Optical Monitoring of Cortical Functions as a Method for Screening of Disease-Linked Genes and Prospective Therapeutic Compounds

Lay Summary

An Optical Imaging Tool for Understanding and Treating Developmental and Psychiatric Disorders

This research will employ two cellular imaging techniques to develop a new method to identify and quantify changes in glutamate synaptic transmission that are related to developmental and psychiatric disorders in a mouse model and to assess the effects of therapies.

Human genetics studies are beginning to uncover many genes that may contribute to neurodevelopmental and psychiatric disorders.  The researchers hypothesize that many of these human genetic mutations directly or indirectly lead to changes in synaptic transmission of glutamate in the cortex.  To test this hypothesis, they will develop an optical imaging method that can measure the distribution of two major types of receptors that receive glutamate signals from neighboring brain cells as the cells communicate. Then they will monitor the receptors’ functions as glutamate is passed from one cell to another. The imaging technique requires combining two-photon cellular imaging with a new fluorescent labeling technique, in which a certain type of antibody binds to, and turns on, a fluorescent organic dye molecule.  Through this process, they will be able to visualize the two types of glutamate receptors in the dendritic spines of brain cells, which receive glutamate signals from their neighboring cells.

They predict that the several mouse models that have been engineered to harbor disease-linked genetic mutations will show changes in the distribution of these two glutamate receptors at the synapses and that these changes can be quantified. Additionally, they predict that they can successfully use this new combined optical imaging technique to screen drugs for their abilities to reverse the observed changes in the distribution of glutamate receptors.

Significance:  The new combined optical imaging approach developed in mouse models may reveal how genetically determined changes in glutamate transmission lead to developmental and psychiatric disorders and become a tool for testing therapies designed to reverse these.

Abstract

Optical Monitoring of Cortical Functions as a Method for Screening of Disease-Linked Genes and Prospective Therapeutic Compounds

We hypothesize that many human genetic mutations linked to neurodevelopmental and psychiatric diseases cause directly or indirectly changes in glutamatergic synaptic transmission in the cortex. Therefore, we propose to develop a method for optical monitoring of glutamatergic synaptic functions in the mouse cortex, based on a combination of in vivo two photon imaging and novel protein tagging with single chain variable fragment (scFv) antibodies binding small organic dyes. This method is designed to be fairly non-invasive and applicable for repeated measurements in order to compare conditions before and after drug applications. We envision that the method can become streamlined as a relatively high-throughput assay for pharmaceutical drug screening.

The specific aims are:

Aim 1: To establish conditions for acute in vivo imaging of scFv-tagged AMPA GluR1 and NMDA NR1 receptors expressed from recombinant adeno-associated virus (rAAV) vectors.

Aim 2: To establish conditions for chronic in vivo imaging of scFv-GluR1 and scFv-NR1 receptors expressed from rAAV vectors.

Aim 3: To generate transgenic mice expressing scFv-GluR1 and scFv-NR1 receptors.

The methods we plan to use to achieve these aims include two-photon imaging of cortical neurons in anesthetized mice and a novel labeling technique, which will allow us to selectively visualize synaptic surface expressed glutamate receptors, GluR1 subunit-containing AMPA receptors, or NR1 subunit-containing NMDA receptors. The technique development will be done in wild type mice, using recombinant adeno-associated vectors for expression of the tagged GluR1 and NR1 protein. The final deliverable from the project are transgenic mice stably expressing the tagged glutamate receptor subunits in cortical neurons.