- Email: [email protected]
Effects of chronic lithium, clorgyline, imipramine, fluphenazine and constant darkness on the α-melanotropin content and circadian rhythm in rat brain
European Journal of Pharmacology, 85 (1982) 1-7
1
Elsevier Biomedical Press
EFFECTS OF CHRONIC LITHIUM, CLORGYLINE, IMIPRAMINE, FLUPHENAZINE AND CONSTANT DARKNESS ON THE a-MELANOTROPIN CONTENT AND CIRCADIAN RHYTHM IN RAT BRAIN THOMAS L. O'DONOHUE l, ANNA WIRZ-JUSTICE 2,, MARIAN S. KAFKA 3, DIETER NABER 3** IAIN C. CAMPBELL 4 and THOMAS A. WEHR 2 J Laboratory of Clinical Science, N1MH, Bldg. 10, Rm. 3D-48, National Institutes of Health, Bethesda, MD 20205, 2 Clinical Psychiatry Branch, NIMH, 3 Biological Psychiatry Branch, N1MH, 4 Clinical Neuropharmacolog; Branch, NIMH, U.S.A.
Received 31 March 1982, revised MS received 27 July 1982, accepted 9 August 1982
T.L. O'DONOHUE, A. WIRZ-JUSTICE, M.S. KAFKA, D. NABER, I.C. CAMPBELL and T.A. WEHR, Effects of chronic lithium, clorgyline, imipramine, jTuphenazine and constant darkness on the c¢-melanotropin content and circadian rhythm in rat brain, European J. Pharmacol. 85 (1982) 1-7.
The effects of constant darkness, chronic lithium, clorgyline, imipramine and fluphenazine treatment on the content and diurnal rhythm of a-MSH in rat forebrain were investigated. The persistence of the a-MSH rhythm in constant darkness demonstrated that the rhythm was circadian in nature. Constant darkness increased the 24 h mean a-MSH concentration in brain while lithium, fluphenazine and imipramine decreased it. In addition, imiparamine and clorgyline delayed the phase of the a-MSH circadian rhythm while lithium advanced it. Circadian rhythm Fluphenazinc
a-MSH rhythm
Lithium, chronic
1. Introduction D i u r n a l variations in the c o n c e n t r a t i o n of am e l a n o c y t e s t i m u l a t i n g h o r m o n e in b r a i n ( O ' D o n o h u e et al., 1979b, 1980; M o n n e t et al., 1981) and b l o o d ( U s a t e g u i et al., 1976; M o n n e t et al., 1981) have been described. W h i l e p i t u i t a r y - d e r i v e d circ u l a t i n g a - M S H regulates a d a p t i v e p i g m e n t a t i o n a l t e r a t i o n s in various species t h r o u g h o u t the a n i m a l Thomas L. O'Donohue is a Pharmacology Research Associate in the National Institute of General Medical Sciences, to whom all correspondence should be addressed. * Anna Wirz-Justice was a Fellow of the Swiss Foundation for Biomedical Research and is currently at Psychiatric Clinic, University of Basel, Basel, Switzerland, CH-4025. ** Dieter Naber was a Fellow of the German Academic Exchange Service and is currently at the Psychiatric Clinic, University of Munich, 7 Nussbaumstrasse, Munich, W. Germany. 4 lain C. Campbell is currently at the Institute of Psychiatry, The Maudsley Hospital, Decrespigny Park, Denmark Hill, London, England SE5 8AF.
Darkness, constant
Clorgyline
lmipramine
k i n g d o m ( B a g n a r a a n d Hadley, 1973), its precise e n d o c r i n e role in m a m m a l s is largely u n k n o w n (cf. T h o d y , 1980). N e u r o n a l a - M S H a p p e a r s to fulfill m a y of the criteria for a n e u r o t r a n s m i t t e r or m o d u l a t o r (cf. O ' D o n o h u e and Jacobowitz, 1980; O ' D o n o h u e et al., 1981; Swaab et al., 1981) that m a y p l a y a role in the processes of attention, arousal, learning a n d m e m o r y (see W i t t e r et al., 1981 ; Bohus a n d D e Wied, 1981 ; G o l d a n d Delanoy, 1981; Riccio a n d C o n c a n n o n , 1981; M o u r a t et al., 1981; G a i l l a r d , 1981). It is possible that the d i u r n a l r h y t h m s of a - M S H in rat b r a i n m a y provide a clue to its function in these behaviors. The p u r p o s e o f this s t u d y was to investigate the p h y s i o l o g y a n d p h a r m a c o l o g y of a - M S H r h y t h m s in brain. To d e t e r m i n e whether the a - M S H d i u r n a l r h y t h m was actually c i r c a d i a n in nature, persisting in the absence of visual cues, the r h y t h m was investigated in rats h o u s e d in chronic darkness. R e c e n t d a t a suggest that altered c i r c a d i a n r h y t h m s was involved in the p a t h o p h y s i o l o g y o f depression
and mania and that the efficacy of antidepressant and antimanic drugs may be due, at least in part, to their ability to alter circadian rhythms in the depressed or manic patient (see Wehr and Goodwin, 1981). Furthermore, antimanic and antidepressant drugs have been shown to alter the circadian rhythms of brain neurotransmitter receptor rhythms (Wirz-Justice et al., 1980a,b; N a b e r et al., 1980; Kafka et al., 1981, 1982; Wirz-Justice, 1982). In this study, we extended those findings and examined the influence of imipramine, a tricylic antidpressant; clorgyline, an MAO inhibitor; fluphenazine, a neuroleptic; and lithium on the rhythm of a - M S H concentrations in brain.
2. Materials and methods
Male Sprague Dawley rats (225-250 g at time of sacrifice) were housed six or seven per cage in a controlled light-dark (LD) cycle with lights on from 7 a.m. to 7 p.m. with food and water freely available. In an experiment performed in October, 1979, the effects of constant darkness and of chronic fluphenazine, lithium and clorgyline on the diurnal rhythm of c~-MSH was investigated. To test the effect of constant darkness, rats were maintained in identical LD conditions until 3 days before killing. After the onset of darkness, they were moved to an isolated room and maintained in constant darkness for 52-72h before sacrifice. Fluphenazine (10 m g / k g ) was administered in a s.c. depot once a week for two weeks before sacrifice. Clorgyline hydrochloride (a gift from May and Baker Pharmaceuticals, Dagenham, U.K.) in distilled deionized water was administered at a constant rate of 4 m g / k g per 14 days using s.c. implanted Alzet minipumps (Model 1701, Alza Corp., Palo Alto, CA). The minipumps were implanted under short-lasting halothane anesthesia during the light phase. Lithium was administered orally for two weeks by mixing it with powdered animal chow (2.26g of lithium carbonate, 1500 g of animal chow and 21 of water). Trunk blood from each lithium-treated rat was collected in heparin-coates tubes, centrifuged and plasma stored at - 2 0 ° C for measurement of plasma
lithium by flame phatometry. In an experiment performed in May-June, 1979, there was an evaluation of the effects of chronic imipramine administration on the circadian rhythm of ~x-MSH in brain. Rats (100 g) were administered imipramine hydrochloride (10 m g / k g per day) (a gift from Ciba-Geigy, Summit, N J) or saline 0.1 ml twice a day for days 1-5 to attain a steady state in the imipramine level and once a day between 15-17 h for days 6-20 to maintain the imipramine level. On day 21, each rat received a final injection 2 h before sacrifice. Although imipramine-treated rats weighed 15% less than controls, they were apparently healthy. Rats were killed by decapitation. There were 7 rats in each group. The forebrain anterior to the cerebellum was divided sagitally and the striata removed. The forebrains minus strata were weighed, frozen on dry ice and stored at - 2 0 ° C until assayed. The brains were homogenized in 20 vol of Tris buffer (pH 7.0, 0.5 M) in the cold. An aliquot was removed, acidified to 1 N acetic acid and boiled for 10 min. The sample was centrifuged for 20 rain at 8000 X g and duplicate aliquots of the supernatant fluid were lyophilzed and assayed for ~x-MSH as described previously (O'Donohue et al., 1979a). In routine measurement of a - M S H in brain, samples are placed directly into 1N acetic acid. In this experiment, we placed the samples in buffer to allow measurement of various receptors (see Kafka et al., 1981, 1982; Naber et al., 1980; Wirz-Justice et al., 1980a,b, 1982). Preliminary experiments demonstrated that a - M S H concentrations were the same in samples that were placed directly in acid and those that were homogenized in buffer and subsequently acidified and boiled. Concentrations of c~-MSH at various times within a treatment group were compared by oneway analysis of variance (ANOVA) and the least-squares difference (LSD) procedure (Snedecor and Cochran, 1967). Differences between two groups were compared by two-way ANOVA. Data was also fitted to a sinecurve by the method of least squares. The phase position (~b) of the rhythm was estimated as the time of the maximum of the best cosive fit (acrophase). Direction of the rhythm shift was determined by assuming that a shift in ~b was less than 12 h. A positive ~b indicated that the
CONTROL r~.~ ~ 24MEAN~_ HOUR ~~~r~'~''r":"
peak was earlier than control; negative ~ - later than control.
3. Results
The effects of drug and light manipulations on the a - M S H rhythm are illustrated in figs. 1-3. The statistical analyses of data are reported in tables 1 and 2. The results of plasma lithium determinations have been described previously (Kafka et al., 1982). Consistent with previously reported results (O'Donohue et al., 1979b; Monnet et al., 1981), the a - M S H concentration in the normal forebrain varied in a diurnal fashion. As shown in fig. l, concentrations of a - M S H are lowest during the end of the light period and the beginning of the dark period in rats sacrificed in October. Fig. 3 shows that a similar rhythm of a - M S H content in forebrain exists in rats sacrificed in June, although the overall a - M S H concentration was actually higher in the rats sacrificed in June when compared to October. A qualitatively similar rhythm has been observed in discrete brain regions of rats sacrificed in July of 1978 (O'Donohue et al., 1979b). Our preliminary results have also shown a similar rhythm in the brains of rats maintained and killed in January, 1980 in Zurich, Switzerland by Drs. A. Borb61y and I. Tobler. The effects of constant darkness on the a - M S H rhythm is shown in fig. 1. The rhythm was qualitatively similar and not significantly different from that of control rats. The mean concentration of a-MSH, in these rats, however, was significantly higher than controls. It is also noteworthy that the standard errors of each time point in the rats housed in chronic darkness were about twice as great as those of control rats. The increased variability of values at each time point may have been due to a lack of precise synchrony between rhythms in different animals in the free-running condition. The effects of chronic lithium are shown in fig. 1. Lithium administration significantly changed both the rhythm and overall concentration of aMSH. The acrophase of the a - M S H rhythm (~p = + 5 . 2 h) occurred 5.2 h earlier than those of control rats. Overall concentrations of a - M S H after
4!5 ± CONSTANT DARKNESS
~ll ]~...
3:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: :::::.......:::'..... : ~
I~i~i~i~iii~i~i~i~i~i~i~ii~i~i~i~i~i~i~ii~i~i~iii~i~i~!~!~i~i~iii~i~i~!~!i~i~i!i!i~i~i~!i~! 2 i~i:iiii~!i:~i~i~ii~i~i~!~}iiiii~i}!~i~}:i)ii~i~!i!~!~}~)~)iii~ii}!i~!~i~)~)i~i~i)!~!iii!~ i:i!!:::i::::::!if:i!.!:iii:i::!i!ii!i!!::il:i::!:!:i:i!.!:::?:::!::i::i!.i :iiiii:!:!i:::: ::::::::::::::::::
i::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: iiiiiiiiiiiiiii!iiiiiiiiiiiiiiiii!iiiiiiiiiiiiiiiiiiiiiiiiiii!!i!!i!!i!iii.. 0
iii!i!:!iii•!iii•i•ii!i!ii!•i••iii•iiii!i•iii•!•!iii!iiii!ii!ii•!•!i!i•i•i•ii!ii!•i•!iii•i•i•ii!iiiiiiii!•ii2i•i•i•!ii IIIII IIII IIII
CHRONICLITHIUM
i
a ~ii~ii::iii~i!iiiiiiiiiiiiii~i!iii!i~iiii~-i~ii~ii!ii! 7e
::::::...H::::.:::.::.
10 14 18 22 2 TIME(hr) 1
+::::::.
6
Fig. 1. R h y t h m o f a - M S H in the f o r e b r a i n s of rats h o u s e d in c o n t r o l c o n d i t i o n s , c o n s t a n t d a r k c o n d i t i o n s or t r e a t e d with l i t h i u m a n d k i l l e d in O c t o b e r . T h e h o r i z o n t a l d a r k g r a y b a r i n d i c a t e s the 24 h m e a n o f c o n t r o l rats. S t a t i s t i c a l d i f f e r e n c e between mean of experimental group and control: **P<0.01, ***P<0.001.
lithium treatment were significantly lower than control. Chronic fluphenazine treatment had qualita-
CONTROL
24 HOUR
.....
IMIRRAMINE CONTROL
24 HOUR MEAN
]
i
iiliiiiiliiiiiiiiiiiiiiiiiliiiiiiiiiiiiiiiiiiiiiiiiiil ii!iii t
.::::
..................
0~
.:< . . . . . . .
k:::+:':':':::::':':':'":'iii'i'i'i"'
/11/ ' ~ \ ' "
iI
**
CHRONIC CLORGYLINE
4
i=
I
I
14
18
22
2
6
TIME (hr) ~N.S.
-r
~2
CHRONIC FLUPHENAZlNE
4
iii! iiiiii iii!~iii~i~i!ill
I
!iiii!i!ii!)iii?iiii!i!?!iiiii!?:i!iiiii!i!!!i!i!ii!!ii
21 ~ ~-
1 _
0
t
10
_
.T_
_
±
10
i!iiiiiii£!!i!ii;iiiil;iiii~s:....~:~ii!i!i::iii i!ii~i!ii!i~i~i~: A
i
::::::::::::::5:: . . . . . . . . . . . . . . . . .
_
1
14
:
::::::::!..::i:ii!ii:i:i:i:iii:i:i:i:i:i:i:i:i:i:i: _
.:::.:.;::.:.:.:-;.:-;.:.:.:.:.:,:.:.:,:::::::.:.:,:.:-
18 22 TIME (hr)
2
6
Fig. 2. Rhythm of a-MSH in the forebrains of control rats and rats treated with clorgyline or fluphenazine and killed during October. The horizontal dark gray bar indicates the 24 h mean of control rats. ***Overall mean is significantly different from control - 24 h mean - - P<0.001.
Fig. 3. Rhythm of ~x-MSH in the forebrains of control rats (solid line) and imipramine-treated rats killed during June. The horizontal dark gray bar indicates the 24 h mean of control rats. ***Overall mean is significantly different from control 24 h mean - P<0.00001.
tively and quantitatively similar effects as lithium on the a-MSH rhythm. Compared to the rhythm in control rats, the amplitude of the a-MSH rhythms of lithium- and fluphenazine-treated rats were both decreased (see figs.), the acrophases were slightly advanced ( ~ - - 2 . 6 h for fluphenazine) and overall concentrations were significantly lowered. Chronic clorgyline treatment had no effect in overall concentrations of a-MSH but had a very dramatic effect on the phase position of the rhythm. In control rats, the nadir was at 18 and 22 h while in clorgyline-treated rats, the peak was at these times. The fitted peak in a-MSH concentration was delayed by 9.3 h. In the case of imipramine treatment, there was a significant change in both overall a-MSH content and the rhythm (fig. 3). Concentrations of c~-MSH in imipramine-treated rats were much lower than controls. The shift in the rhythm was quite similar to that caused by clorgyline, as the fitted peak was also delayed by about 9 h.
TABLE 1 Statistical analyses of circadian rhythms of ot-MSH: One way ANOVA. F
October Controls Lithium Clorgyline Constant darkness Fluphenazine June Controls Imipramine
Least square differences
P
P<0.01**
P<0.01*
2, 6 > 18, 22 10, 14>22 18, 6 < 2 all time p t s > 2 18, 2 2 > 6
151>18
5.59
< 0.0008
4.76 17.21
< 0.0022 < 0.00001
1.77 3.45
<0.14 NS < 0.013
18, 2 2 < 2
10>18 10, 14< 2
26.84 4.53
< 0.00001 <0.003
18, 222, 6, 10, 18
2 2 > 10, 2
10, 14, 18, 6 < 2 10, 14<18, 22 10> 6
TABLE 2 Statistical analyses of circadian rhythms of a-MSH: Two way ANOVA.
Lithium Clorgyline Constant darkness Flupenazine Imipramine
Main effects
Treatment
F
F
8.82 1.32 17.12 8.41 43.85
P
<0.0001 <0.24 ys < 0.0001 <0.0001 <0.0001
15.04 0.15 61.71 24.1 152.9
Time P
<0.001 <0.70 Ns < 0.0001 <0.0001 <0.00001
4. Discussion The results of these experiments confirm the presence of a rhythm in the concentration of aM S H in brain (O'Donohue et al., 1979b; Monnet et al., 1981) and demonstrate that the rhythm is circadian in nature as it persists in the absence of light cues. The overall increase in concentrations of a - M S H in brains of rats housed in constant darkness is similar to the reported increased concentration of a - M S H in the pineal glands of chronically dark-housed rats (O'Donohue et al., 1980). It is unknown, however, whether an increase in a - M S H concentration in the brain corre-
F
7.58 1.61 8.23 5.29 19.05
2 way interaction P
<0.0001 <0.17 ys <0.0001 <0.0001 <0.0001
F
P
3.16 17.73 1.26 1.26 25.87
<0.013 <0.0001 <0.29 ys <0.29 Ns <0.0001
sponds to increased or decreased a - M S H release. The effects of pharmacological manipulations on the a - M S H concentrations and circadian rhythms can be interpreted either as secondary effects on the a - M S H neuronal system via interactions with monoaminergic systems or as a primary effect on the frequency of a putative driving circadian oscillator reflected in changes in the phase-position of many neuronal systems of the brain including the a-melanotropinergic neuronal system. The pharmacologically induced changes in the circadian rhythm of a - M S H by clorgyline, imipramine and lithium can be interpreted in light of
the effects of these drugs on circadian frequency. C o n s i s t e n t with this hypothesis are data d e m o n strating similar temporal r h y t h m alterations not only of a - M S H but also various n e u r o t r a n s m i t t e r receptors by these drugs. Chronic clorgyline or i m i p r a m i n e t r e a t m e n t delayed the peak in the rhythms of c~-MSH c o n c e n t r a t i o n a n d a - a d r e n ergic, /~-adrenergic, cholinergic muscarinic, dopaminergic, opiate and benzodiazepine receptor n u m b e r s by 4 - 1 2 h (Wirz-Justice et al., 1980a,b, 1982: Kafka et al., 1981; N a b e r et al., 1980). The fact that the delay of 8-12 h in a - M S H concentrations and a-adrenergic receptors was quite similar after both a n t i d e p r e s s a n t drugs may indicate a functional relationship between these systems. The similar effects of clorgyline and i m i p r a m i n e on neurochemical rhythms are reflected in similar effects of these drugs on behavioral rhythms both delay circadian r h y t h m s in activity of hamsters (Wirz-Justice et al., 1980b). The ability of lithium to reduce the a m p l i t u d e of neurochemical rhythms has been suggested to be related to its ability to d a m p cycling between m a n i a and depression ( K a f k a et al., 1982). Lithium, f l u p h e n a z i n e and i m i p r a m i n e all have in c o m m o n the effect of decreasing the overall c o n c e n t r a t i o n of a - M S H in brain. These data may indicate an interaction between m o n o a m i n e r g i c a n d a - m e l a n o t r o p i n e r g i c systems. Such an interaction would be expected from the surprising a n a t o m i c a l overlap of m o n o a m i n e r g i c a n d am e l a n o t r o p i n e r g i c systems ( O ' D o n o h u e et al., 1979a). Results of n u m e r o u s other studies support i n t e r a c t i o n between c~-MSH a n d m o n o a m i n e r g i c systems (for references see G o l d a n d Delanoy, 1981). G o l d a n d D e l a n o y (1981) hypothesize that m o n o a m i n e s a n d M S H peptides act in c o n j u n c t i o n to m o d u l a t e m e m o r y processes as well as other behavioral responses. It is u n d o u b t e d l y the interactions between r h y t h m s in m o n o a m i n e r g i c , ~m e l a n o t r o p i n e r g i c a n d other n e u r o t r a n s m i t t e r and e n d o c r i n e systems that drive n o r m a l physiological a n d behavioral rhythms. F u r t h e r definition of these chemical rhythms will allow insight into the physiological processes u n d e r l y i n g the generation of d i u r n a l r h y t h m s a n d the pathological processes which alter them.
References Bagnara, J.T. and M.E. Hadley, 1973, Chromatophores and Color Change (Prentice-Hall, Englewood Cliffs, N.J.). Bohus, B. and D. De Wied, 1981, Actions of ACTH- and MSH-like peptides on learning, performance and retention, in: Endogenous Peptides and Learning and Memory Processes, eds. J.l. Martinez, Jr. et al. (Academic Press, New York) p. 59. Gaillard, A.W.K., 1981, ACTH analogs und human performance, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 181. Gold, P.E. and R.L. Delanoy, 1981, ACTH modulation of memory storage processing, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 79. Kafka, M.S.A. Wirz-Justice, D. Naber, P.J. Marangos, T.L. O'Donohue and T.A. Wehr, 1982, The effect of lithium on circadian neurotransminer receptor rhythms, Neuropsychobiology 8, 41. Kafka, M.S., A. Wirz-Justice, D. Naber and T.A. Wehr, 1981, Circadian acetylcholine receptor rhythm in rat brain and its modification by imipramine, Neuropharmacology 20, 421. Monnet, F., J.-C. Reubi, A. Eberle and W. Lichtensteiger, 1981, Diurnal variation in the release of alpha-MSH from rat hypothalamus and pituitary, Neuroendocrinology 33, 284. Mourat, B.S., J. Micheau and C. Franc, 1981, A C T H 4 , ~ analog (ORG 2766) and memory processes in mice, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 143. Naber, D., A. Wirz-Justice, M.S. Kafka and T~A. Wehr, 1980, Dopamine receptor binding in rat striatum: Uhradian rhythm and its modification by chronic imipramine, Psychopharmacology 68, 1. O'Donohue, T.L., G.E. Handelmann, T. Chaconas, R.L. Miller and D.M. Jacobowitz, 1981, Evidence that N-acetylation regulates the behavioral activityof a-MSH in the rat and human central nervous system, Peptides 2, 333. O'Donohue, T.L. and D.M. Jacobowitz, 1980, Studies on amelanotropin in the central nervous system, in: Polypeptide Hormones, eds. R.F. Beers et al. (Raven Press, New York) p. 203. O'Donohue, T.L., R.L. Miller and D.M. Jacobowitz, 1979a, Identification, characterization and stereotaxic mapping of intraneuronal a-melanocyte stimulating hormone-like immunoreactive peptides in discrete regions of the rat brain, Brain Res. 176, 101. O'Donohue, T.L., R.L. Miller, R.C. Pendleton and D.M. Jacobowitz, 1979b, A circadian rhythm of immunoreactive o~-melanocytestimulating hormone in discrete regions of the rat brain, Neuroendocrinology 29, 281. O'Donohue, T.L., R.L. Miller, R.C. Pendleton and D.M. Jacobowitz, 1980, Demonstration of an endogenous circadian rhythm of ~-melanocyte stimulating hormone in the rat pineal gland, Brain Res. 186, 145.
Riccio, D.C. and J.T. Concannon, 1981, ACTH and the reminder phenomena, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 117. Snedecor, G.W. and W.G. Cochran, 1967, Statistical Methods, 6th edn. (lowa State Univ. Press, Ames, IA). Swaab, D.F., P.W. Achterberg, G.J. Boer, J. Dogterom and F.W. Van Leeuwen, 1981, The distribution of MSH and ACTH in the rat human brain and its relation to pituitary stores, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 7. Thody, A.J., 1980, The MSH Peptides (Academic Press, New York). Usategui, R., C. Oliver, H. Vaudry, G. Lombard, I. Rozenberg and A.M. Mourre, 1976, lmmunoreactive a-MSH and ACTH levels in rat plasma and pituitary, Endocrinology 98, 189. Wehr, T.A. and F.K. Goodwin, 1981, Biological rhythms and
psychiatry, in: American Handbook of Psychiatry, 2nd edn. Vol. 7, eds. S. Arien et al. (Basic Books, New York) p. 46. Wirz-Justice, A., M.S. Kafka, D. Naber, I.C. Campbell, P.J. Marangos, L. Tamarkin and T.A. Wehr, 1982, Clorgyline delays the phase-position of circadian neurotransmitter rhythms, Brian Res. (in press). Wirz-Justice, A., M.S. Kafka, D. Naber and T.A. Wehr, 1980a, Circadian rhythms in rat brain, a- and/3-adrenergic receptors are modified by chronic imipramine, Life Sci. 27, 341. Wirz-Justice, A., T.A. Wehr, F.K. Goodwin, M.S. Kafka, D. Naber, P.J. Marangos and I.C. Campbell, 1980b, Antidepressant drugs slow circadian rhythms in behavior and brain neurotransmitter receptor rhythms, Psychopharmacol. Bull. 16, 45. Witter, A., W.H. Gispen and D. De Wied, 1981, Mechanisms of action of behaviorally active ACTH-Iike peptides, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 37.
1
Elsevier Biomedical Press
EFFECTS OF CHRONIC LITHIUM, CLORGYLINE, IMIPRAMINE, FLUPHENAZINE AND CONSTANT DARKNESS ON THE a-MELANOTROPIN CONTENT AND CIRCADIAN RHYTHM IN RAT BRAIN THOMAS L. O'DONOHUE l, ANNA WIRZ-JUSTICE 2,, MARIAN S. KAFKA 3, DIETER NABER 3** IAIN C. CAMPBELL 4 and THOMAS A. WEHR 2 J Laboratory of Clinical Science, N1MH, Bldg. 10, Rm. 3D-48, National Institutes of Health, Bethesda, MD 20205, 2 Clinical Psychiatry Branch, NIMH, 3 Biological Psychiatry Branch, N1MH, 4 Clinical Neuropharmacolog; Branch, NIMH, U.S.A.
Received 31 March 1982, revised MS received 27 July 1982, accepted 9 August 1982
T.L. O'DONOHUE, A. WIRZ-JUSTICE, M.S. KAFKA, D. NABER, I.C. CAMPBELL and T.A. WEHR, Effects of chronic lithium, clorgyline, imipramine, jTuphenazine and constant darkness on the c¢-melanotropin content and circadian rhythm in rat brain, European J. Pharmacol. 85 (1982) 1-7.
The effects of constant darkness, chronic lithium, clorgyline, imipramine and fluphenazine treatment on the content and diurnal rhythm of a-MSH in rat forebrain were investigated. The persistence of the a-MSH rhythm in constant darkness demonstrated that the rhythm was circadian in nature. Constant darkness increased the 24 h mean a-MSH concentration in brain while lithium, fluphenazine and imipramine decreased it. In addition, imiparamine and clorgyline delayed the phase of the a-MSH circadian rhythm while lithium advanced it. Circadian rhythm Fluphenazinc
a-MSH rhythm
Lithium, chronic
1. Introduction D i u r n a l variations in the c o n c e n t r a t i o n of am e l a n o c y t e s t i m u l a t i n g h o r m o n e in b r a i n ( O ' D o n o h u e et al., 1979b, 1980; M o n n e t et al., 1981) and b l o o d ( U s a t e g u i et al., 1976; M o n n e t et al., 1981) have been described. W h i l e p i t u i t a r y - d e r i v e d circ u l a t i n g a - M S H regulates a d a p t i v e p i g m e n t a t i o n a l t e r a t i o n s in various species t h r o u g h o u t the a n i m a l Thomas L. O'Donohue is a Pharmacology Research Associate in the National Institute of General Medical Sciences, to whom all correspondence should be addressed. * Anna Wirz-Justice was a Fellow of the Swiss Foundation for Biomedical Research and is currently at Psychiatric Clinic, University of Basel, Basel, Switzerland, CH-4025. ** Dieter Naber was a Fellow of the German Academic Exchange Service and is currently at the Psychiatric Clinic, University of Munich, 7 Nussbaumstrasse, Munich, W. Germany. 4 lain C. Campbell is currently at the Institute of Psychiatry, The Maudsley Hospital, Decrespigny Park, Denmark Hill, London, England SE5 8AF.
Darkness, constant
Clorgyline
lmipramine
k i n g d o m ( B a g n a r a a n d Hadley, 1973), its precise e n d o c r i n e role in m a m m a l s is largely u n k n o w n (cf. T h o d y , 1980). N e u r o n a l a - M S H a p p e a r s to fulfill m a y of the criteria for a n e u r o t r a n s m i t t e r or m o d u l a t o r (cf. O ' D o n o h u e and Jacobowitz, 1980; O ' D o n o h u e et al., 1981; Swaab et al., 1981) that m a y p l a y a role in the processes of attention, arousal, learning a n d m e m o r y (see W i t t e r et al., 1981 ; Bohus a n d D e Wied, 1981 ; G o l d a n d Delanoy, 1981; Riccio a n d C o n c a n n o n , 1981; M o u r a t et al., 1981; G a i l l a r d , 1981). It is possible that the d i u r n a l r h y t h m s of a - M S H in rat b r a i n m a y provide a clue to its function in these behaviors. The p u r p o s e o f this s t u d y was to investigate the p h y s i o l o g y a n d p h a r m a c o l o g y of a - M S H r h y t h m s in brain. To d e t e r m i n e whether the a - M S H d i u r n a l r h y t h m was actually c i r c a d i a n in nature, persisting in the absence of visual cues, the r h y t h m was investigated in rats h o u s e d in chronic darkness. R e c e n t d a t a suggest that altered c i r c a d i a n r h y t h m s was involved in the p a t h o p h y s i o l o g y o f depression
and mania and that the efficacy of antidepressant and antimanic drugs may be due, at least in part, to their ability to alter circadian rhythms in the depressed or manic patient (see Wehr and Goodwin, 1981). Furthermore, antimanic and antidepressant drugs have been shown to alter the circadian rhythms of brain neurotransmitter receptor rhythms (Wirz-Justice et al., 1980a,b; N a b e r et al., 1980; Kafka et al., 1981, 1982; Wirz-Justice, 1982). In this study, we extended those findings and examined the influence of imipramine, a tricylic antidpressant; clorgyline, an MAO inhibitor; fluphenazine, a neuroleptic; and lithium on the rhythm of a - M S H concentrations in brain.
2. Materials and methods
Male Sprague Dawley rats (225-250 g at time of sacrifice) were housed six or seven per cage in a controlled light-dark (LD) cycle with lights on from 7 a.m. to 7 p.m. with food and water freely available. In an experiment performed in October, 1979, the effects of constant darkness and of chronic fluphenazine, lithium and clorgyline on the diurnal rhythm of c~-MSH was investigated. To test the effect of constant darkness, rats were maintained in identical LD conditions until 3 days before killing. After the onset of darkness, they were moved to an isolated room and maintained in constant darkness for 52-72h before sacrifice. Fluphenazine (10 m g / k g ) was administered in a s.c. depot once a week for two weeks before sacrifice. Clorgyline hydrochloride (a gift from May and Baker Pharmaceuticals, Dagenham, U.K.) in distilled deionized water was administered at a constant rate of 4 m g / k g per 14 days using s.c. implanted Alzet minipumps (Model 1701, Alza Corp., Palo Alto, CA). The minipumps were implanted under short-lasting halothane anesthesia during the light phase. Lithium was administered orally for two weeks by mixing it with powdered animal chow (2.26g of lithium carbonate, 1500 g of animal chow and 21 of water). Trunk blood from each lithium-treated rat was collected in heparin-coates tubes, centrifuged and plasma stored at - 2 0 ° C for measurement of plasma
lithium by flame phatometry. In an experiment performed in May-June, 1979, there was an evaluation of the effects of chronic imipramine administration on the circadian rhythm of ~x-MSH in brain. Rats (100 g) were administered imipramine hydrochloride (10 m g / k g per day) (a gift from Ciba-Geigy, Summit, N J) or saline 0.1 ml twice a day for days 1-5 to attain a steady state in the imipramine level and once a day between 15-17 h for days 6-20 to maintain the imipramine level. On day 21, each rat received a final injection 2 h before sacrifice. Although imipramine-treated rats weighed 15% less than controls, they were apparently healthy. Rats were killed by decapitation. There were 7 rats in each group. The forebrain anterior to the cerebellum was divided sagitally and the striata removed. The forebrains minus strata were weighed, frozen on dry ice and stored at - 2 0 ° C until assayed. The brains were homogenized in 20 vol of Tris buffer (pH 7.0, 0.5 M) in the cold. An aliquot was removed, acidified to 1 N acetic acid and boiled for 10 min. The sample was centrifuged for 20 rain at 8000 X g and duplicate aliquots of the supernatant fluid were lyophilzed and assayed for ~x-MSH as described previously (O'Donohue et al., 1979a). In routine measurement of a - M S H in brain, samples are placed directly into 1N acetic acid. In this experiment, we placed the samples in buffer to allow measurement of various receptors (see Kafka et al., 1981, 1982; Naber et al., 1980; Wirz-Justice et al., 1980a,b, 1982). Preliminary experiments demonstrated that a - M S H concentrations were the same in samples that were placed directly in acid and those that were homogenized in buffer and subsequently acidified and boiled. Concentrations of c~-MSH at various times within a treatment group were compared by oneway analysis of variance (ANOVA) and the least-squares difference (LSD) procedure (Snedecor and Cochran, 1967). Differences between two groups were compared by two-way ANOVA. Data was also fitted to a sinecurve by the method of least squares. The phase position (~b) of the rhythm was estimated as the time of the maximum of the best cosive fit (acrophase). Direction of the rhythm shift was determined by assuming that a shift in ~b was less than 12 h. A positive ~b indicated that the
CONTROL r~.~ ~ 24MEAN~_ HOUR ~~~r~'~''r":"
peak was earlier than control; negative ~ - later than control.
3. Results
The effects of drug and light manipulations on the a - M S H rhythm are illustrated in figs. 1-3. The statistical analyses of data are reported in tables 1 and 2. The results of plasma lithium determinations have been described previously (Kafka et al., 1982). Consistent with previously reported results (O'Donohue et al., 1979b; Monnet et al., 1981), the a - M S H concentration in the normal forebrain varied in a diurnal fashion. As shown in fig. l, concentrations of a - M S H are lowest during the end of the light period and the beginning of the dark period in rats sacrificed in October. Fig. 3 shows that a similar rhythm of a - M S H content in forebrain exists in rats sacrificed in June, although the overall a - M S H concentration was actually higher in the rats sacrificed in June when compared to October. A qualitatively similar rhythm has been observed in discrete brain regions of rats sacrificed in July of 1978 (O'Donohue et al., 1979b). Our preliminary results have also shown a similar rhythm in the brains of rats maintained and killed in January, 1980 in Zurich, Switzerland by Drs. A. Borb61y and I. Tobler. The effects of constant darkness on the a - M S H rhythm is shown in fig. 1. The rhythm was qualitatively similar and not significantly different from that of control rats. The mean concentration of a-MSH, in these rats, however, was significantly higher than controls. It is also noteworthy that the standard errors of each time point in the rats housed in chronic darkness were about twice as great as those of control rats. The increased variability of values at each time point may have been due to a lack of precise synchrony between rhythms in different animals in the free-running condition. The effects of chronic lithium are shown in fig. 1. Lithium administration significantly changed both the rhythm and overall concentration of aMSH. The acrophase of the a - M S H rhythm (~p = + 5 . 2 h) occurred 5.2 h earlier than those of control rats. Overall concentrations of a - M S H after
4!5 ± CONSTANT DARKNESS
~ll ]~...
3:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: :::::.......:::'..... : ~
I~i~i~i~iii~i~i~i~i~i~i~ii~i~i~i~i~i~i~ii~i~i~iii~i~i~!~!~i~i~iii~i~i~!~!i~i~i!i!i~i~i~!i~! 2 i~i:iiii~!i:~i~i~ii~i~i~!~}iiiii~i}!~i~}:i)ii~i~!i!~!~}~)~)iii~ii}!i~!~i~)~)i~i~i)!~!iii!~ i:i!!:::i::::::!if:i!.!:iii:i::!i!ii!i!!::il:i::!:!:i:i!.!:::?:::!::i::i!.i :iiiii:!:!i:::: ::::::::::::::::::
i::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: iiiiiiiiiiiiiii!iiiiiiiiiiiiiiiii!iiiiiiiiiiiiiiiiiiiiiiiiiii!!i!!i!!i!iii.. 0
iii!i!:!iii•!iii•i•ii!i!ii!•i••iii•iiii!i•iii•!•!iii!iiii!ii!ii•!•!i!i•i•i•ii!ii!•i•!iii•i•i•ii!iiiiiiii!•ii2i•i•i•!ii IIIII IIII IIII
CHRONICLITHIUM
i
a ~ii~ii::iii~i!iiiiiiiiiiiiii~i!iii!i~iiii~-i~ii~ii!ii! 7e
::::::...H::::.:::.::.
10 14 18 22 2 TIME(hr) 1
+::::::.
6
Fig. 1. R h y t h m o f a - M S H in the f o r e b r a i n s of rats h o u s e d in c o n t r o l c o n d i t i o n s , c o n s t a n t d a r k c o n d i t i o n s or t r e a t e d with l i t h i u m a n d k i l l e d in O c t o b e r . T h e h o r i z o n t a l d a r k g r a y b a r i n d i c a t e s the 24 h m e a n o f c o n t r o l rats. S t a t i s t i c a l d i f f e r e n c e between mean of experimental group and control: **P<0.01, ***P<0.001.
lithium treatment were significantly lower than control. Chronic fluphenazine treatment had qualita-
CONTROL
24 HOUR
.....
IMIRRAMINE CONTROL
24 HOUR MEAN
]
i
iiliiiiiliiiiiiiiiiiiiiiiiliiiiiiiiiiiiiiiiiiiiiiiiiil ii!iii t
.::::
..................
0~
.:< . . . . . . .
k:::+:':':':::::':':':'":'iii'i'i'i"'
/11/ ' ~ \ ' "
iI
**
CHRONIC CLORGYLINE
4
i=
I
I
14
18
22
2
6
TIME (hr) ~N.S.
-r
~2
CHRONIC FLUPHENAZlNE
4
iii! iiiiii iii!~iii~i~i!ill
I
!iiii!i!ii!)iii?iiii!i!?!iiiii!?:i!iiiii!i!!!i!i!ii!!ii
21 ~ ~-
1 _
0
t
10
_
.T_
_
±
10
i!iiiiiii£!!i!ii;iiiil;iiii~s:....~:~ii!i!i::iii i!ii~i!ii!i~i~i~: A
i
::::::::::::::5:: . . . . . . . . . . . . . . . . .
_
1
14
:
::::::::!..::i:ii!ii:i:i:i:iii:i:i:i:i:i:i:i:i:i:i: _
.:::.:.;::.:.:.:-;.:-;.:.:.:.:.:,:.:.:,:::::::.:.:,:.:-
18 22 TIME (hr)
2
6
Fig. 2. Rhythm of a-MSH in the forebrains of control rats and rats treated with clorgyline or fluphenazine and killed during October. The horizontal dark gray bar indicates the 24 h mean of control rats. ***Overall mean is significantly different from control - 24 h mean - - P<0.001.
Fig. 3. Rhythm of ~x-MSH in the forebrains of control rats (solid line) and imipramine-treated rats killed during June. The horizontal dark gray bar indicates the 24 h mean of control rats. ***Overall mean is significantly different from control 24 h mean - P<0.00001.
tively and quantitatively similar effects as lithium on the a-MSH rhythm. Compared to the rhythm in control rats, the amplitude of the a-MSH rhythms of lithium- and fluphenazine-treated rats were both decreased (see figs.), the acrophases were slightly advanced ( ~ - - 2 . 6 h for fluphenazine) and overall concentrations were significantly lowered. Chronic clorgyline treatment had no effect in overall concentrations of a-MSH but had a very dramatic effect on the phase position of the rhythm. In control rats, the nadir was at 18 and 22 h while in clorgyline-treated rats, the peak was at these times. The fitted peak in a-MSH concentration was delayed by 9.3 h. In the case of imipramine treatment, there was a significant change in both overall a-MSH content and the rhythm (fig. 3). Concentrations of c~-MSH in imipramine-treated rats were much lower than controls. The shift in the rhythm was quite similar to that caused by clorgyline, as the fitted peak was also delayed by about 9 h.
TABLE 1 Statistical analyses of circadian rhythms of ot-MSH: One way ANOVA. F
October Controls Lithium Clorgyline Constant darkness Fluphenazine June Controls Imipramine
Least square differences
P
P<0.01**
P<0.01*
2, 6 > 18, 22 10, 14>22 18, 6 < 2 all time p t s > 2 18, 2 2 > 6
151>18
5.59
< 0.0008
4.76 17.21
< 0.0022 < 0.00001
1.77 3.45
<0.14 NS < 0.013
18, 2 2 < 2
10>18 10, 14< 2
26.84 4.53
< 0.00001 <0.003
18, 22
2 2 > 10, 2
10, 14, 18, 6 < 2 10, 14<18, 22 10> 6
TABLE 2 Statistical analyses of circadian rhythms of a-MSH: Two way ANOVA.
Lithium Clorgyline Constant darkness Flupenazine Imipramine
Main effects
Treatment
F
F
8.82 1.32 17.12 8.41 43.85
P
<0.0001 <0.24 ys < 0.0001 <0.0001 <0.0001
15.04 0.15 61.71 24.1 152.9
Time P
<0.001 <0.70 Ns < 0.0001 <0.0001 <0.00001
4. Discussion The results of these experiments confirm the presence of a rhythm in the concentration of aM S H in brain (O'Donohue et al., 1979b; Monnet et al., 1981) and demonstrate that the rhythm is circadian in nature as it persists in the absence of light cues. The overall increase in concentrations of a - M S H in brains of rats housed in constant darkness is similar to the reported increased concentration of a - M S H in the pineal glands of chronically dark-housed rats (O'Donohue et al., 1980). It is unknown, however, whether an increase in a - M S H concentration in the brain corre-
F
7.58 1.61 8.23 5.29 19.05
2 way interaction P
<0.0001 <0.17 ys <0.0001 <0.0001 <0.0001
F
P
3.16 17.73 1.26 1.26 25.87
<0.013 <0.0001 <0.29 ys <0.29 Ns <0.0001
sponds to increased or decreased a - M S H release. The effects of pharmacological manipulations on the a - M S H concentrations and circadian rhythms can be interpreted either as secondary effects on the a - M S H neuronal system via interactions with monoaminergic systems or as a primary effect on the frequency of a putative driving circadian oscillator reflected in changes in the phase-position of many neuronal systems of the brain including the a-melanotropinergic neuronal system. The pharmacologically induced changes in the circadian rhythm of a - M S H by clorgyline, imipramine and lithium can be interpreted in light of
the effects of these drugs on circadian frequency. C o n s i s t e n t with this hypothesis are data d e m o n strating similar temporal r h y t h m alterations not only of a - M S H but also various n e u r o t r a n s m i t t e r receptors by these drugs. Chronic clorgyline or i m i p r a m i n e t r e a t m e n t delayed the peak in the rhythms of c~-MSH c o n c e n t r a t i o n a n d a - a d r e n ergic, /~-adrenergic, cholinergic muscarinic, dopaminergic, opiate and benzodiazepine receptor n u m b e r s by 4 - 1 2 h (Wirz-Justice et al., 1980a,b, 1982: Kafka et al., 1981; N a b e r et al., 1980). The fact that the delay of 8-12 h in a - M S H concentrations and a-adrenergic receptors was quite similar after both a n t i d e p r e s s a n t drugs may indicate a functional relationship between these systems. The similar effects of clorgyline and i m i p r a m i n e on neurochemical rhythms are reflected in similar effects of these drugs on behavioral rhythms both delay circadian r h y t h m s in activity of hamsters (Wirz-Justice et al., 1980b). The ability of lithium to reduce the a m p l i t u d e of neurochemical rhythms has been suggested to be related to its ability to d a m p cycling between m a n i a and depression ( K a f k a et al., 1982). Lithium, f l u p h e n a z i n e and i m i p r a m i n e all have in c o m m o n the effect of decreasing the overall c o n c e n t r a t i o n of a - M S H in brain. These data may indicate an interaction between m o n o a m i n e r g i c a n d a - m e l a n o t r o p i n e r g i c systems. Such an interaction would be expected from the surprising a n a t o m i c a l overlap of m o n o a m i n e r g i c a n d am e l a n o t r o p i n e r g i c systems ( O ' D o n o h u e et al., 1979a). Results of n u m e r o u s other studies support i n t e r a c t i o n between c~-MSH a n d m o n o a m i n e r g i c systems (for references see G o l d a n d Delanoy, 1981). G o l d a n d D e l a n o y (1981) hypothesize that m o n o a m i n e s a n d M S H peptides act in c o n j u n c t i o n to m o d u l a t e m e m o r y processes as well as other behavioral responses. It is u n d o u b t e d l y the interactions between r h y t h m s in m o n o a m i n e r g i c , ~m e l a n o t r o p i n e r g i c a n d other n e u r o t r a n s m i t t e r and e n d o c r i n e systems that drive n o r m a l physiological a n d behavioral rhythms. F u r t h e r definition of these chemical rhythms will allow insight into the physiological processes u n d e r l y i n g the generation of d i u r n a l r h y t h m s a n d the pathological processes which alter them.
References Bagnara, J.T. and M.E. Hadley, 1973, Chromatophores and Color Change (Prentice-Hall, Englewood Cliffs, N.J.). Bohus, B. and D. De Wied, 1981, Actions of ACTH- and MSH-like peptides on learning, performance and retention, in: Endogenous Peptides and Learning and Memory Processes, eds. J.l. Martinez, Jr. et al. (Academic Press, New York) p. 59. Gaillard, A.W.K., 1981, ACTH analogs und human performance, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 181. Gold, P.E. and R.L. Delanoy, 1981, ACTH modulation of memory storage processing, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 79. Kafka, M.S.A. Wirz-Justice, D. Naber, P.J. Marangos, T.L. O'Donohue and T.A. Wehr, 1982, The effect of lithium on circadian neurotransminer receptor rhythms, Neuropsychobiology 8, 41. Kafka, M.S., A. Wirz-Justice, D. Naber and T.A. Wehr, 1981, Circadian acetylcholine receptor rhythm in rat brain and its modification by imipramine, Neuropharmacology 20, 421. Monnet, F., J.-C. Reubi, A. Eberle and W. Lichtensteiger, 1981, Diurnal variation in the release of alpha-MSH from rat hypothalamus and pituitary, Neuroendocrinology 33, 284. Mourat, B.S., J. Micheau and C. Franc, 1981, A C T H 4 , ~ analog (ORG 2766) and memory processes in mice, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 143. Naber, D., A. Wirz-Justice, M.S. Kafka and T~A. Wehr, 1980, Dopamine receptor binding in rat striatum: Uhradian rhythm and its modification by chronic imipramine, Psychopharmacology 68, 1. O'Donohue, T.L., G.E. Handelmann, T. Chaconas, R.L. Miller and D.M. Jacobowitz, 1981, Evidence that N-acetylation regulates the behavioral activityof a-MSH in the rat and human central nervous system, Peptides 2, 333. O'Donohue, T.L. and D.M. Jacobowitz, 1980, Studies on amelanotropin in the central nervous system, in: Polypeptide Hormones, eds. R.F. Beers et al. (Raven Press, New York) p. 203. O'Donohue, T.L., R.L. Miller and D.M. Jacobowitz, 1979a, Identification, characterization and stereotaxic mapping of intraneuronal a-melanocyte stimulating hormone-like immunoreactive peptides in discrete regions of the rat brain, Brain Res. 176, 101. O'Donohue, T.L., R.L. Miller, R.C. Pendleton and D.M. Jacobowitz, 1979b, A circadian rhythm of immunoreactive o~-melanocytestimulating hormone in discrete regions of the rat brain, Neuroendocrinology 29, 281. O'Donohue, T.L., R.L. Miller, R.C. Pendleton and D.M. Jacobowitz, 1980, Demonstration of an endogenous circadian rhythm of ~-melanocyte stimulating hormone in the rat pineal gland, Brain Res. 186, 145.
Riccio, D.C. and J.T. Concannon, 1981, ACTH and the reminder phenomena, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 117. Snedecor, G.W. and W.G. Cochran, 1967, Statistical Methods, 6th edn. (lowa State Univ. Press, Ames, IA). Swaab, D.F., P.W. Achterberg, G.J. Boer, J. Dogterom and F.W. Van Leeuwen, 1981, The distribution of MSH and ACTH in the rat human brain and its relation to pituitary stores, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 7. Thody, A.J., 1980, The MSH Peptides (Academic Press, New York). Usategui, R., C. Oliver, H. Vaudry, G. Lombard, I. Rozenberg and A.M. Mourre, 1976, lmmunoreactive a-MSH and ACTH levels in rat plasma and pituitary, Endocrinology 98, 189. Wehr, T.A. and F.K. Goodwin, 1981, Biological rhythms and
psychiatry, in: American Handbook of Psychiatry, 2nd edn. Vol. 7, eds. S. Arien et al. (Basic Books, New York) p. 46. Wirz-Justice, A., M.S. Kafka, D. Naber, I.C. Campbell, P.J. Marangos, L. Tamarkin and T.A. Wehr, 1982, Clorgyline delays the phase-position of circadian neurotransmitter rhythms, Brian Res. (in press). Wirz-Justice, A., M.S. Kafka, D. Naber and T.A. Wehr, 1980a, Circadian rhythms in rat brain, a- and/3-adrenergic receptors are modified by chronic imipramine, Life Sci. 27, 341. Wirz-Justice, A., T.A. Wehr, F.K. Goodwin, M.S. Kafka, D. Naber, P.J. Marangos and I.C. Campbell, 1980b, Antidepressant drugs slow circadian rhythms in behavior and brain neurotransmitter receptor rhythms, Psychopharmacol. Bull. 16, 45. Witter, A., W.H. Gispen and D. De Wied, 1981, Mechanisms of action of behaviorally active ACTH-Iike peptides, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr. et al. (Academic Press, New York) p. 37.