Search

Females have long been underrepresented in preclinical research and clinical drug trials. Directives by the U.S. National Institutes of Health have increased female participation in research protocols, although analysis of outcomes by sex remains infrequent. The long-held view that traits of female rats and mice are more variable than those of males is discredited, supporting equal representation of both sexes in most studies. Drug pharmacokinetic analysis reveals that, among subjects administered a standard drug dose, women are exposed to higher blood drug concentrations and longer drug elimination times. This contributes to increased adverse drug reactions in women and suggests that women are routinely overmedicated and should be administered lower drug doses than men. The past decade has seen progress in female inclusion, but key subsequent steps such as sex-based analysis and sex-specific drug dosing remain to be implemented.

Introduction: Excessive oxidant stress has been shown to damage cardiac tissue and result in impaired cardiac function. Animals with chronic heart failure (CHF) exhibit lower levels of antioxidant enzymes in many tissues including the heart, brain, and skeletal muscle (SkM). Nrf2 is a ubiquitous master transcription factor that regulates antioxidant proteins during enhanced oxidative stress. Earlier studies from this laboratory showed that exercise training was cardioprotective following ischemia reperfusion injury in mice. This protective effect was abrogated in mice in which the Nrf2 gene was deleted selectively in skeletal muscle. Given that skeletal muscle is approximately 40% of body weight and in light of recent findings on skeletal muscle derived extracellular vesicles, we sought to determine if up and downregulation of Nrf2 in skeletal muscle altered cardiac function. We hypothesized that skeletal muscle knockout of Nrf2 would impair cardiac function and the response to the beta 1 agonist dobutamine. Materials and Methods: Experiments were carried out in 37 males iMS-Keap1flox/flox and iMS-Nrf-2flox/flox (i.e. SkM specific) mice. Gene deletion was conditional upon the administration of the estrogen receptor agonist tamoxifen at 3 months of age; floxed mice given vehicle instead of tamoxifen were considered WT. The mice were assigned to 4 groups: Keap1 knockout (KO) (10), Keap1 WT (8), Nrf-2 KO (11), and Nrf-2 WT (8). Results: Keap1 deletion resulted in an increase in skeletal muscle hemoxygenase 1 (HO1) (p = 0.0286) while Nrf2 deletion resulted in a reduction in HO1 (p = 0.0286). However, there were no changes in the level of HO1 or other antioxidant proteins in the heart. Cardiac function was assessed with high frequency echocardiography (Vevo 3100). Systolic and diastolic function were analyzed. Up and downregulation of skeletal muscle Nrf2 did not evoke any change in either systolic or diastolic function. Conclusions: Previous studies have shown that Nrf2 upregulation salvaged cardiac function in mice subjected to either myocardial infarction or thoracic aortic constriction. Therefore, it may be that our lack of an effect of SkM specific Nrf2 manipulation is due to this study being carried out in normal animals and not in a disease setting such as CHF. In future work, this effect will be examined in mice with cardiac dysfunction. Supported by P0162222 and SURF from the APS This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Post-exercise hypotension (PEH) is an immediate reduction in blood pressure (BP) after a single bout of exercise that forms the long-term hypertension amelioration by chronic exercise. The present study aims to explore a novel mechanism of PEH, with a focus of skeletal muscle-derived extracellular vesicles (SkM-EVs). We hypothesize that repetitively contracting-relaxing muscle releases antioxidant-enriched EVs that induce hypotension. Experiments were carried out in 48 male Sprague-Dawley rats. BP was recorded by a Millar catheter inserted into right carotid artery. EVs were isolated from SkM and plasma using ultracentrifugation, and then qualified and quantified using transmission electron microscopy and the ZetaView nanoparticle tracking analyzer. Results: First, in rats undergoing 1-hour of treadmill exercise (15/30/15 m/min for 10/40/10 min), we found an elevated BP during exercise (34.7 ± 5.2 mmHg, p < 0.001; n = 8), followed by a decrease in BP below baseline during the post exercise period (-7.3 ± 1.1 mmHg, p < 0.01). Pretreatment with the EV inhibitor GW4869 (1.5 mg/kg) had no effects on hypertension during exercise, but completely abolished the post-exercise hypotension (1.93 ± 0.64 mmHg, p < 0.001), suggesting a role for SkM-EVs in this response. Bolus i.v. injection of SkM-EVs from exercised rats (400 μg EV protein) induced a dramatic decrease in BP (-16.7 ± 4.4 mmHg; p < 0.01, n = 6/group) in non-exercised rats, which lasted for up to 40 min. Second, 1-hour sciatic nerve electrical stimulation (2.5V, 50Hz, 0.3s/3s)-evoked hindlimb muscle contraction in isoflurane-anesthetized rats induced a biphasic change in BP, with hypertension during the initial 20 min followed by hypotension in the last 40 min. Transection of the sciatic nerve or pretreatment with GW4869 abolished the stimulation-evoked hypertension and hypotension. Bolus i.v. injection of EVs from contracting-muscle significantly reduced BP with a maximal change of -14 ± 3.2 mmHg (p = 0.003; n = 5). Third, mass spectroscopic and western blotting analyses of these EVs identified 827 proteins, including Catalase, GSTMs, PRDXs, EPHX1, HSP90, and many others, which are involved in redox homeostasis and mitochondrial bioenergetics. Finally, using muscle-specific GFP reporter mice (MS-mG), we found increased SkM-EVs in both muscle and plasma following exercise by using flow cytometry. Confocal imaging identified GFP+-particles in the neurons of the rostral ventrolateral medulla (RVLM) of C57BL/6 mice that received an injection of SkM-EVs isolated from the exercised MS-mG mice, suggesting their active uptake across the blood-brain barrier. In summary, the major findings of this study include (1) SkM-EVs are essential for PEH; (2) i.v. administration of SkM-EVs reduced BP; (3) SkM-EVs contain enriched antioxidant enzymes and other cytoprotective proteins. These results suggest a novel mechanism underpinning PEH and provides a potential therapeutic strategy for hypertension. Supported by NIH grant R01HL160820. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background/hypothesis of the study: Acute lung injury (ALI) increases respiratory rate (RR), induces hypoxemia. This stimulates carotid body (CB) chemoreflex to maintain oxygen homeostasis. Our previous study indicates a sensitized chemoreflex during the recovery of ALI. Chronic chemoreflex hyperactivity negatively affects the overall quality of life. The neural mechanisms underlying the sensitized CB chemoreflex during the recovery from ALI remain a gap in knowledge. Electrical stimulation of the superior cervical ganglion (SCG) sensitizes the CB chemoreflex in rats. We hypothesized that the SCG causes the CB chemoreflex sensitization post-ALI. Methods and Results: We performed a bilateral SCG ganglionectomy (SCGx) or sham-SCGx (Sx) in our male SD rats 2 weeks before inducing ALI (W-2). After 2 weeks of recovery, ALI was induced using a single intra-tracheal instillation of bleomycin (day 1) and measured the resting respiratory variables- RR, TV (Tidal Volume), and MV (Minute Ventilation). The chemoreflex response to 10% hypoxia and 5% normoxic hypercapnia were measured on W(-3) (pre-SCGx), W0 (post-SCGx/pre-ALI) and W4 (post-ALI) using whole-body plethysmography (WBP). Ganglionectomy did not alter the resting lung parameters and the chemoreflex response to either gas challenge in normal, healthy rats. An increase in resting RR at W1 post-ALI was not significantly different between Sx and SCGx rats. At W4 post-ALI, there were no significant differences in resting lung parameters between Sx and SCGx rats. Consistent with our previous study, the CB chemoreflex response to hypoxia and normoxic hypercapnia in Sx rats at W4 post-ALI was sensitized (p This study was supported by: NIH Grant- RO1 HL-152160 and RO1 HL-126796; Lieberman Grant 2021- Dept. of Anesthesiology, UNMC; American Heart Association- Pre-doctoral fellowship, AHA. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduction: Excessive oxidant stress has been shown to mediate a variety of cardiovascular pathological conditions, including hypertension. The level of reactive oxygen species (ROS) in tissues is regulated by the balance between the production of ROS and the ability to scavenge superoxide and hydrogen peroxide by a myriad of antioxidant enzymes and small molecules. One of the most important regulators of antioxidant activity is the ubiquitous transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 binds to antioxidant response elements of multiple genes following its release from its inhibitory protein Keap1 and part of the E3 ubiquitin ligase complex. In this study we hypothesized that endothelial Nrf2 is a critical factor in blood pressure regulation following vasoconstrictor stimuli. Methods: Transgenic mice were created using the endothelial Tie2 promoter expressing Cre recombinase (Tie2-Cre) crossed with either Nrf2 floxed or Keap1 floxed mice so that Nrf2 would be deleted or upregulated respectively, and selectively in the endothelium. Cre negative mice were used as wild type (WT) controls. Mice were instrumented with radiotelemetry transducers to chronically record arterial blood pressure and heart rate in the conscious state. Blood pressure and heart rate were recorded for 24 hours once per week. After baseline recordings, animals were challenged with an osmotic minipump infusion of Angiotensin II (Ang II) (600ng/Kg/min) for 7 days or L-nitroarginine methyl ester (L-NAME) (100 mg/kg/d in drinking water). Results: Baseline mean arterial pressure (MAP) was 101.6 ± 2.5 mm Hg (mean±SEM; n=7) in WT mice (daytime =95.4±1.5; nighttime =106.1±1.0). In mice with endothelial Nrf2 deletion baseline MAP was significantly higher(p Supported by NHLBI P01-62222 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic Heart failure (CHF) is a highly prevalent disease that leads to significant morbidity and mortality. Diffuse vasculopathy is a common feature of the morbidity associated with CHF. Increased vascular permeability leading to plasma extravasation (PEx) occurs in surrounding tissues following endothelial dysfunction. Such micro- and macro-vascular complications develop over time and lead to edema, inflammation, and multi-organ dysfunction in CHF. However, a systemic examination of PEx in vital organs among different time windows of CHF has never been performed before. In the present study, we investigated time-dependent PEx in several major visceral organs including heart, lung, liver, spleen, kidney, duodenum, ileum, cecum and pancreas between sham-operated and CHF rats induced by myocardial infarction (MI). Plasma extravasation was determined by evaluating Evans Blue concentrations calorimetrically at fluorescence excitation wavelength of 620 nm (bandwidth 10 nm) and an emission wavelength of 680 nm (bandwidth 40 nm) at 3 days, 8-10 weeks and 4 months following MI. Data show that heart PEx level was initially high at day 3 post MI and then gradually decreased but still remained at a moderately high level at 8-10 weeks and 4 months post MI. Lung PEx began at day 3 and remained significantly high at both 8-10 weeks and 4 months post MI. Spleen PEx level was significantly high at 8-10 weeks and 4 months but not day 3 post MI. Liver PEx occurs early at day 3 and remain significantly high at 8-10 weeks and 4 months post MI. For the gastrointestinal (GI) organs including Duodenum, ileum and cecum, there was a general trend that PEx level gradually increased following MI and reached a statistical significance at 4 months post MI. Different with GI PEx, renal PEx level was initially high at day 3 but back to normal level at 8-10 weeks post MI. However, renal PEx was significantly elevated again at 4 months post MI. In summary, we found that MI generally incites a time-dependent PEx of multiple visceral organs. However, the PEx time window for individual organ in response to the MI challenge was different, suggesting that different mechanisms might be involved in the pathogenesis of PEx in these vital organs during CHF. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Introduction: Endothelial dysfunction is considered a key contributor to vascular pathologies. It is well-accepted that excessive reactive oxygen species and oxidative damage likely contribute to endothelial dysfunction. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is considered the master regulator of endogenous antioxidant responses. Therefore, Nrf2 may be a potential therapeutic target to protect against endothelial dysfunction. However, the roles of endothelial cell (EC)-specific Nrf2 on endothelial function are not known. The major objective of this study was to evaluate the impacts of EC Nrf2 deletion on endothelial-mediated vasodilation, which we hypothesized would be attenuated with EC Nrf2 deletion. Methods: Femoral-to-popliteal artery segments were harvested from 6-mo old C57BL/6 mice (WT, n = 6) and EC-specific Nrf2-knockout mice (Tie2-Cre-Nrf2 floxed-KO, n = 6) using both sexes (3 male and 3 female/group). Vasodilation was assessed in response to flow (30 uL·min-1), acetylcholine (ACh, 10-7-10-3 M) with and without NΩ-Nitro-L-arginine methyl ester (L-NAME), and sodium nitroprusside (SNP, 10-9-10-4 M) using videomicroscopy. Glutathione peroxidase 1 (GPX1) and catalase (CAT) skeletal muscle protein expression was assessed by immunoblotting and normalized to total protein. Results: Endothelium-dependent vasodilation was lower in Nrf2-KO compared to WT induced by flow (WT: 34.8 ± 2.9%, Nrf2-KO: 20.7 ± 3.7%, P < 0.01) and ACh (10-3 M, WT: 68.3 ± 8.2%, Nrf2-KO: 44.5 ± 7.1%, P < 0.01). L-NAME incubation attenuated endothelium-dependent vasodilation in WT mice in response to flow (12.8 ± 4.5%, P < 0.01) and ACh (10-3 M, 19.1 ± 4.4%, P < 0.01) but did not change in Nrf2-KO in response to flow (15.6 ± 6.8%, P = 0.28) or ACh (10-3 M, 37.7 ± 7.0%, P = 0.16). Endothelium-independent vasodilation was not different (SNP 10-4 M, WT: 92.7 ± 3.6%, Nrf2-KO: 81.9 ± 10.2%, P = 0.16). In addition, GPX1 was lower in Nrf2-KO mice (WT: 0.47 ± 0.06, Nrf2-KO: 0.001 ± 0.003, P < 0.01), but CAT was not different (WT: 0.16 ± 0.04, Nrf2-KO: 0.37 ± 0.21, P = 0.08). Conclusions: EC Nrf2 may play a key role in endothelial-mediated vasodilatory function. The nitric oxide synthase inhibitor L-NAME attenuated endothelial-mediated vasodilation in WT but not in EC Nrf2-KO. EC Nrf2 may play a role in skeletal muscle antioxidant homeostasis, which suggests potential systemic implications of EC Nrf2 deletion. These results suggest that the EC antioxidant defense system is linked to endothelial dysfunction and changes in the skeletal muscle redox environment, likely through nitric oxide- and oxidative stress-related mechanisms. NIH (R01 AG034995, R01 AG049868, P01HL62222) NIH National Institute of General Medical Sciences, which funds the Great Plains IDeA-CTR Network (U54GM115458) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Patients suffering from acute lung injury (ALI) are at a high risk of developing cardiac arrhythmias. Our recent study demonstrated that bleomycin (Bleo)-induced ALI in rats exhibited significantly more spontaneous premature ventricular contractions (PVCs) from one to three weeks post induction. Although spontaneous PVCs disappeared at 4 weeks post ALI, PVCs were evoked again by electrical stimulation of the decentralized stellate ganglia in Bleo-exposed rats but not in normal rats. In the current study, we aimed to utilize non-biased RNA-Seq analysis to identify potential gene candidates in cardiac tissues, which may be associated with cardiac arrhythmia during the recovery from ALI. We created an ALI rat model using a single tracheal instillation of bleomycin (2.5 mg/kg), with saline as a vehicle (Veh) control. Blood troponin level was measured between Veh- and Bleo-treated rats at Week 1 (W1) post Veh/Bleo. Cardiac tissues were harvested at W4 post Veh/Bleo for RNA-seq analysis and following PCR validation. At W1 post ALI, cardiac troponin level was significantly increased in Bleo-treated rats whereas it was undetectable in Veh-treated (Control) rats. RNA-seq analysis indicated 11 downregulated (AABR07045427, Habp2, AC130862, Per2, Coch, Phactr3, Angptl7, LOC103690108, Mthfd2, Sorcs1, U6) and 13 upregulated (Calhm5, Metazoa_SRP, Tgm1, Pcare, Drd4, Selplg, AABR07027722; Rn5-8s, 5_8S_rRNA) genes in cardiac tissues at 4 weeks post ALI vs Veh. PCR validated that the gene expressions of Habp2, Coch, Phactr3, LOC103690108, Mthfd2 were significantly downregulated and the gene expressions of Pcare, Drd4 were significantly upregulated in the hearts of Bleo-treated rats at W4 post ALI. Our study demonstrated that multiple gene changes in cardiac tissues are associated with cardiac injury and arrhythmogenesis in bleomycin-induced ALI in male rats. Future studies will further determine the cause-effect relationship between these altered genes and cardiac arrhythmia post ALI. This study was supported by NIH grant R01 HL-152160 and in part, by NIH grants R01 HL126796. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Circadian rhythms are generated by interlocked transcriptional-translational feedback loops of circadian clock genes and their protein products. Mice homozygous for a functional deletion in the Period-2 gene ( Per2m/m mice) exhibit short free-running circadian periods and eventually lose behavioral circadian rhythmicity in constant darkness (DD). We investigated Per2m/m mice in DD for several months and identified a categorical sex difference in the dependence on Per2 for maintenance of circadian rhythms. Nearly all female Per2m/m mice became circadian arrhythmic in DD, whereas free-running rhythms persisted in 37% of males. Remarkably, with extended testing, Per2m/m mice did not remain arrhythmic in DD, but after varying intervals spontaneously recovered robust, free-running circadian rhythms, with periods shorter than those expressed prior to arrhythmia. Spontaneous recovery was strikingly sex-biased, occurring in 95% of females and 33% of males. Castration in adulthood resulted in male Per2m/m mice exhibiting female-like levels of arrhythmia in DD, but did not affect spontaneous recovery. The circadian pacemaker of many gonad-intact males, but not females, can persist in DD for long intervals without a functional PER2 protein; their circadian clocks may be in an unstable equilibrium, incapable of sustaining persistent coherent circadian organization, resulting in transient cycles of circadian organization and arrhythmia.

BackgroundWomen experience adverse drug reactions, ADRs, nearly twice as often as men, yet the role of sex as a biological factor in the generation of ADRs is poorly understood. Most drugs currently in use were approved based on clinical trials conducted on men, so women may be overmedicated. We determined whether sex differences in drug pharmacokinetics, PKs, predict sex differences in ADRs.MethodsSearches of the ISI Web of Science and PubMed databases were conducted with combinations of the terms: drugs, sex or gender, pharmacokinetics, pharmacodynamics, drug safety, drug dose, and adverse drug reaction, which yielded over 5000 articles with considerable overlap. We obtained information from each relevant article on significant sex differences in PK measures, predominantly area under the curve, peak/maximum concentrations, and clearance/elimination rates. ADRs were identified from every relevant article and recorded categorically as female-biased, male-biased, or not sex-biased.ResultsFor most of the FDA-approved drugs examined, elevated blood concentrations and longer elimination times were manifested by women, and these PKs were strongly linked to sex differences in ADRs. Of the 86 drugs evaluated, 76 had higher PK values in women; for 59 drugs with clinically identifiable ADRs, sex-biased PKs predicted the direction of sex-biased ADRs in 88% of cases. Ninety-six percent of drugs with female-biased PK values were associated with a higher incidence of ADRs in women than men, but only 29% of male-biased PKs predicted male-biased ADRs. Accessible PK information is available for only a small fraction of all drugsConclusionsSex differences in pharmacokinetics strongly predict sex-specific ADRs for women but not men. This sex difference was not explained by sex differences in body weight. The absence of sex-stratified PK information in public records for hundreds of drugs raises the concern that sex differences in PK values are widespread and of clinical significance. The common practice of prescribing equal drug doses to women and men neglects sex differences in pharmacokinetics and dimorphisms in body weight, risks overmedication of women, and contributes to female-biased adverse drug reactions. We recommend evidence-based dose reductions for women to counteract this sex bias.

Breastfeeding women are excluded from clinical trials of psychoactive drugs because of ethical concerns. Animal testing, which often is predictive of adverse effects in humans, represents the only avenue available for assessing drug safety for human offspring exposed to drugs during lactation. I determined whether behavioral outcomes for children exposed during breastfeeding to antidepressants, anxiolytics, antipsychotics, anti-seizure medications, analgesics, sedatives, and marijuana can be predicted by rodent studies of offspring exposed to drugs during lactation. Animal data were available for only 10 of 80 CNS-active drugs canvassed. Behavioral deficits in adolescence or adulthood in rats and mice after various drug exposures during lactation included reductions in sexual behavior, increased anxiety, hyperactivity, and impaired learning and memory. Whether similar adverse effects will emerge in adulthood in children exposed to drugs during breastfeeding is unknown. Rodent research has the potential to forecast impairments in breastfed children long before information emerges from post-marketing reports and should be prioritized during preclinical drug evaluation by the FDA for new drugs and for drugs currently prescribed off-label for lactating women.

Many drugs with unknown safety profiles are administered to pregnant women, placing their offspring at risk. I assessed whether behavioral outcomes for children exposed during gestation to antidepressants, anxiolytics, anti-seizure, analgesic, anti-nausea and sedative medications can be predicted by more extensive animal studies than are part of the FDA approval process. Human plus rodent data were available for only 8 of 33 CNS-active drugs examined. Similar behavioral and cognitive deficits, including autism and ADHD emerged in human offspring and in animal models of these disorders after exposure to fluoxetine, valproic acid, carbamazepine, phenytoin, phenobarbital and acetaminophen. Rodent data helpful in identifying and predicting adverse effects of prenatal drug exposure in children were first generated many years after drugs were FDA-approved and administered to pregnant women. I recommend that enhanced behavioral testing of rodent offspring exposed to drugs prenatally should begin during preclinical drug evaluation and continue during Phase I clinical trials, with findings communicated to physicians and patients in drug labels.

Author(s): Smarr, Benjamin; Rowland, Neil; ZUCKER, Irving | Abstract: The exclusion of female rodents from biomedical research is well documented and persists in large part due to perceptions that ovulatory cycles render female traits more variable than those of males, and females must be tested at each of four stages of the estrous cycle to generate reliable data. These beliefs are not empirically based. The magnitude of trait variance associated with the estrous cycle may be sufficiently low and of little impact, or trait variability of males tested on 4 consecutive days may be as great as that of females over the 4 days of the estrous cycle. Here, we analyzed food intake data from mice in 4-day blocks, corresponding to the females 4-day estrous cycle in several schedules of food procurement or reward. Variance was compared within and across individual mice. In no instance did the overall variance differ by sex under any of the food reward schedules. This extends earlier observations of trait variability in body temperature and locomotor activity of mice and supports the claim that there is no empirical basis for excluding female rodents from biomedical research.

Diverse mammalian ultradian rhythms (URs) with periods in the 1-6h range, are omnipresent at multiple levels of biological organization and of functional and adaptive significance. Specification of neuroendocrine substrates that generate URs remains elusive. The suprachiasmatic (SCN) and arcuate (ARC) nuclei of the rodent hypothalamus subserve several behavioral URs. Recently, in a major advance, dopaminergic signaling in striatal circuitry, likely at D2 receptors, has been implicated in behavioral and thermoregulatory URs of mice. We propose a neural network in which reciprocal communication among the SCN, the ARC and striatal dopaminergic circuitry modulates the period and waveform of behavioral and physiological URs.

Alongside the development of sexual characteristics and reproductive competence, adolescents undergo marked cognitive, social, and emotional development [1]. A fundamental question is whether these changes are triggered by activation of the hypothalamic-pituitary-gonadal (HPG) axis at puberty (puberty-dependent), or whether they occur independently of HPG activation (puberty-independent). Disentangling puberty-dependent from puberty-independent mechanisms is difficult because puberty and adolescence typically proceed concurrently. Here, we test a new approach that leverages natural adaptations of a seasonally breeding species to dissociate pubertal status from chronological age. Siberian hamsters (Phodopus sungorus) reared in a long, summer-like day length (LD) exhibit rapid pubertal development, whereas those reared in a short, winter-like day length (SD) delay puberty by several months to synchronize breeding with the following spring [2,3]. We tested whether the SD-induced delay in puberty delays the peri-adolescent decline in juvenile social play and the rise in aggression that characterizes adolescent social development in many species [4–6], and compared the results to those obtained after prepubertal gonadectomy. Neither SD-rearing nor prepubertal gonadectomy altered the age at which hamsters transitioned from play to aggression; SD-reared hamsters completed this transition prior to puberty. SD-rearing and prepubertal gonadectomy, however, increased levels of play in male and female juveniles, implicating a previously unknown role for prepubertal gonadal hormones in juvenile social behavior. Levels of aggression were also impacted (decreased) in SD-reared and gonadectomized males. These data demonstrate that puberty-independent mechanisms regulate the timing of adolescent social development, while prepubertal and adult gonadal hormones modulate levels of age-appropriate social behaviors.

The effects of temperature and food availability on social bonds and group formation are poorly understood. Because seasonal transitions in female social behavior facilitate the assembly of winter groups in meadow voles, we explored the role of same-sex female associations in winter sociality. To examine the effects of winter typical environmental conditions on same-sex female affiliative behavior, paired female meadow voles were housed in varying combinations of day length, temperature, and food availability for 7 weeks and then tested for social preference. In short days (SDs), lower ambient temperature increased huddling with unfamiliar females without interfering with existing social bonds, whereas lower temperature disrupted the retention of bonds in long days (LDs). Mild food restriction with no discernible effects on body mass enhanced affiliative behavior in SDs, but not LDs. A second experiment examined the effects of sex and day length on the propensity to aggregate with unfamiliar same-sex voles. Compared to LD females and SD males, SD females spent more time in group huddles with unfamiliar voles and displayed no social preference. These outcomes indicate that winter-like conditions enhance affiliative behavior between females and that pre-existing social bonds do not preclude integration into new winter social groups. The adaptive value of these behaviors is discussed.

Modern life involves chronic circadian disruption through artificial light and these disruptions are associated with numerous mental and physical health maladies. Because the developing nervous system is particularly vulnerable to perturbation, we hypothesized that early-life circadian disruption would negatively impact offspring development and adult function. Pregnant mice were subjected to chronic circadian disruption from the time of uterine implantation through weaning. To dissociate in utero from postnatal effects, a subset of litters was cross-fostered at birth from disrupted dams to control dams and vice versa. Postnatal circadian disruption was associated with reduced adult body mass, social avoidance, and hyperactivity. In utero disruption resulted in more pronounced social avoidance and hyperactivity, phenotypes not abrogated by cross-fostering to control mothers. To examine whether circadian disruption affects development by acting as an early life stressor, we examined birthweight, litter size, maternal cannibalism, and epigenetic modifications. None of these variables differed between control and disrupted dams, or resembled patterns seen following early-life stress. Our findings indicate that developmental chronic circadian disruption permanently affects somatic and behavioral development in a stage-of-life-dependent manner, independent of early life stress mechanisms, underscoring the importance of temporal structure during development, both in utero and early postnatal life.

Background Females are markedly underinvestigated in the biological and behavioral sciences due to the presumption that cyclic hormonal changes across the ovulatory cycle introduce excess variability to measures of interest in comparison to males. However, recent analyses indicate that male and female mice and rats exhibit comparable variability across numerous physiological and behavioral measures, even when the stage of the estrous cycle is not considered. Hormonal changes across the ovulatory cycle likely contribute cyclic, intra-individual variability in females, but the source(s) of male variability has, to our knowledge, not been investigated. It is unclear whether male variability, like that of females, is temporally structured and, therefore, quantifiable and predictable. Finally, whether males and females exhibit variability on similar time scales has not been explored. Methods These questions were addressed by collecting chronic, high temporal resolution locomotor activity (LA) and core body temperature (CBT) data from male and female BALB/c mice. Results Contrary to expectation, males are more variable than females over the course of the day (diel variability) and exhibit higher intra-individual daily range than females in both LA and CBT. Between mice of a given sex, variability is comparable for LA but the inter-individual daily range in CBT is greater for males. To identify potential rhythmic processes contributing to these sex differences, we employed wavelet transformations across a range of periodicities (1–39 h). Conclusions Although variability in circadian power is comparable between the sexes for both LA and CBT, infradian variability is greater in females and ultradian variability is greater in males. Thus, exclusion of female mice from studies because of estrous cycle variability may increase variance in investigations where only male measures are collected over a span of several hours and limit generalization of findings from males to females.

The dorsomedial nucleus (DMN) of the hypothalamus has been implicated in seasonal control of reproduction. Syrian hamsters with DMN lesions, unlike control hamsters, do not undergo testicular regression after transfer from a long day length (14 h of light per day; LD) to a short day length (8 h of light per day; SD). SDs also markedly reduce hamster locomotor activity (LMA). To assess whether the DMN is a component of the neural circuitry that mediates seasonal variation in LMA, neurologically intact males (controls) and hamsters that had sustained lesions of the DMN (DMNx) were housed in an LD or SD photoperiod for 26 weeks. DMNx that prevented testicular regression counteracted decreases in LMA during 8 to10 weeks of SD treatment; steroid-independent effects of SDs did not override high levels of LMA in DMNx males. As in previous studies, testosterone (T) restoration increased LMA in LD but not SD castrated control males. In the present study, T also failed to increase LMA in SD-DMNx hamsters. The DMN is not necessary to maintain decreased responsiveness of locomotor activity systems to T in SDs, which presumably is mediated by other central nervous system androgen target tissues. Finally, DMNx did not interfere with the spontaneous increase in LMA exhibited by photorefractory hamsters after 26 weeks of SD treatment. We propose that DMN is an essential part of the substrate that mediates seasonal decreases in LMA as day length decreases but is not required to sustain decreased SD responsiveness to T or for development of refractoriness to SDs.

The impact of pregnancy and lactation on ultradian rhythms (URs) and circadian rhythms (CRs) of locomotor activity was assessed in circadian rhythmic and arrhythmic Siberian hamsters maintained in a long-day photoperiod (16 h light/day). Progressive decrements in CR robustness and amplitude over the course of gestation were accompanied by enhanced URs. Dark-phase UR period and amplitude increased during early gestation and complexity and robustness increased during late gestation. The persistence of pregnancy-associated enhancements of URs in circadian arrhythmic (ARR) hamsters suggests that reproductive modulation of the UR waveform is not dependent on coherent circadian organization. The increased incidence of dark-phase URs appeared more rapidly in ARR dams than entrained (ENTR) dams. Throughout gestation, the percentage of dams with dark-phase URs was significantly greater in the ARR group. Gestational increases in UR complexity and robustness emerged earlier and were greater in ARR than ENTR dams. The attenuation of CRs during lactation is correlated with increased expression of URs. Relaxation of circadian control of the dam's behavior may increase fitness by permitting more efficient interactions with circadian arrhythmic pups.

Sex differences in ultradian activity rhythms (URs) and circadian rhythms (CRs) were assessed in Siberian hamsters kept in long day (LD) or short day (SD) photoperiods for 40 weeks. For both sexes URs of locomotor activity were more prevalent, greater in amplitude and more robust in SDs. The UR period was longer in females than males in both day lengths. The reproductive system underwent regression and body mass declined during the initial 10 weeks of SD treatment, and in both sexes these traits spontaneously reverted to the LD phenotype at or before 40 weeks in SD, reflecting the development of neuroendocrine refractoriness to SD patterns of melatonin secretion. Hamsters of both sexes, however, continued to display SD-like URs at the 40 weeks time point. CRs were less prevalent and the waveform less robust and lower in amplitude in SDs than LDs; the SD circadian waveform also did not revert to the long-day phenotype after 40 weeks of SD treatment. Short day lengths enhanced ultradian and diminished circadian rhythms in both sexes. Day length controls several UR characteristics via gonadal steroid and melatonin-independent mechanisms. Sex differences in ultradian timing may contribute to sex diphenisms in rhythms of sleep, food intake and exercise.

Many controllable factors negatively impact fetal development, underscoring the importance of early pregnancy detection and identification of events that reliably predict potential complications. Clinically, core body temperature (CBT) is used to aid family planning and pregnancy detection. However, such temperature data typically are gathered in single, daily measurements. In animal studies, interventions or cell/tissue harvesting at defined stages of fetal development are arduous, requiring timed mating by trained observers. The value of continuous temperature measurements remains largely unexplored, but the advent of small, inexpensive, and increasingly ubiquitous, accurate sensor devices makes continuous measures feasible. Here, using a mouse model, we show that continuous, 1-min resolution CBT measurements reliably allow for the earliest and most accurate detection of pregnancy (100%, within 14 h of initial pairing), without requiring interaction with the animal for data collection. This method also reveals a subset of females that exhibit a pregnancy-like response following pairing that persists for a variable number of days. Application of wavelet analysis that permits frequency analysis while preserving temporal resolution, uncovers significant differences in ultradian frequencies of CBT; these rhythms are significantly increased in the 12 h after the day of pairing for pregnancies carried to term compared to apparent pregnancies that failed. High temporal resolution CBT and wavelet analysis permit strikingly early detection and separation of successful pregnancies and pregnancy-like events.

Dentin is deposited on a circadian basis, and daily layers manifest as bands on the medial surfaces of rodent incisors. Hibernation alters dentin deposition, and a distinct hibernation mark has been described on incisor surfaces of several rodent species; the factors that influence the morphology of this mark are poorly understood. We tested the effects of day length, torpor expression, and ambient temperature on incisor surface morphology in Turkish hamsters housed in one of four conditions: long days (LDs) at 22 °C, short days (SDs) at 22 °C, SDs at 5 °C, and SDs at 13 °C. Body temperature was monitored continuously with implanted radio transmitters, and teeth examined postmortem. Teeth of SD hamsters had narrower, less distinct circadian increments than those of LD hamsters, but the width of ultradian increments was similar in both photoperiods. Hibernation at both 5 and 13 °C was associated in most specimens with very narrow, sharply defined dentin increments and increased tooth heterogeneity. Hamsters in SDs at 5 °C that did not hibernate lacked characteristic hibernation increments. At 5 °C, but not 13 °C, the number and cumulative width of hibernation increments were related to number and cumulative duration of periodic arousals. Our results suggest that incremental deposition of dentin in rodent incisors may be a useful trait for characterizing hibernation behavior in both evolutionary and historical contexts.

Turkish hamsters (Mesocricetus brandti) are a model organism for studies of hibernation, yet a detailed account of their torpor characteristics has not been undertaken. This study employed continuous telemetric monitoring of body temperature (T(b)) in hibernating male and female Turkish hamsters at ambient temperatures (T(a)s) of 5 and 13 °C to precisely characterize torpor bout depth, duration, and frequency, as well as rates of entry into and arousal from torpor. Hamsters generated brief intervals of short (12 h), shallow test bouts (T(b)20 °C), followed by deep torpor bouts lasting 4-6 days at T(a) = 5 °C and 2-3 days at T(a) = 13 °C. Females at T(a) = 5 °C had longer bouts than males, but maintained higher torpor T(b); there were no sex differences at T(a) = 13 °C. Neither body mass loss nor food intake differed between the two T(a)s. Hamsters entered torpor primarily during the scotophase (subjective night), but timing of arousals was highly variable. Hamsters at both T (a)s generated short, shallow torpor bouts between deep bouts, suggesting that this species may be capable of both hibernation and daily torpor.

Yohimbine and apomorphine selectively act on noradrenergic and dopaminergic neural substrates to augment male sexual behavior (MSB) in several rodent species. The present study assessed whether these drugs can overcome the suppressive effects of short winter-like day lengths on MSB. Yohimbine treatments that markedly increase copulatory behavior of male hamsters in long days were completely ineffective in facilitating MSB when injected after gonadal regression induced by 16 wks of short day lengths and after complete gonadal recrudescence after 32 wks of short days; apomorphine was similarly ineffective. The brain circuit that mediates MSB either may be less responsive to yohimbine and apomorphine in short than long days, or these drugs may not produce equivalent neurotransmitter changes in the two day lengths. After 32 wks of short-day treatment, all males had undergone testicular recrudescence and successfully ejaculated on initial tests with sexually receptive females after a hiatus of at least 4 mo during which they were denied mating opportunities. This suggests that overwintering males in the field are in a state of reproductive readiness at the outset of spring conditions favorable for survival of offspring.

Long days stimulate and short days (SDs) inhibit the reproductive axis of photoperiodic rodents. In long-day Turkish hamsters, unlike most other rodents, elimination of pineal melatonin secretion by constant light or pinealectomy initiates a cycle of gonadal involution and recrudescence outwardly similar to that induced by short days. The present study assessed whether short days and constant light induce the seasonal reproductive cycle via common or different interval timing mechanisms. Male hamsters that had undergone gonadal involution in SDs for 8 or 14 weeks were treated with LL for 14 and 8 weeks, respectively. If SDs and LL act via independent mechanisms, then gonadal quiescence of SD-regressed males, which normally lasts 10 weeks, might be extended by LL treatment; alternatively, if SDs and LL act on the same timer, or the timer cannot be retriggered, then LL will not extend the duration of reproductive quiescence. Neither of these outcomes materialized. Instead, male hamsters exposed to LL while reproductively quiescent exhibited accelerated gonadal recrudescence. Extended LL treatment did not restore responsiveness to SDs in photorefractory hamsters. In Turkish hamsters, photoperiodic history determines whether constant light inhibits or stimulates the hypothalamic-pituitary-testicular axis.

Both growth and thermoregulation are energetically costly, and many studies implicate an energetic tradeoff between them. Moreover, fur is known to ameliorate thermoregulatory costs in adult mammals, but its role in maintaining energy balance during growth is unclear. This study tested for an energetic tradeoff between growth and thermoregulation in juvenile Siberian hamsters (Phodopus sungorus) and the effect of an insulative pelage on intrinsic growth rate. Hamsters weaned at 18 days of age and left fully furred or deprived of all dorsal fur by shaving at 20 days of age, were housed at 10 degrees C or 23 degrees C. Body mass, length, and food consumption were measured until hamsters were 35 days old. Thermal challenge, whether by low ambient temperature or shaving, resulted in increased food intake and decreased efficiency at converting food into body mass. Body mass and length were not affected by the thermal challenges. These results suggest that there is no mandatory tradeoff between growth and thermoregulation in this species, particularly when food is in abundant supply. Although fur was not necessary for normal growth to proceed, it ameliorated energetic costs associated with thermoregulation, and may play a role in maintaining energy balance under conditions of limited food availability.

The neuropeptide oxytocin (OT) has been implicated in a range of mammalian reproductive and social behaviors including parent-offspring bonding and partner preference formation between socially monogamous mates. Its role in mediating non-reproductive social relationships in rodents, however, remains largely unexplored. We examined whether OT facilitates same-sex social preferences between female meadow voles-a species that forms social nesting groups in short, winter-like day lengths. In contrast to results from studies of opposite-sex attachment between prairie vole mates, we found that neither OT nor dopamine neurotransmission was required for baseline levels of social partner preference formation or expression. OT enhanced preference formation beyond baseline levels-an effect that was counteracted by treatment with an oxytocin receptor antagonist (OTA). Oxytocin receptor (OTR) density correlated with social behavior in brain regions not known to be associated with opposite-sex affiliation, including the lateral septum and central amygdala. In addition, voles housed in short day lengths (SD) exhibited higher levels of OTR binding in the central amygdala, and voles exposed to high concentrations of estradiol exhibited less binding in the nucleus accumbens (NAcc) and increased binding in the ventromedial nucleus of the hypothalamus. These results suggest that same-sex social behavior shares common elements with other mammalian social behaviors affected by OT, but that the specific neural pathways through which OT exerts its influence are likely distinct from those known for sexual attachments.

The seasonal reproductive cycle of photoperiodic rodents is conceptualized as a series of discrete melatonin-dependent neuroendocrine transitions. Least understood is the springtime restoration of responsiveness to winter-like melatonin signals (breaking of refractoriness) that enables animals to once again respond appropriately to winter photoperiods the following year. This has been posited to require many weeks of long days based on studies employing static photoperiods instead of the annual pattern of continually changing photoperiods under which these mechanisms evolved. Maintaining Siberian hamsters under simulated natural photoperiods, we demonstrate that winter refractoriness is broken within six weeks after the spring equinox. We then test whether a history of natural photoperiod exposure can eliminate the requirement for long-day melatonin signalling. Hamsters pinealectomized at the spring equinox and challenged 10 weeks later with winter melatonin infusions exhibited gonadal regression, indicating that refractoriness was broken. A photostimulatory effect on body weight is first observed in the last four weeks of winter. Thus, the seasonal transition to the summer photosensitive phenotype is triggered prior to the equinox without exposure to long days and is thereafter melatonin-independent. Distinctions between photoperiodic and circannual seasonal organization erode with the incorporation in the laboratory of ecologically relevant day length conditions.

The duration of nocturnal pineal melatonin secretion transduces effects of day length (DL) on the neuroendocrine axis of photoperiodic rodents. Long DLs support reproduction, and short DLs induce testicular regression, followed several months later by spontaneous recrudescence; gonadal regrowth is thought to reflect development of tissue refractoriness to melatonin. In most photoperiodic species, pinealectomy does not diminish reproductive competence in long DLs. Turkish hamsters ( Mesocricetus brandti) deviate from this norm: elimination of melatonin secretion in long-day males by pinealectomy or constant light treatment induces testicular regression and subsequently recrudescence; the time course of these gonadal transitions is similar to that observed in males transferred from long to short DLs. In the present study, long-day Turkish hamsters that underwent testicular regression and recrudescence in constant light subsequently were completely unresponsive to the antigonadal effects of short DLs. Other hamsters that manifested testicular regression and recrudescence in short DLs were unresponsive to the antigonadal effects of pinealectomy or constant light. Long-term suppression of melatonin secretion induces a physiological state in Turkish hamsters similar or identical to the neuroendocrine refractoriness produced by short-day melatonin signals (i.e., neural refractoriness to melatonin develops in the absence of circulating melatonin secretion). A melatonin-independent interval timer, which would remain operative in the absence of melatonin during hibernation, may determine the onset of testicular recrudescence in the spring. In this respect, Turkish hamsters differ from most other photoperiodic rodents.

Non-sexual social bonding between adult mammals remains poorly understood, despite its importance in many species. Female meadow voles are territorial and nest alone in long summer day lengths when circulating estradiol concentrations are high, but cohabit in groups in short winter photoperiods when estradiol secretion is low. The influence of day length and estradiol on same-sex huddling behavior was assessed in adult female pairs housed together in long day lengths (LDs) or short day lengths (SDs) from weaning. The behavior of intact, ovariectomized, and estradiol-treated ovariectomized females from each photoperiod was assessed during 3 hour partner preference tests. Intact SD voles, unlike intact LD voles, spent the majority of the test in proximity to their cage mates. Estradiol treatment of SD voles significantly reduced time spent huddling with the partner. Neither ovariectomy nor estradiol treatment significantly affected the amount of time LD females spent in contact with their partners. Low estradiol availability is therefore a necessary but not sufficient condition for maintenance of high levels of huddling. These results establish that ovarian hormones interact with photoperiod to affect same-sex social behavior.

Short day lengths increase the duration of nocturnal melatonin (Mel) secretion, which induces the winter phenotype in Siberian hamsters. After several months of continued exposure to short days, hamsters spontaneously revert to the spring-summer phenotype. This transition has been attributed to the development of refractoriness of Mel-binding tissues, including the suprachiasmatic nucleus (SCN), to long-duration Mel signals. The SCN of Siberian hamsters is required for the seasonal response to winter-like Mel signals, and becomes refractory to previously effective long-duration Mel signals restricted to this area. Acute Mel treatment phase shifts circadian locomotor rhythms of photosensitive Siberian hamsters, presumably by affecting circadian oscillators in the SCN. We tested whether seasonal refractoriness of the SCN to long-duration Mel signals also renders the circadian system of Siberian hamsters unresponsive to Mel. Males manifesting free-running circadian rhythms in constant dim red light were injected with Mel or vehicle for 5 days on a 23.5-h T-cycle beginning at circadian time 10. Mel injections caused significantly larger phase advances in activity onset than did the saline vehicle, but the magnitude of phase shifts to Mel did not differ between photorefractory and photosensitive hamsters. Similarly, when entrained to a 16-h light/8-h dark photocycle, photorefractory and photosensitive hamsters did not differ in their response to Mel injected 4 h before the onset of the dark phase. Activity onset in Mel-injected hamsters was masked by light but was revealed to be significantly earlier than in vehicle-injected hamsters upon transfer to constant dim red light. The acute effects of melatonin on circadian behavioral rhythms are preserved in photorefractory hamsters.

Temperate and boreal mammals undergo seasonal changes in pelage that facilitate thermoregulation in winter and summer. We investigated photoperiodic influences on pelage characteristics of male Siberian and Syrian hamsters. Fur density (mg fur/cm2 skin) was measured by weighing the shavings of fur patches removed from the dorsal and ventral surfaces of hamsters maintained in long days (LDs) or transferred to short days (SDs). Patches were reshaved 3 wk later to assess fur regrowth (mg regrown fur/cm2 skin). Fur density was greater in SD than in LD Siberian hamsters after 11 wk of differential phototreatment. The onset of increased fur density in SDs was accompanied by a transient increase in fur regrowth (11–14 wk on the dorsal surface and 7–10 and 11–14 wk on the ventral surface), suggestive of a seasonal molting process. Fur density, body mass, and pelage color of Siberian hamsters returned to values characteristic of LD males after a similar duration of prolonged (>27 wk) SD treatment and appear to be regulated by a similar or common interval-timing mechanism. In Syrian hamsters, dorsal fur density, fur regrowth, and hair lengths were greater in SD than in LD males. Castration increased and testosterone (T) treatment decreased dorsal and ventral fur regrowth in LD and SD hamsters, but the effects of T manipulations on fur density were limited to a decrease in dorsal fur density after T treatment. Decreased circulating T in SDs likely contributes to the seasonal molt of male hamsters by increasing the rate of fur growth during the transition to the winter pelage.

Animals have evolved many season-specific behavioural and physiological adaptations that allow them to both cope with and exploit the cyclic annual environment. Two classes of endogenous annual timekeeping mechanisms enable animals to track, anticipate and prepare for the seasons: a timer that measures an interval of several months and a clock that oscillates with a period of approximately a year. Here, we discuss the basic properties and biological substrates of these timekeeping mechanisms, as well as their reliance on, and encoding of environmental cues to accurately time seasonal events. While the separate classification of interval timers and circannual clocks has elucidated important differences in their underlying properties, comparative physiological investigations, especially those regarding seasonal prolactin secretions, hint at the possibility of common substrates.