Theresa M. Lee - US grants

The overall goal of this project is to understand the mechanisms of seasonal rhythms in reproduction and development of mammals. Specifically, I propose to determine the mechanisms by which the mother's photoperiodic history influences the development of her offspring, and to describe the development of social behaviors underlying seasonal variations in fertility. The model species, the meadow vole (Microtus pennsylvanicus), was chosen because there is extensive knowledge of its natural history upon which to base hypotheses for experimental laboratory analysis. A growing body of research indicates that dams communicate photoperiod information to their fetuses and this information influences neonatal development. Meadow vole dams are capable of fetuses and this information to their offspring pre- and postnatally. Additionally, the length of time the mother has been in short days (whether she is still sensitive to short daylengths or has become photorefractory) prior to impregnation influences development of her offspring pre-and postnatally. Communication of maternal photoperiodic history to the offspring presumably allows them to develop appropriately for the season of their birth. This project addresses the mechanism(s) of the maternal photoperiodic information transfer and the influence of photoperiod history on development of social behaviors. Specific aims include: 1) Delineation of the control of maternal-litter contract time as a function of pup thermoregulatory abilities which are influenced by the maternal photoperiodic history. 2) Determining the role of melatonin in communicating information about photoperiodic history between the dam and her offspring by measurement, removal and replacement experiments. 3) Determining the effect of maternal photoperiodic history, current photoperiod, and age of partner on mating behavior and afflictive/aggressive social interactions that control population fertility. Successful completion of the proposed projects will enhanced our understanding of mechanisms in the mothers and pups that result in significant seasonal differences in physiological and social development. A growing body of data demonstrates that humans are also affected by the changing seasons both neonatally and as adults. The extrapolation of data from meadow voles to other short-lived rodents is plausible, and the possibility of similar mechanisms operating in humans must be entertained. Ultimately seasonal effects on development, directed by the human mother, may prove to be important in conjunction with other environmental factors such as adequacy of nutrition.

The primary purpose of the research described is to determine whether the Octodon degus, a South American, diurnal, hystricimorph rodent is a suitable animal model of diurnal circadian rhythms to eventually use in studies of human circadian disorders and research related to the general neurobiological control of circadian rhythms in diurnal mammals. Previous attempts to develop a diurnal animal model have been limited by difficulty obtaining sufficient animals, circadian rhythms were sufficiently noisy so as to exclude studies of the formal properties of the circadian system, behaviors in the laboratory did not agree with those reported in the field, or methodologies were limited for gathering and analyzing data. To date, few researchers have attempted to find a suitable day-active species for extensive description of the formal circadian properties. Development of a diurnal rodent model will allow a more cost-effective means than using human subjects or primates to develop treatments for human disorders such as the problems that result from transmeridian jet travel, shiftwork, illness and hospitalization for traumatic problems. These situations cause deterioration of mental alertness, sleep disruption, ulcers and, in the most severe cases, depression. A good diurnal rodent model will also allow extensive comparative physiological and neurobiological studies. Thus, the main objective of this project is the determination of the formal circadian properties of the degu to determine whether it may prove to be a model diurnal species. The data currently available for diurnal species indicate that circadian function in such species may differ substantially from that of nocturnal species. To determine the formal circadian properties, the following parameters will be examined in adult male and female degus: 1) The stability and precision of entrained circadian activity rhythms. 2) The reentrainment response (time and form) to various size phase-shifts in the light-dark (LD) cycle. 3) Stability of free-running rhythms and their dependence on constant levels of illumination (Aschoff's Rule). 4) Development of phase response curves (PRC's) using 1 hr light pulses for animals free- running in constant darkness (DD). 5) The limits of entrainment (various LD ratios). 6) Relative importance of LD amplitude in entrainment. 7) Comparison of activity and temperature rhythm entrainment and free- running rhythms. 8) Ability of social cues to act as a non-photic entraining agent.

DESCRIPTION (adapted from applicant's abstract): A variety of environmental and life changes alter human circadian rhythms, and in some individuals cause physiological and psychological problems. Variation in entrained circadian rhythms, due to sex and chronotype, and problems of rhythm disruptions are likely the result of differences in sensitivity or responsiveness to entertaining photic and non-photic environmental cues, such as the light/dark cycle and social interactions. Entrainment of the circadian system by the light/dark cycle has been studied at many levels of analysis particularly in nocturnal rodents. In contrast, a number of non-photic stimuli have been identified that alter entrained or free-running circadian rhythms, but we have only a limited idea of how these stimuli reach the circadian system because most non-photic stimuli are complex and employ multiple sensory systems. A better understanding of how non-photic stimuli interact with the circadian system will best be developed with a zeitgeber that engages only one sensory system. Octodon degus serve as an ideal model for exploration of the interaction of non-photic entrainers of the circadian system and determination of the neural underpinnings of those interactions. Degus are a highly social, diurnal rodents, displaying rhythms similar to those of humans , which are entrained by light and social interactions. Hypotheses to be tested by this proposal focus on how social interactions of degus are able to entrain circadian rhythms. Aim 1. Determine whether olfactory cues delivered to the main olfactory system are sufficient to entrain circadian system. Experiments will examine whether airborne odor cues can replace social interactions for accelerated re-entrainment following phase shifts and entrain animals in constant conditions by phase advances and delays. Aim 2: Delineate the critical components of the neural circuitry transmitting odor cues to the circadian system. Experiments will use tract tracing, selective lesions and Fos immunohistochemistry to determine the functional neuroanatomy involved in the entertainment of circadian rhythms by olfactory/social stimuli. Successful completion of the proposed research will produce the first detailed behavioral/neuroanatomical model of the interaction of non-photic zeitgeber and the circadian clock. This work with degus will eventually allow the investigators to analyze the interactions between photic and non-photic entraining cues in the control of circadian rhythms in a diurnal, social mammal with many properties similar to humans, and may lead to non-photic treatments for circadian disruptions.

Microtine rodents are an ideal genus in which to study the role of the environment on the covariation of affiliative social relationships and patterns of paternal and maternal behaviors, and to determine what neurobiological variations are responsible for these behaviors. Extensive field and laboratory data are available on a number of closely related species with different social and parental patterns of behavior. Microtus ochrogaster (prairie voles) are primarily monogamous, with pair-bonding and biparental care. In contrast, Microtus montanus (montane voles) are promiscuous breeders and males rarely share the nest of the female or care for young in the field. Intermediate between these two extremes is Microtus pennsylvanicus (meadow voles), which have a non-monogamous mating system. During the summer breeding season, males are rarely found in the nests of females or seen caring for young. However, during the colder months of the year, males are found in the field nests with the female and pre- weanling young. Successful meadow vole colonies are rare and cost only slightly more than mice to maintain. In contrast, starting a new colony from wild caught individuals is difficult, costly and time consuming. Thus, maintaining a colony for access by other researchers and to maintain our ongoing research examining variation in reproductive behaviors across closely related species is valuable and cost-effective. During a year of support, while seeking additional sources of funding, the PI will continue to test the hypothesis that photoperiod and duration of cohabitation will influence the formation of partner preferences and paternal behavior in non-monogamous meadow voles, and behaviors may be equivalent to those of monogamous prairie voles under some conditions. Dr. Lee will determine the rate of development and the extent of partner preferences demonstrated by male and female meadow voles cohabiting under winter or summer photoperiods and paternal behavior of males. By manipulating the environment (photoperiod), the PI expects to alter the amount of affiliative and paternal behavior of meadow voles and the rate at which these behaviors develop. Because males in summer conditions are aggressive towards unfamiliar animals and not prepared to live in groups, while animals in winter conditions live in groups and are not aggressive, Dr. Lee hypothesizes that full paternal care will be evident in male meadow voles housed in SD without prior exposure to pups. Alternatively, she may find no influence of photoperiod on development of affiliative behaviors or paternal behavior, indicating that seasonal changes in sociality may not correlate with altered development of these behaviors.

Circadian rhythms are cyclic biological activities that recur at the same time every day. In mammals, a part of the brain called the suprachiasmatic nucleus (SCN) is important for these rhythms, and constitutes part of the biological clock mechanism. These rhythms continue to run under constant conditions, but environmental cues such as timing of daylight can set, or entrain, the timing or phase-shifting of the cycles. Sex differences have been described in the entrained circadian rhythms of most mammals that have been examined, and recent data suggest that steroid hormones can influence the function of the circadian clock biochemistry in the SCN. Most animals used for circadian research are nocturnal rodents, but there are some data on two diurnal species, the human and a rodent called the degu. In the degu, sexually dimorphic circadian effects are seen in responses to free-running periods, light stimuli, and modulation by hormones, among other factors. The sexual dimorphism develops just after puberty, and appears to require changes in both sexes. This project uses biochemical, molecular and behavioral approaches to determine whether sexual differentiation occurs when molecular receptors for estrogen and/or androgen are up-regulated in the SCN, and whether sexual differentiation of circadian rhythms is reflected in changed cycles of the gene expression of key cellular proteins (PER and CRY) that generate the cycles.
Results from this project will be important for general biology and psychology as well as neuroendocrinology, for understanding hormonal and genetic mechanisms of circadian rhythms in mammals, for evolutionary comparisons with nocturnal mammals, and potentially for better understanding of how human circadian rhythms develop during adolescence. This project is also in a laboratory with an excellent record of research mentoring.

Exposing female fetuses to excessive testosterone (Pren-T) causes virilization of the external genitalia and permanent alterations in the central nervous system (CNS) control of the reproductive neuroendocrine axis and sex-typical behaviors. The extent of genital virilization is not a good indicator of the prenatal programming by testosterone. Identification of non-genital indicators of future gendered behavior during development would assist pediatricians in developing better treatment protocols for children with ambiguous genitalia. The sheep is an excellent model in which to study these relationships because required doses of Pren-T for achieving varying amounts of masculinization have been determined, and the pre-pubertal period is sufficiently long that behavioral development can be accurately assessed, yet reproductive maturity occurs within 6-7 months. Because differentiation of some behaviors is evident prior to puberty, we propose to determine which early behaviors accurately predict adult gendered behavior. We expect the behavioral differentiation caused by Pren-T to alter steroid hormone receptors, cytoarchitecture and functional responses in key areas of the CNS. Hypothesis: The timing of Pre-T programs a variety of CNS functions independently of virilization of the genitalia, such that the extent ofvirilization does not accurately predict behavior. In addition, some juvenile behaviors will better predict adult social-sexual behaviors than others. The organization and function of key brain areas involved in social-sexual behaviors will also be programmed by Pren-T. Specific Aims: 1) Test the hypothesis that androgenic and estrogenic affects of T cause juvenile sex-differentiated behaviours that predict adult sexually-dimorphic behaviors and neuroendocrine function. 2) Test the hypothesis that postnatal exposure to estrogen further masculinizes the behavior of females exposed to Pren-T. 3) Test the hypothesis that Pren-T exposure alters the functional neuroanatomy of the amygdala, preoptic area and other sexually-dimorphic hypothalamic nuclei.

DESCRIPTION (provided by applicant): Essentially all multicellular organisms demonstrate circadian rhythms of physiology and behavior. These rhythms maintain synchrony (via specific phase relationships) between the organism and the outside world, as well as internal synchrony between physiological functions. A rapid change in the light:dark cycle alters the phase relationship between the organism and the outside world, typically requiring days to weeks for full recovery, during which time the internal synchrony of the individual is disordered. In humans, this phenomenon is referred to as jet lag and has been linked to physical, emotional, and psychiatric problems such as ulcers, depression, and emotional distress. It is common in shift workers and long-distance travelers. This proposal explores the possibility that the delay in recovery of internal and external circadian synchrony in mammals (reflected in activity and cortisol rhythms), as compared with the recovery of melatonin entrainment (thought to best reflect SCN function), is in part due to the activation of the hypothalamic-pituitary-adrenal axis (stress axis) as a result of the shifted light cycle. We hypothesize that the central circadian mechanism, as reflected in the melatonin rhythm, re-entrains faster than activity or cortisol rhythms, and that melatonin rhythm re-entrainment is independent of the stress axis. Secondarily, we expect that manipulating the stress axis so as to elevate or reduce stress will concomitantly increase or delay recovery rates of activity and cortisol rhythms after phase shifts, but that melatonin re-entrainment will be unaffected. To test these hypotheses we will use microdialysis to measure cortisol and melatonin at hourly intervals while avoiding the blood-loss problems for a small mammal. Specific Aim 1 will determine the relationship between re-entrainment rate and cortisol concentrations and/or circadian cortisol rhythm in a series of 4 experiments which independently manipulate entrainment rate or cortisol levels while measuring the other variable. Specific Aim 2 will determine the relationship between melatonin, cortisol and activity rhythms during re-entrainment to test the hypothesis that melatonin rhythm, and therefore the central oscillator mechanism, recovers more rapidly than other rhythms. Providing a better understanding of the interactions between stress and the circadian system will allow for a more thorough investigation of the pathology of desynchronized circadian rhythms and may lead to treatments to reduce the desynchrony.

[unreadable] DESCRIPTION (provided by applicant): We recently observed that performance of a cognitive task (specifically an attention-demanding task, not involving stressors or unexpected events and not requiring noradrenergic activation), declined during recovery from a phase-shift of the light cycle. Furthermore, stable circadian activity patterns were radically altered by daily training on such a task during the subjective rest phase (i.e., rats became day active). As performance on this task depends upon the integrity and activity of the basal forebrain - cortical cholinergic input system, and as activity in this system during performance is controlled by top-down prefrontal regions, the main goal of the proposed research is to determine the neuronal mechanisms allowing cognition- associated cholinergic activity to modulate circadian rhythms. Although the HPA axis and central attention mechanisms interact, the impact of bottom-up and top-down arousal mechanisms on circadian rhythms appears to be quite different. We hypothesize that the effects of cognitive activity on circadian rhythms are mediated via activation of basal forebrain cholinergic projections to the SCN, while stimulation of the HPA axis contributes little to the reorganization of cognition-entrained rhythms. Specific Aim 1 tests the hypothesis that bottom-up, HPA-mediated arousal is not sufficient or necessary for entraining activity during the rest phase. We will test the hypothesis by examining changes in corticosterone release and ACh release following entrainment to a non-photic signal during the day, while intact or after adrenalectomy or basal forebrain lesions. Specific Aim 2 tests the hypothesis that the pattern of circadian re-organization caused by performance of an attention-demanding cognitive task is gradual, learning-related, and time-of-day specific. Specific Aim 3 tests the hypothesis that basal forebrain cholinergic efferents to the SCN are necessary for the effects of attention-demanding cognition on circadian entrainment. Results from these experiments may increase our understanding of circadian changes associated with shift work, Alzheimer's disease, depression and bipolar disorder. Potential treatments for such disorders may include manipulation of the cortical cholinergic system control of circadian entrainment. [unreadable] [unreadable] [unreadable]

Female fetuses exposed to Testosterone (T) produce variable outcomes in reproductive behavior as a function of the timing and dose of exposure. Prenatal T causes hyperinsulinemia and functional hyperandrogenism in ewes, typical of women with polycystic ovarian syndrome who develop severe metabolic and reproductive dysfunction. Prenatal T-treated sheep, exposed to T for 30 days of a critical period are phenotypically female, capable of copulation, but vary in the amount and type of sex behavior exhibited. Because all females were exposed to the same prenatal T treatment, these data suggest that postnatal life history influences behavioral outcomes, not just prenatal exposure to T. Prenatal T-treated sheep produce behavior typical of increased motivation for food intake and reward perception for foodassociated signals. This alteration in goal-directed behavior likely develops because the animals have excess insulin, leading to altered metabolism and increased salience of food cues. Animals vie for access to food, eventually establishing a social hierarchy. Higher ranking T-treated females exhibit more male-typical behavior than controls. Thus, the variation in adult sex behavior of T-treated females may be the result of an interaction between the effects of prenatal T on metabolism, which alters rewarding properties of food and variably influences social hierarchy, with the predisposition towards male-typical behavior also caused by prenatal T. We hypothesize that prenatal T exposure leads directly or indirectly to altered mesolimbic pathways responsible for goal-directed and rewarding behaviors. Proposed experiments will test whether postnatal attenuation of hyperinsulinemia or hyperandrogenism alters goal-directed behaviors associated with reproduction. We will test the hypotheses that prenatal-T treatment 1) alters motivation and reward perception of stimuli during development and in adults, 2) that postnatal treatment of androgenic and insulin sensitivity will alter motivation and reward responses for reproductive and feeding stimuli, and 3) prenatal Ttreatment affects behavior by altering gene and protein expression in the mesolimbic motivation/reward circuitry.

DESCRIPTION (provided by applicant): Endocrine disrupting chemicals (EDC) are pollutants that disturb normal functioning of the endocrine system. Exposure to such pollutants during pre-natal development can have detrimental effects on the developing fetus because the system is highly plastic and responsive during critical periods. There has been much concern recently about the action of these pollutants on fetuses in utero. One such compound that is prevalent in the environment is bisphenol A (BPA). It is used to make polycarbonate bottles, including baby bottles and linings of cans, so exposure is widespread. Studies in rodents have implicated prenatal BPA exposure in a variety of health problems. We propose to use sheep as a model to examine the effects of prenatal BPA on complex behaviors. Similar work with prenatal testosterone exposure has demonstrated that sheep are an excellent model for such studies. Studies at our facility using sheep as a model for prenatal BPA exposure indicate that exposure to BPA leads to reproductive disruptions. However, no work has yet been done to examine the lifespan development of complex behaviors in a long-lived social species, such as ourselves. The goal of this study is to investigate the effects of prenatal BPA exposure and adolescent BPA exposure on complex behaviors in the sheep model across development. These behaviors will be correlated with physiological endpoints including steroid hormone levels, stress reactivity and body weight. Additionally, we will be examining steroid receptor number and distribution in areas of the brain associated with motivation and consummation of mating behavior. We hypothesize that prenatal exposure to BPA, at levels similar to the exposure of human fetuses, will disrupt a variety of complex behaviors including play, aggressive, motivational and sex behaviors over the lifespan of the animal. A second period of vulnerability is adolescence, so we will also examine the effects of BPA exposure during adolescence. Specific Aims 1 and 2 will examine complex behaviors throughout the lifespan of both male and female exposed to either prenatal, adolescent or prenatal and adolescent BPA compared to controls. Specific Aim 3 will examine the effect of BPA exposure during these vulnerable periods on steroid receptor number and distribution in areas of the brain associated with motivation and consummation of mating behavior. Results from this study are important for characterizing the behavioral and developmental effects of prenatal exposure to the endocrine disruptor BPA. This is an important issue in society, since many products that mothers and young children are exposed to contain BPA. PUBLIC HEALTH RELEVANCE: Bisphenol A (BPA), an endocrine disrupting chemical, is widely used in the industrialized world. Of particular concern is the effect during critical periods of reproductive development, including in utero and adolescent exposure to BPA, which can have detrimental physiological and behavioral effects. This project will examine the behavioral and neurochemical effects of prenatal BPA exposure in the sheep model.

Most species undergo at least two life phases, early growth followed by a mature reproductive stage. In mammals these phases are linked by a period of major physiological, neural and behavioral change defined as adolescence. Hormonal changes associated with maturation cause important changes in the brain and behavior. Little attention, however, has been paid to what happens during the period of adolescent change that is unique to that period. One of these changes is a delay in the timing of sleep/wake cycle that this project models in a slow-developing diurnal rodent, the degu, and a faster-developing common lab animal, the rat. This project will determine the role of adolescent hormone changes on the altered timing of the sleep/wake cycle by removing and replacing them. It will also determine whether the neural changes are occurring in the area of the brain that coordinates all daily rhythms, or if the changes are in areas that receive those central signals by examining slices of the tissue collected and analyzed around the clock. Lastly, the project will examine the impact of different lighting levels on the adolescent changes by examining changes in daily behavior under different conditions. Because changes in the daily timing mechanism of the brain impacts nearly all aspects of physiology, these findings will lead to a greater appreciation of the physiological and behavioral state of mammals during adolescence. The project will support the research activity of a graduate student and at least two undergraduates each year. The students and the project director also provide academic and community learning opportunities related to adolescent sleep/wake changes. The project will also support the development of a data base of daily activity and other rhythms collected from animals that can be shared with other researchers in the field.