The overall goal of this in utero electroporation approach is to characterize the structural connectivity and excitability of cortical neurons under normal and experimental conditions. This method can help answer key questions in the developmental neurobiology, such as dissecting the structure and functional connectivity of the brain and the biology of cognitive disorders. The main advantage of this technique is that it allows the marking of a sparse population of neurons and allows you to dress the dendrites and access the individual neurons which leads to more efficient, more fimetic quantitative analysis.
Before the injection, prepare ten microliters of DNA mixture per surgery for either single cell labeling or standard patch clamp study. Then, manipulate the embryos gently with fingers to locate the telencephalon. Next, place a prepared borosilicate capillary in a mouth pipette.
Pass the tip of the needle through the uterus, avoiding blood vessels, until it reaches the lateral ventricle. After that, slowly inject approximately one microliter of Fast Green colored DNA solution until a large blue spot is observed. A critical step in this procedure is the injection of the DNA.
This should be done as gently as possible. Now, place the seven millimeter platinum electrodes laterally on the head of an embryo and apply voltage via the platinum electrodes. In this procedure, cryoprotect the fixed brains in ten milliliters of 30%sucrose in PBS at four degrees Celsius for one to two days until they sink.
Then prepare one cubed centimeter aluminum foil cubes and fill the cubes 2/3 or the way with OCT. Afterward, put the brains in the cubes and freeze them on dry ice. Subsequently store the frozen brains at 80 degrees Celsius.
To section the brains in the cryostat, place a drop of OCT on the surface of the specimen disc. Peel the aluminum foil from the histology block and position the block at the desired orientation on top of the liquid OCT. Apply firm pressure until the histological block is fixed.
Then insert the specimen disc into the specimen head of the cryostat and orient the specimen for sectioning. Then slice 50-100 micrometer thick sections and transfer the floating cryosections to PBS using a fine brush. Subsequently, block the sections for one hour at room temperature with 5%fetal bovine serum in PBS, containing 0.5%Triton X-100.
Next, incubate them overnight at four degrees Celsius with 1:500 primary antibody diluted in blocking solution. The next day, wash the sections three times in PBS. Add 1:500 secondary antibody diluted in blocking solution and incubate the sections for one hour at room temperature.
Then wash the sections three times in PBS. Afterward, counterstain the sections with dapE in PBS containing 0.5%Triton X-100 for ten minutes. Rinse the sections with PBS and mount them with mounting medium.
To reconstruct the neurons, acquire images of the brain sections with high magnification and high resolution. Next, select Tile Scan in the acquisition software to cover the area of interest, spanning all dendrites and axonal processes. Acquire a sufficient number of stacks on the Z axis to avoid information loss.
After that, open the image and select the segmented line option from the menu. Draw a line following the structure of the neuron. Subsequently, go to Analyze, Tools, followed by ROI manager, then Add to save the line.
Repeat this process for every axon or dendrite of the analyzed neuron. In the ROI Manager menu, press Measure to get the length. Export the measurements to a text file or spreadsheet for analysis.
To perform a recording of GFP expressed neuron from a GFP electroporated mouse, place the brain in a chilled culture dish. Cut off the cerebellum with small scissors. Then pick up the brain with a spatula and blot it dry on a paper towel.
Glue the ventral caudal plane of the brain on a Vibratome holder. Place the holder on a Vibratome filled with ice cold ACSF and ensure the Vibratome chamber has continuous carbogenation. Following this, obtain acute slices by cutting 300 micrometer coronal sections within 15 minutes.
Next, incubate the acute slices at 25 degrees Celsius for at least 60 minutes in ACSF while bubbling with carbogen. After 60 minutes, transfer a slice to the recording chamber using a Pasteur pipette or a small brush. Hold down the slice with a harp and profuse it with ACSF at a rate of two milliliters per minute.
To patch a GFP positive neuron, locate the area of interest through the microscope at 10x. Then find a GFP positive cell using the 60x objective. Next, fill the recording electrode with intracellular solution.
Subsequently, place a glass pipette in the pipette holder. Afterward, place the pipette tip in the bath and focus on the tip. Once the pipette is in the bath, apply positive pressure through the back pressure control system.
Approach the cell of interest under visual guidance while maintaining back pressure in the pipette. Upon the appearance of a small dimple on the cell's surface, release the pressure. At this point, a tight seal with a resistance larger than one giga-ohm may be formed.
Otherwise, apply a light negative pressure to facilitate it. While the seal is being formed, bring the holding voltage clamp to 60 millivolts. Once the giga-ohm seal is formed, apply a pulse of suction to rupture the cell membrane and break into the whole cell mode.
Once it is in whole cell mode, switch from voltage clamp to current clamp mode and start recording. These images show the delivery of vectors to layer 2/3 neurons by in utero electroporation at embryonic day 15.5, and the coronal sections were prepared at post-natal day 16. The CAG DsRed2 vector was cotransfected as a control.
GFP is expressed only in those neurons that also incorporated cre, allowing the recombination of the LoxP sites in the CALNL GFP vector. Here is a high magnification confocal image of the dendritic arbors of another sparsely labeled GFP neuron. This is a pyramidal neuron electroporated with GFP which was observed under bright fields and green fluorescence conditions.
Here is a typical, regular spiking response of a CAG-GFP electroporated control neuron at layer 2/3. Once mastered, this technique can be done in 30 minutes for in utero electroporations and half a day for the patch clamp recording if it's performed properly. While attempting this procedure, it's important to remember to do the surgery as fast as possible to reduce the stress of the mother and increase the chances of survival of the pups.
Following this procedure, other methods like expression can be performed in order to answer additional questions, like the relationship between expression of specific genes and their influence in the development of the surrogate. After its development, this technique paved the way for researchers in the field of developmental neuroscience to explore connectivity and morphology of neurons in mice. After watching this video, you should have a good understanding of how to use in utero operation to describe the structure and connectivity of neurons at the single cell level and the excitability of fluorescently labeled neurons.
