William J. Schwartz - US grants

The objective of my research program continues to focus on the neural regulation of circadian rhythms by the suprachiasmatic nuclei (SCN) of the hypothalamus. The first set of experiments will analyze the relationship between energy metabolism and function in the SCN using the 2-deoxy-D-(14C)glucose (DG) autoradiographic method. Fetal SCN energy metabolism is rhythmic in utero, before either SCN synaptogenesis or the circadian rhythm of SCN impulse activity becomes apparent post-natally. Experiments will test the hypothesis that the generation & propagation of action potentials by the SCN is not an integral part of the actual circadian timing device. Mini-osmotic pumps will locally perfuse the SCN with one millionth M tetrodotoxin (TTX) at 0.5 Mul/hr for 14 days; free-running rhythmic drinking behavior of blinded rats will be monitored before, during and after infusion of the toxin. This paradigm will be used to (a) uncouple the circadian clock in the SCN from both its afferents and efferents, (b) determine a dose-response relationship for TTX infusion, (c) demonstrate topographical specificity of the agent, and (d) quantitate the resulting rhythm of SCN energy metabolism. The second set of experiments will use the DG method to determine how the maternal circadian system coordinates the timing of the developing fetal clock. Studies for the coming year include (a) maternal SCN ablation at later gestational ages, (b) maternal oophorectomy, and (c) evaluation of non-endocrine behavioral mechanisms using food restriction, shifting of mealtimes, and continuous intravenous hyperalimentation regimes. The third set of experiments will investigate the physiological role for the circadian vasopressin rhythm in cerebrospinal fluid (CSF). Preliminary data suggest that the circadian rhythm of brain temperature is severely disturbed in homozygous Brattleboro rats. Further characterization of this abnormality will lead to experiments to (a) abolish the normal temperature rhythms of Long-Evans rats by intracerebroventricular administration of vasopressin antibody or antagonist, and (b) restore temperature rhythmicity in Brattleboro homozygotes by artificial simulation of normal CSF vasopressin rhythms or by transplantation of the fetal SCN. Vasopressin is measured by a sensitive and specific radioimmunoassay.

photobiology; environmental adaptation; circadian rhythms; suprachiasmatic nucleus; bioenergetics; tetrodotoxin; arginine vasopressin; cerebrospinal fluid; action potentials; carbachol; brain metabolism; membrane potentials; glucose; neuropharmacology; radioassay; electrophysiology;

We propose to continue the exploration of the effectiveness of computer-based instruction in sickle cell disease. Specifically, we plan to create new computer-assisted educational material, to update existing programs to incorporate new knowledge and utilize the features of advanced equipment technology, to adapt existing material for use in new groups of learners, and to continue the evaluation of the effectiveness of these educational methods. New programs will include an update of the existing Sickle Cell Teacher program to cover detailed information about the effect of sickle cell disease on the renal and cerebral systems, pain management, transfusion therapy and new treatment modalities such as therapy with hydroxyurea. Existing programs will be modified for use by nurses, health care workers and patients. The new technology allows for the inclusion of color illustrations of blood smears and histopathology in these programs. The DELTA program will be modified for use by parents in the home management of problems related to sickle cell disease. The .major effort will be the design and execution of a sophisticated evaluation system to provide new information about the effectiveness of computer-assisted instruction. We propose to test the programs with the established techniques of educational psychologists to determine the reliability and validity of the modules. Using the existing network of interested pediatric departments, large numbers of testees and experts will provide the necessary power to provide information about the effectiveness of these programs.

DESCRIPTION: (Verbatim from the Applicant's Abstract) The discovery of self-renewing, multipotent neural precursor cells has raised hopes for their possible therapeutic utility, either for neural repair through cell replacement or as gene-delivery vehicles. Transplantation is a key strategy for testing the differentiation capacity and functional activity of such cells; when precursor cells obtained from one brain region are transplanted to another region, they appear to differentiate into neuronal and glial phenotypes appropriate to the implantation site. Whether the cells actually integrate in a regionally-specific, physiologically-relevant fashion remains unanswered. In this application, we will address this question by capitalizing on the unique anatomy, physiology, behavior, and molecular genetics of two hypothalamic neuronal systems that have not been previously exploited as engraftment targets for transplanted precursor cells. The suprachiasmatic nucleus (SCN) is the site of an endogenous clock that regulates circadian locomotor rhythmicity, a behavior that is abolished in mice homozygous for a mutation of the clock gene. The magnocellular division of the paraventricular nucleus (PVN) is the site of arginine vasopressin (AVP)-secreting neurons that regulate water balance, a function that is lost in mice with diabetes insipidus bearing a null mutation of the AVP gene. We will transplant a prototypic neural precursor cell line (C17.2; as well as C17.2NT-3, a modified clone overexpressing neurotrophin-3) into the telencephalic vesicle of embryonic mice and then examine the mice postnatally to address three questions: (A) Do engrafted precursor cells assume the morphologies, peptidergic phenotypes, and efferent projections characteristic of neurons indigenous to the SCN and PVN? (B) Can engrafted precursor cells respond to natural stimuli in ways that emulated the response of neurons indigenous to the SCN and PVN? (C) Will engrafted precursor cells in the SCN and PVN restore defective behaviors in homozygous clock and AVP-deficient mutant mice exhibiting circadian arrhythmicity and diabetes insipidus, respectively? The answers to these questions are initial steps towards establishing the circadian and hypothalamo-neurohypophyseal systems as potentially powerful models for analyzing the structural and functional plasticity of implanted precursor cells.

DESCRIPTION (provided by applicant): Daily rhythms are governed by endogenous circadian clocks, and, in mammals, by the suprachiasmatic nucleus (SCN). The circadian system is complex, with an SCN tissue pacemaker containing multiple, coupled single-cell oscillators and a system composed of interlocking and nested auto-regulatory feedback loops. Advances have been made in delineating the main elements of this network, and we can now begin to analyze its stimulus-response properties at the molecular, cellular, tissue, and behavioral levels. What we are finding is that the system's output after a given input may not be linear, with striking consequences for animal behavior. Notable examples of this are some of the effects of constant light (LL) - "splitting" in golden hamsters and circadian rhythmicity in genetically-deficient mice - that dramatically reorganize the circadian system. We propose to study these phenomena experimentally, but from a perspective that is not traditionally applied in research on hamsters and mice. It is known that complex systems often exhibit two or more stable states - like split & unsplit, or rhythm & no rhythm - that are accessible by small, properly timed perturbations. The preferred state of such systems may switch under certain conditions, e.g., if the outside environment or a system component is altered. Using hamsters and mice, we present preliminary evidence for the potential bi-stability of the rodent circadian system and outline an experimental program for investigating its neurobiology. In Aim 1, we test our hypothesis that bi-stability in the hamster circadian system is revealed in split hamsters that are transferred from LL to darkness, a condition in which the normally favored unsplit state becomes less robust and more vulnerable to being switched to the split state. In Aim 2, we test our hypothesis that LL fosters splitting by introducing an element of noise into an inherently bi-stable circadian system, ultimately propelling the switch from an unsplit to split state. We also test our hypothesis that the running wheel itself is a necessary part of the splitting process. In Aim 3, we test our hypothesis that genetically-deficient murine circadian systems, being less robust than wild type in the circadian domain, are vulnerable to being switched to alternative rhythmic or quiescent states, [especially] by the tonic or noisy inputs in LL. We predict that our studies will provide new insights on the organization of complex circadian systems, on their vulnerability to imperfections in system components and environmental changes, and perhaps even on our views of circadian dysrhythmias and how they might be repaired.

DESCRIPTION (provided by applicant): Daily rhythms of physiology and behavior are governed by an endogenous timekeeping mechanism (a circadian clock). In addition to photic signals, social cues may play an important role in the function of the circadian system. The efficacy of social cues may require that cohabitants be in direct physical contact for a relatively long period of time, but the experimental problem has been that the usual methods of recording daily activity fail to distinguish the activities of individual animals housed together in the same cage. We present preliminary data that we believe open new opportunities to pose questions and address mechanisms at this social level of temporal organization. We have found that co-housing pairs of golden hamsters or female laboratory mice can result in a change in a circadian clock property (the free-running circadian period, D). We have also developed and tested miniature implantable devices for longitudinally recording body temperature and general locomotor activity in individual rodents. In this application, we wish to exploit these advances by proposing three specific aims. In Aim 1, we propose to develop a chronobiology of cohabitation in hamsters, asking if the cohabitation-associated change in D is a function of the phase relationship between cohabitants, depends on wheel running, exhibits a sex difference, and leads to an altered phase or distribution of activity during photic entrainment. In Aim 2, we seek to implicate two likely factors - olfaction and gonadal hormones - as candidate neuroendocrine elements in the pathways involved in transducing social cues into signals that ultimately affect the oscillation of the master circadian pacemaker in the suprachiasmatic nucleus (SCN). In Aim 3, we begin to investigate a social clock in female laboratory mice, asking if group synchronization can be a consequence of cohabitation-associated changes in D and how cohabitation outcome is affected by co- housing wild type mice with behaviorally-defective, mutant cage-mates (Clock ?19/?19, Avpr1atm1Dgen).