Researching Cellular, Molecular, and Integrative Mechanisms
in the Brain's Circadian Clock

Dynamics of SCN and Hippocampal Glial Cells Over Day-Night Cycle

Beyond their roles in structural and trophic support of brain tissue, astrocytic glia mediate rapid responses to altered internal and environmental conditions through plastic morphological changes. Astrocytes have been regarded as morphologically similar throughout the brain, however, we found unstimulated daily changes in astrocyte morphology in both the hippocampal dentate gyrus (DG) and suprachiasmatic nucleus (SCN). Branching patterns are quantitatively more elaborate in the SCN relative to the DG. These patterns increase and decrease in complexity throughout the day-night cycle, but with a 180 degree phase difference. Light experienced in the early night changes astrocyte branch morphology in both regions, but in opposite directions. The magnitude of daily changes in astrocyte morphology in the hippocampus and SCN raises questions about their functional consequences. Electrical coupling between astrocytes also changes dynamically. Mapping these neuron-glial dynamics was the target of two recently completed BRAIN Initiative grants from NSF and NIH.

Quantitative Analysis of Peptides in the Suprachiasmatic Nucleus (SCN) and Identification of a New Functional Form of the Neuropeptide VIP

Understanding how a small brain region, the suprachiasmatic nucleus (SCN), can synchronize the body's circadian rhythms is an ongoing research area. This important timekeeping system requires a complex suite of peptide hormones and chemical transmitters that remain incompletely characterized. With our colleague, Jonathan Sweedler, a renowned analytical chemist, we developed novel sample-collection methods and then characterized in the SCN the most complete peptidome (>440 peptides) of any brain region. Of these, nearly half are from propeptides that are cleaved into functional peptides. 207 peptides were analyzed by two label-free methods, spectral count and spectral index. There were 24 peptides with significant (adjusted p-value < 0.01) differential peptide abundance between daytime and nighttime. We determined/are determining functionality of the most salient novel SCN peptides, with surprising results. These discoveries are being made via the National Institute of Drug Abuse (NIDA) Center in the Neuroproteomics of Cell-Cell Signaling (J.V. Sweedler, PI, now its third funding period). We are extending these discoveries to examine the roles of small molecules and peptide neuromodulators, released by the brain's circadian clock in the SCN, in coupling to and synchronizing peripheral body clocks.

Neuroengineering: High Resolution Analysis of Neuronal Development

Understanding and producing ordered networks of neurons with defined connectivity in vitro presents unusual technical challenges because the results must be compliant with the biological requirements of rewiring neural networks. To address this issue, we developed the ability to form stable, instructive surface-bound gradients of laminin that guide postnatal hippocampal neuron development in vitro. Using this microfluidic environment, we are able to direct single dendrites through fluidically isolated channels. Nascent neuronal extensions are initiated by dendritic filopodia. We guide emerging filopodia into distinct, controlled micro-environments. We are using this highly controllable environment to study the function of miR125b, a microRNA implicated in schizophrenia, depression, and fragile X mental retardation, as well as extracellular guidance cues. We have completed 3 innovation grants in this area: one from the Keck Foundation and two from the National Institute of Mental Health (NIMH) and recently been awarded a new R21 from NIMH to study redox regulation in dendrites. We have published highly-cited papers in this domain, including in Lab on a Chip, as well as a book chapter on methodology.

Neuroengineering: Emergent Behavior of Integrated Cellular Systems

The goal of our research under the NSF-funded EBICS Science & Technology Center is to understand and control the emergence of complex neuronal systems. To generate systems-level behaviors, we are studying effects of self-directed differentiation, cell-to-cell signaling, and local environmental cues. We are probing developmental capacities of hippocampus and spinal sensory neurons in order to generate clusters of cells with specific types of functionalities. This comparison includes defining conditions that optimize cell density for innervations. Our near-term goal is to build an oscillatory spinal-skeletal muscle system, the neuronal circuitry that drives oscillatory motor activity. The long-term goal of this research endeavor is to build machines out of cellular parts. In collaboration with the Bashir, Kong, and Popescu labs at UIUC, we continue to develop a living biological machine composed of multiple cell types, characterize the physiology of hippocampal neurons, and optimize the use of Spatial Light Interference Microscopy (SLIM) to image neurons in culture with the eventual aim of using the technique to study the mass dynamics of the bioactuator. With these studies, we have bridged the usual communications gulf between scientists and engineers. We succeeded in publishing these studies well because they are strong in both neuroscience and novel methodologies. We are competitive for funding that fosters cross-disciplinary research applying cutting-edge technology. Additionally, our strong collaborations across disciplines contributed to our success in acquiring an NSF Understanding the Brain Training Grant focused on Miniature Brain Machinery to develop future scientists with expertise ranging from engineering and cognitive sciences. Importantly, these approaches are enabling us to understand the biology in new and unanticipated ways.

Grants

We are working currently under one grant awarded by the National Institutes of Health and two from the National Science Foundation, investigating: the "High Resolution Analysis of Redox Regulation in Dendrites," "Engineering Neuron-Innervated Muscle with Stimulus-Responsive Contraction and Myokine Secretion" in collaboration with PI Hyunjoon Kong, and "Emergent Behavior of Integrated Cellular Systems (EBICS)," an NSF Science & Technology Center grant to a consortium of institutions including the University of Illinois, the Massachusetts Institute of Technology and the Georgia Institute of Technology.

Collaborators

Our collaborators at the University of Illinois include:

Rashid Bashir - Director of Micro and Nanotechnology Laboratory, Dept. of Electrical and Computer Engineering

Rohit Bhargava - Professor of Bioengineering, Electrical & Computer Engineering, Mechanical Science & Engineering, Chemical and Biomolecular Engineering, and Chemistry

Gabriel Popescu - Professor, Electrical and Computer Engineering

Jonathan Sweedler - James R. Eiszner Family Endowed Chair in Chemistry and Director, School of Chemical Sciences

Hyunjoon Kong - Professor of Chemical and Biomolecular Engineering, School of Chemical Sciences