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The aim of this study was to investigate whether maternal cafeteria ingestion interferes with long-term memory-related behaviors and hippocampal brain-derived neurotrophic factor (BDNF) signaling in the offspring, and if there is a cumulative effect with the exposure to cafeteria diet during the life-course of the pups. Female rats were fed a control (CON, n = 20) or cafeteria diet (CAF, n = 24) from their weaning to weaning of their offspring. After that, their male offspring were divided into 4 groups (CON-CON, n = 36; CON-CAF, n = 38, CAF-CON, n = 46 and CAF-CAF, n = 39) so that all litters ingested CON or CAF, irrespective of maternal diet. At 30 days of age, all groups exposed to cafeteria diet at some stage in life showed a decline in performance on one or both object recognition and inhibitory avoidance tasks. At 120 days, CON-CAF and CAF-CAF groups continued to show memory impairment. There were no significant differences between groups in the hippocampal concentrations of BDNF and cAMP Response Element Binding protein (CREB) in puberty or adulthood, but the concentration of hippocampal Ras-Raf-MEK-mitogen-activated protein kinase (MAPK) was higher in CAF-CAF pubescent offspring when compared to the CON-CON group. These data suggest that maternal diet affects the behavior and the molecular signaling related to long-term memory of the offspring, and that its effects are influenced by postnatal diet., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the present study, we investigated whether maternal exposure to a cafeteria diet affects the metabolism and body composition of offspring and whether such an exposure has a cumulative effect during the lifetime of the offspring. Female rats were fed a control (CON) or a cafeteria (CAF) diet from their own weaning to the weaning of their offspring. At 21 d of age, male offspring were divided into four groups by diet during gestation and after weaning (CON-CON, CON-CAF, CAF-CON and CAF-CAF). Blood was collected from dams (after weaning) and pups (at 30 and 120 d of age) by decapitation. CAF dams had significantly greater body weight and adipose tissue weight and higher concentrations of total cholesterol, insulin and leptin than CON dams (Student's t test). The energy intake of CAF rats was higher than that of CON rats regardless of the maternal diet (two-way ANOVA). Litters had similar body weights at weaning and at 30 d of age, but at 120 d, CON-CAF rats were heavier. At both ages, CAF rats had greater adipose tissue weight than CON rats regardless of the maternal diet, and the concentrations of TAG and cholesterol were similar between the two groups, as were blood glucose concentrations at 30 d of age. However, at 120 d of age, CAF rats were hyperglycaemic, hyperinsulinaemic and hyperleptinaemic regardless of the maternal diet. These findings suggest that maternal obesity does not modulate the metabolism of male offspring independently, modifying body weight only when associated with the intake of a cafeteria diet by the offspring.

Maternal obesity is a risk factor for the development of metabolic syndrome and childhood obesity, and early overnutrition seems to induce the development of pathologies in adulthood, including insulin resistance, cardiovascular diseases, type 2 diabetes mellitus, and a higher BMI. In addition, it is known that obesity can negatively affect fertility and reproductive function in men. The objective of this work was to investigate the impact of maternal obesity induced by the consumption of cafeteria diet on metabolic, endocrine and reproductive outcomes in the male offspring. Body weight, abdominal fat content and concentrations of insulin, leptin, glucose and total cholesterol were analyzed in dams. The same parameters were evaluated in pups when in adulthood, in addition to the analysis of sexual behavior, followed by measurement of plasma luteinizing hormone, follicle-stimulating hormone, testosterone, and prolactin. Maternal consumption of cafeteria diet affected reproductive hormone regulation in the offspring and such modifications were reflected on sexual performance. Also, these modifications were independent of time and of the reproductive period during which dams consumed the diet. Our results indicate, for the first time, that maternal nutrition may have a deep impact on the reproductive function of the adult male offspring., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Besides the well-known detrimental effects of obesity on cardiovascular and metabolic function, studies have shown that obesity is also associated with impaired reproductive function in women. Alterations in Angiotensin II (Ang II) have been associated with obesity and with female reproduction. The aim of the present study was to evaluate the reproductive and metabolic effects of Ang II AT1 receptor blockade with losartan in an animal model of obesity, in which female rats were offered a palatable, high calorie diet from weaning to adulthood. Sexual behavior, ovulation rates and preovulatory levels of the hormones estradiol, progesterone, LH and prolactin were analyzed. Retroperitoneal and perigonadal fat pads, triglycerides and cholesterol (total, HDL and LDL), and insulin resistance were analyzed. Losartan prevented increases in fat pad storage, insulin resistance, as well as triglycerides and LDL levels induced by cafeteria diet intake. Losartan also prevented ovulatory deficits and loss of preovulatory surges of progesterone and LH in cafeteria-fed female rats probably through the prevention of the increase in body weight and body fat. No alterations in sexual behavior were observed. These results suggest, for the first time, that Ang II contributes to the development of the deleterious effects of obesity on preovulatory surges of LH and progesterone and on the reduction of ovulation in obese female rats., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Hypertension is a major public health epidemic that is highly associated with sexual dysfunction in both men and women. Despite its high prevalence, clinical and animal literature on the underlying mechanisms of sexual dysfunction in hypertensive women is remarkably limited., Methods: Using a well-established rodent model of renovascular hypertension-the 2-kidney, 1-clip (2K1C) Goldblatt model-we investigated possible reproductive deficits in female rats. We evaluated several aspects of reproductive function in hypertensive female rats: estrous cycle, sexual behavior, ovulation, and plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol at proestrus afternoon., Results: Clipping of the left renal artery resulted in dramatic elevations in systolic blood pressure and heart rate. Renovascular hypertension was associated with a delay for reestablishing estrous cyclicity (50% of 2K1C rats failed to resume cycling by 15 days after surgery). In rats that resumed cycling, 2K1C female rats showed a decrease in sexual behavior, evidenced by a decreased lordosis quotient and a reduction in ovulation, as demonstrated by a decreased number of oocytes. Moreover, plasma levels of LH on the proestrus afternoon were reduced in hypertensive female rats, but no changes in estradiol or FSH were observed., Conclusions: Our results demonstrate that renovascular hypertension induces an overall decrease in reproductive function in female rats. Most important, our results indicate that the animal model of renovascular hypertension could be used as a relevant tool to understand better the pathophysiological mechanisms involved in the reproductive deficits in women with renovascular hypertension.

Obesity affects a large number of people around the world and appears to be the result of changes in food intake, eating habits and physical activity levels. Changes in dietary patterns and physical exercise are therefore strongly recommended to treat obesity and its complications. The present study tested the hypothesis that obesity and metabolic changes produced by a cafeteria diet can be prevented with dietary changes and/or physical exercise. A total of fifty-six female Wistar rats underwent one of five treatments: chow diet; cafeteria diet; cafeteria diet followed by a chow diet; cafeteria diet plus exercise; cafeteria diet followed by a chow diet plus exercise. The duration of the experiment was 34 weeks. The cafeteria diet resulted in higher energy intake, weight gain, increased visceral adipose tissue and liver weight, and insulin resistance. The cafeteria diet followed by the chow diet resulted in energy intake, body weight, visceral adipose tissue and liver weight and insulin sensitivity equal to that of the controls. Exercise increased total energy intake at week 34, but produced no changes in the animals' body weight or adipose tissue mass. However, insulin sensitivity in animals subjected to exercise and the diet was similar to that of the controls. The present study found that exposure to palatable food caused obesity and insulin resistance and a diet change was sufficient to prevent cafeteria diet-induced obesity and to maintain insulin sensitivity at normal levels. In addition, exercise resulted in normal insulin sensitivity in obese rats. These results may help to develop new approaches for the treatment of obesity and type 2 diabetes mellitus.

There is an association between hypertension and reproductive dysfunction. Angiotensin II (Ang II) is involved in the pathogenesis of hypertension and the regulation of reproduction. The present study aimed to determine whether the angiotensinergic system mediates the effects of hypertension on reproductive function in male rats subjected to a two-kidney, one-clip (2K1C) model. Sexual behavior parameters, gametogenesis and plasma concentrations of Ang II, testosterone, prolactin and corticosterone were evaluated in male rats 28days after 2K1C or sham surgery and losartan (Los) treatment (a type 1 angiotensin II (AT1) receptor antagonist) or vehicle (V) treatment. The animals were divided into Sham+V, 2K1C+V, Sham+Los and 2K1C+Los groups. The 2K1C+V group showed a hypertensive response, inhibition of sexual behavior, spermatogenesis dysfunction, and increases in plasma Ang II and prolactin. Conversely, plasma testosterone decreased, and plasma corticosterone remained constant. Losartan treatment normalized blood pressure and prevented the changes in plasma testosterone and prolactin, sexual behavior and spermatogenesis in the 2K1C+Los group. In addition, losartan treatment caused an additional increase in circulating Ang ll in both groups (Sham+Los and 2K1C+Los). Together, these results suggest that Ang ll, acting through the AT1 receptor, modulates behavioral and endocrine parameters of reproductive function during renovascular hypertension. In addition, the effects of circulating Ang II on plasma testosterone and prolactin seem to contribute to the spermatogenic and sexual dysfunctions in hypertensive rats., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The incidence of obesity is increasing rapidly all over the world and results in numerous health detriments, including disruptions in reproduction. However, the mechanisms by which excess body fat interferes with reproductive functions are still not fully understood. After weaning, female rats were treated with a cafeteria diet or a chow diet (control group). Biometric and metabolic parameters were evaluated in adulthood. Reproductive parameters, including estradiol, progesterone, LH and prolactin during the proestrus afternoon, sexual behavior, ovulation rates and histological analysis of ovaries were also evaluated. Cafeteria diet was able to induce obesity in female rats by increasing body and fat pad weight, which resulted in increased levels of triglycerides, total cholesterol, LDL and induced insulin resistance. The cafeteria diet also negatively affected female reproduction by reducing the number of oocytes and preantral follicles, as well as the thickness of the follicular layer. Obese females did not show preovulatory progesterone and LH surges, though plasma estradiol and prolactin showed preovulatory surges similar to control rats. Nevertheless, sexual receptiveness was not altered by cafeteria diet. Taken together, our results suggest that the cafeteria diet administered from weaning age was able to induce obesity and reduce the reproductive capability in adult female rats, indicating that this obesity model can be used to better understand the mechanisms underlying reproductive dysfunction in obese subjects., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neonatal handling reduces the stress response in adulthood due to a feedback mechanism. The present study analyzed the effects of repeated neonatal environmental intervention (daily handling during the first 10 days after birth) on neuron-, astroglial cell density, and cellular proliferation of the hippocampal (CA1, CA2, and CA3) pyramidal cell layers in female rats. Pups were divided into two groups, nonhandled and handled, which were submitted to repeated handling sessions between postnatal days 1 and 10. Histological and immunohistochemical procedures were used to determine changes in neuron density, astroglial cell density, and cellular proliferation. We found an increase in neuron density in each pyramidal cell layer of the hippocampus (CA1, CA2, and CA3) in female rats (11 and 90 day old) that were handled during the neonatal period. Furthermore, we found an increase in astroglial cell density in both hemispheres of the brain in the handled group. Finally, we observed an increase in cellular proliferation in both hippocampi (CA1, CA2, and CA3) of the brain in female pups (11 days old) handled during the neonatal period. This study demonstrates that an early-life environmental intervention may induce morphological changes in a structure involved with several functions, including the stress response. The results of the current study suggest that neonatal handling may influence the animals' responses to environmental adversities later in life.

Females usually display low levels of aggressiveness; however, during lactation, the aggressive behavior against intruders to the nest area is an important component of the maternal behavioral repertoire. The present study aimed to analyze the influence of progesterone (P4) on the maternal aggressive behavior in rats. Lactating rat were ovariectomized on the first day after delivery and, on the 6th postpartum day, aggressive behaviors against a male intruder were recorded. Also in the 6th PPD, the effects of a P4 receptor antagonist (RU 486) as well as of finasteride - which inhibits the conversion of P4 to its metabolite allopregnanolone - on the aggressive behavior of non-ovariectomized lactating rats were analyzed. Finally, plasma concentration of prolactin was measured on the 8th PPD. This study shows, for the first time, that ovariectomy just after parturition reduces some aspects of the maternal behavior (frequency of licking) and the aggressive behavior and increased plasma prolactin. On the other hand, the administration of RU486 induced a marked increase in the aggressiveness of lactating females. No changes were detected after finasteride injection. Gonadal hormones after parturition seem necessary for the development of maternal aggressive behavior. Furthermore, our results suggest that the increase in P4 levels throughout the postpartum period could be one of the causes for the natural reduction of the aggressive behavior in lactating rats., (Copyright 2010 Elsevier B.V. All rights reserved.)

Stress might influence the reproductive behavior in females, and central angiotensin II (Ang II) is a peptide that plays a role in stress response and in the modulation of sexual behavior. The medial amygdala (MeA), an important structure that regulates this behavior, is strongly involved in stress response. The aim of the present study was to evaluate the effect of acute restraint stress on the night of proestrus on sexual receptivity in female rats and the participation of Ang II and MeA in this effect. Adult female Wistar rats with regular estrous cycles were utilized. The acute stress protocol utilized was the restraint stress for 15 min on the night of proestrus. The participation of Ang II was evaluated by injecting Ang II and Ang II receptor antagonists (losartan and PD12319) into the MeA. The lordosis quotient was recorded. The stress or the microinjection of Ang II into the MeA significantly reduced sexual behavior. The blockade of AT(1) or AT(2) receptors in the MeA prevented the effect of stress and the effect of Ang II microinjection into this nucleus on sexual receptivity. We concluded that acute restraint stress on the night of proestrus reduces sexual behavior in rats, and this effect is mediated by both AT(1) and AT(2) receptors in the MeA., (Copyright 2009 Elsevier B.V. All rights reserved.)

Early-life environmental events can induce profound long-lasting changes in several behavioral and neuroendocrine systems. The neonatal handling procedure, which involves repeated brief maternal separations followed by experimental manipulations, reduces stress responses and sexual behavior in adult rats. The purpose of this study was to analyze the effects of neonatal handling on social behaviors of male and female rats in adulthood, as manifest by the results of social memory and social interaction tests. The number of oxytocin (OT) and vasopressin (VP) neurons in the paraventricular (PVN) and supraoptic (SON) nuclei of hypothalamus were also analyzed. The results did not demonstrate impairment of social memory. Notwithstanding, handling did reduce social investigative interaction and increase aggressive behavior in males, but did not do so in females. Furthermore, in both males and females, handling was linked with reduced number of OT-neurons in the parvocellular region of the PVN, while no differences were detected in the magnocellular PVN or the SON. On the other hand, handled males exhibited increased number of VP-neurons in the magnocellular zone of the PVN. We may conclude that the repeated brief maternal separations can reduce affiliative social behavior in adult male rats. Moreover, the disruption of the mother-infant relationship caused by the handling procedure induced long-lasting morphological changes in critical neuroendocrine areas that are involved in social bonding in mammals.

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.

Background/aims: To evaluate the effects of neonatal handling on hydroelectrolytic balance in adult rats., Methods: The litters were divided into two groups: nonhandled and handled. The procedure consisted of handling the pups for 1 min/day in the first 10 days postnatally. When adults, animals had their body weight verified and were housed in individual metabolic cages. After a 24-hour period, urine samples were collected and the urinary and water intake volumes measured. Blood samples to determine osmolality, aldosterone, corticosterone, angiotensin II, creatinine, urea, sodium and potassium levels were collected. The kidneys were removed for histological assessment. Urinary osmolality, sodium, urea and creatinine were also measured and the creatinine clearance (CC) calculated., Results: No difference between groups was found in the body weight. Handled animals showed a reduction in the total kidney wet weight, water intake, urinary volume, CC, plasma angiotensin II, corticosterone and aldosterone when compared to the nonhandled and an increase in the urinary osmolality and sodium excretion fraction. No differences in serum potassium and no evidence of structural changes were demonstrated by histological analysis., Conclusion: Neonatal handling induced long-lasting effects decreasing renal function without evidence of kidney structural changes., ((c) 2009 S. Karger AG, Basel.)

Early-life environmental events that disrupt the mother-pup relationship may induce profound long-lasting changes on several behavioral and neuroendocrine systems. The neonatal handling procedure, which involves repeated brief maternal separations followed by experimental manipulations, reduces sexual behavior and induces anovulatory estrous cycles in female rats. On the afternoon of proestrus, neonatally handled females show a reduced surge of luteinizing hormone (LH) and an increased content of gonadotropin-releasing hormone in the medial preoptic area (MPOA). In order to detect the possible causes for the reduced ovulation and sexual behavior, the present study aimed to analyze the effects of neonatal handling on noradrenaline (NA) and nitric oxide (NO) levels in the MPOA on the afternoon of proestrus. Neonatal handling reduced MHPG (NA metabolite) levels and MHPG/NA ratio in the MPOA, indicating decreased NAergic activity. Additionally, neonatal handling decreased NO levels, as measured by the metabolites (NO(x)), nitrite and nitrate in the same period. We may conclude that the neonatal handling procedure decreased activity of the NAergic and NOergic systems in the MPOA during proestrus, which is involved in the control of LH and FSH secretion, and this may possibly explain the anovulatory estrous cycles and reduced sexual behavior of the neonatally handled female rats., ((c) 2007 S. Karger AG, Basel)

Physical or emotional stress can affect the female reproductive physiology and angiotensin II (Ang II) is a hormone that participates in the stress response and also in the control of reproductive hormones. The present study aimed at evaluating the effects of acute stress in the morning and afternoon of proestrus on sexual behavior and ovulation and the participation of Ang II in the stress-induced effects. Female rats with regular estrous cycles were used. Several different stress protocols were tested in the morning and in the afternoon of proestrus: restraint stress 10 min; restraint stress 1 h and ether stress, respectively. The participation of Ang II was evaluated by injecting Ang II receptor antagonists (losartan and PD123319) 15 min before stress. The lordosis quotient was recorded and the number of oocytes was counted. Plasma levels of luteinizing hormone, progesterone, prolactin and corticosterone were measured. All types of stress in the morning of proestrus induced a reduction in the number of oocytes. Restraint stress (1 h) in the afternoon of proestrus induced a significant reduction in the lordosis quotient. Peripheral and central losartan, but not PD123319, injections partly reverted the effects of stress on ovulation in the morning of proestrus. Acute stress in the morning of proestrus also reduced luteinizing hormone, progesterone and prolactin surges later on the same day. In conclusion, acute stress on the day of proestrus can affect female reproductive physiology. Moreover, the angiotensinergic system, through AT(1) receptors, participates in the effects of acute stress in the morning of proestrus.

Early-life events can exert profound long-lasting effects on several behaviors such as fear/anxiety, sexual activity, stress responses and reproductive functions. Present study aimed to examine the effects of neonatal handling on the volume and number of cells in the hypothalamic paraventricular nucleus (pPVN, parvocellular and mPVN, magnocellular regions) and the supraoptic nucleus (SON) in female rats at 11 and 90 days of age. Moreover, in the same areas, immunohistochemistry for oxytocin (OT) and glial fibrillary acidic protein (GFAP) were analyzed in the adult animals. Daily handling during the first 10 postnatal days reduced the number of cells in the pPVN and SON at both the 11 and 90 days. Handling decreased the number of OT-positive parvocellular cells in the PVN in adult females. No significant differences were detected on the optical density (OD) of GFAP-positive cells between the handled and nonhandled adult females. The effect of handling on cell loss was observed 24 h after the 10-day handling period and persisted into adulthood, indicating a stable morphological trace. Results suggest that neonatal handling can induce plastic changes in the central nervous system.

Objective: The present study investigated the effects of renovascular hypertension (2K/1C model) on the reproductive function of male rats, represented by sexual behavior, plasma prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone, and spermatogenesis., Methods: The present experiments were performed to investigate the reproductive function of 2K/1C male Wistar rats and compare with 2K/1C male rats successfully treated for hypertension with nifedipine and was divided in the following groups: (1) Sham+V (n=10): Sham-operated males with vehicle used; (2) Sham+N (n=13): Sham-operated males treated with nifedipine (10 mg/kg/day); (3) 2K/1C+V (n=14): 2K/1C-operated males with vehicle used; and (4) 2K/1C+N (n=16): 2K/1C-operated males treated with nifedipine., Results: The data indicated an association between hypertension induced by the 2K/1C model and reduction of reproductive function, as demonstrated by an impairment of sexual behavior, higher plasma PRL and lower plasma testosterone and FSH. The treatment with nifedipine prevented the reduction of sexual behavior and the increase of plasma PRL, but did not alter the reduction of plasma testosterone and FSH and spermatogenesis of 2K/1C rats., Conclusions: Reproductive function is adversely affected in the 2K/1C animal model, and high blood pressure plays a role in the modulation of plasma PRL and sexual behavior. Moreover, other events, without high blood pressure, but with high plasma renin activity associated with the 2K/1C model, contribute directly to the reduction of plasma testosterone and FSH and impaired spermatogenesis.

Environmental stimuli in early life may result in permanent behavioral and physiological changes. Present study evaluated the effects of exposing pups to a novel environment on behaviors (open-field test and sexual behavior) and prolactin stress-responses in adult male rats. Half of a litter was daily removed outside (OUT) from the nest and stimulated by handling for 3 min, while the other half remained inside (IN) the nest and was also handled for the same period during the first 10 days postpartum. Maternal behavior after all the pups were returned to the nest was not different between IN and OUT littermates. In adulthood, OUT males showed increased general and central locomotion activity in the open-field test, reduced sexual behavior, and attenuated prolactin secretion in response to restraint stress compared with the IN littermates. The repeated exposition of rat pups to a novel environment is a causal factor for the long-lasting behavioral and endocrine changes. The premature exposition of the pup to unfamiliar environments decreases fear and stress-response, and also reduces sexual behavior. We suggest that the absence of the odor of the mother may be crucial to explain the effects detected in adulthood., ((c) 2007 Wiley Periodicals, Inc.)

Neonatal handling affects the hypothalamus-pituitary-gonadal axis in female rats. Indeed, postnatal handling induces anovulatory estrous cycles and decreases sexual receptiveness. On the other hand, Angiotensin II (Ang II) infused into the medial amygdala (MeA) reduces sexual behavior in male and female rats. Considering this, and that gonadal steroid secretion after copulatory behavior is important for reproductive success, the purpose of the present study was to investigate whether the reduction in sexual receptiveness in neonatally handled female rats is mediated by changes in Ang II receptor density in MeA. Moreover, gonadal steroid secretion after sexual behavior was analyzed. Two groups of female Wistar rats were studied: nonhandled (pups were left undisturbed) and handled (pups were handled for 1 min once a day during the first 10 days of life). Once they were 80-85 days old in the evening of the proestrus day, sexual receptiveness was recorded and after that the animals were killed by decapitation. Trunk blood samples were collected, and plasma estradiol and progesterone were measured by radioimmunoassay. The brains were removed for Ang II receptor autoradiography in MeA. The decreased lordosis quotient in the neonatally handled group was confirmed in the present study. Neonatal handling also reduced the progesterone concentration in the plasma, but did not change the estradiol and the density of Ang II receptors in MeA. The reduced progesterone could be due to the decreased lordosis frequency of handled females. However, this decreased sexual receptiveness is not mediated by changes in Ang II receptors in MeA.

The expression of angiotensin II (Ang II) receptors in the brain is modulated by estradiol and progesterone. Considering that Ang II plays a critical role in controlling prolactin secretion and that neurons in the arcuate nucleus (ARC) are the main regulator of this function, the present study aimed to evaluate ARC Ang II receptor binding in 2 experimental models with different estradiol and progesterone plasma levels. Animals were divided into 4 groups: ovariectomy (OVX) plus oil vehicle, OVX plus estradiol and progesterone replacement, lactating rats on day 7 postpartum, and lactating rats on day 20. Animals were killed by decapitation, and the brains were removed. Ang II receptors were quantified by autoradiography in ARC. Trunk blood samples were collected, and plasma estradiol and progesterone were measured by radioimmunoassay. Treatment of OVX rats with estradiol and progesterone increased Ang II receptor binding when compared to OVX vehicle-treated animals. Plasma estradiol (r = +0.77) and progesterone (r = +0.87) were highly correlated with Ang II receptors in ovariectomized animals. Lactating rats (day 20) showed a significant decrease in Ang II receptor binding and plasma progesterone when compared to lactating rats (day 7), however, no difference was seen in plasma estradiol. Plasma levels of progesterone (r = +0.81), but not estradiol (r = +0.32), were highly correlated with Ang II receptors in lactating rats. In conclusion, present results show that ARC Ang II receptors decreases on day 20 of lactation compared to day 7 and are highly correlated with plasma progesterone, indicating a pivotal role for progesterone in this regulation.

We have previously shown that the locus coeruleus (LC) is essential for triggering surges of LH. Since LC neurons are responsive to estradiol, which induces progesterone receptor (PR) expression, this study aimed to investigate whether LC neurons express the alpha-estradiol receptor (alphaER) and PR as well as comparing such responses to that observed in the preoptic area (POA). Female rats were perfused at 10, 14 and 16 h on each day of the estrous cycle, and a blood sample was collected for estradiol, progesterone and LH measurements. alphaER- and PR immunoreactive (ir) neurons were detected in POA and LC by immunocytochemistry (ICC). Higher plasma estradiol levels were observed on the day of proestrus, when a smaller number of alphaER-ir POA neurons were detected. An increase in the number of alphaER-ir neurons were observed at 16 h of proestrus and estrus. The number of PR-ir neurons increased in POA only at 16 h of proestrus, and remained unchanged during all other days and times. The profile of alphaER-ir and PR-ir neurons in LC changed over the estrous cycle, with a lower expression on metestrus morning and reaching a peak on diestrus afternoon before declining on the day of proestrus. However, on estrus afternoon, alphaER-ir neurons increased, while PR-ir neurons decreased which may be related to the prolactin surge of estrus. These data show that LC neurons express alphaER and PR and seem to be more sensitive to variations in estradiol than POA. Also, the fluctuation in alphaER and PR observed for LC neurons seems to accompany the hormonal events that occur during the estrous cycle. This profile of alphaER and PR expression might be related to the ability of estradiol and progesterone in regulating the activity of LC neurons, which could be associated to the control mechanisms of LH and prolactin release.

This study aimed at identifying the effects of neonatal handling (H) and maternal separation (MS) on two paradigms of fear, learned and innate, and on the tyrosine hydroxylase (TH) immunoreactive cells in adult life. Wistar rats were daily handled with a brief maternal separation, maternal separated for 3 h or left undisturbed during the first 10 days of life. Behavioural responses in the open-field (innate fear) and conditioned fear (learned fear) were evaluated. Moreover, a semi-quantitative analysis of TH immunoreactivity in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) was performed using optical densitometry and confirmed by planar measurements of neuronal density. Early handling decreased behaviour responses of innate and learned fear in adult life, while maternal separation had no significant long-lasting effect on these responses compared to the non-handled group. The behavioural effects of early handling could not be explained by changes in the density of midbrain dopaminergic cells, which were not affected by handling or maternal separation.

Neonatal handling alters the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonads axis (HPG) in adult animals, and angiotensin II (Ang II) modulates the functions in these axes. We tested whether neonatal handling could change the density of Ang II receptors in some central areas in female rats. Results showed decreased density of the Ang II receptors in the medial preoptic area (MPOA) and hypothalamic paraventricular nucleus (PVN) of the neonatal handled group.

Angiotensin II (Ang II) receptors in specific brain areas and in the anterior pituitary are controlled by reproductive hormones. Since Ang II also plays a role in controlling reproductive functions, such as luteinizing hormone and prolactin secretion, the objective of the present study was to evaluate the regulation of Ang II receptors by estradiol (E(2)) and progesterone (P) in areas of the brain involved in homeostatic and reproductive functions, such as the locus coeruleus (LC), median preoptic nucleus (MnPO) and subfornical organ (SFO). Adult female rats were ovariectomized under anesthesia and divided into 2 groups after 2 weeks: OVX plus E(2)/P replacement (OVXE(2)P) and OVX plus oil vehicle (OVX). E(2) was injected for 3 consecutive days followed by an injection of P on the 4th day. Animals were killed by decapitation and the brains were removed and frozen. Consecutive coronal brain sections were cut in a cryostat and Ang II receptors were quantified by autoradiography in the MnPO, LC and SFO. Treatment of OVX rats with E(2) and P induced a significant increase in the Ang II receptor binding (fmol/mg protein) in the MnPO (OVX: 4.48 +/- 0.58 and OVXE(2)P: 9.89 +/- 1.65), LC (OVX: 2.72 +/- 0.37 and OVXE(2)P: 8.03 +/- 0.9) and SFO (OVX: 5.45 +/- 0.66 and OVXE(2)P: 10.73 +/- 1.79) compared to OVX animals treated with the vehicle, P < 0.05. In conclusion, these results show that Ang II receptors are upregulated by E(2) and P in the LC, MnPO and SFO of ovariectomized 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.

Neonatal handling induces behavioral and hormonal changes, characterized by reduced fear in novel environments, and lesser elevation and faster return to basal levels of plasma corticosterone, prolactin and adrenaline, in response to stressors in adulthood. The present study aimed to analyze the effects of neonatal handling from Days 1 to 10 postnatal on prolactin response to ether stress in male and female rats at three life periods: neonatal, peripubertal and adulthood. Moreover, adult females were tested in two different phases of the estrous cycle, i.e., diestrus and estrus. In another set of experiments, the behavior of peripubertal and adult males and females in estrus and diestrus was analyzed in the elevated plus maze test. Pups were either handled for 1 min (handled group) or left undisturbed (nonhandled group) during the first 10 days after delivery. In adults, in the handled females in diestrus, stress induced a lesser increase in plasma prolactin compared with nonhandled ones, as in males. However, in estrus, handled females showed no difference in the prolactin response to stress. In the elevated plus maze, handled females in diestrus, but not in estrus, showed higher locomotor activity compared with nonhandled ones. Peripubertal male and female rats handled during the neonatal period showed no difference in behavior in the elevated plus maze compared with nonhandled animals. Early-life stimulation can induce long-lasting behavioral and stress-related hormonal changes, but they are not stable throughout life and phases of the estrous cycle.

Angiotensin II (Ang II) is a peptide that exerts an inhibitory effect upon pituitary prolactin (PRL) release through the hypothalamic arcuate nucleus (ARC). Since both PRL and Ang II are known to be affected by stress, the experiments reported here were conducted to investigate the possible participation of Ang II in the stress-induced response of PRL in situations in which pre-stress PRL levels are high, as during the PRL surge induced by estradiol (E(2)) and progesterone (P) in ovariectomized rats (OVXE(2)P) and lactating females on day 7 post-partum. Adult female rats were stereotactically implanted with bilateral guide-cannulae in the ARC; 3 days later, they were microinjected with saline or losartan and, after a 15-min interval, they were submitted to stress by ether inhalation during 1 min. Five minutes after stress, trunk blood samples were collected. Plasma PRL was measured by radioimmunoassay (RIA). In OVXE(2)P and lactating rats, a significant reduction in PRL levels was detected after stress compared to non-stressed animals. The microinjection of losartan in the ARC before stress blocked the reduction of PRL in both OVXE(2)P and lactating females. In conclusion, the stress-induced reduction of plasma PRL in OVXE(2)P and lactating rats is mediated by Ang II through AT(1) receptors in the ARC.

Neonatal handling induces long-lasting effects on behaviors and stress responses. The objective of the present study was to analyze the effects of neonatal handling (from the 1st to the 10th day after delivery) on the number of cells and volume of locus coeruleus (LC) nucleus in male and female rats at 4 different ages: 11, 26, 35, and 90 days. Results showed significant reductions in the number of cells and the volume of the LC nucleus in neonatally handled males and females compared with nonhandled rats. Environmental stimulation early in life induced a stable structural change in a central noradrenergic nucleus, which could be one of the causal factors for the behavioral and hormonal alterations observed in adulthood., ((c) 2003 APA, all rights reserved)

Research was undertaken to study the role of central angiotensin in the modulation of male sexual behavior, testing the effect of angiotensin II (Ang II) injections into the medial amygdaloid nucleus (MeA). The sexual behavior of adult male Wistar rats was evaluated, 15 min after bilateral intra-amygdaloid microinjection (0.3 microl) of saline and 5 doses of Ang II: 10; 25; 50; 100, and 150 fmol. The effects of the Ang II receptor blockade were also studied. We tested the effect of coinjection of Ang II (50 fmol) with the AT1 antagonist, losartan (20 pmol) and the AT2 antagonist, CGP 42112 (1 pmol). Ang II inhibited sexual behavior and this inhibition was prevented by the coinjection of AT1 antagonist, losartan, or the AT2 antagonist, CGP 42112. Results show that Ang II has a powerful effect on male sexual behavior, which may be mediated by both AT1 and AT2 receptors., ((c) 2002 Elsevier Science (USA).)

Neonatal handling has long-lasting effects on behavior and stress reactivity. The purpose of the present study was to investigate the effect of neonatal handling on the number of dopaminergic neurons in the hypothalamic nuclei of adult male rats as part of a series of studies that could explain the long-lasting effects of neonatal stimulation. Two groups of Wistar rats were studied: nonhandled (pups were left undisturbed, control) and handled (pups were handled for 1 min once a day during the first 10 days of life). At 75-80 days, the males were anesthetized and the brains were processed for immunohistochemistry. An anti-tyrosine hydroxylase antibody and the avidin-biotin-peroxidase method were used. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted bilaterally in the arcuate, paraventricular and periventricular nuclei of the hypothalamus in 30-microm sections at 120-microm intervals. Neonatal handling did not change the number of TH-IR neurons in the arcuate (1021 +/- 206, N = 6; 1020 +/- 150, N = 6; nonhandled and handled, respectively), paraventricular (584 +/- 85, N = 8; 682 +/- 62, N = 9) or periventricular (743 +/- 118, N = 7; 990 +/- 158, N = 7) nuclei of the hypothalamus. The absence of an effect on the number of dopaminergic cells in the hypothalamus indicates that the reduction in the amount of neurons induced by neonatal handling, as shown by other studies, is not a general phenomenon in the brain.

Since previous work has shown that stimulation early in life decreases sexual receptiveness as measured by the female lordosis quotient, we suggested that neonatal handling could affect the function of the hypothalamus-pituitary-gonadal axis. The effects of neonatal handling on the estrous cycle and ovulation were analyzed in adult rats. Two groups of animals were studied: intact (no manipulation, N = 10) and handled (N = 11). Pups were either handled daily for 1 min during the first 10 days of life or left undisturbed. At the age of 90 days, a vaginal smear was collected daily at 9:00 a.m. and analyzed for 29 days; at 9:00 a.m. on the day of estrus, animals were anesthetized with thiopental (40 mg/kg, ip), the ovaries were removed and the oviduct was dissected and squashed between 2 glass slides. The number of oocytes of both oviductal ampullae was counted under the microscope. The average numbers for each phase of the cycle (diestrus I, diestrus II, proestrus and estrus) during the period analyzed were compared between the two groups. There were no significant differences between intact and handled females during any of the phases. However, the number of handled females that showed anovulatory cycles (8 out of 11) was significantly higher than in the intact group (none out of 10). Neonatal stimulation may affect not only the hypothalamus-pituitary-adrenal axis, as previously demonstrated, but also the hypothalamus-pituitary-gonadal axis in female rats.

The high-affinity serotonin (5-HT) transporter (5-HTT) plays an important role in the removal of extracellular serotonin, thereby modulating and terminating the action of this neurotransmitter at various pre- and post-synaptic serotonergic receptors and heteroreceptors. In order to characterize the anatomical distribution of the 5-HTT in mouse brain, in situ hybridization histochemistry using 35S-labeled riboprobes was performed. These results were compared with 5-HTT binding site distribution as evaluated by [125I]RTI-55 autoradiography. High levels of 5-HTT mRNA were detected in all brain stem raphe nuclei, with variations in labeling among the various subnuclei. Those brain areas known to possess serotonergic cell bodies stained intensely for both 5-HTT mRNA and 5-HTT binding sites. In contrast to previous findings in rat brain, the highest densities of 5-HTT sites were found in areas outside the raphe complex, particularly in the substantia nigra, globus pallidus, and superior colliculi.

Brain angiotensin II (Ang II) inhibits pituitary prolactin release by an indirect mechanism requiring stimulation of dopamine formation and release. We report that [125I]Sar1-Ang II binding to AT1 receptors and AT1A receptor mRNA expression increase selectively in the dorsomedial arcuate nucleus of 17beta-estradiol-primed ovariectomized rats after treatment with progesterone. In hormone-treated rats, arcuate nucleus AT1A receptor mRNA expression is associated with tyrosine hydroxylase-positive neurons. No AT1A receptor mRNA was detected in tyrosine hydroxylase-positive cells of the arcuate nucleus of intact male rats. Conversely, in the anterior pituitary, where local or circulating Ang II stimulates prolactin release, [125I]Sar1-Ang II binding to AT1 receptors and AT1B receptor mRNA expression are decreased in 17beta-estradiol/progesterone-treated ovariectomized rats. Thus, AT1A receptors in the dorsal arcuate nucleus and AT1B receptors in the anterior pituitary are regulated inversely by estrogen/progesterone treatment, supporting the hypothesis of a dual role for brain and pituitary Ang II on prolactin release. The colocalization of AT1A receptor mRNA and tyrosine hydroxylase in neurons of the arcuate nucleus furthermore indicates that within this area central Ang II acts directly on dopaminergic neurons. These results support the hypothesis that central Ang II inhibits pituitary prolactin release indirectly via modulation of dopaminergic activity in the arcuate nucleus.

We studied angiotensin II (ANG II) receptor subtype expression in selected brain nuclei and pituitary gland after water deprivation by in vitro receptor autoradiography using 125I-labeled [Sar1]ANG II and by in situ hybridization using 35S-labeled AT1A, AT1B, and AT2 receptor-specific riboprobes. In control rats we found binding to AT1 receptors in the subfornical organ, paraventricular nucleus, median eminence, and anterior pituitary; AT1A mRNA expression in the subfornical organ and paraventricular nucleus; and AT1B mRNA expression in the anterior pituitary. No receptor mRNA was found in the median eminence. AT1 receptors and AT1A receptor mRNA levels were increased in the subfornical organ, and, in the anterior pituitary, AT1 receptors and AT1B receptor mRNA were increased, only after 5 days of water deprivation. No significant changes occurred after 1 or 3 days of water deprivation, and no regulation of ANG II receptor expression was detected in other brain areas. Our results show that prolonged water deprivation selectively regulates AT1 receptor expression and AT1A and AT1B receptor mRNA levels in the subfornical organ and anterior pituitary, respectively, supporting a role for these receptors during sustained dehydration.

The hypothalamic angiotensin II (Ang II) system plays an important role in pituitary hormone release. Little is known about this system in the mouse brain. We studied the distribution of angiotensin-converting-enzyme (ACE), Ang II, Ang II receptor subtypes, and vasopressin in the hypothalamus of adult male mice. Autoradiography of binding of the ACE inhibitor [125I]351A revealed low levels of ACE throughout the hypothalamus. Ang II- and vasopressin-immunoreactive neurons and fibers were detected in the paraventricular, accessory magnocellulary, and supraoptic nuclei, in the retrochiasmatic part of the supraoptic nucleus and in the median eminence. Autoradiography of Ang II receptors was performed using [125I]Sar1-Ang II binding. Ang II receptors were present in the paraventricular, suprachiasmatic, arcuate and dorsomedial nuclei, and in the median eminence. In all areas [125I]Sar1-Ang II binding was displaced by the AT1 receptor antagonist losartan, indicating the presence of AT1 receptors. In the paraventricular nucleus [125I]Sar1-Ang II binding was displaced by Ang II (Ki = 7.6 X 10(-9)) and losartan (Ki = 1.4 X 10(-7)) but also by the AT2 receptor ligand PD 123319 (Ki = 5.0 X 10(-7)). In addition, a low amount of AT2 receptor binding was detected in the paraventricular nucleus using [125I]CGP42112 as radioligand, and the binding was displaced by Ang II (Ki = 2.4 X 10(-9)), CGP42112 (Ki = 7.9 x 10(-10)), and PD123319 (Ki = 2.2 x 10(-7)). ACE, Ang II, and AT1 as well as AT2 receptor subtypes are present in the mouse hypothalamus. Our data are the basis for further studies on the mouse brain Ang II system.

The difficulty in obtaining cerebrospinal fluid (CFS) in an efficient and simple manner in rats prompts us to introduce a new technique that makes use of a cannula placed in the lateral ventricle. The cannula is implanted with a stereotaxic apparatus and the CSF is collected with a glass capillary tube. The technique has proved to work well for experiments in which the CSF must be free of blood. It also permits the collection of volumes of CSF sufficient for radioimmunoassays, and may be used in chronic experiments.