The effect of nembutal on the estrous activity cycle

Physiology and Behavior. Vol. 4, pp. 963-964. Pergamon Press, 1969. Printed in Great Britain

The Effect of Nembutal on the Estrous Activity Cycle' J A M E S R. W H I T M A N

Veterans Administration Hospital, American Lake, Tacoma, Washington 98493 AND EPHRAIM

PERETZ

Oregon Regional Primate Research Center, Beaverton, Oregon 97006 (Received 7 July 1969) WHITMAN, J. R. ANDE. PERETZ. The effect of nembutal on the estrous activity cycle. PHYSIOL.BEHAV.4 (6) 963-964, 1969.The purpose of this study was to determine whether or not the day of peak activity for rats showing regular estrous activity cycles is delayed by one day when sodium pentobarbital (Nembutal) is administered under the conditions found by Everett and Sawyer to be effective for delaying ovulation. Thirteen rats with regular 4-day estrous activity cycles received Nembutal and saline treatments on days of proestrus. Treatments were administered in a counterbalanced order; and after each, the day of peak activity in subsequent cycles was determined. For 12 of 13 animals treated with Nembutal the day of peak activity was delayed by one day; whereas for 11 of 12 animals, when saline was administared, the day of peak activity was not delayed.

Estrous activity cycle

Sodium pentobarbital

Reproductive cycle

EVERETT and Sawyer [1] showed that a barbiturate administered to rats on the day of proestrus delayed ovulation by one day. Ovulation was delayed only if the barbiturate was given at certain hours determined by the lighting schedule. The period when a barbiturate is effective is referred to as the "critical period". Because the ovary is necessary for the estrous activity cycle and because the amount of activity displayed by the rat over the course of the cycle is greatest on the night of ovulation, it is of interest to know if barbiturates can also delay the period of maximal activity. If such an effect were obtained, a door would be opened to analysis of horrnonal mechanisms of the estrous activity cycle in the "intact" animal by blocking and augmenting the various naturally occurring hormonal changes. The specific purpose of this study then was to determine whether or not the day of peak activity for rats showing regular estrous activity cycles is delayed by one day when sodium pentobarbital (Nembutal, Abbott) is administered under the conditions found by Everett and Sawyer to be effective for delaying ovulation.

start of the experiment, during which they were studied for a period of 55 days. F r o m the time they were introduced into the laboratory until the experiment was terminated, the animals were maintained in Wahmann activity wheels housed in a light-proofed and temperature-regulated room in which the temperature ranged between 70-75°F. Fluorescent overhead lighting controlled by a time switch was provided daily for a 14-hr period starting at 5.00 a.m. F o o d and water were always available. The animals were given a period of 28 days to adapt to the wheel and the maintenance conditions. Following the adaptation period, all the animals were used in a preliminary study in which they received one injection either of saline or Nembutal. The experiment began an average of 21.4 days (range 14-30 days) after the preliminary study. Activity was measured in terms of the number of revolutions of the wheel, which were registered on counters in a room adjacent to the experimental rooms. At each revolution a cam on the wheel closed an electric circuit which activated an impulse counter. Counter readings were recorded daily at 9.00 a.m. The criterion for selection of an animal was that it displayed two successive 4-day activity cycles. Treatment was given on the day ofproestrus of the third cycle. Following this treatment when rat again showed two successive 4-day cycles, the second treatment was given. Treatments were given in counterbalanced order. All treatments were given at 2.00 p.m.,

MATERIALS AND METHOD

Thirteen random-bred Sprague-Dawley albino female rats served as subjects. They were obtained commercially from Bio-Science Animal Laboratories in Oakland, Ca. The estimated average age, based on weight, was 126 days at the

Whe assistance of Mr. N. Roberts in the collection of data is acknowledged. 963

964

WHITMAN AND PERETZ TABLE 1.

EFFECTS

OF

NEMBUTAL

AND

SALINE

ON

ESTROUS

ACTIVITY

CYCLES

the presumed beginning of the critical period. Treatment consisted of the intraperitoneal injection of 0.2 cc of 0.9 per cent saline or one of Nembutal (30 mg/kg of body weight).

Effect

RESULTS

Treatment Order of Number of Number ofAnimals for which Treatment Animals Peak was: Delayed Not Delayed Nembutal

I st 2nd

6 7 13

6 6 12

1 1

I st 2nd

7 5* 12

-1 1

7 4 11

Total Saline Total

* Entry does not include an animal which showed no peak on the cycle in which criterion measure was made. However, on the night following saline injection a peak occurred and from the two following cycles the evidence indicated that the peak had not been delayed.

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In order to evaluate the effect of Nembutal on the estrous activity cycle, the depression of activity produced by the drug had to be taken into account. Since Nembutal was given on the afternoon of proestrus, it would have been no surprise that activity would be lower that evening and hence a peak in the following night could be an artifact of the depressant effect on activity. To overcome this difficulty, it was assumed that if Nembutal does block the peak of activity a new series of 4-day cycles beginning one day later than normal would be initiated. The delay could then be measured on the cycle following Nembutal injection at which time the depressant action would have disappeared: the peak of that cycle would occur one day later than it would occur had Nembutal not been given. The results are summarized in Table 1, which shows that for 12 of the 13 animals treated with Nembutal the peak of activity was delayed, whereas for 11 of the 12 animals treated with saline the peak was not delayed. The delay was constant, in all cases being one day. The same results were obtained regardless of whether Nembutal or saline was given first. It was also found that when a delay occurred, subsequent cycles were 4 days long for at least 2 cycles, after which the experiment was terminated. Figure 1 shows the activity records for 2 animals illustrating the delay following Nembutal treatment and the absence of any delay following saline treatment.

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DAYS

FIG. 1. Daily measurements for two animals plotted illustrating the delay with the Nembutal (N) treatment and the absence of the delay with the saline (S) treatment.

The results show clearly that Nembutal given at a particular time in the light-dark cycle produced a 1-day delay in the peak of the estrous activity cycle. Although no systematic attempt was made to determine the critical period for delaying peak activity, evidence gathered in the preliminary study indicates that when Nembutal was given at 3.00 p.m. on the day of proestrus the delay did not occur. The effect obtained in the experiment is analogous to the delay in ovulation produced by Everett and Sawyer [1], and the inference is that the delay in peak activity and in ovulation occurred together. In this experiment, however, no attempt was made to determine whether ovulation was delayed. The parallelism between the two effects suggests that delay in peak activity is the result of the prolongation of the life of the follicles to be ovulated. Since such a delay is produced in the intact animal, this experiment provides a new approach for the analysis of hormonal mechanisms regulating the estrous activity cycle. By blocking ovulation and extending the life of the follicle we have retarded the hormonal clock by one day without destroying the processes producing cycling. By experimental manipulation of follicular development we should be able to produce parrallel changes in the estrous activity cycle.

REFERENCE 1. Everett, J. W. and C. H. Sawyer. A 24-hour Periodicity in the "LH-release apparatus" of female rats, disclosed by barbiturate sedation. Endocrinology47: 198-218, t950.