A platform for high throughput, cell type-restricted in vivo knockdown of pre- or postsynaptic gene expression

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One of the most challenging problems in science is to understand how the molecules expressed by nerve cells in the brain enable thoughts and actions to take place. Addressing this challenge is of fundamental importance for understanding how brains work. It will also underpin future development of therapies for neurological and psychiatric disorders, and of biologically inspired computing technologies. However, while cells in the brain are very much like those in other organs, figuring out how molecules in cells contribute to brain functions is exceptionally challenging because of the brain's great complexity. Conventionally one could study the function of a molecule by finding drugs that bind to it, or by engineering animals from which the molecule is deleted. However, many brain functions involve signals being passed between different types of cell that are found nearby one another. With conventional approaches it is usually difficult and very time consuming to figure out which cell type mediates a molecules effects. Moreover, with these approaches a single model animal can usually only be used to study one molecular target.To address these issues we propose to develop a system for relatively low cost and efficient investigation of the role of any given molecule in signalling between a particular population of neurons and identified neurons with which they interact. This system will use viruses to introduce two types of genetic sequence into neurons. The first encodes short interfering RNAs. These are short genetic sequences that contain recognition sites that enable them to "knockdown" targeted molecules. They can be designed to knockdown expression of almost any molecule of interest. The second type of genetic sequence encodes proteins that act either as fluorescent labels or as light-sensitive neuronal activators. These proteins can be used to identify and control infected neurons. The particular power of our system comes from a novel approach we will use to control the cells in which these two types of genetic sequence are expressed. This system makes expression of both types of sequence require the presence of marker molecules called Cre and Flp. By introducing viruses into animals in which Cre and Flp label cell populations of interest, we can target expression of the virally delivered proteins and interfering RNAs to these populations.We propose to validate this new approach by developing new tools aimed at studying neural circuits in a brain area called the entorhinal cortex. This region is important for spatial cognition. Focussing on this region allows us to take advantage of well established experimental assays, while generating tools that should enable us to address previously very challenging questions. Our first aim will be to generate and characterise tools that knockdown expression of particular ion channels in only a single type of cell. Our second aim will be to make tools that knockdown expression of receptors at either the up- or downstream side of a connection between two distinct populations of neurons. The tools will also allow the upstream neurons to be activated specifically with light and the downstream neurons to be identified by their fluorescence in order to guide subsequent electrical recordings. Our third aim will be to carry out preliminary work to extend our approach to investigation of multiple molecular targets in parallel in the same animal.On completion of the project we aim to have introduced and validated a new toolset for high throughput and low cost investigation of the roles of signalling molecules at connections between defined neuronal populations. The platforms that we aim to establish will be of general utility for fundamental and applied research into molecular mechanisms of signalling between cell populations. Applications include investigation of mechanisms of cognitive function in the young and ageing brain, and development of novel models for drug development.