Food deprivation in F0 generation and hypercaloric diet in F1 generation reduce F2 generation astrogliosis in several brain areas after immune challenge.

AIMS: The effects of maternal food restriction during gestation in F0 generation followed by hypercaloric diet (HD) during puberty in F1 generation (F1HD) were investigated on astrocyte behavior of F2 generation. Also, the astrocyte behavior, after an immune challenge, was examined by the immunohistochemical expression of glial fibrillary acidic protein (GFAP) in several brain areas. METHODS: The body weight gain (BW) during development and in postnatal day (PND) 90-95, the retroperitoneal fat weight (RPF), and the size of larger and smaller adipocytes in the F1 generation were assessed to observe the effects of HD in female rats. The BW, RPF weight and size of smaller and larger adipocytes was also measured to evaluate the transgenerational effects of F0 and F1 diets on F2 generation, treated or not with lipopolysaccharide (LPS). KEY FINDINGS: The F1HD group exhibited a higher BW gain than the F1 treated with normocaloric diet (ND, group F1ND), from weaning to PND65. In the frontal/parietal cortex, nucleus accumbens, hypothalamic arcuate/periventricular nuclei, molecular/granular layers of the cerebellum areas, excepting the pons, GFAP expression was greater in F1HD group relative to F1ND group. A reduced GFAP expression was observed in both groups born from F1 generation fed with HD (groups F2HDS and F2HDLPS) in relation to F2 generation born from dams fed with ND (groups F2NDS and F2NDLPS), independently of LPS challenge. SIGNIFICANCE: These data show an attenuation of LPS effect on GFAP expression, probably by a transgenerational effect of both maternal food deprivation in F0 generation and HD in F1 generation.

Hypercaloric diet prevents sexual impairment induced by maternal food restriction.

Prenatal undernutrition impairs copulatory behavior and increases the tendency to become obese/overweight, which also reduces sexual behavior. Re-feeding rats prenatally undernourished with a normocaloric diet can restore their physiological conditions and copulatory behavior. Thus, the present study investigated whether a hypercaloric diet that is administered in rats during the juvenile period prevents sexual impairments that are caused by maternal food restriction and the tendency to become overweight/obese. Female rats were prenatally fed a 40% restricted diet from gestational day 2 to 18. The pups received a hypercaloric diet from postnatal day (PND) 23 to PND65 (food restricted hypercaloric [FRH] group) or laboratory chow (food restricted control [FRC] group). Pups from non-food-restricted dams received laboratory chow during the entire experiment (non-food-restricted [NFR] group). During the juvenile period and adulthood, body weight gain was evaluated weekly. The day of balanopreputial separation, sexual behavior, sexual organ weight, hypodermal adiposity, striatal dopamine and serotonin, serum testosterone, and tumor necrosis factor α (TNF-α) were evaluated. The FRH group exhibited an increase in body weight on PND58 and PND65. The FRC group exhibited an increase in the latency to the first mount and intromission and an increase in serum TNF-α levels but a reduction of dopaminergic activity. The hypercaloric diet reversed all of these effects but increased adiposity. We concluded that the hypercaloric diet administered during the juvenile period attenuated reproductive impairments that were induced by maternal food restriction through increases in the energy expenditure but not the tendency to become overweight/obese.

Transgenerational effects of a hypercaloric diet.

The effects of a maternal hypercaloric diet (HD) during puberty and early adulthood on neuroimmune aspects in offspring were investigated. In female rats of the F0 generation and male rats of the F1 generation, bodyweight (BW) gain, retroperitoneal fat (RPF) weight, the number of hypodermic adipocytes (HAs) and expression of glial fibrillary acidic protein (GFAP) were measured in hypothalamic astrocytes. On Postnatal Day 50, the F1 pups were challenged with lipopolysaccharide (LPS, 100µgkg-1, s.c.) or an equal volume of saline (S), and behaviour in the open field test was evaluated, as were plasma neuropeptide and cytokine concentrations. The maternal HD caused the female F0 rats to become overweight. The F1 offspring of dams fed the HD and challenged with saline (HDS group) exhibited increases in BW gain, RPF weight and in the number of large HAs and a decrease in GFAP immunoreactivity. F1 offspring of dams fed the HD and challenged with LPS (HDLPS group) exhibited decreases in BW gain, RPF weight and GFAP immunoreactivity, but no differences were observed in the number of larger and small HAs. Plasma tumour necrosis factor-α concentrations were high in the HDS and HDLPS groups. Thus, the maternal HD during puberty and early adulthood caused the F1 generation to become overweight despite the fact that they received a normocaloric diet. These results indicate a transgenerational effect of the HD that may occur, in part, through permanent changes in immune system programming. The attenuation of neuroinflammation biomarkers after LPS administration may have resulted in a decrease in the number of adipocytes, which, in turn, reduced cytokine, adipokine and chemokine levels, which are able to recruit inflammatory cells in adipose tissue.

Maternal food restriction in rats of the F0 generation increases retroperitoneal fat, the number and size of adipocytes and induces periventricular astrogliosis in female F1 and male F2 generations.

The present study investigated whether male offspring (F2 generation) from female rats (F1 generation) whose mothers (F0 generation) were food restricted during gestation inherit a phenotypic transgenerational tendency towards being overweight and obese in the juvenile period, in the absence of food restriction in the F1/F2 generations. Dams of the F0 generation were 40% food restricted during pregnancy. Bodyweight, the number and size of larger and small hypodermal adipocytes (HAs), total retroperitoneal fat (RPF) weight and the expression of glial fibrillary acidic protein (GFAP) in periventricular hypothalamic astrocytes (PHAs), as determined by immunohistochemistry, were evaluated in both generations. In the female F1 generation, there was low bodyweight gain only during the juvenile period (30-65 days of age), a decrease in the size of small adipocytes, an increase in the number of small adipocytes, an increase in RPF weight and an increase in GFAP expression in PHAs at 90-95 days of age. In males of the F2 generation at 50 days of age, there was increased bodyweight and RPF weight, and a small number of adipocytes and GFAP expression in PHAs. These data indicate that the phenotypic transgenerational tendency towards being overweight and obese was observed in females (F1) from mothers (F0) that were prenatally food restricted was transmitted to their male offspring.

Repeated methylphenidate administration during lactation reduces maternal behavior, induces maternal tolerance, and increases anxiety-like behavior in pups in adulthood.

Methylphenidate (MPD) is a dopamine uptake inhibitor and the most commonly prescribed drug for the treatment of attention-deficit/hyperactivity disorder in children. Several studies have shown that such stimulants as cocaine and amphetamine that are administered during gestation and lactation may disrupt maternal behavior. Also, MPD is used in lactation. Repeated MPD administration can induce either sensitization or tolerance. The aim of the present study was to investigate whether repeated MPD administration alters maternal behavior and promotes tolerance or sensitization in these females. The effects in adult offspring were also examined in models of anxiety. Methylphenidate (5mg/kg) was administered from lactation day 2 to 4, and maternal pup retrieval behavior was assessed. This treatment was continued until lactation day 7. At weaning, the dams received a challenge dose of MPD, and general activity was evaluated in the open field. Striatal monoamine and metabolite levels were also measured to determine whether this treatment promotes behavioral or biochemical plasticity. The long-term behavioral effects of MPD exposure were evaluated in pups in adulthood. The results showed an increase in the latency to retrieve the first, second, and third pups and a decrease in the number of dams that retrieved all pups. After a challenge dose of MPD, the dams exhibited a decrease in locomotion frequency, an increase in immobility duration in the open field, and a decrease in striatal serotonin levels. In pups, anxiety-like behavior increased in the light/dark box test. These results indicate that repeated MPD administration during early lactation impairs maternal behavior, likely by decreasing maternal motivation. Repeated MPD administration induced maternal tolerance at weaning after a challenge dose of MPD, suggesting the development of central nervous system plasticity. In pups, maternal exposure to MPD during early lactation induced long-term effects and increased anxiety-like behavior in adulthood.

Treatment with steroid hormones and morphine alters general activity, sexual behavior, and opioid gene expression in female rats.

AIMS: Previous studies have shown that brain opioid peptides exert an inhibitory influence on gonadotropin secretion. Different types of brain opioids, such as ß-endorphin, enkephalin, and dynorphin, exert their actions by binding to specific opioid receptors (i.e., µ, δ, and κ, respectively). The present study determined the effects of chronic treatment with morphine in female rats with pharmacologically induced estrus on behavior and opioid receptor gene and protein expression in the hypothalamus, striatum, and periaqueductal gray. MAIN METHODS: Female ovariectomized rats treated with estrogen+progesterone received 3.5mg/kg morphine once per day for 6days. We evaluated general activity, sexual behavior, Oprm1, Oprd1, and Oprk1 gene expression, and µ opioid receptor (MOR), δ opioid receptor (DOR), and κ opioid receptor (KOR) protein expression in the hypothalamus, striatum, and periaqueductal gray in adult virgin female ovariectomized rats. KEY FINDINGS: Chronic morphine treatment increased locomotion and grooming behavior, decreased immobility time, decreased sexual behavior, and decreased the lordosis quotient. The molecular biology results showed that morphine treatment increased Oprm1 gene and MOR protein expression in the striatum and decreased KOR protein expression in the hypothalamus in animals that were assessed for general activity. The animals that were evaluated for sexual behavior exhibited an increase in Oprm1 expression in the periaqueductal gray and increase in KOR expression in the striatum. SIGNIFICANCE: These results suggest that both opioid system activation and sex hormones alter behavioral and molecular patterns in ovariectomized rats within a relatively short period of time.

Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid ...

Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression.

The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female reproduction.

Reproductive experience modifies dopaminergic function, serum levels of prolactin, and macrophage activity in female rats.

Reproductive experience (RE), i.e. pregnancy and lactation, induces physiological changes in mammals. Recent data show that neuroimmune interactions are modulated by a diversity of events involving neurotransmitters and neuropeptides. These molecules, particularly dopamine (DA), were reported to mediate the relevant cross talk between immune and neuroendocrine systems. Moreover, DA-mediated regulation of leukocyte function is a reasonable approach to investigate the DA-operated regulatory switch for immune-competent cells, such as macrophages. Therefore, the goals of the present study were to determine the effects of RE on: (1) dopaminergic function through hypothalamic levels of DA, dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA); (2) basal levels of circulating prolactin (PRL); and (3) activity of peritoneal macrophage (phagocytosis and oxidative burst). A total of 16 adult (200-250 g) female Wistar rats were used, divided in two groups: nulliparous and primiparous. Approximately 2-3 weeks after weaning pups from the primiparous group, both groups of rats were tested. The findings indicate that: (1) DOPAC concentrations, DOPAC/DA and HVA+DOPAC/DA ratios decreased in primiparous rats as compared to virgin rats, (2) primiparous rats showed significantly lower serum PRL levels, and (3) phorbol miristate acetate (PMA)-induced oxidative burst was decreased in peritoneal macrophage from primiparous rats as compared to virgin rats. To test the possible positive correlation between serum levels of PRL and the intensity of oxidative burst by peritoneal macrophage, an extra experiment was done with adult virgin female rats treated with domperidone, an antagonist of DA receptors. Domperidone-treated animals showed increased serum levels of PRL and simultaneous increase in peritoneal macrophage oxidative burst. Thus, suggesting an indirect participation of hyperprolactinemia, induced by this treatment in peritoneal macrophage activity of female rats. These results suggest that a previous RE can modulate the activity of dopaminergic hypothalamic systems, while decreasing PRL serum levels and the oxidative burst of peritoneal macrophage. The neurochemical and hormonal RE-induced changes correlate with the immune alterations.

Preliminary study of the effects of morphine treatment on opioid receptor gene expression in brain structures of the female rat.

Opioid receptors play an important role in female physiology. They modulate directly and indirectly neuroendocrine phenomena that influence pregnancy maintenance, pain threshold, parturition, lactation, maternal behavior, rewarding and addiction. Thus understanding the gene expression levels of the three major opioid receptors, mu, delta and kappa in different brain regions is essential for investigating dynamic mechanisms of opioidergic transmission. Adult virgin female rats were treated acutely with morphine sulfate (3.5 mg/kg or 20 mg/kg s.c.) or chronically for 5 days (3.5 mg/kg). Rats were killed 1 h after the last injection. In the acute treatment, expression levels for the encoded mu-opioid receptor Oprm1, as detected by reverse transcription-polymerase chain reaction, were significantly decreased in the periaqueductal gray. In chronic treatment, both Oprk1 and Oprm1 expression levels, that encoded kappa and mu-opioid receptor respectively, showed significant decreases in the periaqueductal gray and striatum. Regional changes in opioid receptor gene expression levels might reflect highly specialized roles for these receptors with possible functional meaning for the plasticity of the opioidergic transmission.

Prenatal treatment with picrotoxin promotes heterotypical sexual behavioral and neurochemical changes in male rat offspring.

The effects of maternal prenatal exposure to picrotoxin (0.75 mg/kg S.C. days 16-19 of pregnancy) in male rat offspring were observed. Adult sexually experienced and inexperienced animals were evaluated for heterotypical sexual behavior, as well as the testosterone plasma levels and striatal neurotransmitters. In relation to sexual behavior and analysis of sexual organs, the results showed that animals treated with picrotoxin exhibited a more intense reproductive behavior, and this could be expressed by a significant decrease in the number of mounts and intromissions and increase in the numbers of ejaculation, showed that these males are most motivate for sexual behavior. Testosterone levels as well as weight for sexual organs did not differ from control group. The neurochemical analysis showed that picrotoxin did not alter DA, 5-HT, 5-HIAA and GABA in animals. The DOPAC/DA and HVA/DA relation showed that the treatment increased the DA system activity in animals sexually experienced, as well as promote a decrease in 5-HT/5-HIAA relation, that is known was an inhibitory neurotransmitter system, blockade a male sexual behavior. There are no alterations observed in GABA levels. It's could be explained by suggests that picrotoxin modification DA system activity through GABAergic system, permitting that DA system to be freely active and facilitate the heterotypical behavior of male rats. These results show that the maternal prenatal exposure to picrotoxin produced changes in the neurochemical and sexual behavior of the adult male rats. Also previous heterotypical experience leads to changes in biogenic amine concentrations in these animals.

Perinatal maternal exposure to picrotoxin: effects on sexual behavior in female rat offspring.

A previous study in our laboratory showed that perinatal maternal picrotoxin exposure (0.75 mg/kg) in rats improved heterosexual behavior in male offspring. In the present study, we examined the effects of this maternal treatment on sexual behavior in the female offspring. The dams received 0.75 mg/kg picrotoxin treatment (PT) once a day on the 18th and 21st day of pregnancy, 2 h after parturition and once a day during the first 4 days of lactation. The results showed that (1) at birth, the body weight and anogenital distance were not modified by treatment; (2) female sexual behavior was improved in experimental animals. These results demonstrate that perinatal picrotoxin exposure improves adult sexual behavior in female rat offspring as suggested by increase in the lordosis quotient.

Effects of maternal exposure to picrotoxin during lactation on physical and reflex development, square crossing and sexual behavior of rat offspring.

The effects of maternal exposure during the first 10 days of lactation to picrotoxin (0.75 mg/kg sc) on maternal behavior, offspring physical and neurobehavioral development as well as sexual behavior were studied. Results showed that (1) dam food and water consumption, maternal behavior and body weight were not different between control and experimental animals, (2) male and female pup body weight and the development of physical landmarks did no differ between control and experimental groups, (3) negative geotaxis was improved in female experimental offspring and palmar grasp reflex did not differ between groups, (4) at 75 days of age the square crossing by female rats of the experimental group was increased in relation to the control group; no differences were observed between male control and experimental animals, (5) male experimental rats exhibited a significant increase in the number of mounts, intromissions and ejaculations parallel to a decrease in latency to first mount, intromissions and ejaculation as well as in the latencies of first postejaculatory mount and intromission and (6) the intromission frequency per minute (hit rate) was increased in these animals. These results suggest that postnatal exposure to picrotoxin improved the sexual behavior of rats. Three hypotheses were proposed to explain the mechanisms underlying this effect: (1) the development of subsensitivity of GABAergic receptors, (2) an interference with early receptor development or (3) with neurotransmitter balance, mainly involving the dopaminergic system.