Today in the lab, I was analysing the calcium activity of the white matter in the corpus callosum (the area of the brain that connects the two hemispheres). I was using recordings of the brain collected with a 2-photon microscope. The blood vessels were visualised using a red fluorescent dye, and the white matter (aka the oligodendrocytes surrounding the neurons) were visualised using a green fluorescent dye. My aim was to analyse the spontaneous calcium activity in the white matter, something which was never attempted before. The process involved a lot of coding using Matlab and many complicated mathematical functions to extract the relevant information from the microscope recordings.
An example of what the actual recordings looked like under the microscope is shown below.
Timelapse: Example of a 2-photon imaging session, showing blood vessels in red, and brain neurons in green. You can see the individual red blood cells moving through the vessels as black shadows. The increase in green signal shows the calcium activity of the oligodendrocytes (aka the white matter) that insulate the neurons.
Detailed methods of today’s experiment
For this experiment, a genetically-encoded calcium indicator was expressed in oligodendrocytes, allowing the recording of calcium activity in myelin in vivo, using 2-photon imaging, through a cranial window implanted over the corpus callosum. Computer code was then used to identify individual oligodendrocytes and extract their basal calcium activity, which was then compared in different conditions (3 weeks post-surgery, 5 weeks post-surgery, during locomotion, and at rest). A lot of mathematical functions were used for data analysis, such as principal component analysis (PCA) and independent component analysis (ICA) to extract cellular signals from the imaging data sets, and to calculate the firing rate of the cells.