Pharmacological probes of the mammalian circadian clock: use of the phase response curve approach

TIPS - June 1987 [Vol. 81

harmacological probes of the mammalian circadian clock: use of the phase response curve approach Fred W. Turek A useful approach to examine whether or not a specific pharmacological agent can influence a c&a&an clock is to determine if the acute administration of the hg can induce a permanent phase shift of rhythm driven by the clock under constant environmental conditions. As Fred Turek describes in this review. recent studies utilixing this approach indicate a role for acetytcholine, NPY, GABA and protein synthesis in the entrainment and/or generation of circadian rhythms in mammals. In addition to providing insight into the neurochemical and molecular events underlying circadian rhythmicity, pharmacological probes of the circadian system might prove useful in identifying drugs which can be utilized in the treatment of various physical and mental illnesses that have been associated with a disorder in arcadian time-keeping. One of the most obvious charac-

teristics of life is the daily change in behavior of plants and animals that is correlated with the daily change in the physical environment due to the rotation of the earth on its axis. In addition, many biochemical and physiological events within the organism are observed to fluctuate dramatically on a regular basis throughout the 24hour day. A remarkable feature of these daily rhythms is that they are not simply a response to the 24-hour changes in the physical environment imposed by the principles of celestial mechanics, but instead arise from an internal time-keeping system. The ‘biological clock’ is synchronized to the physical environment by external entraining agents, primarily the light-dark cycle. Evidence for the endogenous nature of this clock includes the observation that most daily rhythms persist under constant environmental conditions in a laboratory setting with the period Fred Turek is Professor in the Department of Neumbiology and Physiology, Northwestern University, Evanston, lllinois 60201, USA, and institute of Interdisciplinary Research and Luboratoy of Neuropathology and Neuropeptide Research, Free University of Brussels, B-1070 Brussels, Belgium.

of the rhythm being close to, but rarely exactly equal to, 24 h (i.e. circadian). Thus, the rhythms become desynchronized from all 24hour signals generated in the physical environment (For reviews see Refs l-4). In mammals, a circadian clock regulating many circadian rhythms has been located within the suprachiasmatic nucleus (SCN), a bilaterally paired structure in the anterior hyEothalamus1’3 receiving both direct and indirect afferents from the retina that relay synchronizing light-dark information to the SCN. Although there is some experimental evidence that neural circadian clocks may residue outside the SCN region, no other structure within the mammalian brain has been identified as a circadian oscillator. Thus, a great deal of attention is focused on understanding the neurobiology of the SCN. A variety of techniques have been, and are presently, used to determine the anatomy, neurophysiology and neurochemistry of the SCN in anticipation that such information will eventually be used to answer the questions: (1) How do sensory signals, relaying information about the physical environment, entrain circadian oscillators? (2) How can neural

tissue generate circadian signals? ar,d (3) How are neural oscillators coupled to the circadian rhythms they drive? The importance of circadian rhythmicity for pharmacology has been recognized for many years since the effects of many drugs can vary dramatically as a function of the time of day the drug is delivered4. However, only relatively recently have pharmacological approaches become irnportant for examining the physiological mechanisms underlying circadian rhythmicity. While a decade ago the statement, The circadian clock is insensitive to all but a few chemical agents’, could be made safely, today this is no longer the case. A number of central acting drugs and endogenous substances have been indentified which can infhrence the mammalian circadian system. This paper reviews those studies in mammals which have used drugs to ‘phase shift’ a central circadian pacemaker (i.e. induce a permanent advance or delay in the output rhythm of the clock) regulating an easily measurable circadian rhythm (see Box). The major goal of experiments using this approach is to elucidate the neurochemical and celhtlar events that are associated with the entrainment, generation and expression of circadian rhyehms in mammals. Experimental approaches Two general strategies have been employed to determine if a specific drug can alter a central circadian clock in mammals. Because the results of numerous experiments indicate that a circadian pacemaker resides within the SCN, drugs have been applied to SCN tissue, either in an in-vitro or an in-vivo preparation, to determine if such treatment can alter neural firing rates and/or neurochemical activ$@. Such experiments usually involve the monitoring of SCN activity over short time periods (e.g. l-3 h). While this approach a!lows one to determine if individual or groups of neurons respond to specific pharmacological agents, little information is provided as to whether these agents actually induce any change in the circadian clock itself since there is currently no information about which neurons in the SCN are actually part of the clock

TlPS -June 1987 fVol.81 machinery. A second approach involves monitoring, for many circadian days, a measurable rhythm before and after drug treatment to determine if the drug can alter an aspect of the rhythm which is known to be a property of the clock driving that rhythm (see below). A particularly promising approach is the study of circadian rhythms in SCN tissue itself, such as is presently being done using hypothalamic explants that are maintained in culture for many days while the rhythm of vasopressin release is monitored6. Theoretically, any rhythm coupled to a circadian clock can be utilized as a marker of the state of the clock. However, some rhythms appear to be more regular than others, and for technical reasons, some rhythms are more easilv measured. For example, circadian rhythms in various brain neurotransmitter receptor levels have been reported and there is some indication that these rhythms can be altered by drug treatment7~8. However, in order to evaluate the state of such a receptor rhythm, the animal must be sacrificed and thus the rhythm cannot be followed in a single animal but instead is only represented on a population level. Rhythms that can be monitored continuously in a single animal through time include a number of behavioral (e.g. feeding, drinking, locomotor activity) and endocrine rhythms. The behavioral rhythms have the obvious advantage in that they can be monitored frequently (e.g. at one minute intervals) for many days by automatic recording equipment with little disturbance to the animal. However, measurable circadian rhythms only represent the ‘hands’ of the clock. In attempting to use the rhythmic output of the clock to determine if a particular drug treatment has altered the clock itself, it is necessary to monitor a property of the rhythm which is also a property of the clock. Only two properties of a rhythm are reliable indicators of the state of the clock: the period length and the steady-state phase of the oscillator’. In order to determine if a particular drug can induce a change in period, it must be delivered chronically over a relatively long period of time. Such studies must take into account the fact that the response

213

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Fig. i. Activity rewtin~ tir,lii *hi iExiiSi~i.5 &I- a i&i2 diiji p$ti h,%,z &7,1 s,&: 25 mg triamkm (i.p.) at vadous circadian times. Each line repmsents a single 24-h period and successive days are plotted from top to bottom in each record. The tir animals depicted on the left (HI) were exposed to constant darkness while the four animals shown on tile right (e-h) were exposed to constant light throughout the study. T. day of the trtazolam injection; l , exact time of injection. Using the daily onsets of activity for five days preceding the injection as phase reference points for the pre-injection rhythm, a time of activity onset on the succeeding day has been graphically projected. In addition, the dai/y onsets of activity for S7 days after the injection (once a newsteadystata was achieved) were usedas phasereference points for thepost-injectionrhythm. The subtraction of the estimatsdphase of the rhythm after the treatment from the projected phase of the rhythm before treatment, y&Ids the magnitude and direction of the phasezshift. Regardless of the lighting conditions, an injection of tdazolam 3-6 h before the onset of activity induces a phase advance in the activity rhythm (a, b,e,Q while injkctionof triazok?m fH3 h atter the onset of activityinduces phase delays (c,d,g, h). As shown hem and in fig. 2, the magnitude and amplitude of the phase shit?induced by triazo/am is dependent on the circadian time at which the drug is administered. (Reproduced with permission from Ret. 19.)

to the drug may change during continuous administration. Furthermore, it is not possible to determine if there is a circadian rhythm in responsiveness to the drug if it is delivered in a chronic fashion. However, there is clear evidence that the chronic administration of specific drugs or hormones can alter the period of ‘free-running’ circadian rhythms under constant environmental conditionsa*ro. The studies described below involves attempts to alter the steady-state phase of the oscillator by determining if the acute administration of a drug can induce a permanent phase-shift in the circadian clock of mammals. Phase response curves For the last 25 years, one of the most widely used methods to examine how the light-dark cycle influences the circadian system has been to expose animals to a brief pulse of light (e.g. l-60 minutes in duration) during

maintenance in constant darkness (DD). The effects on a phase reference point of a circadian rhythm (e.g. onset of locomotor activity or peak of d&inking rhythm, see Fig. 1) by light p=Jses is then determined. This approach has demonstrated that light pulses can induce phase advances, phase delays or may have no effect on circadian rhythms1,39. The direction and magnitude of the shifts are strongly dependent on the circadian time at which the light pulse occurs (Fig. 2). A plot of the shift induced by an phase environmental perturbation as a function of the circadian time at which the perturbation is given is called a ‘Phase Response Curve’ (PRC). The PRC approach has also been used to determine if various drugs or putative neurotransmitters can alter the hase of the P‘, and has circadian clock’,3,1** yielded new information regarding the neurotransmitters and cellular events that may be involved

TIPS - Jtrne 1987 IVol. 81

2

in the entrainment and generation of czircadian rhythms in mammals. R-t information obtained from pharmacologiical PRCs is reviewed below.

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SCN’P~~,it might be expected that administration of these neurotransmitters, or drugs which act as agonists and/or antagonists to them, could alter clock function and thus be able to induce phase shifts in the circadian clock. However, very few detailed studies of the type required to test the phase shifting abilities of a particular drug (see Box} have actually been performed. Evidence obtained from the pharmacological PRC approach indicates that an alteration in acetylcholine, neuropeptide Y (NPY) or GABA activity within the CNS can induce phase shifts in a central circadian pacemaker of mammals. Using the PRC approach, Z&z’4 was the first to provide evidence that a specific neurotransmitter, acetylcholine, may be involved in relaying light information to or within the circadian clock of a mamma). There are now a number of reports3 indicating that a single intraventriciiar injection of carbachol, a cholinergic agonist, can mimic the phase shifting effects of brief light pulses on various rhythms in rats, mice and hamsters (Fig. 2). Furthermore, the phase shifting effects of a light pulse on the circadian rhythm of Incomotor activity in hamsters can be blocked by an injection of the cholinergic antagoI?iPt, necamylanine, ten minutes prior to the presentation of the light pulse’5. These results, coupled with the observations that acetylcholine can alter the firing rate of SCN neurons and that light can alter acetylcholine concentration within the SCN3*16, suggest that acetylcholine may be involved in relaying light information from the retina to the SCN and/or in the integration of light information within the SSN itself. A second neurotransmitter, NPY, may also play a role in the transfer of light information to the biological clock. Administration of NPY directly into the SCN region at various circadian times to hamsters free running ir. constant light phase shifts the rhythm of wheelrunning behavior in a manner similar to the presentation of dark pulses”. There is anatomical support for such a roti ior NPY since NPY immunozactivity in the SCN originates in part from cells in the geniculate area which receives direct retinal afferentsl*. Oneofthemostabundantneuro-

TIPS -June

1987 lVoL8J

In the design of any phannamlogical experiment, it is r;n.a m?cessL,-. & t& jE& -9-t --O .4U __-ge. r, ph5E!W mlogicai experiments aimed at studying the &cadian clock, a number of factors other than dosage need do bs mnsidered in order to accurately determine if a paftiCllkEdWgCanaltWtheduCk.FailupetOt&?ilUO acmunt these factors can lead to erroneous&mcl&iuns. Below is a brief description of some cf the issues 6hid1 are critical for both the design and the integetation of experiments which use pham~~+$cal prdbes to investigate the nature of the circ&an dock merhanian Pnxently, it Is not possib!e to assay tha state ob the circadiando&directlyinanyqerimentalmodeL Therefore, the test of whether or not a pa&c&r dq can alter the dock re.qui=~ t%at meazurable rhythms representing the ‘hands’ cf the dock bemonitured. Not all features of a given overt rhythm represent a prop&y of the ‘master’ cirrztdian dock undedying the genera&n of that rhythm. For axample, while the amplitude of a rh~mmaybeinnuencedbythedodritself,itislbfoa property of a rhythm which is highly sensitive to f24c&s ‘downstmam’ fnnn the dock, i.e. between the dock itself and the final events associated with the ClrpRessiotr of the overt rhythm. Similarly the abolishment of a particular rhythm, i.e. the induction of arrhythxnitity, might also be due to effects on the output pathway a& not qm the dock mechanism itself. There are only two pn+rties of circadian rhythms which azc thought to be pa~~~~etms n=omsentative of the status of theckxk itself: the steady s&e phase ofthe oscillation and its period leqth. In examining whether ornotadnagcanindueeachangeintheperiodofthe rhythm, the drug may be given on either a chronic or acute basis, and the rhythm must be monitctxed under mnditions in which the dock is not entrained by soma environmentalagent.Theacuteadminishationofa drugcanalsobeusedtodetermineifthedrughas induced a ‘phase-shift’ in the undedying dock The inducedphas? shift must still be yt a 8ubsequent days without drug Watment before it can be mnduded that a drug has altered the clock itself. A temp%uy phaseshiftmaybedue~oan~nofthe,diugonan output pathway between the dock and the Enal expression of the rhythm. As eIaborated below, attempts to determine if a paxticular drug can induce a permanent phase shii in the circadian dock must take into acmunt the timing of drug adiidGstatin, the lighting conditions, and the sampling lnaerval for monitoring the rhythm. Timingofdrugadminiska6on TheeffectsotaIidnqswhichhavebeenfoundto phase shift 1 central cimadian pacemaker in mammals are dependent on the time of adxninistrath~ of the drug. Thus, the same drug can phase adWa!Me,phase de-lay or have no ef&ct on a particuiar Circadian l%z depending on the circadian time of e negative result from a study which only invdves~dkg adminisftation at one ckadian time pmvides little information on theability of that drug to alter dock function since the drug may be administered at a time

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TZPS-June 1987 1VoZ.4

216 transmitters within the SCfi of ndcnts is CA8A13. Recent pharmacological experiments involving a short-acting benzodiazepine, triazolam, which is thought to act by potentiating the action of GABA, suggest a functional ro!e for GABA in the organization of the mammalian circadian systemlg. A single intraperitoneal injection of triazolam induces a phase shift in the onset of the rhythm of wheelrunning behavior (Fig. 1) that is dependent on the circadian time of administration in hamsters exposed to eitherDD or constant light (LL).AplotofthePRCstotriazolam in either DD or LL reveals that the general shapes of these curves are sin&r but dramatically different from the PRCs to light pulses or injections of carbacholj (Fig. 2), demonstrating that triazolam is not mimickingtheeffectsoflightonthe circadian clock. The effects of triazolam appear to be mediated through the benzodiazepine/GABA receptor complex since a single injection of the specific benzodiazepine antagonist, Ro 15-1788 (fhunazepil), can completely block the phase-shifting effects of triazolam on the circadian clock of hamsters (Van Reeth and Turek, unpublished data). Other recent experiments also suggest a possible role for GABAergic neurons in the circadian clock system of mammals since treatment with muscimol, a GABA agonist, has similar effects on the circadian rhythm of activity in

hamsters as does triaz~larn~. GARAergic nerrrons may also be involved in mediating the effects of light on the circadian clock since the systemic administration of bicuculline, a selective antagonist of GABA, can block the phase delaying, but not the phase advancing effects of light pulses on the circadian rhythm of locomotor activity of hamster$*, while the benzodiazepine, diazeparn, does not block light-induced phase delays at doses that si ificantly block phase advances2??. Further studies are necessary before a more precise role for GABA in the entrainment and/or generation of circadian rhythms can be determined.

Cellularevents Pharmacological manipulations of circadian rhythms have been utilized extensively to identify key cellular and biochemical steps in the circadian organization of such diverse preparations as the eye of the Aplysia and the avian pineal gland, as well as in unicellular organisms,1,12,23.z4. These studies indicate that key elements in the regulation of circadian rhythmicity include membrane potentials, cyclic nucleotides and protein synthesis. Recent pharmacological studies involving protein synthesis inhibitors also indicate a role for protein synthesis in the circadian organization of mammals. A single systemic injection of

either of two inhibitors of protein synthesis an 8OS ribosomes, anisomycin or cicloheximide, induce phase-dependent phase shifts in the circadian rhythm of locomotor activity in hamsters free-running in either DD or LL (Fig. 2, Ref. 25). Similar phase shifts are observed when anisomycin is microinjected directly into the SCN region and the magnitude of the phase shift is proportional to the dose and the distance of the guide cannula from the center of the SCN (Inouye, Takahashi and Turek, unpublished data). importantly, drugs which mimic some of the sideeffects of protein synthesis inhibitors do not have the same effects on the circadian activity rhythm=. It is perhaps noteworthy that the PRC to protein synthesis inhibitors in the hamster is similar to the PRC to triazolam (Fig. 2), although the functional significance of these similarities, if any, is not known. These results suggest that a specific protein@ is produced at one time of the day which influences the timing of the cimadian activity rhythm. A useful approach to the study of the molecular basis for the generation of circadian rhythms may be to isolate and characterize proteins in SCN neurons which are produced at specific times of the day. Of particular interest is the observation that the PRCs for protein synthesis inhibitors in Aplysia, N~UYOS~OYU and hamsters share a number of common features2*25 indicating that the biochemical mechanisms generating circadian oscillations in mammals may share common features with those found in very distantly related phylogenetic groups. Implications for medicine There is now a large body of evidence indicating that at least some forms of depression and insomnia may involve a disruption of normal circadian rhythmicity4,73,26, including an abnormal phase relationship of various circadian rhythms to the external environment. It might be possible to treat patients with abnormally phased circadian rhythms so that normal rhythmic patterns are restored by appropriately timed administrationof drugs which can induce phase shifts in the human circadian system. Of

TlPS -June 1987 iVol.81 particular interest is the observation that triazolam can induce phase shifts in the mammalian circadian clock since the benzodiazepines are at present the drug treatment of choice for the management of anxiety and stress-related conditions as well as insomnia19. The recent finding that treatment with valproate, a drug which appears to elevate GABA levels, can induce changes in the period of the temperature and drinking rhythms in monkeysz7, also indicates that drugs which can alter the activityofGABAergicneuronsmay be useful in the treatment of patients with abnormal circadian rhythmic profiles. Similarly, drugs which can induce phase shifts in the human circadian system might aid in shortening the time of reentrainment of various circad.ian rhythms following a shift in the sleep-wake cycle, such as occurs following rapid transit across time zones (i.e. ‘jet lag’), or in shift workers. However, as is clear from Fig. 2, any attempt to design an appropriate drug regime for phase shifting the human circadian clock must take into account the time of administration since any drug which can induce phase shifts may have no effect, a delaying or an advancing effect on the clock depending on the time of drug delivery (see Box). Q

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Obtaining PRCs to pharmacological probes is an important step in elucidating the neurochemical and cellular events associated with the entrainment and generation of circadian rhythms. In addition to indicating a role for specific neurotransmitters and biochemicalevents in the functioning of the circadian clock, PRCs to drugs also provide information on a possible ‘window of time’ during the circadian cycle when a particular substance is playing an active role. lhis information can then be utilized to elucidate the endogenous changes in known biological clocks, such as the mammalian SCN, that are part of the clock mechanism.

Acknowledgements Unpublished work presented in this paper and the preparation of this review were supported by NIH Grant HD-09885, the Upjohn

217 Company and a Senior International Fogarty Award (TW 01099). I am grateful to Dr Oliver Van Reeth and MS Susan LoseeOlson for their collaboration on a number of projects reviewed in this paper. References 1 Takahashi, J. S. and Zatz, M. (1982) science 217,1104-1111 2 Moore, R. Y. and Card, J. P. (1985) Ann. N.Y. Acad. Sci. 453,123-133 3 Turek, F. W. (1985) Annu. Rev. Physiol. 47,49-64 4 Moore-Ede, M. C., S&man, F. M. and Fuller, C. A. (1982) The clocks that time us, pp. l-448, Harvard University Press 5 Liou, S. Y., Shibata, S., Shiratsuchi, A. and Ueki, A. (1986) Neurosci. L&t. 67, 339-343 6 Earnest, D. J. and Sladek, C. D. (1986) Brain Res. 382, 129-133 7 Kafka, M. S., Win-Justice, A., Naber, D., Moore, R. Y. and Benedito, M. A. (1983) Fed. Proc. 42,2796-2801 8 Wirz-Justice, A. Prog. in Neurobiol. (in press) 9 Pittendrieh. C. S. 11981) in Handbook of Behaviors Neurobiology Biologicif Rhythms (Aschoff, J., ed.), Vol. 4, pp. 5780, Plenum Press 10 Zucker, I. (1979) Biological Rhythms and their Central Mechanisms (Suda, M., Hagaishi, 0. and Nakagawa, H.. eds), pp. 369-381, Elsevier Biomedical Press

11 Ehret, C. F., Potter, V. R. and Dobra. K. W. (1975) Science 188,1212-1215 12 Eskin, A., Takahashi, J. S., Zatz, M. and Block, G. D. (1984) J. Neuroscience 4, 2466-2471 13 van den Po!, A. H. azd Tsujimoi9, K. L. (1985) Neuroscience 15. 1849-86 14 Zatz,.M. (1979) Fed. Pioc. 38,2596-2681 15 Keefe, D. L., Earnest, D. J., Nelson, D., Takahashi, J. S. and Turek, F. w. (i!386j Brain Res. 403, 308-312 16 Murakami, N., Takahashi, K. and Kawashima, K. (1984) Bruin Res. 311, 358-360 17 Albers, H. E. and Ferris, C. F. (1984) Neurosci. Mt. 58.163-168 18 Harrington, M. E., Nance, D. M. and Rusak, B. (1985) Brain Res. Bull. 15,s 472 19 Turek, F. W. and Losee-Olson, S. (1986) Nature 321,167-168 20 Smith, R. D. and Turek, F. W. (1986) Neurosci. Abstr. l2,209 21 Ralph, M. R. and Menaker, M. (1985) Brain Res. 325,362-365 22 Ralph, M. R. and Menaker, M. (1986) Brain Res. 372,405-408 23 Johnson, C. H. and Hastings, J. W. (1986) Am. Sci. 74,29-36 24 Jacklet, J. W. (1984) Int. Rev. Cytology 89, 251-294 25 Takahashi, J. S. and Turek, F. W. (1987) Brain Res. 405.199-203 26 Van Cauter, E. and Turek, F. W. (1986) Perspectives Biol. Med. 29.510-519 27 Borsook, D., Richardson, G. S., MooreEde, M. C. and Brennan, M. J. W. (1986) Med. Hypotheses 19,185-198

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