The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied.
This method can help classify neuronal subtypes and extend our understanding of the functional map of cortical circuitry. This protocol has been optimized to preserve the ultrastructure of the neurons and obtain excellent recovery of both dendritic and axonal arbors, allowing high quality reconstructions. At the end of an electrophysiological recording, transfer the slice containing the recorded cell to a small dish of ACSF.
While working in a fume hood, roll the slice onto a small piece of fine quality filter paper and place another piece of moistened filter paper onto to the slice. Place the tissue in a small plastic pot containing 5 to 10 milliliters of fixative solution and store at four degrees Celsius overnight. The next day, transfer the tissue from the container with fixative solution to a container with two milliliters of 0.1 molar phosphate buffer in order to rinse, and cut away any excess tissue with a scalpel blade.
Remove excess tissue by placing the slice in a Petri dish, and cut away the excess tissue with a scalpel blade. Transfer the trimmed tissue to a clean Petri dish, ensuring that it lies flat with no folds or creases. Remove the excess buffer using a dry paint brush.
Cover the tissue with warm gelatin solution and place the Petri dish onto a frozen block to quickly cool the solution. Then, move the dish of setting gelatin to 4 degrees Celsius for 30 to 60 minutes. In a fume hood, use a scalpel blade to cut out a small, 1 X 1 centimeter block containing the gelatin embedded tissue.
Lift the block using a small spatula and carefully transfer to fresh fixative solution. Allow to fix for at least 30 minutes at four degrees Celsius. Following this second fixation, wash the gelatin block in five milliliters of PB three times.
After washing, dry the block using a piece of paper tissue. Then use a small amount of cyanoacrylate glue to stick the block, oriented with the tissue at the top, onto a vibratome truck using super glue. Section the slice at 50 micron thickness using a vibratome and place each section carefully in a glass vial containing 10 percent sucrose.
After sectioning, carefully pick up a section from the vial and place it flat into a Petri dish lid. Then use a fresh scalpel blade to remove the gelatin from around the section. Place the sections into a vial containing 2 milliliters of fresh 10 percent sucrose in PB, and agitate for 10 minutes to begin the cryoprotection process.
Following cryoprotection, place the sections flat onto a small rectangle of aluminum foil using a paint brush. Remove any excess liquid from the sections and carefully fold the foil into a parcel. Hold the aluminum foil close to the surface of liquid nitrogen without touching the surface for 30 seconds.
And then allow the sections to thaw completely for approximately 30 seconds. Repeat the freeze thaw another two times. Remove all the sections from the foil with a paintbrush and place them into a glass vial containing two milliliters of PB to wash off excess sucrose under constant agitation.
After immunostaining for fluorescence imaging, place the slide into a glass Petri dish containing PBS, and carefully remove the cover slip. Then wash the sections off the slide using gentle pulses of PBS from a Pasteur pipette. Place the sections into a clean glass vial containing PBS.
Perform the Avidin HRP reaction by first incubating the sections in Avidin biotin complex or ABC, for at least two hours to amplify the HRP reaction product. Next, wash the sections with PBS 3 times for 10 minutes, and then with tris buffer twice for 10 minutes. After removing the last tris buffer wash, quickly add one drop of eight percent nickel chloride solution to the diaminobenzidine solution or DAB solution.
Then pipette the solution in and out to mix and quickly add one milliliter of this solution over the sections. Incubate the sections in this solution for 15 minutes. Then add 10 microliters of 1 percent hydrogen peroxide to the DAB solution.
Allow the reaction to proceed in the dark under constant agitation for about one to two minutes until the cells are labeled. In a fume hood, place a small circle of filter paper into a Petri dish and dampen it with PB.Lift the sections one at a time from the glass vial using a paint brush and carefully place them flat on the paper. Cover the sections with another moistened circle of filter paper and remove excess buffer by gently touching tissue paper to the surface.
Apply eight or nine drops of one percent osmium tetroxide in PB to the top paper. Cover the dish and retain in the fume hood for at least 30 minutes, but no more than 1 hour. After rinsing, mounting, and coverslipping the sections, dehydrate through a series of 50 percent, 70 percent, 95 percent, and 100 percent alcohol solutions.
Following the dehydration step, transfer the sections to a glass vial containing 100 percent ethanol on a shaker for approximately five minutes. Replace the alcohol solution with propylene oxide and wash three times for five minutes. Following the last wash, keep around two milliliters of propylene oxide in the vial and add resin at a 1:1 ratio.
Ensure that the resin is dissolved and keep the sections under constant agitation for 30 minutes. After 30 minutes, use a wooden stick to place each section in an aluminum planchette containing epoxy resin, and incubate overnight. The next day, place the planchette on a hot plate for approximately 10 minutes.
Then pick up each section with a wooden stick and place on a clean slide. Once all the sections have been transferred onto the slide, view the slide under a dissecting microscope to check the orientation of the slices and place a coverslip over the sections. Cure in the oven for 48 hours at 56 degrees Celsius.
Use a low magnification objective to focus on the home section containing the cell body. Then use a 100x objective, focus on the cell body, and click in the center to mark the reference point. To trace the soma in 3D using 100x objective, select contour from the trace tab and select the cell body contour.
Use the joystick to move the focus to the very top of the cell body. Place the points by clicking around the perimeter of the part that is currently in focus. Right-click and select close contour to finish this first outline.
Repeat this process at different z positions until the bottom of the cell body is reached. To trace the dendritic arbor, select dendrite or apical dendrite, in the neuron menu. First, trace a short initial segment for each dendrite using the mouse scroll wheel to adjust the diameter of the cursor to match the diameter of the dendrite.
Trace along each dendrite. When a node in the tree is reached, right-click and select bifurcating node or trifurcating node from the dropdown menu. To identify matching points between the dendrites in a section that match as the completed section, click on the move tab and select match points.
Select the number of points needed to be matched, and then press OK.Click on the ending of a completed branch and then click on the branch. Repeat this for each match point. Once all of the dendrites of each section have been traced, trace the axon using the same process Shown here is a reconstruction of a CA2 basket cell with restricted dendritic and axonal arbor using a drawing tube.
The dendrites are in black and the axon is in red. This is an example image of the biocytin filled basket cell following the Avidin HRP protocol described here. Shown here is a video of a 3D neuronal reconstruction of the CA2 basket cell.
Dendrites are in dark pink and the axon is in white. Lastly, this image shows a dendrogram of the CA2 basket cell. It will take time becoming proficient with this protocol.
However, this optimized approach can generate high-resolution images of complex cortical and hippocampal structures in-vitro.
