Dynamics of Neurons and Glia in Developing Mammalian Brain Tissues

Research in our laboratory addresses the nature, mechanisms and functional roles of dynamic changes in cell structure during mammalian central nervous system (CNS) development and following CNS tissue injury.  Cellular dynamics are studied using multi-dimensional (4-D and 5-D) time-lapse fluorescent confocal imaging in live brain tissue slices.

Our projects currently span three major areas:

Microglial activation and motility following brain tissue injury.

• Defining motility phenotypes of activated microglia using time-lapse imaging in live tissue slices.
• Determining signaling cascades underlying microglial activation.

Astrocyte development and remodeling.

• Factors regulating the development of astrocyte structure.
• Localization and dynamic remodeling of astrocyte proteins including glutamate transporters.

Neuronal synapse formation and plasticity.

• Developmental formation and plasticity of dendritic spines and synapses.
• Remodeling of dendritic spines and synapses in epileptic tissues.

Methodology

These dynamic events are being studied in rat and mouse hippocampal tissue slices utilizing techniques that include quantitative immunohistochemistry, electron microscopy, calcium imaging, biolistic gene transfection, and time-lapse fluorescence confocal imaging.  For general reviews on the use of these technical approaches in our lab, see:

• Dailey ME, Manders E, Soll D, Terasaki M. (2005) Confocal microscopy of live cells. In, Handbook of Biological Confocal Microscopy, 3rd Edition (Pawley J, ed.) (Chapter 19) in the press.
  
  Preprint (0.8MB PDF file)
• Dailey ME, Marrs GS, Kurpius D. (2005) Maintaining living cells and tissue slices in the imaging setup. In, Imaging in Neuroscience and Development: A Laboratory Manual, (Yuste R and Konnerth A, eds.) Cold Spring Harbor Lab Press: Cold Sprig Harbor, NY. (Chapter 1) pp. 1-8.
  
  Preprint (2.5MB PDF file)
• Kurpius D, Dailey ME. (2005) A practical guide to imaging microglia in live brain slices and slice cultures. In, Imaging in Neuroscience and Development: A Laboratory Manual, (Yuste R and Konnerth A, eds.) Cold Spring Harbor Lab Press: Cold Spring Harbor, NY. (Chapter 55) pp. 425-429
  
  Preprint (0.5MB PDF file)
• Dailey (2002)  Optical imaging of neural structure and physiology: Confocal fluorescence microscopy in live brain slices.  In, Brain Mapping: The Methods, 2nd Edition, (A. Toga & J. Mazziotta, eds.) Elsevier Science, pp 49-76.
  
  Amazon page    Full text (1.0MB PDF file)
• Dailey et al. (1999)  Concepts in imaging and microscopy: Exploring biological structure and function with confocal microscopy.  Biological Bulletin. 197(2):115-122.
  
  Medline abstract    Full text (5.0MB PDF file)
• Dailey and Waite (1999)  Confocal imaging of microglial cell dynamics in hippocampal slice cultures. Methods. 18(2):222-230.
  
  Medline abstract    Full text (343K PDF file)    Fig. 4 Color Plate (165K JPEG file)

I. Microglial Activation and Motility

Microglia are resident brain cells that mediate responses to CNS tissue injury.  Microglia become "activated" following brain tissue injury, and rapidly transform from a resting, ramified form to a highly mobile, macrophage-like form.  This transformation appears to be essential for expression of the full repertoire of microglial functions.  We seek to better understand the cellular and molecular mechanisms of microglial activation, and to define the functional behavior of activated microglia in relation to dead and damaged neurons.  We are using time-lapse imaging of microglia in live rat and mouse brain slices to study the cellular dynamics underlying activation, and to explore the diversity of microglial behaviors following activation.  Dual-channel fluorescence imaging is being utilized to study cell-cell interactions between activated microglia and dead/dying neurons.  We also are investigating whether particular signaling mechansisms and cascades, such as NF-κB dependent cell adhesion molecule expression, regulates changes in microglial motility.  A better understanding of the cellular and molecular bases of microglial activation and motility may reveal avenues for regulating microglial function in relation to neuro-pathological conditions in humans.

Publications on Microglia:

• Dailey and Waite (1999)  Confocal imaging of microglial cell dynamics in hippocampal slice cultures.  Methods.  18(2):222-230.
  
  Medline abstract    Full text (343K PDF file)    Fig. 4 Color Plate (165K JPEG file)
• Stence et al. (2001)  Dynamics of microglial activation:  a confocal time-lapse analysis in hippocampal slices.  Glia.  33(3):256-266.
  
  Medline abstract    Full text (803K PDF file)
• Grossmann et al. (2002)  Juxtavascular microglia migrate along brain microvessels following activation during early postnatal development.  Glia.  37(3):229-240.  [with journal cover.]
  
  Medline abstract    Full text (3.8MB PDF file)
• Dailey ME, Fuller L, Hoffman A, and Kurpius D (2003)  Microglial activation in brain slices:  Time-course of nuclear translocation of NF-κB and changes in surface expression of cell adhesion molecules.  (Annual Society for Neuroscience Meeting abstract).
  
  Full text of abstract
• Petersen MA, Dailey ME (2004)  Diverse Microglial Motility Behaviors During Clearance of Dead Cells in Hippocampal Slices.  Glia 46:195-206.
  Full text (1MB PDF file)
• Kurpius D, Wilson N, Fuller L, Hoffman A, Dailey ME (2006)  Early activation, motility, and homing of neonatal microglia to injured neurons does not require protein synthesis.  Glia Jul;54(1):58-70.
  Medline abstract  Preprint (3MB PDF file)
  

• Kurpius D, Nolley E, Dailey ME (2007)  Purines induce directed migration and rapid homing of microglia to injured pyramidal neurons in developing hippocampus.  Glia 55:873-884.
  Medline abstract  Preprint (3MB PDF file) Cover Illustration

Click here to view movies of microglia.

II. Astrocyte Development and Remodeling

For many years, astrocytes were thought of as supporting "glue" for CNS tissues, but they are now emerging as intimate partners with neurons in a wide variety of physiological functions.  Indeed, recent work suggests that astrocytes, like neurons, may contain local signaling domains, or "micro-domains."  However, little is known about the functional organization of these highly complex cells.  We are interested in understanding structure-function relationships in astrocytes, that is, how their unique structure (at the cellular and molecular levels) contributes to their diverse functions.  Existing in vitro models have limited the progress on these topics somewhat because astrocytes in dissociated cell culture display an abnormal, flattened shape.  In contrast, astrocytes in tissues in vivo have diverse and highly complex forms.  To facilitate studies on astrocyte development and remodeling, we are utilizing an in vitro tissue slice model in which astrocyte structure is much more akin to that seen in vivo (see 3D view of an astrocyte in slice culture).  To visualize the structure and physiology of individual astrocytes in tissue slices, we are using ballistic (gene gun) mediated labeling to introduce fluorescent membrane dyes (DiI), proteins (e.g., LCK-GFP, GLT1a-GFP), or physiological indicators (e.g., calcium probes).  Time-lapse confocal imaging of live, labeled astrocytes is enabling a dynamic analysis of astrocyte structure and physiology in the context of a more intact brain tissue environment.  These technical approaches are being used to study some fundamental questions related to astrocyte cell biology, such as:

• Factors regulating the development of astrocyte structure.
• Spatio-temporal patterns of calcium signaling in astrocytes in situ. (Click here to see a movie.)
• Localization and dynamic remodeling of astrocyte proteins, including glutamate transporters.  Work on glutamate transporters in astrocytes is being done in collaboration with Dwight Bergles (Johns Hopkins) and Jeffrey Rothstein (Johns Hopkins).

Publications on Astrocytes:

• Benediktsson AM, Schachtele SJ, Green SH, Dailey ME.  (2005) Ballistic Labeling and Dynamic Imaging of Astrocytes in Organotypic Hippocampal Slice Cultures. 
  Journal of Neuroscience Methods 141:41-53.
  Preprint (752KB PDF file)    Link to Supplemental Video (Fig. 2G)
  

• AM Benediktsson¹, GS Marrs¹, JC Tu³, PF Worley³, JD Rothstein³, DE Bergles³, and ME Dailey^1,2 (2003)  Clustering and dynamic remodeling of a glutamate transporter, Glt-1a, in hippocampal astrocytes.  (Annual Society for Neuroscience Meeting abstract).
  
  [Affiliations:  ¹Program in Neuroscience, ²Dept. of Biological Sciences, Univ. of Iowa, Iowa City, IA, and ³Dept. of Neuroscience, Johns Hopkins Univ. Med. Sch., Baltimore, MD, USA.]
  
  Full text of abstract

Click here to view movies of astrocytes.

III.  Neuronal Synapse Formation and Plasticity

1. Developmental formation of dendritic spines and synapses.  Most excitatory synapses in the mammalian brain are formed at tiny dendritic protrusions, termed dendritic spines.  Dynamic changes in spine structure are known to occur during normal brain development, and they likely contribute to synaptic plasticity underlying processes such as learning and memory.  Our research seeks to better define the cellular and molecular mechanisms regulating synaptic spine development. Dendritic spines and synapses are being visualized in living neurons by gene transfection techniques that yield fluorescent fusion proteins.  Time-lapse imaging of transfected cells is enabling direct observation of the dynamics of synaptic structures, and this data is being used to construct a model of CNS synaptogenesis.

   Based on time-lapse analyses of developing hippocampal dendrites in slice cultures, spiny dendritic protrusions can be classified on the basis of spine lifetime and motility into one of three categories:  filopodia, protospines, or spines (Dailey & Smith, 1996).  Mature dendritic spines are formed from dynamic spine precursors (filopodia and protospines) that become stabilized, or occasionally by direct extension from the dendrite shaft.  By imaging a GFP fusion protein of PSD95, a PDZ-domain scaffolding molecule, we found that the conversion of filopodia to protospines coincides with the formation of a postsynaptic density (PSD) containing PSD95 (Marrs et al., 2001).  We also found that PSD95-containing spines can form independent of neural activity or glutamate receptor activation (Qin et al., 2001).  Results from these studies show that:

   1. Conversion of filopodia to spines involves assembly of a core post-synaptic density (PSD) scaffold (time-course: ~0.5-2hr).
   2. PSDs in developing spines (proto-spines) are highly dynamic:  they can rapidly appear or disappear, as well as grow, shrink, move and possibly split and merge.
   3. The presence of a presynaptic axon terminal can trigger assembly of a core PSD scaffold, but this is not mediated by neurotransmitters.

   CaMKII.  Calcium-calmodulin dependent protein kinase II (CaMKII) is another major component of the postsynaptic density (PSD) at CNS synapses.  We are examining CaMKIIα recruitment and trafficking at nascent synapses in developing neurons transfected biolistically with a GFP-CaMKIIα fusion cDNA.  Time-lapse observations show that CaMKIIα is recruited to spines at early stages of spine development, during the maturation of protospines into mature, stable spines.  Experimental treatments in slice cultures indicate that recruitment of CaMKII to spines involves synaptic activity-dependent mechanisms and (synaptic activity-independent) F-actin based mechanisms.  Ongoing studies are focused on defining a role for CaMKIIα in spine assembly and maintenance, such as whether CaMKII promotes spine maturation and recruitment of other synaptic proteins in developing spines.  This work on CaMKII is being performed in collaboration with our Iowa colleagues, Steven Green (Biological Sciences), Stefan Strack (Pharmacology), and Johannes Hell (Pharmacology).

   Publications on Dendrite and Synapse Formation:

   • Dailey & Smith (1996)  The dynamics of dendritic structure in developing hippocampal slices.  Journal of Neuroscience.  16(9):2983-2994.  [with journal cover.]
     
     Medline abstract    Full text (1.3MB PDF file)
   • Marrs et al. (2001)  Rapid formation and remodeling of postsynaptic densities in developing dendrites.  Nature Neuroscience.  4(10):1006-1013.
     
     Medline abstract    Full text (4.2MB PDF file)
   • Qin et al. (2001)  Hippocampal mossy fibers induce assembly and clustering of PSD95-containing postsynaptic densities independent of glutamate receptor activation.  Journal of Comparative Neurology.  440(3):284-298.
     
     Medline abstract    Full text (1.1MB PDF file)
   • Ahmed et al. (2006)  Synaptic activity and F-actin coordinately regulate CaMKIIa localization to dendritic postsynaptic sites in developing hippocampal slices.  Molecular and Cellular Neuroscience.  31:37-51.
     
     PubMed abstract    Full text (1.1MB PDF file)
   • Marrs et al. (2006)  Dendritic arbors of developing retinal ganglion cells are stabilized by b1-integrins.  Molecular and Cellular Neuroscience.   32:230-241.
     
     PubMed abstract    Full text (1.1MB PDF file)

 

2. Remodeling of dendritic spines and synapses in epileptic tissues.  Abnormal patterns of neural activity, which occur during epilepsy, can induce changes in neuronal structure and synaptic connectivity.  We use hippocampal slice cultures as an in vitro model to explore the cellular and molecular mechanisms by which epileptiform activity induces changes in dendritic branch, spine, and synaptic structure.  Currently, we are testing whether CaMKII activation plays a role in epilepsy-induced dendritic spine remodeling.  Calcium imaging in hippocampal slice cultures shows synchronized, epileptiform patterns of calcium transients in pyramidal neurons that develop spontaneously or following treatment with GABA_A receptor antagonists, gabazine or picrotoxin (Dailey, 2002; see Figure at right).  In slice cultures, epileptiform patterns of activity that persist for 48-hr induce withdrawal of ~50% of spines.  Preliminary evidence also indicates that epileptiform activity enhances the expression and activation (phosphorylation) of CaMKIIα.  Moreover, expression of specific molecular inhibitors of CaMKII activation reduce or prevent spine withdrawal, indicating that CaMKII activation is essential for structural remodeling of spines in response to epileptiform activity.  This work on CaMKII and epilepsy is being done in collaboration with Steven Green (Biological Sciences).

   Publications on Epileptiform Activity and Spines:

• Zha X-M, Green SH, Dailey ME. (2005) Regulation of hippocampal synapse remodeling by epileptiform activity. Molecular and Cellular Neuroscience. 29(4):494-506. *

* "Recommended" by Faculty of 1000. (http://www.facultyof1000.com/article/15953736).

Preprint (966KB PDF file) Link to Supplemental Video


Click here to view another movie of an epileptic hippocampal tissue slice.