Implication of the estrous cycle on conditioned avoidance behavior in the rat

Physiology & Behavior, Vol. 21, pp. 441--446. Pergamon Press and Brain Research Publ., 1978. Printed in the U.S.A.

Implication of the Estrous Cycle on Conditioned Avoidance Behavior in the Rat ANASTASIA SFIKAKIS, CHRISTINA SPYRAKI, NIKOLAS SITARAS AND DENIS VARONOS

Laboratory of Experimental Pharmacology, School of Medicine, University of Athens, Goudi, 609 Athens, Greece (Received 4 August 1977) SFIKAKIS, A., CH. SPYRAKI, N. SITARAS AND D. VARONOS. Implication o f the estrous cycle on conditioned avoidance behavior in the rat. PHYSIOL. BEHAV. 21(3) 441--446, 1978.--The two-way conditioned avoidance response, serum estradiol, food daily consumption, water intake and urine output were simultaneously studied in rats during the various stages of the estrous cycle. Rats in proestrus exhibited: (a) a significant facilitation of conditioned avoidance response in relation to the intervening stages of the cycle (diestrus and estrus), (b) a highly significant rise in serum estradiol concentration, and (c) a significant depression in food intake and urine output. An impairment of conditioned avoidance response occurred at diestrus when compared with metestrous rats and castrated rats. Rats in metestrus exhibited a significant decrease of serum estradiol concentrations. The rise in serum estradiol at proestrus suggests a negative control upon food intake, a decrease in urine output and involvement in the facilitation of conditioned avoidance response.

Estrous cycle

Conditionedavoidance behavior

Estradiol

ALTHOUGH a number of investigators have determined the effect of gonadal hormones upon acquisition of conditioned avoidance behavior, either by injecting hormones [1,20] or surgically removing the gonads [19], the effect of the physiological hormonal changes, due to ovarian cyclicity, upon conditioned avoidance behavior has not been studied. The present experiments were designed to provide this information in correlation with estradiol levels in blood. The second purpose of the study was to relate variations in food intake during the estrous cycle [2, 9, 18] to endogenous estradiol levels, since lack of estrogens [12, 15, 18] or estrogen replacement [12,21] exert important regulatory influences on eating behavior. Furthermore, considering the effect of vasopressin (antidiuretic hormone [5] upon acquisition of the avoidance behavior and the variations in urine output during the cycle (unpublished observations), we determined whether cyclic variations in urine output and water intake are correlated with the results of the avoidance test. METHOD

Animals Female rats (Wistar strain) reared in our laboratory were used. Animals were separated from their male litter mates at 12 days of age, weaned at 24 days and kept in plastic cages in groups of eight animals. Food and water were provided ad lib and animals were kept under constant lighting conditions (14 hr light-10 hr dark, the dark cycle beginning at 8:00 p.m,) Vaginal smears were taken daily during at least five consecutive estrous cycles. Vaginal smears were obtained between 11 and 12 a.m. and colored by Giemsa solution 1:2 for 30 min [23].

Food intake

Urineoutput

Experiment 1 Fifty five rats 2-5 '/2 months of age were tested for acquisition of a conditioned avoidance behavior. Animals exhibiting two or more consistent 4 day estrous cycles and ovariectomized animals were utilized. A cycle was represented by vaginal smears with the typical cell population characteristic of the various stages of the cycle [23] and in the consecutive order diestrus (D) proestrus (P), estrus (E), metestrus (M). Rats were divided into 4 groups, each group representing one phase of the estrous cycle. Ovariectomized animals were tested ten days after surgical removal of the ovaries. The test was performed between 2 and 5 p.m. in a room isolated from external noise. The apparatus used for the study of the conditioned avoidance behavior was a two-way shuttle box described previously [14]. Each animal was placed in the standard avoidance conditioning apparatus for 70 min. An initial 10 min adaptation period was allowed to elapse. During the 60 min testing period the animal was presented every min with the conditioned visual stimulus (CS, light) for 6 sec, immediately followed by 4.24 sec of both the CS and unconditioned stimulus (UCS, 45 V electric shock) and a 49.7 sec rest period. The following parameters were utilized for analysis of these data: (1) Rate of performance (RP): The mean time in sec of the 60 efforts. (2) Error score (ES): The number of unsuccessful efforts in avoidance response. (3) Conditioned stimulus latency (CSL): The mean time in sec of the avoidance response. (4) Unconditioned stimulus latency (USL): The mean time in sec of escape response. The statistical analysis of the data was performed according to Wilcoxon-White test.

'We appreciate the Technical Assistance of M. Baitazani and F. Kapetopoulou.

Copyright © 1978 Brain Research Publications Inc.--0031-9384/78/090441-06502.00/0

442 Rats exhibiting estrous cycles were tested only once, since the purpose of the study was to investigate whether the day to day variations in hormonal milieu during the cycle might influence the avoidance behavior. Ovariectomized rats that had not achieved criterion on the first session were tested on consecutive days until criterion was achieved (criterion: 12 consecutive successful trials). All rats were autopsied at the end of the study; the uteri of intact rats were removed and weighed on an electric balance. Serum estradiol concentrations were determined in the above rats during the various stages of the estrous cycle at 2--4 p.m., 4 days following avoidance testing. Blood was obtained via the orbital sinus of the rat without general anaesthesia (24) with microhematocrit tubes (length 0.075 m internal dia. 0.0011 m) containing 2 u.s.p, units of ammonium Heparin in order to prevent clotting during the time of blood withdrawal. Only specimens obtained within one min of immobilization of the rat were used in order to avoid possible interference of stress upon estradiol levels in blood. Blood was collected into centrifuge tubes and after clotting at room temperature and centrifugation, serum was recovered and stored at -20°C until assayed.

Estradiol-I 7~ Assay Serum samples (1 ml) were extracted with 3 ml of diethyl ether for 2 rain with a vortex mixer and centrifuged in a freezing centrifuge. Diethyl ether (analytical grade; Merck Darmstadt) was used after being freed from peroxides by silver nitrate before distillation. The radioimmunoassay was performed on 1.5 ml of the extract dried under nitrogen gas at 35--40°C. The standard estradiol curve (12.5--400 pg) was prepared in the presence of 1.5 ml of solvent in Order to account for solvent blank interference. Estradiol radioimmunoassay was performed using kits supplied by CEAIRE-SORIN. The antiserum provided by the kit was specific enough [11] to permit the assay on dried solvent extracts without prior chromatography, however an overestimation of estradiol values by 5 percent due to cross reaction with estrone should be taken into account. The antiserum was obtained in rabbit immunized with estradiol-6-human thyroglobulin-conjugate (prepared by condensation of estradiol-6-(o-carboxymethyl) oxime, which was obtained from 6-keto estradiol, manufactured by Steraloid). The tracer 2,4,6,7 3H-estradiol, specific activity 350-400 /xCi//~g was diluted at the time of the assay 1:20. The diluent was a phosphate buffer, ph 7.4, containing EDTA sodium salt and bovine serum albumin. The incubation volumes were 0.3 ml of diluent with standard or with dried extract, 0. l ml of antiserum and 0. l ml of :~H estradiol. After mixing with a vortex the tubes were incubated for 30 min at 37°C and 2 hr at 4°C. Bound :~H-estradiol was determined after removal of unbound ligand with dextran coated charcoal. After 10 min centrifugation 0.5 ml of the supernatant was removed, transfered into a vial containing 10 mi of the scintillation coktail (PPO, POPOP, naphtalene, dioxane) and counted for 10 min in a Tri carb scintillation spectrometer (Packard). The recovery of '~H estradiol added to the blood serum was 97%. When 200 pg, 100 pg and 50 pg standard estradiol were added to the serum pool and assayed the values were 82.5, 80 and 7 8 ~ respectively of those expected. The sensitivity of the assay was 3.2 -+ 1.2 pg. The inter assay coefficient of variation for the standard and intra assay coefficient of variation for rat serum pool were 10.3 and 11.3% respectively.

S F I K A K I S , SPYRAKI. SITARAS AND VARONOS F o r statistical evaluation of estradiol and uterine data the Student t test was used.

Experiment 2 Food consumption, water intake and urine output were monitored over 24 hr and between 8:45 and 9:15 a.m., in 12 rats 3-3 ~/2 months of age during 4 to 5 consecutive estrous cycles starting two or three cycles after the avoidance test. Rats were kept in individual metabolic cages (techniplast Art 1700) and fed laboratory chow ad lib. Vaginal smears were examined daily. Mean values of the three parameters studied during the different stages of the cycle were statistically evaluated by the Student t test. RESULTS

Experiment 1 The results of the conditioned avoidance behavior at the four different stages of the estrous cycle and in ovariectomized rats are summarized in Fig. 1. Rate performance and error score were significantly decreased at proestrus (P) in relation to the intervening stages of the cycle diestrus (D) and estrus (E), indicating a facilitation for the aquisition of the conditioned avoidance response. Rats in diestrus had impaired performance compared to animals in proestrus or metestrus (M) or castrated rats. The stages of the cycle and castration had no significant effect on conditioned stimulus latency, while the rapidity of escape was significantly decreased during diestrus and enhanced at proestrus. The number of rats that achieved criterion were: 1/! 1 (9%) at diestrus, 2/10 (20%) estrus, 10/14 (71.4%) at proestrus, 6/11 (54.5%) at metestrus and 5/9 (55%) in ovariectomized rats. The number of trials to criterion in ovariectomized rats was 59 -+ 13.56. The weight o f the estrous cycling rats on the day of the avoidance test was quite similar (D:207 ~ 12: P:196 ± 13; E:202 -~ 13.9; M:207 _+ 17.5;) and did not markedly differ from that of the castrated rats/234.6 - 12.10). Serum estradiol concentrations ranged from 50 to 174 pg/ml and were greater (p<0.005) on proestrus, than during the other three stages of the cycle (Table 1). A decrease (p<0.05) in estradiol levels were found on metestrus. The uterine weight was significantly greater (Table 1) on proestrus than on the other three stages of the cycle and significantly lower on metestrus.

Experiment 2 Clear consecutive 4 day cycles were obtained in 7/12 rats and food and water intake and urine output data are presented for these animals. The results of the remaining rats are not presented because in 3/12 rats the 4 day estrouscycles were followed by five-day estrous cycles (two consecutive estrus smears) or (two consecutive diestrus smears) while in 2/12 rats the cycles deteriorated with the appearance of diestrus smears, possibly, because of stimulation of the cervix due to improper manipulation. A significant decrease in food intake and urine output occurred on proestrus when compared to diestrus in 5 of 7 rats (Fig. 2). Significant day to day variation occurred for each rat but the decline at proestrus persisted. A significant decline in food (p<0.001) and water (p<0.05) intake and in urine output (p<0.01) occurred in proestrus when compared to the mean level of the four stages (Table 2).

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FIG. 1. The effect of the estrous cycle (D; diestrus, P; proestrus E; estrus, M; metestrus) and castration (CA) on conditioned avoidance behavior (vertical bars: means _+ SE). Numbers of animals are in parentheses. *Significantly diferent (/7<0.01) from proestrus **Significantly different (p <0.05) from diestrus

444

SFIKAKIS, SPYRAKI, SITARAS AND VARONOS TABLE 1 SERUM ESTRADIOLCONCENTRATIONSAND UTERINEWEIGHTSIN RATSDURINGTHE ESTROUS CYCLE§ Stage of estrous cycle

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Estradiol (pg/ml)

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83.3 _+ 6.27 123.5 _+ 9.32* 79.5 + 5.89 63.2 _+ 4.28:~

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B.W. (g)

(9) 147.9 _+ 7.06 (10) 313.3 -+ 18.317 (8) 191.7 -* 12.07 (8) 126.2 -* 6.33~:

(9) 210.3 + 10.10 (10) 209.3 _+ 18.92 (8) 225.6 + 15.24 (8) 234.6 + 14.25

§ Means __- SEM Number of animals in parentheses * Significantly different (p<0.005) from the other stages of the estrous cycle t Significantly different (p<0.0005) from the other stages of the estrous cycle ~t Significantly different (p<0.05) from diestrus and estrus TABLE 2 DALLYFOOD AND WATERINTAKEAND URINE OUTPUTIN RATSDURINGTHE ESTROUS CYCLE§ Stage of estrous cycle Diestrus Proestrus Estrus Metestrus Mean level of the 4 stages

Food Intake (g)

Water Intake (ml) Urine Output (ml)

(7) 20.66 _+ 0.79 (7) 16.23 ___0.36 (7) 19.53 _+ 0.41 (7) 20.76 _+ 0.87

32.55 _+ 0.88 27.54 ___ 1.08 32.37 _+ 1.09 32.73 _+ 0.61

13.51 _+ 0.81 10.32 _+ 0.76 13.22 +- 0.92 13.83 _+ 0.83

(28) 18.90 _+ 0.52*

31.30 _+ 1.39t

12.72 _+ 0.475

§ Mean _+ SEM * Significantly different from proestrus p<0.001 t Significantly different from proestrus p<0.05 $ Significantly different from proestrus p<0.01

Figure 3 depicts the average cycle based on the percentage transformed deviation from the mean level, of food, water intake and urine output data, from 30 estrous cycles. The depression as per cent deviations from the mean on the day of proestrus with respect to food, water and urine were 14.12, 12.0 and 18.86 respectively. DISCUSSION The present study indicates that the estrous cycle in rats influences the two way conditioned avoidance performance. This influence determined between 14.00 and 17.00 hr seems to be a facilitation of the response at proestrus and a deterioration at the intervening phases of the cycle diestrus and estrus. The avoidance response 10 days after gonadectomy appears similar to the avoidance at metestrus but significantly different from that during the diestrns stage of the cycle. Telegdy et al., [ 19] using the established technique for the acquisition of conditioned avoidance behavior found no difference between estrous cycling and castrated rats. The results of the present study would agree with this report [19] if the cycling rats were considered as an homogenous group, irrespective of day to day variations of the estrous cycle. Significant differences in estrous cycling animals would not be revealed when compared collectively with ovariectomized rats, because the lower performance at diestrus and

estrus would be obscured by the higher performance at proestrus and metestrus. Serum estradiol concentrations in rats, of the present study ranged from 50 to 174 pgtml with the highest levels on the day of proestrus. These values are similar to the range (80-211 pg/ml) obtained by Shaikh and Shaikh [16] using radioimmunoassay, but higher than those obtained by Butcher et al., [3]. An overestimation of our estradiol values by 5% due to cross reaction with estrone may account for these differences. Despite differences in ab~alute levels of our estradiol data when compared with studies using competitive protein binding assays [13], or radioimmunoassays [3,7], the rise in estradiol concentrations on proestrus is consistent with these studies [3, 7, 13]. The decrease of estrogen levels during metestrus (1400 to 1600 hr) agrees with Butcher et al., [3] who observed the lowest level of estradiol between midnight ofestrus and 1500 hr of metestrus. Estradiol was undetectable in ovarian vein plasma on the morning of the day of metestrns [17], which adds further support to our finding. The effect of estradiol levels on avoidance behavior is unknown. In the present study proestrus was associated with a significant rise in serum estradiol and facilitation of the avoidance response. Diestrus and estrus were associated with lower serum estradiol levels and reduction in avoidance response. The mechanism of action of estradiol on behavioral patterns is unclear since our knowledge with respect to

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FIG. 2. The individual cyclical variations in food consumption water intake and urine output during 4 day estrous cycles in 7 rats. Diestrus; D, Proestrus; P, Estrus; E, Metestrus; M. *Significantly different (p<0.0.~-p<0.001) from proestrus tSignificantly different (0.05
estrogens and to different neurotransmitters which might influence behavior is very limited. Estrogen might facilitate avoidance behavior indirectly by stimulating corticotrophin (ACTH) release [8]• ACTH might explain the augmentation of avoidance behavior through its fah'ly well established action on avoidance acquisition in rat [4, 5, 6] and possibly through release of mineralocorticoids [22]• However, the possible relation of other hormones [3] cannot be overlooked. Thus, increased levels of progesterone at estrus [16], might be responsible in part for the deterioration of behavior at this stage of the cycle [1]. The fact that the time to escape shock was signitlcantly lower on proestrus than on diestrus, suggests that pain

thresholds were lower on proestrus and the emotional response possibly higher• These factors and thus more rapid responses to electric shock stimuli might be responsible for the higher avoidance on proestrus. However, as there were differences in avoidance response, but no differences in the interval required for shock escape between diestrus and metestms and proestrus and estrus, the above mentioned factors could not be further implicated in the observed differences in avoidance. A depression in food consumption, water intake and urine output occurred on proestrus in the present study in rats exhibiting four day estrous cycles, which appeared more significant with respect to food and urine than to water. A

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FIG. 3. Food and water intake and urine output in 7 rats during the various stages of the estrous cycle expressed as percent deviation from the mean. Diestrus; D, Proestrus; P, Estrus; E, Metestrus; M.

significant d e c r e a s e in food and water intake at estrus in rat.~ exhibiting five days' estrous cycles have been demonstrated by Tartellin and Gorski [18] under a r e v e r s e d lighting schedule. Since this period in terms of conventional experiments would be the night of vaginal proestrus, the results of the present study are in good a g r e e m e n t with those of Tartellin and Gorski [18]. A significant d e c r e a s e in food intake on proestrus has been noticed also by ter H a a r [9]. Our finding of a significant rise in serum estradiol levels and depression in food intake during proestrus and previous data [12, 15, 18, 21], point to an association role of endogenous estradiol and eating behavior. O f further interest in the present study was the correlation of a depression in urine output and i m p r o v e d avoidance response during proestrus, and increase in urine output and a deficit in response at diestrus and estrus. The reason for the d e c r e a s e in urine output at proestrus is u n k n o w n at present. The d e c r e a s e in food and w a t e r certainly are contributing factors. It is n o t e w o r t h y h o w e v e r that Legros et al., [10] d e m o n s t r a t e d an increase in neurophysin (carrier o f o x y t o c i n and vasopressin) in w o m e n , during the ovulatory as compared to the follicular phase. Possibly, the rise in antidiuretic h o r m o n e (vasopressin) may explain the l o w e r urine output at proestrus, and better performance, in view o f the well established facilitatory effect of lysine vasopressin on conditioned avoidance response [51.

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