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Influence of deuterium oxide on circadian activity rhythms of hamsters: Role of the suprachiasmatic nuclei
Brain Research, 376 (1986) 149-154 Elsevier
149
BRE 11F89
Influence of Deuterium Oxide on Circadian Activity Rhythms of Hamsters: Role of the Suprachiasmatic Nuclei GARY E. PICKARD and IRVING ZUCKER
Department of Psychology, Universityof California, Berkeley, CA 94720 (U.S.A.) (Accepted October 29th, 1985)
Key words: circadianperiod length - - locomotoractivity
The period of the free-running Circadian activity rhythm of Syrian hamsters was measured before and during treatment with 10% deuterium oxide (D20). Deuteration increased period length by approximately 0.5 h per cycle both pre- and postoperativelyin hamsters sustaining complete, incomplete or no unilateral lesions of the suprachiasmatic nuclei (SCN). Neither couplingbetween the bilaterally paired SCN, nor elimination of 50% of SCN tissue affected period length during D20 treatment. However, variability of the response to D20 was much greater in lesioned than in intact hamsters. We propose that a small percentage of the normal complement of SCN neurons is sufficient to permit full responsiveness of the circadian system to D20 and that there is substantial redundancy in the neural system that responds to deuterium. Stability of the circadian system appears to be increased by the full complement of SCN neurons.
INTRODUCTION The bilaterally paired suprachiasmatic nuclei (SCN) are important for the expression of circadian rhythms in mammals. Destruction of the SCN abolishes coherent circadian rhythmicity in several physiological and behavioral parameters12,19,22. Furthermore, the SCN and individual SCN cells show circadian variations in their in vivo and in vitro firing rates 7,9 and in their metabolic activity20. Each of the suprachiasmatic nuclei can function as an independent circadian pacemake#,16,17. Reciprocal neural connections between the SCN14,23 provide a pathway for physiological coupling of these independent oscillators in the two halves of the brain. By this means the two SCN can remain mutually coupled and express a unified circadian output either in the presence of external synchronizing stimuli or in the absence of such zeitgebers. Coupling between the paired SCN may increase the stability or 'strength' of SCN regulation of circadian rhythms. Although a single suprachiasmatic nucleus may be sufficient for the generation of circadian
activity rhythms and their entrainment to the illumination cycle, variability in the free-running period or the phasing of rhythms may be greatly increased. Thus, the response of a reduced system to light-induced phase shifts or to chemically mediated changes in the period of the free-running rhythm might be different from that of an intact system. Deuterium oxide (D20) is one of the few chemicals that reliably alters the period of mammalian circadian oscillations. The period of the circadian activity rhythm increases in a linear fashion as the tissue concentration of D2O increases 1,3,5,11,21. If coupling between the two SCN or the full complement of SCN neurons is required for normal responsiveness to D20, then unilateral ablation of the SCN, by reducing the number of D20 'target' neurons, might be expected to decrease the period lengthening effects of deuterium oxide. Alternatively, the neural system that presumably mediates the effects of D20 on the period of the activity rhythm may permit a normal response as long as sufficient SCN tissue is available to generate a coherent circadian rhythm. This hypothesis implies that each 'target' neuron is affected by
Correspondence: G.E. Pickard. Present address: Institute of Neuroscience, Universityof Oregon, Eugene, OR 97403, U.S.A. 0006-8993/86/$03.50(~) 1986Elsevier SciencePublishers B.V. (BiomedicalDivision)
150 D20 to the same degree and would be indicative of
TABLE I
substantial redundancy in the neural regulation of circadian rhythms. A third possibility is that the effects of D20 on the circadian period of the activity rhythm are independent of the SCN. To discriminate among these hypotheses the present experiment determined the effects of deuteration on the period of the circadian activity rhythm of hamsters with varying degrees of damage to the SCN.
Experimentalprocedures and testsequence
MATERIALS AND METHODS
Adult male Syrian hamsters (Mesocricetus auratus), obtained from Simonsen Laboratories, Gilroy, CA, were housed individually in polypropylene cages, each equipped with an activity wheel. Room temperature was kept at 23 +_ 2 °C. Food (Simonsen rat pellets, maintenance diet) and tap water were available ad libitum. Each revolution of the activity wheel was recorded on an Esterline Angus event recorder operating at a chart speed of 45.7 cm/24 h. Chart paper was removed at irregular intervals, cut into 24 h segments and activity records assembled in the traditional manner. The period (r) of the circadian rhythm of activity was calculated by eyefitting a straight line through the activity onsets for at least 10 consecutive days. Judgements of r were made without knowledge of the animals' group membership. The experimental design, including photoperiod, lesion and deuterium oxide treatments is summarized in Table I. Neurologically intact hamsters originally were entrained to a light:dark (L:D) 14:10 h photoperiod (14 h light/day beginning at 08.00 h Pacific Standard Time). Illumination was provided by 4 overhead cool white fluorescent lamps with light intensity at cage level ranging from 1500 to 400 lux from the top to the bottom of the rack holding the cages. Hamsters also were maintained in constant dim light (LL); illumination intensity varied from 100 to 20 lux depending on cage position in the holding rack. Each animal remained in the same cage position throughout the experiment. Several times in the course of the experiment (Table I) a 10% deuterium oxide solution (D20), prepared by mixing 100 ml of 'pure' D20 (Sigma Chemical Co, St. Louis, MO) with 900 ml tap water, replaced tap water as the only source of fluids available to hamsters.
L:D 14:10, 14 h light/day; LL, constant dim illumination;D20, 10% deuterium oxide; H20, tap water.
Preoperative testing Days 1-14 15-60 61-84 85-97 98-99 (surgery)
Photoperiod
Drinking fluid
L:D 14:10 LL LL L:D 14:10 L:D 14:10
H20 H20 DzO H20 H20
LL LL
H20 D20
Postopera~vetes6ng 100-138 139-164
At the completion of preoperative testing with D20 (Table I, Day 84), the original L:D 14:10 h cycle was reinstated and 12 days were allowed for animals to re-entrain to this photoperiod and to clear their bodies of D20 (ref. 11). Hamsters received unilateral lesions aimed at the SCN as previously described 16 or were sham-operated. Briefly, the animals were positioned in a Kopf stereotaxic apparatus under deep pentobarbital sodium anesthesia (80 mg/kg). Using coordinates determined empirically, a tungsten wire electrode (diameter 0.2 mm), coated with Epoxylite except for the cross-sectional area of the tip, was angled laterally, 10° to the vertical and lowered into the SCN. A current of 2 mA was applied for 10 s. Sham operations were performed by lowering the electrode to a position 2 mm above the SCN and withdrawing the electrode without applying current. Surgery was completed on all animals over the course of 2 days. Nine operated animals were removed from the experiment because they either ran too infrequently in the wheels to permit accurate estimation of activity onsets or because their activity rhythm split into two components 1. The postsurgical testing procedure included estimations of r in LL while hamsters were drinking tap water and subsequently D20. Fluid intake was measured to the nearest milliliter during the last week of each phase of the experiment. At the termination of behavioral testing, a histochemical marker was used to delineate the SCN. Retinal processes infiltrate almost the entire 3-dimensional boundary of the hamster SCN 6,14,15 and there-
151 fore can help in determining the amount of SCN tissue remaining after placement of brain lesions. To label retinal processes, animals were deeply anesthetized with pentobarbital sodium and 4.0 ~1 of a 30% solution of horseradish peroxidase (3 mg HRP/10 #1 buffer; Sigma type VI H R P ) in 0.05 M Tris buffer (pH 7.4) was injected into the vitreous of each eye 24-48 h before the animals were killed. Brains were prepared for the histochemical demonstration of anterogradely transported H R P as described previously 15. Extent of SCN damage was determined without knowledge of the animals' behavioral data. The lesions were categorized as involving: (1) no damage to either SCN; (2) complete unilateral SCN destruc-
¸
tion; (3) partial unilateral SCN damage; and (4) bilateral SCN damage. Differences between and within groups were evaluated with two-tailed t-tests for independent and dependent means, respectively. RESULTS
Histological data The SCN, as demonstrated by labeled retinal processes, were completely intact in 6 sham-operated hamsters and in two animals that sustained lesions anterior to the SCN (Fig. 1A). Nine hamsters had only a single intact SCN as indicated by the total ab-
nl
Fig. 1. Bright-field photomicrographs in the coronal plane illustrating labeled retinal processes in the suprachiasmatic nucleus (SCN) after the injection of HRP into the vitreous humor of each eye. In a sham-lesioned animal with intact suprachiasmatic nuclei, each SCN is heavily labeled (A). In hamsters judged to have complete unilateral SCN destruction, HRP-filled retinal processes are restricted to a single SCN (B and C). The animal in B manifested only a slight decrease in r (0.05 h) after the lesion whereas the r of the hamster sustaining the lesion illustrated in (C) was decreased by 0.82 h. Labeled retinal processes are present in both the intact and lesioned SCN (arrow) in a hamster with partial unilateral SCN ablation (D). OC, optic chiasm; V, third ventricle.
152 TABLE If
Influence of DeO on the period of the circadian activity rhythm of hamsters with varying amounts of unilateral damage to the SCN Group Intact Unilateral SCN-x Partial Unilateral SCN-x
n 8 7 9
Preoperative testing
Postoperative testing
r-H20
T-De0
AT
T-H20
T-DeO
AT
24.36 + 0.04 24.37 + 0.05 24.36 + 0.04
24.85+ 0.09 24.90 + 0.13 24.96 + 0.11
0.49 + 0.07 0.53 + 0.11 0.60 + 0.09
24.20 + 0.08" 24.02+ 0.15"* 24.22 + 0.04*
24.72+ 0.09 24.53 + 0.21 24.68 + 0.05
0.52 + 0.05*** 0.51 + 0.09*** 0.46 + 0.04***
* P < 0.05. ** P < 0.025. *** Not significantly different from preoperative values.
sence of labeled retinal processes on the side of the lesion (Fig. 1B, C). A n additional 10 hamsters incurred sub-total destruction of one of the S C N that involved < 30% tissue d a m a g e in 7 animals and approximately 67% of the nucleus in 3 hamsters (Fig. 1D); for purposes of analysis these animals were treated as a single group. The SCN were d a m a g e d bilaterally in two hamsters, but their erratic wheel-running behavior p r e c l u d e d assessment of responsiveness to D 2 0 .
Preoperative measures The baseline (tap water) free-running p e r i o d (r) of the activity rhythm in L L for animals that c o m p l e t e d the experiment was 24.36 + 0.02 h; r was increased by 0.56 + 0.05 h ( P < 0.001) during ingestion of D 2 0 . Baseline r and responsiveness to D 2 0 did not differ significantly during p r e o p e r a t i v e testing for animals assigned to the different lesion groups (Table II). Postoperative testing Baseline r (tap water) was shorter p o s t o p e r a t i v e l y (Days 100-138) than p r e o p e r a t i v e l y (Days 15-60), irrespective of the amount of d a m a g e to the SCN. F o r the 6 s h a m - o p e r a t e d animals the decrease was 0.11 + 0.07 h (P > 0.05); for the 20 lesioned animals for which accurate pre- and p o s t o p e r a t i v e measures were available, r decreased by 0.31 + 0.08 h ( P < 0.001). D a t a for individual hamsters are shown in Fig. 2. The postoperative decrease in baseline r values was substantially greater for a subset of animals that sustained complete unilateral ablation of the SCN than for o t h e r hamsters. Thus, 4 of 9 h a m sters with complete unilateral SCN lesions manifested large postoperative decreases in r that were
out of the range of changes o b s e r v e d in o t h e r animals. The p o s t o p e r a t i v e decrease in baseline r among hamsters with c o m p l e t e unilateral SCN lesions did not correlate with the extent or locus of damage to the SCN or surrounding tissue (Fig. 1B, C).
Response to deuteration The response to D 2 0 was similar pre- and postoperatively for each of the groups of hamsters (Table II) and was i n d e p e n d e n t of the baseline free-running period or of the degree of d a m a g e to the SCN. H o w ever, responsiveness to D 2 0 was m o r e variable in animals sustaining SCN lesions than in neurologically intact hamsters (Table III); when expressed as the absolute difference in r attributable to d e u t e r a t i o n p r e o p e r a t i v e l y minus that r e c o r d e d postoperatively,
24.4
24.0.
23.6
23.2
iNTACTSCN
COMPLETE UNILATERAL SCN LESION
iNCOMPLETE UNILATERAL SCN LESION
Fig. 2. Free-running period (T) of individual hamsters maintained in constant light (LL) and drinking tap water. The postoperative r was decreased in each group of animals irrespective of the amount of SCN damage. The horizontal and vertical bars to the left and right of each set of lines indicate the group mean and S.E.M. r before and after surgery, respectively.
153 TABLE III Increase in the period of the circadian activity rhythm (h) induced by D20 in individual hamsters before and after SCN lesions
A, absolute value of postoperative minus preoperative effects of DzO. Intact
Unilateral SCN-x
Partial SCN-x
Preoperation
Postoperation
A
Preoperation
Postoperation
A
Preoperation
Postoperation
A
0.81' 0.61" 0.36* 0.53* 0.18" 0.49* 0.58 0.35
0.74 0.64 0.44 0.54 0.31 0.50 0.55 0.44
0.07 0.03 0.08 0.01 0.13 0.01 0.03 0.09
0.74 0.57 0.24 0.16 0.79 0.44 0.79
0.62 0.90 0.57 0.28 0.52 0.25 0.43
0.12 0.33 0.33 0.12 0.27 0.19 0.36
0.20 0.72 0.93 0.90 0.56 0.47 0.66 0.29 0.73
0.33 0.24 0.44 0.59 0.56 0.47 0.55 0.46 0.46
0.13 0.48 0.49 0.31 0.00 0.00 0.11 0.17 0.27
* Sham-operated.
the value for animals without damage to the SCN was 0.06 + 0.02 h as compared to values of 0.25 + 0.04 and 0.22 + 0.06 h, respectively for animals with complete and incomplete unilateral SCN lesions (P < 0.005 and P < 0.025 for intact hamsters vs those with complete and incomplete unilateral SCN damage, respectively). Postoperatively, all animals drank significantly less deuterium oxide; intact hamsters consumed 85.2% of the preoperative level (P < 0.05), unilaterally SCN-lesioned animals 84.1% (P < 0.01) and animals with partial lesions of the SCN, 82.7% (P < 0.01). Percentage differences in postoperative D20 intake relative to preoperative baselines did not differ among the groups. DISCUSSION The extent to which D20 lengthens the period of the circadian activity rhythm was unaffected by unilateral ablation of the hamster SCN: increases in period length attributable to ingestion of D20 were similar in neurologically intact hamsters and those sustaining complete or partial ablation of one of the bilaterally paired SCN. Neither coupling between the two SCN nor elimination of one half of the normal complement of SCN neurons influenced responsiveness of the circadian system to D20. We tentatively conclude that if sufficient neural tissue remains to generate a coherent circadian rhythm, the periodlengthening response to deuterium will be normal. This conjecture might best be evaluated in hamsters
with the minimal number of SCN neurons consistent with normal circadian organization. It is not known whether partial bilateral destruction of the SCN has a greater influence on responsiveness to D20 than an equivalent amount of unilateral damage. The circadian behavior of two animals with partial bilateral damage to the SCN was too erratic to permit accurate determination of the effects of deuteration on the circadian period. Mutual coupling between the two SCN appears to influence the period of the circadian activity rhythm. The r was reduced by 0.92 + 0.09 h postoperatively in 4 of 9 hamsters with complete unilateral SCN lesions; for two of these hamsters r was in the range of 23.3 h. Five animals with similar SCN damage manifested only a slight decrease in r (0.14 + 0.04 h), equivalent in magnitude to the changes observed in sham-operated animals. These findings confirm those of a prior study in which circadian period was reduced by 0.61 h in 5 of 7 hamsters incurring complete unilateral SCN destruction 17. Unilateral ablation of the SCN also altered the period of circadian rhythms in birds 4. Unilateral excision of the optic lobes of cockroaches consistently affected the period of circadian oscillations 13and the two bilaterally symmetric circadian pacemakers in the eyes of Aplysia became desynchronized from each other in constant light8. Taken together, these findings suggest that mutual coupling between bilaterally paired pacemakers contributes to circadian organization. We were unable to discern any correlation between locus and extent of unilateral lesions and post-
154 operative shortening in the p e r i o d of the circadian activity rhythm. Extensive individual variation remains a perplexing p h e n o m e n o n in several types of circadian investigations and is particularly characteristic of the splitting p h e n o m e n o n in constant illumination 18. Unilateral ablation of the S C N can abolish splitting of the circadian activity r h y t h m into distinct c o m p o n e n t s 16. W e speculate that mutual coupling between the bilateral SCN p a c e m a k e r s m a y decelerate each individual oscillator system in some hamsters, t h e r e b y producing a longer p e r i o d in the coupled state. U n i l a t e r a l ablation of the SCN may shorten the p e r i o d of the overt circadian r h y t h m in these animals. Evidence that the two SCN are not always mutually synchronized comes from a study of rats showing a significant phase difference in simultaneously r e c o r d e d multiple unit activity of the two SCN 10. A l t h o u g h there is no effect of unilateral SCN ablaREFERENCES 1 Daan, S. and Pittendrigh, C.S., A functional analysis of circadian pacemakers in nocturnal rodents. III. Heavy water and constant light: homeostasis of frequency? J. Comp. Physiol., 106 (1976) 267-290. 2 Davis, F.C. and Gorski, R.A., Unilateral lesions of the hamster suprachiasmatic nuclei: evidence for redundant control of circadian rhythms, J. Comp. Physiol., 154 (1984) 221-232. 3 Dowse, H.B. and Palmer, J.D., The chronomutagenic effect of deuterium oxide on the period and entrainment of a biological rhythm, Biol. Bull., 143 (1972) 513-524. 4 Ebihara, S. and Kawamura, H., The role of the pineal organ and the suprachiasmatic nucleus in the control of circadian locomotor rhythms in the Java sparrow, Padda oryzivora, J. Comp. Physiol., 141 (1981) 207-214. 5 Fitzgerald, K., Zucker, I. and Rusak, B., An evaluation of the homeostasis of circadian periodicity in the golden hamster, J. Comp. Physiol., 123 (1978) 265-269. 6 Frost, D.O., So, K.-F. and Schneider, G.E., Postnatal development of retinal projections in Syrian hamsters: a study using autoradiographic and anterograde degeneration techniques, Neuroscience, 4 (1979) 1649-1677. 7 Green, D.J. and Gillette, R., Circadian rhythms of firing rate recorded from single cells in the rat suprachiasmatic brain slice, Brain Research, 245 (1982) 198-200. 8 Hudson, D.J. and Lickey, M.E., Internal desynchronization between two identified circadian oscillators in Aplysia, Brain Research, 183 (1980) 481-485. 9 Inouye, S.T. and Kawamura, H., Persistence of circadian rhythmicity in a mammalian hypothalamic 'island' containing the suprachiasmatic nucleus, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5962-5966. 10 Inouye, S.T. and Kawamura, H., Characteristics of a circadian pacemaker in the suprachiasmatic nucleus, J. Cornp. Physiol., 146 (1982) 153-160. 11 Katz, J.J. and Crespi, H.L., Isotope effects in biological systems. In C.J. Collins and N.S. Bowman (Eds.), Isotope Effects in Chemical Reactions, Van Nostrand Reinhold,
tion on the period-lengthening effects of D 2 0 , variability in the response to D 2 0 is much g r e a t e r in lesioned than in intact hamsters (Table III). The full c o m p l e m e n t of SCN neurons and/or coupling between cells in the SCN a p p a r e n t l y increases stability of the circadian system in the face o f chemical perturbations. O u r findings also confirm and extend earlier observations that coupling b e t w e e n the two SCN plays a significant role in determining the p e r i o d of the circadian activity rhythm.
ACKNOWLEDGEMENTS This research was s u p p o r t e d by N I H G r a n t s H D 02982 and NS-20537. W e are grateful for the technical support of D a r l e n e Frost, Patricia Sollars, Eve Sloan, Ruth BreMiller and Steve Balsma and to John D a r k for comments on the manuscript. New York, 1970, pp. 286-363. 12 Moore, R.Y., Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus, Fed. Proc. Fed. Am. Soc. Exp. Biol., 42 (1983) 2783-2789. 13 Page, T.L., Caldarola, P.C, and Pittendrigh, C.S., Mutual entrainment of bilaterally distributed circadian pacemakers, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 1277-1281. 14 Pickard, G.E., The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retinohypothalamic projection, J. Comp. Neurol., 211 (1982) 65-83. 15 Pickard, G.E. and Silverman, A.J., Direct retinal projections to the hypothalamus, piriform cortex and accessory optic nuclei in the golden hamster as demonstrated by a sensitive anterograde horseradish peroxidase technique, J. Comp. Neurol., 196 (1981) 155-172. 16 Pickard, G.E. and Turek, F.W., Splitting of the circadian rhythm of activity is abolished by unilateral lesions of the suprachiasmatic nuclei, Science, 215 (1982) 1119-1121. 17 Pickard, G.E. and Turek, F.W., The suprachiasmatic nuclei: two circadian clocks?, Brain Research, 268 (1983) 201-210. 18 Pinendrigh, C.S. and Daan, S., A functional analysis of circadian pacemakers in nocturnal rodents. V. Pacemaker structure: a clock for all seasons, J. Cornp. PhysioL, 106 (1976) 333-355. 19 Rusak, B. and Zucker, I., Neural regulation of circadian rhythms, Physiol. Rev., 59 (1979) 449-526. 20 Schwartz, W.J. and Gainer, H., Suprachiasmatic nucleus: Use of 14C-labeled deoxyglucose uptake as a functional marker, Science, 197 (1977) 1089-1091. 21 Suter, R.B. and Rawson, K.S., Circadian activity rhythm of the deermouse, Peromyscus: effect of deuterium oxide, Science, 160 (1968) 1011-1014. 22 Turek, F.W., Neural circadian rhythms in mammals, Annu. Rev. Physiol., 47 (1985) 49-64. 23 Van den Pol, A.N., The hypothalamic suprachiasmatic nucleus of rat: intrinsic anatomy, J. Comp. Neurol., 191 (1980) 661-702.
149
BRE 11F89
Influence of Deuterium Oxide on Circadian Activity Rhythms of Hamsters: Role of the Suprachiasmatic Nuclei GARY E. PICKARD and IRVING ZUCKER
Department of Psychology, Universityof California, Berkeley, CA 94720 (U.S.A.) (Accepted October 29th, 1985)
Key words: circadianperiod length - - locomotoractivity
The period of the free-running Circadian activity rhythm of Syrian hamsters was measured before and during treatment with 10% deuterium oxide (D20). Deuteration increased period length by approximately 0.5 h per cycle both pre- and postoperativelyin hamsters sustaining complete, incomplete or no unilateral lesions of the suprachiasmatic nuclei (SCN). Neither couplingbetween the bilaterally paired SCN, nor elimination of 50% of SCN tissue affected period length during D20 treatment. However, variability of the response to D20 was much greater in lesioned than in intact hamsters. We propose that a small percentage of the normal complement of SCN neurons is sufficient to permit full responsiveness of the circadian system to D20 and that there is substantial redundancy in the neural system that responds to deuterium. Stability of the circadian system appears to be increased by the full complement of SCN neurons.
INTRODUCTION The bilaterally paired suprachiasmatic nuclei (SCN) are important for the expression of circadian rhythms in mammals. Destruction of the SCN abolishes coherent circadian rhythmicity in several physiological and behavioral parameters12,19,22. Furthermore, the SCN and individual SCN cells show circadian variations in their in vivo and in vitro firing rates 7,9 and in their metabolic activity20. Each of the suprachiasmatic nuclei can function as an independent circadian pacemake#,16,17. Reciprocal neural connections between the SCN14,23 provide a pathway for physiological coupling of these independent oscillators in the two halves of the brain. By this means the two SCN can remain mutually coupled and express a unified circadian output either in the presence of external synchronizing stimuli or in the absence of such zeitgebers. Coupling between the paired SCN may increase the stability or 'strength' of SCN regulation of circadian rhythms. Although a single suprachiasmatic nucleus may be sufficient for the generation of circadian
activity rhythms and their entrainment to the illumination cycle, variability in the free-running period or the phasing of rhythms may be greatly increased. Thus, the response of a reduced system to light-induced phase shifts or to chemically mediated changes in the period of the free-running rhythm might be different from that of an intact system. Deuterium oxide (D20) is one of the few chemicals that reliably alters the period of mammalian circadian oscillations. The period of the circadian activity rhythm increases in a linear fashion as the tissue concentration of D2O increases 1,3,5,11,21. If coupling between the two SCN or the full complement of SCN neurons is required for normal responsiveness to D20, then unilateral ablation of the SCN, by reducing the number of D20 'target' neurons, might be expected to decrease the period lengthening effects of deuterium oxide. Alternatively, the neural system that presumably mediates the effects of D20 on the period of the activity rhythm may permit a normal response as long as sufficient SCN tissue is available to generate a coherent circadian rhythm. This hypothesis implies that each 'target' neuron is affected by
Correspondence: G.E. Pickard. Present address: Institute of Neuroscience, Universityof Oregon, Eugene, OR 97403, U.S.A. 0006-8993/86/$03.50(~) 1986Elsevier SciencePublishers B.V. (BiomedicalDivision)
150 D20 to the same degree and would be indicative of
TABLE I
substantial redundancy in the neural regulation of circadian rhythms. A third possibility is that the effects of D20 on the circadian period of the activity rhythm are independent of the SCN. To discriminate among these hypotheses the present experiment determined the effects of deuteration on the period of the circadian activity rhythm of hamsters with varying degrees of damage to the SCN.
Experimentalprocedures and testsequence
MATERIALS AND METHODS
Adult male Syrian hamsters (Mesocricetus auratus), obtained from Simonsen Laboratories, Gilroy, CA, were housed individually in polypropylene cages, each equipped with an activity wheel. Room temperature was kept at 23 +_ 2 °C. Food (Simonsen rat pellets, maintenance diet) and tap water were available ad libitum. Each revolution of the activity wheel was recorded on an Esterline Angus event recorder operating at a chart speed of 45.7 cm/24 h. Chart paper was removed at irregular intervals, cut into 24 h segments and activity records assembled in the traditional manner. The period (r) of the circadian rhythm of activity was calculated by eyefitting a straight line through the activity onsets for at least 10 consecutive days. Judgements of r were made without knowledge of the animals' group membership. The experimental design, including photoperiod, lesion and deuterium oxide treatments is summarized in Table I. Neurologically intact hamsters originally were entrained to a light:dark (L:D) 14:10 h photoperiod (14 h light/day beginning at 08.00 h Pacific Standard Time). Illumination was provided by 4 overhead cool white fluorescent lamps with light intensity at cage level ranging from 1500 to 400 lux from the top to the bottom of the rack holding the cages. Hamsters also were maintained in constant dim light (LL); illumination intensity varied from 100 to 20 lux depending on cage position in the holding rack. Each animal remained in the same cage position throughout the experiment. Several times in the course of the experiment (Table I) a 10% deuterium oxide solution (D20), prepared by mixing 100 ml of 'pure' D20 (Sigma Chemical Co, St. Louis, MO) with 900 ml tap water, replaced tap water as the only source of fluids available to hamsters.
L:D 14:10, 14 h light/day; LL, constant dim illumination;D20, 10% deuterium oxide; H20, tap water.
Preoperative testing Days 1-14 15-60 61-84 85-97 98-99 (surgery)
Photoperiod
Drinking fluid
L:D 14:10 LL LL L:D 14:10 L:D 14:10
H20 H20 DzO H20 H20
LL LL
H20 D20
Postopera~vetes6ng 100-138 139-164
At the completion of preoperative testing with D20 (Table I, Day 84), the original L:D 14:10 h cycle was reinstated and 12 days were allowed for animals to re-entrain to this photoperiod and to clear their bodies of D20 (ref. 11). Hamsters received unilateral lesions aimed at the SCN as previously described 16 or were sham-operated. Briefly, the animals were positioned in a Kopf stereotaxic apparatus under deep pentobarbital sodium anesthesia (80 mg/kg). Using coordinates determined empirically, a tungsten wire electrode (diameter 0.2 mm), coated with Epoxylite except for the cross-sectional area of the tip, was angled laterally, 10° to the vertical and lowered into the SCN. A current of 2 mA was applied for 10 s. Sham operations were performed by lowering the electrode to a position 2 mm above the SCN and withdrawing the electrode without applying current. Surgery was completed on all animals over the course of 2 days. Nine operated animals were removed from the experiment because they either ran too infrequently in the wheels to permit accurate estimation of activity onsets or because their activity rhythm split into two components 1. The postsurgical testing procedure included estimations of r in LL while hamsters were drinking tap water and subsequently D20. Fluid intake was measured to the nearest milliliter during the last week of each phase of the experiment. At the termination of behavioral testing, a histochemical marker was used to delineate the SCN. Retinal processes infiltrate almost the entire 3-dimensional boundary of the hamster SCN 6,14,15 and there-
151 fore can help in determining the amount of SCN tissue remaining after placement of brain lesions. To label retinal processes, animals were deeply anesthetized with pentobarbital sodium and 4.0 ~1 of a 30% solution of horseradish peroxidase (3 mg HRP/10 #1 buffer; Sigma type VI H R P ) in 0.05 M Tris buffer (pH 7.4) was injected into the vitreous of each eye 24-48 h before the animals were killed. Brains were prepared for the histochemical demonstration of anterogradely transported H R P as described previously 15. Extent of SCN damage was determined without knowledge of the animals' behavioral data. The lesions were categorized as involving: (1) no damage to either SCN; (2) complete unilateral SCN destruc-
¸
tion; (3) partial unilateral SCN damage; and (4) bilateral SCN damage. Differences between and within groups were evaluated with two-tailed t-tests for independent and dependent means, respectively. RESULTS
Histological data The SCN, as demonstrated by labeled retinal processes, were completely intact in 6 sham-operated hamsters and in two animals that sustained lesions anterior to the SCN (Fig. 1A). Nine hamsters had only a single intact SCN as indicated by the total ab-
nl
Fig. 1. Bright-field photomicrographs in the coronal plane illustrating labeled retinal processes in the suprachiasmatic nucleus (SCN) after the injection of HRP into the vitreous humor of each eye. In a sham-lesioned animal with intact suprachiasmatic nuclei, each SCN is heavily labeled (A). In hamsters judged to have complete unilateral SCN destruction, HRP-filled retinal processes are restricted to a single SCN (B and C). The animal in B manifested only a slight decrease in r (0.05 h) after the lesion whereas the r of the hamster sustaining the lesion illustrated in (C) was decreased by 0.82 h. Labeled retinal processes are present in both the intact and lesioned SCN (arrow) in a hamster with partial unilateral SCN ablation (D). OC, optic chiasm; V, third ventricle.
152 TABLE If
Influence of DeO on the period of the circadian activity rhythm of hamsters with varying amounts of unilateral damage to the SCN Group Intact Unilateral SCN-x Partial Unilateral SCN-x
n 8 7 9
Preoperative testing
Postoperative testing
r-H20
T-De0
AT
T-H20
T-DeO
AT
24.36 + 0.04 24.37 + 0.05 24.36 + 0.04
24.85+ 0.09 24.90 + 0.13 24.96 + 0.11
0.49 + 0.07 0.53 + 0.11 0.60 + 0.09
24.20 + 0.08" 24.02+ 0.15"* 24.22 + 0.04*
24.72+ 0.09 24.53 + 0.21 24.68 + 0.05
0.52 + 0.05*** 0.51 + 0.09*** 0.46 + 0.04***
* P < 0.05. ** P < 0.025. *** Not significantly different from preoperative values.
sence of labeled retinal processes on the side of the lesion (Fig. 1B, C). A n additional 10 hamsters incurred sub-total destruction of one of the S C N that involved < 30% tissue d a m a g e in 7 animals and approximately 67% of the nucleus in 3 hamsters (Fig. 1D); for purposes of analysis these animals were treated as a single group. The SCN were d a m a g e d bilaterally in two hamsters, but their erratic wheel-running behavior p r e c l u d e d assessment of responsiveness to D 2 0 .
Preoperative measures The baseline (tap water) free-running p e r i o d (r) of the activity rhythm in L L for animals that c o m p l e t e d the experiment was 24.36 + 0.02 h; r was increased by 0.56 + 0.05 h ( P < 0.001) during ingestion of D 2 0 . Baseline r and responsiveness to D 2 0 did not differ significantly during p r e o p e r a t i v e testing for animals assigned to the different lesion groups (Table II). Postoperative testing Baseline r (tap water) was shorter p o s t o p e r a t i v e l y (Days 100-138) than p r e o p e r a t i v e l y (Days 15-60), irrespective of the amount of d a m a g e to the SCN. F o r the 6 s h a m - o p e r a t e d animals the decrease was 0.11 + 0.07 h (P > 0.05); for the 20 lesioned animals for which accurate pre- and p o s t o p e r a t i v e measures were available, r decreased by 0.31 + 0.08 h ( P < 0.001). D a t a for individual hamsters are shown in Fig. 2. The postoperative decrease in baseline r values was substantially greater for a subset of animals that sustained complete unilateral ablation of the SCN than for o t h e r hamsters. Thus, 4 of 9 h a m sters with complete unilateral SCN lesions manifested large postoperative decreases in r that were
out of the range of changes o b s e r v e d in o t h e r animals. The p o s t o p e r a t i v e decrease in baseline r among hamsters with c o m p l e t e unilateral SCN lesions did not correlate with the extent or locus of damage to the SCN or surrounding tissue (Fig. 1B, C).
Response to deuteration The response to D 2 0 was similar pre- and postoperatively for each of the groups of hamsters (Table II) and was i n d e p e n d e n t of the baseline free-running period or of the degree of d a m a g e to the SCN. H o w ever, responsiveness to D 2 0 was m o r e variable in animals sustaining SCN lesions than in neurologically intact hamsters (Table III); when expressed as the absolute difference in r attributable to d e u t e r a t i o n p r e o p e r a t i v e l y minus that r e c o r d e d postoperatively,
24.4
24.0.
23.6
23.2
iNTACTSCN
COMPLETE UNILATERAL SCN LESION
iNCOMPLETE UNILATERAL SCN LESION
Fig. 2. Free-running period (T) of individual hamsters maintained in constant light (LL) and drinking tap water. The postoperative r was decreased in each group of animals irrespective of the amount of SCN damage. The horizontal and vertical bars to the left and right of each set of lines indicate the group mean and S.E.M. r before and after surgery, respectively.
153 TABLE III Increase in the period of the circadian activity rhythm (h) induced by D20 in individual hamsters before and after SCN lesions
A, absolute value of postoperative minus preoperative effects of DzO. Intact
Unilateral SCN-x
Partial SCN-x
Preoperation
Postoperation
A
Preoperation
Postoperation
A
Preoperation
Postoperation
A
0.81' 0.61" 0.36* 0.53* 0.18" 0.49* 0.58 0.35
0.74 0.64 0.44 0.54 0.31 0.50 0.55 0.44
0.07 0.03 0.08 0.01 0.13 0.01 0.03 0.09
0.74 0.57 0.24 0.16 0.79 0.44 0.79
0.62 0.90 0.57 0.28 0.52 0.25 0.43
0.12 0.33 0.33 0.12 0.27 0.19 0.36
0.20 0.72 0.93 0.90 0.56 0.47 0.66 0.29 0.73
0.33 0.24 0.44 0.59 0.56 0.47 0.55 0.46 0.46
0.13 0.48 0.49 0.31 0.00 0.00 0.11 0.17 0.27
* Sham-operated.
the value for animals without damage to the SCN was 0.06 + 0.02 h as compared to values of 0.25 + 0.04 and 0.22 + 0.06 h, respectively for animals with complete and incomplete unilateral SCN lesions (P < 0.005 and P < 0.025 for intact hamsters vs those with complete and incomplete unilateral SCN damage, respectively). Postoperatively, all animals drank significantly less deuterium oxide; intact hamsters consumed 85.2% of the preoperative level (P < 0.05), unilaterally SCN-lesioned animals 84.1% (P < 0.01) and animals with partial lesions of the SCN, 82.7% (P < 0.01). Percentage differences in postoperative D20 intake relative to preoperative baselines did not differ among the groups. DISCUSSION The extent to which D20 lengthens the period of the circadian activity rhythm was unaffected by unilateral ablation of the hamster SCN: increases in period length attributable to ingestion of D20 were similar in neurologically intact hamsters and those sustaining complete or partial ablation of one of the bilaterally paired SCN. Neither coupling between the two SCN nor elimination of one half of the normal complement of SCN neurons influenced responsiveness of the circadian system to D20. We tentatively conclude that if sufficient neural tissue remains to generate a coherent circadian rhythm, the periodlengthening response to deuterium will be normal. This conjecture might best be evaluated in hamsters
with the minimal number of SCN neurons consistent with normal circadian organization. It is not known whether partial bilateral destruction of the SCN has a greater influence on responsiveness to D20 than an equivalent amount of unilateral damage. The circadian behavior of two animals with partial bilateral damage to the SCN was too erratic to permit accurate determination of the effects of deuteration on the circadian period. Mutual coupling between the two SCN appears to influence the period of the circadian activity rhythm. The r was reduced by 0.92 + 0.09 h postoperatively in 4 of 9 hamsters with complete unilateral SCN lesions; for two of these hamsters r was in the range of 23.3 h. Five animals with similar SCN damage manifested only a slight decrease in r (0.14 + 0.04 h), equivalent in magnitude to the changes observed in sham-operated animals. These findings confirm those of a prior study in which circadian period was reduced by 0.61 h in 5 of 7 hamsters incurring complete unilateral SCN destruction 17. Unilateral ablation of the SCN also altered the period of circadian rhythms in birds 4. Unilateral excision of the optic lobes of cockroaches consistently affected the period of circadian oscillations 13and the two bilaterally symmetric circadian pacemakers in the eyes of Aplysia became desynchronized from each other in constant light8. Taken together, these findings suggest that mutual coupling between bilaterally paired pacemakers contributes to circadian organization. We were unable to discern any correlation between locus and extent of unilateral lesions and post-
154 operative shortening in the p e r i o d of the circadian activity rhythm. Extensive individual variation remains a perplexing p h e n o m e n o n in several types of circadian investigations and is particularly characteristic of the splitting p h e n o m e n o n in constant illumination 18. Unilateral ablation of the S C N can abolish splitting of the circadian activity r h y t h m into distinct c o m p o n e n t s 16. W e speculate that mutual coupling between the bilateral SCN p a c e m a k e r s m a y decelerate each individual oscillator system in some hamsters, t h e r e b y producing a longer p e r i o d in the coupled state. U n i l a t e r a l ablation of the SCN may shorten the p e r i o d of the overt circadian r h y t h m in these animals. Evidence that the two SCN are not always mutually synchronized comes from a study of rats showing a significant phase difference in simultaneously r e c o r d e d multiple unit activity of the two SCN 10. A l t h o u g h there is no effect of unilateral SCN ablaREFERENCES 1 Daan, S. and Pittendrigh, C.S., A functional analysis of circadian pacemakers in nocturnal rodents. III. Heavy water and constant light: homeostasis of frequency? J. Comp. Physiol., 106 (1976) 267-290. 2 Davis, F.C. and Gorski, R.A., Unilateral lesions of the hamster suprachiasmatic nuclei: evidence for redundant control of circadian rhythms, J. Comp. Physiol., 154 (1984) 221-232. 3 Dowse, H.B. and Palmer, J.D., The chronomutagenic effect of deuterium oxide on the period and entrainment of a biological rhythm, Biol. Bull., 143 (1972) 513-524. 4 Ebihara, S. and Kawamura, H., The role of the pineal organ and the suprachiasmatic nucleus in the control of circadian locomotor rhythms in the Java sparrow, Padda oryzivora, J. Comp. Physiol., 141 (1981) 207-214. 5 Fitzgerald, K., Zucker, I. and Rusak, B., An evaluation of the homeostasis of circadian periodicity in the golden hamster, J. Comp. Physiol., 123 (1978) 265-269. 6 Frost, D.O., So, K.-F. and Schneider, G.E., Postnatal development of retinal projections in Syrian hamsters: a study using autoradiographic and anterograde degeneration techniques, Neuroscience, 4 (1979) 1649-1677. 7 Green, D.J. and Gillette, R., Circadian rhythms of firing rate recorded from single cells in the rat suprachiasmatic brain slice, Brain Research, 245 (1982) 198-200. 8 Hudson, D.J. and Lickey, M.E., Internal desynchronization between two identified circadian oscillators in Aplysia, Brain Research, 183 (1980) 481-485. 9 Inouye, S.T. and Kawamura, H., Persistence of circadian rhythmicity in a mammalian hypothalamic 'island' containing the suprachiasmatic nucleus, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5962-5966. 10 Inouye, S.T. and Kawamura, H., Characteristics of a circadian pacemaker in the suprachiasmatic nucleus, J. Cornp. Physiol., 146 (1982) 153-160. 11 Katz, J.J. and Crespi, H.L., Isotope effects in biological systems. In C.J. Collins and N.S. Bowman (Eds.), Isotope Effects in Chemical Reactions, Van Nostrand Reinhold,
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