Environmental prenatal stress eliminates brain and maternal behavioral sex differences and alters hormone levels in female rats.

Environmental prenatal stress (EPS) has effects on fetuses that are long-lasting, altering their hormone levels, brain morphology and behavior when they reach maturity. In previous research, we demonstrated that EPS affects the expression of induced maternal behavior (MB), the neuroendocrine system, and morphology of the sexually dimorphic accessory olfactory bulb (AOB) involved in reproductive behavior patterns. The bed nucleus of the accessory olfactory tract (BAOT) is another vomeronasal (VN) structure that plays an inhibitory role in rats in the expression of induced maternal behavior in female and male virgins. In the present study, we have ascertained whether the behavioral, neuroendocrine, and neuromorphological alterations of the AOB found after EPS also appear in the BAOT. After applying EPS to pregnant rats during the late gestational period, in their female offspring at maturity we tested induced maternal behavior, BAOT morphology and plasma levels of testosterone (T), estradiol (E2), progesterone (P), adrenocorticotropic hormone (ACTH) and corticosterone (Cpd B). EPS: a) affected the induction of MB, showed a male-like pattern of care for pups, b) elevated plasma levels of Cpd B and reduced E2 in comparison with the controls, and c) significantly increased the number of BAOT neurons compared to the control females and comparable to the control male group. These findings provide further evidence that stress applied to pregnant rats produces long-lasting behavioral, endocrine and neuroanatomical alterations in the female offspring that are evident when they become mature.

Maternal care interacts with prenatal stress in altering sexual dimorphism in male rats.

The present study analyzes the interaction between prenatal stress and mother's behavior on brain, hormonal, and behavioral development of male offspring in rats. It extends to males our previous findings, in females, that maternal care can alter behavioral dimorphism that becomes evident in the neonates when they mature. Experiment 1 compares the maternal behavior of foster mothers toward cross-fostered pups versus mothers rearing their own litters. Experiment 2 ascertains the induced "maternal" behavior of the male pups, derived from Experiment 1 when they reached maturity. The most striking effect was that the males non-exposed to the stress as fetuses and raised by stressed foster mothers showed the highest levels of "maternal" behavior of all the groups (i.e., induction of maternal behavior and retrieving behavior), not differing from the control, unstressed, female groups. Furthermore, those males showed significantly fewer olfactory bulb mitral cells than the control males that were non-stressed as fetuses and raised by their own non-stressed mothers. They also presented the lowest levels of plasma testosterone of all the male groups. The present findings provide evidence that prenatal environmental stress can "demasculinize" the behavior, brain anatomy and hormone secretion in the male fetuses expressed when they reach maturity. Moreover, the nature of the maternal care received by neonates can affect the behavior and physiology that they express at maturity.

Maternal care counteracts behavioral effects of prenatal environmental stress in female rats.

There is extensive evidence in rats that prenatal environmental stress (PES) exposure and early postnatal altered maternal care, as a consequence of stress during gestation, can detrimentally affect the brain and behavioral development of the offspring. In order to separate the effect of PES on the fetuses from that on the behavior of the mother, in the present study, we used a cross-fostering procedure in which PES-fetuses were raised by non-stressed mothers and non PES-fetuses were raised by stressed mothers. In Experiment 1, non-stressed mothers showed significantly more maternal behavior than stressed mothers. In Experiment 2, when the female offspring from Experiment 1 reached maturity, they were tested for: (1) induced maternal behavior (MB), (2) plasma levels of corticosterone (Cpd B), progesterone (P), and estradiol (E(2)), (3) number of accessory olfactory bulb (AOB) mitral cells, and (4) c-fos expression measured in AOB and medial preoptic area (MPOA) neurons. We replicated our previous findings that the PES group reared by their own stressed mothers, when adult, attacked the young, expressed disorganized MB and showed altered Cpd B, P and E(2) levels, plus a male-like neuro-morphological pattern in the AOB, by comparison with the non-PES group, reared by their own non-stressed mothers. By contrast, when adult, the PES group reared by non-stressed mothers showed hormonal and morphological neuronal alterations, but they displayed appropriate (full) MB. The non-PES group raised by stressed mothers also showed altered hormone levels, but showed full MB and no morphological neuronal changes. Significant differences in the AOB and MPOA c-fos activity, related to whether or not MB was expressed, were found in the non-PES groups, but not in the PES group reared by non-stressed mothers. To our knowledge, this is the first study to document that adequate maternal care, early in development, can shape the subsequent expression of induced MB, overcoming neuro-morphological and hormonal alterations that are produced by prenatal environmental stress. We conclude that maternal care during early postnatal development can counteract detrimental effects of prenatal environmental stress, exerting long-lasting effects that modulate the behavioral phenotype of the offspring.

The development of brain sex differences: a multisignaling process.

In order to account for the development of sex differences in the brain, we took, as an integrative model, the vomeronasal pathway, which is involved in the control of reproductive physiology and behavior. The fact that brain sex differences take place in complex neural networks will help to develop a motivational theory of sex differences in reproductive behaviors. We also address the classic genomic actions in which three agents (the hormone, the intracellular receptor, and the transcription function) play an important role in brain differentiation, but we also point out refinements that such a theory requires if we want to account of the existence of two morphological patterns of sex differences in the brain, one in which males show greater morphological measures (neuron numbers and/or volume) than females and the opposite. Moreover, we also consider very important processes closely related to neuronal afferent input and membrane excitability for the developing of sex differences. Neurotransmission associated to metabotropic and ionotropic receptors, neurotrophic factors, neuroactive steroids that alter membrane excitability, cross-talk (and/or by-pass) phenomena, and second messenger pathways appear to be involved in the development of brain sex differences. The sexual differentiation of the brain and reproductive behavior is regarded as a cellular multisignaling process.

Estradiol masculinizes the number of accessory olfactory bulb mitral cells in the rat.

Orchidectomized males injected with a single dose of estradiol benzoate on the day of birth (D1) showed mitral cell numbers in the accessory olfactory bulb similar to those of control males. However, orchidectomized males that received no additional estradiol benzoate treatment and those orchidectomized and given a single dose of dihydrotestosterone on D1 showed decreases in the number of accessory olfactory bulb mitral cells compared with control males. These results support the notion that the presence of estradiol immediately after birth induces the masculinization of mitral cells number in the accessory olfactory bulb.

Role of GABAA receptors in the organization of brain and behavioural sex differences.

Sex differences in GABA neurotransmitter have been described. We have studied the involvement of the GABAA receptor in sex differences in the brain and reproductive behaviour. Neonatal administration of the GABAA agonist diazepam to male rats facilitated the induction of maternal behaviour in adults, while the antagonist picrotoxin disrupted it in females. Sex differences in the accessory olfactory bulb were also reversed, but gonadal function remained unaltered in both sexes. This suggests that neonatal changes in neuronal membrane permeability to Cl- ions may play a role in the organization of sex differences. Our study constitutes a new model for understanding the early neurobiological organization of sex differences.

Early postnatal diazepam exposure facilitates maternal behavior in virgin female rats.

Virgin female rats do not display maternal behavior if they are not exposed to the pups during several days. This exposure is called induction. In this work we have studied the effects of early postnatal (PO-P16) diazepam (DZ) administration (1 and 2.5 mg/kg, SC) on the display of maternal behavior of virgin female rats when adults. Although we did not find statistically significant differences between P0-P16 DZ treated and control females with respect to the latency of retrieval, P0-P16 DZ administration resulted in a statistically significant increase of the percentage of female rats that became maternal, showing retrieval behavior. This early postnatal treatment with DZ also increased other variables that are currently measured in maternal behavior tests, such as: time of physical contacts, grooming, crouching, and nest building quality. No statistically significant differences were found in the body weight of treated versus control animals during development, nor during adulthood. Our results provide further evidence that the GABAA-BDZ-Cl- receptor complex is implicated in the development of maternal behavior in female rats.

Perinatal administration of diazepam alters sexual dimorphism in the rat accessory olfactory bulb.

The present study examines the effects of pre and/or early postnatal administration of diazepam on the mitral cell and on the light and dark granule cell populations in the sexually dimorphic accessory olfactory bulb of the rat. Quantitative differences related to sex were observed in the numbers of the three types of neurons, with vehicle males showing greater numbers of cells than vehicle females. The number of mitral cells in males decreased to the levels shown by female rats following prenatal and pre-postnatal diazepam treatments, whereas the DZ treatments did not affect the females. In addition, the diazepam administration during the prenatal, postnatal and pre-postnatal periods decreased the numbers of both light and dark granule cells in males, while these two granule cell subpopulations were not affected in diazepam treated females. These results indicate that perinatal administration of diazepam can alter the sexual dimorphism in the accessory olfactory bulb and that the GABAA/benzodiazepine receptor complex is involved in the sexual differentiation this part of the brain.

Effects of estradiol on the development of sexual dimorphism in the bed nucleus of the accessory olfactory tract in the rat.

Orchidectomized males injected with a single dose of estradiol benzoate (EB) on the day of birth (D1) showed a volume and neuron number in the nucleus of the accessory olfactory tract (BAOT) similar to that of control males. However, orchidectomized males and those orchidectomized and given a single dose of DHT on D1 showed a decrease in BAOT volume and neuron number with respect to control males. These results support the notion that estradiol induces the morphological masculinization of this structure. The inability of DHT in counteracting the effect of orchidectomy is addressed taking into account the inhibitory action of androgens.

Effects of perinatal diazepam exposure on the sexually dimorphic rat locus coeruleus.

Diazepam (DZ) administration over prenatal, postnatal, and pre plus postnatal periods altered the normal expression of the morphological sex differences of the LC. Males were affected only by the prenatal exposure and the effect of this exposure produced an increase in the volume and neuron number of male's LC. By contrast, females were affected by both pre and postnatal treatments and the effect of this exposure resulted in a decrease in the volume and neuron number of female's LC. However, pre plus postnatal treatment did not affect female's LC.

Postnatal administration of dihydrotestosterone to the male rat abolishes sexual dimorphism in the accessory olfactory bulb: a volumetric study.

The regulatory action of the non-aromatizable androgen dihydrotestosterone (DHT) on sexual differentiation of the volume of the rat accessory olfactory bulb (AOB) was studied. Postnatal treatment with DHT (180 micrograms/day) carried out daily between days 6 and 20 produced a drastic reduction in overall AOB size and that of its constituent neural layers in genetic males with respect to intact and control males. The volumetric measures found in DHT-treated males did not differ from those shown by the intact females. These results, which indicate a demasculinization and a feminization of the AOB volume in gonadally intact male rats induced by DHT, are discussed in relation to the presumably regulatory role of DHT on neuron populations during the sexual organizational process of the brain.

Effects of early postnatal sex steroids on acquisition and extinction of a continuously reinforced lever-pressing response.

The effects of early postnatal dihydrotestosterone (DHT) and estradiol on the sexually dimorphic continuously reinforced lever-pressing response were investigated. 90-day-old male rats postnatally treated (during the first eight days of postnatal life) with cyproterone acetate (CA), tamoxifen (TX) or vehicle, and 90-day-old females treated with estradiol benzoate (EB), DHT or vehicle in the same postnatal period, were studied during the acquisition and extinction of the continuously reinforced lever-pressing response using a free-operant procedure. During acquisition, the control males made more responses per minute than the control females, and also reached the extinction criterion significantly sooner than the females. CA treatment impaired the male's performance at the levels of that shown by females, whereas TX treatment affected neither acquisition nor extinction. Inversely, in both experimental phases females treated with DHT performed like control females, whereas the acquisition and extinction performances of the EB-females were similar to those obtained in the control or TX male groups.

Female's DHT controls sex differences in the rat bed nucleus of the accessory olfactory tract.

In the present study the regulatory action of the non-aromatic androgen dihydrotestoterone (DHT) on the volume of the sexually dimorphic bed nucleus of the accessory olfactory tract (BAOT) was investigated. Postnatal treatment with DHT (180 micrograms day-1) between days 6 and 20 (D6-D20) induced, in gonadally intact male rats, a drastic reduction in the overall volume to levels typical in control females. Conversely, the postnatal administration of the anti-androgen cyproterone acetate (CA) to the females from D6-D20 produced an increment in the BAOT volume not dissimilar to that found in control males. These findings reveal that sexual organization in this vomeronasal structure is dependent on the presence of DHT in females during postnatal development.

Early postnatal diazepam exposure alters sex differences in the rat brain.

The volume and neuron number of the sexually dimorphic accessory olfactory bulb and locus coeruleus are altered by early postnatal exposure (from the day of birth to postnatal day 16) to diazepam. After diazepam treatment, both volume and neuron number were decreased in the male accessory olfactory bulb and in the female locus coeruleus. These results indicate that early postnatal diazepam administration can bear gender-dependent teratogenic effects upon sexually dimorphic nuclei and suggest that endogenous benzodiazepines may be involved in the sexual differentiation of the brain.

Effects of perinatal diazepam administration on two sexually dimorphic nonreproductive behaviors.

The effects of prenatal and/or early postnatal diazepam (DZ) administration on open field activity and continuously reinforced lever-pressing response were studied. Rat pups of both sexes were prenatally (during the last week of pregnancy) and/or postnatally (from the day of birth to day 16) daily exposed to a 2.5 mg/kg dose of DZ. At the age of 60 days all groups were tested in the open field for 5 consecutive days and thirty days later they were studied in a continuously reinforced lever-pressing situation during four consecutive days. In the open field test, females showed greater activity than males and prenatal and/or early postnatal DZ treatments did not alter this sexual dimorphism, although all treatments decreased the open field activity in both male and female 60-day-old rats. In the Skinner box, 90-day-old males presented higher rates of lever-pressing response than females, and only the early postnatal DZ treatment was effective in altering this sexual dimorphism, by decreasing the male's but not female's rates of response. These results are discussed in regard to the possible interaction between DZ and gonadal hormones during the early sexual differentiation period.