Circadian acetylcholine receptor rhythm in rat brain and its modification by imipramine

Neurophurmoroloyy

Vol.

20. pp.

421

to 425.

0028.3908/81/050421-05802.00/O

1981

Prmted m GreatBritain

Pergamon

Press Ltd

CIRCADIAN ACETYLCHOLINE RECEPTOR RHYTHM IN RAT BRAIN AND ITS MODIFICATION BY IMIPRAMINE MARIAN S. KAFKA, ANNA WIRZ-JUSTICE*, D. NABERt and T. A. WEHR Biological Psychiatry Branch and Clinical Psychobiology Branch, National Institute of Mental Health, Bethesda, MD 20205, U.S.A. (Accepted

15 December

1980)

Summary-A circadian rhythm was found in the number of muscarinic acetylcholine receptors of the rat forebrain. The daily rhythm was endogenous and changed throughout the year. Chronic administration of the antidepressant drug imipramine altered the shape (wave form) of the rhythm, amplitude, 24-hr mean, and the timing of its peak (phase). As neuronal transmission is altered by both pre- and postsynaptic receptors, circadian and seasonal rhythms in a number of muscarinic receptors may play a role in the temporal organization of brain synaptic transmission.

Increasing evidence for functionally significant circadian changes in mammalian biological and behavioral variables has accumulated in recent years. The suprachiasmatic nuclei of the h)pothalamus, considered to be the locus of a “biological clock” or “master oscillator” coupling circadian oscillators and synchronizing brain rhythms to time cues in the environment (Moore, 1978; Rusak and Zucker, 1979), may drive many of these circadian rhythms. Recent work has shown that there are circadian rhythms in the number of c(- and B-adrenergic (Kalka, Wirz-Justice and Naber, 1981) dopamine (Naber, Wirz-Justice, Kafka and Wehr, 1980) opiate (Naber, Wirz-Justice and Kafka, 1981), and benzodiazepine (Marangos, WirzJustice, Kafka, Naber and Wehr, unpublished) receptors in the rat brain. The antidepressant drug imipramine altered the rhythm parameters of shape (wave form), timing (phase), amplitude (increase in peak over trough) and 24-hr mean in these receptor rhythms. The present authors investigated whether there is also a circadian rhythm in muscarinic cholinergic receptors and if so, whether imipramine changes this rhythm. METHODS Male Sprague-Dawley rats (100 g) were maintained in a controlled lightdark cycle (LD 12: 12, lights on from 7 a.m. to 7 p.m.). After 1 week, imipramine hydrochloride (10 mg/kg per day) (a gift from CibaGeigy, Summit, NJ) or saline (0.1 ml) was injected intraperitoneally twice a day for days l-5 to attain a steady state in the imipramine level, and once a day between 15-17 hr for days 6-20 to maintain the imipramine level (carried out in May-June, 1979). On day 21, each group received a final injection of saline *Fellow Research. t Fellow

of the

Swiss

of the German

Foundation Academic

for

Exchange

Biomedical Service.

or imipramine 2 hr before sacrifice by decapitation, and paired control and imipramine-treated groups were sacrificed beginning at 10 hr and then at 4-hr intervals afterwards until 6 hr the next day. Although the imipramine-treated animals weighed 15% less than controls (250 g compared with 280 g on day 21). they were apparently healthy. The forebrain (anterior to the cerebellum and without striata) was divided sagitally, weighed and rapidly frozen. Left and right forebrains were assayed alternately. Additional measurements over 24-hr in control rats were made in the preceding February and April and in the subsequent October. Parallel to control rats trained to the usual LD cycle in October, 42 rats were kept under the same LD conditions until 3 days before sacrifice. After the onset of darkness, this separate group was moved to an isolated room where they were maintained in constant darkness (DD) for 48-72 hr. They were sacrificed in the dark in groups of 7 at 4-hr intervals with the LD control group and their brains removed and prepared in the same way as those of the controls. An experiment to investigate the effect of light on binding to the muscarinic receptor was carried out in April. Light was extended 7 hr after the usual onset of darkness (from 7 p.m. to 2 a.m.) for a group of rats at which time the animals maintained in light were sacrificed with those maintained in darkness. In June, an experiment was carried out to measure the effect of darkness on binding to the muscarinic receptor. Darkness was extended for 7 hr after the usual onset of light (from 7 a.m to 2 p.m.) for a group of rats which was sacrificed with the group maintained in light. Acetylcholine muscarinic receptors were measured by the specific binding of C3H]QNB (quinuclidinyl benzilate, New England Nuclear, specific activity 29.4Ci/mmol) by the method of Yamamura and Snyder (1974). Specific binding, defined as the difference between C3H]QNB binding with and without 421

MARIAN S.KAFKA et al.

422

a&opine (2 x 10e6 M), was assessed by thawing and incubating for 1 hr at 37”C, frozen brain homogenates, which had been prepared previously by washing and centrifuging 3 times with final suspension in Tris buffer (pH 7.7, 0.05 M) at a concentration of 1 g in 10ml. The incubation was terminated by filtration through Whatman GF/B filters under vacuum with 3 Tris (pH 7.0. 0.05 M, 4°C) washes of 6 ml each. Filters were removed and counted in a scintillation counter. As a change in binding at the concentration of C3H]QNB used (0.16 nM) could indicate a change in receptor affinity, number, or both, saturation curves (using C3H]QNB at concentrations 0.05-6nM) were run at time points of maximal and minimal binding. Scatchard analyses of the data were used to calculate the affinity (the KD or apparent dissociation constant, viz. the concentration of C3H]QNB required for halfmaximal saturation) and the receptor density at saturation (Scatchard, 1949). The reliability of the assays was estimated by coefficients of variation (SD x 100 + mean). The average method error for triplicate determinations was 7 + 1%. The average inter-animal coefficient of variation (7 rats per time point for 6 experiments) was 8.6 f 1.5%. The inter-assay coefficient of variation (using internal standards across assays and on separate days N = 14) was 6.4 f 1.5%. For a given study, all time points were assayed in random order on a single day. All homogenates for a single time-point were filtered at the same time.

The daily rhythm persisted in the absence of light cues, with a change in wave form and 24-hr mean (Fig. 2, lower part). In the absence of light the rhythm showed a single broad peak from 2 through 14 hr and pmol [*HI ONE/g Wet Wt fonbmn January

22 20

FEBRUARY

16 16

March

14 12

APRIL

10

Mav

JUNE

16

July

August 16 September

16 14

OCTOBER

RESULTS

Binding of C3H]QNB to muscarinic acetylcholine receptors in rat forebrain showed a clear and significant daily rhythm. In experiments carried out in February, April and June a broad single peak began late in the light phase and extended into the dark phase. A trough late in the dark phase followed. The amplitude (peak:trough as %) of the peak in April was 39%; in June, 26%; and in February, 28%, accompanied by a minor (8%) single point peak at 10 hr (Fig. 1). In October a different pattern was observed. The peak in the dark phase (amplitude 35%) was accompanied by a second peak in the light phase (amplitude 45%) (Figs 1 and 2). A common feature of rhythms in all 4 months was a peak at 22 or 2 hr followed by a distinct minimum late in the dark phase. The average amplitude of the cholinergic rhythms was 35 f 8%, and the mean 24-hr binding, 17.1 + 2.0 (mean f SEM) pmol/g wet wt of tissue. Scatchard analysis of C3H]QNB saturation curves, run on pooled forebrain samples from groups of 3 animals sacrificed at times of maximal and minimal binding, revealed a single class of binding sites. The apparent K,‘s were 0.022 and 0.026nM and the densities of the binding sites, 61 and 49pmol/g wet wt, respectively. Further Scatchard analyses (n = 4) showed no differences in affinity at any time point (0.021 f 0.002, n = 4).

24

November

December

12

i L--i 10

14

16 22 2 6 Time of Day (hr)

10

14

16

Fig. 1. Circadian rhythms in specific binding to forebrain m&arinic acetylchbline receptors throughout the year. Soecific binding was measured with f3H10NB (New Eigland Nuclear. specific activity 29.4 LCi/&&ol). Each rhythm was measured at 6 time points over a 24-hr period, and each point represents the mean +SEM of 6-7 rats. with the exception of the rhythm measured in Aprd in which a pooled homogenate of 7 rats was measured at each time point and is expressed, accordingly, without SEM. The dark area represents the dark phase of the 12: 12 LD cycle and the dotted lines, the times of dawn and dusk in the external environment (drawn from the Nautical Almanac for the latitude of Washington, DC. 1979-1980). An entire light phase is repeated in each to clarify the rhythmic patterns. Statistical analyses with l-way analysis of variance (ANOVA) was followed by calculations of leastsquares difference to distinguish significant differences between time points. **P < 0.01; *P < 0.05. Each rhythm (except for that in April which could not be analyzed) was highly significant by l-way analysis of variance. The F-values were 9.26 (P i O.OQOOl) in February, 10, 18 and 22 hr being higher than 14 and 2 hr (P < 0.01) and 6 hr higher than 14 and 2 hr (P i 0.05); 3.85 in June (P i 0.0068), l&22 hr higher than 2 and 6 hr (P< 0.01); F = 27.23 in October (P i 0.00001). 10 and 2 hr higher than 18, 22 and 6 hr (P < 0.01) and 14 hr higher than 18 and 22 hr (P < 0.05).

423

ACh circadian rhythm *+P
2

6

IO

I4

I6

22

2

6

IO

TIME OF DAY (hr) **p
I

01

I

16. P 2

=3

14.

depressant drug, imipramine hydrochloride, (in June) was strongly modified, as expressed in the significant interaction term measured by the 2-way ANOVA (Fig. 3). The single broad peak in binding to control brains (l&22 hr) was replaced by 2 peaks occurring at 6 and 14-18 hr, respectively. The change from unimodal to bimodal form made it difficult to estimate a change in phase. The amplitude of 26% in controls was diminished to 15% in imipramine-treated animals. The 24-hr mean of binding was decreased by 20% (18.8 f 0.96 pmol/g wet wt to 15.7 + 0.23, P < 0.0025, Student’s t-test). The K,‘s in pooled brain homogenates from time points of maximal and minimal binding in imipramine-treated and control rats were similar (0.024 k 0.002 nM, n = 2, in imipraminetreated rats and 0.021 &-0.002 nM, n = 6, in control rats). DISCUSSION

2

6

IO

14

I6

22

2

6

IO

TIME OF DAY (hr)

Fig. 2. Specific binding to the muscarinic acetylcholine receptor measured at 6 points throughout the 24-hr day. Each point represents the mean k SEM of 67 rats, 3 points being repeated without SEM to clarify the rhythm by removing the 24-hr cut-off time. The shaded area represents the dark phase of the light-dark (LD) cycle. Upper: Circadian rhythm of specific binding to forebrain muscarinic acetylchbhne receptors in October under a 12: 12 LD cvcle. ANOVA showed F = 27.2. P -c 0.00001. Bindina at lb and 2 hr was significantly higher than binding at 18: 22 and 6 hr (P < 0.01) and bmding at 14 hr. higher than that at 18 and 22 hr. Lower: Circadian rhythm in binding in DD following a previous trammg to the 12: 12 LD cycle (delineated by vertical lines). ANOVA showed F = 34.27, P < 0.00001. Binding at 10, 14, 2 and 6 hr IS higher than binding at 18-22 hr. P < 0.01. Comparison of the 2 rhythms by 2-way analysis of variance (2-way ANOVA) indicated significant interacttons between the rhythms in the 2 lighting regimens (F = 11.53, P < 0.0001). The 24-hr means of bmdrng were srgnificantly different (P < 0.01). 729; of the variance is explained by the interactions between time and light.

These studies demonstrate the existence of an endogenous circadian rhythm in binding to the muscarinic acetylcholine receptor. As the affinity of the receptor for C3H]QNB over the 24-hr period remained the same, the change in binding reflects a change in the number of receptors. Even under controlled LD conditions, the wave form of the rhythm changed during the year. The changes during the year could arise from changes in the environment (e.g. the content of food pellets, constituents of the atmosphere, electromagnetic fields) or could result from a seasonal rhythm, as l*

P
c PC005

2

6

IO

14

18

22

2

6

IO

TIME DF DAY (hr)

a trough, as under LD conditions, from 18 to 22 hr. The 24-hr mean under DD was significantly lower than under LD (14.7 f 0.44 and 12.5 + 0.44 pmol/g wet wt. P < 0.01, respectively); the amplitudes of the peaks (45 and 35% under LD) increased to 71% under DD conditions (Fig. 2). Binding was similar in forebrains from rats maintained in extended light and in the usual darkness (15.47 f 0.78 pmol/g, in light and 17.25 f 0.68 pmol/g in darkness, n = 3. respectively). Binding was also similar in forebrains from rats maintained in extended darkness and in the usual light (18.07 f l.l7pmol/g we wt of tissue, n = 3, and 21.12 f 8.9, n = 3, respectively). The circadian rhythm in the muscarinic receptor in a group of rats treated chronically with the anti-

Fig. 3. Circadian rhythm in binding to muscarmic acetylcholine receptors in controls (0.1 ml NaCI) and rats treated chronically with imipramine (lOmg/kg per day for 3 weeks). Details are as in Figure 1. One-way analysts of variance for each group indicated significant changes with time (controls: F = 3.85, P < 0.0068; imipramine-treated: F = 5.24, P < 0.001). The least-squares difference analysis showed that, in the controls, binding at 14. 18 and 22 hr was higher than at 6 hr (P < 0.01) and binding at 10 hr higher than at 6 hr (P < 0.05); in imipramine-treated rats, binding at 14, 18 and 6 hr was higher than at 22 and 2 hr (P < 0.01) and 6 hr higher than 10, 22 and 2 hr (P -C 0.05). Two-way ANOVA showed that the interaction of time and imipramine (the effect of imipramine on the circadian rhythm) was significant with F = 4.776, P < 0.001). The 24hr mean was lower by 20% in imipramine-treated rats (P -c 0.0025, Student’s r-test). 47% of the variance is accounted for by interactions between time and imipramine.

424

MARIAN S. KAFKA et al.

seasonal modifications of circadian rhythms have been found under circumstances in which environmental factors have been controlled (von Mayersbach, 1967; Scheving, Pauly, Mayersbach and Dunn. 1974). Evidence suggesting a seasonal rhythm was obtained in a separate experiment carried out in a different environment (Switzerland) m December-January, 1979 (unpublished observations). In this experiment a bimodal rhythm similar to that measured in the October Bethesda experiment was found. Secondary peaks seemed to occur at certain times of year in the cholinergic muscarinic receptor rhythm. Analogously. a behavioral rhythm, the mouse rest-activity cycle, also showed apparent seasonal changes m the occurrence and amplitude of secondary peaks in a unimoda1 rhythm (Pittendrigh and Daan, 1976). Not only the acetylcholine muscarinic receptor. but the brain concentration of acetylcholine showed a daily rhythm. One group (Friedman and Walker, 1972) found that maximal levels occured in the dark, whereas two other groups (Saito. Yamashita, Yamazaki. Okada, Satomi and Fujieda, 1975; Hanin, Massarelli and Costa 1970) found maxima in the light. the latter measurements being made in October to February. The muscarinic receptor number in the present experiments was inversely related to the brain acetylcholine concentration as measured by the latter two groups. A seasonal rhythm in brain acetylcholine production has been measured in the rabbit (Monnier and Herkert, 1972). A single study of circadian rhythms in muscarinic receptor binding in post-mortem human brain showed peak binding in the light compared with rat brain peak binding at the beginning of the dark phase (Perry, Perry and Tomlinson. 1977). In the 2 species the rhythm showed a similar phase relationship to the rest-activity (sleepwake) cycle. Although brief changes in exposure to light or darkness (7-hr extensions) did not change the receptor additional experiments are needed to number, measure the direct effects of light. Light has been shown to increase muscarinic receptors in the visual cortex of dark-reared rats (Rose and Stewart, 1978). The daily rhythmic pattern in the muscarmic cholinergic receptor number shares some features with the pattern in r-adrenergic receptors in rising throughout the light phase, and with the pattern in b-adrenergic receptors in displaying a pre-dawn minimum. The interactions between the circadian receptor rhythms in the cholinergic and adrenergic systems may contribute to the central effects of these systems which sometimes operate in opposition to one another (Decsi and Nagy, 1977; Buccafusco and Brezeneff, 1980; Gillin, Mendelson. Sitaram and Wyatt. 1978; Jacobs and Jones, 1978; Sitaram, Moore and Gillin, 1979; Monnier and Herkert. 1972; Clark and Straughan, 1977; Mason, 1979) and sometimes in concert (Bianchi, Spiadlieri, Guadalini, Tangenelli and Beani, 1979; Kuhar, Atweh and Bird, 1978). Chronic administration of antidepressant drugs has been shown not to alter the number of muscarinic

cholinergic receptors in rat brain (Maggi, UPrichard and Enna, 1980) and to increase their number in mouse brain (Rehavi. Ramot. Yavetz and Sokolovsky, 1980). The present data show that imipramine caused a marked change in the circadian rhythm of the muscarinic receptor, so that the time of day at which the animals are sacrificed dictates whether a difference is found between control and imipramine-treated animals. Daily rhythmic changes, as well as species differences, may underlie the apparent contradictory findings in rat and mouse brain. The present authors have found that large concentrations of imipramine and its major metabolite, desmethylimipramine (l-1.5 /IM) remained in the membranes even after 6 washes (and were similar to the concentrations remaining after the 2 washes used in these experiments). The presence of imipramine in membranes must be considered in interpreting the effect of the drug on muscarinic receptors. as imipramine antagonizes binding to the muscarinic receptor (Yamamura and Snyder. 1974; Golds, Przylso and Strange, 1980). Imipramine did not appear to be competing with C3H]QNB for receptor sites, however, as the affinity of the sites was not decreased in the presence of the drug. It is possible that the daily injection of imipramine served as a time cue or zeitgeber, synchronizing the muscarinic receptor rhythm. If so, the modification found in the rhythm may result from the timing of the drug dose, as well as from the presence of the drug itself. Slight diurnal fluctuations m membrane drug concentration were not correlated with the muscarinic receptor rhythm (r = 0.52, NS). The change in wave form from a unimodal to a bimodal rhythm after imipramine treatment complicates the estimation of a change in phase. A phase shift, accompanied by a change in wave form. frequently results from an interaction between the endogenous circadian rhythm and “masking”. a direct effect of an environmental stimulus on the rhythm (Wever. 1979). After chronic imipramine administration. instead of a single peak from 10 to 22 hr, there were 2 peaks, one at 6 hr and another at 14-18 hr. As reported elsewhere, the peak numbers of CL-and fl-adrenergic (Wirz-Justice, Kalka, Naber and Wehr, 1980). dopaminergic (Naber et al.. 1980), and benzodiazepine (Marangos et al., unpublished) receptors shift to a later time of day and coincide at 6 hr after chronic imipramine administration. Perhaps the acetylcholine muscarinic receptor peak at 6 hr is also due to the delaying action of the drug. If the phase of the rhythm has been delayed by imipramine treatment, it is possible that the second peak results from a direct masking effect of the environmental lightdark cycle on the phase-shifted circadian rhythm. On the other hand. peak muscarinic binding may not be delayed by imipramine treatment as it is not delayed by another antidepressant drug, clorgyline. The existence of circadian rhythms in the number of muscarinic acetylcholine, as well as other neurotransmitter. receptors provides additional neuro-

ACh circa dian rhythm chemical evidence that transmission in the brain is temporally organized. Cycles in the numbers of preand postsynaptic receptors may be important features of the neuronal mechanisms that underlie daily cycles in behavior. Acknowledgements-Our appreciation to Drs W. Z. Potter and A. Zavadil of the NIMH for measuring brain membrane imipramine and desmethylimipramine by gas chromatography mass spectrometry, to Dr M. Zatz for useful experimental advice and to MS Dawn McBrien for typing this manuscript. REFERENCES

Bianchi. C., Spiadheri. G., Guandalim, P., Tangenelli, S. and Beani, L. (1979). Inhibition of acetylcholine outflow from guinea-pig cerebral cortex following locus coeruleus stimulation. Neurosci. ht. 14: 97-100. Buccafusco, J. J. and Brezenoff. H. E. (1980). Opposing influences on behavior mediated by muscarinic and nicotinic receptors in the rat posterior hypothalamic nucleus. Psychopharmacology 67: 249-254. Clarke, G. and Straughan, D. W. (1977). Evolution of the selectivity of antagonists of glutamate and acetylcholine applied microiontophoretlcally onto cortical neurones. Neuropharmacology 16: 391-399. Becsl, L. and Nagy, J. (1977). Adrenergic modulation of a cholinerglc emotlonal reaction in the cat’s thalamus. Psychopharmacology 54: 303-305.

FrIedman. A. H. and Walker, C. A. (1972). The acute toxicity of drugs acting at cholinoceptive sites and twentyfour hour rhythms in brain acetylcholine. Arch. Toxik. 29: 39-10.

Gillin, J. C., Mendelson, W. B., Sitaram, N. and Wyatt, R. J. (1978). The neuropharmacology of sleep and wakefulness. A. Rev. Pharmac. Toxic. 18: 563-579. Golds, P. R., Przyslo, F. R. and Strange, P. G. (1980). The binding of some antidepressant drugs to brain muscarinic acetylcholme receptors. Br. J. Pharmac. 68: 541-549. Hamn, I.. Massarelli, R. and Costa, E. (1970). Acetylcholine concentrations in rat brain: diurnal oscillation. Science 170: 341-342. Jacobs, B. L. and Jones, B. E. (1978). The role of central monoamine and acetylcholine systems in sleep-wakefulness states: mediation or modulation? In: CholinergicMonoaminergic Interactions rn the Bram (Butcher, L. L.. Ed.), pp. 271-290. Academic Press, New York. Kafka, M. S.. Wirz-Justice, A. and Naber, D. (1981). Circadian and seasonal rhythms m z- and /?-adrenergic receptors in the rat brain. Brain Res. In press. Kuhar, M. J., Atweh. S. F. and Bird, S. J. (1978). Studies of cholinergic-monoaminergic interactions in rat brain. In& Cholrnergic-Monoaminergic

Interactions

in the

Brain

(Butcher, L. L.. Ed.), pp 21 l-227. Academic Press, New York. Maggi, A.. U’Prichard. D. C. and Enna, S. J. (1980). Differ-

425

ential effects of antidepressant treatment on brain monoaminergic receptors. Eur. J. Pharmac. 61: 91-98. Mason, S. T. (1979). Central noradrenergic-cholinergic interaction and locomotor behavior. Eur J. Pharmac. 56: 131-137.

von Mayersbach, H. (1967). Seasonal influences on biological rhythms of standardized laboratory animals. In: Cellular Aspects of Biorhythms (von Mayersbach. H.. Ed.), pp. 87199. Spiinger-Verlag, New York. __.Monnier. M. and Herkert. B. (1972). Concentration and seasonal variations of a&tylcgohne in sleep and waking dialysates. Neuropharmacology 11: 479-489. Moore, R. Y. (1978). Central control of circadian rhythms. Frontiers Neuroendocr. 5: 185-206.

Naber, D., Wirz-Justice, A. and Kafka, M. S. (1981). Circadian rhythm m brain opiate receptor. Neurosci. Left. In press. 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. Psychopharmacology 68: l-5.

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.

Pittendrigh, C. S. and Daan, S. (1976) A functional analysis of circadian pacemakers m nosturnal rodents. J. camp. Physiol. 106: 333-355.

Rehavi, M., Ramot, D., Yavetz, B. and Sokolovsky, M. (1980). Amitryptyline: long-term treatment elevates x-adrenergic and muscarinic receptor bmdmg in mouse brain. Brain Res. 194: 443-453. Rose, S. P. R. and Stewart, M. G. (1978). Transient Increase in muscarinic acetylcholine receptor and acetylcholine esterase in visual cortex on first exposure of dark-reared rats to light. Nature 271: 169-170. Rusak+ B. and Zucker, I. (1979).Neural regulation of circadian rhythms. Physiol. Rev. 59: 449-526. Saito. Y.. Yamashita. I.. Yamazaki, K., Okada, F., Satomi. R. gnd Fujieda. T. (1975). Circadian fluctuation of brain acetylcholine. Life Sci. 16: 281-288. Scatchard, G. (1949). The attractions of proteins for small molecules and ions. N.Y. Acad. Sci. 51: 660-672. Scheving, L. E., Pauly. J. E.. von Mayersbach. H. and Dunn, J. D. (1974). The effect of continuous light or darkness on the rhythm of the mitotic index in the corneal epithelium of the rat. Acta Anat. 88: 411423. Sitaram, N., Moore, A. M. and Gillin, C. J. (1979). Scopolamine-induced muscarinic supersensitivity in normal man: changes in sleep. Psychiat. Res. 1: 9-16. Wever, R. (1979). The Cwcadian System of Man. SpringerVerlag, New York. Wirz-Justice, A., Kafka, M. S., Naber, D. and Wehr, T. A. (1980). Circadian rhythms in rat brain alpha- and betaadrenergic receptors are modified by chronic imipramine. Life Sci. 27: 341-347. Yamamura, H. I. and Snyder, S. H. (1974). Muscarinic cholinergic binding in rat brain, Proc. natn. Acad. Sci. U.S.A. 71: 1725-1729.