Mark S. Blumberg, Ph.D. - US grants

Mammalian thermoregulation is typically divided into two components, autonomic and behavioral. The characteristics of both components are well known in adults, but their coordination in adults makes it difficult to discern the ways in which animals integrate and coordinate responses. Developmental approaches are useful in this regard because altricial newborn mammals possess immature responses that become mature and coordinated during relatively brief developmental transitions. One such transition, recently identified in this laboratory, involves a diminution in thermoregulatory responsiveness between the ages of two and eight days. This transition is important because its occurrence suggests mechanisms of thermoregulatory activation that are different from those currently suspected. Specifically, it is believed that heat production in newborn rats is activated solely by direct neural activation of brown adipose tissue (BAT). It is hypothesized here, however, that circulating catecholamines, released from chromaffin tissue in the adrenal medulla and/or the organ of Zuckerkandl, contribute to BAT activation. It is further hypothesized that the transition from robust BAT activation at birth to muted BAT activation within one week results from developing sympathetic innervation as well as involution of the organ of Zuckerkandl. These hypotheses will be tested using pharmacological and surgical manipulations of these organs and systems during development. In parallel with these physiological investigations, a novel head-turning paradigm involving radiant heat reinforcement will be used to investigate the development of thermal preference over the same postpartum period. In addition, because a synthesis of these two approaches is one primary goal of this project, interactions between autonomic and behavioral responses will be investigated as pups develop. It is hypothesized that coordination of physiological and behavioral responses will emerge as both thermoregulatory components mature during the first week postpartum. Finally, these interdisciplinary studies of week-old rats may provide valuable information regarding the comparable developmental stage of two to four months in human infants, at which time infants are most at risk for Sudden Infant Death Syndrome.

Recent work in the Principal Investigator's laboratory has shown that infant rats, contrary to a long-held view, exhibit many signs of successful thermoregulation when tested under appropriate conditions. Their success depends on the internal production of heat using brown adipose tissue (BAT), as well as the delivery of this heat to the heart. If BAT thermogenisis is not adequate and heart temperature decreases, cardiac rate decreases as well, resulting in significant challenges to the pup's ability to maintain arterial pressure and venous return to the heart. This application examines this thermoregulation-cardiovascular interaction in detail, and also addresses the hypothesis that the ultrasonic vocalizations emitted by infant and adult rats, typically assumed to be purposeful communicatory signals of emotional distress, are instead the acoustic by-products of a physiological maneuver that is recruited to maintain cardiovascular function during extreme cold exposure and in other physiologically challenging contexts. Aim 1 of this application addresses the neural and hormonal mechanisms by which infant rats increase peripheral resistance to the cold when cardiac output is decreasing. Specific mechanisms to be examined are alpha-1 adrenoreceptors, angiotensin II and vasopressin. Through selective blockade of each of these systems, as well as simultaneous blockade of all three systems, the interactions between cardiac rate, arterial pressure, venous return and ultrasound production are examined. Aim 2 addresses the proximate physiological stimuli that elicit ultrasound production and the associated changes in cardiovascular function that accompanies ultrasound production. For these experiments, venous return is (a) measured using Doppler flow probes and (b) manipulated using pharmacological and mechanical techniques. Finally, Aim 3 addresses the hypothesis that the infant and adult rat's ultrasonic vocalizations are homologous. This hypothesis, based on a variety of similarities between the two vocalizations is tested in adults by determining whether experimental decreases in venous return are sufficient to evoke ultrasonic vocalizations and by assessing cardiovascular changes during fever and copulation, two contexts associated with ultrasound production. This application addresses basic aspects of homeostatic regulation in infants and adults using behavioral, developmental, physiological approaches. Through these approaches, an integrated view of the varied mechanisms by which infant and adult mammals regulate cardiovascular function during physiological and behavioral challenges will emerge.

Infant mammals devote considerable resources to thermal, fluid, and cardiovascular homeostasis. Recent work in the PI's laboratory has shown that infant rats, long considered poor thermoregulators because of their high rates of heat loss during cold exposure, actually exhibit many signs of successful thermoregulation when tested under appropriate conditions. Their success depends on the ability to produce heat internally using brown adipose tissue (BAT), as well as the delivery of this heat to the heart and other temperature-sensitive organs. We have learned that infants that possess endothermic capabilities (i.e., rats) exhibit significantly different behavioral and physiological responses to cold than infants that do not (i.e., hamsters). We still do not understand, however, how infants of either species orient and locomote toward warmth and whether the ability to produce heat internally interferes with the expression of thermoregulatory behavior. Therefore, this proposal represents the logical next step in a research program that addresses basic issues in biobehavioral research and aims to develop a better understanding of the myriad physiological and behavioral mechanisms by which infants regulate their internal thermal environment and select their external thermal environment. This work has important implications for the thermal management of preterm and full-term human infants, sick or healthy, who differ in their abilities to produce heat endogenously and about whose thermoregulatory behavior we know very little. First, with our new appreciation of the thermoregulatory capabilities of individual infant rats, the behavioral and physiological responses of huddling rat pups during cold exposure are examined. Group regulatory behavior is examined in infant rats after ganglionic blockade, after selective activation of BAT thermogenesis in ganglionically blocked pups, and in mixed huddles comprised of infant rats and hamsters. These experiments will reveal how endothermy contributes to the expression and effectiveness of huddling behavior. Second, thermoregulatory behaviors in isolated individuals are examined using a newly- developed, novel apparatus - a multi-tiled "checkerboard" apparatus composed of peltier diodes - that provides fine control over the thermal environment. In addition, the use of infrared thermography provides essential thermal data without interfering with behavioral expression. The combination of these approaches places us in an ideal position to critically examine behavioral arousal, locomotion, orientation, and thermal preference in infant rats and hamsters during cold exposure, as well as the contributions of endothermy to each of these behavioral processes. Finally, subsequent experiments will address the sensory and neural mechanisms that mediate behavioral responses to cold.

[unreadable] DESCRIPTION (provided by applicant): An Independent Scientist Award would support on-going NIH-funded investigations of basic biobehavioral processes in infant rodents, especially concerning the mechanisms and function of sleep. Over the past 5 years, the PI has directed increasing resources toward one of the central questions concerning sleep: Why do infants sleep more than adults? Despite the obvious implications of this question for human health, the vast majority of sleep research continues to focus on adults. Building on recent progress in the PI's laboratory, including the development of techniques for recording neural activity in unanesthetized infant rats as they cycle between sleep and wakefulness, the current proposal has two primary aims. The first aim is to examine the sensory and neural mechanisms that underlie development of ultradian and circadian sleep- wake rhythms. A related aim is to describe the mechanisms that produce the dramatic quantitative and qualitative developmental changes in the statistical distributions of sleep and wake bouts that have now been demonstrated in rats and mice over the first three postnatal weeks. The PI is in the unique position of being able to examine the development of the neural mechanisms that generate these fundamental sleep-wake rhythms, and will do so using a variety of experimental approaches. For example, the developmental contributions of the visual system and its anatomical and functional connections with the suprachiasmatic nucleus and optic tectum will be examined. The second aim of this proposal is to employ non-traditional species to address mechanistic and functional questions concerning the development of sleep and wakefulness. For example, as a precocial species, cotton rats (Sigmodon hispidus) may help us to explain why infants born in a state of relative maturity exhibit less sleep throughout life; and as a diurnal species, Nile grass rats (Arvicanthis niloticus) may help us to identify the critical developmental changes in neural circuitry that underlie the diurnal circadian pattern of humans. The developmental framework adopted in this proposal avoids comparison of infant sleep against an adult standard but rather seeks to understand the processes of change over developmental time. One long-term goal of this project, which renewal of this award will make possible, is to further lay a foundation for our understanding of the mechanisms of sleep development so as to enable identification of the mechanisms underlying sleep dysregulation later in life. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Sleep occupies one-third of our adult lives and yet its function is still not known. In infants, sleep is even more prominent, as are the spontaneous myoclonic twitches that are a defining feature of active (or REM) sleep. Over the past decade, research in the Principal Investigator's laboratory has helped dispel the notion - which held sway until recently - that the brain plays no role in the control of infant sleep. Moreover, it is now widely accepted that sensory feedback (i.e., reafference) from twitching is closely monitored at all levels of the neuraxis. Of particular importance for this application is the recent discovery of oscillatory events-called spindle-bursts (SBs)-that are produced in primary sensory cortices in response to endogenously and exogenously generated sensory stimulation. It is thought that twitching, its associated reafference, and cortical SBs participate together in the development and maintenance of topographic organization. The Principal Investigator's laboratory has been engaged in this effort by monitoring neurophysiological activity in unanesthetized newborn rats as they cycle spontaneously between sleep and wakefulness and respond to specific peripheral tactile and proprioceptive stimulation. This application addresses two broad aims. First, a transient period during the first postnatal week in rats has been discovered when corpus callosotomy disinhibits spontaneous cortical activity and when recovery of function after callosotomy is possible. It is hypothesized that sleep-related twitching contributes to this recovery of function, just as it contributes to normal development. To test this hypothesis, neurophysiological and neuropharmacological approaches will be used, including the novel application of amperometry to unanesthetized infant rats for the measurement of real-time extracellular changes in cortical choline, glutamate, and GABA levels in relation to behavioral state, myoclonic twitching, and peripheral stimulation. Second, recent work suggests that SBs are differentially regulated depending on whether they are produced spontaneously (during active sleep) or evoked by peripheral proprioceptor stimulation. Because the ability of individuals to differentiate self-produced from externally produced sensory input relies upon the production of an efference copy, it is important to identify in newborn rats the neural circuitry that transmits spontaneous and evoked sensory information to somatosensory cortex. This aim will be accomplished using a combination of methods, including surface EEG, amperometry, and selective unilateral inactivation of brain regions in the midbrain and forebrain. Preliminary results from the Principal Investigator's laboratory now demonstrate the feasibility of using amperometry in unanesthetized infant rats. The NIH Blueprint for Neuroscience emphasizes the need for more basic research to understand neurodevelopment, neurodegeneration, and neuroplasticity. This application is compatible with the Blueprint, focusing as it does on sleep, neural function, and brain plasticity across early infancy under normal conditions and after neural insult. Moreover, the approach that informs this work is contributing to a fundamental reconceptualization of infant sleep that will soon provide the foundation for a broader understanding of the function of sleep in both infants and adults. PUBLIC HEALTH RELEVANCE: The NIH Blueprint for Neuroscience and the NIMH Blueprint for Change emphasize how basic, interdisciplinary research into the development of the brain and its response to injury is critical for advancing our understanding of the causes of the high rates of mental illness among children and adolescents. The current application focuses on the role that sensory feedback from sleep- related "twitch" movements, which are especially prominent during the prenatal and early postnatal period, plays in the construction of the nervous system, especially as concerns the cerebral cortex and its interconnections. By understanding how the brain interprets these movements and how early injury and sensory disruption alter the brain's responses, we will begin to shed light on how early sensory experiences during sleep and wakefulness help to shape both normal and pathological outcomes.

DESCRIPTION (provided by applicant): Sleep occupies one-third of our adult lives and yet its function is still not known. In infants, sleep is even more prominent, as are the spontaneous myoclonic twitches that are a defining feature of active (or REM) sleep. In infant rats, twitches are produced tens of thousands of times each day, and sensory feedback from twitching produces substantial stimulation to the primary somatosensory cortex. In fact, each day in neocortex, sleep- related twitches trigger thousands of cortical oscillatory events - called spindl bursts. This sleep-related activation of neocortex is channeled subsequently to the hippocampus, whose activity in early infancy appears to be driven primarily during sleep. Accordingly, it has been suggested that spontaneous, sleep-related motor activity contributes to the development of neural circuits within and between neocortex and hippocampus, just as retinal waves are thought to contribute to the development of visual cortex and related structures. Interestingly, work in our laboratory suggests that it may be the proprioceptive feedback from limb twitches that specifically trigger spindle bursts; tactile stimulation of the limbs appears insufficient for producing a spindle burst. Thus, this R21 exploratory/developmental application aims to investigate the specific contributions of proprioceptive feedback from twitching to neocortical and hippocampal activity and development. Because surgical and pharmacological methods of disrupting proprioception are not sufficiently specific to that modality - and may also disrupt motor outflow and thus disrupt twitching itself - we propose here to test infant mutant mice that are genetically engineered such that they fail to develop muscle spindles, the sensory organs essential for proprioception. These mutants develop neuromuscular junctions and Ia afferent connections from muscle to spinal cord. Importantly, we have also confirmed the presence of twitching in these infant mutant mice and its similarity to that in wild-types. Using methods that were developed in our laboratory for recording neurophysiological activity in unanesthetized infant rats as they cycle spontaneously between sleep and wakefulness and respond to experimenter-controlled delivery of peripheral tactile and proprioceptive stimuli, we will record sleep-wake activity and neocortical and hippocampal activity in infant mutant and wild-type mice across early development. The innovation of this application lies in the use of state-of-the-art recording techniques in conditional knockout mice to test specific, mechanistic hypotheses concerning the phenomenology and function of sleep-related motor activity in somatosensory development. Also, this application will provide a foundation for future developmental studies of neurophysiological and sleep-wake activity in mice that can take full advantage of the molecular tools that are readily available in that species. Finally, this application is compatible with the IH Blueprint for Neuroscience, which emphasized the need for more basic research to understand neurodevelopment, neurodegeneration, and neuroplasticity. PUBLIC HEALTH RELEVANCE: The NIH Blueprint for Neuroscience emphasized the need for interdisciplinary basic research that addresses critical issues concerning neurodegeneration, neurodevelopment, and neuroplasticity. The current application fits well with the Blueprint by focusing on the role of sensory feedback from sleep-related twitch movements - which are especially prominent during the prenatal and early postnatal period - in the construction of the nervous system, including the cerebral cortex and hippocampus. By understanding how the brain interprets these twitch movements and how interrupting sensory feedback from twitches alters the course of development, we will begin to shed light on how early somatosensory experiences during sleep and wakefulness help to shape both normal and pathological outcomes.

DESCRIPTION (provided by applicant): Every animal must distinguish sensations that arise from its own movements from those arising from stimuli in the external world (e.g., lifting your arm vs. having your arm lifted for you). This distinction between self- and other-produced movements requires precise self-monitoring. Self-monitoring is instantiated mechanistically by copies of motor commands-corollary discharges-that prepare the nervous system for the arrival of sensations triggered by self-produced movements. Corollary discharge signals can gate, cancel, or otherwise modify incoming sensory signals. We propose to investigate two novel aspects of corollary discharge: Its expression in early infancy and its modulation by sleep-wake state. One impetus for this proposal is the observation that, in newborn rats, sensory feedback from self-produced limb twitches during active (or REM) sleep triggers spindle bursts in sensorimotor cortex and increased Purkinje cell activity in cerebellar cortex. In contrast, during wakefulness when vigorous, self-produced limb movements typically occur, cortical spindle bursts are surprisingly absent and Purkinje cells are largely silent. This paradoxical masking of neural activity during wake suggests the novel hypothesis that corollary discharge mechanisms are regulated in a state- dependent fashion. Importantly, the hundreds of thousands, if not millions, of twitches produced by infant rats each day during sleep trigger substantial brain activity that is ideally suited to promote activity-dependent development in the sensorimotor system. Specific Aim 1 will characterize state-dependent neural activity in sensorimotor cortex and cerebellum across the first two postnatal weeks in rats, a period of rapid change in those structures. Critically, by comparing spontaneous and evoked neural activity during sleep and wake, this Aim will establish two new models for exploring the neural mechanisms and developmental origins of corollary discharge. Specific Aim 2 will provide critical new data regarding sensorimotor processing in early infancy through systematic comparison of state-dependent activity in brainstem nuclei implicated in (a) the production of limb twitches, (b) the reception of proprioceptive input from limbs, and (c) the processing of corollary discharge. We will also test the novel hypothesis that the locus coeruleus, a brainstem nucleus that is both wake-active and a primary source of norepinephrine to the cerebral cortex and cerebellum, contributes to the state-dependent modulation of corollary discharge. Finally, Specific Aim 3 will explore the developmental emergence of reciprocal and state-dependent modulation of sensorimotor cortex and cerebellum. Such reciprocal interactions are essential for mature sensorimotor integration throughout the brain. The NIH Blueprint for Neuroscience emphasizes the need for more basic research to understand neurodevelopment and neuroplasticity. This proposal meets that need by uniquely integrating several innovative conceptual and methodological approaches to provide new insights into the functional development of critical sensorimotor systems.

PROJECT SUMMARY Active (or REM) sleep is an abundant and ubiquitous feature of mammalian life early in development: Human newborns sleep 16 hours each day and 8 of those hours are spent in active sleep. The relatively high proportion of active sleep in early development inspired the hypothesis that active sleep is important for early brain development. But what is it about active sleep that might make it especially important for brain development? One feature, especially notable in newborns, is the abundant phasic activity that characterizes active sleep, comprising twitches of the limbs, head, face, and eyes. Over the past decade, research in infant rats has demonstrated that sensory feedback from twitching limbs triggers pronounced activity throughout the brain, including events (called spindle bursts) in sensorimotor cortex. Moreover, in a first-ever systematic study focusing on twitching in human infants, the PI and Co-I found abundant twitching in human infants that parallels that seen in rats. In addition to finding that human infants produce dozens of twitches per hour, they also found that patterns of twitching evolve differently in distinct muscle groups, thus supporting the notion that twitching reflects the neurological development of specific action systems. As proposed here, the next step is to determine whether twitches trigger brain activity in newborns and whether this twitch-dependent activity persists over the early postnatal period: This step is essential for determining whether there may be diagnostic and explanatory value in monitoring and assessing twitching in human newborns. Accordingly, this proposal combines behavioral analysis, motion sensing, electrooculography (EOG), and high-density electroencephalography (EEG) in sleeping human infants monitored cross-sectionally at 2 weeks and 1, 2, 3, 4, 5, and 6 months of age. Specific Aim 1 focuses on whether twitches of the arms and legs trigger EEG events in topographically appropriate regions of sensorimotor cortex. In addition, one of the analytical challenges of this proposal is to determine whether twitch-triggered cortical events can be detected if, as expected, background EEG activity increasingly obscures them over the first six months. Specific Aim 2 parallels the first but focuses on twitches of the facial muscles (e.g., mouth, cheeks) and associated rapid eye movements (measured using EOG). One major outcome of this aim will be to place the facial sensorimotor system within a broader context that will allow comparisons with sensorimotor development in the limbs. Overall, the work proposed here will lay a foundation for understanding the role that sleep plays in the developmental origins of the sensorimotor deficits that are known to accompany many neurodevelopmental disorders (e.g., cerebral palsy, autism). Ultimately, this research may lead us toward an understanding of how sleep deprivation or restriction (resulting from malnutrition, environmental factors, or neglect) can initiate a cascade of effects that begins with sensorimotor development and continues on to impair cognitive and emotional development.