Imaging PKA Activation Inside Neurons in Brain Slice Preparations
互联网
490
Cyclic-AMP dependent protein kinase (PKA) is present in most branches of the living kingdom, and as an example in the nervous
system where PKA integrates the cellular effects of various neuromodulators. The recent development of fluorescence resonance
energy transfer (FRET) biosensors such as AKAR now allows a direct measurement of PKA activation in living cells by simply
ratioing the fluorescence emission at the CFP and YFP wavelength. This novel approach provides data with a temporal resolution
of a few seconds at the cellular and even sub-cellular level, opening a new possibility for understanding the integration
processes in space and time.
Our protocol was optimized to study morphologically intact mature neurons and we describe how simple and cheap wide-field
imaging as well as more elaborate two-photon imaging allow real-time monitoring of PKA activation in pyramidal cortical neurons
in neonate rodent brain slices. In addition, many practical details presented here also pertain to image analysis in other
cellular preparations such as cultured cells. This protocol also applies to the various other CFP–YFP-based FRET biosensors
for other kinases or other intracellular signals, and it is likely that this kind of approach will generalize in a near future.
