Behavioural food anticipation in clock genes deficient mice: confirming old phenotypes,describing new phenotypes

Animals fed daily at the same time exhibit circadian food‐anticipatory activity (FAA), which has been suggested to be driven by one or several food‐entrainable oscillators (FEOs). FAA is altered in mice lacking some circadian genes essential for timekeeping in the main suprachiasmatic clock (SCN). Here, we confirmed that single mutations of clock genes Per1^?/? and Per2^Brdm1 alter FAA expression in constant darkness (DD) or under a light–dark cycle (LD). Furthermore, we found that Per1^?/?;Per2^Brdm1 and Per2^Brdm1;Cry1^?/? double mutant animals did not display a stable and significant FAA either in DD or LD. Interestingly, rescued behavioural rhythms in Per2^Brdm1;Cry2^?/? mice in DD were totally entrained to feeding time and re‐synchronized after phase‐shifts of mealtime, indicating a higher SCN sensitivity to feeding cues. However, under an LD cycle and restricted feeding at midday, FAA in double Per2^Brdm1;Cry2^?/? mutant mice was absent. These results indicate that shutting down one or two clock genes results in altered circadian meal anticipation. Moreover, we show that in a genetically rescued SCN clock (Per2^Brdm1;Cry2^?/?), food is a powerful zeitgeber to entrain behavioural rhythms, leading the SCN to be more sensitive to feeding cues than in wild‐type littermates.     

Shifting the Circadian Rhythm of Feeding in Mice Induces Gastrointestinal,Metabolic and Immune Alterations Which Are Influenced by Ghrelin and the Core Clock Gene Bmal1

Background

In our 24-hour society, an increasing number of people are required to be awake and active at night. As a result, the circadian rhythm of feeding is seriously compromised. To mimic this, we subjected mice to restricted feeding (RF), a paradigm in which food availability is limited to short and unusual times of day. RF induces a food-anticipatory increase in the levels of the hunger hormone ghrelin. We aimed to investigate whether ghrelin triggers the changes in body weight and gastric emptying that occur during RF. Moreover, the effect of genetic deletion of the core clock gene Bmal1 on these physiological adaptations was studied.

Methods

Wild-type, ghrelin receptor knockout and Bmal1 knockout mice were fed ad libitum or put on RF with a normal or high-fat diet (HFD). Plasma ghrelin levels were measured by radioimmunoassay. Gastric contractility was studied in vitro in muscle strips and in vivo (¹³C breath test). Cytokine mRNA expression was quantified and infiltration of immune cells was assessed histologically.

Results

The food-anticipatory increase in plasma ghrelin levels induced by RF with normal chow was abolished in HFD-fed mice. During RF, body weight restoration was facilitated by ghrelin and Bmal1. RF altered cytokine mRNA expression levels and triggered contractility changes resulting in an accelerated gastric emptying, independent from ghrelin signaling. During RF with a HFD, Bmal1 enhanced neutrophil recruitment to the stomach, increased gastric IL-1α expression and promoted gastric contractility changes.

Conclusions

This is the first study demonstrating that ghrelin and Bmal1 regulate the extent of body weight restoration during RF, whereas Bmal1 controls the type of inflammatory infiltrate and contractility changes in the stomach. Disrupting the circadian rhythm of feeding induces a variety of diet-dependent metabolic, immune and gastrointestinal alterations, which may explain the higher prevalence of obesity and immune-related gastrointestinal disorders among shift workers.     

Sex chromosome complement affects social interactions in mice

Sex differences in behavior can be attributed to differences in steroid hormones. Sex chromosome complement can also influence behavior, independent of gonadal differentiation. The mice used for this work combined a spontaneous mutation of the Sry gene with a transgene for Sry that is incorporated into an autosome thus disassociating gonad differentiation from sex chromosome complement. The resulting genotypes are XX and XY⁻ females (ovary-bearing) along with XXSry and XY⁻Sry males (testes-bearing). Here we report results of basic behavioral phenotyping conducted with these mice. Motor coordination, use of olfactory cues to find a food item, general activity, foot shock threshold, and behavior in an elevated plus maze were not affected by gonadal sex or sex chromosome complement. In a one-way active avoidance learning task females were faster to escape an electric shock than males. In addition, sex chromosome complement differences were noted during social interactions with submissive intruders. Female XY⁻ mice were faster to follow an intruder than XX female mice. All XY⁻ mice spent more time sniffing and grooming the intruder than the XX mice, with XY⁻ females spending the most amount of time in this activity. Finally, XX females were faster to display an asocial behavior, digging, and engaged in more digging than XXSry male mice. All of these behaviors were tested in gonadectomized adults, thus, differences in circulating levels of gonadal steroids cannot account for these effects. Taken together, these data show that sex chromosome complement affects social interaction style in mice.     

Severe pulmonary hypertension in aging female apolipoprotein E-deficient mice is rescued by estrogen replacement therapy

Corticotropin-releasing factor overexpressing (CRF-OE) male mice showed an inhibited feeding response to a fast, and lower plasma acyl ghrelin and Fos expression in the arcuate nucleus compared to wild-type (WT) mice. We investigated whether hormones and hypothalamic feeding signals are impaired in CRF-OE mice and the influence of sex. Male and female CRF-OE mice and WT littermates (4–6 months old) fed ad libitum or overnight fasted were assessed for body, adrenal glands and perigonadal fat weights, food intake, plasma hormones, blood glucose, and mRNA hypothalamic signals. Under fed conditions, compared to WT, CRF-OE mice have increased adrenal glands and perigonadal fat weight, plasma corticosterone, leptin and insulin, and hypothalamic leptin receptor and decreased plasma acyl ghrelin. Compared to male, female WT mice have lower body and perigonadal fat and plasma leptin but higher adrenal glands weights. CRF-OE mice lost these sex differences except for the adrenals. Male CRF-OE and WT mice did not differ in hypothalamic expression of neuropeptide Y (NPY) and proopiomelanocortin (POMC), while female CRF-OE compared to female WT and male CRF-OE had higher NPY mRNA levels. After fasting, female WT mice lost more body weight and ate more food than male WT, while CRF-OE mice had reduced body weight loss and inhibited food intake without sex difference. In male WT mice, fasting reduced plasma insulin and leptin and increased acyl ghrelin and corticosterone while female WT showed only a rise in corticosterone. In CRF-OE mice, fasting reduced insulin while leptin, acyl ghrelin and corticosterone were unchanged with no sex difference. Fasting blood glucose was higher in CRF-OE with female > male. In WT mice, fasting increased hypothalamic NPY expression in both sexes and decreased POMC only in males, while in CRF-OE mice, NPY did not change, and POMC decreased in males and increased in females. These data indicate that CRF-OE mice have abnormal basal and fasting circulating hormones and hypothalamic feeding-related signals. CRF-OE also abolishes the sex difference in body weight, abdominal fat, and fasting-induced feeding and changes in plasma levels of leptin and acyl ghrelin.     

The number of x chromosomes causes sex differences in adiposity in mice

Sexual dimorphism in body weight, fat distribution, and metabolic disease has been attributed largely to differential effects of male and female gonadal hormones. Here, we report that the number of X chromosomes within cells also contributes to these sex differences. We employed a unique mouse model, known as the "four core genotypes," to distinguish between effects of gonadal sex (testes or ovaries) and sex chromosomes (XX or XY). With this model, we produced gonadal male and female mice carrying XX or XY sex chromosome complements. Mice were gonadectomized to remove the acute effects of gonadal hormones and to uncover effects of sex chromosome complement on obesity. Mice with XX sex chromosomes (relative to XY), regardless of their type of gonad, had up to 2-fold increased adiposity and greater food intake during daylight hours, when mice are normally inactive. Mice with two X chromosomes also had accelerated weight gain on a high fat diet and developed fatty liver and elevated lipid and insulin levels. Further genetic studies with mice carrying XO and XXY chromosome complements revealed that the differences between XX and XY mice are attributable to dosage of the X chromosome, rather than effects of the Y chromosome. A subset of genes that escape X chromosome inactivation exhibited higher expression levels in adipose tissue and liver of XX compared to XY mice, and may contribute to the sex differences in obesity. Overall, our study is the first to identify sex chromosome complement, a factor distinguishing all male and female cells, as a cause of sex differences in obesity and metabolism.     

Sex difference in thermal preference of adult mice does not depend on presence of the gonads

Background

The thermoneutral zone (TNZ) is a species-specific range of ambient temperature (T _a), at which mammals can maintain a constant body temperature with the lowest metabolic rate. The TNZ for an adult mouse is between 26 and 34 °C. Interestingly, female mice prefer a higher T _a than male mice although the underlying mechanism for this sex difference is unknown. Here, we tested whether gonadal hormones are dominant factors controlling temperature preference in male and female mice.

Methods

We performed a temperature preference test in which 10-week-old gonadectomized and sham-operated male and female C57BL/6J mice were allowed to choose to reside at the thermoneutral cage of 29 °C or an experimental cage of 26, 29, or 32 °C.

Results

All mice preferred a T _a higher than 26 °C, especially in the inactive phase. Choosing between 29 and 32 °C, female mice resided more at 32 °C while male mice had no preference between the temperatures. Hence, the preferred T _a for female mice was significantly higher (0.9?±?0.2 °C) than that for male mice. However, gonadectomy did not influence the T _a preference.

Conclusions

Female mice prefer a warmer environment than male mice, a difference not affected by gonadectomy. This suggests that thermal-sensing mechanisms may be influenced by sex-specific pathways other than gonadal factors or that the thermoregulatory set point has already been determined prior to puberty.

     

Gonadal hormone effects on entrained and free-running circadian activity rhythms in the developing diurnal rodent Octodon degus

The slowly maturing, long-lived rodent Octodon degus (degu) provides a unique opportunity to examine the development of the circadian system during adolescence. These studies characterize entrained and free-running activity rhythms in gonadally intact and prepubertally gonadectomized male and female degus across the first year of life to clarify the impact of sex and gonadal hormones on the circadian system during adolescence. Gonadally intact degus exhibited a delay in the phase angle of activity onset (Psi(on)) during puberty, which reversed as animals became reproductively competent. Gonadectomy before puberty prevented this phase delay. However, the effect of gonadal hormones during puberty on Psi(on) does not result from changes in the period of the underlying circadian pacemaker. A sex difference in Psi(on) and free-running period (tau) emerged several months after puberty; these developmental changes are not likely to be related, since the sex difference in Psi(on) emerged before the sex difference in tau. Changes in the levels of circulating hormones cannot explain the emergence of these sex differences, since there is a rather lengthy delay between the age at which degus reach sexual maturity and the age at which Psi(on) and tau become sexually dimorphic. However, postnatal exposure to gonadal hormones is required for sexual differentiation of Psi(on) and tau, since these sex differences were absent in prepubertally gonadectomized degus. These data suggest that gonadal hormones modulate the circadian system during adolescent development and provide a new model for postpubertal sexual differentiation of a central nervous system structure.     

The Clock Genes Period 2 and Cryptochrome 2 Differentially Balance Bone Formation

Background

Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype.

Methodology/Principal Findings

We found that Per2^Brdm1 mutant mice as well as mice lacking Cry2^−/− displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2^Brdm1 mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2^−/− displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2^−/− mutants despite the simultaneous inactivation of Per2.

Conclusions/Significance

This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters.     

Nippostrongylus brasiliensis: effect of host hormones on helminth ingestion in vivo

Nippostrongylus brasiliensis: effect of host hormones on helminth ingestion in vivo. International Journal for Parasitology16: 77–80. Ingestion of dye in vivo by both sexes of Nippostrongylus brasiliensis was increased in gonadectomized male, but not female, mice that were implanted with testosterone-filled capsules. The nematodes increased their ingestion of dye in both sexes of gonadectomized hosts that were implanted with 17 β-estradiol and decreased their uptake of dye in progesterone-implanted animals. Implantation of intact male mice with capsules of hydrocortisone and cholesterol suggested that ingestion by N. brasiliensis was decreased and increased, respectively, in these animals. Ligation of the bile duct in gonadectomized male mice that were implanted with testosterone capsules reduced the ingestion of dye by both sexes of nematode.     

Sex chromosome complement contributes to sex differences in coxsackievirus B3 but not influenza A virus pathogenesis

Background

Both coxsackievirus B3 (CVB3) and influenza A virus (IAV; H1N1) produce sexually dimorphic infections in C57BL/6 mice. Gonadal steroids can modulate sex differences in response to both viruses. Here, the effect of sex chromosomal complement in response to viral infection was evaluated using four core genotypes (FCG) mice, where the Sry gene is deleted from the Y chromosome, and in some mice is inserted into an autosomal chromosome. This results in four genotypes: XX or XY gonadal females (XXF and XYF), and XX or XY gonadal males (XXM and XYM). The FCG model permits evaluation of the impact of the sex chromosome complement independent of the gonadal phenotype.

Methods

Wild-type (WT) male and female C57BL/6 mice were assigned to remain intact or be gonadectomized (Gdx) and all FCG mice on a C57BL/6 background were Gdx. Mice were infected with either CVB3 or mouse-adapted IAV, A/Puerto Rico/8/1934 (PR8), and monitored for changes in immunity, virus titers, morbidity, or mortality.

Results

In CVB3 infection, mortality was increased in WT males compared to females and males developed more severe cardiac inflammation. Gonadectomy suppressed male, but increased female, susceptibility to CVB3. Infection with IAV resulted in greater morbidity and mortality in WT females compared with males and this sex difference was significantly reduced by gonadectomy of male and female mice. In Gdx FCG mice infected with CVB3, XY mice were less susceptible than XX mice. Protection correlated with increased CD4+ forkhead box P3 (FoxP3)+ T regulatory (Treg) cell activation in these animals. Neither CD4+ interferon (IFN)γ (T helper 1 (Th1)) nor CD4+ interleukin (IL)-4+ (Th2) responses differed among the FCG mice during CVB3 infection. Infection of Gdx FCG mice revealed no effect of sex chromosome complement on morbidity or mortality following IAV infection.

Conclusions

These studies indicate that sex chromosome complement can influence pathogenicity of some, but not all, viruses.     

Circadian Regulation of Food-Anticipatory Activity in Molecular Clock–Deficient Mice

In the mammalian brain, the suprachiasmatic nucleus (SCN) of the anterior hypothalamus is considered to be the principal circadian pacemaker, keeping the rhythm of most physiological and behavioral processes on the basis of light/dark cycles. Because restriction of food availability to a certain time of day elicits anticipatory behavior even after ablation of the SCN, such behavior has been assumed to be under the control of another circadian oscillator. According to recent studies, however, mutant mice lacking circadian clock function exhibit normal food-anticipatory activity (FAA), a daily increase in locomotor activity preceding periodic feeding, suggesting that FAA is independent of the known circadian oscillator. To investigate the molecular basis of FAA, we examined oscillatory properties in mice lacking molecular clock components. Mice with SCN lesions or with mutant circadian periods were exposed to restricted feeding schedules at periods within and outside circadian range. Periodic feeding led to the entrainment of FAA rhythms only within a limited circadian range. Cry1^−/− mice, which are known to be a “short-period mutant,” entrained to a shorter period of feeding cycles than did Cry2^−/− mice. This result indicated that the intrinsic periods of FAA rhythms are also affected by Cry deficiency. Bmal1 ^−/− mice, deficient in another essential element of the molecular clock machinery, exhibited a pre-feeding increase of activity far from circadian range, indicating a deficit in circadian oscillation. We propose that mice possess a food-entrainable pacemaker outside the SCN in which canonical clock genes such as Cry1, Cry2 and Bmal1 play essential roles in regulating FAA in a circadian oscillatory manner.     

Methamphetamine-induced conditioned place preference is facilitated by estradiol pretreatment in female mice

Ovarian hormones were well documented to modulate the dopamine release in the central dopaminergic systems. The dopamine-releasing effects in the nucleus accumbens, a major target of the mesolimbicortical dopaminergic system, were closely associated with the reinforcing effects of two psychomotor stimulants, cocaine and methamphetamine. This study aimed to examine the sex differences in the cocaine- and methamphetamine-reinforcing behavior, conditioned place preference. In addition, the modulating effects of estradiol and progesterone on methamphetamine-induced conditioned place preference were investigated in both sexes of adult gonadectomized mice. There was no sex difference in the sensitivity to the cocaine (5 mg/kg)-induced conditioned place preference. However, female mice exhibited a more potent methamphetamine (1 mg/kg)-induced conditioned place preference than did male mice. Moreover, pretreatment with estradiol for two consecutive days before the beginning of the conditioning and throughout the four daily conditionings (0.47 microg/day for totally six days) effectively facilitated methamphetamine-induced conditioned place preference in gonadectomized female mice, but not in gonadectomized male mice. Progesterone, under a similar treatment regimen (0.47 microg/day for six consecutive days), did not alter the methamphetamine-induced conditioned place preference in either sex of gonadectomized mice. Taken together, we conclude that the facilitating effects of estradiol on methamphetamine-induced conditioned place preference could be sex-dependent with an eminent sensitivity associated with the adult female mice.     

Nippostrongylus brasiliensis: effects of testosterone on reproduction and establishment

Gonadectomy of the host reduced the establishment of Nippostrongylus brasiliensis in male mice, but elevated the recovery of worms from female hosts. Similarly, production of eggs by individual females of N. brasiliensis decreased and increased in gonadectomized male and female animals, respectively. Implantation of testosterone-filled capsules caused a dosage-dependent release of eggs by helminths in gonadectomized hosts of both sexes. Maximal production of eggs by nematodes was found when plasma testosterone in the host exceeded 20 ng/ml. Treatment of gonadectomized mice of both sexes with testosterone implants gave recoveries of worms that were similar to the number of helminths in intact male mice. Ligation of the bile duct of male mice that were implanted with testosterone reduced both establishment and reproduction of N. brasiliensis.     

Sex-related difference of kinin level in rat pituitary gland

The kinin level in the pituitary glands was compared in adult male and female rats. A sex-related difference in the bradykinin (BK)-like immunoreactivity was found in the posterior lobe. The posterior pituitaries of female rats contained a higher concentration of the immunoreactive kinin than those of males. Ovariectomy of female rats resulted in the disappearance of a sex difference in the posterior kinin level and about a 3-fold increase in the anterior one. Orchidectomy of adult male rats failed to alter the kinin levels in both lobes. Moreover, the constitution of pituitary kinins was determined using HPLC. The pituitary kinins consisted of BK, Lys-BK (L-BK) and Met-Lys-BK (ML-BK) in different proportions in both lobes of male and female rats. The gonadectomy altered the proportions of these kinins. These results suggest that the pituitary kinin system may be regulated by circulating gonadal steroid hormones.     

Glucose and lactate utilization by the amygdala of male and female rats

Gonadal hormones appear to modulate brain energy metabolism, and morphological and functional sexual differences are found in the amygdaloid complex (AC) of rats. Our aim was to study the CO₂ production and lipid synthesis, measured by the rate of L-[U-¹⁴C]lactate or D-[U-¹⁴C]glucose utilization (in pmol.hr^–1.mg^–1), by AC slices in vitro of male and female rats. Lactate was more used than glucose as energy substrate (p < 0.01) but no sex-related difference was observed in glucose or lactate oxidation to CO₂ (p > 0.05) or on lipid synthesis obtained from both substrates (p > 0.05). In addition, there was no effect of the estrous cycle on lactate oxidation to CO₂ by the AC of females (p > 0.05). Based on the present data, it appears that the endogenous normal levels of gonadal hormones are not able to promote sex-related differences in the in vitro glucose or lactate utilization by the AC of rats.     

Hormonal differentiation of sexual behavior in Japanese quail

The effect of hormones on the development of Japanese quail during the postembryonic period was examined. First, subcutaneous implants of estradiol monobenzoate (EB) and testosterone propionate (TP) were implanted 6–12 hr after hatching. EB and TP had no effect on the differentiation of sexual behavior in genetic males or females. However, EB had marked feminizing effects on plumage in genetic males. Second, the role of gonadal hormones during development was examined by gonadectomizing males and females 6–12 hr after hatching and treating them intramuscularly with EB or TP as adults. EB-treated adult females displayed sexual behavior typical of the genetic female and developed female plumage. A significant proportion of TP-treated females (57%) displayed male sexual behavior patterns. Cloacal gland development and male-type vocalizations were induced. EB-treated males displayed either male or female sexual patterns depending on the stimulus conditions. Third, to test whether bisexuality in gonadectomized males and females is maintained despite steroid treatment and expression of sexual behavior in adulthood, gonadectomized quail which were originally treated with EB received TP and vice versa. The results indicate that in the absence of gonadal hormones after hatching female quail remain bisexual until exposed to estrogen, whereas gonadectomized male quail retain behavioral bisexuality irrespective of prior estrogen or androgen exposure.     

Unique food-entrained circadian rhythm in cysteine414-alanine mutant mCRY1 transgenic mice

Food availability is a potent environmental cue that directs circadian locomotor activity in rodents. Daily scheduled restricted feeding (RF), in which the food available time is restricted for several hours each day, elicits anticipatory activity. This food-anticipatory activity (FAA) is controlled by a food-entrainable oscillator (FEO) that is distinct from the suprachiasmatic nucleus (SCN), the master pacemaker in mammals. In an earlier report, we described generation of transgenic (Tg) mice ubiquitously overexpressing cysteine414-alanine mutant mCRY1. The Tg mice displayed long locomotor free-running periods (approximately 28 h) with rhythm splitting. Furthermore, their locomotor activity immediately re-adjusted to the advance of light–dark cycles (LD), suggesting some disorder in the coupling of SCN neurons. The present study examined the restricted feeding cycle (RF)-induced entrainment of locomotor activity in Tg mice in various light conditions. In LD, wild-type controls showed both FAA and LD-entrained activities. In Tg mice, almost all activity was eventually consolidated to a single bout before the feeding time. The result suggests a possibility that in Tg mice the feeding cycle dominates the LD cycle as an entrainment agent. In constant darkness (DD), wild-type mice exhibited robust free-run activity and FAA during RF. For Tg mice, only the rhythm entrained to RF was observed in DD. Furthermore, after returning to free feeding, the free-run started from the RF-entrained phase. These results suggest that the SCN of Tg mice is entrainable to RF and that the mCRY1 mutation alters the sensitivity of SCN to the cycle of nonphotic zeitgebers.