Neonatal handling increases fear and aggression in lactating rats

Physiology & Behavior 86 (2005) 209 – 217

Neonatal handling increases fear and aggression in lactating rats Marcia Giovenardi a,b, Marcia S. de Azevedo a,b, Simone P. da Silva a, Erica do E.S. Hermel b, Carmen M. Gomes b, Aldo B. Lucion b,* a b

Laborato´rio de Neurocieˆncias, Universidade do Vale do Rio dos Sinos, Sa˜o Leopoldo, RS 93022-000, Brazil Departamento de Fisiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90050-170, Brazil Received 12 June 2004; received in revised form 5 July 2005; accepted 12 July 2005

Abstract Neonatal handling reduces fear in male and cycling female rats, but increases maternal aggressive behavior against intruders to the nest area. Present study aimed to analyze the effects of neonatal handling on the maternal aggressive behavior and the activity in the open field with a predator of lactating rats on the 8th and the 18th postpartum days (periods of high and low aggressiveness). As pups, animals were divided into two groups: nonhandled (no neonatal manipulation) and handled (handling for 1 min during the first 10 days after delivery). As adults, females of both groups were impregnated and tested against a male intruder for aggressive behavior and in the open field with a cat inside a wire-meshed cage. Results showed that on the 8th day frequency of aggressive behaviors of handled females was higher than that of the nonhandled ones, but on the 18th day, no significant difference was detected. Surprisingly, in the open field test, handled females showed decreased locomotion and increased freezing on the 8th day compared to the nonhandled ones. The opposite relationship between increased aggressiveness with reduced fear is observed in the nonhandled control females in early and late lactation periods. However, neonatal handling abolishes this relationship. Apparently, the increased aggressiveness in neonatal handled lactating females does not depend on a decrease in fear. Our findings support the hypothesis that long lasting effects of early life stimulation is a dynamic function depending on the behavioral system and the period of life analyzed. Moreover, they caution the relationship between aggressive behavior and fear. D 2005 Elsevier Inc. All rights reserved. Keywords: Neonatal stress; Maternal aggression; Fear; Open field; Predator; Gonadal steroids; Female rats

1. Introduction At the end of pregnancy and during the first days of lactation, the female undergoes several important hormonal and behavioral changes that lead and enable her to protect and nurture the offspring [for reviews see Refs. 1,2]. In the lactating rat, high levels of aggressive behaviors against intruders are displayed during the first 10 days after delivery, and thereafter they decline to very low levels, even though lactation continues [3– 6].

* Corresponding author. Departamento de Fisiologia, Instituto de Ciencias Basicas da Saude, UFRGS, Sarmento Leite 500, Porto Alegre, RS 90050-170, Brazil. Fax: +55 51 33163656. E-mail address: [email protected] (A.B. Lucion). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.07.011

Environmental experiences during the postnatal period exert a remarkably profound impact on behaviors and stress reactivity in adulthood [7– 11]. In rats, early postnatal handling reduces emotional responses in adulthood expressed by increased exploratory activity, which is interpreted as attenuated fear of novel environments [8,12,13]. Besides reducing fear, a core effect of neonatal handing, other behaviors can be affected by the early life stimulation. Previous study [9] showed that the aggressive behavior of lactating rats on the 7th day after delivery against male intruders was greatly increased by early handling. The increased aggressiveness and the reduction in fear occur concomitantly during the lactation period in an inversely correlated manner [14 –16]. Since, reduced fear has been considered a core effect of neonatal handling, it is

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plausible to suppose that lactating females would also show reduction in fear. Thus we may hypothesize that the reduction of fear would be the cause for the increased aggressiveness. The aggressive behavior of lactating rats against male intruders seems to involve a complex regulatory action of several hormones [6,17] that would turn a previous nonaggressive female more reactive to strangers in the nest area. On the other hand, besides inducing the welldescribed long lasting changes on the hypothalamus – pituitary –gonads axis, neonatal handling also alters ovarian and pituitary hormones [18]. Considering the neonatal handling can alter these hormones that are involved with the onset and the maintenance of aggression during the lactating period, we hypothesize that the increased aggressiveness observed on the 8th day [1] could be due to a change in the pituitary and gonadal hormones in this period. However, previous study [19] has shown that hypophysectomy induced no change on the maternal aggressive behavior. On the other hand, maternal aggressive behavior seems to require both gonadal steroid hormonal changes at the end of pregnancy and the presence of the pups [20,21]. Ovariectomized females that were sensitized by continuous exposure to pups displayed attacks toward intruders, although the aggression levels were significantly lower than those of lactating dams [14]. Moreover, previous study [22] showed that ovariectomy of lactating females did not alter the maternal aggressive behavior against unfamiliar female conspecific on the 3rd postpartum week. On the other hand, ovariectomy decreased territorial aggressive behavior against female intruders compared with those treated with gonadal hormones [23]. On the other hand, we have recently shown that the behavioral and the stress responses in neonatal handled male and female rats are not observed in the peripubertal period and also in certain phases of the estrous cycle, which could indicate that the expected changes in emotional behavior and stress responses induced by neonatal handling are not observed with the same intensity, or are not present at all, during lifetime [24]. Thus, the present study tests whether the effects of neonatal handling would lead to stable and uniform effects or vary over time during the lactation period of a female manipulated in the neonatal period. Present study aimed to analyze the effects of neonatal handling on the maternal aggression against a male intruder and the behavior in the open field with a predator at two different postpartum periods: 8th (period of high aggressiveness against intruders) and 18th (period of lower aggressiveness) day. We tested whether the increased aggressiveness induced by the handling procedure and detected on the 8th day would also occur on the 18th day postpartum. Besides that we aimed to verify whether the behavioral inhibition in a novel an aversive environment, as a measurement of fear, would be affected be the

neonatal handling in the lactating female in both days after delivery. Plasma prolactin (Prl), estradiol (E2), progesterone (P4), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were also analyzed on the 8th and 18th day after delivery.

2. Materials and methods 2.1. Subjects Primiparous lactating female Wistar rats from the stock of the Federal University of Rio Grande do Sul, weighing 240 T 20 g. The females were divided into two groups: nonhandled and handled. Each group was studied at two postpartum periods: on the 8th day (period of higher maternal aggressiveness) and on the 18th day postpartum (period of lower maternal aggressiveness). In all the behavioral tests and hormone measurements, different animals were used in the two postpartum periods (8th and 18th day) and they were used solely in one test. Moreover, the animals in each experimental group (nonhandled and handled) were non-siblings (each subject from a different litter), but females in each postpartum period (8th and 18th day) came from the same litter. At all times, animals were maintained on a light : dark (12 : 12 h) cycle with the lights off at 4:00 p.m.; room temperature was kept at 22 -C and the animals had free access to food (Nuvilab Cr2, Colombo, Brazil) and water at all times. 2.2. Neonatal handling The females used in the experiments were raised in our animal facility room. The dams of the experimental animals were taken to our laboratory about 7 days before delivery. The pregnant females were housed individually, the day of birth (day 0) was controlled and the number of pups was culled to 7 per dam. From the 1st to the 10th day after birth, the whole litter (males and females) was submitted to the handling procedure, as previously applied [9,25,26]. The litter and the mother in their home-cage were taken to a quiet room next to the animal facility, with the same light period and temperature. The mother was placed in another cage next to the home-cage and then the experimenter gently handled all pups at the same time using both hands covered with fine latex gloves for 1 min. After handling, the pups were returned to the nest at the same time and the mother was placed back in the homecage. The pups were handled at a distance of about 1 to 2 m from the mother and the total time of mother – infant separation was about 2 min. This procedure was repeated from the 1st to the 10th postnatal day, during the light period of the daily photoperiod cycle. The pups remained with their mothers until weaning (21 days old). Nest material and cage bedding of both groups were not

M. Giovenardi et al. / Physiology & Behavior 86 (2005) 209 – 217

changed during the first 10 days. In the nonhandled group, the dams and pups were left undisturbed during the first 10 days after delivery. After weaning, the females were housed in groups of 3– 5 per cage (41  34  17 cm) according to body weight until the tests procedures. Experiments were performed in accordance with the NIH Guide for Animal Research and were approved by the Research Committee of the University. 2.3. Experimental procedure At approximately 75 days of age, colonies of 5 virgin females (nonhandled or handled) and 1 male were established in order to obtain the lactating females. Three weeks later, the females were visually and manually inspected for pregnancy. Pregnant females were housed individually in the Plexiglas observation cages (50  45  25 cm) with wood shavings for bedding and nest building. During the experiment, we observed that a significantly smaller number of handled females were impregnated compared to the nonhandled group. Indeed, previous study showed that neonatal handling induces a reduction in the number of oocytes [25]. In the present experiment, approximately 3 out of 5 females that were joined with a male during 3 weeks got pregnant. Only dams with at least 7 pups were used. 2.4. Maternal aggressive behavior test On the 8th and 18th days postpartum from 4:00 p.m. to 6:00 p.m., the behaviors of the female in the presence of an intruder male were videotaped for 10 min beginning immediately after placing the intruder into the cage where the female was with her litter. The intruders were young males smaller than the females and were used only once. In the present study and others in the laboratory using the same rat strain, no pup was attacked by the intruder [27 – 29]. The behaviors recorded during tests of maternal aggression were [29]: sniffing (the female approaches the intruder and sniffs its body and/or genitals); lateral attack (the female moves laterally orientated towards the intruder, usually in association with piloerection and biting the back of the intruder); frontal attack (female with the body frontally orientated lunges towards the intruder and this is usually followed by biting the head or shoulders of the male); and biting (female bites any part of the intruder’s body). The frequency of each behavior during the 10-min recording session was the parameter analyzed. The number of animals in each group and postpartum period was: nonhandled 8th day (n = 11); handled 8th day (n = 13); nonhandled 18th day (n = 14) and handled 18th day (n = 11). 2.5. Open field with a predator Animals were tested in a modified open field test, in which a predator (a cat) inside a wire-meshed cage is placed in the open field, a procedure that tends to increase

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the behavioral inhibition of the rats [29 – 31]. The apparatus was a square 1 m side arena that was even lighted with overhead fluorescent lights. The experiment was divided in three sequential periods, during which the behavior of the rat was continuously videotaped. First, the rat was placed in a corner of the open field and the activity was recorded for 5 min. With the rat still in the arena, a tame adult female cat inside a wire-meshed cage was placed for another 5 min at the opposite corner of the open field from where the rat was initially placed. After that, the cat was removed and the behavior of the rat was recorded for another 5 min period. The rat had visual, olfactory and hearing interaction with the cat that remained inside a wire-meshed cage and had no direct physical contact with it [9,29]. The open field was thoroughly cleaned after each rat was tested. The animals were tested individually and only once in the 15-min videotape recordings. Testing was from 8:30 a.m. to 12:00 p.m. The following behaviors of the rat were analyzed in each 5-min period: frequency and duration (s) of locomotion; duration (s) of sniffing and/or exploring the area where the cat actually is (cat period) or was (post-cat period); and duration (s) of freezing. Exploration of the cat’s area consists of approaching the area and usually sniffing the cage and/or the ground. In all cases, frequency is the sum of numbers of times that a behavior occurs and duration is the sum of the time of the each behavior during the session. The number of animals in each group and postpartum period was: nonhandled 8th day (n = 11); handled 8th day (n = 10); nonhandled 18th day (n = 10) and handled 18th day (n = 9). 2.6. Pup retrieval test In the retrieval test, the female was removed from the home-cage and the pups were spread around in the cage. The latency (s) to retrieve the 1st and the 7th pup was recorded only on the 8th day after delivery in nonhandled (n = 10) and handled (n = 10) groups. After the test, the whole litter (7 pups in each) was weighed. 2.7. Hormone measurements From 4:00 p.m. to 6:00 p.m., females of the two groups (nonhandled and handled) and the two postpartum periods (8th and 18th day) were killed by decapitation and blood was collected from the trunk. Females had not been used in the other experiments nor were siblings. Samples were centrifuged for 10 min at 3000 rpm; the plasma was separated and stored frozen ( 70 -C). Plasma Prl (ng/mL), LH (ng/mL) and FSH (ng/mL) were determined by double antibody radioimmunoassay using reagents provided by the National Institute of Arthritis, Digestive Diseases and Kidney. The lowest detected amount of Prl was 0.09 ng/ mL, LH was 0.04 ng/mL and FSH was 0.39 ng/mL. The concentration of E2 in the plasma (pg/mL) was determined by radioimmunoassay using a DPC Coat-a-Count Estradiol

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kit. The lowest value detected was 0.5 pg/mL. Plasma concentration of P4 (ng/mL) was determined by radioimmunoassay using the Biochem ImmunoSystems (Bologna, Italy) MAIAR kit and the lowest value detected was 0.075 ng/mL. The hormone measurements were performed in the laboratory of Dr. Celso R. Franci (Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Brazil). 2.8. Statistical analysis In the maternal aggressive behavior test, mean (TSEM) frequency of the behaviors was compared between the two groups (nonhandled and handled) and the two postpartum periods (8th and 18th day after delivery) using a two-way ANOVA.

In the modified open field test, mean (T SEM) duration and frequency of the behaviors were compared between the two groups of females, the two postpartum periods, and among the three recording sessions (pre-cat, cat and post-cat, repeated measurements) using a three-way ANOVA. The mean (T SEM) concentration of plasma hormones was compared between the two groups and the two postpartum periods using a two-way ANOVA. In all cases, when appropriate, ANOVA was followed by the Newman– Keuls test. The median latency (interquartile range) to retrieve the pups (8th day postpartum) was compared between the handled and nonhandled groups using the Mann –Whitney U test. The mean (TSEM) weight of the litter (8th day postnatal) was compared between the handled and non-

MATERNAL AGGRESSION Sniffing intruder 40

Lateral attack 25 20

Frequency

30

* 15

20 10 10

5 & 0

0 8th

18th

8th

& 18th

Postpartum day

Postpartum day Frontal attack

Bite

8

30 25

Frequency

6

*

20 4

15 10

2 5 &

& 0 8th

18th

Postpartum day

0

8th

18th

Postpartum day

Nonhandled females on the 8th and 18th postpartum day (n=11, n=14) Handled females on the 8th and 18th postpartum day (n=13, n=11)

* p<0.05 compared to the nonhandled group at the same postpartum day &

p<0.05 compared to the 8th postpartum day in the same group

Fig. 1. Effects of neonatal handling on the maternal aggressive behavior in females on the 8th and 18th postpartum day. Data are plotted as mean T SEM. The number of animals (n) is given in parentheses. Two-way ANOVA (between the groups and between postpartum day) followed by Newman – Keuls test was employed. *Indicates significant difference between the groups (nonhandled and handled) within the same postpartum day. &Indicates significant difference between the postpartum days (8th and 18th day) within the same group. In all cases, significance accepted at p < 0.05.

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handled and handled) and postpartum periods [ F(1, 45) = 1.688]. Significant main effects for group [ F(1, 45) = 7.209, p < 0.01] and postpartum period [ F(1, 45) = 55.781, p < 0.0001] were detected in the frequency of lateral attack. Significant interaction was shown between groups and postpartum periods [ F(1, 45) = 8.918, p < 0.01]. Newman– Keuls post hoc analysis revealed that handled females on the 8th day postpartum showed increased frequency of lateral attack as compared to the nonhandled group in the same period; and both handled and nonhandled females on the 18th day showed a decrease in the behavior compared to the 8th day, respectively. The main effect for group [ F(1, 45) =

handled groups by the Student’s t test. In all comparisons, p < 0.05 was considered statistically significant.

3. Results Fig. 1 represents the maternal aggressive behavior of handled and nonhandled females on the 8th and the 18th day postpartum. No significant main effects for group [ F(1, 45) = 0.271] and postpartum period [ F(1, 45) = 1.168] were detected in frequency of sniffing the intruder. No significant interaction was shown between groups (non-

NEONATAL STIMULATION : OPEN FIELD WITH A PREDATOR TEST IN LACTATING FEMALE RATS LOCOMOTION

LOCOMOTION 150

60

120 DURATION (s)

FREQUENCY

50 40 30 20

*

90

* 60

& &

30

10

* 0

0 PRE

CAT

PRE

POST

300

250

250

200 150 100 50

POST

FREEZING

300

DURATION (s)

DURATION (s)

TIME IN CAT AREA

CAT

200 150 100 50

0

0 PRE

CAT

POST

PRE

CAT

POST

Nonhandled 8th day (n=11) Nonhandled 18th day (n=10) Handled 8th day (n=10) Handled 18th day (n=9)

* p<0.05 comparing the nonhandled and handled groups at the same postpartum day and session &

p<0.05 comparing the 8th and the 18th day postpartum in each group and session

Fig. 2. Effects of neonatal handling on the behavior of lactating female rats (8th and 18th postpartum day) in the open field with a cat. Females were analyzed in three different sessions of 5 min each: before (PRE), during (CAT) and after (POST) the cat. Data are plotted as mean T SEM. The number of animals (n) is given in parentheses. Three-way ANOVA (between the groups, between postpartum days and among sessions) followed by Newman – Keuls test was employed. Significant main effect for group was detected in the frequency of locomotion, duration of exploring the cat area and freezing. Significant main effects for postpartum periods and sessions were detected in all behaviors, but a significant interaction (groups, postpartum days and sessions) was only shown in the duration of locomotion. *Indicates significant difference between the groups (nonhandled and handled) within the same postpartum day and session. & Indicates significant difference between the postpartum days (8th and 18th day) within the same group and session. In all cases, significance accepted at p < 0.05.

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3.679] almost reached significance ( p = 0.06) and a main effect for postpartum period [ F(1, 45) = 17.334, p < 0.0001] was detected in frontal attack. No significant interaction was shown between groups (nonhandled and handled) and postpartum periods [ F(1, 45) = 1.980]. Significant main effects for group [ F(1, 45) = 10.929, p < 0.01] and postpartum period [ F(1, 45) = 50.759, p < 0.0001] were detected in the frequency of bite. Significant interaction was shown between groups and postpartum periods [ F(1, 45) =10.609, p < 0.01]. Newman – Keuls post hoc analysis revealed that handled females on the 8th postpartum day showed increased frequency of bite as compared to the nonhandled group in the same period; and both handled and nonhandled females on the 18th day showed a decrease in the behavior compared to the 8th day, respectively. Fig. 2 represents the behavior of neonatal handled and nonhandled females on the 8th and the 18th day postpartum in the open field test. Significant main effects for group [ F (1, 36) = 31.150, p < 0.001], postpartum period [ F(1, 36) = 17.240, p < 0.001] and cat [ F(2, 72) = 45.740, p < 0.001] were detected in frequency of locomotion. The interaction among groups, postpartum periods and cat almost reached significance [ F(2, 72) = 2.720, p = 0.07]. No significant main effect for group [ F(1, 36) = 2.491] was detected in the duration of locomotion in the open field. However, significant main effects for postpartum period [ F(1, 36) = 10.781, p < 0.01] and cat [ F(2, 72) = 94.748, p < 0.001] were detected in that behavior. A significant interaction was shown between groups, postpartum periods and cat [ F(2, 72) = 3.608, p < 0.05]. Newman– Keuls post hoc analysis revealed that handled females on the 8th postpartum day showed decreased duration of locomotion in the pre-cat and cat recording sessions as compared to the nonhandled group in the same period and session. On the 18th postpartum day, handled females showed increased duration of locomotion only in the pre-cat session as compared to the nonhandled group in the same period and session. Nonhandled females on the 18th day, but not the handled ones, showed a decrease in the duration of locomotion compared to the 8th day in the pre-cat and cat sessions. Significant main effects for group [ F(1, 36) =

30.110, p < 0.0001], postpartum period [ F(1, 36) = 20.720, p < 0.0001] and cat [ F(2, 72) = 9.200, p < 0.001] were detected in the duration of exploring the cat area. No significant interaction among groups, postpartum periods and cat was reached [ F(2, 72) = 1.872]. Significant main effects for group [ F(1, 36) = 50.820, p < 0.0001], postpartum period [ F(1, 36) = 11.350, p < 0.001] and cat [ F(2, 72) = 21.920, p < 0.001] were detected in duration of freezing. No significant interaction among groups, postpartum periods and cat was reached [ F(2, 72) = 1.469]. Table 1 shows the plasma concentrations of hormones of neonatal handled and nonhandled females on the 8th and the 18th day postpartum. Main effect for group [ F(1, 34) = 3.271] almost reached significance ( p = 0.07), but no s ig nificant ma in effect fo r po stp artum p eriod [ F(1, 34) = 2.656] was detected in E2. No significant interaction was shown between groups and postpartum periods [ F(1, 34) = 0.890]. A significant main effect for group [ F(1, 32) = 4.715, p < 0.05], but no significant main effect for postpartum period [ F(1, 32) = 0.005] was detected in P4. No significant interaction was shown between groups and postpartum periods [ F(1, 32) = 0.001]. No significant main effect for group [ F(1, 33) = 1.381], but significant main effect for postpartum period [ F(1,33) = 6.640, p < 0.05] was detected in Prl. No significant interaction was shown between groups and postpartum periods [ F(1, 33) = 0.001]. No significant main effect for group [ F(1, 28) = 0.004], but significant main effect for postpartum period [ F(1, 28) = 12.183, p < 0.001] was detected in FSH. No significant interaction was shown between groups and postpartum periods [ F(1, 28) = 0.208]. No significant main effects for group [ F(1, 28) = 1.826] and postpartum period [ F(1, 28) = 0.507] were detected in LH. No significant interaction was shown between groups and postpartum periods [ F(1, 28) = 0.682]. The median (interquartile range) latency (s) to retrieve the 1st [25.5(6.0 / 57.0)] pup by the neonatal handled dam was not different (Mann – Whitney test, U = 37.0) from the nonhandled dams [36.0(12.0 / 255.0)]. Furthermore no difference was detected comparing the latency to retrieve the last pup [578.5(81.0 / 900.0)] and 158.0(70.0 / 900.0)], respectively (Mann –Whitney test, U = 44.5). No significant

Table 1 Basal plasma E2 (pg/mL), P4 (ng/mL), Prl (ng/mL), FSH (ng/mL), and LH (ng/mL) in neonatal handled (from the 1st to the 10th day) and nonhandled lactating females Groups

Postpartum day

Plasma hormones E2 (pg/mL)

P4 (ng/mL)

Prl (ng/mL)

FSH (ng/mL)

LH (ng/mL)

Nonhandled

8th 18th 8th 18th

13.3 T 1.9 10.4 T 0.5 10.1 T 0.8 9.3 T 0.5

33.0 T 10.0 (n = 10) 33.6 T 11.4 (n = 9) 14.6 T 4.5 (n = 8) 15.4 T 3.6 (n = 9)

189.2 T 61.2 (n = 10) 59.0 T 10.2 (n = 8) 248.0 T 67.5 (n = 9) 118.6 T 32.9 (n = 10)

0.7 T 0.1 1.2 T 0.2 0.8 T 0.1 1.1 T 0.1

0.5 T 0.1 0.4 T 0.1 0.6 T 0.1 0.8 T 0.2

Handled

(n = 10) (n = 9) (n = 9) (n = 10)

(n = 8) (n = 8) (n = 8) (n = 8)

(n = 9) (n = 9) (n = 6) (n = 8)

Data are plotted as mean T SEM. The number of females in each group is given in parentheses. Two-way ANOVA (between the groups and between postpartum day) followed by Newman – Keuls test was employed. Significant main effect for group in P4 (handled groups are lower than nonhandled ones); significant main effect for postpartum day in Prl (the 18th day in both groups is lower than the 8th day); and significant main effect for postpartum day in FSH (the 18th day in both groups is higher than the 8th day) were detected.

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difference (t = 0.862) was detected between the mean (TSEM) weight (g) of the pups from the neonatal handled dams [106.5 T 5.2] and the nonhandled dams [115.4 T 8.9].

4. Discussion Present study analyzes two factors: lactation and neonatal handling. The behavioral and the hormonal changes induced by pregnancy and lactation were studied in neonatal handled females. Pregnancy and lactation occurred in females that probably had their neuroendocrine basis altered by the early life stimulation. Results showed that handling increased maternal aggression on the 8th day after delivery (high aggressiveness period), which is in agreement with previous studies [9]. However, handling induced no change on the 18th day, a period when aggression against intruders to the nest area is naturally lower. Since neonatal handling increased aggression during a period in which it had already developed and did not reinstate it in the low aggressive period (18th day after delivery), we may conclude that handling did not alter the expected decline in aggressiveness in the late lactation period. Thus, neonatal handling was not able to alter the normal course of the aggressive behavior against intruders throughout the lactating period. This conclusion agrees with previous studies, demonstrating that neonatal handling affected the behaviors in a novel environment or the hormone change after a stressor, whereas basal levels did not change [8,9,11,12]. It is also noteworthy that handling affected specifically the aggressiveness against the intruder but not against the pups. Moreover, the retrieving latency and body weight of the pups were not different between the groups. In agreement with previous study [32], present results suggest that handling does not affect maternal care taking behaviors. On the other hand, behavioral inhibition in the open field with a predator increased during both postpartum periods (8th and 18th day) as compared to the nonhandled females. Differences between the groups on the behavioral inhibition differences between the groups and postpartum periods were observed before the cat’s presence, probably due to the novel environment. As expected [31], the presence of the cat (innate fear) further reduced locomotion in the arena with different degrees between groups and periods. The neonatal handling procedure induced unexpected results on the behavioral inhibition of lactating rats in the open field test. The increased behavioral inhibition in the open field with a cat was detected on the 8th and not as clearly on the 18th day after delivery compared to the nonhandled females. Previous studies [14,33] showed that lactating female rats on the 6th and the 7th day after delivery that had not been submitted to any systematic environmental stimulation during the neonatal period show reduced fear in the elevated plus maze and freezing tests as compared to nonlactating females. Neonatal handling did not decrease fear either on the 8th or 18th day postpartum,

215

as it does in cycling females [9,24] and males [8,9,24]. On the contrary, handled females particularly on the 8th day postpartum showed increased fear compared to the nonhandled ones. In fact, the duration of locomotion in the open field in the lactating handled females on the 8th day is quite similar to the virgin nonhandled ones, as previously shown [9]. Thus, handling appears to abolish the expected decrease of fear observed during the early lactation period. Several studies have suggested an inverse correlation between fear and aggression in the postpartum period [for reviews see Refs. 1,34]. However, present results challenge the hypothesis that increased aggressiveness during postpartum period is determined by the reduced fear. Here we show increased aggression and increased fear concurrently in the lactating females handled in the neonatal period. We may infer that maternal aggression could be mediated by systems that, at least in part, are independent of fear, and thus the neonatal handling procedure increased aggressive behavior independently from fear reduction. However, since neonatal handling appears to reduce ability to impregnate, due to a reduced ovulation [25] and sexual behavior [9], the population of handled females analyzed was not random. Only those that had at least 7 oocytes fecundated were used. Reduced fear has been shown in both neonatal handled cycling [9,24] and lactating females [14,33], however, when we have a lactating female that had been handled in the neonatal period, an opposite result emerges, and no reduction in fear is observed. On the other hand, maternal aggressive behavior of neonatal handled females increased, but only in the early lactation period. Another result to be discussed is the relationship between fear and maternal aggression with hormonal changes observed in the early postpartum period. Previous studies have shown that P4 is related to a reduction in anxiety-like behaviors when administered systemically or into the amygdala and this effect is higher when P4 is associated with E2 [35]. Moreover, during the proestrus, when both P4 and E2 are high, female spend more time in the open arms compared to other phases of the estrous cycle [36]. P4 and its metabolites exert an anxiolytic effect by modulating GABA receptors in rats [37 – 39]. Fluctuations in plasma level of P4 appear to determine the expression and function of GABAA receptors [40]. According to these studies, P4 seems to reduce anxietylike behaviors. Present results showed that neonatal handling decreased plasma P4 in both postpartum periods, but no change was detected on E2 levels. In the handled females on the 8th day after delivery, a reduced plasma P4 is related to an increased anxiety-like behaviors. In the nonhandled females, the increased plasma P4 compared to a cycling female could be one of the causes for the decreased fear in the early postpartum period as predicted. In the neonatal handled lactating females, P4 did not increase after delivery, as compared to the nonhandled ones, which could prevent the expected fear decrease.

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The effect of P4 on maternal aggression remains to be determined. It is noteworthy that plasma P4 in handled females is lower on the 8th and on the 18th day after delivery compared to nonhandled ones, but aggressiveness increased only on the 8th day. In cycling females, neonatal handling also reduces P4, besides the reduction of LH, E2 and Prl [18]. In lactating females, P4 seems to be related but not determinant to the onset of aggressive behavior in the postpartum period, although, previous study [22] showed that ovariectomy did not alter maternal aggression. In conclusion, the opposite relationship between increased aggressiveness with reduced fear is observed in the nonhandled control females in early and late lactation periods. However, neonatal handling abolishes this relationship. Apparently, the increased aggressiveness in neonatal handled lactating females does not depend on a decrease in fear. In fact, during the early postpartum period females show high levels of aggressiveness and fear at the same time. Although the reduction of fear in novel environments is a core effect of neonatal handling in adult male and females, this expected decrease was not demonstrated in the both early and late postpartum periods. We may conclude that handling can induce long lasting effects on several behaviors possibly having different effects on the behavioral neural systems. Our findings support the hypothesis that long lasting effects of early life stimulation is a dynamic function depending on the behavioral system and the period of life analyzed. Moreover, they caution the relationship between aggressive behavior and fear.

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