This protocol makes it possible to evaluate potential morphological alterations in neurons and dendritic spines that may underline neurochemical and behavioral abnormalities. It is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction. Begin by preparing PVP solution according to manuscript directions.
Fill the tubing with PVP and leave it for 20 minutes. And expel it through the other end with a 10 milliliter syringe. Combine 170 milligrams of tungsten microcarrier beads with 250 microliters of methylene chloride.
Then thoroughly vortex the suspension. Next, add 6 milligrams of lipophilic DilC18(3)dye to 300 microliters of methylene chloride and vortex. Pipet 250 microliters of the tungsten bead suspension onto a glass slide.
Wait for the suspension to air dry and add 300 microliters of the dye solution. Once dried, use a razor to split the mixture into two 1.5 milliliter centrifuge tubes and fill the tubes with water. Sonicate the mixture in a water bath until homogenous.
Making sure that the sonicator tip lies directly on the tubes. Combine the two mixtures in a 15 milliliter conical tube. And sonicate for another three minutes, making sure that no large clumps of dye coated beads remain.
After sonication, draw the mixture into the PVP coated tubing with a 10 milliliter syringe. And feed the tubing into the preparation station. Rotate the tubing for one minute.
Carefully remove all the water using a syringe. Turn on the nitrogen gas and adjust the nitrogen flow to approximately 0.5 liters per minute. Rotate the tubing in the preparation station and dry it with nitrogen for 30 minutes.
Remove the tubing from the station and cut it into 13 millimeter segments with a tubing cutter. Make sure that the rat is not responsive to noxious stimuli and reflexes are absent. Then secure it in the supine position.
Make an incision along the thoracic midline. Separate the diaphragm and open the chest with scissors. Then insert a 20 gauge 25 millimeter needle into the left ventricle.
Immediately cut the right atrium with scissors and perfuse 15 milliliters of 100 millimolar PBS with a 5 millimeters per minute flow rate. Then perfuse 100 milliliters of 4%PFA buffered in PBS. After perfusion, remove the entire rat brain.
And postfix it with 4%PFA for 10 minutes. Use a rat brain matrix to cut 500 micrometer thick coronal section. Make a fist cut and keep the blade in place.
Then make a second cut with a second blade and vertically remove the first blade, keeping the tissue on the blade surface. Place the brain slices in the 24-well plate with 1 milliliter of PBS in each well. And repeat the process until all slices have been cut.
Remove PBS from each targeted well. Load the cartilage with piece of Dil/tungsten tubing and place it into the applicator. Put a piece of filter paper between the two mesh screens.
And connect the applicator to the helium hose. Then adjust the output pressure of helium to 90 pounds per square inch. Place the applicator vertically on the center of the targeted well 1.5 centimeters between the sample and the mesh screen.
Then fire the Dil/tungsten tubing. Load the cartilage with the next tubing and continuously fire the beads from the tubing on the remaining slices. Fill the 24-well plate with 100 millimolar PBS.
And wash the slices three times with 500 microliters of fresh PBS. Making sure that the slices do not flip over during the wash. When finished, add 500 microliters of fresh PBS to the slices.
And incubate them for three hours at four degree Celsius in the dark. After the incubation, use a fine brush to transfer the brain slices onto glass slides. And immediately add one milliliter of antifade mounting medium to each section.
Place a 22 by 15 millimeter coverslip over the sections and dry the slides in the dark. Turn on the confocal microscope system and switch to a 60X objective. Adjust the imaging settings according to manuscript directions.
And obtain Z-stack images for the targeted neuron type based on brain region boundaries and morphological characteristics of neurons. Isolate primary cortical neuron from F344/N rats at postnatal day one and culture them in a 35 millimeter glass bottom dish for one week. Refreshing half of the medium on the third day after isolation.
Wash the dish twice with 1 milliliter of 100 millimolar PBS. And fix the cells with 4%PFA for 15 minutes at room temperature. Ballistically label the cells as previously described.
And wash them three more times with 1 milliliter of PBS. Add 500 microliters of PBS to the cells and incubate them for three hours at four degree Celsius in the dark. Then add 200 microliters of antifade mounting medium.
And obtain Z-stack images for each targeted neuron. Typical pyramidal neurons in hippocampal region in the rat brain sections were identified with ballistic labeling technology characterized by one large apical dendrite and several smaller basal dendrites around the soma. Neuronal reconstruction quantitative analysis software was used to trace dendritic branches and detect spines.
Subsequently, the software was used to assess the dendritic branching complexity and neuronal arbor complexity. Morphological changes in dendritic spines were assessed using length, volume, and head diameter. Then spines were classified into thin, stubby, and mushroom spines.
And the relative frequency of the number of the spines between each radius was examined. Further more the ballistic labeling technique was validated on a primary pyramidal neuron in cell culture. Pyramidal neurons were identified based on the triangle shape of the soma and large apical dendrite.
Then neuronal reconstruction software was used to analyze the distribution of thin dendritic spines and dendritic spine length. When attempting this protocol, it is important to avoid large clumps or clusters of ballistic dye coated tungsten beads starting preparation. Clumps would not allow individual neurons to be distinguished.
Combined with neuronal reconstruction software this method allows us to examine neuronal and dendritic spine morphology in hippocampal pyramidal neurons. Neuronal reconstruction software utilize an algorithm to offer automatical assisted classification of dendritic spines. The quantification of multiple neuronal parameters offers an opportunity to better understand the mechanism underlying neuronal cognitive dysfunction.
