Neuronal Development

 

The nervous system is a fantastically complex organization of neurons and non-neuronal cells that allows organisms to sense the environment, initiate movement and produce consciousness.  We are interested in understanding how the nervous system develops and functions; specifically the central nervous system (CNS) (i.e. the brain and spinal cord).  Two regions of the brain that we focus on are the cortex and hippocampus.  When plated in culture, these neurons undergo a stereotyped developmental pattern.  From a spherical cell body neurons first extend lamellipodia and filopodia (see figure below).  This is termed stage 1 of hippocampal/cortical development.  Neurons then extend minor processes (stage 2) that will eventually develop into dendrites (stage 4).  In stage 3 one minor process begins to extend rapidly to become the axon.  In stage 4 the other minor processes elongate and thicken to become dendrites.  The dendrites then mature to produce filopodia and dendritic spines (stage 5).  Our interest lies in understanding how the underlying structure of the neuron forms and functions.

Projects

Advances in Neurobiology

Dent Lab

Cytoskeletal Dynamics in Neuritogenesis, Axon Branching and Guidance

 

We are interested in understanding how the cytoskeleton is regulated in the developing and adult nervous systems.  The cytoskeleton is composed of dynamic protein polymers that give cells shape, allow cells to divide, traffic intracellular components, and are instrumental for cell motility.  In neurons the cytoskeleton is composed of filamentous actin (f-actin), microtubules and neurofilaments.  We focus on microtubules and f-actin (see figure below).  Although the name cytoskeleton suggests that these are stable polymers that act as struts or tracks, they are anything but static.  Microtubules and actin filaments  have the amazing ability to polymerize and depolymerize rapidly, which allows cells to quickly respond to the their environment.  Our working hypothesis is that f-actin/microtubule interactions serve crucial roles throughout both axonal and dendritic development.  These two cytoskeletal polymers appear to coordinate their dynamics, resulting in important developmental changes in the neuron, such as neuritogenesis, axon outgrowth, branching and growth cone guidance.  The coordination of actin/microtubule dynamics is mediated by a myriad of microtubule- and actin-associated proteins. 

Cytoskeletal Dynamics in Dendrites and Dendritic Spines

 

In addition to our longstanding interest in earlier developmental events we are also pursuing projects aimed at understanding how microtubule and f-actin dynamics are regulated in mature neurons.  At present we are studying microtubule dynamics in dendrites and dendritic spines.  It has long been held that dendritic spines, the points of communication between excitatory neurons in the CNS, contain dynamic actin filaments, but are devoid of microtubules.  Our most recent study (Hu et al., 2008 – see Publications) shows that microtubules remain dynamic in mature dendrites and rapidly extend into and retract from dendritic spines, sometime producing transient spine head protrusions (See also a study by James Zheng lab, www.jneurosci.org [Full Text] ).  We have also discovered that these microtubule dynamics in spines are regulated by activity.  The more active the neuron, the more microtubules target dendritic spines.  We are following up on these studies to understand how microtubule invasions affect dendritic spine structure and function.  

Exploiting High-Resolution Live-Cell Imaging of Neurons

 

Although we use molecular, biochemical and immunocytochemical methods, we specialize in exploiting high-resolution, live-cell imaging of neurons.  We have a standard inverted wide-field epifluorescence and transmitted light microscope (Nikon TE300) that we share with Dr. Tim Gomez in the Anatomy Department.  We use this scope for time-lapse imaging of neurons at low magnification and standard epifluorescence of fixed specimens.  Additionally, we have assembled a custom designed total internal reflection fluorescence (TIRF) microscope (http://www.nikoninstruments.com/lasertirf/) that we use for high resolution time-lapse imaging and epifluorescence in fixed specimens (also shared with the Gomez lab).  TIRF imaging only illuminates a few hundred nanometers into the sample.  Thus, for imaging dendritic spines we have to image dendrites that are directly attached to the substrate (not growing on top of other neurons/glia).  However, TIRF affords high signal-to-noise imaging and allows us to image two fluorophore-labeled proteins within neurons at high resolution for extended periods of time.  In fact, we have imaged EGFP-tubulin/DsRed2 at 30 second intervals for over 19 hours or 10 second intervals for over 8 hours!  Presently, we are developing transfected hippocampal slice cultures (via biolistics transfection with the Biorad Helios Gene Gun) to image the cytoskeleton in an intact system with scanning confocal and two photon confocal microscopy.