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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.anbehav.2004.09.014 Byline: P. Palanza, D. Della Seta, P.F. Ferrari, S. Parmigiani Abstract: We assessed the effects of different situational or social determinants on the regulation of female-female competition. We carried out a laboratory study to examine aggression and reproductive success of pairs of wild female mice, Mus musculus domesticus spp., as a function of the timing of settlement of females relative to that of males and the genetic relatedness and familiarity between females (sibling versus nonsibling females). After a few days of cohabitation with a male, females were highly aggressive towards, and intolerant of, any intruder female, regardless of relatedness and familiarity. In this condition, monogamy was the resulting mating pattern in approximately 80% of cases. Conversely, pairs of females who made contact with each other at the same time, or prior to cohabitation with a male, showed comparatively little aggression and a high degree of reciprocal tolerance. Only in these latter conditions did genetic relatedness and familiarity between females influence their behavioural interactions and reproductive success. Although nonsibling pairs showed higher frequencies of aggressive interactions than siblings, polygyny resulted in 97% of cases. However, in most sibling groups both the females weaned young and had greater reproductive success than nonsiblings. Nonsibling females appeared to compete for reproduction through the inhibition of reproduction or infanticide. These findings suggest that the timing of male/female settlement in a deme determines the level of female competition, which, in turn, affects the resulting mating pattern. Only when females showed social tolerance did genetic relatedness and familiarity influence reproductive success. Author Affiliation: (*) Dipartimento di Biologia Evolutiva e Funzionale, Universita degli Studi di Parma, Italy (a ) Dipartimento di Fisiologia, Universita degli Studi di Siena, Italy Article History: Received 6 February 2003; Revised 30 April 2003; Accepted 5 September 2004 Article Note: (miscellaneous) MS. number: 7614R

Juvenile social play contributes to the development of adult social and emotional skills in humans and non-human animals, and is therefore a useful endpoint to study the effects of endocrine disrupters on behavior in animal models. Ethinylestradiol (EE 2 ) is a widely produced, powerful synthetic estrogen that is widespread in the environment mainly because is a component of the contraceptive pill. In addition, fetuses may be exposed to EE2 when pregnancy is undetected during contraceptive treatment. To understand whether exposure to EE 2 during gestation or lactation affects social play, we exposed 72 female Sprague-Dawley rats to EE 2 or vehicle either during gestation (gestation day (GD) 5 through GD 20) or during lactation (from postnatal day (PND) 1 through PND 21). Two doses of EE 2 were used to treat the dams: a lower dose in the range of possible environmental exposure (4 ng/kg/day) and a higher dose equivalent to that received during contraceptive treatment (400 ng/kg/day). Behavioral testing was carried out between PND 40 and 45. A principal component analysis of frequencies of behavioral items observed during play sessions identified three main components: defensive-like play, aggressive-like play, and exploration. Aggressive-like play was significantly increased by both doses of EE 2 , and the gestational administration was in general more effective than the lactational one. Defensive-like play and exploration were not significantly affected by treatment. This research showed that low and very low doses of EE 2 that mimic clinical or environmental exposure during development can affect important aspects of social behavior even during restricted time windows.

17α-Ethinylestradiol (EE), the main component of the contraceptive pill, is a synthetic estrogen found in rivers of the United States and Europe as an environmental contaminant. It is one of the most studied xenoestrogens due to its possible effect on the reproductive system. In the present study we evaluated the modulation of pain responses induced by formalin injection (licking, flexing, paw-jerk) in 8-month-old male and female offspring of female rats treated with two different doses of EE (4ng/kg/day or 400ng/kg/day) during pregnancy and lactation. Spontaneous behaviors and gonadal hormone levels were also determined. Both concentrations of EE induced an increase of pain behaviors in males only, i.e. higher flexing and licking of the formalin-injected paw than in OIL-exposed rats, during the second, inflammatory, phase of the formalin test. Grooming duration was increased by EE exposure in both males and females. Prenatal EE exposure (both concentrations) decreased estradiol plasma levels in the formalin-injected females but not in the males. These results underline the possibility that exposure to an environmental contaminant during the critical period of development can affect neural processes (such as those involved in pain modulation) during adulthood, indicating long-term changes in brain circuitry. However, such changes may be different in males and females., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this study, we aimed at comparing the effect of the social versus the physical enrichment of the environment on inflammatory pain. Hence, a rat model of carrageenan-induced knee inflammation was used. Four housing conditions were investigated: a physically enriched environment (PE), a socially enriched environment (SE), an enriched environment (EE) (i.e. physically and socially enriched) and a restricted environment (RE) (i.e. non-physically or socially enriched housing). Mechanical allodynia was assessed using the von Frey test preoperatively and at day post-operative (DPO) 1, 3, 7, 10, 14, 17, 21, 24 and 28. Besides, anxiety was evaluated at DPO29, using the Elevated Plus-Maze test. Results show that RE housing resulted in a duration of mechanical allodynia of 4 weeks and of only 3 weeks in EE housing. Housing in a physically enriched environment also resulted in a reduction of the duration of mechanical allodynia of 1 week. Finally, if housed in a SE, the mechanical allodynia lasted for 3 weeks and an half. From these data, we conclude that both physical and social aspects of the environment are involved in the reduction of inflammatory pain duration, although the PE has a larger effect than the SE in this experimental setting. Interestingly, an inter-dependent relationship was noted between the PE and SE. Moreover, no significant difference in the rat anxiety was measured between groups, suggesting that the pain outcomes are likely not biased by the mean of potential housing condition-induced anxiety., (Copyright 2009 Elsevier B.V. All rights reserved.)

Estrogens have a variety of effects in addition to their action on reproductive structures, including permanent effects on the Central Nervous System (CNS). Therefore environmental chemicals with estrogenic activity (xenoestrogens) can potentially affect a number of CNS functions. In the present experiment, female rats receiving ethynylestradiol (EE) or methoxychlor (MXC) via the mothers during pregnancy (pre) or lactation (post) were tested in comparison with females born from mothers treated with OIL. The Object Recognition, Plantar and Formalin tests were carried out to evaluate the effects of these compounds on integrated functions such as memory and pain. Testosterone and estradiol plasma levels were determined by RIA. The results of the Object Recognition and Plantar tests did not differ among groups. However the groups differed in the Formalin test since flexing duration was higher in the EE- and MXC-pre groups than in the EE- and MXC-post and OIL groups. Estradiol plasma levels were higher in EE-pre than in the other groups. These results confirm the possibility that estrogen-like compounds (EE and MXC) can affect complex neural processes like pain when taken during critical stages of CNS development.

Xenoestrogens are endocrine-disrupting chemicals that mimic the action of endogenous estrogen hormones. Effects of xenoestrogen on aquatic wildlife are well documented, whereas the experimental evidence for impairment of reproductive behavior and physiology in mammals after exposure to xenoestrogens has been debated. The strongest arguments against such studies have been that the route, time course, and intensity of exposure did not simulate environmental exposure and that the chemicals tested have additional nonestrogenic toxic effects, hindering generalization of actual xenoestrogenic effects. Here we show that environmental-like exposure to the pure estrogen 17alpha-ethinylestradiol during development alters reproductive behavior and physiology in adult female Sprague-Dawley rats. We simulated environmental exposure by giving low doses (0.4 and 0.004 microg/kg.d) of 17alpha-ethinylestradiol orally to pregnant females from conception to weaning of the pups, which continued to receive the treatment until puberty. We studied the sexual behavior, estrous cycle, and estradiol plasma levels of intact female rats when they reached 3 months of age. Exposure to the higher dose strongly affected female sexual behavior and physiology, with suppression of lordosis and the estrous cycle and enhanced aggression toward males. The lower dose disrupted appetitive components of sexual behavior that influence the rate of copulation. Estradiol plasma levels were not affected by the treatment. Our study revealed that exposure to low oral doses of a pure estrogen during development alters female sexual behavior and physiology. These results suggest potential risks of reproductive failure from xenoestrogen exposure in realistic ecological conditions.

Endocrine-disrupting compounds (EDCs) have the capacity of altering the normal function of the endocrine system. EDCs have shown dramatic effects on the reproductive biology of aquatic wildlife and may affect human reproduction as well. Studies on EDCs in mammalian species have often investigated the effects of short-term, high doses on male and female reproductive physiology. However, it is difficult to predict from such studies the effects of EDC on populations that are exposed to very low doses throughout their life via contaminated food and water. We studied the effects of EDC on mammalian reproduction with an environmental-like protocol where the endpoint is the reproductive success of exposed pairs. We focused on a subclass of EDC, the xenoestrogens, which mimic the action of natural oestrogen hormones. Male and female rats were exposed to low doses of the pure oestrogen, ethynyloestradiol, during development, by oral administration to their mothers during pregnancy and lactation, and to them until puberty. We evaluated the effects of the exposure on development and reproductive physiology of individuals, and on fertility and fecundity of pairs in which both members had been exposed to the same treatment. We found that low doses caused major reproductive deficits in the experimental animals. Very low, environmentally relevant doses did not have evident effects on exposed animals; however, the fecundity of exposed pairs was substantially altered. Environmentally relevant doses of xenoestrogens which have no evident physiological effects can alter the reproductive success of exposed pairs in natural populations.

Steroid hormones have profound effects on the development and function of the nervous system. Environmental estrogens or xenoestrogens are manmade or are natural compounds, which mimics the action of estrogen hormones. The experimental evidence for impairment of cognitive functions in humans and mammals following exposure to xenoestrogens has been fiercely debated. The strongest arguments against such studies have been that the route, time course, and intensity of exposure did not simulate environmental exposure, and that the chemicals tested have additional, non-estrogenic toxic effects, hindering a generalization of actual "xenoestrogenic" effects. Here we show that an environmental-like exposure to the pure estrogen, 17alpha-ethynylestradiol (EE2) during development enhances spatial learning abilities in adult male Sprague-Dawley rats. To simulate an environmental exposure, we used a very low dose (4 ng/kg/day) of EE2 equivalent to concentrations measured in European and US streams which was given orally with a non-invasive method, and we extended the treatment for the entire course of development, from conception to puberty. The animals were tested in a Morris water maze protocol at 6 months of age. Male rats treated with EE2 during development showed a faster learning during the training phase, and remembered better the position of the hidden platform in the short term. Our study demonstrates that actual levels of exposure to xenoestrogens can permanently alter cognitive abilities of a mammalian species.

The effects of two environmental endocrine disruptors, the synthetic pharmaceutical estrogen 17-ethinylestradiol (EE) and bisphenol-A (BPA), were analysed in male and female rats in a very sensitive developmental period, puberty. Immunohistochemistry was used to evaluate changes in the number of cells expressing estrogen receptors (ER-alpha) in the arcuate nucleus (ARC), ventromedial nucleus (VMH) and medial preoptic area (MPA) of the hypothalamus. Animals were treated during early puberty, from PND 23 to PND 30, with EE and BPA given orally every day. They were then sacrificed and perfused on PND 37 or PND 90, and blood and brains were collected for hormonal determination (testosterone and estradiol) and immunohistochemistry (estrogen receptors, ER). At PND 37, ER-labelled neurons were higher in males than in females in the ARC and MPA. EE and BPA increased ER-labelled neurons in the ARC and MPA. At PND 90, females showed higher ER-labelled neurons in the VMH. EE and BPA increased ER-labelled neurons in the MPA in females. EE increased testosterone in males at PND 37 and estradiol in females at PND 90. These results indicate the ability of estrogenic chemicals to change the reproductive neural circuits during puberty in male and female rats.

In this paper, we tested the hypothesis that exposure to estrogens of different source and estrogenic potency at early puberty could affect the development of socio-sexual behavior in the male rat. Puberty is regarded as a second stage of the ontogenetic period, in the sexual maturation of mammals, particularly sensitive to gonadal hormone milieu. We treated animals orally, from postnatal day 23 to 30, with an environmentally compatible dose of bisphenol A (BPA, 40 microg/kg/day) and with a dosage of ethinylestradiol (EE, 0.4 microg/kg/day) comparable to the human oral contraceptives. Exposure to EE altered the temporal pattern of male sexual activity, reducing performance, in the adult animals; slight modifications, in the same direction, were observed with BPA. Short-term behavioral effects were observed in the treated animals, both with BPA and EE: the exploratory drive, directed to a stimulus object and to the environment, as well as to conspecifics, was reduced in the juveniles. Modifications in the circulating T levels were observed after treatments: T was reduced in the juveniles, both with BPA and EE. The decrement persisted in the adult animals but reached significance only in the BPA group. On the whole, effects of pubertal exposure on behavior are more marked with EE than BPA. This can be due to the much higher estrogenic potency of EE; the direction of the behavioral effects of BPA, compared with EE, is however indicative of an estrogenic mechanism.

In mammals, endogenous estrogens are crucial for sexual differentiation during the perinatal period, and the modulation in adulthood of many neuroendocrine and behavioral functions involved in reproduction. In rats, the estrogenic environment during pregnancy and lactation affects directly maternal behavior. This experiment was aimed to test whether the exposure to the estrogenic compound bisphenol-A (BPA; 0.040 mg/kg/die, orally) of adult female rats, from mating to weaning of the pups, could alter maternal behavior. An appropriate methodology was applied to reveal differences in the behavior of dams directed to male and female pups, testing the dams on postnatal days 3-4 and 8-9. Results show different maternal behavioral patterns towards male and female pups of control mothers, with more ano-genital licking to males than to females. Exposure of mothers to BPA modified their behavior, reducing specific components of maternal behavior, both active and passive, irrespective of the sex of pups and the period of observation. This experiment shows that maternal behavior is affected by a prolonged exposure to a low dose of BPA during pregnancy and lactation, thus suggesting an effect on neural circuits in adulthood.

Play behavior is affected by alteration of the hormonal environment during development. In fact, congenital adrenal hyperplasia or early administration of diethylstilbestrol are able to modify female play behavior in mammals. In this research, play behavior of female rats was used to explore the effects of perinatal exposure to low, environmentally relevant dose of bisphenol A (BPA), a xenoestrogen widely diffused in the environment. We used 18 females born to mothers exposed to 40 microg/kg/day BPA during pregnancy and lactation, and 18 control females. The subjects were observed in a heterosexual social situation from 35 to 55 days of age. Six main behaviors were identified by principal component analysis (PCA): exploration, defensive behavior to males, play behavior with males, play behavior with females, low-intensity mating behavior, social grooming. Early administration of BPA was responsible for a significant increase of exploration (including social investigation) (p<0.001), as well as a decrease of play with males (p<0.02) and social grooming (p<0.01) at 45 days of age, indicating a general decrease of playful interactions. In general our results suggest that BPA does not induce a clear masculinization of female behavior, but is able however to defeminize some aspects of female behavior. This result is compatible with the estrogenic properties of BPA, and suggests caution in the use of a chemical that, in the range of human exposure, is able to influence the development of the brain during a critical period, resulting in long-term effects on behavior.

Perinatal action of estrogens or aromatizable steroids at the central nervous system level is responsible for brain sexual differentiation. Through early contact with the central nervous system, the estrogenic compound bisphenol A (BPA) could alter the processes affecting sociosexual behavior. To test this hypothesis, we studied agonistic and sexual behavior of adult female and male rats whose mothers were administered BPA (40 microg/kg/day) during pregnancy or lactation. An intruder test revealed in males but not in females an increase in defensive behavior due to BPA. We studied the effect of BPA on sexual behavior by testing sexual orientation and sexual activity. Male sexual orientation toward a stimulus female was not affected by BPA, whereas the sexual activity test revealed a slight impairment of sexual performance due to BPA in terms of latency and frequency of intromissions. In females, BPA produced a small increase in sexual motivation and receptive behavior. In conclusion, BPA administration, both during pregnancy and during lactation, does not masculinize female behavior or potentiate masculinization processes of males. On the contrary, we observed a potentiation of female behavior in females and a depotentiation of male behavior in males.

We investigated the effects of perinatally administered bisphenol A (BPA), an environmental contaminant with estrogenic activity, on formalin-induced nociceptive responses. Male and female offspring of mother rats treated with BPA or oil were cross-fostered after birth to obtain three homogeneous groups: BPA-prenatal, receiving BPA via the placenta; BPA-postnatal, receiving BPA through suckling; OIL, control, from mothers receiving only peanut oil (vehicle). All groups underwent a pain test with s.c. formalin injection (50 microl, 10%) or were sham injected (pricking with a syringe needle) in the dorsal hind paw. They were immediately placed in an open field apparatus where pain responses (licking, flexing and paw-jerk) were recorded for 60 min. Corticosterone, testosterone and estradiol serum levels were determined in blood obtained at the end of the experiment. BPA-prenatal treatment induced an increase in licking duration in females and in flexing duration in both sexes in the first half of the test (0-30 min after formalin injection). BPA-postnatal treatment induced a decrease in paw-jerk frequency in males and females during the second part of the test (30-60 min after formalin injection). Plasma concentrations of corticosterone, estradiol and testosterone did not differ significantly between groups. These results indicate that exposure to BPA modified the activity of neural pathways and/or centers involved in nociception and pain in a sex-related and exposure-related manner.

The effect of long-term exposure to bisphenol-A (BPA) on estrogen receptor-alpha (ER) immunoreactivity was studied in the medial preoptic area, arcuate nucleus and the ventromedial nucleus of the hypothalamus of estrous cycling and lactating female rats. Pregnant/lactating or estrous cycling rats were exposed to BPA (40 mg/Kg/day) or peanut oil. Lactating females showed fewer ER-immunoreactive cells than non-lactating females in the medial preoptic area and ventromedial nucleus of the hypothalamus. BPA induced an increase in ER-immunoreactive cells in the medial preoptic nucleus irrespective of lactation. BPA only induced a decrease in ER-immunoreactive cells in the arcuate nucleus of the lactating group; oil induced an increase in ER-immunoreactive cells in the lactating with respect to non-lactating group. The results demonstrate that exposure of adult females to BPA modifies the number of ERs.

1. In a first study different doses of haloperidol (0.6, 1.2, 2.5, 5, 10, or 20 mg/kg; intraperitoneally) were administered to adult male mice (CD-1 strain) and tested for their ability to induce catalepsy. 2. The minimal haloperidol dose inducing complete catalepsy was found to be the 10 mg/kg dose and selected for the second experiment. 3. Using an immunoenzymatic assay (ELISA) hypothalamic nerve growth factor (NGF) level was measured 20 or 180 min following haloperidol injection (10 mg/kg). 4. Haloperidol treatment decreased NGF levels in mouse hypothalamus and this effect did not differ at the two time points tested. 5. The role of hypothalamic NGF in stress-related events is discussed.

In the present study, serum levels of nerve growth factor (NGF) were assessed in virgin and in lactating female CD-1 mice. In the case of the lactating females, NGF levels were assessed both under basal conditions and 60 and 180 min following a 10 min encounter with a male or a nonlactating female mouse. Basal serum NGF levels of lactating females were higher than those of virgin females but did not increase significantly above base after an aggressive encounter with a male or a female conspecific. Female intruders were attacked in a ritualized manner. In contrast, males received numerous bites to vulnerable regions of their body. A positive correlation was found between serum NGF levels and pattern of aggression in females confronting male conspecifics. Thus, in lactating mice, serum NGF levels following an aggressive encounter relate to the specific pattern of behavior the female uses to defend the offspring.

Adult CD-1 male mice were injected intravenously with 2.5 micrograms/g of highly purified murine NGF and then assessed for hot plate responding (52 degrees C) at 15, 30, 60, 180, and 360 min (repeated test) or at 30, 60, or 360 min (single test, i.e., exposure to hot plate only once). Control animals received cytochrome c (2.5 micrograms/g). In the repeated test, NGF produced hyperalgesia, increasing the number of jumps (particularly at 30 and 60 min postinjection), while in the single test the pain reaction of NGF-treated animals remained unaffected. Sensitization of C-fibers in the periphery or release of bioactive mediators from mast cells may account for NGF-induced changes in nociception.