Cbmp. Biochem. Physiol. Vol. 90.4, NO. 3, pp. 43W4Q 1988 Ok-%29/88 s3.00 + 0.00 Printed in Great Britain @ 1988 Pergamon Press plc

ACTION OF DEUTERIUM OXIDE UPON THE ERG CIRCADIAN RHYTHM IN CRAYFISH,

PROCAMBARUS BOUVIERI

BEATRIZ FUJZNTEZS PARDO and ENRIQUE MORENO SAENZ Departamento de Fisiologia, Division de Posgrado e Investigacibn, Facultad de Medicina, UNAM,

Apartado Postal 70-250, 04510 Mexico, D.F. Mexico

(Received 10 Nooember 1987)

Abstract-l. We studied the action of deuterium oxide upon the rhythm of the ERG’s amplitude recorded in intact animals and in isolated eyestalks.

2. In the isolated eyestalks the circadian rhythm of the ERG prevails. However, this rhythm differs in some features of the intact animal. The most prominent difference consists of the appearance of high frequency (ultradian) cycles superimposed on the circadian oscillation.

3. The deuterium oxide effect upon the circadian rhythm of the ERG, both in the intact animal, as well as in the isolated eyestalk, consists of (a) a decrease of the ERG’s amplitude; (b) decrease in the amplitude of the oscillation; (c) alteration of the a : p ratio; and (d) lengthening of the period. The lengthening of the ultradian and circadian periods is proportional to the deuterium oxide doses applied.

4. Deuterium oxide has a greater effect upon the isolated eyestalk than upon the intact animal, 5. Deuterium oxide does not affect the number of high frequency cycles in the isolated eyestalk; it only

increases their duration and probably enhances the cohesion of the corresponding oscillators. 6. We suggest that the lengthening of the circadian period produced by deuterium oxide is due to its

action upon the high frequency oscillators. This action, in turn, could be due to the dissolving characteristics or higher molecular weight of the deuterium oxide.

INTRODUCTION

In a great deal of papers published in the last 20 years, the circadian rhythms have been manipulated to obtain (and sometimes to predict) modifications in the period, amplitude and phase of the rhythm in response to external signals, such as light or tem- perature changes, or by variations in the concen- tration of a component of the bathing medium, or by application of chemical substances. Some treatments interfering with the cellular metabolism, and thereby stopping or suppresssing the circadian rhythm, have also been applied, but no significant advance in the knowledge regarding the mechanism involved in the oscillation has been obtained. There is a large list of substances applied to different circadian rhythms that have no significant effect on the amplitude or period of the cyclic activity, such as inhibitors of protein synthesis, NaCN, 2, 4-dinitrophenol. CuSO,, ether, chloroform (Btihnemann, 1955; Hastings, 1960; Hastings and Bode, 1962; Biinning, 1967). However, it is well known that some chemical substances greatly influence the synchronizing mechanisms of the circadian rhythm (Sachs et al., 1959; Strum- wasser, 1965; Eskin and Corrent, 1977; Woolum and Strumwasser, 1983).

Deuterium oxide seems to be an exception to the rule that the circadian rhythms are intractable to chemical perturbations (~ttend~gh, 1960). Heavy water has been found to noticeably enhance the period in the circadian activity rhythm of rodents, leave movement rhythm in Phaseolw, phototaxis in Euglena, etc. (Bruce and Pittendrigh, 1960; Biinning and Baltes, 1963; Suter and Rawson, 1968; Palmer and Dowse, 1969; Richter, 1970).

It is possible that the similarities in the effect of deuterium oxide on such diverse circadian rhythms could be due to action mechanisms common to all these rhythms, a fact that would enable an effective but indirect study of these mechanisms. The aim of the present study was to analyse the deuterium oxide effect upon a previously established circadian rhythm, i.e. the amplitude rhythm of the photo- receptors’ electrical response to light (electro- retinogram, ERG) of the crayfish recorded from intact animals and from isolated eyestalks. In the isolated eyestalk, the ERG circadian rhythm retains its essential characteristics, although it differs from the rhythm recorded in intact animals, the main difference being the presence of high frequency cycles (about 1 cycle every 3 hr) superimposed on the 24-hr oscillation (Sanchez and Fuentes-Pardo, 1977; Fuentes-Pardo ef al., 1985). We analysed the effect of the deuterium oxide upon these high frequency (ultra- dian) oscillations and upon the circadian oscillations obtained both from the isolated eyestalks and the intact animals. These comparisons provided informa- tion on the mechanisms involved in the coupling of the oscillators, as a source of the electroretinographic circadian rhythm of the crayfish.

MATRRIALS AND METHODS

We used the crayfish P. ~~~~er~ (O~ann) brought from Uruapan, Mich. (Mexico). The animals were submitted to cycles of 12 hr light and 12 hr darkness (L : D, 12 : 12) before starting the experiments. The first part of the study consisted of analysing the deuterium oxide (D,O) effect upon the ERG of intact animals. For the second part, we analysed the D,O effect upon the ERG recorded from isolated eyestalks.

435

436 BEATR~Z FWNTFS PARW, and ENRIQI;E MORENO SAmz

The D,O doses used for the first part were: 0, 1.25,2.5, 5.0, 10, 20, 30, and 50%, for which the required volume of I&O was added to the tap water in which the animals remained immersed during the whole experiment. For isolated eye- stalk experiments, the D,O doses were: 0, 1.25, 2.5, 5.0, IO and 30%, for which the necessary amount of distilled water, used to prepare the saline solution (Van Harreveld, I936), was substituted by D20. The eyestalk was sectioned from its base and immersed in the saline solution. Each group was formed by 10 intact animals or 10 eyestalks. The experi- ments were performed under constant dark conditions (interrupted only by the light flashes applied every 3 min) and constant temperature (19OC for intact animals and 14°C for isolated eyestalks).

The ERG was obtained in response to a light flash delivered from a Grass photostimulator every 3min and recorded with a metal electrode, l-5 pm tip diameter, inserted through the cornea at one end. The other end was connected to a Tektronix Mod. 122 preamplifier, which generated a signal that was fed to a polygraph (Grass Mod. 7). The recordings from the intact animals lasted for no less than seven consecutive days; the isolated eyestatk recordings lasted no less than three consecutive days. The results were analysed by measuring the ERG amplitude at regular intervals (every 20min). These measurements were plotted against time. When necessary, current statistical techniques were used.

RESULTS

Experiments with intact animals

Figure 1 depicts a typical recording of the ampli- tude changes in the ERG, detected at the 20-min interval measurements during 3 consecutive days chosen from the 7 days duration of the experiment, under constant darkness and temperature con- ditions. The ERG amplitude shows daily fluctuations corresponding to a circadian rhythm from a noc- turnal animal, i.e. the one having its phase of greatest activity at night. The average period, obtained by measuring the time interval peak-to-peak in 10 ex~rimcnts with seven cycles each, was of 23.1 + 1.8 hr (Table 1). The rhythm’s amplitude, defined as the ratio between the amplitude value attained at the peak and the value at the valley (night:day ratio), had values close to three; and the ratio between the active and the resting periods (a :p ratio) presented mean values close to one (1.2 f 0.43). When D,O was applied to the water in which the

Table 1. Intcct crayfish

V Circadian a:p doses period ratio (%I (hr + SD*) (hr + SD*) --- 0 23.1 + 1.8 1.2 * 0.43 1.25 23.4 F 2.1 I .6 * 0.82 2.5 24.6 _+ 0.9 I.92 I.22 5.0 26.1 * 1.2 1.8+0.31

10.0 26.7 rt 1.9 1.8 f 0.62 20.0 27.6 + 2.4 1.9 ItO. 30.0 21.9 + I .9 2.1 * 1.01

*Standard deviation.

crayfish. was immersed, we observed that the cir- cadian rhythm of the ERG’s amplitude persisted, although it showed some important modifications. This finding is illustrated in the lower part of Fig. 1, in which a lengthening to 26.7 + 1.9 hr of the cir- cadian rhythm period can be observed at 10% D,O dose. A decrease not only in the ERG’s amplitude but in the oscillation per se can also be observed, which proves that the night: day ratio decreased to 2: 1. The a :p ratio showed very irregular modifications from cycle to cycle, although, in general, it can be said that it showed a tendency to increase by the DzO action. In Fig. 1, the a :p ratio is 1.4. Table I includes the average values of period, and a : p ratio, obtained from all the D,O doses used in the intact crayfish. Figure 2 depicts the graph obtained by plotting the mean value of the ERG circadian rhythm against the D,O doses applied. This graph shows an increase in the circadian rhythm period, which attained a value of 27.9 -+_ 1.9 at 30% 40, representing an increase of 20.77% of the original period value. The slope of the curve was 0.13 with a correlation coefficient of 0.86. Although an overlapping of the values among the experiments occurred with some of the doses, the average period values and the a :p ratio shows a tendency to increase depending on the D20 dose. At 50% D,O dose, the circadian rhythm was very irregular in all its parameters, the period reaching values from 31 to 45 hr.

Experiments with isolated eyestalks

The upper part of Fig. 3 shows a recording chosen from 10, obtained under the same conditions. This recording shows that the application of a test light flash, every 3 min during three consecutive days to

I I

12 12 12 12 TlME(hr)

Fig. 1. Effects of D,O upon the rhythm of the ERG in the intact animal. The recordings obtained in two intact animals immersed in tap water (0) or in water containing 10% 40 ( x ) are presented. It can be observed that both the amplitude of the ERG and the amplitude of the rhythm are bigger in the animal immersed in tap water than in the animal immersed in water containing 40. Regarding the period, its

lengthening is evident in the animal immersed in the 40 solution.

Effect of D,O on crayfish ERG 437

i 5 io i0

D20 DOSE (percent)

j0

Fig. 2. Effects of the different doses of D,O used upon the period of the circadian rhythm in the intact animal. The increase in the dose produces lengthening in the period of the cycles. Note that this increment is bigger at low doses, the effect being reduced with the higher doses. The mean and its standard deviation were plotted. The value of the slope was 0.13 and that of the correlation index was 0.86.

the isolated eyestalk, produces responses (measured every 20 min) showing periodic changes of amplitude every 22.5 hr, approximately. The ERG’s amplitude, although noticeably reduced as compared with the

Table 2. Isolated eyestalk

D-0 do-ses (%I 0

Circadian period

hr k SD’

22.5 f 1.50

ratio hr k SD*

1.5kO.8

Ultradian perid

hr f SD*

2.82 f 0.09 1.25 22.6 + 0.70 2.4 + 1.8 3.05 f 0.03 2.5 25.0 f 0.63 2.3 f 1.6 3.30 f 0.06 5.0 26.6 f 1.00 2.1 & 1.9 3.63 k 0.08

10.0 29.6 + 0.70 1.9 + 1.8 3.86 f 0.08 20.0 32.2 k 0.80 2.4 f 1.7 4.03 + 0.06 30.0 33.4 * 0.95 2.6 f 1.8 4.20 k 0.06

*Standard deviation.

ERG obtained from intact animals, is maxima1 at night and minima1 during the day. The same trace shows that the night: day ratio is also reduced. The ct : p ratio is irregular, with a 1.5 value in the recording shown. A more detailed observation of this recording (see insert in Fig. 3) indicates the presence of higher frequency oscillations (approximately 1 cycle every 3 hr) superimposed on the circadian oscillation and with an amplitude that seems to depend on the circadian time of the system. In other words, during the ascending phase of the cycle, the amplitude of these oscillations is much greater than at any other moment of the cycle.

By applying D,O to the Van Harreveld solution at doses between 1.25 and 30% and recording the ERG

-2.4 . i2 ’ ;4 TIME(hr)

Ii 1‘2 12 TIMEM

Fig. 3. Effects of 40 upon the ERG rhythm in the isolated eyestalk. The upper trace (0) depicts the oscillation of the ERG obtained in the isolated eyestalk placed in Van Harreveld saline solution; the lower trace ( x ) was obtained from an isolated eyestalk placed in a Van Harreveld saline solution, prepared with 10% 40 and 90% distilled water. The differences in the ERG amplitude, the circadian rhythm amplitude, and the D,O effect upon the period of the circadian oscillation, are prominent. The insert shows a circadian oscillation that shows the presence of high frequency cycles (ultradian), characteristic of this preparation. The number of high frequency cycles superimposed on the circadian oscillation remained constant in the preparations placed in normal Van Harreveld solutions, as well as in those placed in Van

Harreveld solutions with different D,O doses.

BEATRIZ FUENTES PARIIO and ENRIQIJE MORENO SAENZ

P

I 5 IO 20

DzO DOSE (percent) 30

Fig. 4. Effects of the different 40 doses upon the period of the circadian rhythm in the isolated eyestalk. The increase in dose produces, as in the intact animal, a lengthening of the circadian period. Note that, as in the intact animal, the D,O effect is bigger at low doses than at higher ones. However, by comparing the periods obtained in the isolated eyestalk (this figure) with those obtained in the intact animal (Fig. 2) it can be noted that the 40 has a greater effect on the isolated eyestalk, than on the intact animal. The slope had a value of 0.34 and the correlation index was of 0.93.

from the isolated eyestalks immersed in it, we ob- served mo~~cations in the circadian cycles that depended on the D,O doses used. The lower part of Fig. 3 shows a recording of the ERG circadian rhythm obtained from an isolated eyestalk immersed in Van Harreveld solution containing 10% D20. It is evident that the amplitudes of the ERG and of the

34 1 1

i 5 Ib 2b

DtO DOSE (percent)

3b

Fig. 5. E&&s of the different doses of 40 upon the period of the u&radian cycles in the isolated eyestalk. The number of ultradian cycles, superimposed on the circadian oscil- lation, was approximately the same for the Van Harreveld solution without D,O and that containing different D,O

doses.

3.4 3.8 ULTRAOIAN PERIOD (hr)

42

Fig, 6. Relation of the &radian and the circadian cycles in the isolated eyestalk obtained with different D,O doses. The data plotted here are the same as those shown in Figs 4 and 5. It can be observed that, when the period of the ultradian cycles increases, the period of the circadian cycles obtained

from the isolated eyestalk also increases.

circadian rhythm are reduced as compared with eyestalks placed in normal Van Harreveld solution. An increase to a mean value of 29.6 f 0.7 hr also occurred in the circadian period. The high frequency cycles also increased their duration from 2.82 & 0.09 in normal solution to 3.86 k 0.08 in the 10% DzO (see insert in Fig. 3) solution. The a :p ratio showed great irregularity, but was always greater than that obtained from isolated eyestalks immersed in normal solution. Both the circadian period and the super- imposed ultradian periods increased proportionally as compared to those obtained in normal Van Harreveld solution. As a result, the total number of high frequency cycles on the circadian rhythm re- mained unchanged, that is, around 8.5 ultradian cycles in each circadian oscillation. This constant relation was observed with all the D,O doses used. The value of the circadian and ultradian periods and the a :p ratio, obtained from the isolated eye- stalks, change according to the D,O doses used (Table 2).

Figure 4 shows the relation between the 40 doses used and the period duration of the high frequency cycles. The slope of the curve was 0.03 with a correlation coefficient of 0.90. This relation indicates that, at low doses, the effect on the period is propor- tionally higher than with higher doses, although no saturation of the system could be observed with these higher doses. Somewhat similar observations can be made by relating the D,O concentration to the duration of the ERG circadian periods (Fig. 5) recorded from isolated eyestalks. Here, propor- tionally, a greater effect is also found with lower D,O doses. The slope of the curve was 0.34 and the correlation coefficient was of 0.93. Figure 6 shows the linear relation between the duration of the ultradian periods and the circadian periods containing them, for each D,O dose used. The relation is linear, with a slope of 0.34 and a correlation coefficient of 0.937, which could be interpreted as if the change in the

Effect of D,O on crayfish ERG 439

ultradian periods determined, effectively, an equiv- alent change in the circadian period.

DlSCUSSfON

The visual photor~ceptor’s response of the crayfish, evoked by light stimulation, recorded for several consecutive days under constant temperature and light conditions, shows periodical changes in ampli- tude, which correspond to the circadian rhythm of a nocturnal animal. The period is shorter than 24 hr, the activity peak appears at night and the b[ :p ratio is greater than one. The ERG recorded from crayfish immersed in water, partially substituted with 40 also showed the circadian rhythm expected from a nocturnal animal, although with significant differences in the amplitude, the o! :p ratio and mainly in the period, this latter change causing the activity peak to appear promptly at daytime. The D20 action upon the ERG occurred immediately after the animal was placed in the HrCLD,O mixture, and both the response amplitude and the lengthening of the period presented modifications which were maintained throughout the whole recording time The length- ening of the circadian period depended on the D,O dose used, and reached a value of 20.77% more than that obtained with pure water when the mixture was prepared with 30% I.&O. The dose-response curve is similar to that obtained by other authors in different animal or plant species. The dose-response curve also shows that DrO induces greater changes in the period at low concentrations (1.55%) than at higher ones (30%); however, no ~turation of the system was observed, as inferred from the great variability showed by the circadian cycles at 50% D,Q dose. The linear relation, without threshold shown in Fig. 2, supports the idea that D20 diffuses to all cells of the organism. The physicochemi~l changes of the H20-DZO mixture with regard to pure water, such as less mobility, less electrical conductivity and greater viscosity, could be the due to explain the modifications in the circadian rhythms caused by the isotope, which involve a change in membrane trans- port activity. (Njus et al., 1974). Since the duration of the circadian rhythm is the evident manifestation of the oscillators velocity on which the considered rhythm is based, the lengthening produced by 40 could result from the diffusion of this substance to ail the cells of the organism, particularly to the cells involved in the generation and expression of the ERG’s circadian rhythm. These cells would actually be the oscillators that reduce their oscillation velocity and thereby lengthen the circadian period, as a result of a decrement in their reaction velocity produced by the phys~co~h~ical changes of the HrO--D,U mix- ture. This explanation led us towards a possible cellular origin of the ERG circadian rhythm.

The isoiated eyestalk experiments reinforce this interpretation, since the oscillator groups (cells) that partially uncouple due to the eyestalk section in the presence of DZO, also show a decrement in the oscillation velocity that causes a le~g~e~ug of the &radian cycles superimposed on the circadian cycles.

It is important to mention that the DZO action was even greater on the isolated eyestalk than on the

intact animal. This greater action was exerted through diverse effects. For example, the life-span of the isolated eyestalks increased notably, extending over the 3.5-day average life-span of the eyestalks immersed in normal saline solution, to up to 7 days, on average, when ~fferent H,O-D,O proportions were used to prepare the Van Harreveld solution. The lengthening of the circadian cycfes in the isolated eyestalk was greater (m = 0.34) with all the D20 doses used than in that intact animals (m = 0.13). This greater effect on the isolated eyestalk could be because the sectioned structure is in greater contact with the D,O than the eye of the intact animal. Another explanatian could be the different tem- peratures to which the intact animals and the sec- tioned eyestalks were submitted (19C and 14°C respectively}, since it is known that D,O has specific temperatures for optimal action.

Another effect of D,O in the isolated eyestalks relates to the great stability that the isotope seems to have conferred to the oscillators responsible for the high frequency cycles (2.8-4.2 hr), and consequently to the corresponding circadian cycles. None of the D,O doses used produced uncoupling of these cycles nor loss of the oscillators’ cohesion. These results agree with the findings of Daan and Pittendrigh (19761, regarding the activity rhythm of some ro- dents. These authors observed a dissociation of the motor rhythms as a result of the application of certain light schedules, but never related to the addition of D,O to the drinking water.

As in intact animals, D20 action had no threshold, was proportionally greater at low doses than at higher ones, and no saturation was observed with the latter ones. It is worth emphasizing that the length- ening of a circadian cycle is precisely equivalent to the sum of the lengthening percents of the &radian cycles that it comprises. This fact supports the inter- pretation that the &radian cycles are in fact part of the circadian cycles. Even more, it allows us to propose that DrO affects the oscillatory machinery per se by slowing down the activity of the cellular groups due, probably, to its omnipresence as a uni- versa1 dissolving agent (just as water). This sug- gestion is supported by the studies of Aleksandrov er al. (19661, who applied I&O to a whole variety of cellular types in which they worked in vitro, and found that D,O reduces enzymatic activity, as well as cellular permeability and growth inhibits mitosis etc. and, together with this reduction, in cellular func- tions, an increase in the structural stability of the proteins occurs, granting the cell a greater resistance to deletereous agents.

Much has been said about the D,O effect in the reduction of the system’s apparent temperature. This proposition has led some authors (Pittendrigh et a[., 1973; Caldarola and Pittendrigh, 1974; McDaniel et al., 1974; Pittendrigh and Cosby, 1974) to prove the hypothesis that the 40 effect on the circadian rhythm could be based on its action upon the homeo- static machinery of the biological clocks. The latter possess tem~rature com~nsating mmhanisms that practically avoid changes of the circadian period due to temperature changes. However, there is not enough conclusive evidence to accept or disregard this hypothesis, since it has often been found that the

440 BEATRIZ FUENTES PAR~ and ENR~QUE MORENO S~NZ

D,O effect depends on the temperature at which it was worked. It is possible that the greater D,O effect upon the circadian rhythm in the isolated eyestalk, as compared to the intact animal, could be due, at least partiaily, to the low temperature (14°C) at which the recordings were made in this preparation.

Acknowledgemenfs-The authors thank MS Ingrid Mascher for her help in revising the manuscript. This work was partially supported by a grant from the Ricardo J. Zevada Foundation.

REFERENCE

Aleksandrov V. Y., Arronet N. I., Den’ko E. I, and Konstantinova M. F. (1966) Effect of heavy water D,G on resistance of plant and animal cells, cell models and protein to certain denaturing factors. Fed. Proc. 25, T128-T134.

Bruce V. G. and ~ttendrigh C. S. (1960) An effect of heavy water on the phase and period of the circadian rhythm in Euglena. J. ce(l. camp. Physiol. 56, 25-31.

Biihnemann F. (1955) Das Endodiumale System der Oedogonium Zelle III. Uber Den Temperature Influss. Zeit. Naturforsch. lob, 305-310.

Biinning E. (1967) The Physiological Clock. Springer, Berlin. Biinning E. and Baltes J. (1963) Zur Wirkung Von Schweren

Wasser auf die Endogene Tag~rhythmik. Na~ur~iss. 50, 622.

Caldarola P. C. and Pittendrigh C. S. (1974) A test of the hypothesis that D,O affects circadian oscillations by diminishing the apparent temperature. Proc. natn. Acad. Sci. 71, 4386-4388.

Daan S. and Pittendrigh C. S. (1976) A functional analysis of circadian pacemakers in nocturnal rodents. III. Heavy water and constant light: homeostasis of frequency? J. camp. Physiol. 104, 267-290.

Eskin A. and Corrent G. (1977) Effects of divalent cations and metabolic poisons on the circadian rhythm from the Aplysia eye. J. camp. Physiol. 117, 1-21.

Fuentes-Pardo B., Verdugo-Diaz L. and Inclan-Rubio V. (1985) Effect of external level of calcium on ERG cir- cadian rhythm in isolated eyestalk of crayfish. Comp. Biochem. Physiol. 82A, 385-389.

Hastings J. W. (1960) Biochemical aspects of rhythms: phase shifting by chemicals. In Biological Clocks, Cold

Spring Harbor, New York and Symposium on Quanti- iative Biology 25, 131-143.

Hastings J. W. and Bode V. C. (1962) Biochemistry of Rhythmic Systems. Ann. N.Y. Acad. Sci. 98, 876-889.

McDaniel M., Sulzman F. M. and Hastings J. W. (1974) Heavy water slows the Gonyaufax clock. A test of the hypothesis that 40 affects circadian oscillations by diminishing the apparent temperature. Proc. nom. Acad. Sci. 71. 43894391.

Njus D., Sulzman F. M. and Hastings J. W. (1974) Mem- brane model for the circadian clock. Nature 248. 116-120.

Palmer J. D. and Dowse H. B. (1969) Preliminary findings on the effect of D,O on the period of circadian activity rhythms. Bioi. Bull. 137, 388.

Pittendrigh C. S. (1960) Circadian rhythms and the cir- cadian organization of living systems. In Biological Clocks, Cold Spring Harbor, New York and Symposium on Quantirative Biology 2S, 159-184.

Pittendrigh C. S. and Cosbey E. S. (1974) On the very rapid enhancement by D,O of the temperature-tolerance of adult Dro.~ophi~a. Proc. natn. Acad. Sci. 71, 540-543.

~ttend~gh 6. S., Caldarola P. C. and Co&y E. S. (1973) A differential effect of heavy water on temperature- dependent and temperature-compensated aspects of the Drosophila pseudoobscura circadian system. Proc. natn. Acad. Sci. 70, 2037-2041.

Richter C. D. (1970) Blood-clock barrier: its penetration by heavy water. Proc. natn. Acad. Sci. 66, 244.

Sachs R. M., Brets C. and Lang A. (1959) Cell division and Gibberelic acid. Exp. Cell. Bees. 18, 231t244.

Sanchez J. A. and Fuentes-Pardo B. (1977) Circadian rhythm in the amplitude of the electroretinogram in the isolated eyestalk of the crayfish. Comp. Biochem. Physiol. %A, 601605.

Strumwasser F. (1965) The demonstration and manipu- lation of a circadian rhythm in a single neuron. In Circadian Clocks (Edited by Aschoff J.), pp. 442462.

Suter R. B. and Rawson K. S. (1968) Circadian activity rhythm of the deer mouse Peromyscus: effect of deu- terium oxide. Science 160, 101 l-1014.

Van Harreveld A. (1936) A physiological solution for freshwater crustaceans. Proc. Sot. exp. Biol. Med. 34, 428432.

Woolom J. C. and Strumwas~r F. (1983) Is the period of the circadian oscillator in the eye of ApZysia directly homeostatically regulated? J. camp. Physiol. 151, 253-259.