We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.
This protocol can be used to correlate calcium activity patterns with gene expression at the single cell level empowering researchers to investigate novel questions about the relationships between these two features. In contrast to previous approaches, which typically study whole tissues, our technique zooms in to the single cell level enabling us to assess cell autonomous relationships between calcium activity and gene expression. Begin by UV sterilizing two 35 millimeter plastic Petri dishes and one 35 millimeter cell culture dish for approximately 30 minutes.
Working in the laminar flow hood, add 10 milliliters of two millimolar calcium solution to each of two 50 milliliter plastic conical tubes, one UV-sterilized Petri dish, and the UV-sterilized cell culture dish. Add two milliliters of calcium and magnesium free solution to the other UV-sterilized Petri dish and fill two 100 millimeter plastic Petri dishes and one 35 millimeter plastic Petri dish with 0.1X MMR supplemented with gentamicin. Then fill a 60 millimeter plastic Petri dish with 70%ethanol.
Immediately before the dissection, thoroughly mix 0.01 grams of collagenous B into one tube of calcium solution. Add the solution to a new 60 millimeter plastic Petri dish and use a dissecting microscope to identify embryos of the desired developmental stage. Use a sterile transfer pipette to move at least six appropriate embryos into one 100 millimeter plate containing MMR plus gentamicin.
Then move one embryo from the holding plate into the second 100 millimeter plate of MMR plus gentamicin and place this dissection plate under the microscope. Using a pair of blunt forceps to stabilize the embryo, carefully peel away the vitelline membrane surrounding the embryo with a pair of fine forceps. When all of the membrane has been removed, use the fine forceps to pinch the embryo along the anterior posterior axis to separate the dorsal and ventral regions.
Use the new sterile transfer pipette to transfer the dorsal portion to the 60 millimeter plate of collagenous solution for one to two minutes before carefully transferring the specimen back to the dissection plate. To complete the dissection, carefully remove all of the residual endodermal and mesodermal contamination from the presumptive neural tissue of the ectoderm and gently transfer the explant to the 35 millimeter plate of calcium solution. When three more explants have been collected, use a P1000 micropipette to transfer all four of the explants to the 35 millimeter plate of calcium and magnesium free solution taking care to avoid any contact between the explants and the air water interface.
Then gently swirl the dish so that all of the explants cluster in the center of the plate and incubate the specimens for one hour at room temperature to allow the the tissues to dissociate. During this incubation, transfer the last two embryos to the second dish of MMR with gentamicin, and cover the dish to allow these specimens to develop undisturbed. At the end of the incubation, use super glue to attach a micro-ruled coverslip to the bottom of the 35 millimeter cell culture dish and use a P100 micropipette to collect the dissociated explants.
Holding the pipette at a shallow angle close to the surface of the cell culture dish, position the pipette tip in the corner of the grid facing inwards and firmly expel the cell suspension across the gridded area. Ideally, the cells will settle in a tight, dense cluster. Allow the cells to adhere to the plate for one hour at room temperature.
While the cells are settling, determine and record the developmental stage of the sibling control embryos. At the end of the incubation, move the sample dish to a light-protected location and aspirate 100 microliters of solution from the edge of the dish. Mix this solution with seven microliters of a freshly prepared Fluo-4 AM Pluronic F127 acid solution with up and down pipetting before returning the full volume to the sample dish.
Swirl gently to mix and cover the plate with aluminum foil. After one hour at room temperature, replace one milliliter of the explant culture dish supernatant with three milliliters of fresh two millimolar of calcium solution. Then replace three milliliters of supernatant with three milliliters of fresh calcium solution two more times.
For calcium activity imaging within the explants, place the sample plate onto the stage of an inverted confocal microscope protected from ambient light exposure and use a marker to label the front point of the plate so that the same field of view can be found at subsequent imaging sessions. Use the 10 and 20 times objectives to locate a sample under the microscope and select an appropriate field of view that is cell dense but not so dense that the cells are clumped or difficult to distinguish individually. Adjust the microscope focus so that the grid-ruled coverslip is visible, changing the original field of view until an identifiable number is in frame as necessary.
Obtain a bright field image of the selected field of view with the grid-ruled coverslip in focus and a bright field image of the selected field of view with the cells in focus. Without a clear image of the gridded coverslip and the field of view, you won't be able to co-register your cells with those in the fish images you'll generate later. Then illuminate the samples with a 488 nanometer laser.
For a two hour image, modify the imaging configuration to record 901 frames with a scan time of 3.93 seconds in an interval of eight seconds before running the configuration to acquire the image. Once the imaging is complete, remove the plate from the microscope stage and replace the culture supernatant with one milliliter of 1X MEMFA for a two hour incubation at room temperature taking care to record the developmental stage of the sibling control embryos at the beginning of the incubation. The visualization of a composite plot containing traces for all of the cells recorded in an experiment reveals the degree to which bulk or population measurements can obscure more nuanced patterns of spiking behavior.
When the recorded profiles of individual cell are isolated, examples of the irregular spiking activity characteristic of neural progenitor cells can be clearly identified. To quantify this complexity, different data analysis methods can be applied including diverse parameters to define a spike. If the plates are handled roughly or the washes are performed too forcefully during fluorescent in situ hybridization, the cells can be dislodged from the plate surfaces making it impossible for the cells to be matched across images.
If this disruption affects only some of the cells in the field of view, it may still be possible to detect and assign some of the cells within the image. However, the maximum amount of data is gained from an experiment in which the hybridization is performed carefully and few cells are lost or repositioned between images. Results from experiments collating calcium activity with the expression of neural progenitor marker genes can reveal numerous associations between specific patterns of calcium activity and neuro-transmitter phenotypes.
With this data, researchers can probe more complex features of these dynamic calcium patterns and the relationships to gene expression rather than being restricted to simple spike counting.
