The authors recorded calcium signals in spiny dendrites.
Like Crayola consumers, molecular biologists know the difference between cyan and cerulean. Fluorescent proteins, which are often described by their hue, have distinct excitation and emission spectra and vary in brightness. Heim et al. report that variant fluorescent proteins enable in vivo calcium imaging in transgenic mice in a recent article in Nature Methods.
The authors imaged calcium dynamics using a fluorescence resonance energy transfer (FRET) technique. They designed a calcium indicator that contained the calcium-binding protein troponin C between two modified fluorescent proteins called the 'donor' and the 'acceptor'. When calcium binds to troponin, the donor transfers fluorescence to the acceptor, allowing calcium concentration to be measured as a ratio of acceptor to donor fluorescence. For the donor, the authors chose cerulean fluorescent protein, which is brighter than other cyan fluorescent proteins. They mutated citrine, a modified version of yellow fluorescent protein, to enhance its brightness and used it as the acceptor. The authors placed the entire troponin construct under the control of the Thy-1 promoter.
Transgenic mice showed stable expression of the troponin calcium indicator in the cortex, hippocampus, cerebellum, brain stem, retina and spinal cord for at least 11 months. Primary cultures of hippocampal and cortical neurons from transgenic mice and hippocampal neurons transfected with the troponin calcium indicator showed similar emission spectra in the absence of extracellular calcium. In the presence of extracellular calcium, high bath levels of potassium induced reduced donor and increased acceptor emission spectra in transfected neurons and neurons from transgenic mice.
In cortical slices from transgenic mice, depolarization and glutamate induced changes in the ratio of citrine to cerulean fluorescence that corresponded to electrophysiological response. According to the authors, two to three action potentials reliably produced changes in the ratio of citrine to cerulean fluorescence.
The authors recorded calcium signaling in vivo with a surgically implanted two-photon imaging chamber. Glutamate induced similar changes in the ratio of citrine to cerulean fluorescence in neurons in layer 1 of the visual cortex and cortical slices. However, neurons in layers 2/3 of the visual cortex required longer pulses of glutamate to respond than did more superficial neurons in layer 1, suggesting higher thresholds to signal detection in deep cortical neurons.
The authors recorded calcium signals on the subcellular level. They measured calcium signals from spiny secondary and tertiary dendrites in vivo. Targeted expression of the troponin calcium indicator might help dissect the cellular circuits involved in neural signaling.