The figure above was kindly provided by Dr. Bruno Pichler at The National Institute for Medical Research in London.
According to the paper in Nature Neuroscience:
Single-cell genetic manipulation is expected to substantially advance the field of systems neuroscience. 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. In the mouse brain in vivo, we found that neurons remained intact after 'blind' whole-cell recording, that DNA vectors could be delivered through the patch-pipette during such recordings and that these vectors drove protein expression in recorded cells for at least 7 d. To illustrate the utility of this approach, we recorded visually evoked synaptic responses in primary visual cortical cells while delivering DNA plasmids that allowed retrograde, monosynaptic tracing of each neuron's presynaptic inputs. By providing a biophysical profile of a cell before its specific genetic perturbation, this combinatorial method captures the synaptic and anatomical receptive field of a neuron.
Figure 3: Multiple gene delivery.
(a) Native fluorescence images of a layer 5 neuron in somatosensory cortex 3 d after patching with pCAGGS-Cerulean (top right) and pCAGGS-tdTomato (50 ng Î¼lâ1 each, bottom right). Scale bars, 824 Î¼m (left) and 20 Î¼m (right). (b) Gallery of native fluorescence images for a layer 2/3 cell in somatosensory cortex 5 d after recording with pCAGGS-DsRed2, pCAGGS-Venus and pCAGGS-ChR2-Cerulean. Scale bar, 20 Î¼m.
From Dr. Pichler's e-mail:
Breakthrough in Neuroscience - new method allows characterization of neuronal networks on single-cell level
An international team led by neuroscientist Troy Margrie has developed a new method, which will shape the future of cellular neuroscience. The researchers from MRC National Institute for Medical Research in London, Columbia University in New York and Max-Planck-Institute for Medical Research in Heidelberg succeeded in determining the function of individual nerve cells in the brain and identify those neurons from which a given cell receives its signals. "The new method enables us for the first time to identify a neuronal networks on the level of individual cells and characterize it functionally", explains Ede Rancz. This study is now published in Nature Neuroscience.
A genetically modified rabies virus leads the way
The scientists combined two existing methods, "whole-cell patch clamp recording" and "monosynaptic retrograde virus tracing". They use the patch-clamp technique to determine the exact stimuli to which a given brain cell responds. Through the glass micropipette, which is used to record electrical signals, they simultaneously inject plasmid DNA into this cell. In the vicinity of the cell they later inject a rabies virus, which is lacking proteins necessary for entering a cell and spreading through neuronal pathways. These missing proteins are provided by the plasmid DNA injected previously into the cell. Therefore, the virus can only infect this single cell and then spread across synapses to only those neurons which are exactly one step upstream in the signaling chain. There it stops because these presynaptic cells do not contain the necessary plasmid DNA, which the modified virus needs for spreading.
Cellular networks in the living organism
The plasmid DNA and the virus both produce fluorescent proteins, which are then visualized through specialized microscopes. In this way, the functionally characterized cell as well as its connected 'neighbours', from which the cell receives information - let them be in close proximity or in a different brain area -can be identified. As this technique can be used in a living organism, cellular networks can be identified and then subjected to further experiments. The researchers are convinced that this method opens up the door for answering a plethora of very important but previously unapproachable questions.
The original paper is available online:
Short video clips of original microscopy images are available at:
Prof. Troy Margrie
The Division of Neurophysiology
The National Institute for Medical Research
London NW7 1AA
PS After this was posted, I found another blog posting (Feb. 23) that shares Dr. Pichler's e-mail.
Oh my goodness! an amazing article dude. Thank you However I am experiencing issue with ur rss . Donât know why Unable to subscribe to it. Is there anyone getting identical rss problem? Anyone who knows kindly respond. Thnkx
A bit off-topic, but still relevant for neuroscience:
"Swedish scientists create three arm illusion" http://www.thelocal.se/32234/20110224/
"And this is your Brain on Drugs."
I cannot understand how people can read this and not think âgenius alertâ. Great post. Iâll keep coming back to read even more.