04 Jan 2008

One neuron can make a difference, especially if it sheds light on how its billions of neighbors conspire to form functional neural circuits in the brain. In the December 23 Nature Methods online, researchers led by Michael Häusser, University College London, England, report a new technique for measuring electrical activity in single neurons in the living brain. Unlike currently used methods, which rely on expression of transgenic fluorescent proteins to identify single cells, this one is applicable to wild-type cells. Called “shadowpatching,” the technique can also be used for electroporation and is poised to be a valuable tool in the study of neuronal circuitry and plasticity. “The technique allows you to look at the effect of a single gene in a single cell, and do it in small, defined populations,” said Häusser in an interview with ARF. The work was carried out in collaboration with Winfried Denk’s lab at the Max Planck Institute for Medical Research, Heidelberg, Germany, and Masanobu Kano’s lab at Osaka University, Japan.

Shadowpatching is a variation of two-photon targeted patching (TPTP), which uses the three-dimensional, pinpoint accuracy of the two-photon microscope to guide patch clamps to neurons expressing a fluorescent marker—often green fluorescent protein. TPTP has helped researchers to overcome a major hurdle in recording neurons in vivo, namely the uncertainty in knowing exactly what type of neuron has been clamped. However, because TPTP requires the expression of transgenes, it is restricted to specific animal lines or those transfected by viruses or other vectors. Shadowpatching needs no such manipulation.

The key to shadowpatching is turning the fluorescent marker idea inside out. Instead of labeling the neurons, first authors Kazuo Kitamura, Benjamin Judkewitz, and colleagues labeled the extracellular space. They flooded the neocortex or cerebellum of rodents with a fluorescent dye, Alexa 594, which is not taken up by cells, and found that neurons could be seen as dark cells, or shadows, against the bright background. This “shadowing” could even identify specific neuronal types, such as pyramidal cells and interneurons, by their gross morphology. Guided by the two-photon microscope, the researchers were then able to patch-clamp electrodes to individual shadowed cells. In fact, they used the same micropipette that delivers the fluorescent dye as the patch clamp. This turns out to be more than just convenient. Because there is a constant flow of dye from the pipette, the researchers were able to visualize when the pipette came in contact with a neuron because of a fluorescent dimple made in the cell membrane. Applying suction at just that moment creates a gigaseal or patch clamp in the cell membrane. Shadowpatching is a more controlled version of the “blow and seal” technique originally developed for patch-clamping cells in brain slices.

Kitamura and colleagues shadowpatched and recorded activity from a variety of cells in living mice. They made reliable electrical recordings from pyramidal cells in layers 2/3 of the barrel cortex, Purkinje neurons in the cerebellum, and interneurons in both regions. The success rate of around 70 percent is very high and is similar to that seen in isolated preps, said Häusser. Typical success rates for patch-clamping in vivo are about 20 percent. The researchers were also able to clamp dendrites, though at a reduced efficiency.

What is perhaps most useful about this technical development is the combination of patch-clamp recordings and electroporation. “This gives us a much more precise and targeted way to change properties of specific cell types in the brain,” said Häusser. Other techniques target entire groups or populations of neurons, with more system-wide consequences. “If you knock out a critical gene, for example, an NMDA receptor, then the whole system may compensate for the loss, but if you simply do the knockout in a single cell, then the network will not notice,” he said. Häusser predicts that the technique will give researchers new means to study mouse models of disease.—Tom Fagan