Chronic phase advances reduces recognition memory and increases vascular cognitive dementia-like impairments in aged mice.

Disrupted or atypical light-dark cycles disrupts synchronization of endogenous circadian clocks to the external environment; extensive circadian rhythm desynchrony promotes adverse health outcomes. Previous studies suggest that disrupted circadian rhythms promote neuroinflammation and neuronal damage post-ischemia in otherwise healthy mice, however, few studies to date have evaluated these health risks with aging. Because most strokes occur in aged individuals, we sought to identify whether, in addition to being a risk factor for poor ischemic outcome, circadian rhythm disruption can increase risk for vascular cognitive impairment and dementia (VCID). We hypothesized that repeated 6 h phase advances (chronic jet lag; CJL) for 8 weeks alters cerebrovascular architecture leading to increased cognitive impairments in aged mice. Female CJL mice displayed impaired spatial processing during a spontaneous alternation task and reduced acquisition during auditory-cued associative learning. Male CJL mice displayed impaired retention of the auditory-cued associative learning task 24 h following acquisition. CJL increased vascular tortuosity in the isocortex, associated with increased risk for vascular disease. These results demonstrate that CJL increased sex-specific cognitive impairments coinciding with structural changes to vasculature in the brain. We highlight that CJL may accelerate aged-related functional decline and could be a crucial target against disease progression.

Dim light at night shifts microglia to a pro-inflammatory state after cerebral ischemia, altering stroke outcome in mice.

Circadian rhythms are endogenous biological cycles that regulate physiology and behavior and are set to precisely 24-h by light exposure. Light at night (LAN) dysregulates physiology and function including immune response; a critical component that contributes to stroke pathophysiological progression of neuronal injury and may impair recovery from injury. The goal of this study is to explore the effects of dim LAN (dLAN) in a murine model of ischemic stroke to assess how nighttime lighting from hospital settings can affect stroke outcome. Further, this study sought to identify mechanisms underlying pathophysiological changes to immune response after circadian disruption. Male and female adult Swiss Webster (CFW) mice were subjected to transient or permanent focal cerebral ischemia, then were subsequently placed into either dark night conditions (LD) or one night of dLAN (5 lx). 24 h post-stroke, sensorimotor impairments and infarct sizes were quantified. A single night of dLAN following MCAO increased infarct size and sensorimotor deficits across both sexes and reduced survival in males after 24 h. Flow cytometry was performed to assess microglial phenotypes after MCAO, and revealed that dLAN altered the percentage of microglia that express pro-inflammatory markers (MHC II+ and IL-6) and microglia that express CD206 and IL-10 that likely contributed to poor ischemic outcomes. Following these results, microglia were reduced in the brain using Plexxikon 5622 (PLX 5622) a CSFR1 inhibitor, then the mice received an MCAO and were exposed to LD or dLAN conditions for 24 h. Microglial depletion by PLX5622 resulted in infarct sizes that were comparable between lighting conditions. This study provides supporting evidence that environmental lighting exacerbates ischemic injury and post-stroke mortality by a biological mechanism that exposure to dLAN causes a fundamental shift of activated microglial phenotypes from beneficial to detrimental at an early time point after stroke, resulting in irreversible neuronal death.

Time of day bias for biological sampling in studies of mammary cancer.

Despite its demonstrated biological significance, time of day is a broadly overlooked biological variable in preclinical and clinical studies. How time of day affects the influence of peripheral tumors on central (brain) function remains unspecified. Thus, we tested the hypothesis that peripheral mammary cancer tumors alter the transcriptome of immune responses in the brain and that these responses vary based on time of day; we predicted that time of day sampling bias would alter the interpretation of the results. Brain tissues collected at mid dark and mid light from mammary tumor-bearing and vehicle injected mice were analyzed using the Nanostring nCounter immune panel. Peripheral mammary tumors significantly affected expression within the brain of over 100 unique genes of the 770 represented in the panel, and fewer than 25% of these genes were affected similarly across the day. Indeed, between 65 and 75% of GO biological processes represented by the differentially expressed genes were dependent upon time of day of sampling. The implications of time-of-day sampling bias in interpretation of research studies cannot be understated. We encourage considering time of day as a significant biological variable in studies and to appropriately control for it and clearly report time of day in findings.

Chronotherapeutics for Solid Tumors.

Circadian rhythms are internal manifestations of the 24-h solar day that allow for synchronization of biological and behavioral processes to the external solar day. This precise regulation of physiology and behavior improves adaptive function and survival. Chronotherapy takes advantage of circadian rhythms in physiological processes to optimize the timing of drug administration to achieve maximal therapeutic efficacy and minimize negative side effects. Chronotherapy for cancer treatment was first demonstrated to be beneficial more than five decades ago and has favorable effects across diverse cancer types. However, implementation of chronotherapy in clinic remains limited. The present review examines the evidence for chronotherapeutic treatment for solid tumors. Specifically, studies examining chrono-chemotherapy, chrono-radiotherapy, and alternative chronotherapeutics (e.g., hormone therapy, TKIs, antiangiogenic therapy, immunotherapy) are discussed. In addition, we propose areas of needed research and identify challenges in the field that remain to be addressed.

Acute exposure to artificial light at night alters hippocampal vascular structure in mice.

The structure and function of the cardiovascular system are modulated across the day by circadian rhythms, making this system susceptible to circadian rhythm disruption. Recent evidence demonstrated that short-term exposure to a pervasive circadian rhythm disruptor, artificial light at night (ALAN), increased inflammation and altered angiogenic transcripts in the hippocampi of mice. Here, we examined the effects of four nights of ALAN exposure on mouse hippocampal vascular networks. To do this, we analyzed 2D and 3D images of hippocampal vasculature and hippocampal transcriptomic profiles of mice exposed to ALAN. ALAN reduced vascular density in the CA1 and CA2/3 of female mice and the dentate gyrus of male mice. Network structure and connectivity were also impaired in the CA2/3 of female mice. These results demonstrate the rapid and potent effects of ALAN on cerebrovascular networks, highlighting the importance of ALAN mitigation in the context of health and cerebrovascular disease.

Functions of Steroid Hormones in the Male Reproductive Tract as Revealed by Mouse Models.

Steroid hormones are capable of diffusing through cell membranes to bind with intracellular receptors to regulate numerous physiological processes. Three classes of steroid hormones, namely androgens, estrogens and glucocorticoids, contribute to the development of the reproductive system and the maintenance of fertility. During the past 30 years, mouse models have been produced in which the expression of genes encoding steroid hormone receptors has been enhanced, partially compromised or eliminated. These mouse models have revealed many of the physiological processes regulated by androgens, estrogens and to a more limited extent glucocorticoids in the testis and male accessory organs. In this review, advances provided by mouse models that have facilitated a better understanding of the molecular regulation of testis and reproductive tract processes by steroid hormones are discussed.

Ritual Closure: Rites De Passage and Apotropaic Magic in an Animate World.

Magic and witchcraft, classic topics in the anthropology of religion, involve everyday things such as ashes, ceramics, minerals, shell, and projectile points. In many cultures, people attribute agency to such artifacts, as well as architecture, begging the question what is the archaeological record of such animate beings? To understand past human lifeways more fully, we need to explore the formation processes associated with the interaction between people and other non-human actors. For example, what might we learn from a burned pueblo whose rooms contain ash, projectile points, crystals, and other items? In this paper we argue that deposits in ritually closed pueblos of the North American Southwest, like many other Neolithic villages, likely contain purposely deposited objects in an effort to neutralize the anima left in these places and to prophylactically protect their former inhabitants from future witchcraft. We present Cottonwood Spring Pueblo, New Mexico, as a case study.

Chronic exposure to dim light at night disrupts cell-mediated immune response and decreases longevity in aged female mice.

Circadian rhythms are endogenous biological cycles that regulate physiology and behavior for optimal adaptive function and survival; they are synchronized to precisely 24 hours by daily light exposure. Disruption of the daily light-dark (LD) cycle by exposure to artificial light at night (ALAN) dysregulates core clock genes and biological function. Exposure to ALAN has been associated with increased health risks in humans, and elderly individuals are at elevated risk for poor outcome from disease and often experience elevated exposure to ALAN due to increased care requirements. The role of disrupted circadian rhythms in healthy, aged animals remains unspecified; thus, we hypothesized that disrupted circadian rhythms via chronic exposure to dim ALAN (dLAN) impair immune response and survival in aged mice. Twenty-month-old C57BL/6 male and female mice were exposed to 24 weeks of LD conditions or dLAN (5 lux); then, cell-mediated immune response was assessed using a delayed-type hypersensitivity test. Aged female mice exposed to dLAN displayed dysregulated hypersensitivity and inflammation as a measure of cell-mediated immune response and decreased lifespan compared to females housed in dark nights. Nighttime lighting did not affect cell-mediated immune response or lifespan in males but dysregulated body mass and increased adrenal mass after immune challenge after chronic exposure to dLAN. Together, these data indicate that chronic exposure to dLAN affects lifespan in aged females and suggest that females are more susceptible to the detrimental consequences of disrupted circadian rhythms.

Identification and characterization of novel abdominal and pelvic brown adipose depots in mice.

Brown adipose tissue (BAT) generates heat through non-shivering thermogenesis, and increasing BAT amounts or activity could facilitate obesity treatment and provide metabolic benefits. In mice, BAT has been reported in perirenal, thoracic and cranial sites. Here, we describe new pelvic and lower abdominal BAT depots located around the urethra, internal reproductive and urinary tract organs and major lower pelvic blood vessels, as well as between adjacent muscles where the upper hind leg meets the abdominal cavity. Immunohistochemical, western blot and PCR analyses revealed that these tissues expressed BAT markers such as uncoupling protein 1 (UCP1) and CIDEA, but not white adipose markers, and ß3-adrenergic stimulation increased UCP1 amounts, a classic characteristic of BAT tissue. The newly identified BAT stores contained extensive sympathetic innervation with high mitochondrial density and multilocular lipid droplets similar to interscapular BAT. BAT repositories were present and functional neonatally, and showed developmental changes between the neonatal and adult periods. In summary, several new depots showing classical BAT characteristics are reported and characterized in the lower abdominal/pelvic region of mice. These BAT stores are likely significant metabolic regulators in the mouse and some data suggests that similar BAT depots may also exist in humans.

Time of day as a critical variable in biology.

BACKGROUND: Circadian rhythms are important for all aspects of biology; virtually every aspect of biological function varies according to time of day. Although this is well known, variation across the day is also often ignored in the design and reporting of research. For this review, we analyzed the top 50 cited papers across 10 major domains of the biological sciences in the calendar year 2015. We repeated this analysis for the year 2019, hypothesizing that the awarding of a Nobel Prize in 2017 for achievements in the field of circadian biology would highlight the importance of circadian rhythms for scientists across many disciplines, and improve time-of-day reporting. RESULTS: Our analyses of these 1000 empirical papers, however, revealed that most failed to include sufficient temporal details when describing experimental methods and that few systematic differences in time-of-day reporting existed between 2015 and 2019. Overall, only 6.1% of reports included time-of-day information about experimental measures and manipulations sufficient to permit replication. CONCLUSIONS: Circadian rhythms are a defining feature of biological systems, and knowing when in the circadian day these systems are evaluated is fundamentally important information. Failing to account for time of day hampers reproducibility across laboratories, complicates interpretation of results, and reduces the value of data based predominantly on nocturnal animals when extrapolating to diurnal humans.

The Ventral Tegmental Area and Nucleus Accumbens as Circadian Oscillators: Implications for Drug Abuse and Substance Use Disorders.

Circadian rhythms convergently evolved to allow for optimal synchronization of individuals' physiological and behavioral processes with the Earth's 24-h periodic cycling of environmental light and temperature. Whereas the suprachiasmatic nucleus (SCN) is considered the primary pacemaker of the mammalian circadian system, many extra-SCN oscillatory brain regions have been identified to not only exhibit sustainable rhythms in circadian molecular clock function, but also rhythms in overall region activity/function and mediated behaviors. In this review, we present the most recent evidence for the ventral tegmental area (VTA) and nucleus accumbens (NAc) to serve as extra-SCN oscillators and highlight studies that illustrate the functional significance of the VTA's and NAc's inherent circadian properties as they relate to reward-processing, drug abuse, and vulnerability to develop substance use disorders (SUDs).

Regulation of mammalian spermatogenesis by miRNAs.

Male fertility requires the continual production of sperm by the process of spermatogenesis. This process requires the correct timing of regulatory signals to germ cells during each phase of their development. MicroRNAs (miRNAs) in germ cells and supporting Sertoli cells respond to regulatory signals and cause down- or upregulation of mRNAs and proteins required to produce proteins that act in various pathways to support spermatogenesis. The targets and functional consequences of altered miRNA expression in undifferentiated and differentiating spermatogonia, spermatocytes, spermatids and Sertoli cells are discussed. Mechanisms are reviewed by which miRNAs contribute to decisions that promote spermatogonia stem cell self-renewal versus differentiation, entry into and progression through meiosis, differentiation of spermatids, as well as the regulation of Sertoli cell proliferation and differentiation. Also discussed are miRNA actions providing the very first signals for the differentiation of spermatogonia stem cells in a non-human primate model of puberty initiation.

Light at night disrupts biological clocks, calendars, and immune function.

Light at night is a pervasive problem in our society; over 80% of the world's population experiences significant light pollution. Exacerbating this issue is the reality that artificially lit outdoor areas are growing by 2.2% per year and continuously lit areas brighten by 2.2% each year due to the rapid growths in population and urbanization. Furthermore, the increase in the prevalence of night shift work and smart device usage contributes to the inescapable nature of artificial light at night (ALAN). Although previously assumed to be innocuous, ALAN has deleterious effects on the circadian system and circadian-regulated physiology, particularly immune function. Due to the relevance of ALAN to the general population, it is important to understand its roles in disrupting immune function. This review presents a synopsis of the effects of ALAN on circadian clocks and immune function. We delineate the role of ALAN in altering clock gene expression and suppressing melatonin. We review the effects of light at night on inflammation and the innate and adaptive immune systems in various species to demonstrate the wide range of ALAN consequences. Finally, we propose future directions to provide further clarity and expansion of the field.

Nonclassical androgen and estrogen signaling is essential for normal spermatogenesis.

Signaling by androgens through androgen receptor (AR) is essential to complete spermatogenesis in the testis. Similarly, loss of the main estrogen receptor, estrogen receptor 1 (ESR1; also known as ERα), results in male infertility, due in part to indirect deleterious effects on the seminiferous epithelium and spermatogenesis. Effects of steroid hormones are induced primarily through genomic changes induced by hormone-mediated activation of their intracellular receptors and subsequent effects on nuclear gene transcription. However, androgens and estrogens also signal through rapid nonclassical pathways involving actions initiated at the cell membrane. Here we review the data that nonclassical androgen and estrogen signaling pathways support processes essential for male fertility in the testis and reproductive tract. The recent development of transgenic mice lacking nonclassical AR or ESR1 signaling but retaining genomic nuclear signaling has provided a powerful tool to elucidate the function of nonclassical signaling in the overall response to androgens and estrogens. Results from these mice have emphasized that nonclassical signaling is essential for full responses to these hormones, and absence of either nonclassical or classical AR or ESR1 pathways produces abnormalities in spermatogenesis and the male reproductive tract. Although additional work is required to fully understand how classical and nonclassical receptor signaling synergize to produce full steroid hormone responses, here we summarize the known physiological functions of the classical and nonclassical androgen and estrogen signaling pathways in the testis and reproductive tract.

Time-restricted feeding alters the efficiency of mammary tumor growth.

Disruption of circadian rhythms has detrimental host consequences. Indeed, both clinical and foundational science demonstrate a clear relationship between disruption of circadian rhythms and cancer initiation and progression. Because timing of food intake can act as a zeitgeber (i.e., entrainment signal) for the circadian clock, and most individuals in the developed world have access to food at all times of the day in a "24/7" society, we sought to determine the effects of timing of food intake on mammary tumor growth. We hypothesized that restricting access to food to during the inactive phase would accelerate tumor growth. Adult female Balb/C mice received a unilateral orthotopic injection of murine mammary carcinoma 4T1 cells into the ninth inguinal mammary gland. Beginning on the day of tumor injection and continuing until the end of the experiment, mice were food restricted to their active phase (ZT12 (lights off)- ZT0 (lights on), inactive phase (ZT0 - ZT12), or had ad libitum access to food. Mice that were food restricted to their inactive phase displayed a significant increase in body mass on days 7 and 14 of tumor growth relative to active phase or ad libitum fed mice. Additionally, mice fed during their inactive phase demonstrated a 20% reduction in food consumption relative to mice fed during their active phase and a 17% reduction in food consumption relative to ab libitum fed mice. Tumor volume was not significantly different between groups. However, food restricting mice to their inactive phase increased mammary tumor growth efficiency (i.e., mg of tumor mass per gram of food intake) relative to mice fed during the active phase and approached significance (p = .06) relative to ad libitum fed mice. To determine a potential explanation for the increased tumor growth efficiency, we examined rhythms of activity and body temperature. Mice fed during the inactive phase displayed significantly disrupted daily activity and body temperature rhythms relative to both other feeding regimens. Together, these data demonstrate that improperly timed food intake can have detrimental consequences on mammary tumor growth likely via disrupted circadian rhythms.

Circadian Influences on Chemotherapy Efficacy in a Mouse Model of Brain Metastases of Breast Cancer.

Chemotherapy is more effective in the treatment of peripheral tumors than brain metastases, likely reflecting the reduced ability of chemotherapy to cross the blood-brain barrier (BBB) and blood-tumor barrier at efficacious concentrations. Recent studies demonstrate circadian regulation of the BBB. Thus, we predicted that optimally timed chemotherapy would increase anti-tumor efficacy in a model of brain metastases of breast cancer (BMBC). First, we characterized novel daily alterations in BBB permeability to a commonly used chemotherapeutic, 14C-paclitaxel, within BMBC following injections given at four time points across the day. Peak and trough 14C-paclitaxel concentrations within BMBC occurred during the mid-dark phase and at the beginning of the light phase, respectively. Notably, chemotherapy injections during the dark phase increased cell death within BMBC and delayed onset of neurological symptoms relative to injections during the light phase. These data provide strong evidence for the beneficial effects of chrono-chemotherapy for the treatment of BMBC.

Artificial Light at Night Reduces Anxiety-like Behavior in Female Mice with Exacerbated Mammary Tumor Growth.

Artificial light at night (ALAN) is a pervasive phenomenon. Although initially assumed to be innocuous, recent research has demonstrated its deleterious effects on physiology and behavior. Exposure to ALAN is associated with disruptions to sleep/wake cycles, development of mood disorders, metabolic disorders, and cancer. However, the influence of ALAN on affective behavior in tumor-bearing mice has not been investigated. We hypothesize that exposure to ALAN accelerates mammary tumor growth and predict that ALAN exacerbates negative affective behaviors in tumor-bearing mice. Adult (>8 weeks) female C3H mice received a unilateral orthotropic injection of FM3A mouse mammary carcinoma cells (1.0 × 105 in 100 µL) into the fourth inguinal mammary gland. Nineteen days after tumor inoculation, mice were tested for sucrose preference (anhedonia-like behavior). The following day, mice were subjected to an open field test (anxiety-like behavior), followed by forced swim testing (depressive-like behavior). Regardless of tumor status, mice housed in ALAN increased body mass through the first ten days. Tumor-bearing ALAN-housed mice demonstrated reduced latency to tumor onset (day 5) and increased terminal tumor volume (day 21). Exposure to ALAN reduced sucrose preference independent of tumor status. Additionally, tumor-bearing mice housed in dark nights demonstrated significantly increased anxiety-like behavior that was normalized via housing in ALAN. Together, these data reaffirm the negative effects of ALAN on tumorigenesis and demonstrate the potential anxiolytic effect of ALAN in the presence of mammary tumors.

Androgen Actions in the Testis and the Regulation of Spermatogenesis.

Testosterone is essential for spermatogenesis and male fertility. In this review, topics related to testosterone control of spermatogenesis are covered including testosterone production and levels in the testis, classical and nonclassical testosterone signaling pathways, cell- and temporal-specific expression of the androgen receptor in the testis and autocrine and paracrine signaling of testis cells in the testis. Also discussed are the contributions of testosterone to testis descent, the blood-testis barrier, control of gonocyte numbers and spermatogonia expansion, completion of meiosis and attachment and release of elongaed spermatids. Testosterone-regulated genes identified in various mouse models of idsrupted Androgen receptor expression are discussed. Finally, examples of synergism and antagonism between androgen and follicle-stimulating hormone signaling pathways are summarized.

Male fertility in mice requires classical and nonclassical androgen signaling.

Molecular mechanisms by which androgens signal through the androgen receptor (AR) to maintain male fertility are poorly understood. Transgenic mice were produced expressing mutant ARs that can only (1) alter gene transcription through the classical response pathway (AR-C) or (2) activate kinase signaling cascades via the nonclassical pathway (AR-NC). AR-C is sufficient to produce sperm and fertility. Haploid germ cell production, the blood-testis barrier, and spermatid migration are supported by AR-NC. Gene expression essential for chromosome synapsis during meiosis requires AR-C. We identify targets of androgen signaling required for male fertility and provide a mechanistic explanation for meiotic germ cell arrest in the absence of androgen signaling. Prostate differentiation occurs with AR-C alone, but full development requires synergistic nonclassical signaling. Both AR signaling pathways are necessary for normal male reproductive tract development and function, validating our mouse models for studies of AR functions in other target tissues.

Circadian rhythms and pain.

The goal of this review is to provide a perspective on the nature and importance of the relationship between the circadian and pain systems. We provide: 1) An overview of the circadian and pain systems, 2) a review of direct and correlative evidence that demonstrates diurnal and circadian rhythms within the pain system; 3) a perspective highlighting the need to consider the role of a proposed feedback loop of circadian rhythm disruption and maladaptive pain; 4) a perspective on the nature of the relationship between circadian rhythms and pain. In summary, we propose that there is no single locus responsible for producing the circadian rhythms of the pain system. Instead, circadian rhythms of pain are a complex result of the distributed rhythms present throughout the pain system, especially those of the descending pain modulatory system, and the rhythms of the systems with which it interacts, including the opioid, endocrine, and immune systems.