The overall goal of this procedure is to study the mechanism by which singularly injured neuronal cells degenerate and regenerate by monitoring the dynamics of a single dissected neuron in vivo. This is accomplished by first labeling a subset of inferior ary neuron and their axonal elongation through anter grade tracing. The second step is to perform a cranial window on the cerebellar cortex to monitor repeatedly the climbing fibers at arbitrary time points under a cranial window.
Next image, and dissect single axonal branches by laser ax otomy in vivo. The final step is to monitor the degeneration and eventual remodeling of the axon in the days that follow the laser ex sodomy with two photon in vivo imaging. One of the main advantages of using laser to photon taxotomy is being very, very specific.
You can abl even a single spine while with a massive damage. At that point, you are not able to ablate precise region. Furthermore, in this case, with in vivo imaging, you can study the dynamics which is not possible with a static view.
Finally, it's very important to connect the ablated fibers with the rest of the fibers to show how the regeneration process and the plasticity occurs. So this method can provide insight into the structure of plasticity of CLA Fs in the cerebellar coex. It can be applied to dissect any fluorescent structure.
In live brain las edition indeed has been previously targeted to disruption of single cell bodies. Action dendritic branches or even single spines of pyramidal neurons in the cortex of TH one GFPM mice Begin this procedure by loading the capillary with one to two microliters of the dextran conjugated dye. Next anesthetize a mouse by intraperitoneal injection of 90 milligrams per kilogram ketamine and nine milligrams per kilogram Xylazine ensure that the animal is fully sedated using a tail or toe pinch.
Then use ointment on both eyes to prevent dryness while under anesthesia, shave the hair above the neck with a razor blade. Swab the surgical site twice with Betadine alternated with 70%ethanol. After that, place the mouse on a stereotaxic holder position the head to form a 140 degree angle with the body.
Then apply several drops of lidocaine on the skin between the ears. Subsequently, make an incision of about one centimeter on the skin below the occiput under the dissecting microscope. Gently separate the subcutaneous tissue and muscles with blunt tweezers.
Then push down the horizontal muscle bundle and hold apart the two vertical muscle sles in order to expose the dura over the foramen magnum. Next, dissect the dura with a syringe needle or the very sharp forceps with caution. To expose the brainstem on the stereotactic micro manipulator, rotate the holder of the capillary 45 degrees from the vertical.
Place the capillary at the midpoint between the coddle edge of the cerebellar cortex and the first cervical vertebra. Insert the pipette to a depth of two millimeters from the surface, deliver 0.5 to 1.5 microliters of dye over 10 to 15 minutes. Leave the capillary in place for another 15 minutes before removing it.
Slowly, delicately realign the muscles and suture the skin above. Keep the animal in a heated cage until fully recovered from anesthesia. To visualize the sparsely labeled cerebellar climbing fibers under the permanent cranial window, first anesthetize the mouse as before and use toe pinches to ensure the animal is fully sedated.
Next, place the mouse in a stereotaxic frame and position the ear bars in order to firmly hold the head subcutaneously, administer 0.2 milligrams per kilogram, dexamethasone, and five milligrams per kilogram carprofen to prevent swelling of the brain and possible inflammation at the cranial window site. After that, apply a drop of lidocaine on the skin above the skull. Cut a flap from between the ears to above the eyes.
Then apply lidocaine solution again onto the periosteum before scraping it with a scalpel. Now use the dental drill to thin a semicircular region of skull above the cerebellum. Cut a cover glass in two halves with the help of a razor blade and tweezers.
Then make sure that the cover glass is slightly bigger than the aisle of bone and has a similar shape. Next, remove the flap of bone with the tweezers. Carefully lay the cover glass down upon the dura.
Afterward, seal the window with a mixture of acrylic glue and dental cement. Keep the animal on a heating pad in a recovery chamber until fully recovered from anesthesia two to seven days after the surgery, performed the first imaging session by positioning the head of the anesthetized animal in a stereotactic holder so that the cranial window lays parallel to the objective focal plane. Look at the cortex under the cranial window with two photon fluorescence.
Imaging open the lab view software and acquire a Zack in order to generate a 3D reconstruction of the targeted axon. Next, increase the laser power five to 10 times more than the power used for imaging. Subsequently irradiate the selected point on the distal portion of a cerebellar climbing fiber with a high energy dose, acquire a stack of the same region a few minutes after the irradiation to ensure the ablation has been well performed.
If the region started swelling and the axon formed bead like structures, the neuron has been successfully dissected. In this case, the distal portion of the axon will degenerate and disappear completely in a few hours. Afterward, monitor the eventual remodeling of the axon in the days that follow the laser ex sodomy with two photon in vivo imaging.
This figure shows the time-lapse images of a cerebellar climbing fiber labeled with LOR 4 88 dextran and visualized in vivo by two photon fluorescence microscopy from day 16 to day 18. And this figure shows the laser ex sodomy on a single branch of a climbing fiber. The neuron was irradiated.
Here on day 16, these red arrowhead highlight the disappearance of the distal portion of the axon after laser ax sodomy on day 18. And here is a newly formed transverse branch. Following this procedure are the methods like immuno fluorescent staining or electro microscopy can be applied in order to answer additional questions like the possible formation of new synaptic contact on theones, or to evaluate the effective impact of laser induced damage on nearby structures.
After this video, you can understand how to label image and laser dissects climbing fibers with laser taxotomy.
