Synergistic effects of diet and exercise on hippocampal function in chronically stressed mice.

Severe chronic stress can have a profoundly negative impact on the brain, affecting plasticity, neurogenesis, memory and mood. On the other hand, there are factors that upregulate neurogenesis, which include dietary antioxidants and physical activity. These factors are associated with biochemical processes that are also altered in age-related cognitive decline and dementia, such as neurotrophin expression, oxidative stress and inflammation. We exposed mice to an unpredictable series of stressors or left them undisturbed (controls). Subsets of stressed and control mice were concurrently given (1) no additional treatment, (2) a complex dietary supplement (CDS) designed to ameliorate inflammation, oxidative stress, mitochondrial dysfunction, insulin resistance and membrane integrity, (3) a running wheel in each of their home cages that permitted them to exercise, or (4) both the CDS and the running wheel for exercise. Four weeks of unpredictable stress reduced the animals' preference for saccharin, increased their adrenal weights and abolished the exercise-induced upregulation of neurogenesis that was observed in non-stressed animals. Unexpectedly, stress did not reduce hippocampal size, brain-derived neurotrophic factor (BDNF), or neurogenesis. The combination of dietary supplementation and exercise had multiple beneficial effects, as reflected in the number of doublecortin (DCX)-positive immature neurons in the dentate gyrus (DG), the sectional area of the DG and hippocampal CA1, as well as increased hippocampal BDNF messenger ribonucleic acid (mRNA) and serum vascular endothelial growth factor (VEGF) levels. In contrast, these benefits were not observed in chronically stressed animals exposed to either dietary supplementation or exercise alone. These findings could have important clinical implications for those suffering from chronic stress-related disorders such as major depression.

Circulating and urinary adrenal corticosterone, progesterone, and estradiol in response to acute stress in female mice (Mus musculus).

In studies of stress, it can be difficult to obtain blood rapidly enough to avoid confounding steroid measures. Noninvasive urinary steroid measures may provide an alternative insofar as they reflect systemic steroids. In Experiment 1, we profiled urinary corticosterone, progesterone, and estradiol in ovariectomized female mice following 1 h on an elevated platform. This increased urinary corticosterone for 3 h and progesterone for 4 h. In Experiment 2, blood and urine samples were obtained at 0-6 h following stressor offset. Females showed increased serum corticosterone and progesterone immediately after stressor offset. Urinary corticosterone was increased at both 0 and 2 h post-stress, while an increase in progesterone 2-6 h after stressor offset was not significant. Estradiol was not influenced by this mild stressor. In Experiment 3, mice were exposed to a more severe 1 h stressor, a rat across a wire-mesh grid. In serum, both corticosterone and progesterone were elevated immediately after stressor offset and returned to baseline within 2 h. In urine, this severe stressor elevated corticosterone immediately and 2 h after stressor offset, and in progesterone 2 h after stressor offset. Estradiol in serum was not dynamic, but it was significantly elevated in urine 4 h after stressor offset. Urinary measures generally reflected systemic measures; however, with a different time course resulting in a longer return to baseline. We suggest that the relative value of serum or urinary steroid measures in mice depends upon the experimental design, and that estradiol may only respond when the stressor is severe.

Circadian rhythm and response to an acute stressor of urinary corticosterone, testosterone, and creatinine in adult male mice.

In small laboratory species, steroid measures can be obtained more frequently and less invasively from urine than blood. Insofar as urinary levels reflect systemic levels, they could provide advantages particularly for measurement of glucocorticoids, whose blood levels react rapidly to handling and stress. In Experiment 1, urinary samples were collected from male mice every second hour over a 14:10 h light:dark cycle. Samples were analyzed via enzyme immunoassay for corticosterone, testosterone, and creatinine. Corticosterone had peak concentrations 1 h after light offset and a trough 1 h after light onset. Testosterone showed peak concentrations 5-7 h after light onset and lowest concentrations during the dark phase of the cycle. Creatinine showed some variation over the light-dark cycle, but steroid measures showed similar trends with and without adjustment for creatinine. In Experiment 2, mice were stressed via an injection at times close to the determined peak and trough levels of corticosterone. In urinary samples taken 90 min after injection, corticosterone was significantly higher in injected animals at both times relative to levels in control animals, but testosterone was unaffected by injection stress. In Experiment 3, serum and urine samples were collected from mice every sixth hour across the diurnal cycle. Corticosterone peaked in urine and serum immediately after light offset, and urinary measures predicted those in serum. These data indicate that urinary corticosterone reflects systemic levels in mice, document circadian variation in urinary testosterone, and indicate that circadian variation in creatinine is minimal, but potentially relevant in stressed animals.

Behavioral and molecular changes in the mouse in response to prenatal exposure to the anti-epileptic drug valproic acid.

Experiments in rodents have indicated that maternal valproic acid (VPA) exposure has permanent adverse effects upon neurological and behavioral development. In humans, prenatal exposure to VPA can induce fetal valproate syndrome, which has been associated with autism. The present study examined mouse pups exposed in utero to VPA, measuring physical development, olfactory discrimination, and social behavior as well as expression of plasticity-related genes, brain derived neurotrophic factor (BDNF) and NMDA receptor subunits NR2A and NR2B. VPA-exposed mice showed delayed physical development, impaired olfactory discrimination, and dysfunctional pre-weaning social behavior. In situ hybridization experiments revealed lower cortical expression of BDNF mRNA in VPA animals. These results support the validity of the VPA mouse model for human autism and suggest that alterations in plasticity-related genes may contribute to the behavioral phenotype.

Prenatal exposure to valproic acid leads to reduced expression of synaptic adhesion molecule neuroligin 3 in mice.

In rodents, a single administration of valproic acid (VPA) in utero leads to developmental delays and lifelong deficits in motor performance, social behavior, and anxiety-like behavior in the offspring. Recently, we have demonstrated that VPA mice show alterations in postnatal growth and development, and deficits in olfactory discrimination and social behavior early in development. Based on behavioral and molecular parallels between VPA rodents and individuals with autism, maternal challenge with VPA has been suggested to be a good animal model of autism. Neuroligins (NLGN) are a family of postsynaptic cell-adhesion molecules that play a role in synaptic maturation through association with their presynaptic partners, the neurexins (NRXN). Both NLGNs and NRXN members have been implicated in genetic studies of autism. In the present study, we examined changes at the level of expression of NLGN and NRXN mRNAs in the adult brain from mice exposed in utero to VPA. Mouse brain tissue was processed using in situ hybridization and analyzed with densitometry to examine expression of three NLGN genes (NLGN1, NLGN2, and NLGN3) and three NRXN genes (NRXN1, NRXN2, and NRXN3). Expression levels of NLGN1, NLGN2, NRXN1, NRXN2, and NRXN3 were observed to be similar in VPA and control mice. NLGN3 mRNA expression was found to be significantly lower in the VPA mice relative to control animals in hippocampal subregions, cornu ammonis (CA1) and dentate gyrus, and somatosensory cortex. This lowered expression may be linked to autistic-like behavioral phenotype observed in the VPA mice.

Enzyme immunoassay of testosterone, 17beta-estradiol, and progesterone in perspiration and urine of preadolescents and young adults: exceptional levels in men's axillary perspiration.

Enzyme immunoassays for testosterone, 17beta-estradiol, and progesterone were validated for human facial and axillary perspiration and compared to levels in urine. In study 1, these assays were applied to samples from preadolescent girls and boys and young women and men. Men's axillary perspiration contained substantially higher levels of steroids than seen in other substrates from men or in any sample from women, boys, and girls. Male axillary steroid levels were very variable across individuals, and on average they exceeded levels in facial perspiration by 90-fold for testosterone and 45-fold for estradiol. Men's urinary testosterone also exceeded urinary levels of the other subjects. In study 2, axillary perspiration, urine, and saliva were collected from young men. Substantial axillary levels of testosterone and estradiol were again observed. Correlations of the same hormone among the different substrates were generally very low, except for a small correlation between estradiol levels measured in axillary perspiration and urine in study 2. High unconjugated steroid content in men's axillary excretions could, if absorbed by women during intimacy, be implicated in pheromonal activity.

Urinary sex steroids during sexual development in female mice and in proximate novel males.

The experiments described here were designed to determine whether males' capacity to accelerate female pubertal development is reflected in females' urinary steroid levels in mice, and whether steroids in males' urine are influenced by exposure to developing females. In the first experiment, measures from urine collected daily from female mice aged 31-59 days showed a gradual rise in 17beta-estradiol levels and a distinct linear rise in progesterone levels. In a second experiment, daily steroids were measured in females aged 30-42 days while they were either housed alone or underneath two novel outbred males. Females exposed to males showed accelerated development at day 43 in uterine weight, and to a lesser extent in ovarian and whole-body weights. Average steroid levels did not significantly differ between conditions, but intra-individual variance in estradiol measures was greater in male-exposed than in isolated females. Creatinine levels were higher in isolated females. Males exposed to developing females excreted higher levels of estradiol in their urine compared to isolated males. These data suggest that excreted steroids can reflect general pubertal development, but may not fully reflect substantial morphological impacts of exposure to novel males. Elevations of estrogen levels in males exposed to developing females could help to account for precocious puberty in such females.

Enzymeimmunoassay of oestradiol, testosterone and progesterone in urine samples from female mice before and after insemination.

ELISA measurements of 17beta-oestradiol, testosterone and progesterone were determined for urine samples collected non-invasively from female mice. Initial samples were collected during 5 successive days while mature female mice were isolated and cyclic. Subsequently, female mice were inseminated and additional urine samples were collected during days 2-6 after observation of copulatory plugs. Measurements of oestradiol and testosterone showed variance over days within individuals and did not significantly differ in measurements taken before or after insemination. Progesterone concentrations were significantly higher after insemination compared with before mating. In a second sample of inseminated females, urinary progesterone was measured during days 2-18 of pregnancy. Most females showed high urinary progesterone up to day 10 of pregnancy and lower concentrations during the remainder of gestation. These results indicate that urinary progesterone reflects established systemic increases of this hormone during pregnancy.

Enzyme immunoassay of 17 beta-estradiol, estrone conjugates, and testosterone in urinary and fecal samples from male and female mice.

ELISA measures of 17 beta-estradiol, estrone conjugates, and testosterone were adapted for fecal and urinary samples from laboratory mice. We will report on validations of these assays and data from interacting males and females. Unconjugated gonadal steroids were consistently measurable in urine and feces of both males and females. Females that were parturient following insemination excreted relatively low levels of urinary testosterone compared to non-parturient females. The results are consistent with evidence that elevated androgens and estrogens are incompatible with intrauterine implantation of fertilized ova, and suggest that steroids in male urine could contribute to pheromonal action. These methods permit repeated noninvasive measurement of steroid activity in this species.

Novel male mice show gradual decline in the capacity to disrupt early pregnancy and in urinary excretion of testosterone and 17 beta-estradiol during the weeks immediately following castration.

Novel male mice can disrupt intrauterine implantation of fertilized ova in inseminated females. Evidence indicates mediation by androgen-dependent excretions. This study was designed to examine the time course of males' ability to disrupt pregnancy following castration and relate this to their urinary excretion of testosterone and 17 beta-estradiol. During days 1-5 of pregnancy, previously inseminated females were housed underneath castrated novel males at various intervals after the surgery. Castrated males generally continued to disrupt pregnancy during the initial weeks after surgery. Progressively, the probability of retention of pregnancy increased as a linear function of time since castration. There was an apparent asymptote, where the majority of females remained pregnant, beginning at about 6 weeks following surgery. Males' excretion of testosterone and 17 beta-estradiol in urine, measured via ELISA procedures, diminished gradually during the weeks after castration.

Administration of minute quantities of 17beta-estradiol on the nasal area terminates early pregnancy in inseminated female mice.

It is well established that chemical emissions of novel males disrupt intrauterine implantation of fertilized ova in inseminated female mice, but the specific nature of these chemicals is not known. Given that novel males excrete androgens and estrogens in their urine and feces, the current experiments were designed to determine whether nasal application of these steroids could disrupt pregnancy. Nasal application of testosterone propionate to females during early pregnancy had no impact on gestation. However, nasal application of 17beta-estradiol terminated all pregnancies in females at all doses greater than or equal to approximately 1 microg/day. Nasal application of 17beta-estradiol benzoate similarly terminated all pregnancies in females at very low doses. In subcutaneous administration, 17beta-estradiol is also the most potent steroid in disrupting pregnancy compared to other estrogens and androgens. These data suggest the possibility that males' emission of estrogens is among factors mediating the Bruce effect.

Differential sexual activity of isolated and group-housed male mice: lack of substantial influence of acute or chronic naloxone administration.

Influences of naloxone upon male sexual behavior were examined using two different baseline activity levels: individually and group-housed mice. In Experiment 1, single injections of 0,12.5, or 50 micrograms per animal were administered before testing. Isolated mice showed more sexual activity than did grouped mice; naloxone failed to alter those differences. In Experiment 2, a similar result was obtained despite administration of 50 or 150 micrograms per animal of naloxone. In Experiment 3,0 or 50 micrograms of naloxone was administered to isolated or grouped males daily on the 5 days before testing. Isolated mice showed performance superior to that of grouped mice, but there was no effect of the drug. In Experiment 4, doses of 0. 12.5, or 50 micrograms of naloxone were given to isolated or grouped males twice daily for 7 days prior to testing, producing little effect. These results suggest that the influences of prior social condition on male sexual activity are robust in the face of naloxone administration.

Differential sexual activity of isolated and group-housed male mice: influence of acute d-amphetamine sulfate administration.

It is established that group housing can impair sexual activity of male mice, and that central catecholamines are involved in male sexual response, but it is not known whether catecholamine mechanisms are involved in sexual impairment in grouped males. Injections of 0, 0.22, 0.67, 2.0, or 6.0 mg/kg of d-amphetamine sulfate were administered 1 h before testing to individually and group-housed male C57BL/6J mice. Isolated mice showed more mounts, intromissions, and ejaculations and shorter response latencies than did group-housed mice. The latencies to first mount, intromission, and ejaculation were nonmonotonically related to dosage, being shortest at the lowest dosage in isolated mice, but significantly elevated by the higher dosages in both isolated and grouped males. The number of ejaculations was significantly elevated by moderate dosages in both isolated and grouped mice, peaking at the 2.0 mg/kg dosage in isolated mice and at 0.67 mg/kg in grouped mice. Nevertheless, amphetamine treatment generally failed to eliminate differences between males from isolated and grouped backgrounds.

Interactions of contact, odor cues, and androgens in strange-male-induced early pregnancy disruptions in mice (Mus musculus).

Novel males can disrupt early pregnancy in female house mice (Mus musculus). In Experiment 1, exposure to novel males disrupted pregnancy, but exposure to male urine did not. In Experiment 2, urine from male or female mice or rats painted on females' noses did not influence pregnancy. In Experiment 3, the conjunction of urine painted on female's noses and vulval stimulation did not affect pregnancy more than water with similar stimulation. In Experiment 4, males housed above females were separated from them by a wire mesh grid; intact males disrupted pregnancy, but castrated ones did not. In Experiment 5, such housing of castrated males or ovariectomized females produced a strong disruption of pregnancy if the stimulus animal was given testosterone but not if it was given oil injections. In Experiment 6, transfers of odorous emissions failed to disrupt pregnancy. Contact and androgen activity are necessary for strange males to disrupt pregnancy.

Estrogen antibodies reduce vulnerability to stress-induced failure of intrauterine implantation in inseminated mice.

In Experiment 1, inseminated mice were randomly assigned to either an undisturbed control condition or four conditions involving exposure to restraint stress on days 1 through 5 of pregnancy. Restrained animals received one of three doses of estrogen antibodies or just vehicle injections on each day of restraint. Restrained animals receiving vehicle only showed significantly fewer uterine implantation sites than did unrestrained controls, while restrained animals given estrogen antibodies showed more implantation sites than did vehicle-treated restrained animals. In Experiment 2, varied dosages of refined estrogen antibodies were administered to inseminated females concurrent to restraint-stress on days 1 through 5 of pregnancy. More females receiving the higher dosages of antibodies produced litters than did restrained females with just vehicle injections, and at the highest dose the number of litters was similar to that produced by undisturbed control females. These results converge with other evidence to suggest that stress-induced pregnancy blocks are mediated by estrogens.

Influences of exogenous epinephrine on two reproductive parameters in female mice: disruption of receptivity but not implantation.

Diverse stressors impede female receptivity and fertility. Since epinephrine is released from the adrenal during stress, it might play a role in stress-induced disruptions of female reproductive parameters. Experiment 1 examined whether exogenous epinephrine would disrupt lordosis behavior in estrogen- and progesterone-treated ovariectomized female mice. Single dosages of 20 and 40 micrograms almost eliminated sexual receptivity. Experiments 2 and 3 examined whether chronic dosages of epinephrine shortly after insemination would lead to a failure of implantation of mouse embryos. Results indicated that epinephrine had little effect on the number of dams delivering pups, the number of pups born, litter weight, or the number of stillbirths.

Chronic stress increases estrogen and other steroids in inseminated rats.

Physical restraint, like many other stressors, can block early pregnancy, but the underlying physiological mechanisms have not been established. Exogenous estrogens in minute doses will also block early pregnancy. In the present study, female rats were exposed to 5 h of restraint daily for the first 5 days after insemination. A subset of animals was sacrificed after 1, 2, 3, 4, and 5 days of restraint, and blood was collected for radioimmunoassay. Blood was also collected from unstressed control animals on each of the first 5 days after insemination. Plasma concentrations of estradiol, as well as those of corticosterone and progesterone, were increased in the stressed animals as compared to the controls. The finding of significantly enhanced maternal estrogen suggests the possibility that estrogens mediate psychogenic pregnancy blocks.

Psychogenic pregnancy disruptions in mammals.

Evidence scattered over the literatures of zoology, psychology, agricultural science, and medicine indicates that diverse stressors will interfere with pregnancy in its early stages. It is probable that the most sensitive period is around the point of intrauterine implantation of fertilized ova. Although there is some indication that conventional "stress" hormones of the pituitary-adrenal axis can inhibit implantation, this evidence is too weak and inconsistent to suggest that these hormones are primary mediators of early pregnancy disruptions. Increasingly, evidence indicates that the balance of ovarian steroids is most important for pregnancy maintenance. It is well known that minute amounts of exogenous estrogens can completely disrupt pregnancy, and some new evidence suggests that endogenous estrogens may be released from the adrenals and/or ovaries during psychological stress.

Duration of mating relates to fertility in mice.

In normal mating, house mice vary dramatically in the number and duration of intromissions that precede ejaculation. Also, some proportion of inseminations typically fails to yield litters. The present studies involved systematic observations of matings of females in natural estrus with males of proven fertility. After standardized observation of mating behavior culminating in ejaculation and a sperm plug, females were allowed to produce litters in undisturbed conditions. In two replications, greater numbers of intromissions and a longer latency from first intromission to ejaculation both correlated positively with litter size.

Antagonism of estrogen-induced lordosis by corticosterone in adrenalectomized-ovariectomized female rats and mice.

Previous experimentation has established that adrenalectomy can facilitate lordosis in ovariectomized estrogen-primed female rats. Experiment 1 examined the role of adrenal steroids in this effect, the results indicating an attenuation with chronic corticosterone but not with desoxycorticosterone or progesterone administration. Experiment 2 established a dose-response curve for this corticosterone effect. Experiments 3 and 4 indicated that corticosterone administration inhibits lordosis when it precedes estrogen administration. Experiment 5 demonstrated that corticosterone also inhibits estrogen-induced lordosis in mice. These data suggest that corticosterone may modulate estrogen-mediated behavior in rodents.