that are also important in single time point studies of receptor binding: age and gender, species, strain and line, light: dark cycle and its intensity, time of year, correlation with amine release or receptor stimulated post-synaptic events. Outside the scope of this review, but recognised as a real source of variance between different assays, are: membrane preparation (centrifugation speed, number of washings); differences in timing of the assay after collection (storage effect); buffer systems and their concentrations; addition of the antioxidant ascorbate or a monoamineoxidase inhibitor; binding of radioligand to glass filters or plastic tubes; filtration set-up (multiple ports with vacuum gradient); differences in protein content used in the assay or methods used to calculate protein concentration (Leslie et al., 1980; Kraeuchi et al., 1981; Weiner et al., 1982; Muscettola et al., 1986). Such methodological factors at the level of the binding assay itself are not trivial, as pedantic neurochemists who try to standardise and optimise their assay well know. Much time needs to be invested to attain the level of reproducibility that is necessary for measuring relatively small differences in receptor binding. Most pharmacological and lesion studies of receptor populations deal with changes of the order of magnitude of 15-40% , rarely more. Further methodological issues in 24-hour studies of receptor binding are found in Section 5.2. Circadian rhythms are defined as having a period of ca. 24 hours, ultradian rhythms a period of less than 20 hours, and infradian, long-term rhythms a period of greater than 28 hours (usually days, months, years). There are practical limitations to the experimental measurement of circadian rhythms in vitro. Receptor binding studies are of necessity invasive, cross-sectional measures of values from individual animals, and therefore subject to greater variance than if intraindividual receptor sensitivity could be measured over time. The requirement that the number of animals per study be kept to a reasonable minimum has led to various strategies to increase information yield. Too few time points can skew the shape of a rhythm and the conclusions drawn: wave form is defined by the interval between time points. Studies using four or less time points are most likely to be confounded by type II errors, when no temporal variation is found. A single 24-hour period may also not be representative: more convincing (but more tiring to do) are studies repeating the first time point (Brennan et al., 1985; Di Lauro et al., 1986), or the entire 24-hour span (Kafka et al., 1986a; Campbell et al., 1986) and finding similar patterns. A further methodological issue involves the assay itself: if information is to be attained about whether circadian changes are in receptor number or affinity, it is necessary to carry out saturation or displacement curves. However the more precise the dissection, the less tissue is available for complete characterisation of binding. Additionally, if each membrane preparation is used for triplicate assays of total and nonspecific binding, the daunting aspect of such circadian studies--where all time points should be measured in random order in a single assay--becomes clear: an average day of around 600 samples. There is not only the qualitative aspect of changes in number or affinity. Quantitatively, for statistical analysis, an adequate number of individual animals needs to be measured at a given time point: a compromise has been to measure binding at one concentration of radioligand, followed by saturation or displacement curves in pooled homogenates at the times of maxima or minima. A few heroic studies have done both, thus establishing simultaneously the 24-hour time course of the number of binding sites together with appropriate statistics (Lee et al., 1984; Brennan et al., 1985; Di Lauro et al., 1986) (see Tables 3-6; Figs 8, 11). Receptor binding is followed by alterations of cellular metabolic events, such as enzyme activities or ion fluxes, that are ultimately expressed as characteristic physiologic or pharmacologic effects. In general, receptor binding studies are currently moving from phenomenology to this more difficult but more relevant aspect of receptor occupancy and its functional correlates. Circadian studies of receptors are still, however, largely at the descriptive level.
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4. The Beta-Adrenergic Receptor of the Pineal Gland The pineal gland has proved to be a useful model for studying the interaction of environmental and neural influences. The large amplitude rhythm in nocturnal secretion of the neurohormone melatonin is generated by a large rhythm in serotonin N-acetyltransferase activity. This in turn is regulated by a neural circuit from the retina via the SCN that stimulates rhythmic noradrenaline release from pineal sympathetic nerve endings: binding to fl-adrenergic receptors, cyclic-AMP and isoproterenol-stimulated increase of cyclic-AMP (Romero et al., 1975; Mikuni et al., 1981) all undergo circadian rhythms. In contrast, there is no large change in pineal ~tl-adrenergic receptor binding with time of day (Sudgen and Klein, 1985) (Fig. 1). In the pineal, fl-adrenergic receptor binding rhythms, as measured by conventional techniques, are correlated with physiological and pharmacologic response. A rapid change in the number of binding sites occurs within a few hours. It is surprising that the study of pineal fl-adrenergic receptors, one of the very first applications of the new technique of using highly labelled antagonist drugs to measure high affinity specific binding to membranes, did not lead to any further investigation of circadian variation in other receptor populations, nor was the pineal used as a model for studying the mechanisms of rapid and large amplitude changes (ca. 100%) in fl-adrenergic receptor binding. PINEAL ADRENERGIC RECEPTOR BINDING 120(]
111X
E lO :
i".
p
TiME OF DAY (hr) FIG. I. A comparison of the pattern of specific binding to cq- and fl-adrenergic receptors of rat pineal gland homogenates. The former is redrawn from Sudgen and Klein (rats kept on a 14:l0 LD cycle; radioligand: [t25I]-HEAT; N = 5-7 pineals; ANOVA n.s.); the latter, unpublished data, Wirz-Justice e t a / . (rats kept on a 12:12 LD cycle and then released into DD; radioligand: [3H]-DHA; N = 4 pools of 3 pineals; ANOVA p < 0.01).
5. The Mammalian CNS 5.1. CHANGESIN RECEPTOR BINDING OVER 24 HOURS: FIRST CNS STUDIES Although circadian and ultradian rhythms in all aspects of CNS neurotransmitter metabolism, from enzyme activity to release, have been repeatedly demonstrated, the possibility that neurotransmitter receptors also show circadian rhythmicity was considered seriously only for the fl-adrenergic receptor on the pineal gland. The classical concepts of long-term change in receptor number, as found after denervation or chronic treatment with drugs, prevailed. Thus the first CNS receptor rhythm studies emphasised the use of straightforward, established binding techniques (Table l), wholebrain or forebrain homogenates from rats that had been entrained to the usual laboratory 12:12 LD cycle,
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TABLE 1. LIGANDS USED FOR MEASUREMENT OF SPECIFIC BINDING TO RECEPTORS IN TISSUE HOMOGENATES
Receptor fl-adrenergic
3H-Ligand DHA
~q-adrenergic
WB-4101 prazosin clonidine QNB spiroperidol
ct2-adrenergic muscarinic cholinergic dopaminergic
serotonergic opiate GABA benzodiazepine adenosine
5HT spiroperidol ketanserin naloxone morphine GABA diazepam flunitrazepam Ro 15-1788 1-PIA
Displacing drug propranolol alprenolol phentolamine phentolamine atropine ( + )butaclamol spiroperidol ( - )sulpiride haloperidol LSD, 5HT ketanserin methysergide naloxone levorphanol morphine muscimol diazepam clonazepam clonazepam 1 -PIA
and binding studies carried out in a single run (using randomised sampling) to minimise systematic errors, variance across days and across assays. The strategy was to have groups of animals similar to those that compare different treatment paradigms, the treatment being simply time of day. Statistical analysis in the former case is usually that of Student's t-test for independent variables; in the latter, multiple t-tests across the different time points cannot be used (although permitted for comparing peak to trough values). Furthermore, it is important not to make assumptions as to the wave form of the rhythm (as done in cosinor analysis), since wave form is modified by the interval between time points taken. It is more important to choose a conservative test of significance likely to be biologically relevant, than obtaining significance itself (e.g. analysis of variance, ANOVA). As a first step in defining methodology, a summary of the variation coefficients (SD/mean x 100) at three levels of experimentation can be made. First, for a given ligand, the assay variance of triplicate samples; second, for each time point, the interindividual variance; and third, the variance in ligand binding across the 24-hour time span, which reflects the amplitude of rhythmic change (Table 2). Table 3 clearly demonstrates that the variation coefficients of receptor binding over 24 hours exceed the single time point interindividual variance by a factor of 3-10. Thus an
TABLE 2. COEFFICIENT OF VARIATION* OVER A SERIES OF BINDING STUDIES
Inter-individual Amplitudeof Receptor Method (N = 7 rats/ rhythm binding (triplicates) timepoint) peak : trough % fl-adrenergic 5.2% 9.5% 35% cq-adrenergic 3.9% 10% 27% ~t2-adrenergic~ 7.0% 9% 39% muscarinic cholinergic 6.5% 7.9% 28% dopaminergic 6.7% 15.6% 75% opiate 10.4% 23. 1% 87% benzodiazepine 3. 1% 3.8% 40% imipramine:~ 5.7% 10% 35% * SD x 100/mean. Analysed from data summarised in Kafka et al., 1983; tKrauchi et al., 1984 (N = 3 pools of 2/time point); :~Wirz-Justiceet aL, 1983b(N = 4 pools of 9/time point).
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TABLE 3. 24-HouR RECEPTOR BINDING IN WHOLE FOREBRAIN HOMOGENATESFROM MALE RATS (IN A 12:12 LD CYCLE UNLESS OTHERWISE INDICATED)
Ligand/assay*/strain/ age/conditions
Rhythm (wave form)t/ amplitude (peak : trough %)/ N (time points) [hi (animals)/ significance (ANOVA unless otherwise noted)
Reference
DHA/I Sprague-Dawley 250 g/LD DD
C/26%/6 [7]/p < 0.01 C/28%/6 [7]/p < 0.001
Kafka et al., 1981a Kafka et al., 1981a
DHA/I Sprague-Dawley 200 g/DD SCN lesion
U/73%/6 [8]/p < 0.05 U/100%/6 [8]/,0 < 0.05
Kafka el al., 1985 Kafka et al., 1985
U/21%/8/n.s.
Lemmer et al., 1985
C/65%/8/p
< 0.001 C/120%/8/p < 0.0001 U / 2 2 % / 8 / p < 0.01
Lemmer et al., 1985 Lemmer et al., 1985 Lemmer et al., 1985
WB-4101/I Spragne-Dawley 250 g/LD DD
C/45%/6 [7]/p < 0.001 C/22%/6 [7]/p < 0.01
Kafka et al., 1981a Kafka et al., 1981a
WB-4101/I Sprague-Dawley 200 g/DD SCN lesion
C/28%/6 [8]/p < 0.01 U/14%/6 [8]/p < 0.05
Kafka et al., 1985 Kafka et al., 1985
QNB/I Sprague-Dawley 250 g/LD DD
U/56%/6 [7]/p < 0.001 C/71%/6 [7]/p < 0.001
Kafka et al., 1981b Kafka et al., 1981b
QNB/1 Sprague-Dawley 200 g/DD SCN lesion
U/20%/6 [8]/p < 0.05 U/28%/6 [8]/p < 0.05
Kafka et al., 1985 Kafka et al., 1985
QNB/Bm.~ Sprague-Dawley 200 g (receptor occupation)/LD
C/100%/4 [6]/p < 0.001
Mash et al., 1985
5HT/2 Wistar-Wu 200 g/LD
C/215%/12 [3]/p < 0.001
Wesemann et al., 1986a
DHA/Bm, x Wistar-TNO, 150 g (adenylate cyclase, c-AMP, phosphodiesterase)
Spiroperidol/l Sprague-Dawley LD 14:10 Ontogeny: Postnatal day 15 prepubertal day 31 adult day 90
- / - / 8 [3-10]/n.s. t-test U/28%/10 [3-17]/p < 0.01 C/93%/13 [3-13]/p < 0.01
Morphine/Bronx Sprague-Dawley 200g/LD DD
C/106%/6 [6]/p < 0.05 C/314%/6 [6]/p < 0.01
Lee et al., 1984 Lee et al., 1984
Naloxone/l Sprague-Dawley 250 g/LD DD
C/78%/6 [7]/p < 0.001 C/89%/6 [7]/p < 0.001
Naber et al., 1981 Naber et al., 1981
- / 2 0 8 % / 2 [5]/p < 0.001 t-test
Hendrie et al., 1983
U/25%/6 [7]/p < 0.001 C/31%/6 [7]/p < 0.001
Kafka et aL, 1983 Kafka et aL, 1983
Naloxone/B~x Hooded-Lister 300 g/LD Diazepam/1 Sprague-Dawley 250 g/LD DD
Bruinink et al., 1983
Continued
A. WIRZ-JUSTICE
226
TABLE 3---continued
Rhythm (wave fo~'m)t/ amplitude (peak : trough %)/ N (time points) [n] (animals)/ significance (ANOVA unless otherwise noted)
Ligand/assay*/strain/ age/conditions Diazepam/1 Sprague-Dawley 200 g/DD SCN lesion N6-(l-phenyl-isopropyl) adenosine/B=ax Sprague-Dawley/LD
Reference
C/33%/6 [8]/p < 0.01 U/34%/6 [8]/p < 0.01
Kafka et al., 1985 Kafka et al., 1985
C/74%/6 [6]/p < 0.02
Virus et al., 1984
* Number of ligand concentrations = 1, 2, etc; or Scatchard Plot = B ~ . * Circadian (unimodal) = C; Ultradian (bimodal) = U. LD = light:dark cycle; DD = continuous darkness.
explanation of these daily changes as mere "biological noise" is, at least in our studies, insufficient. Furthermore, the argument that a 20-40% change over 24 hours is unlikely to be relevant to receptor function should also be as exigently applied to non-circadian studies: this range is precisely that consistently demonstrated with most drug or lesion protocols. The first studies of binding to forebrain homogenates from rats on a 12:12 LD cycle showed significant changes throughout 24 hours. Although single concentrations of radioligand were used to measure specific binding, in further experiments pooled homogenates were subjected to saturation curves (at each time point, or at the times of maximum and minimum). In none of the experiments was a change in Kd observed (e.g. Fig. 2), indicating that the changes in specific binding throughout the day were changes in the apparent number of receptors (Kafka et al., 1983). 5.2. REPLICATION OF RECEPTOR BINDING RHYTHMS AND POSSIBLE SEASONAL MODULATION
Similar experiments in the same laboratory using the same rat strain and assays were repeated four times: each time, significant rhythms were found in st and fl-adrenergic, muscarinic cholinergic, opiate and benzodiazepine receptor binding in forebrain and in dopaminergic receptor binding in striatum (summarised in Kafka et al., 1983). One problem was immediately made clear: a given rhythm was not as reproducible in its wave-form and phase characteristics as was the pineal fl-adrenergic receptor rhythm. Since the animals were strictly entrained to the same LD cycle, the data suggested that either the changes were due to random variance, or due to an effect of season that was not one of photoperiod. The latter explanation would require finding the same pattern during the same month of consecutive years--a somewhat mammoth undertaking. The former was considered unlikely for reasons of methodology described above. A similar variability of circadian patterns had been found in repeated measurement of noradrenaline concentration in rat brain (Davies et al., 1972). In this context, some observations of apparent seasonal changes in number of receptor binding sites had already been made in single time point studies (in the light phase). For example, the density of naloxone binding sites in mouse brain homogenates was 73% higher in winter than in summer (Codd and Byrne, 1981); similarly, the number of muscarinic cholinergic receptors was 54% higher in spring than in summer (Kastrup and Christensen, 1984). In the seasonally reproductive ewe, pineal fl-adrenergic receptor density and affinity peak in May and November (Foldes et al., 1985). A phase-shift could explain such seasonal changes just as could a change in the 24-hour mean. In our studies, QNB binding in the early light phase could be high or low, depending on the rhythm at that time of year; additionally, changes in the 24-hour mean across
CIRCADIAN RHYTHMS
227
SCATCHARD PLOT OF OPIATE RECEPTORBINDING
10 hr
0.015 0.010i
? ~ !
0.015 0.010
0.006
14 hr • t
•
0.005 0.05 0.10 0.15
B/F
,
i
r• 0.015~
18 hr
0.010' • •
0.05 0.10 0.15 B/F L z\ 0.015g 0.010i
0.06 0.10 0.15
B/F 0.015
2 hr
0.05 0.10 0.15
6 hr
0.015
•
0.010
0.005
0,006 0.10 0.15
•e'X~
B/F
0.010
0.06
22 hr
"
0.06 0.10 0.15
[ IH]-NALOXONE SPECIFICALLYBOUND (pmol / 10 mO wet weigkt) FIG. 2. Scatchard plot of opiate receptor binding at 6 different times of day. Male Sprague-Dawley rats were kept on an LD cycle, and the entire forebrain assayed for specific naloxone binding (pool of N = 6 at each time point, experiment carried out in April) (from Naber et al., 1981).
seasons were found. This meant that although the lowest 24-hour mean QNB binding occurred in October, the value in the early light phase was higher in October than in April (Fig. 3) (Kafka et al., 1981b). In those receptors where binding showed both uni- and bimodal patterns [such as muscarinic cholinergic, Fig. 3, or ~l- and ~-adrenergic, benzodiazepine, or striatal dopamine receptors (Kafka et al., 1983)] no straightforward statement as to at which time maximum and minimum binding occurs could be made with respect to whole for•brain. However a unimodal rhythm in opiate receptor binding did show replicable characteristics of peak binding occurring in the dark phase (Naber et al., 198!; Wirz-Justice et al., 1981; Lee et al., 1984) (Figs 2, 18, 22). All data available for whole for•brain receptor rhythms are summarised in Table 3. 5.3. PERSISTENCEOF CIRCADIAN RHYTHMS UNDER CONSTANT CONDITIONS BUT NOT AFTER SCN LESIONS
The previous studies had been carried out in animals kept on an LD cycle. It is well known that light can directly modify behaviour and physiology by "masking" (Aschoff et al., 1981). By studying animals that had been kept in constant darkness (DD) during the last day(s) of the experiment, possible masking effects of light itself on receptor binding could be eliminated. In addition, a rhythm persisting under conditions without time cues satisfies an important criterion for an endogenous circadian rhythm generated within the brain itself.
228
A. WIRZ-JUSTICE pmol3H-QNB/ gramwetweight 24 forebrain January
i %
2O FEBRUARY
18
t
March 12 APRIL
10
I
I
22
/
May
S
J
JUNE
i 1/
July
August
18
*~
,
**
i
September 14] OCTOBER
November December
12:
* *p<0.01
I
* p<0.05 ,,e,,,i,A, 10
14
18
22
2
10
14
18
Timeof Day (hr) F I G . 3. 24-Hour patterns of [3H]-QNB specific binding to muscarinic cholinergic receptors in rat forebrain in four separate experiments (mean + s . e . m , o f N = 7 animals at 6 time points, except for April, where the 7 animals were pooled for Scatchard analyses). The shaded area indicates the dark phase to which the animals were exposed, the dotted lines the changing external photoperiod (from Kafka et al., 198 l b ) . A fifth experiment (data not shown) carried out in December-January in Switzerland with the Sprague-Dawley Ivanovas rat strain showed a bimodal pattern with peaks in the mid-dark and mid-light phase intermediate between the experiments in October and February ( W i r z - J u s t i c e et al., 1981).
Indeed, it was found that all binding rhythms remained significant in DD, and their wave form was greatly simplified to a unimodal pattern in most cases (Table 3, Fig. 4). Thus the persistence of significant changes over 24 hours in the absence of time cues indicates their endogenous nature (see also Naber et al., 1981; Lee et al., 1984 for similar patterns of opiate receptor binding in LD and DD). To address the even more crucial question of whether these circadian rhythms in receptor binding are driven by the putative circadian pacemaker in the SCN, a difficult study with SCN lesioned animals was undertaken by the Bethesda group (Kafka et al., 1985). The endogenous circadian rhythm in ~-adrenergic and benzodiazepine receptor binding found in controls was no longer present after SCN lesions: the patterns became ultradian with two peaks in 24 hours (Fig. 5). The/~-adrenergic and muscarinic cholinergic receptor rhythms were ultradian in control animals and this pattern persisted after SCN lesions. Thus SCN ablation appeared to cause a selective loss of circadian function in those receptors that showed unimodal rhythmicity: ultradian rhythms were not affected. This experiment indicates that the SCN is necessary for the maintenance of circadian expression of receptor binding, as it is for most other circadian functions that have been investigated.
229
CIRCADIAN RHYTHMS
RECEPTOR BINDING IN DO 12
BZP
Z L--J D .c
I~
1
•-~ ~
|,.~
Z FIG. 4. Circadian rhythm of binding to e~- and ~-adrenergic and benzodiazepine receptors in forebrain homogenates from rats kept under LD 12:12 for three weeks (as indicated by the vertical lines at 7 hr for lights on, at 19 hr for lights off), and then maintained for three days in DD. They were sacrificed under red light (N = 6 rats at 6 time points). All rhythms were significant by ANOVA; the peak values indicated are significantly different from the troughs (redrawn from Kafka et al., 1983). 5.4. REGIONAL DIFFERENCES IN RECEPTOR RHYTHMS M o n o a m i n e s , their enzymes and metabolites undergo circadian variations in the C N S that vary f r o m region to region and even a m o n g the nuclei o f a given region (e.g. N A and 5 H T metabolism in different brainstem nuclei ( K a n et al., 1977; Daiguji et al., 1978; Natali et al., 1980a, b; Chevillard et al., 1981; D a s z u t a et al., 1982; Semba et al., 1984; K o u l u et al., 1985; Rosenwasser et al., 1985). Thus the rhythms measured in whole forebrain are the sum o f all nuclei included in the dissection. F o r this reason, the focus shifted to studies o f regional differences in receptor rhythms. 5.4.1. Frontal cortex and hypothalamus Frontal cortex is the region most often used for studies o f drug effects on receptor binding: for this reason a n u m b e r o f groups have begun their investigations o f 24-hour rhythms in a brain area not noted for high amplitude rhythmicity. These results are summarised in Table 4. cc c A D R E N O C E P T O R B I N D I N G mCONTROLS ....SCN LESION
a O
,
o
L+
I 3
i 7
I | 11 D + 3
I 7
| 11
TIME OF DAY (hr)
FIG. 5. Circadian rhythm in ~q-adrenergic receptor binding in controls and ultradian rhythm in SCN lesioned animals (redrawn from Kafka et aL, 1985).
230 TABLE 4.
A. WmZ-JUSTtCE 24-Hour RECEPTORBINDINGIN CORTEX(OR REGIONSTrmREOF)FROMMALERATS(IN 12:12 LD CYCLE UNLESSOTHERWISEINDICATED)
Ligand/Assay/Strain/ Age/Conditions DHA/Bmu Sprague-Dawley 200 g/DD (fl-stimulated c-AMP) DHA/I Sprague-Dawley 200 g/DD
Rhythm (wave form)t/ amplitude (peak:trough %)/ N (time points) In] (animals)/ significance (ANOVA unless otherwise noted)
Reference
U/40%/6 [6]/p < 0.01 U/80%/6 [6]/p < 0.05 C/18-47%/6 [8]/p < 0.05 in 5/9 discrete cortical regions
Benedito and Kafka, 1982 Benedito and Kafka, 1982
DHA/B~x Wistar-King 250 g/LD
- / - / 4 [?]/n.s.
Matsubara et al., 1982
DHA/Bmx /
-/20%/7 [4]/n.s.
Di Lauro et al., 1986
- / 13%/4 [3-5]/n.s.
Fowler et al., 1985
DHA/l Wistar-Kyoto SHR 300 g/DD
- / - / 8 [6]/n.s. - / - / 8 [6]/n.s.
Wirz-Justice et al., 1983a
DHA/1 Wistar-Fuellinsdor f/LD 3 months 12 months 24 months
-/33%/6 [4]/n.s. -/23%/6 [4]/n.s. -/29%/6 [4]/n.s.
Jenni-Eiermann et al., 1985
- / 7 % / 6 [3]/n.s.
Wirz-Justice et al., unpubl, data
-/28%/2 [8]/p < 0.05 t-test
Mogilnicka et al., 1985
C/26%/6 [6]/p < 0.05 C/150%/6 [6]/p < 0.05
Benedito and Kafka, 1982 Benedito and Kafka, 1982
-/17%/7 [4]/n.s.
Di Lauro et al., 1986
-/14%/6 [4]/n.s. -/18%/6 [4]/n.s. -/22%/6 [4]/n.s.
Jenni-Eiermann et al., 1985
Kafka et al., 1986a
Wistar-Morini 300 g/DD DHA/Bmx Sprague-Dawley/LD
DHA/1 Wistar-Fuellinsdorf 350 g/LD DHA/Bmax Wistar 200 g/LD WB-4101/Bm,x Sprague-Dawley 200 g/DD or-stimulated c-AMP WB-4101/B~x Wistar-Morini 300 g/DD WB-4101/l Wistar-Fuellinsdorf/LD 3 months 12 months 24 months Prazosin/Bmax Wistar 200 g/LD Prazosin/l Sprague-Dawley 200 g/DD
-/19%/2 [8]/p < 0.05 t-test
Mogilnicka et al., 1985
C/44-117%/6 [8]/p < 0.01 in 7/7 discrete cortical regions
Kafka et al., 1986a
p-aminoclonidine/l Sprague-Dawley 200 g/DD
C/49-92%/6 [8]/,o < 0.05 in 3/6 discrete cortical regions
Kafka et al., 1986a
Clonidine/l Wistar-Fuellinsdorf 350 g/DD QNB/1 Fischer 10 weeks/LD
C/61%/8 [4]/,o < 0.05
Kraeuchi et al., unpubl, data
- / 10%/6 [3-4]/n.s.
Por and Bondy, 1981 Contmued
CIRCADIAN RHYTHMS
231
TABLE 4---continued
Ligand/Assay/Strain/ Age/Conditions
Rhythm (wave form)t/ amplitude (peak:trough %)/ N (time points) In] (animals)/ significance (ANOVA unless otherwise noted)
Reference
C/25-85%/6 [8]/p < 0.01 in 2/4 discrete cortical regions
Kafka et aL, 1986a
-/29%/6 [4]/n.s. -/19%/6 [4]/n.s. -/32%/6 [4l/n.s. -/14%/8 [4]/n.s. occipital cortex
Jenni-Eiermann et al., 1985
- / 9 % / 6 [3]/n.s. occipital cortex
Wirz-Justice et al., unpubl, data
25% increase in temporal 65% in visual cortex of dark-reared rats after 3 hr light/p < 0.01 t-test
Murphy et al., 1980
LSD/I Wistar-Fuellinsdorf/LD 3 months 12 months 24 months
U/29%/6 [41/p < 0.05 - / 1 ! %/6 [4]/n.s. U/24%/6 [4]/p < 0.05
Jenni-Eiermann et al., 1985
Spiroperidol/1 Wistar-Fuellinsdorf/LD 3 months 12 months 24 months
--/41%/6 [4]/n.s. -/17%/6 [4]/n.s. --/31%/6 [4]/n.s.
Jenni-Eiermann et al., 1985
Spiroperidol/1 Wistar-Fuellinsdorf 350 g/DD
-/20%/8 [4]/n.s. occipital cortex
Kraeuchi et al., unpubl, data
Spiroperidol/1 Wistar-Fuellinsdorf 350 g/LD
C/36%/6 [3l/p < 0.05 occipital cortex
Wirz-Justice et al., unpubl, data
Spiroperidol/Bmax Wistar-Morini 300 g/DD
-/10%/7 [4]/n.s.
Di Lauro et al., 1986
Ketanscrin/B~,ax Wistar (UK) 300 g/LD
-/50%/6 [6]/n.s.
Campbell et al., 1986
Imipramine/B~x Wistar-Morini 300 g/DD
-/16%/7 [4]/n.s.
Di Lauro et aL, 1986
Imipramine/1 Wistar-Fuellinsdorf 350 g/DD
U/36%/8 [4]/p < 0.05 occipital cortex
Kraeuchi et al., 1986
Imipramine/1 Wistar-Fuellinsdorf 350 g/LD
U/31%/6 [3]/p < 0.01 occipital cortex
Kraeuchi et al., 1986
Naloxone/l Sprague-Dawley/ 9, 15, 26, 30 days Female/Male
- / - / 4 / n . s . t-test
Jacobson and Wilkinson, 1985
Flunitrazepam Sprague-Dawley 200 g/DD Flunitrazepam/Bmx Sprague-Dawley 350 g/LD 14:10
C/45-125%/6 [81/p < 0.05 in 3/5 discrete cortical regions
Kafka et al., 1986a
C/50%/6 [3]/p < 0.001 t-test frontal cortex
Brennan et al., 1985
QNB/1 Sprague-Dawley 200 g/DD QNB/I Wistar-Fuellinsdorf/LD 3 months 12 months 24 months QNB/1 Wistar-Fuellinsdorf 350 g/DD QNB/I Wistar-Fuellinsdorf 350 g/LD 5HT/1 CFHB 55 days
Kraeuchi et aL, unpubl, data
Continued
232
A. WIRZ-JUSTICE TABLE 4 - - c o n t i n u e d
Ligand/Assay/Strain/ Age/Conditions Human Autopsy Material Scopolamine/1 5HT, Spiperone/Bmax
Rhythm (wave form)t/ amplitude (peak:trough %)/ N (time points) [n] (animals)/ significance(ANOVA unless otherwise noted) C/137%/8 [345]/p < 0.02 t-test - / - / 4 [18-20]/n.s. t-test
Reference Perry et al., 1977 Wester et al., 1984
Whereas in whole forebrain significant 24-hour variations were found in all studies (Table 3), only about 40% of measurements in cortex (or defined areas thereof) evidenced significant rhythmicity (Table 4). The hypothalamus more often (60%) showed large amplitude rhythms (Table 5).
5.4.2. Discrete brain regions The diversity of results with respect to the existence or not of circadian rhythms in neurotransmitter receptors predicates caution. More detailed dissection has revealed circadian or ultradian receptor rhythms in some, but not all regions, even within a given area such as the cortex (e.g. Fig. 6). This complexity is entirely analogous to that found in the numerous studies on circadian rhythms in neurotransmitters themselves. We have been able to replicate receptor patterns when precisely defined punched-out brain areas have been used. For example, the SCN itself shows a robust circadian rhythm in imipramine binding with a minimum at dusk and a maximum at dawn (Fig. 7). A smaller amplitude, ultradian rhythm in imipramine binding is found in the occipital cortex, whereas the caudate putamen shows no significant changes over 24 hours (Wirz-Justice et al., 1983b; Kraeuchi et al., 1986).
5.4.3. Negative results Some groups have carefully executed studies that show no rhythm in maximal number of binding sites (Fowler et al., 1985; D'i Lauro et al., 1986; Campbell et al., 1986) (e.g. Fig. 8). These negative findings need to be included in any discussion of receptor rhythms, but cannot be used to refute categorically their existence; there are a number of experimental factors apart from brain regions that can account for such differences, such as strain, LD or DD conditions, the number of time points and the number of animals per time point.
5.4.4. Regional differences in rhythmicity: relationship to regional function? It is only when repeated studies under adequate conditions consistently show no change over 24 hours, that a negative finding could be as functionally relevant for the given receptor system as a positive finding in another region. A detailed example illustrating this concept is therefore presented: ct2-adrenergic receptor binding in the locus coeruleus (LC) and in the medial hypothalamus (MH). The LC contains the largest clusters of noradrenaline-containing neurons in the CNS. There is evidence for ~tE-adrenergic receptors in the LC mediating inhibitory responses (Aghajanian and VanDer Maelen, 1982), and being involved in regulation of the sleep-wake cycle (Drew et al., 1979). There is no circadian modification in binding to ct2-adrenergic receptors in the LC of control Wistar rats nor in animals whose sleep-wake cycle is phase shifted by chronic methamphetamine treatment, nor after one or four weeks drug withdrawal (Wirz-Justice et al., 1985a and unpublished data) (Fig. 9). Noradrenaline
CIRCADIANRHYTHMS
233
TABLE 5. 24-HouR RECEPTORBINDINGIN HYPOTHALAMUS(OR REGIONS* THEREOF) FROMMALE RATS(IN 12:12 LD CYCLE UNLESSOTHERWISEINDICATED)
Ligand/Assay/Strain/ Age/Conditions
Rhythm (wave form)f/ amplitude (peak : trough %)/ N (time points) [hi (animals)/ significance (ANOVA unless otherwise noted)
Reference
DHA/1 Sprague-Dawley 200 g/DD
C/100%/6 [81/p < 0.05
Kafka et al., 1986a
DHA/1 Wistar-Fuellinsdorf 350 g/LD
U/40%/6 [3]/LH, p < 0.05
Kraeuchi et al., 1984
DHA/Bm~/
-/29%/7 [41/n.s.
Di Lauro et al., 1986
Matsubara et al., 1982
Wistar-Morini 300 g/DD DHA/Bm,~
- / -/4
Prazosin/1 Sprague-Dawley 200 g/DD
C/70%/6 [8]/p < 0.05
Kafka et al., 1986a
WB-4101/Bma~ Wistar-Morini 300 g/DD
-/27%/7 [4]/n.s.
Di Lauro et al., 1986
U/21%/8 [1/pool of 6 ] / C/39%/8 [l/pool of 6 ] / -
Wirz-Justice et al., 1983a
p-Amino-clonidine/1 Sprague-Dawley 200 g/DD
C/50%/6 [8]/n.s.
Kafka et al., 1986a
p-Amino-clonidine/1, B~x Sprague--Dawley 300 g/LD
C in SCN, PVN, SON, LH (of 8 discrete nuclei)
Jhanwar-Uniyal et al., 1986
Clonidine/1 Wistar-Fuellinsdorf 350 g/LD
C/39%/6 [31/p < 0.05
Kraeuchi et al., 1984
Clonidine/1 Wistar-Fuellinsdorf 350 g/DD
U/18%/8 [4I/VMH, p < 0.05 C/46%/8 [4]/PVN, p < 0.05
Kraeuchi et al., unpubl, data
U/20%/8 [pool of 6]/ C/31%/8 [pool of 6]/
Wirz-Justice et al., 1983a
QNB/I Fischer 10 weeks
C/20%/6 [3-4]/p < 0.06
Por and Bondy, 1981
QNB/1 Wistar-Fuellinsdorf 350 g/DD
- / 11%/8 [4I/POM, n.s.
Kraeuchi et al., unpubl, data
Spiperone/Bmax Wistar-Morini 300 g/DD
-/45%/7 [4l/n.s.
Di Lauro et al., 1986
Spiperone/1 Wistar-Fuellinsdorf 350 g/LD
C/23%/6 [3]/LH, p < 0.05
Wirz-Justice et al., unpubl, data
Imipramine/Bm,x Wistar-Morini 300 g/DD Imipramine/l Wistar-Fuellinsdorf 350 g/DD
-/15%/7 [4]/n.s.
Di Lauro et al., 1986
C/35%/8 [4I/SCN, p < 0.02 C/27%/8 [4]/POM, p < 0.05 C / 4 1 % / 8 [4]/AH, p < 0.05
Wirz-Justice et al., 1983b
C/30%/6 [3]/SCN, p < 0.05
Kraeuchi et al., 1986
WB-4101/I Wistar-Kyoto SHR 300 g/DD
•
Clonidine/l Wistar-Kyoto SHR 300 g/DD
lmipramine/1 Wistar-Fuellinsdorf 350 g/LD
[?]/n.s.
Continued J,PN 293--B
234
A. WIRZ-JUSTICE TABLE 5--continued
Rhythm (wave form)t/ amplitude (peak:trough %)/ N (time points) [n] (animals)/ significance (ANOVA unless otherwise noted)
Ligand/Assay/Strain/ Age/Conditions Naloxone/l Sprague-Dawley 9, 15, 26, 30 days
C/
Flunitrazepam/Bma x Sprague-Dawley
- / - / 6 [3]/n.s. t-test
Reference Jacobson and Wilkinson, 1985
/ 4 / V M H / p < 0.01
in females only Brennan et al., 1985
350 g/LD 14:10
* Abbreviations for hypothalamic regions: SCN, suprachiasmatic nucleus; PVN, paraventricular nucleus; SON, supraoptic nucleus; POM medial preoptic area; VMH, ventral medial hypothalamus; LH, lateral hypothalamus; AH, anterior hypothalamus.
]H-DIHYOROALPRENOLOL ($H-OHA) BOUNO
*=p<005 "* = o , ~ 0 , 0 1
50 OLFACTORY BUL8
ANOVA
40 30 SO
FRONTAL CORTEX
50
ENTORHINAL CORTEX
40 30
CINGULATE CORTEX
SO
.
.
.
.
.
.
4
7O
SO PIRIFORM CORTEX
SO 70 SO
INSULAR CORTEX
PARIETAL CORTEX
SO 5O
70 SO 8O
TEMPORAL 70 CORTEX SO 5O SO OCCIPITAL CORTEX HYPOTHALAMUS
50 40 20 50
HIPPOCAMPUS
40 3O
CEREBELLUM
30 22
PONS-MEDULLA
20 12
CAUOATE-PUTAMEN
50
THALAMUS SEPTUM
40 3O 20 10
14
18 22 TIME (hi)
2
6
FIG. 6. [3H]-DHA (0.8 riM) specific binding to fl-adrenergic receptors at 6 time-points throughout 24 hours in discrete brain regions. Sprague-Dawley rats were maintained on a 12:12 LD cycle as indicated (dark phase from 19-7 hr), and enucleated 2-3 days before the experiment (functionally without time cues). Each time point is the mean from 6-8 pools of regions from 3 rats. Significance rhythms were found in 7 of the 15 regions investigated (from Kafka et al., 1986a).
CIRCADIAN RHYTHMS
235
I,a~ Exp.1 e~Q Exp2 800
750
f .g
7OO
/ i
550 500=
550. 5o0
i
I
SCN
t
350. 300=
~
250=
30C | ~
25C
'
%
OC
200 |
*2
i
*6
|
|
*8 D *2
|
•
*6
*8
FIG. 7. A comparison between 24-hour rhythms and overall means of specific [3H]-imipramine binding in three different brain regions in two separate experiments. Exp. 1 was carried out in DD, with 6 month-old male Fuellinsdorf-Wistar rats in October; mean + SEM of 3--4 pools of 9 animals; N = 36 for the 24-hour mean. Exp. 2 was carried out in LD, with 3 month-old rats in March; mean + SEM of 3 pools of 2 animals (and a larger punch for the SCN area); N = 18 for the 24-hour mean. The black line represents the dark phase of the entraining LD cycle (from Kraeuchi et al., 1986).
in the LC also shows no circadian rhythmicity in one study of Wistar rats (Semba et al., 1984), which is consistent with these findings, but a significant rhythm in another study using Sprague-Dawley rats (Koulu et al., 1985). It is surprising that a drug that increases wake time by 4 hours does not even change the 24-hour mean binding to ~2-adrenergic receptors in the LC. This overall stability indicates that these noradrenergic receptors are involved neither in the rhythmicity of the sleep-wake cycle, nor in the homeostasis of sleep regulation. A different pattern is shown in another region studied in the same experiment. The medial hypothalamus, in particular the paraventricular nucleus, is involved in the regulation of feeding and corticosterone rhythms, both via an ~2-adrenergic receptor mechanism (Marino et al., 1983; Jhanwar-Uniyal et al., 1986). In this region we found a significant rhythm in [3H]-clonidine binding (Kraeuchi et al., 1984). After chronic methamphetamine treatment, ~2-adrenergic receptor binding increased at dawn together with increased feeding at this time of day. After drug withdrawal, the rhythm gradually returned to the control pattern (Fig. 10). Moreover, at one week's withdrawal--a time characterised by rebound eating and weight gain--an increase in the 24 hour mean of • 2-adrenergic receptor binding was found. Thus both the circadian and homeostatic aspects of feeding are reflected in parallel changes in medial hypothalamic ~-receptors (Kraeuchi et al., 1984).
236
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Recently, Leibowitz' group has observed decreased ~(2-adrenergic receptor binding sites in the medial hypothalamus after 48 hours food deprivation; furthermore receptor change was circadian phase dependent and unusually rapid (Jhanwar-Uniyal et al., 1985). Three hours food deprivation at the beginning of the dark phase reduced binding by 65%, and six hours food deprivation at the end of the dark phase increased binding by 54%. These studies support our contention that it should be possible to correlate rapid changes in receptor number in specific regions with the function of the neurotransmitter in that region. A similar circadian rhythm of ~2-adrenergic receptor binding has been found in the paraventricular nucleus itself (Jhanwar-Uniyal et al., 1986). This study in punched-out brain areas is the most detailed up until now and beautifully illustrates the phenomenon: in only three out of fourteen brain areas--and these all in the hypothalamus--could a circadian rhythm in ct2-adrenergic receptor binding be demonstrated. However these three areas are precisely those with an important role in rhythmic function: the SCN, showing a rhythm with the same pattern as in the paraventricular nucleus, and the supraoptic nucleus, with a rhythm 180 ° out of phase with the other two regions (Jhanwar-Uniyal et al., 1986).
CIRCADIAN RHYTHMS
237
locus coeruleus [3HI - clonidine binding
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Thus an adequate description of receptor rhythms still remains to be established. Neurotransmitter receptors in discrete brain areas with known functional correlates are most likely to show replicable rhythmicity, or a lack thereof; in addition, parallel measurement of amine turnover or second messenger rhythms may be necessary (see Section 5.7).
238
A. WIRZ-JUSTICE medial hypothalamus
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5.5. STRAIN DIFFERENCES IN RECEPTOR RHYTHMS
The initial studies on receptor rhythms were carried out in the same line of SpragueDawley rats (Kafka et al., 1983). Naturally, replication studies in different laboratories have used different strains and/or lines of animals. Differences in sleep-wake or restactivity rhythms have been shown between strains of mice and rats (Oliverio and Malorni, 1979; Valatx, 1984; Lemmer et al., 1981), and differences in neurotransmitter rhythms found in the nuclei involved in sleep regulation (Natali et al., 1980a, b). Thus it is not surprising that such differences also exist for neurotransmitter receptors. Single time point studies have shown differences in catecholamine receptors between rat strains (Wolf et al., 1980; Helmeste et al., 1982; Chiu et al., 1982; Helmeste, 1983); another study found no differences in cholinergic receptors (Blaker et al., 1983). The circadian approach has been applied to the Wistar-Kyoto strain of spontaneous hypertensive rats (SHR) and their normotensive controls (WKY). Although blood pressure is higher in SHRs at all times of day, there are marked differences in many circadian rhythms: the rest-activity cycle shows earlier onset of nocturnal locomotion; plasma prolactin and stress-induced corticosterone secretion begins earlier in the light phase in SHR than in WKY rats; and adrenergic receptors show circadian phase dependent differences (Kraeuchi et al., 1983; Wirz-Justice et al., 1983a).
CIRCADIAN RHYTHMS
239
The mean 24-hour binding to ~-, ~2- and fl-adrenergic receptors was higher in SHR than WKY in all regions investigated, but greatest differences were found at the end of the dark phase. Again, there were pronounced regional differences• In the cerebellum, fl-adrenergic receptor binding was higher in SHR rats at all times of day, with an identical rhythm to that in WKY rats. Displacement curves indicated no difference in affinity either between time points or between strains (Fig. 11) (Wirz-Justice et al., 1983a). These detailed studies in WKY and SHR rats indicate that the differences between strains in most brain regions are small, and very dependent on time of day, the increase in adrenergic receptor binding being most consistently seen at the end of the dark phase. Furthermore, with respect to most efficacious therapy, it has been observed in rats that the anti-hypertensive drug, ct-methyl-DOPA, produces the greatest and most long-lasting decrease in blood pressure when administered in the dark phase (Post and Nair, 1975). A less obvious source of differences in receptor rhythms is not the strain of rats but the separate lines of a given strain that are used. There is as yet only one systematic investigation of this issue (Jenni-Eiermann et al., 1986). Large differences in a number of receptor rhythms were found in many brain regions from two rat lines of Wistar origin (Kfm: WlST and Fuellinsdorf Albino)• The 24-hour mean of QNB and spiroperidol binding in striatum, fl-adrenergic receptor binding in hippocampus, and GABA-receptor binding in cerebellum was more than double in Kfm compared with Fuellinsdorf rats. In seven out of eleven assays the Kfm line had rhythm amplitudes greater than 50%, whereas this was the case in only one out eleven assays for the Fuellinsdorf line. Finally, marked differences in phase position were observed between the two lines in six out of eleven assays• Since most of the studies of receptor rhythms in Basel have used the FueUinsdorf line, our increased number of negative findings compared with earlier studies (Kafka et al., 1983) may be related to this chance choice of one of the less rhythmic lines in the available sources of animals. An example of this difference between Wistar lines is shown in Fig. 12A. Such major differences in two closely related rat lines underline the difficulties in determining the patterns and timing of receptor rhythms. This problem is not unique to receptors: diurnal fluctuations of acetylcholine (Saito et al., 1975) and choline acetyl-
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transferase activity varied across region and between two strains of Sprague-Dawley rats (Nordberg and Wahlstroem, 1980) as well as Wistars (Figure 12b) (Jenni-Eiermann et al., 1986). 5.6. AGE AND GENDER
Most studies of receptor binding are carried out in adult male rats of ca. 200-300 g body weight. Similarly, there are as yet few studies investigating the modification of receptor rhythms by gender (in particular possible differential effects of puberty), or ontogeny and senescence. The only study comparing male and female rats at different ages found no opiate receptor rhythms in cortical slices (Jacobson and Wilkinson, 1985). However, in mediobasal hypothalamic slices, a complex developmental pattern was seen in both sexes. Female hypothalamus by 26 and 30 days of age showed a clear diurnal decrease in opiate receptor binding. These binding changes in prepubertal female rats have a functional correlate: the diurnal variation in LH response to opiate receptor blockade is maximal in the morning and minimal in the late afternoon.
CIRCADIAN RHYTHMS
241
A detailed study of the ontogeny of high affinity spiperone binding sites in forebrain (Bruinink, 1983) found no marked diurnal variations at postnatal day 15. At 30 days a sharp peak in the mid-dark phase was found for both dopaminergic and serotonergic binding sites. This pattern inversely reflected the bimodal activity-rest cycle at this age, and was similar to earlier findings of a bimodal rhythm of spiroperidol binding in striatum from another line of Sprague-Dawley rats (Naber et al., 1980). In adult rats (90 days) a different rhythm of spiperone binding was observed, with a decrease in the dark phase again inverse to the unimodal peak of nocturnal activity. A similar pattern was found in striatal spiroperidol binding in adult rats of the same strain (Wirz-Justice et al., 1981). Thus during ontogeny, certain receptor rhythms increase in 24-hour mean and amplitude, and change in wave form from ultradian to circadian. It is therefore of interest to note that ageing can induce the reverse: a decrease in 24-hour mean and amplitude, and dispersal of phase. Yet there is no linear trend with ageing that could be used as a generalisation (Jenni-Eiermann et al., 1985). Many ligands were used to measure regional receptor binding in 3, 12 and 24 month old male Fuellinsdorf Albino rats. In general, but not always, 24-hour mean binding was lowest in old rats. The amplitude of receptor rhythms was considerably less pronounced in adult than in young rats; however aged rats again had many rhythms of high amplitude. The phase position of peak binding of a given ligand in a given region also varied with age, with respect to each other and to the LD cycle. Noticeable was the increased dispersion of phase with age. An example is shown in Fig. 13. 5.7. RECEPTOR RHYTHMS: CORRELATIONWITH NEUROTRANSMITTERTURNOVER OR SECOND MESSENGER With this list of problematic issues related to replicability of receptor rhythms, a more general problem is introduced. For all studies involving receptor binding, there is no functional correlate of changes in apparent number. Thus the importance of a 30% change in binding can be assessed only with respect to statistical, but not biological significance. This is a fundamental drawback of the elegant and deceptively easy technique of radioligand binding. It is only in the pineal gland that the chain of metabolic events triggered by noradrenaline release from the superior cervical ganglion has been extensively studied (see Section 4). The rhythms in the pineal are of large amplitude. Since rapid changes in apparent number of fl-adrenergic receptors have been replicably found in the pineal, and linked to the sensitivity of adenylate cyclase stimulation, parallel phenomena should be measurable in CNS tissue.
CIRCADIAN RHYTHMS OF GABA-RECEPTOR BINDING WITH AGE
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242
A. WIRZ-JUSTICE 5.7.1. Presynaptic events
The relationship between neurotransmitter turnover and receptor occupancy is not at all clear. Initially the concept prevailed that increased release of a neurotransmitter would lead to gradual development of post-synaptic receptor down-regulation. However this direct relationship is in reality complicated by differential changes in pre- and post-synaptic receptor populations to transmitter release. The circadian rhythms in the pineal gland do show this relationship. But already here, in well-defined systems, receptor differences are seen. Noradrenaline release over 24 hours and fl-adrenergic receptor binding show an inverse relationship, with only small changes in ~-adrenergic receptor binding throughout the day (Fig. 1). We have found CNS adrenergic receptor rhythms that follow a parallel, not inverse, time course. For example, the functional importance of noradrenaline in feeding regulation has been established. Feeding is regulated in the lateral hypothalamus via a fl-adrenergic receptor mechanism, and in the medial hypothalamus via an ~2-adrenergic receptor mechanism (Leibowitz, 1980) (Figs 10, 14 and Sections 5.4.4; 6.3). In the lateral hypothalamus, the patterns of presynaptic noradrenaline uptake (Van der Gutgen and Slangen, 1975), rate of neuronal firing (Schmitt, 1973), and fl-adrenergic receptor binding (Kraeuchi et al., 1984) are all bimodal. More important, the functional response of feeding induced by local injection of noradrenaline in the lateral hypothalamus also shows a bimodal rhythm (Margules et al., 1972): thus maximal fl-adrenergic receptor binding occurs at dusk and dawn when the feeding response is also highest (Fig. 14). Here begins the difficulty of interpretation: whereas in the pineal, high noradrenaline release led to decline in the number of fl-adrenergic binding sites, in the lateral hypothalamus, high I,J~T[RAL HYPOTHALAI'~ RHYThRS
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CIRCADIAN
RHYTHMS
243
noradrenaline release (as indirectly measured by reuptake and firing rates) occurs at the same times as high fl-adrenergic receptor binding. In this same region we have also measured serotoninergic parameters (Fig. 15A, unpublished data). First, two putative serotonergic binding sites using the ligands spiroperidol (5HT-2 postsynaptic receptors) and imipramine (presynaptic 5HT-uptake sites) follow identical patterns: indeed, there is a high correlation between the amount of binding to the two ligands in individual animals (r = 0.75, p < 0.001). Second, the circadian rhythms of 5HT and its metabolite 5HIAA in the tissue extract also follow a remarkably similar pattern. In this context, it is perhaps interesting to note that a third serotonergic binding site, using 5HT as ligand (5HT-1 postsynaptic receptors) shows an inverse rhythm to that of 5HT itself (at least, when measured in forebrain) (Weseman et al., 1986a) (Fig. 16). Third, the pattern of 5HT-parameters in the LH is inverse to that found for fl-adrenergic receptors in the same region (Fig. 14). Do these patterns indicate that synthesis and release of 5HT and NA in the lateral hypothalamus are not directly responsible for receptor density, as in the classical model? Interactions between the levels of neurotransmitter regulation are still incompletely understood at the single time point level. Studies of circadian rhythms may appear more
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244
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complex, yet on the other hand could provide a physiological model to unravel the functional process of change unperturbed by pharmacological intervention. That pharmacological intervention itself may disrupt circadian organisation of these interrelated parameters is shown by the chronic methamphetamine study (Fig. 15B). Whereas in control animals (Fig. 15A) all 5HT parameters measured showed parallel rhythmicity in the LH, this was not the case after drug treatment. Peak 5HT and 5HIAA were both shifted to the light phase. Imipramine binding was not significantly different from controls. Spiroperidol binding showed quite a different wave-form and timing from controls that had no obvious correlation with any of the other parameters. Thus maximum release of 5HT and maximum receptor binding were no longer in phase. 5.7.2. P o s t s y n a p t i c events When ~j- and /~-adrenergic receptor binding in cerebral cortical slices, measured over 24 hours, are compared with noradrenaline-stimulated cyclic-AMP production in the slices, the circadian rhythms are similar (Benedito and Kafka, 1982) (Fig. 17). Thus it appears that the number of ~1- and/~-adrenergic receptors stimulated by noradrenaline in vitro can regulate the magnitude of cyclic-AMP production dependent on time of day. Yet in vivo, if discrete brain regions are analysed, the patterns become quite complex. First, the rhythms in noradrenaline release and metabolism do not confer the rhythms measured in adrenergic receptors; and second, the rhythms of adrenergic receptors and cyclic-AMP concentration are not in phase in every region (Kafka et al., 1986a,b). In the cingulate cortex, the peak cyclic-AMP concentration parallels the peak binding to the cq-adrenergic receptor; in contrast, in occipital, parietal, and temporal cortices, the rhythms are 180 ° out of phase. A further study of such relationships has recently been carried out by Lemmer, who had characterised circadian rhythms in the /3-receptor-adenylate cyclase-eyclic-AMPphosphodiesterase system in rat heart ventricles that varied with season (Lemmer and Lang, 1983; Lang et al., 1983; Lemmer et al., 1983). In forebrain,/~-adrenergic receptor binding showed little rhythmicity. However brain adenylate cyclase activity and cyclicAMP showed highly significant circadian rhythms of large amplitude ( > 1 0 0 % peak: trough), and phosphodiesterase an ultradian rhythm (Lemmer et al., 1985). Thus it appears that in brain the existence of circadian rhythms in functional response of receptors is
245
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probably far more pronounced than the rhythms in the receptors themselves. As in all binding studies, the issue remains unsolved of how much change in receptor number is necessary for a functional modification of response.
6. Receptor Rhythms and Receptor-Mediated Function 6. l. OPIATE RECEPTORS AND PAIN SENSITIVITY We have described regional dependence of receptor rhythms and related function (Section 5.4.4). An example of a more general phenomenon is the correlation of opiate receptors and behavioural pain sensitivity. There are well-defined circadian rhythms in tail-pinch or paw-lick nociceptive responses in mice and rats (summarised in Kavaliers and Hirst, 1983). In nearly all studies, peak sensitivity was found in the dark phase. Circadian variations in brain endorphin content (Simantov et aL, 1980; Kerdelhue et aL, 1983; Jung et aL, 1984) also show maximal concentrations in the dark phase. Whole brain opiate receptor binding, whether measured with [3HI-morphine (Lee et al., 1984) or [3H]-naloxone (Naber et al., 1981; Wirz-Justice et al., 1981) showed a large peak in the dark phase. Some examples of opiate receptor binding and opiate analgesia are shown in Fig. 18. Measurement in brain regions such as the pontomesencephalic region (Lopez-Colome and Drucker-Colin, 1984) or hypothalamus (Jacobson and Wilkinson, 1985) (Fig. 18A) have indicated that a bimodal rhythm can also be present, with peaks in both the dark and light phase. However opiate receptor changes in these specific brain areas may be more related to other functions such as ultradian R E M - N R E M sleep rhythms or gonadotrophic hormone release, respectively. Only one study has directly compared receptor binding and nociception: greatest pain sensitivity occurs at the end of the dark phase when the maximal number of naloxone binding sites are found (Hendrie et al., 1983).
246
A. WIRZ-JUSTICE
6.2. SEROTONIN RECEPTOR STIMULATIONAND BEHAVIOURAL RESPONSE [3H]-Serotonin is considered to bind to 5HT-1 receptor sites. A significant circadian rhythm has been found in rat brain with maximum in the dark phase (Wesemann et al., 1983, 1986a). [3H]-Spiroperidol is the ligand used to measure 5HT-2 receptor sites in frontal cortex. A bimodal pattern of binding is seen in both frontal cortex and lateral hypothalamus: these rhythms are correlated with those found for [3H]-imipramine binding--a putative presynaptic site related to 5HT uptake. None of these receptor rhythms have been directly related to function. However 5HT behavioural rhythms have been investigated separately. The head twitch response induced in mice and rats when 5HT-2 receptors are stimulated by 5-MeODMT shows a circadian rhythm (Singleton and Marsden, 1981; Moser and Redfern, 1985, 1986). In contrast, the 5HT-syndrome that has been suggested to be mediated by 5HT-1 receptors shows no circadian rhythm (Moser and Redfern, 1985, 1986). In order to provide direct correlations between 5HT-induced behaviours and receptor rhythms, parallel studies should be made of 5HT receptors in that brain region relevant for the 5HT-induced behaviour measured. 6.3. ALPHA2-ADRENERGIC RECEPTOR STIMULATED FEEDING Clonidine elevates food intake in rats after peripheral or intra-hypothalamic application through an cq-adrenergic receptor mechanism in the paraventricular nucleus (McCabe et
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al., 1984). ct2-Adrenergic receptors in the PVN (and medial hypothalamus) show a circadian rhythm with peak binding at dusk and a trough at dawn (Leibowitz et al., 1984; Kraeuchi et al., 1984) (Fig. 10). We carried out a functional test of this rhythm: low doses
of i.p. clonidine stimulated feeding only at dusk when ~2-adrenergic receptors are high and not at dawn when they are low (Kraeuchi et al., 1985).
6.4. ALPHA I- AND BETA-ADRENERGIC STIMULATED BEHAVIOURS IN THE OPEN FIELD
Many behaviours in the open field show circadian rhythmicity (Vassout et al., 1982; Gentsch et aL, 1982). ~t- and //-Adrenergic receptors together mediate exploratory behaviour: an gragonist can enhance, fl-agonists suppress ambulation at the beginning of the dark but not at the beginning of the light phase. Parallel measurements indicated that both ~ - and fl-adrenergic receptors are higher at this sensitive phase (Mogilnicka et ai., 1985).
248
A. WIRZ-JUSTICE
7. Retinal Input to the Circadian System We have considered circadian rhythms in the pineal and in the CNS areas that drive them. Largely neglected has been consideration of the sensory organ that mediates visual information synchronising these rhythms, that itself is of CNS origin: the retina. The retinal-hypothalamic tract to the SCN directly relays LD information to the circadian pacemaker (Moore, 1983). However the retina does not merely passively code for luminance (that aspect of visual information primarily required for entrainment; Groos, 1982). The mammalian retina itself may contain a self-sustaining circadian pacemaker (Terman and Terman, 1985), modulating the light signal to the SCN through its own circadian rhythm of visual sensitivity. The visual cells manifest endogenous circadian rhythms in metabolic activity on the morphological level e.g. disk-shedding from outer segments (Besharse, 1982) and organelle turnover in inner segments (Rem~ et al., 1985). The retinal neurotransmitters, dopamine and melatonin, that are considered to be involved in the regulation of these phenomena, also undergo circadian variation (Pang et al., 1980; Besharse, 1982; Dubocovich, 1983; Wirz-Justice et al., 1984). Retinal dopamine synthesis is stimulated by light; this is mimicked by GABA antagonists and blocked by benzodiazepines (Morgan, 1982). Even though these retinal neurotransmitters show circadian rhythms and respond to light, neither dopamine nor benzodiazepine receptors in the retina do so (Dubocovich et al., 1985; Wirz-Justice et al., 1985b), although both are modified by longer exposure to light or darkness. Thus a negative finding in receptor binding can occur with a positive finding of a rhythm in the neurotransmitter itself (Fig. 19). It may be particularly important in the retina for the system to be buffered against rapid changes in receptor sensitivity related to the external LD cycle (see Section 8, Table 6 for drug effects).
8. Antidepressant Drugs and Receptor Rhythms Our specific interest in the action of antidepressant drugs on receptor rhythms arose from behavioural studies of the circadian rest-activity cycle. Lithium was the first psychopharmacological agent to have been shown to lengthen period and delay phase of the rest-activity cycle in many species (for review see Wirz-Justice, 1983). Clorgyline, a monoamine oxidase inhibitor, also had similar effects (Wirz-Justice and Campbell, 1982; Duncan et al., 1986), as did amphetamine (summarised in Wirz-Justice et al., 1985a). Since a great deal of work on the mechanisms of antidepressant drug action has focussed on receptor binding, it seemed important to evaluate such drug effects across 24 hours. RETINAL
CIRCADIAN
RHYTHMS
e-me
o,,,,,,o
-80
DA
Ro 15-1788 SPECIFIC BINDING • 60 fmolJmg prot.
SYNTHESIS ng/retina/min
20-
I
LI 2.5 5
11 D
4
11
FIG. 19. Circadian rhythm of DA synthesis (measured using AMPT) in retina from rats kept in DD after entrainment to the 12:12 LD cycle shown. DA synthesis practically ceases in the dark phase, and rises rapidly at the beginning of the light phase (even without the direct stimulus of light). The diurnal pattern of specific binding of the benzodiazepine antagonist Ro 15-1788 is not statistically significant, even though the pattern bears some resemblance to the DA synthesis rhythm. N = 5 retinae/time point (redrawn from Wirz-Justice et al., 1984; 1985b).
CIRCADIANRHYTHMS
249
TABLE 6. DRUG EFFECTSON CIRCADIANRECEPTORRHYTHMS
Drug/dosage/duration
Radioligand
Modification of rhythm wave form*/ ampl./mean/phaset
Reference
Uptake lnhibitors
Imipramme I0 mg/kg/day i.p., 21 days
QNB
C--* U / - 11%/-20%/
Kafka et al., 1981b
Imipramme 10 t*mol/kg oral, 21 days
DHA
-/-/-35%/
Fowler et al., 1985
Imipramlne 10 mg/kg/day i.p., 21 days
DHA
C ~ U / + 19%/-30%/
Wirz-Justice et al., 1980
Imipramme I0 mg/kg/day i.p., 21 days
WB-4101
C / + 2 9 % / - 1 5 % / - 8 hr
Wirz-Justice et al., 1980
Imipramme 10 mg/kg p.o., 14 days
Prazosin
N = 2/ /+30%/8 hr + 10%/20 hr
Maj et al., 1985
Imipramlne 10 mg/kg/day i.p., 21 days
Spiroperidol (striatum)
U/-14%/-20%/-4 - 60%
Naber et al., 1980
Imipramlne 15 mg/kg/day water, 14 days
5HT
C/=/-54%/-2hr
Wesemann et al., 1986b
Imipramme I0 mg/kg/day i.p., 21 days
Diazepam
C/+20%/=/=
Marangos et al., unpubl. data
Imipramme 10 mg/kg/day Alzet, 14 days
Flunitrazepam Ro 1 5 - 1 7 8 8 (retina)
O ~ C/+ / - 23 %/ O~C/+/-16%/
Wirz-Justice et al., 1985b
Imipramlne 10 mg/kg/day i.p., 14 days
Opiate
C --*U / + 100%/- 7%/
Lee et al., 1984
Zimelidine 10/~mol/kg p.o., 21 days
DHA
-/-/-20%/
Fowler et al., 1985
Amitryptiline I 0 mg/kg p.o., 14 days
Prazosin
N=2/
+18% 8hr/ +3% 20hr/
Maj et al., 1985
Citalopram 10 mg/kg p.o., 14 days MAOI
Prazosin
N=2/
/+20% 8hr/ +9% 20 hr/
Maj et al., 1985
Pargyline 5 mg/kg i.p., 14 days
Opiate
C~U/+82%/=/
Lee et al., 1984
Clorgyline (irreversible MAO-A inhibitor) 4 mg/kg/day Alzet s.c. 14 days
Opiate DHA WB-4101 QNB Spiroperidol (striatum) Diazepam
C --*U / + 83 % / = / C--*/=/=/ U ~ C/= / = / U ---,C / - 26%/+ 25%/ C / + 1 4 % / - 7 3 % / + 6 hr
Wirz-Justice Wirz-Justice Wirz-Justice Wirz-Justice Wirz-Justice
U --*C / = / = /
Wirz-Justice et al., 1982
Ro 15-1788 (retina)
-/-/-/
Wirz-Justice et al., unpubl, data
Ketanserin
-/-/-17%/
Campbell et aL, 1986
DHA
-/-/-
Fowler et al., 1985
Clorgyline I mg/kg/day p.o., 25 days Amiflamine (reversible MAO-A inhibitor) 3 #mol/kg p.o., 21 days JPN
29'3~-
14%/
hr
et et et et et
al., al., al., al., al.,
1982 1982 1982 1982 1982
Continued
250
A. WIRZ-JUSTICE
TABLE6~continued
Modification of rhythm Drug/dosage/duration Lithium Lithium 0.06% food 14 days
wave form*;;' ampl./mean/phaset
Radioligand DHA WB4101 Diazepam Opiate QNB
Reference
C-*O/-13%/+28%/ U-*0/-36%/+ 13%/ C --* O / - 2 2 % / - 11%/ C/-39%/+43%/-4 hr U/-28%/+45%/-6 hr C --* U / + 1 6 % / - 31%/
Kafka Kafka Kafka Kafka Kafka Kafka
et et et et et et
al., al., al., al., al., al.,
1982 1982 1982 1982 1982 1982
C--*U/=/+55%/ U ~ C / + 2 2 % / + 17%/ U ~ C / - 2 4 % / + 37%/ C -* U / + 3 3 % / - 195%/
Naber Naber Naber Naber
et et et et
al., al., al., al.,
1982 1982 1982 1982
Opiate
C/-47%/+34%/12hr
Naber et al., 1982
Opiate
C / + 7 7 % / - 1 6 % / + 4 hr
Lee et al., 1984
Opiate
C/+23%/-16%/=
Lee et al., 1984
DHA (LH)
U -~ C/= / =/
Kraeuchi et al., 1984
Clonidine
C~O/-23%/+
Spiroperidol (striatum) Neuroleptica
Fluphenazine decanoate 2 x 10mg in 14 days
DHA WB4101 QNB Spiroperidol
(striatum) Chlorpromazine 10 mg/kg i.p., 14 days Amphetamines
Amphetamine 5 mg/kg i.p., 14 days Methamphetamine 100 mg/l water 28 days
13%/
Kraeuchi et al., 1984
(MH)
Imipramine
C -* O / - / = /
Kraeuchi et al., 1986
C or U/decreased
Wirz-Justice et al., 1981
(SCN, LH)
Non-Pharmacological Antidepressant: Sleep Deprivation 12-24 hr SD
DHA WB4101 QNB Diazepam Opiate Spiroperidol
amplitude in 5/6 rhythms/ = /=
(striatum) 24-48 hr SD
5HT
U --* C / = / + 20%/
Wesemann et al., 1986b
* C = circadian; U = ultradian; O = no significant rhythm; -~ change of wave form. t Phase: - = DELAY; + = A D V A N C E (in hours).
In a series of detailed studies using many ligands and a number of antidepressant drugs, we found that inclusion of the temporal framework precluded any simple explanation of drug effects. The number of investigators who have carried out circadian rhythm studies with antidepressants is increasing. The main points are summarised in Table 6. A replication of findings from single time points studies was made with respect to most receptors (e.g. fl-adrenergic down-regulation with the tricyclic antidepressant imipramine). An example of the methodological problem raised by such circadian studies is shown in Fig. 20. a,-Adrenergic binding showed two main peaks at the end of the light and beginning of the dark phase. Chronic imipramine treatment changed the wave form, and the main peak occurred ca. 8 hours later. However comparison of control and treated animals at each time point yields different results depending on time of day: higher 0q-adrenergic receptor binding after imipramine treatment at dawn; no difference at the beginning of the light phase; and a marked decrease at all other times of day. A similar result is found with 0q-adrenergic receptor binding after chronic clorgyline (Wirz-Justice
CIRCADIAN
RHYTHMS
251
I-ADRENOCEPTOR BINDING 4.5
~nCONTROLS ""CHRONIC IMIPRAMINE
4'01 ~
3.5
**-x-
m ~ 3.0
--
~
|
L÷ 3
!
!
|
|
7 11D+ 3 7 TIME OF DAY (hr)
|
11
FIG. 20. Effect of chronic imipramine treatment (10 mg/kg/day, i.p. for 3 weeks) on binding to the cq-adrenergic receptor in forebrain homogenates. Rats were kept on a 12:12 LD cycle; N = 7/time point; both rhythms significant (ANOVA) (Redrawn from Wirz-Justice et al., 1980).
1982). Maj has also found that chronic imipramine significantly increases Ctl-adrenergic receptor binding at dawn but has no effect at dusk (two time points only) (Maj et al., 1985). Similarly, zimeline-induced down-regulation of cortical fl-adrenergic receptors was dependent on time of day (Fowler et al., 1985). The time-dependent modification of receptor binding is not limited to brain tissue: in the retina itself, imipramine may modify light input information via down-regulation of benzodiazepine receptors (Fig. 21) (Wirz-Justice et al., 1985b). Binding to both agonist and antagonist drugs was reduced only in the dark phase. This suggests the possibility that active antidepressant drugs may affect circadian rhythms by modulating visual sensitivity to light (since the photoreceptor response to light is dependent on the DA-GABAbenzodiazepine receptor complex). At the photoreceptor level, the few drugs investigated do reduce light responses (Rem6 et al., 1984; Rem6 and Wirz-Justice, 1985); at the behavioural level, lithium changes the sensitivity of hamster rhythms to light pulses (Han, 1984), and in man, characteristics of the EOG (Emrich et al., 1984). In contrast to the marked effects on different receptor rhythm characteristics by psychopharmacological agents, sleep deprivation, a non-pharmacological antidepressant modality, only slightly decreases amplitude. Essentially, all receptor rhythms showed a convincing replication (e.g. opiate receptor binding, Fig. 22) (Wirz-Justice et al., 1981). et al.,
9. Human Receptor Rhythms Postmortem studies of monoamines in human hypothalamus have documented large amplitude rhythms (Carlsson et al., 1980). Perry et al. (1977) were the first to demonstrate circadian variation in any CNS receptor: muscarinic cholinergic receptor binding in cerebral cortex was 100% lower during the day than during the night. No variation in 5HT-1 or -2 receptor binding could be demonstrated when 6-hour intervals were analysed (Wester et al., 1984). There are now a number of studies documenting diurnal rhythms of catecholaminergic binding sites on blood cells, fl-Adrenergic receptors on intact human lymphocytes show a circadian rhythm in males and an ultradian rhythm in females (Pangerl et al., 1986). The peak number of binding sites occur at the same time as maximal blood pressure. Two other studies show higher fl-adrenergic receptor binding during the day than night (Froehler et
252
A. W1RZ-JusTICE
RETINAL BENZODIAZEPINE RECEPTOR BINDING (Scatchard plots) Effect of chronic imipramine depends on time of day , - , D+IO
L*IO 3H - F L U N I T R A Z E P A M
006
0.04
002_
,-
tOO
200
300
400
. . . . . . .
500
KX~
200
300
400
,,.
'J,-:
Imcl~/mt m
3H-Ro 15-1788 F
%%
006
%% °%%%~ %%
0.04
°% 002
Q
"%%~ 0 %%
J
,
"i
FIG. 21. Scatchard plots from two time points (end of the dark and end of the light phase) to illustrate (a) the similarity of agonist ([3H]-flunitrazepam) and antagonist ([3H]-Ro 15-1788) binding characteristics in retinal homogenates after chronic imipramine treatment; (b) that imipramine reduces the apparent number and not affinity of benzodiazepine receptor binding and; (c) that the effect of imipramine is dependent on time of day. Rats were kept on a 12:12 LD cycle, given imipramine via Alzet osmotic minipumps (10 mg/kg/day for 2 weeks) and transferred to DD on the experimental day (from Wirz-Justice et al., 1985b).
al., 1985; Titinchi et al., 1984). There appear to be both direct effects of noradrenaline or
agonists, as well as an independently regulated circadian rhythm. For example, stress of highly active control subjects raises plasma catecholamines in the morning and lowers fl-adrenergic receptor number; however both active and inactive subjects have lower binding in the evening independent of catecholamine levels (Krawietz et al., 1985). The agonist salbutamol induces down-regulation in the morning but not in the evening (Titinchi et al., 1984). A biphasic effect of i.v. agonist (doubled fl-adrenergic receptor binding after 30 minutes; halved after 4 hours) has not been investigated at different times of day (Tomeh and Cryer, 1980). One study of ~2-adrenergic receptor binding in platelets showed a 30% amplitude rhythm (Froehler et al., 1985), another found no modulation by time of day (Jones et al., 1983). The number of "peripheral type" benzodiazepine receptor binding sites in platelets did undergo significant circadian variation (Levi et al., 1986).
C I R C A D I A N RHYTHMS
253
OPIATE RECEPTOR RHYTHM • Controls
2.0
o Sleep Deprived
!
f= 1.6
'
_-
-6
E
v a z
1,2
o
2
rn
0x
q
0.8
<
z "1-
0.4 ~//'///////'///Z////~///~///JJ~'////////Z//A~ I
18
[-
22
I
2 6 8 10 TIME OF DAY (houri
I
I
14
18
EXPERIMENTAL PROTOCOL FOR SLEEP DEPRIVED RATS 24 Hours of Sleep Deprivation -[~ RecoverySleep
•[
Specific binding of [3H]-naloxone to the opiate receptor during the last 13 hours of a sleep deprivation protocol and the first 12 hours of recovery sleep, together with non-sleep-deprived control groups at the same time points in the light and dark phase. N = 6/time point; both rhythms highly significant by ANOVA (from Wirz-Justice et aL, 1981). F I G . 22. 24-hour
A well known hormonal receptor manifests both a circadian rhythm in afffinity--insulin binding to erythrocytes is 25% less at midnight than during the day--and insulin sensitivity is higher in the afternoon than at night (Schultz et al., 1983a,b). 10. Hormone Receptors The analogy with hormone receptors in the brain may also be made. The rhythm in rat brain glucocorticoid receptor binding in hippocampus and septum persists independent of the pituitary (Angelucci et al., 1980); the number of cytoplasmic estrogen receptors from rat and hamster hypothalamus also show a circadian rhythm in the absence of circulating estrogens (Roy and Wilson, 1981; Wilson et aL, 1983; O'Connor et aL, 1985). These results support the idea that there are centrally driven diurnal fluctuations in the sensitivity of target cells to their respective steroids. It has even been postulated that the diurnal variations in catecholaminergic transmission might be a factor underlying the hormonal receptor rhythm (O'Connor et al., 1985). 11. Mechanisms of Rapid Receptor Change The number of receptors on cells is very dynamically regulated. Altered receptor concentration often determines altered tissue sensitivity to drug or hormone action, since the concept of a biologic receptor includes two distinct functions: not only must a receptor bind biologically active agonist hormones or their functionally inert antagonists, but it must also activate enzymes and ion channels to lead to a characteristic physiologic response. Mechanisms of membrane receptor regulation have been extensively investigated for adrenergic receptors (Harden, 1983; Lefkowitz et al., 1984). Two possible mechanisms have been proposed for adenylat¢ cyclase coupled receptor desensitisation. Within minutes of exposure of cells to isoproterenol, the receptors become functionally uncoupled i.e.
254
A. WIRZ-JUSTICE
incapable of forming the high affinity complex and therefore of stimulating adenylate cyclase. Subsequently the uncoupled receptors are removed from the cell surface and sequestered within the cell--the receptors in this internalised compartment are removed from contact with the effector components of the system. Their functional inadequacy is due to their physical separation: when agonist is removed, the receptors recycle to the cell surface and become recoupled to the adenylate cyclase. The demonstration of such a rapid and reversible functional change, resulting in changes in the apparent number of fl-adrenergic receptor binding sites, provides a potential model for circadian rhythms in CNS receptors. The second mechanism of desensitisation is phosphorylation of fl-adrenergic receptors--this is irreversible and thus not able to be invoked for short term changes. Not only in model systems (Lefkowitz et al., 1984) are rapid changes in receptor binding found: incubation of cortical slices with isoproterenol decreases both fl-adrenergic receptors and increase 5HT-2 receptors within 30 minutes (Scott and Crews, 1985); 5HT-2 receptors are rapidly down-regulated with a combination of ~2-antagonists and antidepressant drugs (Scott and Crews, 1983). One exception to the general finding that agonists induce a decrease in receptor number is the doubling of 13-adrenergic receptors on lymphocytes during the first 30 minutes of agonist infusion, followed by large decreases after 4-6 hours further infusion (Tomeh and Cryer, 1980). An agonist-induced increase in receptor number potentially could be related to the observation that phospholipid methylation unmasks "cryptic" fl-adrenergic receptors in rat reticulocytes (Strittmatter et al., 1979). Further evidence that circadian variations in radioligand binding may be dependent on agonist occupation has been found (Mash et al., 1985). Since endogenous, high affinity agonist-receptor complexes can prevent the assay of muscarinic receptors, even with potent antagonists, assay conditions were selected to fully uncouple such complexes and yield "total" receptor levels. Binding was then constant over 24 hours. Under normal assay conditions in Krebs buffer, binding assays measure only unoccupied receptors. Free QNB binding sites varied 100% over 24 hours. This interesting study indicates that agonists in binding experiments offer a new approach to investigating mechanisms of neurotransmitter receptor rhythms. It also indicates that circadian changes in cholinergic receptors may be due to alterations in the use of proteins rather than to changes in the amount of protein, a conclusion applicable to the studies of fl-adrenergic receptors, where no new synthesis of receptors is involved (Lefkowitz et al., 1984). 12. Conclusions
Lefkowitz concludes "Important clinical ramifications of this new information about agonist-induced receptor alterations leading to desensitisation is that whenever possible, such agonist therapy should be as intermittent as possible to minimise the development of desensitisation to drug effects" (1984). Precisely analogous statements form the basis of chronopharmacology (Reinberg and Smolensky, 1983). Since there are circadian rhythms in sensitivity to drugs, clinical application should, where possible, take this into account in designing treatment schedules. Sensitivity to drugs is itself mediated by receptor function: a drug binds to a receptor and activates, or blocks, a biological process. Thus circadian rhythms in receptor number may underlie pharmacological rhythms in response; intermittent drug application could fulfil both criteria--dosage at the right time of day for maximum effect, pulse dosage to minimise development of desensitisation. However, as this review notes, research into dynamic changes of receptors with respect to circadian physiology has a short history, in spite of the demonstration of pineal fl-adrenergic receptor rhythmicity over a decade ago. The approach to molecular mechanisms of receptor desensitisation induced by agonist drugs could also be applied to mechanisms of endogenous receptor rhythms. Given the caveats delineated above (with respect to age, strain, gender, LD and housing conditions, and in particular the importance
CIRCADIANRHYTHMS
255
of precise brain regions) the non-pharmacological model of circadian rhythms in CNS receptors may provide a new approach to receptor mechanisms and receptor function in neurobiology. Acknowledgements I thank all my collaborators in these studies, and in particular Kurt Kraeuchi, Marian Kafka, Thomas Wehr, Iain Campbell and Gianni Muscettola for extensive discussions over the years. A fellowship from the Swiss Foundation for Biomedical Research, and Twinning Grants from the European Science Foundation (with I.C. and G.M.) are gratefully acknowledged. Further thanks are due to the many researchers who sent preprints of their recent data on receptor rhythms. All previously published Figures are reprinted with permission.
References AGHAJANIAN,G. K. and MANNERMAELEN, C. P. (1982) ~t2-Adrenoceptor-mediated hyperpolarization of locus coeruleus neurons: intracellular studies /n vivo. Science 215, 1394-1396. ANGELUCCI,L., VALERI,P., PALMERY,M., PATACCHIOLI,F. R. and CATALANI,A. (1980) Brain glucocorticoid receptor: correlation of in vivo uptake of corticosterone with behavioral, endocrine, and neuropharmacological events. In: Receptors for Neurotransmitters and Peptide Hormones, pp. 391-406. Eds. G. PEPEU, i . J. KUHAR and S. J. ENNA. Raven Press: New York. ASCHOFF,J., DAAN,S. and HONMA,K. I. (1982) Zeitgebers, entrainment and masking: some unsettled questions. In: Vertebrate Circadian Systems, pp. 13-24. Eds. J. ASCHOFF,S. DAANand G. GROOS.Springer: Heidelberg. BENEDITO, M. A. and KAFKA, i . S. (1982) Circadian rhythms in alpha- and beta-adrenergic receptor number and cyclic-AMP production in rat cerebral cortical slices. Soc. Neurosci. Abstr. 8, 660. BESHARSE, J. C. (1982) The daily light,lark cycle and rhythmic metabolism in the photoreceptor-pigment epithelial complex. In: Progress in Retinal Research, Vol. 1, pp. 82-124. Eds. N. OSBORNEand G. CHADER. Pergamon Press: Oxford. BLAKER, W. D., CHENEY, D. L. and STOFF, D. M. (1983) Interstrain comparison of avoidance behavior and neurochemical parameters of brain cholinergic function. Pharmac. Biochem. Behav. 18, 189-193. BRENNAN,M. J. W., VOLICER,L., MOORE-EDE,M. C. and BORSOOK,D. (1985) Daily rhythms of benzodiazepine receptor numbers in frontal lobe and cerebellum of the rat. Life Sci. 36, 2333-2337. BRUININK,A., LICHTENSTEIGER,W. and SCHLUMPF,i . (1983) Ontogeny of diurnal rhythms of central dopamine, serotonin and spirodecanone binding sites and of motor activity in the rat. Life Sci. 33, 31-38. BUCKETT,W. R. (1981) Stimulation-produced analgesia is dependent upon the influence of both circadian and circannual rhythms. Br. J. Pharmac. 281P. CAMPBELL, I. C., DURCAN, M. J., DUNN, G., KOSHIKAWA,F. and KOSHIKAWA,N. (1986) Circadian studies of central neurotransmitter populations in the rat. Abstract, 2nd International Conference o f Chronopharmacology, Montreux. CARLSSON, A., SVENNERHOLM,L. and WINBLAO, B. (1980) Seasonal and circadian monoamine variations in human brains examined post mortem. Acta psychiat, scand. Suppl. 280, 75-85. CHEVILLARD, C., BARDEN,N. and SAAVEDRA,J. M. (1981) Twenty-four hour rhythm in monoamine oxidase activity in specific areas of the rat brain stem. Brain Res. 223, 205-209. CHIU, P., RAJAKUMAR,G., CHIU, S., CHIu-YIN, K. and MISHRA,R. K. (1982) Enhanced [aH]-spiroperidol binding in striatum of spontaneously hypertensive rat (SHR). Eur. d. Pharmac. 82, 243-244. COOD, E. E. and BYRNE,W. L. (1981) Seasonal variation in the apparent number of binding sites for 3H-opioid agonists and antagonists. Life Sci. 28, 2577-2583. DIAGUJI, i . , MIKUNI, M., OKADA, F. and YAMASHITA, I. (1978) The diurnal variations in dopamine-flhydroxylase activity in the hypothalamus and locus coeruleus of the rat. Brain Res. 155, 409-412. DASZUTA,A., FAUOON, M. and TERNAUX,J.-P. (1982) Uptake of [3H]-serotonin and [3H]-noradrenaline in the raphe nuclei and the locus coeruleus of C57BL/6RHOLCO and BALB/cCENCLO mice at three times of the day. Neurosei. Lett. 29, 141-146. DAVIES,J. A., ANCILL,R. J. and REDFERN,P. H. (1972) Hallucinogenic drugs and circadian rhythms. In: Progress in Brain Research, pp. 79-95. Eds. P. S. BRADLEYand R. W. BRIMBLECOMBE.Elsevier: Amsterdam. Dl LAURO, A., GIANNINI, C. P., MUSCETTOLA,G., GRECO, A. M. and DE FRANCISCIS,P. (1986) No circadian rhythms of serotoninergic, alpha-, beta-adrenergic and imipramine binding sites in rat brain regions. Chronobiol. Int. 3, 123-126. DREW, G. M., GOWER, A. J. and MARRIOT,A. S. (1979) ~t2-Adrenoceptors mediate clonidine-induced sedation in the rat. Br. J. Pharmac. 67, 133-141. DUBOCOVlCH, M. L. (1983) Melatonin is a potent modulator of dopamine release in the retina. Nature 306, 782-784. DUBOCOVICH,M. L., LUCAS,R. C. and TAKAHASHI,J. S. (1985) Light-dependent regulation ofdopamine receptors in mammalian retina. Brain Res. 335, 321-325. DUNCAN,W. C., TAMARKIN,L. and WEHR, m. A. (1986) Clorgyline increases the circadian period, the activity-rest ratio and total activity in syrian hamsters. A. Rev. Chronopharmac. 3, 17-20. EMRICH, H. M., ULLRICH, A., ADAMCZYK,J. and STEIN, R. (1984) Lithium affects EOG in humans. Psychiat. Res. 13, 355-357.
256
A. WIRZ-JUSTICE
FOLDE$, A., MAXWELL, C. A., SCARAMUZZ1,R. J., DONNELLY, J. B., HOSKINSON, R. M. and RINTOUL, A. J. (1985) Seasonal changes in the sensitivity of ovine pineal fl-adrenoceptors to steroids. Neuroendocrinol. 41, 494---498. FOWLER, C. J., HALL, H., SAAF, J., ASK, A.-L. and Ross, S. B. (1985) Pharmacological manipulation of biochemically measured rhythms in the mammalian central nervous system. In: Circadian Rhythms in the Central Nervous System, pp. lll-120. Eds. P. H. REDFERN, I. C. CAMPBELL,J. A. DAVIES and K. F. MARTIN. Macmillan: London. FROEHLER, i . , SAITO, Y., ACKENHEIL, i . , BAK, R., BONDY, B., FEISTENAUER, E., HOFSCHUSTER, E., VAKIS, A. and WELTER, n . (1985) Catecholaminergic binding sites on blood cells of healthy volunteers with special respect to circadian rhythms. Pharmacopsychiat. lg, 147-148. GARFINKEL, A. (1983) A mathematics for physiology. Am. J. Physiol. 245, R455-R466. GENTSCH, C., LICHTSTEINER, M. and FEER, H. (1982) Behavioural comparisons between individuallyand group-housed male rats: effects of novel environments and diurnal rhythm. Behav. Brain Res. 6, 93-100. GROOS, G. (1982) The neurophysiology of the mammalian suprachiasmatic nucleus and its visual afferents. In: Vertebrate Circadian Systems pp. 96-105. Eds. J. ASCHOFF, S. DAAN and G. GROOS. Springer: Heidelberg. HAN, S.-Z. (1984) Lithium chloride changes sensitivity of hamster rhythm to light pulses. J. interdiscipl. Cycle Res. 15, 139-145. HARDEN, T. K. (1983) Agonist-induced desensitization of the fl-adrenergic receptor-linked adenylate cyclase. Pharmac. Rev. 35, 5 32. HELMESTE, D. M. (1983) Spontaneous and apomorphine-induced locomotor changes parallel dopamine receptor differences in two rat strains. Pharmacol. Biochem. Behav. 19, 153-155. HELMESTE, D. M., SEEMAN, P. and COSClNA D. V. (1981) Relation between brain catecholamine receptors and dopaminergic stereotypy in rat strains. Fur. J. Pharmac. 69, 465-470. HENDRIE, C. A., NOHRIA, V. and ROOGERS, R. J. (1983) Circadian variation in pain sensitivity in rats may be related to alterations in opiate receptor density. Br. J. Pharmac. 78, 46P. JACOBSON, W. and WILKINSON, M. (1985) Diurnal changes in the opiatergic control of LH release in prepubertal rats are associated with diurnal variations in hypothalamic but not cortical [3H]-naloxone binding sites. Soc. Neurosci. Abstr. 11. JENNI-EIERMANN, S., YON HAHN, H. P. and HONEGGER, C. G. (1985) Circadian variations of neurotransmitter binding in three age groups of rats. Gerontology 31, 138-149. JENNI-EIERMANN, S., VON HAHN, H. P. and HONEGGER, C. G. (1986) Diurnal rhythms in neurotransmitter receptor binding and CAT activity: different patterns in two rat lines of Wistar origin. Brain Res. 370, 54-60. JHANWAR-UNIYAL, M., DARWlSH, M., BEITMIRZA,G. and LEIBOWITZ, S. F. (1985) Impact of food deprivation on hypothalamic and cortical cq- and ~t2-noradrenergic receptors. Soc. Neurosci. Abstr. l l, No. 21.1. JHANWAR-UNIYAL, M., ROLAND, C. R. and LEIBOWITZ,S. F. (1986) Diurnal rhythm of ct2-noradrenergic receptors in the paraventricular nucleus and other brain areas: relation to circulating corticosterone and feeding behavior. Life Sci. 38, 473-482. JONES, S. B., BYLUND, D. B., RIESER, C. A., SHEKIM, W. O., BYER, J. A. and CAgR, G. W. (1983) ~-Adrenergic receptor binding in human platelets: alterations during the menstrual cycle. Clin. Pharmac. Ther. 34, 90-96. JUNG, C.-Y., KIM, K.-W., CHO, K.-P. (1984) A study on the diurnal variation of endorphin in rat brain. Korean J. Pharmac. 20, 35-47. KAFKA, M. S., WIRZ-JUSTICE,A. and NABER, n. (1981a) Circadian and seasonal rhythms in ~t- and fl-adrenergic receptors in the rat brain. Brain Res. 207, 409-419. KAFKA, M. S., WIRZ-JUSTICE, A., NABER, D. and WEHR, T. A. (1981b) Circadian acetylcholine receptor rhythm in rat brain and its modification by imipramine. Neuropharmacol. 20, 421-425. KAFKA, M. S., WIRZ-JUST1CE,A., NABER, D., MARANGOS,P. J., O'DONOHUE, T. L. and WEHR, T. A. (1982) Effect of lithium on circadian neurotransmitter receptor rhythms. Neuropsychobiol. 8, 41--50. KAFKA, i . S., WIRZ-JUSTICE, A., NABER, D., MOORE, R. Y. and BENEDITO, M. A. (1983) Circadian rhythms in rat brain neurotransmitter receptors. Fedn Proc. 42, 2796-2801. KAFKA, m. S., MARANGOS,P. J. and MOORE, R. Y. (1985) Suprachiasmatic nucleus ablation abolishes circadian rhythms in rat brain neurotransmitter receptors. Brain Res. 327, 344-347. KAFKA, i . S., BENEDITO,M. A., BLENDY,J. A. and TOKOLA, N. S. (1986a) Circadian rhythms in neurotransmitter receptors in discrete rat brain regions. Chronobiol. Int. 3, 91-100. KAFKA, M. S., BENEDITO, M. A., ROTH, R. H., STEELE, L. K., WOLFE, W. W. and CATRAVAS,G. N. (1986b) Circadian rhythms in catecholamine metabolites and cyclic nucleotide production. Chronobiol. Int. 3, 101-115. KAN, J. P., CHOUVET, G., HI~RY, F., DEBILLY, G., MERMET, A., GLOWINSKI, J. and PUJOL, J. F. (1977) Daily variations of various parameters of serotonin metabolism in the rat brain. 1. Circadian variations of tryptophan-5-hydroxylase in the raphe nuclei and the striatum. Brain Res. 123, 125-136. KASTRUP,J. and CHRISTENSEN,J. (1984) Lack of effects of thyroid hormones on muscarine cholinergic receptors in rat brain and heart. Scand. J. clin. Lab. Invest. 44, 33 38. KAVALIERS, M. and HIRST, M. (1983) Daily rhythms of analgesia in mice: effects of age and photoperiod. Brain Res. 279, 387-393. KERDELHUE, B., KARTESZl, M., PASQUALINI, C. REINBERG, A., MEZEY, E. and PALKOVITS, M. (1983) Circadian variations in fl-endorphin concentrations in pituitary and in some brain nuclei of the adult male rat. Brain Res. 261, 243-248. KOULU, M., BJELOGRLIC, N., AGREN, H., SAAVEDRA, J. M. and L1NNOILA, M. (1985) Circadian changes in the concentrations of monoamines in discrete rat brain stem nuclei. Acts physiol, scand. 124, Suppl. 542. 268. KRAEUCHI, K., ALTHAUS, A. and FEER, H. (1981) Easier binding studies: no more washing! Experientia 37, 672.
CIRCADIANRHYTHMS
257
KRAEUCHI, K., WIRZ-JUSTICE, A., WILLE~R, R., CAMPBELL, I. C. and FEER, H. (1983) Spontaneous hypertensive rats: behavioral and corticosterone response depend on circadian phase. Physiol. Behav. 30, 35~,0. KRAEUCHI, K., WIRZ-JUSTICE, A., MORIMASA, T., WILLENER, R. and FEER, H. (1984) Hypothalamic cq- and /~-adrenoceptor rhythms are correlated with circadian feeding: evidence from chronic methamphetamine treatment and withdrawal. Brain Res. 321, 83-90. KRAEUCm, K., WIRZ-JUSTICE, A., SUETTERLIN-WILLENER, R. and FEER, H. (1985) Clonidine stimulation of food intake is circadian phase dependent. IRCS Med. Sci. 13, 561. KRAEUCHI, K., WIRZ-JUSTICE, A., MORIMASA, T., SUETTERLIN-WILLENER, R. and FEER, H. (1986) Temporal distribution of [3H]-imipramine binding in rat brain regions is not changed by chronic methamphetamine. Chronobiol. Int. 3, 127-133. KRAWIETZ, W., KLEIN, E. M., UNTERBERG, CH. and ACKENHEIL, M. (1985) Physical activity decreases the number of/~-adrenergic receptors on human lymphocytes. Klin. Wschr. 63, 73-78. LANG, P.-H., BISSINGER, H. and LEMMER, B. (1983) Circadian rhythm in cAMP content and in the activities of basal adenylate cyclase and phosphodiesterase in rat heart ventricles, d. Rev. Chronopharmae. I, 219-222. LEE, M.-S., KIM, K.-W. and CHO, K.-P. (1984) A study on the circadian rhythm of opiate receptor in rat brain. Korean J. Pharmac. 20, 23-34. LEFKOWITZ, R. J., CARGN, M. G. and STILES, G. L. (1984) Mechanisms of membrane-receptor regulation. Biochemical, physiological, and clinicalinsights derived from studies of the adrenergic receptor. New Engl. J. Med. 310, 1570-1579. " LEmOWITZ, S. F. (1980) Neurocbemical systems of the hypothalamus: control of feeding and drinking behavior and water-electrolyte excretion. In: Handbook of the Hypothalamus, Vol. I, Part A, Behavioral Studies of the Hypothalamus, pp. 299-437. F_As. P. J. MORGANE and J. PANKSEPP. Marcel Dekker: New York. LnM~R, B. and LANG,P.-H. (1983) Circadian-stage-dependency in antagonist binding of [3H]-dihydroalprenolol to rat heart ventricular membranes. A. Rev. Chronapharmac. 1, 321-324. LEMMER, B., CASPARI-IRVING, G. and WEIMER, R. (1981) Strain dependency in motor activity and in concentration and turnover of catecholamines in synchronized rats. Pharmac. Biochem. Behav. 15, 173-178. LEMMER, B., LANG, P.-H. and BISSINGER,H. (1983) Circannual peak-shift in circadian rhythm in basal cAMP content in rat heart ventricles..4. Rev. Chronopharmac. 1, 223-225. LEMMER, B., LANG, P.-H., GORKA, Z., SCHMIDT, S. and BAERME1ER,H. (1985) Circadian rhythms in the beta-receptor-adenylate-cyclase-cAMP-phosphodiesterase-system in heart ventricles and brain of the rat. J. Interdiscipl. Cycle Res. 16, 142. LESLIE, F. M., DUNLAPIII, C. E. and Cox, B. M. (1980) Ascorbate decreases ligand binding to neurotransmitter receptors. J. Neurochem. 34, 219-221. LEVI, F., BANAVIDES,J., TOUITOU,V., QUARTERRONET,D., CANTON,T. UZAN, A., GUEREMY,C., LE FUR G. and REINBERG,A. (1986) Circadian rhythm in peripheral type benzodiazepine binding sites in human platelets. Biochem. Pharmac. 35, 2623-2625. LOPEZ-COLOME,A. M. and DRUCKER-COLIN,R. (1984) Ultradian rhythm of pontomesencephalic opiate receptor: modification by chloramphenicol. Exp. Neurol. 85, 209 215. MAJ, J., KLIMEK, V. and NOWAK, G. (1985) Antidepressant drugs given repeatedly increase the binding to cq-adrenoceptors in the rat cortex. Fur. J. Pharmac. 119, 113-116. MARGULES,n. L., LEWIS, M. J., DRAGOVICH,J. A. and MARGULES,A. S. (1972) Hypothalamic norepinephrine: circadian rhythms and the control of feeding behavior. Science 178, 640~43. MARINO, L. A., DEBELLIS,M. and LEIBOWlTZ,S. F. (1983) ct2-Adrenergic receptors in the paraventricular nucleus mediate feeding induced by norepinephrine and clonidine. Soc. Neurosci. Abstr. 9, 467. MASH, n., FLYNN, D. D. KALINOSKI,L. and POTTER, L. T. (1985) Circadian variations in radioligand binding to muscarinc receptors in rat brain dependent upon endogenous agonist occupation. Brain Res. 331, 35-38. MATSUBARA,S., KAZAWA,T., OKA, I., KOYAMA,T. and YAMASHITA,[. (1982) Circadian variation in rat brain fl-adrenergic receptor. Folia psychiat, neurol, jap. 36, 478-479. MCCABE, J. T., DEBELLIS,M. and LEIBOWITZ,S. F. (1984) Clonidine-induced feeding: analysis of central sites of action and fiber projections mediating this response. Brain Res. 309, 85-104. MIKUNI, M., SAITO,Y., KOYAMA,T. and YAMASHITA,I. (1981) Circadian variation of cyclic AMP in the rat pineal gland. J. Neurochem. 36, 1295-1297. MOGILNICKA, E., WEDZONY,K., KLIMEK, V. and NOWAK, G. (1985) The importance of timing for the action of ctI- or fl-agonists: possible relationship with the density of [3H]-prazosin and [3H]-dihydroalprenolol binding sites. Eur. J. Pharmac. I13, 279-282. MOORE, R. Y. (1983) Organisation and function of a CNS circadian oscillator: the suprachiasmatic nucleus. Fedn Proc. 42, 2783-2789. MORGAN, W. W. (1982) Dopamine neurons in the retina: a new pharmacological model. In: Cell Biology o f the Eye, pp. 533-554. Ed. D. S. McDEVITT. Academic Press: New York. MOSER, P. C. and REDFERN,P. H. (1985) Circadian variation in behavioural responses to central 5HT receptor stimulation in the mouse. Psychopharmac. 86, 223-227. MOSER, P. C. and REDFERN,P. H. (I 986) Circadian rhythms in behaviours mediated by 5HT receptor stimulation in the rat. A. Rev. Chronopharmac. 3, 13-16. MURPHY, S., UZBEKOV,M. G. and ROSE,S. P. R. (1980) Changes in serotonin receptors in different brain regions after light exposure of dark-reared rats. Neurosci. Left. 17, 317-321. MUSCETTOLA,G., DI LAURO,A. and GIANNINI,C. P. (1986) Platelet 3H-imipramine binding in bipolar patients. Psychiat. Res. 18, 343-353. NABER, D., WIRZ-JUSTICE,A., KAFKA,M. S. and WEHR, T. A. (1980) Dopamine receptor binding in rat striatum: ultradian rhythm and its modification by chronic imipramine. Psychopharmac. 68, 1-5.
258
A. WIRZ-JusTICE
NABER, D., WIRZ-JUSTICE,A. and KAFKA, M. S. (1981) Circadian rhythm in rat brain opiate receptor. Neurosci. Lett. 21, 45-50. NAaER, D., WmZ-JUSTICE, A. and KAFKA, M. S. (1982) Chronic fluphenazine treatment modifies circadian rhythms of neurotransmitter receptor binding in rat brain. J. Neural Transm. 55, 277-288. NATALI, J. P., MCRAE-DEGOEURCE,A., CHOUVET, G. and PUJOL, J. F. (1980a) Genetic studies of daily variations of first-step enzymes of monoamine metabolism in the brain of inbred strains of mice and hybrids. I. Daily variations of tryptophan hydroxylase activity in the nuclei raphe dorsalis, raphe centralis and in the striatum. Brain Res. 191, 191-203. NATALI, J. P., MCRAE-DEGUEURCE, A., KEANE, P., DEBILLY, G. and PUJOL, J. F. (1980b). II. Daily variations of tyrosine hydroxylase activity in the locus coeruleus. Brain Res. 191, 205 213. NORDBERG,A. and WAHLSTROEM,G. (1980) Diurnal fluctuation in striatal choline acetyltransferase activity and strain difference in brain protein content of the rat. Acta physiol, scand. 108, 385-388. O'CONNOR, L. H., MORIN, L. P. and FEDER, H. H. (1985) A diurnal fluctuation in medial basal hypothalamicpreoptic area cytosol estrogen receptors in ovariectomized hamsters. Brain Res. 347, 376-380. OUVERIO, A. and MALORNI, W. (1979) Wheel running and sleep in two strains of mice: plasticity and rigidity in the expression of circadian rhythmicity. Brain Res. 163, 121-133. OLIVERIO,A., CASTELLANO,C. and PUGLISI-ALLEGRA,S. (1982) Opiate analgesia: evidence for circadian rhythms in mice. Brain Res. 249, 265-270. PANG, S. F., YU, H. S., SUEN, H. C. and BROWN, G. M. (1980) Melatonin in the retina of rats: a diurnal rhythm. J. Endocr. 87, 89-93. PANGERL, A., REMIEN, J. and HAEN, E. (1986) The number of fl-adrenoceptor sites on intact human lymphocytes depends on time of day, on season, and on sex. ,4. Rev. Chronopharmac. 3, 331-334. PERRY, E. K., PERRY, R. H. and TOMLINSON, B. E. (1977) Circadian variations in cholinergic enzymes and muscarinic receptor binding in human cerebral cortex. Neurosci. Lett. 4, 185-189. PILCHER, C. W. T., JONES, S. M. and BROWNE, J. (1982) Rhythmic nature of naloxone-induced aversions and nociception in rats. Life Sci. 31, 1249-1252. POR, S. B. and BONDY, S. C. (1981) Regional circadian variation of acetylcholine muscarinic receptors in the rat brain. J. Neurosci. Res. 6, 315-318. POST, J. and NAIR, V. (1975) Circadian variations in the action of antihypertensive drugs. In: Proceedings of the XII International Conference of the International Society for Chronobiology, pp. 375-386. PUGLIS1-ALLEGRA,CASTELLANO,C. and OLIVERIO,A. (1982) Circadian variations in stress-induced analgesia. Brain Res. 252, 373-376. RAPP, P. E. (1979) Bifurcation theory, control theory and metabolic regulation. In: Biological Systems, Modelling and Control, pp 1-83. Ed. D. A. LtNKENS. Peter Peregrinus Ltd: New York. RAPP, P. E. (1987) Why are so many biological systems periodic? Prog. Neurobiol. 29, 261 273. REINaERG, A. and SMOLENSKY, M. (1983) Biological rhythms and medicine. Springer Verlag: New York, Heidelberg. REML C. and WIRZ-JUST1CE,A. (1985) Circadian rhythms of the retina and their pharmacological modulation. In: Circadian Rhythms in the Central Nervous System, pp. 135-145. Eds. P. H. REDFERN, I. C. CAMPBELL, J. A. DAVIES, K. F. MARTIN. MacMillan: London. REMI~, C., WIRZ-JusTICE, A., AEBERHARD, B. and RHYNER, A. (1984) Chronic clorgyline dampens rat retinal rhythms. Brain Res. 298, 99 106. REMIt, C., AEBERHARD,B. and SCHOCH, M. (1985) Circadian rhythm of autophagy and light responses of autophagy and disk-shedding in the rat retina. J. comp. Physiol. A, 156, 669-677. ROMERO, J. A., ZATZ, M., KEBABIAN,J. W. and AXELROD,J. (1975) Circadian cycles in binding of [3H]-alprenolol to fl-adrenergic receptor sites in rat pineal. Nature 258, 435-436. ROSENWASSER, A. M., TRUBOWITSCH, G. and ADLER, N. T. (1985) Circadian rhythm in metabolic activity of suprachiasmatic, supraoptic and raphe nuclei. Neurosci. Lett. 58, 183 187. Roy, E. J. and WILSON, M. A. (1981) Diurnal rhythm of cytoplasmic estrogen receptors in the rat brain in the absence of circulating estrogens. Science 213, 1525-1527. SAITO, Y., YAMASHITA,I., YAMAZAKI, K., OKADA, F., SATOMI, R. and FUJIEDA, T. (1975) Circadian fluctuation of brain acetylcholine in rats. I. On the variations in the total brain and discrete brain areas. Life Sci. 16, 281-288. SCHMITT,M. (1973) Circadian rhythmicity in responses of cells in the lateral hypothalamus. Am. J. Physiol. 225, 1096-1101. SCHULZ, B., RATZMANN, K. P., ALBRECHT, G. and BIBERGEIL, H. (1983a) Diurnal rhythm of insulin sensitivity in subjects with normal and impaired glucose tolerance. Exp. clin. Endocr. 81, 263-272. SCHULZ, B., GREENFIELD, M. and REAVEN, G. M. (1983b) Diurnal variation in specific insulin binding to erythrocytes. Exp. clin. Endocr. 81, 273-279. SCOTT, J. A. and CREws, F. T. (1983) Rapid decrease in rat brain beta-adrenergic receptor binding during combined antidepressant alpha-2 antagonist treatment. J. Pharmac. exp. Ther. 224, 640-646. SCOTT, J. A. and CREWS, F. T. (1985) Increase in serotonin-2 receptor density in rat cerebral cortex slices by stimulation of beta-adrenergic receptors. Biochem. Pharmac. 34, 1585-1588. SEMBA,J., TORU, M. and MATAGA,N. (1984) Twenty-four hour rhythms of norepinephrine and serotonin in nucleus suprachiasmaticus, raphe nuclei, and locus coeruleus in the rat. Sleep 7, 211-218. SIMANTOV,R., ZANG,D. and YEHUDA,S. (1980) Regulation of pituitary endorphins under steroids treatment and during day-night cycle. Adv. bioehem. Psychopharmac. 22, 247-256. SINGLETON, C. and MARSOEN, C. A. (1981) Circadian variation in the head twitch response produced by 5-methoxy-N,N-dimethyltryptamine and p-chloroamphetamine in the mouse. Psychopharmac. 74, 173-176. STRITTMATTER, W. J., HIRATA, F. and AXELROD, J. (1979) Phospholipid methylation unmasks cryptic fl-adrenergic receptors in rat reticulocytes. Science 204, 1205-1207. SUDGEN, D. and KLEIN, D. C. (1985) Regulation of rat pineal cq-adrenoceptors. J. Neurochem. 44, 63-67. TERMAN, M. and TERMAN, J. (1985) A circadian pacemaker for visual sensitivity? Ann. N.Y. Acad. Sci. 453, 147-161.
CIRCADIAN RHYTHMS
259
TITINCHI, S., AL SHAMMA,M., PATEL, K. R., KERR, J. W. and CLARK,B. (1984) Circadian variation in number and affinity of fl~-adrenoceptors in lymphocytes of asthmatic patients. Clin. Sci. 66, 323-328. TOMEH, J. F. and CRYER, P. E. (1980) Biphasic adrenergic modulation of beta-adrenergic receptors in man: agonist-induced early increment and late decrement in beta-adrenergic receptor number. J. clin. Invest. 65, 836-840. VALAXX,J. L. (1984) Genetics as a model for studying the sleep-wake cycle. In: Sleep mechanisms, pp. 135-145. Eds. A. BORBELYand J. L. VALATX.Springer: Berlin. VAN DER GtrrGEN, J. and SLANGEN,J. L. (1975) Norepinephrine uptake by hypothalamic tissue from the rat related to feeding. Pharmac. Biochem. Behav. 3, 855-860. VASSOUT,A., FIDALGO, D., HtmN, CH. WIRZ-JUsTICE,A. and DELINI-STULA,A. (1982) Circadian activity in socially-raised and isolated rats as assessed in an open-field device. Experientia 38, 720. VIRUS, R. M., BAOLA~WSKI, T. and RADULOVACKI,M. (1984) Circadian variation of [aH]N6-(l-phenylisopropyl)adenosine binding in rat brain. Neurosei. Lett. 46, 219-222. WEINER, N., AROLD, N. and WESEMANN,W. (1982) Possible hazards in serotonin binding assays in human and rat brain: effects of ascorbate, pargyline, and buffer concentration. J. Neurosci. Meth. 5, 41-45. WESEMANN, W., WEINER, N., ROXSCH, M. and SCHULZ, E. (1983) Serotonin binding in rat brain: circadian rhythm and effect of sleep deprivation. ]. Neural Transm. Suppl. 18, 287-294. W~SEUANN, W., ROXSCH, M., SCHULZ, E., STURM, G. and ZOEFEL,P. (1986a) Circadian rhythm of serotonir] binding in rat brain: I. Effect of the light/dark cycle. Chronobiol. Int. 3, 135-139. WESEMANN,W., ROTSCH,M., SCnULZ, E. and ZOEFEL,P. (1986b) Circadian rhythm of serotonin binding in rat brain: II. Influence of sleep deprivation and imipramine. Chronobiol. Int. 3, 141-146. WESTER, P. HARDY, J. A., MARCUSSON,J., NYBERG, P. and WINBLAD,B. (1984) Serotonin concentrations in normal aging human brains: relation to serotonin receptors. Neurobiol. Aging 5, 199-203. WILSON, M. A., CLARK,A. S., CLYDE,V. and RoY, E. J. (1983) Characterisation of a pineal-independent diurnal rhythm in neural estrogen receptors and its possible behavioral consequences. Neuroendocr. 37, 14-22. WIRZ-JUSTICE, A. (1983) Antidepressant drugs: effects on the circadian system. In: Circadian Rhythms in Psychiatry, pp. 235-264. Eds. T. A. WEHR and F. K. GOODWIN. The Boxwood Press: Pacific Grove. W1RZ-JUSTICE,A. and CAMPBELL,I. C. (1982) Antidepressant drugs can slow or dissociate carcadian rhythms. Experientia 38, 1301-1309. WIRZ-JUSTICE, A., KAFKA, M. S., NABER, D. and WEHR, T. A. (1980) Circadian rhythms in rat brain ~t- and fl-adrenergic receptors are modified by chronic imipramine. Life Sei. 27, 341-347. WIRZ-JUSTICE, A., TOBLER, I., KAFKA, U. S., NABER, D., MARANGOS,P. J., BORBELY, A. A. and WEHR, T. A. (1981) Sleep deprivation: effect on circadian rhythms of rat brain neurotransmitter receptors. Psychiat. Res. 5, 67-76. WIRZ-JUSTICE, A., KAFKA, M. S., NABER, D., CAMPBELL, I. C., MARANGOS,P. J., TAMARKIN,L. and WEHR, T. A. (1982) Clorgyline delays the phase-position of circadian neurotransmitter receptor rhythms. Brain Res. 241, 115-122. WIRZ-JUSTICE,A., KRAEUCHI,K., CAMPBELL,I. C. and FEER, H. (1983a) Adrenoceptor changes in spontaneous hypertensive rats: a circadian approach. Brain Res. 262, 233-242. WIRZ-JusxICE, A., KRAEUCHI, K., MORIMASA,T., WILLENER,R. and FEER, H. (1983b) Circadian rhythm of [3H]-imipramine binding in the rat suprachiasmatic nuclei. Eur. J. Pharmac. 87, 331-333. WIRZ-JUSTICE,A., DAPRADA,M. and REM/LC. (1984) Circadian rhythm in rat retinal dopamine. Neurosci. Left. 45, 21-25. WIRZ-JUSTICE,A., KRAEUCHI,K., MORIMASA.T. and RIETVELD,W. (1985a) Circadian feeding rhythms: central mechanisms and methamphetamine modulation. In: Circadian Rhythms in the Central Nervous System, pp. 123-133. Eds P. H. REDFERN,I. C. CAMPBELL,J. A. DAVIESand K. F. MARTIN. Macmillan: London. WIRZ-JUSTICE, A., SUETTERLIN-WILLENER,R., REMIt, C., MUSCETTOLA,G. and DI LAURO, A. (1985b) Retinal benzodiazepine receptor binding. Experientia 41, 793. WOLF, M. D., WILCOX,R. E., RIFFLE,W. H. and ABRAHAM,L. n. (1980) Strain differences in dopamine receptor function and the initiation of movement. Pharmacol. Biochem. Behav. 13, 5-7.