Development of a fetal circadian rhythm after disruption of the maternal circadian system

DevelopmentalBrain Research, 41(1988)313-317 Elsevier

313

BRD60271

Development of a fetal circadian rhythm after disruption of the maternal circadian system Shigenobu Shibata 1'3 and Robert Y. Moore 1'2 Departments of 1Neurologyand 2Neurobiologyand Behavior, State Universityy of New York at Stony Brook, Stony Brook, NY 11794 (U.S.A.) and ~Departmentof Pharmacology, Faculty of PharmaceuticalSciences, Kyushu University, Fukuoka (Japan) (Accepted 8 March 1988) Key words: Circadian rhythm; Suprachiasmatic nucleus; Development; 2-Deoxyglucose method; Maternal influence

The role of maternal circadian rhythms in the development of the fetal circadian system was investigated in the rat. Pregnant females were subjected to procedures known to disrupt circadian function, ablation of the maternal suprachiasmatic nuclei (SCN) or housing in constant illumination, on gestational day 10. Circadian function was assessed in fetuses at gestational day 22 by analysis of glucose utilization in hypothalamic slices in vitro using the 2-deoxyglucose method. Fetuses from control females exhibit a robust rhythm in glucose utilization in the SCN. In contrast, the SCN of fetuses from females with SCN lesions, or housed in constant illumination, show no significant day-night difference in glucose utilization. Analysis of individual brains indicates, however, that this apparent disruption in the development of circadian rhythmicity in metabolism in the fetal SCN is due to a desynchronization of individual fetuses resulting from the loss of maternal entraining influences. Thus, the fetal SCN is capable of developing a circadian rhythm in glucose utilization independent of the maternal circadian system.

The development of circadian rhythms is a genetically determined event. In mammals the only established circadian pacemaker is the suprachiasmatic hypothalamic nucleus (SCN). The SCN is formed in the rat from the diencephalic germinal matrix between embryonic days 13 (El3) and 17 (ref. 1). The SCN exhibits a rhythm in glucose metabolism that has been demonstrated by the 2-deoxyglucose (2DG) method both in vivo 11't2 and in vitro 6'ta'tS. This rhythm is the first circadian rhythm known to develop in the rat 3'8,9, appearing on El9. There are two interesting features of the development of the rhythm in glucose metabolism in the fetal SCN. First, it develops in a nucleus that is extremely immature and appears to have no intrinsic or extrinsic connections as it contains few, if any, synapses ~. Second, it is entrained to maternal rhythmicity 2'7's by mechanisms that are unknown 7. A circadian rhythm in neuronal activity has been observed at E22 and into the early postnatal period 13. This also appears entrained to

maternal rhythmicity. In a prior study, Reppert and Schwartz 1° demonstrated an apparent loss of the SCN 2-DG rhythm at E 2 0 - E 2 1 in vivo in fetuses from mothers subjected to SCN lesions at E7. The apparent loss of the rhythm occurred with individual pups exhibiting either high values or low values of relative optical density in the SCN autoradiograms at both subjective day and subjective night points. This suggests that the pups had independent, free-running rhythms. This is important because it addresses the issue of whether maternal influences are necessary both to initiate circadian function in and entrain the fetal SCN. For that reason we have chosen to repeat this experiment with two significant changes in experimental paradigm. First, we use constant illumination as well as SCN lesions to eliminate maternal rhythmicity because that does not mechanically disrupt hypothalamic function. Continuous light abolishes overt circadian rhythms in the rat and also eliminates a circadian variation in sensitivity of single

Correspondence: R.Y. Moore, Department of Neurology, HSC, T-12, SUNY at Stony Brook, Stony Brook, NY 11794-8121, U.S.A. 0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

314 units in the SCN and other brain areas to iontophoresed serotonin 5. This, then, provides an important

Pregnant S p r a g u e - D a w l e y rats were housed in a normal (lights on 07.00-19.00 h) or a reversed (lights

control for incidental effects of anterior hypothalam-

on 19.00-07.00 h) l i g h t - d a r k cycle from gestational

ic lesions that ablate the SCN as those lesions might affect endocrine or metabolic functions other than

days 2 - 2 2 except for the constant illumination (EL) group that was kept in constant light from gestational

circadian rhythm regulation. Second, we have car-

days 10-22. The light intensity was in the range of

ried out the 2-DG studies using a sensitive in vitro method that permits a direct m e a s u r e m e n t of local

mal in the cage. Bilateral lesions of the maternal SCN

glucose utilization.

were made during the light period on day 10 of gesta-

100 lux depending upon the exact location of the ani-

A

LD

LD

B

LL

LL

C

SCN

Lesion

SCN

Lesion

Fig. 1. Autoradiographs of coronal sections from hypothalamic slices containing the SCN from fetuses from control, continuous light (LL) and SCN lesion females obtained during either subjective day (A left, B right, C left) or subjective night (A right, B left, C right). Thus, the autoradiographs show the typical subjective day-subjective night difference in control brain in A. In B (LL group), the subjective day value is low and the subjective night high whereas in C (SCN lesion group) a subjective day brain with a high value and a subjective night brain with a low value are shown. The autoradiographs with high grain density over the SCN also show an apparent rim of high grain density along the chiasm. This is variable from slice to slice and is not a consistent finding. The location of the SCN on the sections was verified histologically. Marker bar = 500/~m.

315 from the chamber and rinsed for 30 min before sections are cut on a cryostat at 20 #m and dried on slides. The dry sections are exposed to Kodak 0M1 film for 2 weeks with radioactive standards (Amersham) included in each cassette for analysis. After exposure, sections are stained with Cresyl violet for histological analysis and the autoradiographs are analyzed by densitometry to permit a calculation of local glucose utilization (LGU, #mol glucose/100 g tissue/ min) as described previously6'15. The results of the in vitro 2-DG experiments are presented in Figs. l and 2 and in Tables I and II. Autoradiographs from control fetuses show a high density of silver grains over the SCN in slices studied during subjective day as compared to those studied during subjective night (Fig. 1A). The location of the SCN was confirmed histologically in all cases. Autoradiographs from fetuses of mothers with incomplete lesions show differences between subjective day and subjective night that do not differ from'controls and, for that reason, no individual examples from this group are shown. In contrast, fetuses from mothers with histologically confirmed, complete SCN lesions do not show a consistent pattern. In some cases, the SCN from subjective day or subjective night slices show a high grain density whereas in others it is low (Fig. 1C). The same is true of fetuses obtained from mothers maintained in LL (Fig. 1B). Individual values for LGU in SCN and A H A of fetuses from control, LL and complete SCN lesion mothers are shown in Fig. 2. Mean values for L G U in the SCN

tion (El0) under deep anesthesia with a mixture ot ketamine and xylazine. An insulated stainless steel wire electrode (tip length of 0.5 mm, diameter of 0.3 mm) was inserted into the brain at the following coordinates ( A - P , -0.2 mm from bregma; ventral 9.5 mm from the skull surface and lateral 0.3 mm from the midline). The lesion was made on each side by passing a direct current of 2 mA for 10 s. The location and extent of the lesions were verified by histological examination of 20 #m, Cresyl violet-stained coronal sections at the conclusion of the experiment. Control animals were sham-operated. At E22 the animals were decapitated and the brain quickly removed. Coronal hypothalamic slices (400-600 #m thick) including the SCN and anterior hypothalamus were prepared using a tissue chopper. In subsequent sections CT refers to circadian time in which lights-on is arbitrarily designated as CT 0000. For slice preparation, animals were sacrificed at CT 0500 for subjective day or at CT 1700 for subjective night experiments. Preparations were pre-incubated with a Krebs solution 6'15 continuously flowing at a rate of 2.2 ml/min for 1 h and then transferred to an incubation chamber. Incubations with isotope are carried out for 20 min in a recirculating system with a total chamber/reservoir volume of 13.3 ml with a flow rate of 4.4 ml/min. The preincubation and incubation buffers are identical except that the latter contains 1/.tCi/ml of 2-DG (1-14C-2-deoxy-o-glucose, spec. act. 50 mCi/mmol; Amersham). The slices are then removed Control (L r--I

~-40

= E

SCN

D)

LL

AHA

SCN

SCN

AHA

Lesion

SCN

AHA

0 0

-: _ 3 0 , 5 o

~ 9,,20,

~10

_-

g .

o

,.i I

l

•

o

"

oo

0 •

Subject=re

Day

0

Subiective

Night

Fig. 2. Localglucose utilization values for AHA and SCN from individual fetuses from control, EL and SCN lesion females. Each point represents the value from a single fetus. Fetuses from at least two litters were used to obtain the subjective day values and the subjective night values from each experimental group.

316 TABLE I Suprachiasmatic nucleus (SCN) and anterior hypothalamic area (AHA) metabolism in pregnant female rats

LGU is expressed as/~m glucose/100 g tissue/min + S.E.M. The number of animals in each sample is given in parentheses. Group

Local glucose utilization (L G U) Subjective day

Control Continuous light

Subjective night

SCN

AHA

SCN

AlIA

43.9 _+4.6 (4)* 31.3 _+4.2 (2)

14.7 ± 0.7 (4) 15.4 _+ 1.9 (2)

22.5 + 0.8 (4) 33.9 + 5.5 (2)

16.(1 _+0.4 (4) 17.8 +_0.3 (2)

* The LGU for subjective day control SCN is significantly higher than that for subjective night (P < 0.01, t-test). No other differences among SCN values or AHA values are statisticallysignificant.

and A H A from pregnant females in the control and LL groups are shown in Table I. Control females show a significant difference between subjective day and subjective night in the SCN, but not,in the A H A . Continuous light females show no significant d a y night difference and the values are intermediate between day and night values for controls. M e a n L G U values for SCN and A H A for all groups of fetuses are shown in Table II. Control and incomplete lesion SCN values for L G U show typical d a y - n i g h t differences. It should be noted, however, that the mean subjective day values for these groups are significantly lower than those for the control maternal SCN (P < 0.01) but the m e a n subjective night values are only slightly lower. This indicates that the amplitude of the rhythm is lower in the fetuses. In contrast to these controls, the SCN from fetuses from the complete maternal SCN lesion group and the maternal LL group do not show significant d a y - n i g h t differences in mean L G U values. In contrast to the controls, there is a wide range of L G U values in both groups with m a r k e d overlap of subjective day and

subjective night L G U values. No d a y - n i g h t differences are seen in any group in L G U values in A H A . The data obtained in this study indicate that rat fetuses can develop a rhythm in metabolism, as shown by the 2 - D G m e t h o d , in the SCN even when maternal rhythmicity is seriously disrupted. A s would be expected from prior studies 8-m, fetuses from control mothers show a rhythm in glucose utilization in the SCN which is identical to the rhythm of the mother, and this holds true even when the glucose utilization is measured in vitro in a hypothalamic slice. Thus, even though maternal rhythmicity is entraining the fetal SCN, it is not driving the rhythm. Disruption of maternal rhythmicity, either by SCN ablation at E l 0 , well before the birth dates of fetal SCN neurons, or by exposure of the m o t h e r to constant light, appears to abolish the relationship between maternal rhythmicity and fetal rhythmicity. That is, the loss of maternal rhythms p r o d u c e d by these treatments does not prevent the d e v e l o p m e n t of fetal rhythms but does disrupt the coordination of individual fetal rhythms as best this can be judged when each animal

TABLE II Suprachiasmatic nucleus (SCN) and anterior hypothalamic area metabolism in embryonic day 22 rat fetuses

See legend for Table I. Group

Local glucose utilization (L G U) Subjective day

Control SCN lesion - - incomplete SCN lesion - - complete Continuous light

Subjective night

SCN

AlIA

SCN

AHA

28.8-+ 1.4 (14)* 27.2 + 1.5 (10)* 21.1 +--2.0 (7) 19.7 + 2.8 (8)

11.2 + 0.9 (14) 11.0 _+0.8 (10) 10.5 + 1.1 (7) 8.9 +_0.6 (8)

18.0 + 0.9 (12) 16.0 + 0.8 (9) 20.3 --- 1.8 (12) 23.1 +_ 1.7 (8)

12.1 -+ 0.4 (12) 10.0 _+0.9 (9) 9.4 +_0.5 (12) 11.8 _+0.8 (8)

317 provides only one data point. If m e a n values are examined, the fetuses a p p e a r to have no rhythms but, when individual values are examined, the rhythm in glucose utilization in the SCN appears to be present. A t both time points e x a m i n e d , the L G U of individual SCNs varies from high to low. This strongly suggests that the fetuses are free-running i n d e p e n d e n t of each other. Thus, although fetuses may be e n t r a i n e d by maternal rhythms, no rhythm other than that in the SCN has d e v e l o p e d in the fetuses that allows them to be mutually entrained. Again, since fetal rhythmicity is expressed in vitro, it is i n d e p e n d e n t of any m a t e r nal influences at the time it is actually measured. This also indicates that any maternal rhythmicity that might be m a i n t a i n e d following the SCN lesion or exposure to continuous light is not sufficient to entrain the fetal SCN. Finally, can we state definitively that fetal rhythmicity can develop in the absence of m a t e r n a l rhyth-

micity? P r o b a b l y not as it is possible that some maternal influence might d e t e r m i n e the d e v e l o p m e n t of the ventral diencephalic germinal matrix in some critical way prior to E l 0 . This seems very unlikely, however, and our results are most consistent with the view that the d e v e l o p m e n t of circadian function in the SCN is a genetically d e t e r m i n e d process. This process is normally controlled by maternal influences to provide a coordination of maternal and fetal circa-

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dian function that fosters optimal fetal and postnatal d e v e l o p m e n t but the influences are not necessary for the initiation of circadian function in the fetal SCN.

This work was The manuscript ( R . Y . M . ) was a ences Institute, New York, NY.

s u p p o r t e d by N I H G r a n t NS-16304. was p r e p a r e d while one of us Fellow-in-Residence, the NeurosciNeurosciences Research Program,