Hormonal programming across the lifespan.

Hormones influence countless biological processes across an animal's lifespan. Many hormone-mediated events occur within developmental sensitive periods, during which hormones have the potential to cause permanent tissue-specific alterations in anatomy and physiology. There are numerous selective critical periods in development with different targets being affected during different periods. This review outlines the proceedings of the Hormonal Programming in Development session at the US-South American Workshop in Neuroendocrinology in August 2011. Here we discuss how gonadal steroid hormones impact various biological processes within the brain and gonads during early development and describe the changes that take place in the aging female ovary. At the cellular level, hormonal targets in the brain include neurons, glia, or vasculature. On a genomic/epigenomic level, transcription factor signaling and epigenetic changes alter the expression of critical hormone receptor genes across development and following ischemic brain insult. In addition, organizational hormone exposure alters epigenetic processes in specific brain nuclei and may be an important mediator of sexual differentiation of the neonatal brain. Brain targets of hormonal programming, such as the paraventricular nucleus of the hypothalamus, may be critical in influencing the development of peripheral targets, such as the ovary. Exposure to excess hormones can cause abnormalities in the ovary during development leading to polycystic ovarian syndrome (PCOS). Exposure to excess androgens during fetal development also has a profound effect on the development of the male reproductive system. In addition, increased activity of the sympathetic nerve and stress during early life have been linked to PCOS symptomology in adulthood. Finally, we describe how age-related decreases in fertility are linked to high levels of nerve growth factor (NGF), which enhances sympathetic nerve activity and alters ovarian function.

Acute stress anticipates and amplifies the luteinizing hormone pre-ovulatory surge in rats: Role of noradrenergic neurons.

The stress experienced during rape seems to facilitate ovulation since the pregnancy rate in raped women is higher than that resulting from consensual intercourse. Adrenal progesterone, as well as central norepinephrine, is released in stressful situations. At adequate estrogenic levels, one of the main actions of progesterone is to anticipate the preovulatory LH surge through noradrenaline release. We aimed to investigate whether acute stresses that mimic those of rape (exposure to predator, restraint and cervix stimulation) applied on the proestrus morning in female rats could release progesterone, activate the noradrenergic neurons and facilitate the occurrence of the LH surge. Female rats were submitted to jugular vein cannulation immediately following acute stress: restraint (R), exposure to cat (P), uterine cervix stimulation (CS) applied individually or in association (SA). Non-stressed rats were used as control. Blood samples were collected from 11:00-18:00 h for LH, progesterone, corticosterone and estradiol measurements. Double labeling for c-Fos and tyrosine hydroxylase (TH) was examined in A1, A2 and A6 noradrenergic neurons after stresses. The SA group showed a greater stress-induced increase in progesterone compared to the other groups and the preovulatory LH surge was anticipated and amplified. This effect of SA seems to be related to the higher number of c-Fos/TH + neurons in the A1 and A2. The effect of anticipating the preovulatory surge of LH could in part elucidate why, in raped women, conception can occur in phases of the menstrual cycle other than the ovulatory phase facilitating the occurrence of pregnancies.

Neonatal handling and the maternal odor preference in rat pups: involvement of monoamines and cyclic AMP response element-binding protein pathway in the olfactory bulb.

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic-pituitary-adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age.

Both infantile stimulation and exposure to sweet food lead to an increased sweet food ingestion in adult life.

We have reported that neonatal handling leads to increased sweet food preference in adult life. Our aim was to verify if these differences in feeding behavior appear before puberty, and whether other types of intervention in periadolescence (such as exposure to toys) could interfere with sweet food consumption later in life. Nests of Wistar rats were (1) non-handled or (2) handled (10 min/day) on days 1-10 after birth. Males from these groups were subdivided in two subgroups: one was habituated to sweet food (Froot Loops-Kellogs) in a new environment for 4 days and tested for sweet food preference at age 27 days, before submitting to a new habituation and test for sweet food ingestion again in adult life. The other subgroup was habituated and tested only in adulthood. In another set of experiments, neonatally non-handled rats were exposed or not to a new environment with toys in periadolescence, and tested for sweet food ingestion as adults. Neonatal handling increases sweet food consumption only if the habituation and tests are performed after puberty. Interestingly, infant exposure to sweet food had a similar effect as neonatal handling, since controls that were exposed to sweet food at age 22 to 27 days increased their ingestion as adults. Exposure to toys in periadolescence had the same effect. We suggest that an intervention during the first postnatal days or exposure to an enriched environment later in the pre-pubertal period leads to behavioral alterations that persist through adulthood, such as increased sweet food ingestion.

Effect of 5-HT1B receptor agonists injected into the prefrontal cortex on maternal aggression in rats.

Serotonin (5-HT1B) receptors play an essential role in the inhibition of aggressive behavior in rodents. CP-94,253, a 5-HT1B receptor agonist, can reduce aggression in male mice when administered directly into the ventro-orbitofrontal (VO) prefrontal cortex (PFC). The objective of the current study was to assess the effects of two selective 5-HT1B receptor agonists (CP-94,253 and CP-93,129), microinjected into the VO PFC, on maternal aggressive behavior after social instigation in rats. CP-94,253 (0.56 microg/0.2 microL, N = 8, and 1.0 microg/0.2 microL, N = 8) or CP-93,129 (1.0 microg/0.2 microL, N = 9) was microinjected into the VO PFC of Wistar rats on the 9th day postpartum and 15 min thereafter the aggressive behavior by the resident female against a male intruder was recorded for 10 min. The frequency and duration of aggressive and non-aggressive behaviors were analyzed using ANOVA and post hoc tests. CP-93,129 significantly decreased maternal aggression. The frequency of lateral attacks, bites and pinnings was reduced compared to control, while the non-aggressive behaviors and maternal care were largely unaffected by this treatment. CP-94,253 had no significant effects on aggressive or non-aggressive behaviors when microinjected into the same area of female rats. CP-93,129, a specific 5-HT1B receptor agonist, administered into the VO PFC reduced maternal aggressive behavior, while the CP-94,253 agonist did not significantly affect this behavior after social instigation in female rats. We conclude that only the 5-HT1B receptor agonist CP-93,129 administered into the VO PFC decreased aggression in female rats postpartum after social instigation.

Neonatal handling and reproductive function in female rats.

Neonatal handling induces anovulatory estrous cycles and decreases sexual receptivity in female rats. The synchronous secretion of hormones from the gonads (estradiol (E2) and progesterone (P)), pituitary (luteinizing (LH) and follicle-stimulating (FSH) hormones) and hypothalamus (LH-releasing hormone (LHRH)) are essential for the reproductive functions in female rats. The present study aimed to describe the plasma levels of E2 and P throughout the estrous cycle and LH, FSH and prolactin (PRL) in the afternoon of the proestrus, and the LHRH content in the medial preoptic area (MPOA), median eminence (ME) and medial septal area (MSA) in the proestrus, in the neonatal handled rats. Wistar pup rats were handled for 1 min during the first 10 days after delivery (neonatal handled group) or left undisturbed (nonhandled group). When they reached adulthood, blood samples were collected through a jugular cannula and the MPOA, ME and MSA were microdissected. Plasma levels of the hormones and the content of LHRH were determined by RIA. The number of oocytes counted in the morning of the estrus day in the handled rats was significantly lower than in the nonhandled ones. Neonatal handling reduces E2 levels only on the proestrus day while P levels decreased in metestrus and estrus. Handled females also showed reduced plasma levels of LH, FSH and PRL in the afternoon of the proestrus. The LHRH content in the MPOA was significantly higher than in the nonhandled group. The reduced secretion of E2, LH, FSH and LHRH on the proestrus day may explain the anovulatory estrous cycle in neonatal handled rats. The reduced secretion of PRL in the proestrus may be related to the decreased sexual receptiveness in handled females. In conclusion, early-life environmental stimulation can induce long-lasting effects on the hypothalamus-pituitary-gonad axis.

8-OH-DPAT in the median raphe nucleus decreases while in the medial septal area it may increase anxiety in female rats.

The experiment evaluated the effects of 8-OH-DPAT on the activity of virgin female rats (diestrus) in the elevated plus maze. The 5-HT1A receptor agonist was infused into the median raphe nucleus (N = 60) and medial septal area (N = 68) 10 min before the test. Five groups for each brain area were analyzed: intact, saline (0.2 microl) and 8-OH-DPAT (0.2; 0.5 and 2.0 microg rat(-1)). The following measures were recorded: number of entries onto open and enclosed arms and time spent on the open and enclosed arms. In addition, the frequency of stretch-attend and head-dipping were also evaluated. The results showed that in the median raphe nucleus only the highest dose of 8-OH-DPAT (2.0 microg) increased the percentage of time spent on the open arms. On the other hand, in medial septal area 8-OH-DPAT in the dose of 0.5 microg decreased the percentage of time spent on the open arms, while the doses of 0.2 and 2.0 microg had no significant impact on anxiety. Data suggest that 8-OH-DPAT acting on 5-HT1A somatodendritic autoreceptors decreases anxiety. However, at a specific dosage and acting on postsynaptic receptors of the medial septal area, 8-OH-DPAT can increase anxiety.

Central oxytocin mediates stress-induced tachycardia.

To address the role of oxytocin in the control of cardiovascular reactivity, we examined the effect of central injection of oxytocin, vasopressin and mixed base antisense oligodeoxynucleotides on stress-induced cardiovascular and endocrine changes. Antisense oligomers were injected into the paraventricular nucleus (PVN), 4 h prior to the stress test. The oxytocin antisense abolished the tachycardia produced by 5 min of shaker stress. The blood pressure and plasma oxytocin responses were not different between the groups. PVN levels of OT were reduced in the oxytocin antisense-treated group while brain stem levels were increased. These results demonstrate the importance of a specific peptide system, the PVN/oxytocin axis, in stress-induced tachycardia. Further, the data illustrate the effectiveness of short-term treatment with antisense oligomers on physiological responses.

Long-lasting effects of neonatal stimulation on the behavior of rats.

The present study aimed to analyze the effects of neonatal stimulation on species-specific behaviors (defensive reactions to a predator and social interactions) in adult male and female rats. Handling and an unpredictable sequence of aversive stimuli were applied to male and female pups from the 1st to the 10th day after delivery; behavioral inhibition, aggression, and sexual behavior were evaluated in adulthood. Results showed that either neonatal handling or aversive stimulation decreased behavioral inhibition in a novel and potentially harmful situation (open field with a predator) in both male and female rats and increased maternal aggressive behavior. Sexual behavior in both males and females decreased, which could affect reproductive capability. The results could cast doubts on the generalization of beneficial effects of neonatal stimulation on the behavior of adult rats.

8-OH-DPAT in the median raphe, dorsal periaqueductal gray and corticomedial amygdala nucleus decreases, but in the medial septal area it can increase maternal aggressive behavior in rats.

The purpose of the present study was to analyze the role of somatodendritic autoreceptors and postsynaptic 5-HT1A receptors in the modulation of maternal aggressive behavior. The 5-HT1A receptor agonist 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) was microinjected (0.2, 0.5 and 2.0 microg/0.2 microl) in different brain areas of female Wistar rats: median raphe nucleus (MnR); medial septal area (MS); anterior corticomedial amygdaloid nucleus (ACoM); and dorsal periaqueductal gray (DPAG). The behaviors of lactating female rats with pups against a conspecific male intruder were recorded on day 7 post-partum. Results showed that in the median raphe nuclei, in the dorsal periaqueductal gray and in the corticomedial amygdaloid nucleus 8-OH-DPAT decreased maternal aggression; while in the medial septum, the intermediate dose (0.5 microg/0.2 microl) of the 5-HT1A receptor agonist increased the aggressive behavior of the lactating female rat. It is concluded that the main role of the 5-HT1A somatodendritic autoreceptors and the post-synaptic receptors of the brain areas studied is to decrease maternal aggression, however, at a specific dosage, 8-OH-DPAT acting on postsynaptic receptors of the medial septal area can increase aggressiveness.

Hypothalamic paraventricular nucleus, oxytocin, and maternal aggression in rats.

Both the lesion of the parvocellular region of the PVN (FIG. 1) and the acute reduction of OT synthesis in that nucleus (FIG. 2) increase maternal aggression in rats. Previous work showed that ibotenic acid as well as the OT antisense in the PVN reduced the level of OT in the brainstem, but not in the pituitary. Therefore, the oxytocinergic parvocellular neurons of the PVN appear to exert an inhibitory effect on the aggressive behavior of the lactating female rat against an adult intruder. In a relationship of a different nature, mother-infant, a facilitatory effect of OT has been shown. Previous work showed a significant decrease of OT mRNA levels in the PVN of female rats during the first 10 days after delivery compared to late pregnancy, which is the inverse ratio of the natural temporal evolution of maternal aggressive behavior. Furthermore, in the present work, a functional decrease of OT mRNA was probably the effect of the antisense in the PVN. In conclusion, OT cells in the PVN appear to play different roles on maternal care and maternal aggression.

Pre-training adrenaline recovers the amnestic effect of Met-enkephalin in demedullated rats.

The intracerebroventricular administration of 25 ng of Met-enkephalin causes retrograde amnesia for a shuttle avoidance task in intact rats. In demedullated rats, this effect of Met-enkephalin was lost. Intraperitoneal injection of 1.2 micrograms/kg adrenaline recovered the amnestic effect of Met-enkephalin. These results confirm the idea that the amnestic effect of Met-enkephalin is centrally mediated and that adrenal enkephalins do not seem to be important to the amnestic effect of Met-enkephalin since adrenaline recovers this effect in demedullated rats.

Intracerebroventricular administration of nanogram amounts of beta-endorphin and Met-enkephalin causes retrograde amnesia in rats.

The intracerebroventricular (icv) administration of 5.0 or 25.0 ng of beta-endorphin or Met-enkephalin causes retrograde amnesia for a shuttle avoidance task ion rats. In both cases, the higher dose was more effective than the lower one. The present results confirm previous similar findings obtained using systemic administrations of these compounds, and suggest that the amnestic effect of beta-endorphin and Met-enkephalin is mediated centrally.

The effect of testosterone and DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) on male sexual behavior of rats.

The effects of a 5-HT2 receptor agonist, DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; 0.5 mg/kg), on the behavior of male rats at different ages when given alone or with different levels of testosterone, in the presence of sexually receptive and non-receptive females are presented. DOI increased mounting and/or mount plus thrusting behavior in adult males with receptive females. In pre-pubertal males, DOI increased the frequency of pursuit and genital sniffing in the presence of receptive females, but not of non-receptive ones, when no mounts or thrustings were recorded. In castrated rats treated with testosterone and tested with receptive females, DOI increased the frequency of thrusting behavior, but in castrated rats without testosterone treatment, DOI produced no change. DOI did not induce mounting in pre-pubertal or castrated rats without testosterone substitution therapy. These results suggest that DOI influences male sexual behavior through a neural system that is modulated by testosterone.

Effects of intracerebroventricular administration of 5-HT receptor agonists on the maternal aggression of rats.

This study attempted to analyze the effects of 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), TFMPP (1-(3-trifluoromethyl-phenyl)piperazine hydrochloride), and DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) on maternal aggressive behavior. Female Wistar rats were divided into 4 groups of 12 animals each. They received an intracerebroventricular (i.c.v.) injection of: (1) saline, (2) 8-OH-DPAT (20 micrograms/rat), (3) TFMPP (100 micrograms/rat), and (4) DOI (100 micrograms/rat). 5-HT1A (8-OH-DPAT) and 5-HT2 (DOI) receptor agonists decreased the frequency of attack 15 but not 55 min after i.c.v. injection. The 5-HT1B/D receptor agonist (TFMPP), in the dose studied, showed no significant difference as compared to saline. Pup care and non-aggressive social interaction with the intruder were not affected by any drug. These data suggest that 5-HT1A and 5-HT2 receptor agonists can specifically inhibit maternal aggression without affecting maternal care; however, this effect is of short duration.

Technique for collecting cerebrospinal fluid in the cisterna magna of non-anesthetized rats.

We developed a technique for collecting cerebrospinal fluid (CSF) from the cisterna magna in non-anesthetized adult and young pup rats. In the adults, CSF was collected through a previously implanted guide cannula without previous disruption of the cisterna magna. In the pups, CSF was directly aspirated through a syringe from the cisterna in awake animals without previous surgery. In the adults, the volume of CSF collected varied from 50 to 120 microl, and in pups 7 to 10 days old, it was approximately 25 microl. The technique can easily be done by anyone who is familiar with stereotaxic surgery, and the material needed is cheap and easy to obtain commercially. A simple procedure to calculate the parameters for the implantation of guide cannula in rats other than Wistar ones is also presented.

Lesion of hypothalamic paraventricular nucleus and maternal aggressive behavior in female rats.

Lactating female rats on the 3rd to 12th day postpartum are more aggressive towards an intruder male than are nonlactating females. In this study, maternal aggressive behavior was recorded by introducing a strange male in the territory of the female and her offspring, on the fifth, seventh, and ninth day postpartum. Electrolytic lesions of the paraventricular nucleus of the hypothalamus (PVN) were performed on the fifth day postpartum. The results showed that the PVN lesion reduced the frequency and duration of attacks on the intruder. In addition, the lesion caused reduced weight gain in the pups compared to pups of the sham lesion group. The results suggest that PVN participates in the modulation of maternal aggression in rats. A possible role of oxytocin in that behavior is discussed.

Hypothalamic paraventricular nucleus modulates maternal aggression in rats: effects of ibotenic acid lesion and oxytocin antisense.

Central oxytocin (OT) appears to be crucial for maternal behavior. OT, through the parvocellular neurons of the hypothalamic paraventricular nucleus (PVN), can exert its physiological and behavioral effects by acting on OT receptors in nonpituitary projections of the PVN. The purpose of the present study was to analyze the role of the PVN and OT on maternal aggressive behavior in two different periods after delivery: on the fifth day (period of high aggressiveness) and on the eighteenth day postpartum (period of low aggressiveness). In the first experiment, ibotenic acid was injected into the PVN in order to lesion the parvocellular neurons. A second experiment was designed to study more specifically the effects of OT using the antisense technique. On the fifth day postpartum, both the PVN lesion by the ibotenic acid and a possible acute reduction of OT synthesis by the antisense administration in that nucleus increased maternal aggressive behavior, while on the eighteenth day postpartum no effect was recorded. We may conclude that central projections of the PVN modulate maternal aggression during a restricted period after delivery, only when lactating females show naturally high levels of aggressive behaviors.

Influence of the mother on development of aggressive behavior in male rats.

The present experiments investigated pre- and postnatal maternal effects on aggressive behavior in rats. Resident-intruder aggressive behavior of male rats in colonies (two males and two females) was studied in five experimental groups: 1 = WWY (n = 7) the two males of each colony were wild (biological father and mother were wild) fostered by a wild mother; 2 = WAY (n = 11) the two males were wild fostered by an albino Wistar mother; 3 = AAY (n = 11) the two males were albino (biological father and mother were Wistar) fostered by an albino mother; 4 = AWY (n = 12) the two males were albino fostered by a wild mother; and 5 = HWX+HAX (n = 9) one of the males was hybrid born and reared by a wild mother (the father was albino) and the other was also hybrid but born and reared by an albino mother (the father was wild). Each test lasted 10 min and the intruder was always a Wistar male. Aggression of wild rats was higher than the laboratory ones, independently of the mother (albino or wild) they were fostered by. However, hybrid males born and reared by a wild mother were more aggressive than those that were born and reared by an albino mother, in spite of the father being wild. In conclusion, crossfostering has little effect on territorial aggression, but prenatal maternal effects seem to play a major role on the ontogeny of aggressive behavior of male rats.

Influence of early postnatal gonadal hormones on anxiety in adult male rats.

Behavioral sex differences have been linked to the presence of testosterone secretion during a critical perinatal period. The present experiment tested whether or not castration at different ages (early postnatal period and adulthood) would alter performance in the plus maze, a behavioral test of anxiety. Intact adult male rats (n = 17) were compared to intact adult females (n = 17); adult castrated males (n = 7) to sham-operated adult male rats (n = 9); and newborn castrated males (n = 7) to sham-operated male offspring (n = 8). When adult, the subjects were left on an elevated plus maze for 5 min. Females made a higher percentage of entries onto the open arms and showed a greater number of scans over the edge of an open arm than males. There were no differences in the percentage of arm entries or time spent on the open arms when adult castrated males were compared to sham-operated rats. On the other hand, newborn castrated males showed a significantly higher number of open arm entries and spent a greater percentage of time on the open arms than sham-operated offspring. The results demonstrate that the absence of male gonadal hormones during the perinatal period decreases anxiety, as assessed in the elevated plus maze, leading to a behavioral pattern that resembles that of females. These data provide evidence for the organizational role of gonadal hormones in the development of behavioral inhibitory systems.