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e-learning VI - Neuroscience
Patch clamping in brain slices
In your lectures you have heard about the power and versatility of patch clamp methods and you should be awed by its time resolution and ability to look at the behaviour of single protein complexes.
Standard patch clamp techniques are carried out on cultured cell lines or cells that have been extracted from nervous system tissue. Clearly, this is not a very realistic way of looking at things;
the brain is much more complex than a collection of single cells, and neurones frequently behave very differently to the types of cells that can be routinely cultured in the lab.
Well, fortunately we can apply patch-clamping on a larger scale, as patch clamp seals can be made on cells in thin slices of brain tissue, or even in whole brains.
This type of recording is made by applying positive pressure to the fine glass recording-electrode. A jet of solution comes out of the tip of the electrode and is used like a pressure washer to "blast" debris out of the way as the electrode is pushed into the tissue.
In slices, it is sometimes possible to use a microscope to see when the electrode is close to a neuron, but frequently the technique is done "blind" with the neuron hunter relying on changes in electrical resistance to indicate when a cell is under the tip of the electrode. Suction is then applied and recordings can be made just as you would with a cultured cell.
Patch Clamp/Single Cell RT PCR
A further extension to the patch clamp technique comes when it is combined with a powerful molecular biology technique: single cell reverse transcription polymerase chain reaction (RT PCR).
Just as patch clamp gives investigators the ability to measure the activity of ion channels and receptors in a single cell, so single cell RT PCR allows them to measure the levels of individual mRNA molecules and thus infer the expression levels of particular proteins.
This technique is quite simple in principle. At the end of a patch clamp recording experiment the contents of the cell are sucked into the patch clamp electrode.
The contents of the electrode are then transferred into a test tube for RT PCR.
The first step in RT PCR is the conversion of the mRNAs present in the sample into cDNAs using a reverse transcriptase enzyme. Primers are added for the protein of interest and the corresponding cDNA is amplified and measured using quantitative PCR (qPCR).
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