A platform for high throughput two-photon-targeted in vivo cellular physiology

The whole-cell patch clamp recording technique has had much impact in the Life Sciences by allowing the currents in ion channels to recorded: this has been crucial to advances in our understanding of cellular function. Use of the patch-clamp technique in vivo, however, has been difficult, because it essentially has to be carried out "blind", with the only feedback obtained by monitoring the impedance of the micropipette used to make the recording.

Recent developments in multiphoton microscopy now allow us to make targeted recordings from cells that have been fluorescently labelled. It has also recently been shown that in vivo patch-clamp recordings can be automated. In this project, we will combine these principles to develop a new approach for whole cell patch clamp electrophysiology. Our platform will allow genetically targeted classes of cells to be visually selected ("point and click") by a human operator, and then automatically patched by a group of robotic micromanipulators capable of obtaining recordings from up to six cells simultaneously. As well as allowing high throughput characterization of cells in vivo for basic scientific or drug discovery purposes, our platform will allow new scientific questions to be asked involving interactions between cells that have not hitherto been addressable. Finally, the precision afforded by the automated robotic control system will allow the patch-clamping of subcellular structures in vivo, which has not previously been systematically achievable. We will demonstrate the utility of our two-photon targeted robotic patch clamp platform by using it to target two particular classes of pyramidal cell in the mouse cerebral cortex, in order to ascertain their respective roles in processing sensory information.

Our objectives are
1. To develop a platform for making high yield robotically assisted whole cell patch clamp recordings from multiple genetically targeted and visually selected cells in vivo.
2. To demonstrate the utility of this technology by using it to resolve an unanswered question about mammalian cortical circuit function: what are the respective roles of slender and thick-tufted layer V pyramidal neurons in processing sensory information?

We will achieve our first objective by building a customised two-photon microscope, utilising ScanImage, an open source toolbox for microscope control. An additional module will be developed for ScanImage which allows point-and-click acquisition of cellular target locations, resulting in automated patch-clamp recording from the targeted cell. The robotic patch-clamp control algorithm will monitor impedance and an electropneumatic converter to control micropipette pressure. Pressure will be maintained at a high level until the pipette is in the target location. Multiple pipettes will be operated simultaneously.

Once the platform has been developed, it will be used to address the second objective, by making use of two available Cre mouse lines, Etv1 and Glt25d2, together with an AAV vector made in house, to obtain targeted patch-clamp recordings from layer 5 pyramidal cells of defined class, while somatosensory stimulation is performed.

The project will impact upon:
Life Scientists (see Academic Beneficiaries)
The UK Life Science Instrumentation Industry
This will be of immediate benefit to our industrial partner, Scientifica Ltd, a rapidly growing UK Life Sciences instrumentation company, who will be involved in commercializing developments from the project. However, we expect the benefits to extent to the wider UK Life Sciences industry, who will be galvanized by our developments.
The advances gained over competing Life Sciences instrumentation companies in other countries will improve the economic competitiveness of the UK
These benefits are likely to follow reasonably quickly after the end of the project (within 1-3 years), as the potential product takes advantage of a number of separate things we already know how to do, and could be brought to market quickly
Drug discovery companies
This includes both "Big Pharma" and the new breed of smaller, more dynamic drug discovery companies.
Products focused on drug discovery may require scaling up our system ("higher throughput"), and thus are likely to follow 3-5 years from the end of the project.
Some parts of the Charity Sector will in the longer run reap many benefits from the technology we propose, as many problems of interest to Charities such as the Alzheimer's Trust involve specific cell types that are not well physiologically characterized in vivo, and can be genetically targeted for basic physiology studies using our platform
In the longer run, Society, through economic benefits ensuing from the use of our technology to advance the understanding of basic processes underlying disease mechanisms, aging, and the discovery of new drugs.