MRC National Institute for Medical Research, London
Science for Health
Bridging single-cell physiology, genetics and connectomics
02 March 2011
NIMR scientists have shown that the whole-cell patch clamp method can be used to transfect single cells during neuronal recording in vivo. With this combined technique they have been able to record the synaptic input and sensory function of a neuron then use a single cell transfection-based viral approach to trace the upstream brain circuits. The research is published in Nature Neuroscience.
A central goal for neuroscience is to understand how neurons in the brain and spinal cord process information. Ultimately this requires information from combined electrophysiological, neuroanatomical, optical and genetic methods that together can address the necessary broad range of questions, from the level of genes and molecules up to single cells and circuits. Electrical recording methods, such as the whole-cell patch-clamp technique, can provide a relatively complete biophysical characterization of an individual neuron. This method has been especially useful for determining the synaptic receptive fields of single cells so that synaptic activity can be linked directly to sensory processing and behaviour.
Concurrently, developments in optogenetics and virus-based circuit tracing have provided effective tools for manipulating and tracing neuronal circuits both in vitro and in vivo. Viral strategies for expression are also becoming increasingly useful for spatially, temporally and genetically controlling network function and even tracing the anatomical connectivity of a single cell. However, existing gene delivery techniques do not allow researchers to electrophysiologically characterize cells and to thereby establish an experimental link between physiology and genetics for understanding neuronal function.
Troy Margrie (pictured), from NIMR's Division of Neurophysiology, has examined the feasibility of combining in vivo whole-cell recording and gene delivery by answering four specific questions.
- Do cells survive for substantial periods after whole-cell recording in vivo?
- What is the likelihood and longevity of whole-cell recordings with internal solutions containing plasmid DNA?
- Can plasmid DNA dialyze into the cell and drive protein expression?
- Is protein expression restricted to the patched cell?
The delivery of DNA into neurons through an intracellular recording method should be considered useful for any experimental system including neuronal and non-neuronal cultures where cells can be maintained for extended periods of time. In combination with electrical recording from the cell, the list of potential applications of this method includes genetic perturbation of intracellular signaling cascades and up- or downregulation of ion channels, membrane receptors, light-activatable ion channels or genetically encoded indicators, expressed across functionally related presynaptic circuits.
Only by probing both the function and connectivity of specific cells and circuits can we obtain a complete understanding of the functional architecture of the brain. The method of DNA delivery by whole-cell recording described here provides a versatile tool for both assaying and manipulating single cells and probing the functional connectivity of specific local and long-range networks.
Troy Margrie
Click image to view at full-size
Classical neuronal staining approaches (like Golgi staining, left) have revealed the mind-boggling complexity of the brain. The new method (right) allows scientists to record the function of a single cell (red) and identify its presynaptic connectivity (white) which gives rise to its function.
Original article
Ede A Rancz, Kevin M Franks, Martin K Schwarz, Bruno Pichler, Andreas T Schaefer & Troy W Margrie (2011)
Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics
Nature Neuroscience, Epub ahead of print. Publisher abstract.
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