Effects of prenatal stress on avoidance acquisition, open-field performance and lordotic behavior in male rats

Physiology & Behavior, Vol. 22, pp. 527-530. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.

Effects of Prenatal Stress on Avoidance Acquisition, Open-Field Performance and Lordotic Behavior in Male Rats R O B E R T L. M E I S E L , z G A R Y P. D O H A N I C H 3 A N D I N G E B O R G L. W A R D P s y c h o l o g y D e p a r t m e n t , Villanova University, Villanova, P A 19085 ( R e c e i v e d 7 April 1978) MEISEL, R. L., G. P. DOHANICH AND I. L. WARD. Effects of prenatal stress on avoidance acquisition, open-field performance and lordotic behavior in male rats. PHYSIOL. BEHA V. 22(3) 527-530, 1979.--Prenatal stress enhanced lordotic behavior potentials in male rats but did not feminize patterns of active avoidance acquisition or open-field performance. These results suggest that prenatal stress selectively feminizes some but not all behavior patterns shown to differentiate under the influence of perinatal gonadal hormones. In the rat, the critical period for the differentiation of active avoidance behavior appears to span prenatal and early neonatal ontogeny. Prenatal stress Lordotic behavior Sexual differentiation

Open-field behavior

P R E N A T A L stress has been shown to feminize and demasculinize the copulatory behavior of male rats [20,22]. The present study sought to assess the effect of this treatment on other sexually dimorphic behaviors. Specifically, open-field behavior and active avoidance performance differ reliably between adult male and female rats. Males perform significantly fewer avoidance responses and display faster rates of extinction than females [2, 3, 7, 13, 15], while females display higher levels of activity in an open-field than males [4, 6, 13]. Like copulatory behavior (see review by Ward, [22]), normal patterns of active avoidance and open-field behavior can be altered by manipulating circulating androgen levels during perinatal development. For instance, neonatal castration of male rats, particularly if combined with prenatal exposure to the antiandrogen cyproterone acetate, will feminize adult avoidance and open-field performance [8, 16, 18]. Conversely, injections of testosterone propionate to neonatal females will masculinize these same behavior patterns [2, 5, 6, 8, 12, 16]. It is not known whether a prenatal stress treatment shown to reduce plasma testosterone titers in fetal male rats [23], affects the development of sexually dimorphic behaviors other than patterns of copulation. METHOD

Animals Twenty-four male and 24 female rats (Sprague-Dawley,

Active avoidance acquisition

Inc., Madison, WI) were bred in the Villanova laboratory. The gestation period of this strain is 23 days. The animals were housed in a vivarium in which a reversed 12 hr light-12 hr dark cycle prevailed (lights off 0800 hr). Ad lib access to food and water existed at all times in the home cages. Apparatus Mothers were stressed in Plexiglas animal holders (13×6x8 cm) illuminated by two 150 W floodlights. The open-field apparatus consisted of a black square wooden box ( l l 6 x 116x41 cm). White lines divided the floor into 16 equal 29 cm squares. A removable start box fitted into the corner squares. The two way shuttleboxes used in this study were identical to the apparatus described by Schwartzbaum, Green, Beatty and Thompson [17]. This is a Plexiglas box (56x 19×31 cm) with a grid floor consisting of 3.2 mm aluminum bars spaced 16 mm apart. A central partition divided the box into two identical compartments. A 10 cm wide aperture in the central partition extended from floor to ceiling and allowed the rats to shuttle freely between the two compartments. Shuttling responses were recorded by photoelectric relays located in the side walls of each compartment, 10 cm from the partition and 19 mm above the floor. A 6 W light was positioned unilaterally in the side wall of each compartment opposite the relays, 14 cm from the end and 11 cm above the

1This study was supported by Grant HD-04688 from the National Institute of Child Health and Human Development and by Research Scientists Development Award, Type II I-K2MH-0049 from the National Institute of Mental Health. 2Now at Department of Psychology, University of Connecticut. aNow at Department of Zoology, Michigan State University.

C o p y r i g h t © 1979 Brain R e s e a r c h Publications Inc.--0031-9384/79/030527-04502.00/0

528 floor. These lights served as the CS and as a light source for the photoelectric relays. Shock was delivered to the grid floor by a high voltage Foringer AC shock source (Model 1154) through a Foringer grid shock scrambler (Model 1925). The UCS was a constant current 1.0 mA scrambled shock delivered through an external 820 k ~ resistor in series with the rat. Each shuttlebox was housed in a sound-attenuated wooden box (61x43x52 cm) equipped with an exhaust fan and a one-way vision mirror which allowed observation of the animals. Testing for sexual behavior took place in semicircular arenas (61x31 x31 cm) equipped with a Plexiglas front.

Procedure Six females were time-mated. Three of these were stressed daily from Day 14--21 of gestation. Day 1 of gestation is the day following mating. The stress procedure consisted of placing each mother into a Plexiglas restrainer for 45 min three times per day (0900, 1300, 1700 hr). Two hundred footcandles of light were directed onto the surface of the restrainers. Control mothers were not handled. All stress and control mothers delivered normally. Offspring were weighed and weaned at 23 days of age into group cages housing 2-3 animals of the same sex and treatment. Animals were eliminated randomly to create equal sized groups of 12 animals. A single open-field test was given at 65 days of age. The test apparatus was located in a dimly illuminated room (less than 5 ft-c.). The illumination level within the open-field was 3 fl-c. At the beginning of the test, the animal was restrained for 30 sec in a corner square of the open field by a barrier. The barrier then was removed allowing free access to the field for a 5 min period. The number of squares crossed and the number of boli excreted were recorded. The apparatus was thoroughly cleaned with 95% ethanol after each animal. All animals were tested between 1200 and 1900 hr of the same day. At approximately 80 days of age, each rat received six daily sessions of avoidance training. Before each training session the animal was placed into the shuttlebox for 5 min and allowed to habituate to the two compartments. Twenty-five avoidance trials on an FI-60 sec schedule then were given. The CS was the offset of light in the occupied compartment. Each CS was followed after 5 sec by gridshock. Shock could be terminated or avoided if the animal crossed to the opposite compartment. The number of avoidance responses per five trial block was recorded. All healthy males were castrated at approximately 115 days of age under Chloropent (Fort Dodge Labs) anesthesia. Body weight was taken at this time and the testes and epididymides were weighed on an analytic balance. Beginning one week later, each male was injected IM with 0.1 mg estradiol benzoate followed 44 hr later by 1.0 mg progesterone. This treatment was repeated once per week for three consecutive weeks. Tests for lordotic behavior were given 4.5 hr after the progesterone injection during the second and third week of injection. The test room was illuminated by a 100 W red light. Sexually vigorous stud males were allowed to adapt to the observation boxes for 5 min before an experimental male was added. The pair remained together until 7-10 mounts had been emitted by the stud male. A lordosis quotient (LQ), consisting of the ratio of lordotic responses to the number of times mounted x 100, was computed for each of the two test sessions.

MEISEL, D O H A N I C H AND WARD RESULTS

The mean number of squares entered by each group in the open field apparatus is presented in Table 1. Analysis of variance (sex x treatment) revealed that females entered a significantly larger number of squares than males, F(1,44)=10.84, p<0.01. Neither the treatment differences between prenatally stressed and control animals nor the sex xtreatment interaction were significant. Very little defecation occurred in any of the four groups, thus this measure was not analyzed. TABLE 1 SUMMARY OF BEHAVIOR EMITrED DURING OPEN-FIELD TESTING AND DURING AVOIDANCE TRAINING (MEAN -+ SEM)

Open-Field

Avoidance Training

Treatment

N

No. squares entered

% achieving criterion*

Trials to criterion

Control Females

12

64 ___6t

83:~

90 _+ 10

Stress Females

12

79 ___8t

67~

81 --- 8

Control Males

12

41 _ 8

50

84 _ 9

Stress Males

12

48 ~ 9

33

98 -+ 21

*Criterion=20 avoidance/25 trials. tp<0.01 compared to male. ~:p<0.05 compared to male. The results of active avoidance training are shown in Table 1 and Fig. I. Analysis of variance (sex×treatmentxtrials) of the number of avoidance responses revealed that the prenatal stress treatment had no effect on this measure. The main effects of sex, F(1,44)=6.86, p<0.05, and trials, F(5,220)=47.24, p<0.01, as well as the sex x trials interaction, F(5,220) = 3.15, p < 0.01, were significant. Males showed significantly higher levels of performance on Days 3, 4, 5 and 6 as compared to Day 1 (Newman-Keuls, p <0.01). Day 2 was significantly different from Days 4, 5 and 6 (/9<0.01). Females showed significantly higher levels of avoidance responding on Days 3, 4, 5, and 6 compared to either Day 1 or 2 (p<0.01). There was a significant improvement between Day 1 and 2 in females (p<0.05). There were no differences in performance among Days 3, 4, 5, and 6 in either males or females. Females showed significantly more avoidance responses than males on all but the first two days of testing (t=2.23; df=23; p<0.05). Furthermore, the proportion of females which achieved a criterion of 20 avoidances out of 25 trials was significantly larger compared to males (X2=4.2, df= I; p<0.05). However, if only the animals that achieved criterion are considered, there were no significant differences between males and females in the number of avoidance responses required before criterion was achieved (Table t). The morphological data taken at the time the males were castrated are presented in Table 2. Prenatally stressed males had a significantly lighter body weight, F(13)=2.59,/9<0.05,

PRENATAL STRESS AND AVOIDANCE ACQUISITION

18-

16s~

14

u•12 Z

or) LIJ

n-

IO-

bJ L) Z

s~

r~

8-

z

6-

bJ

=E 4-

te 1-25

I 26-50

I 51-75

I 76-100

I 101-125

I 126-150

529 p <0.01). There was no significant correlation (r=-0.398) between body weight and LQ of responding males. To verify that the results were not due to litter effects [1,19], all behavioral and morphological measures were reanalyzed using only litter means. In the open-field test, females entered significantly more squares than males (MannWhitney U=6; p<0.03). There were no significant differences as a function of the prenatal stress treatment in either males (Mann-Whitney U=4; p<0.5) or females (Mann-Whitney U=3; p<0.35). Females emitted a significantly larger overall number of avoidance responses than males (t=2.7642; dr= 10; p<0.02). There was no effect due to the prenatal stress treatment in either males or females. Similarly, a significantly larger proportion of females than males achieved a criterion of 20 avoidance responses (Mann-Whitney U=6; p<0.03) but there were no differences as a function of the prenatal stress treatment. Mann-Whitney U tests revealed that males and females did not differ in mean number of avoidance responses emitted during the first 50 trials. However, females responded more frequently than males on trials 51-75 (p<0.03), trials 76-100 (p<0.01), trials 101-125 (p<0.06) and trials 126-150 (p<0.047). There were no differences between stress and control males or between stress and control females. Finally, stress males showed significantly higher lordosis quotients than control males (t=2.886; df=4; p<0.05). With regard to morphology, stress males had lighter body weights (t=2.042; df=4; p<0.05), testis weights (t=3.858; df=4; p<0.02), and epididymis weights (t=4.4132; df=4; p<0.01) than control males. The conclusions to be drawn from the litter mean analyses were identical to those using individual animal scores.

TR I ALS DISCUSSION

FIG. 1. Mean number of avoidance responses performed by prenatally stressed and control male and female rats on 6 consecutive days of testing. Twenty-five trials were given daily. TABLE 2 SUMMARY OF MORPHOLOGICAL MEASURES TAKEN IN MALES AT THE TIME OF CASTRATION (MEAN -- SEM)

Treatment

N

Body weight (g)

Epididymis weight (g)

Testis weight (g)

Control

8

376 ___ 14

0.627 -+ 0.011

1.678 ___ 0.005

Stress

7

329 _+ 12"

0.521 --- 0.032t

1.329 _+ 0.099I

*p<0.05. tp<0.001.

epididymis weight (Mann-Whitney U = 1; p <0.001) and testis weight (Mann-Whitney U=3; p<0.001) than control males. Body weight differences were not significantly different at the time of weaning. Two of eight control males and six of seven prenatally stressed males showed lordosis behavior on at least one of three tests for female behavior (p<0.025; Fischer test). Stressed males had a significantly higher mean LQ (LQ=51) than did control males (LQ=7) (Mann-Whitney U=8;

Prenatal stress feminized copulatory behavior in male rats but did not alter open-field or active avoidance performance. The most parsimonious explanation for such a dissociation in sexually dimorphic behavior is that the prenatal stress treatment did not reduce testosterone levels for a sufficiently long period to demasculinize behaviors which differentiate during both prenatal and neonatal development. Either prenatal [10, 21, 24] or neonatal [9, 11, 24] manipulations of androgen titers have been shown to alter adult potentials for lordosis. Very limited data exist regarding the critical time period during which open-field and active avoidance performance differentiate. Beatty and his collaborators [18] have demonstrated that neonatal castration of male rats combined with prenatal exposure to an anti-androgen feminized open-field and avoidance performance more effectively than did neonatal castration alone. The effect of prenatal cyproterone acetate alone was not assessed. The results of the present study indicate that in the rat suppression of endogenous androgen during prenatal ontogeny is not a sufficient condition to alter normal masculinization of active avoidance and open-field behavior. The failure to demonstrate feminized open-field behavior in our prenatally stressed males must be qualified. Masterpasqua, Chapman and Lore [12] found that the male offspring of rats exposed to a conditioned emotional response paradigm during pregnancy showed higher levels of activity in an open field and lower levels of male copulatory behavior than control males. However, the differences in open-field performance were apparent only after the first day of testing. Since the animals in the present study were tested only

530

ME1SEL, DOHANICH AND WARD

o n c e , it is possible t h a t feminized p a t t e r n s of activity might h a v e b e e n d e t e c t e d if a r e p e a t e d testing p r o c e d u r e h a d b e e n employed. While i n c r e a s e d lordotic p o t e n t i a l s are in a g r e e m e n t with p r e v i o u s c h a r a c t e r i z a t i o n of the p r e n a t a l stress s y n d r o m e [20,23], the a l t e r a t i o n s in male m o r p h o l o g y are not. In a p r e v i o u s study from this l a b o r a t o r y [23], p r e n a t a l l y s t r e s s e d males s h o w e d i m p a i r e d e j a c u l a t o r y b e h a v i o r a n d e n h a n c e d lordosis but, unlike the p r e s e n t study, b o d y , e p i d i d y m u s , and testis weight w e r e n o t significantly different from c o n t r o l males. Since t h e s t r e s s p a r a m e t e r s to w h i c h t h e m o t h e r s had

b e e n e x p o s e d were the same in the two studies, it woukl a p p e a r that the effects of p r e n a t a l s t r e s s on r e p r o d u c t i v e m o r p h o l o g y are not as p r e d i c t a b l e as on sexual b e h a v i o r . H o w e v e r , the failure to d e t e c t b o d y weight differences bet w e e n stress a n d control males at the time of w e a n i n g suggests an a l t e r n a t i v e e x p l a n a t i o n . Stress during a d u l t h o o d r e d u c e s t e s t o s t e r o n e levels in males (see r e v i e w [22]). If prenatally s t r e s s e d males w e r e more s e n s i t i v e t h a n control m a l e s to the stress i m p o s e d by the a v o i d a n c e c o n d i t i o n i n g p r o c e d u r e , it could be reflected in a d e c r e a s e d size o f wLrious androgen dependent structures.

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