The overall goal of the following experiment is to assess the effect of the protein of interest on neuronal migration and morphology. This is achieved by electroporated, the cerebella of P four rat pups. As a second step immunohisto chemistry of sections from GFP positive cerebella is performed, which allows the visualization of the electroporated neurons.
Next, the immuno stain sections are imaged in order to measure the distance of neuronal migration and the length of neuronal processes. Results obtained will show the effect of the protein of interest on neuronal migration and morphology in the developing cerebellar cortex. The main advantage of this technique over existing techniques, like in utero aspiration, is that the postnatal cerebellum is easily accessible.
This method can help answer key questions in the neurodevelopmental field as it allows to unravel the mechanisms underlying neuronal migration and morphogenesis Visual demonstration of this technique is critical as the reliable targeting of the injection site requires practice. Prior to showing the in vivo electroporation of the cerebellum, we will first demonstrate a morphological assay that uses cultured cerebellar granule neurons as a model system. Begin this procedure by collecting the conditioned media and subjecting the neurons to starvation in DMEM.
Then mix DNA solution and calcium chloride. Add two X-H-B-S-S and gently mix the solution by making air bubbles. Then add 40 microliters of DNA precipitate to each well and incubate the neurons for 18 minutes in the incubator at 37 degrees Celsius.
After that, remove the D-M-E-M-D-N-A mix and wash the neurons twice with 500 microliters of Prewarm DMEM. Next, add the collected conditioned media back to the neurons if the neurons will be in culture for more than three days. Supplement the media with 25 millimolar glucose to replenish the carbon source after one to five days.
Subject the neurons to immunochemistry using GFP antibodies. Image at least 30 individual neurons per condition in a blinded manner using a fluorescent microscope to measure the axons and dendrites. Convert images to eight bit with image J by opening the image.
Then choose image, click type, then eight bit and click save to save the image. Now run the neuron J plugin and open the eight bit image. Use the add tracings option to track the axon first, click the left mouse button once at the beginning of the axon and move the mouse along the process.
Then double click on the tip of the axon if the trace matches the axonal shape. If the suggested trace differs from the axonal shape, click once on the axonal process to anchor the trace. Then double click on the tip of the axon.
Next, click on measure tracings. Choose the display trace measurements option and press run axon measurements are all displayed in a new window. Save them as a separate file that can be opened in any spreadsheet program.
To measure total dendrite length, click on measure tracings. Choose the display group measurements and press run. Total dendrite measurements are all displayed in a new window.
Save them as a separate file that can be opened in any spreadsheet program in this procedure. After anesthetizing a P four rat puff with iso fluorine, sterilize the back of its head with 70%ethanol. Next, locate the cerebellum of an albino pup under a gooseneck lamp.
The transverse sinus, which sharply demarcates the midbrain from the cortical hemispheres should be visible. The cerebellum is located adjacent to the midbrain and appears in a darker shade. Keep the pup in a fixed position and use a permanent marker to indicate the cerebellum with a dot.
Then insert a needle and slowly inject three microliters of DNA into the cerebellum. Should anesthesia wear off during this procedure, expose pup to iso fluorine Prior to injecting the DNA, let the DNA solution diffuse for 30 seconds to one minute. After the injection of DNA place the pup's head between the tweezer TROs so that the minus pole makes contact with the cerebellar region at the back of the head, and the plus pole contacts the opposite side of the head.
Adjust the voltage according to the pup's weight to ensure good electroporation efficiency without compromising their survival. Then subject the pup to five electrical pulses. After the electroporation, let the pup recover on a heated pad or underneath an infrared lamp before returning it to the dam.
For an in-depth analysis, subdivide the IGL into halves, resulting in an upper IGL facing the molecular layer and a lower IGL facing the white matter and count the GFP positive neurons residing in each half. To measure the dendrite length, open the image series in Amaris to generate a 3D image of the dendrites. Next, click on surpass mode to view the neuron in 3D.
Then select add new filament and click skip Automatic creation to start semi-automatic tracing. Afterward, select draw tab and auto path. Move the mouse cursor over the cell body and shift plus mouse Right click to select the cell body to perform auto calculation.
Then add paths to the filament using shift plus mouse left click and visualize them in real time. Finally, go to the filament statistics window and click on detailed specific values and F mite dendrite length sum for the sum total dendrite length. This figure shows the analysis of axon and dendrite growth in the cerebellar granule neurons.
The cerebellar granule neurons that had been transfected with a plasmid encoding GFP at div zero were cultured for one, two, or three days in either full, medium or basal. Medium Eagle supplemented with insulin after fixation. Neurons were subjected to immuno cyto chemistry using the GFP antibody and axon and dendrite lengths were measured.
Here, white arrowheads indicate axons and yellow arrowheads indicate dendrites. In this experiment, the cerebella of P four rat pups were electroporated with the psin GFP plasmid and isolated. Five days later, 40 micrometer coronal sections were subjected to immunohistochemistry using the GFP antibody Localization of cerebellar granule neurons in the cerebellum was assessed and the total dendrite length was measured using a Mars software.
The arrows indicate the cell bodies of cerebellar granule neurons and the arrowhead indicate the dendrites Once mastered. This technique allows to finish the exploration of a pup in five minutes if it is performed properly. While attempting this procedure, it is important to adjust the voltage according to the P'S weight.
Using this technique, several proteins of interest can be manipulated simultaneously to delineate a pathway during cerebellar development.
