Time-lapse, In Vivo Imaging of Neural Circuit Formation in the Visual System

Justin Crowley, Ph.D.

Carnegie-Mellon University

Funded in December, 2006: $293000 for 3 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Cellular Imaging May Reveal How Neurons Connect Into Brain Circuits During Development

Carnegie Mellon scientists will use cellular imaging in laboratory animals to understand how normal brain circuits are formed during development.

There are two competing explanations for how brain cells find the right neighbors to connect up with, to form the neural circuits that enable the brain to function normally.  One theory, an “activity dependent” explanation, suggests that there is an initial exuberance of connections, followed by retraction of all connections that are found, by experience, to be inappropriate.  The contrasting explanation suggests that, rather than guided by experience, neural connections are guided in a rapid and precise manner by patterns of gene expression.  Preliminary results from the Carnegie Mellon researchers’ initial studies favor this latter explanation.

They will differentiate between the two explanations by using time-lapse two-photon laser scanning microscopy in the animals’ visual cortex.  The imaging will enable them to see the actions of the same brain cells over time, in combination with physiological imaging techniques that will detect the activity patterns of large groups of neurons.  In parallel with these imaging techniques, the investigators will work to determine which molecular patterning cues may be involved in structuring neural circuits.  The will identify candidate genes, and then image the consequences to neural connections when these genes are either over-or-under expressed in the laboratory animals.

Significance:  This research may identify the fundamental clues underlying brain circuit formation, which would represent a major advance in our understanding of neural development.  Clinically, this could lead to development of approaches to prevent and treat disorders of neural development and to therapies to improve recovery from trauma and neurodegenerative diseases that destroy brain connections.

ABSTRACT

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Time-lapse, In Vivo Imaging of Neural Circuit Formation in the Visual System

Establishing a thorough understanding of how neurons connect to form the circuits of the brain
in normal development will be a critical step toward prevention of and treatment for a variety of central nervous system pathologies. The research proposed here focuses on the formation of neural circuitry in the visual system, specifically the development of the neural processing modules in primary visual cortex for ocular dominance columns and orientation preference columns. The patterning forces underlying the formation of these circuits have been hypothesized to be patterns of gene expression or patterns of neural activity or a combination of these influences. Currently, the relative importances of these patterning forces are a subject of debate.

This research employs in vivo two-photon laser scanning microscopy and optical imaging of intrinsic signal to explore both the structure and function of neural circuitry during development. Two-photon imaging enables the examination of fine scale neuronal structure, in real time, as neurons develop and change.This enables time-lapse assessment of the magnitude of axonal pruning and the specificity of axonal outgrowth in both normally reared animals and those with manipulations of neural activity.

In order to assess gene expression differences correlated with cortical column identity, we will determine proteome-level differences between physiologically defined samples using differential gel electrophoresis and identify differently expressed candidates by mass spectroscopy. Candidate patterning genes will be tested for their competence to influence cortical circuit organization by manipulating their expression levels using viral transfection and electroporation. In parallel with these studies, we will continue to refine the use of laser scanning microscopy in larger mammals, which possess cortical columns. This approach, combined with physiological and other anatomical techniques, will shed light on the dynamic interplay between structure and function in the developing visual system and allow the examination of the mechanisms underlying the development of both ocular dominance and orientation preference.

HYPOTHESIS

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Hypothesis:
The formation of precise patterns of neural circuitry has been argued to result from either neural activity-dependent influences or genetically programmed patterning cues.  This research is based on the hypothesis that the initial development of columnar circuitry in visual cortex occurs in a rapid and precise manner more consistent with guidance by patterns of gene expression than patterns of neural activity.

Goals:
The goal of this work is to determine the mechanisms underlying the formation of columnar circuitry in the visual cortex.  We will directly observe the formation of the neural circuits that give rise to ocular dominance and orientation columns and determine the proteomic correlates of these structures in development.  Taken together, these data will provide new information on the forces that act on neocortical axons as they grow, eventually determining the functional organization of the neocortex.

Methods:
In order to assess the specificity of axonal outgrowth as columnar circuits in visual cortex are formed, we will image developing axons in a time-lapse experimental design as they grow toward their targets.  These data will allow us to ascertain the relative importance of axonal pruning and selective, nonrandom process outgrowth in the formation of cortical circuits.  In addition, we will directly explore the proteomic correlates of columnar circuitry in visual cortex using differential protein gel analysis of physiologically identified cortical columns.

SELECTED PUBLICATIONS

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Kawasaki H., Crowley J.C., Livesey F.J. and Katz L.C.  Molecular organization of the ferret visual thalamus. J Neurosci. 2004 Nov 3;24(44):9962-70.

Mizrahi* A., Crowley* J. C., Shtoyerman E., and Katz L. C.  High resolution in vivo imaging of hippocampal dendrites and spines.  J Neurosci. 2004 Mar 31;24(13):3147-51.

Crowley J.C. and Katz L.C.  Early development of ocular dominance columns. Science. 2000 Nov 17;290(5495):1321-4.