fosGFP Mouse: A Novel Transgenic Mouse for Identifying Subsets of Activated Cells

Applications

• Analyzing neural activation caused by a stimulus, including:
• 1) Identifying which subsets of neurons are activated by administration of a drug
• 2) Quantifying the level of neural activation caused by a drug
• 3) Analyzing a drug's neural activation over time
• 4) Visualizing neural activation caused by depression, anger, pain or other emotional stimuli
• 5) Indentifying drugs that penetrate the blood/brain barrier
• Identifying activation without immunochemisty: The fosGFP Mouse can be used to identify cfos-expressing cells without immunohistochemistry. This simplifies the procedure for examining cfos induction in that tissue does not require fixation, block, primary or secondary antibody application, or any washes. It allows a much higher throughput for examination of cfos expression patterns under a variety of experimental conditions.
• Using DNA microarrays to generate expression profiles of labeled cells: cfosGFP labeled cells could be used with microdissection to look at expression profiles of labeled cells using DNA microarrays. An advantage over current technology here is it does not require you to fix or permeablize the tissue to find "activated" cell, processes which decrease the amount of mRNA that you can recover from a cell.

Advantages

• Detecting GFP expression in vivo without additional manipulation (e.g. fixation, dye administration).
• Performing live-cell assays on subset of activated cells.
• Examining in vivo the electorphysiological properties of neurons.
• Previously neuroscientists have been able to identify an *area* of the brain that may have been activated by a stimulus, but they were unable to isolate the particular subset of cells that are directly involved in mediating the behavior or the "memory." The fosGFP Mouse enables direct identification of this subset of cells without killing the cells. Thus, the electrophysiological properties of the neuron can be examined and other in vivo properties of the cell (for example, its response to a pharmacological compound) can be assayed.

Scientific Context:

To determine the neural basis for behavior and memory, scientists have employed a variety of techniques to allow the identification of brain areas that may be involved. For example, tissue lesion, either by stroke, surgery or toxin exposure, has been used to verify the role of a CNS area in a particular task. This has been a fruitful method in some respects, as it has allowed identification of the hippocampus, for example, as a site of short-term memory storage. However, lesion analysis has not provided a direct test of functional involvement, and usually involves the disruption of an enormous number of neurons, many of which may not be involved in the behavior being assayed.

In a similar attempt to uncover the neural basis of memory and behavior, other scientists have recorded from single neurons in vivo, in an attempt to look for changes in neuronal firing properties after training or during a particular behavioral state. However, this technology is also limited in that it is difficult to distinguish the activated subset of neurons (signal) that are directly involved in a behavior from those that are not (noise). For this reason, sophisticated statistics and large data sets are required to detect any changes between trained and untrained animals, for example.

The fosGFP Mouse addresses this issue in a novel way. Based upon the long-standing observation that immediate-early genes such as c-fos are expressed by individual cells in a regionally specific manner after a stimulus, we have created a reporter gene construct that allows in vivo identification of activated subsets of cells. The fosGFP Mouse, carrying the c-fos gene coupled to GFP, thus expresses GFP when and wherever the c-fos gene is expressed. Using whole-cell recording techniques, this allows us to functionally identify individual cells (neurons) that are activated following a given stimulus or learning event, as well as detailed analysis of the types of changes in this subset of cells that accompany learning.

The fosGFP Mouse offers the ability to determine the neural basis of behavioral states, learning, and memory by allowing us to assay function, in living brain cells. An important benefit of this technology is that it enables functional characterization of cellular subtypes involved in pathological states, as well as the ability to directly examine drug efficacy against an identified target population of cells using whole-cell recording techniques or other assays of cell function.

Patents

• Published Application: 20040031065