Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

The overall goal of this procedure is to visualize migrating neurons in living brain slices. This is accomplished by first dissecting the cerebellum from a P 10 rat. The next steps are to prepare acute cerebellar slices and to label the neurons with a fluorescent probe.

Then the time-lapse experiment is performed through macro confocal imaging or confocal microscopy. The final step is to track the neurons in the resulting movies. Ultimately, ex vivo microscopy is used to show the role of endogenous factors or toxic substances that regulate neuronal migration.

The main advantage of this technique of existing methods like confocal microscopy, is that macro confocal imaging increases the feel of view of leaving cerebral art slices directly placed on the groin insert. I first, I had the idea for this method when I had difficulties to transfer and stabilize cerebral art slices for confocal microscopy experiments Begin by having access the skull of a decapitated P 10 rat pup head, using fine iris scissors delicately make two lateral incisions from the base to the rostral region of the skull. Then remove the dissected skull with two number three forceps, breaking any adhesion between the brain and the skull.

Now, scoop the brain out with the spoon end of a spatula, and put it into a 35 millimeter Petri dish with two milliliters of cold HBSS on ice. Now, under a stereo microscope, isolate the cerebellum from the brain by D lacerations using two number three forceps. Spoon the cerebellum to a new dish with ice cold buffer.

Then using the same tools, remove and discard the residual spinal cord and the peel membrane. Prepare a number three solid scalpel handle with a number 15 surgical blade. Using the blade under a stereo microscope, cut the cerebellum between the verus and the right hemisphere.

Now at the vibrato, put a drop of cyanocrylate glue on the specimen disc and wait 15 to 25 seconds for the solvent vapor to dissipate before transferring the cerebellum to the disc. Next, fix the edge of the cerebellum to the specimen disc and wait 10 seconds. Now using the manipulator, insert the specimen disc into the buffer tray so the transverse axis of the cerebellum is perpendicular to the knife holder.

Be sure to secure the disc using an alum wrench. Then carefully cover the cerebellum with HBSS and load crushed ice into the cooling bath. To slice the specimen position a cleaned blade, so its edge is right behind the back edge of the specimen.

Define this as the starting point for icing. Then use the forward command to define the endpoint as just past the front edge of the specimen. Now set the slice thickness to 180 microns.

Then set the sectioning speed to 2.5 and the frequency to eight, and start sectioning the specimen. Collect up to five slices per cerebellum. Pick up each section using a truncated wide boar glass pipette.

Transfer the sections to a dish containing cold HBSS on ice. After collecting the slices under a stereo microscope, carefully remove the meninges using two Number five forceps. Also, gently separate the LOEs for better probe loading.

After slicing the specimen, use a truncated wide boar pipette to transfer them with some HBSS to a six Well plate load up to three slices in each well. Next, after emptying the media in each loaded, well add five milliliters of loading solution containing 10 micromolar of the fluorescent dye to protect the samples from light, cover the plate with foil. Then put the plate on a mutator set to 35 RPM.

Let the plate incubate for 10 minutes at room temperature to let the dye label the cells wells. Now transfer the slices as before to the membrane of a transwell insert that has three micron pores. Then aspirate the loading medium, leaving just the slices.

Now remove the insert and slices to fill the well with 1.9 milliliters of DMEM. Then put the insert back and add another 100 microliters of DMEM to cover the tissues. Incubate this preparation for two hours after which granule cells will be visible.

Transfer the plate without the lid to an environmental chamber with constant gas flow on a confocal microscope. Then place glass cover on the plate insert of the confocal microscope for time-lapse experiments. Let the cells incubate in the chamber for two additional hours before proceeding.

To visualize the granule cells migrating in the tissue slices, illuminate the preparation with 488 nanometer light and use a two x dry objective. Detect fluorescent emissions from 500 to 530 nanometers using image J.For each image of the time-lapse movie, perform a Zack projection using the standard deviation mode, adjust the contrast and the brightness levels of each image so the labeled granule cells are easy to see. Now, choose the manual tracking plugin from the particle analysis menu.

Use the plugin to click on the central point of each labeled cell body throughout the time lapse image sequence. Then export the raw tracking data to a spreadsheet for further analysis. Reorganize the exported raw tracking data from Image J with a smart homemade tool that identifies each cell and associated positions using the program.

Calculate the total travel distance and the average speed of migration for each cell. In the early postnatal cerebellum, granule cells exhibit significant changes in their mode and speed of migration as they crossed different cortical layers. CTG labeled granule cells in PM rat cerebellar tissue slices were examined.

As described, the granule cells migrated radially in the molecular layer with an average speed of 18 microns per hour. Another preparation was then used to test the effect of drugs expected to alter the rate of migration. Application of pay cap 38 to the culture medium resulted in a 79%speed decrease of the granule cells in the molecular layer, a drop to 2.5 microns per hour.

Administration of PI one, an inhibitor of endogenous TPA reduced the speed of granule cell migration by 78%from the control speed to 4.2 microns per hour. After watching this video, you should have a good understanding of how to track cells in the developing theory balloon. While I attempting this procedure, it's important to remember to provide appropriate and constant environmental parameters essential for cell migration After its development.

This technique paved the way for researcher in the field of neurosciences to explore the fact atory processes in the brain.