Horacio O. de la Iglesia - US grants

Living organisms are exposed to environmental cycles that result from the movement of the planet, as well as the movement of the moon. Most species have adapted to the solar day and the alternation of seasons and show oscillations in their physiology and behavior that are in synchrony with these cycles. Furthermore, species living at the seashore show biological oscillations in synchrony with the tidal cycle. Many of these daily and tidal biological oscillations are generated by biological clocks, which are endogenous biological processes that can oscillate in synchrony with the environment. Very little is known about the neural and molecular components that constitute these biological clocks in crustaceans of coastal habitats. The main goal of this project is to characterize the biological clocks responsible for daily and tidal rhythmicity in crustaceans living in coastal areas. Techniques will be used that involve isolation of specific genes involved in the clock mechanism and the study of behavioral rhythmicity in the laboratory. The studies will contribute to the understanding of biological timing systems and it will more specifically establish how species inhabiting the seashore have adapted to the environmental changes that result from the solar day and the tidal cycles. This project provides an excellent opportunity to study biological rhythms in a unique costal environment, namely the Pacific Northwest Coast and will provide a forum for both graduate and undergraduate student research training, by exploiting the research facilities at University of Washington and at Friday Harbor Laboratories.

[unreadable] DESCRIPTION (provided by applicant): Circadian rhythms are oscillations of behavior and physiology with a period of approximately 24 hours that are generated by an endogenous biological timing mechanism, that is, a biological clock. In mammals the master circadian clock is located within the suprachiasmatic nucleus of the hypothalamus (SCN). The SCN is composed of thousands of single cell circadian oscillators (neurons) that together produce a rhythmic output that sets the time for a diversity of rhythms like hormonal release cycles, sleep and wakefulness, and body temperature rhythms. We have recently shown that the SCN of rats exposed to artificially short days (22 h) can be dissociated into two subregions, the SCN core and shell, that oscillate independently and that are associated with two rhythms of locomotor activity, one with a period of 22 hours and the other with the rat endogenous period of 24.9 hours. Specific Aim A.1 proposes experiments to determine whether this animal model presents features of human forced desynchronization and may therefore represent a good animal model for human internal desynchronization. Experiments in Specific Aim A.2 exploit the rat forced desynchronization model to identify output pathways by which the SCN regulates endocrine rhythms that are likely to rely differentially on the circadian activity of the SCN core or that of the SCN shell. For Specific Aim A.3 we propose to develop an in vitro model of the forced desynchronized rat SCN to study the coupling mechanisms between the SCN core and shell. The experiments proposed in this application will unmask specific output pathways by which the master circadian oscillator of mammals times specific rhythmic outputs. Furthermore, the experiments will establish the foundations for the potential use of the forced desynchronized rat model to study the neural bases of human internal synchronization and intercellular coupling in the SCN. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The mammalian hypothalamic suprachiasmatic nucleus (SCN) contains a master circadian clock that governs physiological and behavioral rhythms. The SCN is constituted of single-cell neuronal oscillators that are coupled and generate a coherent circadian output. The mechanisms and transmitters that are responsible for interneuronal coupling within the SCN have not been completely elucidated. The overall goal of this proposal is to study the role of nitric oxide (NO), a gaseous transmitter present in SCN cells, on SCN intercellular communication. Specific Aim C.1 seeks to establish the role of NO-mediated coupling on the determination of the free-running period of the pacemaker within the SCN. Specific Aim C.2 will assess the role of NO intercellular signaling within the SCN in light-induced phase resetting of circadian rhythms. Finally, Specific Aim C.3 will evaluate -both in vitro and in vivo - the role of NO signaling in the resynchronization between the ventrolateral and dorsomedial SCN after they have been desynchronized by exposing animals to an abrupt phase advance of the light-dark cycle. The proposed experiments will provide insights into the mechanisms and signals responsible for synchronization between SCN cells. Furthering our knowledge of how SCN cells work together to constitute a master clock is key to understand and treat circadian pathologies. PUBLIC HEALTH RELEVANCE: The physiology and behavior of mammals, including humans, show robust 24-hour oscillations that are generated and coordinated by an area within the brain's hypothalamus called the suprachiasmatic nucleus. This nucleus contains a biological clock that is made up by several thousand neurons that are themselves clock neurons capable of oscillating independently. Thus, the synchronization between neurons within the suprachiasmatic nucleus is essential for the normal timing of physiological and behavioral rhythms such as the release of hormones and the sleep-wake cycle. Revealing how these neurons communicate with each other is critical to understand the neural bases of some disorders causing abnormal timing of these functions as well as to treat pathologies that result from challenges associated with our modern around-the- clock society, such as traveling across time zones or nocturnal shift work schedules.

Over the last 30 years, it has become clear that female reproductive cycles, including the timing of ovulation, depend on a both hormonal signals from the ovaries (e.g. estradiol, progesterone), and on precise timing signals from the brain's internal, daily clock - the suprachiasmatic nucleus (SCN); without an SCN, animals stop ovulating, and disturbances of the clock (e.g. shifting light schedules, such as in jetlag) cause reproductive disorders. The importance of the SCN in ovulation is clear, but how the SCN connects to and communicates with reproductive centers remains unknown. Using specific light schedules that challenge the clock of rats, researchers are now constructing the first map linking the SCN "clock" to the reproductive system. This brain map will include specific cells within the SCN responsible for the timing signals of ovulation, the reproductive brain centers they connect to, and the chemical signals used in these connections. If successful, this map will provide a more complete understanding of how ovulation is controlled by the brain, as well as how the SCN clock controls other timing events through the brain. The study will not only answer the question of how the clock controls ovulation, but it will also further our general knowledge of how the brain controls female reproduction, opening further questions for these fields.

The researchers involved have a history of scientific outreach activities. They help to organize and run three Brain Awareness Week annual festivals, each bringing 500+ local K-12 students to the University of Washington campus for a day of lectures and educational games and interactive exhibits. Over the past two years, the researchers have made twelve visits to local Seattle classrooms in public, private and special needs schools, where they exposed potential future scientists to behavioral neuroscience research. From these experiences they have developed free on-line lesson plans to help other scientists and educators, which to date have been downloaded by more than 3,000 visitors to the web site.

Ovulation depends on a surge in the release of luteinizing hormone (LH), which in turn depends on a surge of gonadotropin-releasing hormone (GnRH). In recent years, kisspeptin (KISS) has emerged as the most potent stimulator of GnRH and a key regulator of reproductive development and health in vertebrates, including humans. In females, KISS signaling to GnRH cells is critical for the induction of the LH surge. Despite the central role of KISS in reproduction and specifically in female reproductive development and fertility, little is known about the upstream regulators of neurons expressing Kiss1, the gene coding for KISS. Here we present preliminary results that indicate that Kiss1 expression and the expression of c-fos within Kiss1 neurons in female mice is under circadian regulation, and this regulation is dependent on the presence of high ovarian estrogen levels. The overall goal of this proposal is to determine the pathways by which the circadian system may regulate the activity of Kiss1 neurons. Our laboratory has developed a rat model of circadian desynchronization in which independent circadian outputs are associated with the desynchronized activity of anatomically identifiable subregions of the hypothalamic suprachiasmatic nucleus (SCN), the site of the mammalian master circadian pacemaker. Our preliminary data on this forced desynchronized rat model indicates that the gating of the luteinizing hormone (LH) surge is associated with the activity of the dorsomedial (dm) SCN irrespective of the activity of the ventrolateral (vl) SCN. Because the dmSCN is the main source of vasopressinergic efferent fibers, our hypothesis is that vasopressin (VP) release is a critical SCN signal to induce the LH surge and therefore to activate Kiss1 neurons in a circadian pattern. We propose experiments that test specific predictions of this hypothesis. We will test the prediction that SCN vasopressinergic fibers innervate Kiss1-expressing cells and that innervation of the Kiss1 neuronal network by SCN efferent fibers is critical to sustain the circadian regulation of Kiss1 and of c-fos expression within Kiss1 neurons, which are concomitant with the LH surge. We will use unilateral lesions of the SCN to ipsilaterally deplete the anteroventral periventricular nucleus of SCN efferent fibers. In these animals we will assess the level of asymmetric VP innervation of Kiss1 neurons as well as the asymmetry in the circadian regulation of Kiss1 expression and c-fos expression within Kiss1 cells. Our proposed studies will characterize the pathways and mechanisms by which the activity of the Kiss1 neuronal network is regulated. Specifically, we will determine how a critical component of the mechanisms leading to ovulation such as the circadian system regulates gene expression within Kiss1 cells. Because the activity of these neurons and the release of KISS are essential for normal ovulation, understanding the upstream regulators of Kiss1 neurons will be key to developing therapies to treat disorders of the hypothalamo-pituitary-ovarian axis.

Despite the fact that most animals sleep, it is still unclear what function sleep serves. In humans and other mammals, one of the key functions of sleep is likely to improve learning and the ability to retain what was learned. This is indicated by the fact that sleep deprivation leads to learning and memory deficits. In humans, sleep is made up of sleep cycles that last approximately 90 minutes. Within these cycles, sleep alternates between different types of sleep or sleep stages, such as rapid-eye-movement sleep (REMS) and non-REMS. These stages are present in a specific sequence within each cycle and it is believed that these sequences are important for memory. For this research, we will study a laboratory animal model (the rat) in which we can experimentally disrupt the normal sequence of sleep stages without inducing sleep deprivaation. We predict that this disruption will result in deficits in memory, providing evidence for the role of a normal sequence of sleep stages in learning processes. We will also study the molecular mechanisms by which this effect on learning may take place. Our studies will help understand how challenges to normal sleep, that are a common feature of our modern society, affect learning and may help develop strategies to improve learning performance in people that are commonly exposed to sleep disruptions. This project will also include the training of undergraduate and graduate students in research methods and techniques.

Dr. de la Iglesia is one of four investigators who have organized a Chronobiology Workshop that will take place on October 29, 2013 in Mendoza, Argentina, one day before the XII Latin American Symposium of Chronobiology (XII LASC). This Workshop will bring together Latin American (Argentina, Brazil, Chile, Mexico and Uruguay) and US Chronobiology researchers to discuss and challenge the latest ideas in the field and to promote future international collaborations. Chronobiology has grown tremendously in the last decade in Latin American countries, particularly Argentina, Brazil, Chile and Mexico. In most cases, research teams are headed by Principal Investigators who were trained in the US or Europe and who have returned to their home countries to establish independent research laboratories. Exposure of US and European research teams to projects taking place in Latin America offers a unique opportunity to foster intellectual and ethnic diversity, to achieve access to new biological models, and to develop strong academic links. Latin American upper-undergraduate, graduate and postodoctoral students will participate in the Workshop and in a specially designed Chronobiology course that will be held the day before the Workshop.

The one-day Workshop on Chronobiology in Sao Paulo, Brazil, brings together junior and senior scientists from countries throughout the Americas, and offers an opportune environment for the exchange and discussion of the latest topics in chronobiology, the study of biological rhythms such as sleep-wake adaptations to solar cycles. The workshop also serves to identify relevant research topics and approaches that are addressed particularly well in the context of international collaborative work. The award provides support to US-based principal investigators to participate in this workshop. The intellectual merit of the workshop derives from its small size, which promotes interactions between participants, the assembly of many top scientists in chronobiology, the international make-up of the participants, and its temporal and spatial contiguity with the 13th biannual Latin American Symposium on Chronobiology. With the Latin American foreign partnership as well as a strong representation of women among session chairs and speakers, the workshop provides opportunity for outreach to populations underrepresented in science. The close, intensive interactions with senior members in the field afforded by this highly interactive workshop offer learning and networking opportunity for the many trainees and junior faculty participating in this event.

The workshop includes 4 roundtable sessions, each consisting of 4 brief presentations followed by a thorough discussion, and one keynote presentation. The roundtable topics include molecular as well as network, and circadian as well as sleep-focused themes. The collection of presentations spans a complementary range of topics and experimental systems with considerable promise for impactful activities. In addition to engendering a fertile ground for the development of new collaborations between US and Latin American laboratories, the format of the workshop and the composition of speakers and roundtable chairs foster broadening participation in STEM disciplines as well as exchange of ideas across professional levels and ethnic diversity. These aspects, combined with the workshop's central goal of exchanging and advancing exciting new ideas in chronobiology, constitute long-term benefits for the larger Chronobiology community.

? DESCRIPTION (provided by applicant): Animals carrying a heterozygous loss-of-function mutation in the Scn1a gene (Scn1a+/- mice), which encodes a subunit of the voltage-gated Na+ channel NaV1.1, show deficits in both homeostatic regulation of sleep and circadian regulation of rest-activity cycles. The NaV1.1 channel is the primary voltage-gated Na+ channel in adult GABAergic interneurons and its reduced activity results in a decrease of GABAergic tone, suggesting that sleep regulatory deficits in Scn1a+/- mice emerge from reduced GABAergic activity. However, Scn1a+/- mice are a model of a severe form of epilepsy known as Dravet syndrome (DS) and, as DS patients, show not only dysregulation of sleep but also generalized seizures. Epileptic activity complicates the interpretation of sleep disorders in DS, as sleep regulatory deficits could be the result of sleep disruption by seizures or of seizure-associated neural damage. Our hypothesis is that sleep disorders in DS are the consequence of reduced GABAergic tone within sleep regulatory centers that is independent of the presence of seizures. To address this hypothesis, we propose to conditionally target the Scn1a+/- mutation to specific neurons and brain regions. Specific Aim 1 will determine whether sleep abnormalities in global Scn1a+/- mice emerge from the effect of the mutation specifically on GABAergic neurons. We will target the Scn1a+/- mutation to these cells using an Scn1alox/- mouse and a Cre driver mouse line that targets GABAergic neurons throughout the brain, and will assess the integrity of the circadian and homeostatic regulation of sleep in these mutants. This approach will unequivocally determine whether sleep regulatory deficits in Scn1a+/- mice are the result of reduced NaV1.1 channel activity within GABAergic cells or whether non- GABAergic cells that express the channel also contribute to this phenotype. Specific Aim 2 will target the Scn1a+/- mutation to cells in the suprachiasmatic nucleus (SCN), the site of the central circadian pacemaker that regulates sleep. Viruses expressing Cre recombinase, targeting either all SCN cells or specifically vasoactive intestinal polypeptide (VIP)-containing cells, will be injected wihin the SCN of Scn1alox/+ mice. We will also target the mutation to the SCN VIPergic cells by crossing Scn1alox/+ mice with a mouse line in which the VIP promoter drives the expression of Cre. Because VIP neurons are essential for the integrity of the SCN oscillatory network, we expect that these VIP-specific Scn1a+/- mutants will show similar effects to mutants in which all SCN cells are targeted. Specific Aim 3 will virally target the Scn1a+/- mutation to the reticular nucleus of the thalamus (RNT), which is essential for the generation of slow-wave sleep and spindles during non- REM sleep, both compromised in Scn1a+/- mice. None of the conditional mutant approaches in Aims 1 and 2 is expected to induce seizures, offering a unique opportunity to assess the effect of reduced NaV1.1 channel activity in sleep regulatory regions, in the absence of seizures. We predict that the conditional targeting of the Scn1a+/- mutation to the SCN and RNT will lead to deficits in circadian and homeostatic regulation of sleep, respectively. These results would provide direct support for the role of the NaV1.1 channel within these brain regions in the regulation of sleep. They would also directly support our hypothesis that both circadian and homeostatic sleep deficits in DS emerge from seizure-independent reduced GABAergic activity in specific brain regions, providing new avenues for the treatment of sleep disorders in DS.

In this RAPID project, the investigators will measure melatonin levels and timing of sleep in two non-industrialized populations that are similar except for access to electricity, to understand how sleep is affected by artificial light and the interaction of artificial and natural light exposures. Because power lines are quickly being extended in the research area, this experimental design is possible during a limited window of time before electricity reaches all communities. The research will contribute to our understanding of underlying biological mechanisms and variability of human sleep patterns. The project will support student training, international research collaborations, and public and community science outreach activities, and has potential relevance for understanding sleep problems in industrialized settings.

The investigators will collect data on melatonin levels and sleep timing for individuals in each of two Toba/Qom communities. Participants will wear activity watches and keep diaries to document sleep patterns and quality. Saliva samples will be assayed to determine each individual's daily melatonin onset. The main predictions are that individuals in the community with artificial light will have a later time of daily melatonin onset, that differences will be greatest during the winter, and that any effect of moonlight would be to decrease sleep quality, more so in the population without artificial light. The project will expand our understanding of the spectrum of sleep patterns and sleep-regulatory pressures that may have played distinct roles in human history and evolution, and the ways in which modern societies have modified human sleep patterns.

General Abstract

The biological mechanisms that generate biological rhythms are critical for life, as most organisms on earth have to adapt to cyclical changes in their environments. The time-scales associated with these changes vary widely, and thus so do the mechanisms that allow organisms to use them to predict their environments and survive. Identifying the fundamental principles associated with such diversity requires a broad range of intellectual inputs. The conference supported by this award will therefore bring a diverse group of investigators together from across the United States and Latin America, including senior scientists who are leaders in the field of biological rhythms and young investigators just beginning their training, to foster the exchange of ideas and to provide training and mentorship opportunities for the young investigators from Latin America. Senior US investigators will run in-depth discussion groups with young scientists from Latin America during a one-day workshop, and pairs of senior-junior scientists will then interact one-on-one throughout the following week at an associated conference. This not only provides excellent mentorship opportunities for the young scientists, but also the potential for new ideas for the senior scientists that come from fresh perspectives. A high proportion of the US scientists that will be supported are women, and the international collaborations between US and Latin American scientists will promote growth in the field for investigators across the Americas.

Technical Abstract

This is a small meeting that will pair senior US and Latin American scientists with graduate students and postdoctoral associates from Latin America during an intense, one-day workshop in Valparaiso, Chile, followed by continuing interactions between mentors and trainees during a subsequent meeting on the topic of biological rhythms. There will be four roundtable discussions on 1) Neural Circuits Associated with Sleep; 2) Sleep and Human Performance, 3) Neural Models of Rhythmic Physiology, and 4) Mechanisms of Entrainment. Each will be jointly chaired by US and Latin American senior scientists, and attended by graduate students and postdoctoral associates from Latin America. The senior scientists will briefly discuss their research, followed by intense discussion groups with the students. Each senior scientist will be paired with a young scientist and expected to have follow-up interactions with that student during a subsequent meeting Chronobiology that all participants will attend. In the past, these interactions have led to multiple, collaborative papers that include both the senior and junior scientists. This meeting, like those, is expected to greatly benefit both senior and junior scientists across the Americas and lead to significant discoveries in the field of biological rhythms.

SUMMARY Epilepsy is among the most common neurological disorders globally. Symptoms in severe childhood epilepsies such as Dravet syndrome (DS) are not limited to seizures but typically involve increased anxiety, autism spectrum disorder, intellectual disability, and circadian and sleep disorders. Most treatments for epilepsies target seizure severity and frequency but do not necessarily focus on remaining symptoms, which greatly diminish the quality of life of patients and caregivers. In this proposal we test the hypothesis that improving circadian and sleep regulation in DS will not only result in decreased seizure frequency and severity but also in an improvement of other symptoms of the disease. To test predictions of this hypothesis we capitalize on a mouse model of DS. As DS patients, DS mice have a heterozygous loss-of-function mutation in the SCN1A gene, which codes for the primary voltage-gated Na+ channel in adult GABAergic neurons. DS mice show most symptoms of the disease including febrile and spontaneous seizures, increased anxiety, autistic behavior, cognitive deficits and dysregulation of circadian rhythms and sleep. Thus, DS mice represent a reliable genotypic and phenotypic model of the disease. Here we propose to increase the 24-h temporal structure of the environment to improve both circadian and homeostatic regulation of sleep, and in turn to determine whether these improvements result in reduced seizure severity and frequency, reduced anxiety, improved social behavior and cognitive performance. Aim 1 will assess the ability of wheel-running restriction to the dark phase, food restriction to the dark phase or a combination of both routines, to improve behavioral, physiological and molecular circadian rhythms, to increase sleep hygiene, and to reduce seizure frequency in both male and female DS mice. Aim 2 will determine whether the best routine in improving these outcomes will be also effective in reducing anxiety, increasing social behavior and improving memory consolidation. Until now, most treatments of childhood epilepsies, including DS, involve pharmacological treatments that seek to reduce seizure severity and frequency. Many of these treatments are ineffective in treating DS and the development of non-pharmacological, non-invasive treatments that improve symptoms holistically offers great promise to improve the quality of life of DS patients as well as patients with other forms of severe epilepsy. Our proposal exploits a reliable animal model of DS to test potential treatments that, because of their minimal invasiveness, could be implemented in DS patients.

PROJECT SUMMARY We request partial support for the 2018 Society for Research on Biological Rhythms Conference, to be held at the Omni Amelia Island Plantation Resort, in Florida, on May 12-16, 2018. This meeting, which attracts around 700 attendees, will focus on the breadth of topics that represent key research areas in chronobiology, including molecular biology, genetics, cell biology, neurobiology, physiology, metabolism, cancer, aging, infectious disease, immunology, behavior, sleep, mathematical modeling, environmental change and applied research. The theme of the meeting will be ?The Implications of Biological Rhythms for Health and Society?. This theme reflects the extent to which biological rhythmicity affects all aspects of life; as such, it has implications for all areas of biology and health, and has consequences and bears promises for various areas of medicine, industry, policy-making and governance. The meeting will feature 19 symposia of invited speakers, and 16 sessions of short talks, that combine the best of basic clock research with research that translates this information into human applications. The symposium speakers and session chairs are recognized leaders in their fields, and were chosen to represent our breadth and realize our goal of bridging basic and applied circadian clock research. Large efforts are being made to recruit scientists from other fields, not usually attending SRBR. Special attention has been given to cultural and geographical diversity, as well as gender balance. We aim to attract scientists from diverse backgrounds through targeted advertisement, and, with NIH support, to offer of travel fellowships to trainees, prioritizing those from under-represented groups. Various new communication initiatives will ensure a broader dissemination of knowledge. Training aspects of the meeting are fully developed, and include a highly subscribed, free, one-day Trainee Professional Development Day, Junior Faculty Workshops, and a new mentoring program.

SUMMARY Recent work in our laboratory has shown that cyclic fear can prevail over entrainment of circadian rhythms by the light-dark (LD) cycle and lead to diurnal foraging and feeding in nocturnal rodents. When mice or rats are housed in a cage setup in which they need to leave a safe nesting area to access a foraging area for food and water, they forage and feed during the dark phase of the LD cycle. If the foraging area is rendered dangerous with random footshocks during the active dark phase, the animals? foraging and feeding activity shifts to the light phase. This switch to diurnal behavior represents the output of a circadian oscillator, which is entrained by the cyclic fear stimulus, and is dependent on an intact suprachiasmatic nucleus (SCN), where the master clock resides, and an intact amygdala, which encodes fear perception. Our working hypothesis for this proposal is that a circadian oscillator within the amygdala is entrained by cyclic fear and leads to a shift in the timing of foraging and feeding behavior. Specific Aim 1 will determine whether cyclic nocturnal fear entrains the circadian oscillators in the amygdala and/or the SCN in animals entrained to cyclic fear under LD or constant darkness (DD) conditions. Specific Aim 2 will determine whether cyclic fear can also entrain the circadian rhythms of foraging and feeding in female mice, and whether sex differences are explained by the ovarian hormone regulation. Specific Aim 3 will determine whether the evocation of fear by electrical or optogenetic stimulation of the basolateral amygdala (BLA) during the dark phase is sufficient to induce entrainment of feeding and foraging, and whether the activation of the BLA is necessary for fear entrainment by optogenetically inhibiting it while animals are exposed to the fear stimulus. This Aim will also assess whether in vitro optogenetic stimulation of the amygdala is sufficient to locally entrain the amygdala circadian oscillator. Specific Aim 4A,B will exploit region-specific KOs of the clock gene Bmal1 or global KOs of the clock genes Per1 and Per2 to determine whether canonical molecular clock of mammals is part of the fear-entrained oscillator. Aim 4C will assess whether amygdala-specific Bmal1 KOS fail to entrain to cyclic fear. Our finding that nocturnal fear entrains circadian rhythms indicates that limbic centers that encode fear are part of the circuitry that orchestrates circadian rhythms. It also provides a uniquely tractable system to unmask how the master circadian clock within the SCN and the amygdala integrate photic and fear cues to time complex behavioral processes. Circadian and sleep disorders are a hallmark of anxiety and fear disorders such as post- traumatic stress disorder; our findings could shed light into the neural mechanisms that link trauma to the regulation of sleep and circadian rhythms.

PROJECT SUMMARY Circadian rhythms are 24-hour biological cycles, and are evident as both behavioral and physiological outputs of biological clocks distributed throughout the brain and peripheral organs. In mammals, coordination between rhythms and clocks is attained primarily by the function of a master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which is in turn entrained to the light- dark (LD) cycle. When mice need to leave a nesting area and access a foraging area for food and water, they primarily forage and feed during the dark phase of the LD cycle. However, when uncued footshocks occur randomly in the foraging area during the active dark phase, activity shifts to the light phase resulting in avoidance of the dangerous environment. Behavioral rhythms can also be shifted by uncued footshocks applied with a 24-h cycle in constant darkness (DD), but not if these shocks are cued by a tone. After both LD and DD exposure to cyclic unpredictable fear, the rhythms of foraging and feeding persist upon removal of all cyclic environmental time cues, indicating that these rhythms are the output of a fear-entrained oscillator. The Aims of our funded R01 Award are to identify the location of this oscillator and its underlying molecular mechanism, and to determine whether cyclic-fear entrainment differs between female and male mice. Experiments in the parent award test our underlying hypothesis that a circadian oscillator in the amygdala, relying on the canonical clock gene transcriptional-translational loop, is entrained by fear, leading to a shift in foraging and feeding activity. This Supplement proposal seeks to extend these goals in three experimental Specific Aims, which will serve as the framework for graduate training of Asad Beck, a graduate student of African American descent, at the University of Washington Graduate Program in Neuroscience. Specific Aim 1 will determine whether the recall of the circadian time-stamped contextual fear memory of the foraging area is sufficient to reinstate light phase foraging and feeding even in the absence of actual fear. The second Aim will determine whether sleep architecture during the first stages of exposure to nocturnal fear, which is predictive of how effective contextual fear learing is, predicts the speed of entrainment to nocturnal fear. Finally, the third Aim will characterize the sleep architecture in fear-entrained animals, which in animals entrained to nocturnal fear under an LD cycle should reflect the internal misalignment between SCN master clock and circadian oscillators within fear-coding centers. Our proposal also includes a thorough mentoring plan for Asad Beck. A detailed timeline for the 2.5 years of funding is intended to thoroughly train the candidate to accomplish the following goals: acquire quantitative and computational skills, develop mentoring abilities, develop critical thinking, written and oral communication skills, and establish a sense of self-confidence compatible with a leadership role. The mentoring plan takes advantage of the ample resources available for graduate training at the Program in Neurocience and the University of Washington as a whole.

PROJECT SUMMARY We request partial support for the 2020 Society for Research on Biological Rhythms Meeting, to be held at the Omni Amelia Island Plantation Resort, in Florida, from May 30 to June 3, 2020. The conference, which attracts more than 700 attendees, will focus on several topics that represent key research areas in the field of chronobiology, including molecular biology, genetics, cell biology, neurobiology, physiology, metabolism, cancer, aging, infectious disease, immunology, behavior, sleep, mathematical modeling, environmental science and drug development. The theme of the meeting will be ?Rhythms in the Real World.? This theme reflects the extent to which biological rhythmicity affects all aspects of life in all organisms living in their natural environment. The meeting will feature 19 symposia of invited speakers, 16 slide sessions with short talks, and ~350 posters that combine the best of basic clock research with research that translates this information into the prevention, diagnosis and treatment of disease. The symposium speakers and session chairs are recognized leaders in their fields, and were chosen to represent our the breadth of the field and realize our goal of bridging basic and applied circadian clock research. Slide session speaker include a majority of trainees (postdocs or graduate students). Large efforts are being made to recruit scientists from other fields, not usually attending SRBR, as speakers. Special attention has been given to cultural and geographical diversity as well as gender balance. We aim to attract scientists from diverse backgrounds through targeted advertisement and, with NIH support, to offer travel fellowships to trainees, prioritizing those from under- represented groups. Various new and previously successful communication initiatives will ensure a broader dissemination of knowledge. Training aspects of the meeting are fully developed, and include a highly subscribed, free, one-day Trainee Professional Development Day, Junior Faculty Workshops, and a Mentoring Program. Finally, the new 2020 Chronobiology School, directed to graduate students and postdocs new to the field, will be offered.

Diseases of the nervous system represent an existing and growing emotional and economic burden to society. Neuroscientific research is critical to the discovery of new therapies and treatments for these disorders. With this goal in mind, it is imperative that we train research scientists who represent US society, including ethnic minorities, people from economically disadvantaged groups and people with disabilities. Underrepresentation of minorities in science and technology emerges in part from the fact that underrepresented minority students either fail to enter or leave the pathway that starts with a STEM college education and continues with graduate school in science-related fields. The University of Washington Enhancing Neuroscience Diversity through Undergraduate Research Education Experiences (UW-ENDURE) program will capitalize on the NIH Blueprint Program to help community college students in the Puget Sound region transition into graduate research careers by exposing them to summer and academic-year mentored research experiences in the field of neuroscience at the University of Washington. Our first Specific Aim will be to recruit each year 8 undergraduate students from underrepresented backgrounds to participate in academic-year and summer research as well as training in the field of neuroscience. Our second Specific Aim to assure that each recruited student is incorporated into the research program of a neuroscience laboratory within the UW, and is part of a comprehensive set of educational and mentoring opportunities including summer workshops and academic-year courses, and a one-on-one mentoring plan that starts during their stage at UW-ENDURE and continues for year after their completion of the Program. Finally, our third Specific Aim is to thoroughly evaluate UW-ENDURE?s immediate impact on students? quantitative skills and understanding of neuroscience principles, and on the students? success in applying to and entering competitive STEM graduate careers. The Puget Sound region represents an ideal combination of underrepresented minorities in STEM careers within community colleges and a top neuroscience research institution like the UW. UW-ENDURE will capitalize on this scenario to help underrepresented minorities transition into and remain in the fascinating world of neuroscience research.

SUMARY Circadian rhythms depend on the molecular transcription/translation negative feedback loop (TTL) operating in clock neurons, and on the network properties of these neurons. Among the properties that could be recruited by the circadian clock are changes in the identity of pre/post synaptic partners and/or strength of the connectivity between clock neurons, a property collectively termed as circadian structural plasticity. Our central hypothesis is that circadian structural plasticity within the central circadian clocks of mammals and Drosophila are part of the time-encoding mechanisms. We will employ mouse and fly genetics combined with state-of-the- art quantitative 3D light and electron microscopy techniques to address the extent of structural plasticity within specific neurons of the mouse suprachiasmatic nucleus (SCN) and the Drosophila circadian network. Specific aim 1 will assess how widespread structural plasticity is in the Drosophila circadian network as well as which are the functional consequences of those structural changes. We will explore the extent of circadian neuronal remodeling of subsets of PDF and non-PDF pacemaker neurons using CM and SBEM (sub-aims 1A i and ii). We will examine time-of-day dependent functional connectivity changes among clock neurons through chemogenetic GCamP6-reporting (sub-aim 1B). Sub-aim 1C will examine the behavioral consequences of impairing structural remodeling; sub-aim 1D will further investigate the molecular mechanisms underlying circadian structural plasticity. Specific aim 2 will examine the degree of circadian structural remodeling in SCN VIPergic neurons, which are an essential component of the timekeeping mechanism, through virally mediated sparse-labeling (CM) (sub- aim 2A), or serial block-face scanning electron microscopy (SBEM) with a marker that enables the analysis of dendritic ultrastructure (sub-aim 2C). Finally, we will assess if circadian oscillations in VIP neuronal processes rely on the TTL by repeating experiments in 1A in VIP-specific Bmal1-/- mice (sub-aim 2B). Specific aim 3 will explore if connectivity of VIPergic neurons changes throughout the 24-h cycle. Using GFP reconstitution across synaptic partners (GRASP), we will investigate if these connections change with circadian time through immunocytochemistry and CM analysis in fixed tissue (sub-aim 3A) as well as ex vivo in SCN slices (sub- aim 3B). We will also determine whether GRASP-detected rhythms depend on the canonical TTL by repeating experiments in 2A and 2B in VIP- or SCN astrocyte-specific Bmal1-/- mice (sub-aim 3C). Our experiments test predictions of the hypothesis that circadian structural plasticity represents a defining feature of central neuronal circadian pacemakers. Support for this hypothesis would provide a critical new perspective to understand how these pacemakers encode time at the network level. Furthermore, the experiments we propose represent a unique opportunity for research capacity building in Argentina, where the foreign principal investigator is located, and where students and postdocs will be trained in techniques that are still not fully developed in that country.