My students and I are interested in the details of brain development. Our work is focused mainly on the visual system. The purpose of much of our research is to reveal how neurons in the visual system respond to perturbations of visual input during the formative months of early life. We use a multitude of techniques, some new and some conventional, to pursue our interests. The following provides a few examples of what we are doing.

Immunofluorescence:

We have examined many molecules within the developing brain in an attempt to understand how neurons effect major changes in their gross structure in the context of abnormal visual input.


Sections of thalamus cut 14 microns thick. Each section is reacted for 3 neuronal features: neurofilament protein (red), Nissl substance (green), and nucleic acid (blue). This reaction protocol permits quantification of the relationship between neuron gross structure and protein composition.

Microelectrophoresis:

Using this technique we are investigating the fine structure of neurons in the developing brain to better understand the molecular events that underlie aberrant development of neurons that ultimately leads to a severe impairment in visual function – called amblyopia.


This video demonstrates the microelectrophoresis technique on a thalamic neuron. A sharp glass pipette is filled with a blue fluorescent dye (Alexa350) and is maneuvered with an electronic controller so that its tip impales a selected neuron. A green dye, Alexa488, is ejected from the pipette tip and into the neuron by a small current applied to the pipette. The green dye comes to fill the entire neuron, including its axon and dendrites, and thereby reveals its 3-dimensional structure.

 

Zymography:

We are developing techniques to examine enzymatic activity, in situ and in vivo, within the developing visual system. Results from several of our past experiments implicate protease activity as a key contributor to the neuronal structural changes that parallel vision impairments brought about by disrupted sensory input.



Protease activity is revealed in neurons by application of a substrate that fluoresces (in the above case green) when fragmented. This is a powerful technique to evaluate the activity of enzymes in the nervous system.

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