The aim of this procedure is to image calcium activity of cells in the postnatal subventricular zone. This is accomplished by first carefully removing brain tissue from a postnatal mouse. The second step is to make live acute brain slices containing the subventricular zone.
Next, the brain slices are loaded with a calcium sensitive dye. The final step is to image activity with a time-lapse confocal microscope. Ultimately, time-lapse imaging of calcium activity in ventricular zone cells is used to show intercellular activity dynamics to reveal communication patterns among different cell types and the vasculature.
This method can help answer key questions in the neurogenesis field, such as how different cell types communicate with one another, and how cells may influence the vasculature After sacrificing and decapitating a P 20 to P 30 mouse pup. According to institutionally approved guidelines, place the head in a tray field with dissection solution. Stabilize the head with forceps and use micro scissors to make continuous cuts around the skull and midline.
Ensure that the olfactory bulb remains attached to the brain and is not damaged. Make cuts on the dorsal side and then use forceps to remove the overlying skull from the brain. Then, while still holding the head with forceps, use a surgical blade to make a coronal cut at the level of lambda.
Use the same razor blade to make sagittal cuts on both sides of the brain about two to three millimeters lateral to the midline. Then use the blade to scoop underneath the brain and remove it completely from the skull. The procedure to this point should be done in less than three minutes.
Place a dab of cyanoacrylate based super glue on the plate of the vibram. Then pick up the brain tissue and mount it on one of the flat sides created by the lateral sagittal cut. Mounting an agarose block to stabilize the tissue during cutting is optional.
Rinse the vibram blade with ethanol to remove oils and then rinse with distilled water. Place the blade into the blade holder and mount the blade holder onto the vibrator. Set the vibrator so that slices of tissue are cut to thickness of 250 to 350 microns at high frequency vibration and low speed progressively section through the tissue.
The appearance of the olfactory bulb is a good indicator that the sagittal sections are approaching the subventricular zone. The appearance of the ventricles and the thickening of the corpus callosum are also good indicators of the subsequent appearance of the subventricular zone. Once the subventricular zone is reached, use a plastic pasta pipette with the tip cutoff to remove each slice and transfer it to a slice holding chamber.
With A CSF. The slices require one hour of incubation in A CSF. During this time, the pipettes, dye and microscope can be prepared for the subsequent imaging steps.
To prepare the pipettes, pull six to eight glass pipettes so that each has a tip length of around two millimeters and a diameter of two to three microns. In addition, the pipette holder is preset at an angle of around 16 degrees so that the pipette can be mounted without contacting the perfusion chamber or interfering with the placement of the objective. To prepare the calcium dye, add 4.6 microliters of onic F1 27, 20%solution in DMSO to a 50 microgram aliquot of flu oh four.
Finally, to prepare the peristaltic pump, the use of which helps minimize image drift, run a CSF through the perfusion lines and ensure that the lines are bubble free. Now, position the vacuum tip to ensure that the solution is being aspirated from the perfusion chamber. This image shows the setup of the perfusion system.
The solution should be at an adequate level to immerse the objective at the proper working distance, but not so high that the solution spills over the sides of the chamber. Listen for steadiness to verify a constant aspiration to apply loading dye via the bath method. First, make one to two milliliters of the four to five micromolar dye working solution by diluting the stock solution.
In A CSF, transfer the slices to a 35 millimeter cultured dish with a mesh bottom filled with A CSF. Use a three milliliter plastic transfer pipette to gently remove the A CSF while simultaneously adding the calcium dye working solution. Then incubate at 37 degrees Celsius for 30 to 60 minutes in a 5%carbon dioxide gas controlled environment.
After the incubation, place the slice back into the A CSF filled holding chamber to wash away dye that has not entered cells. After 30 minutes in A CSF, transfer the slice to the perfusion chamber for pressure application of the dye. Transfer a slice from the holding chamber to the perfusion chamber on the microscope set up.
Then backfill the glass pipette with 250 micromolar dye working solution. Ensure that the pipette tip is free of bubbles and placed on the micro manipulator. Now lower the pipette to the region of interest.
The pipette can be placed so that the tip is several micrometers above the sliced surface, or buried several micrometers below the surface, but away from the recorded region. Set the pico spritzer to deliver less than three pounds per square inch and apply the dye for one to two minutes. Repeated applications may be necessary depending on labeling as assessed by eye.
When applied to the surface of the slice, this protocol will preferentially label neuroblast when applied below the surface, this technique will preferentially label astrocytes, but if applied for more than three minutes at a concentration of 500, micromolar neuroblast will be labeled. Regardless of the placement of the pipette tip, Allow the slice to wash and recover in A CSF for 30 to 45 minutes. Locate the region of interest with a lower power objective, then switch to a higher power water immersion objective and assess the health of the cells in the region of interest.
Under a differential interference contrast, healthy cells appear round and plump and display faint flu oh four loading. After verifying that the perfusion and vacuum are working adequately, set up the time-lapse resolution and spatial resolution to the desired rates. In this case, one frame is imaged approximately every second with a five 12 by five 12 pixel resolution.
After setting the duration of two to 10 minutes, initiate the run. Pharmacological agents are washed on for five to 10 minutes and then washed out for further five to 10 minutes. This representative averaged image from a time-lapse recording of calcium Activity was taken after astrocytes in the subventricular zone.
Were loaded with pressure. Application of flu oh 4:00 AM deep into the slice. Movies were acquired at 0.5.
Second time steps, the regions of interest are placed over cells exhibiting activity. Here are the traces from the numbered regions of interest shown Previously. The traces were filtered with a moving average and normalized.
The vertical scale represents two times delta F over F zero where F is the signal intensity, and F zero is the average baseline signal and delta F equals F minus F zero. Once mastered. This technique can be done in four to five hours if done correctly.
