The overall goal of this procedure is to prepare healthy acute slices from dorsal intermediate region of hippocampus for long-term recordings and reconstruction of neurons in adult mice. Many laboratories like ours, studying dorsal hippocampus for its role in special learning and navigation. Common techniques employed for this purpose are behavioral experiments, anatomical tracing, and regional specific manipulations.
We have developed a brain slicing method to combine electrophysiology with this techniques to allow for direct correlation of invivodata within video recordings. The advantage of this protocol is that it combines a simple procedure to obtain transversal slices with the use of protecting solution to enhance the viability of brain tissue. This approach is particularly suited when mature animals are used as in behavioral experiments.
To begin this procedure, prepare next to the sink the necessary equipment for this section. Prepare on ice a 150 milliliter beaker and two glass Petri dishes of 10 centimeter diameter each. Draw three part of the lines until the bottom of a plastic Petri dish and position this dish close to the ice tray.
You will also need glue and the specimen holder for the vibratome. Then equip the surgery bench with big scissors, small scissors, rounded tip tweezers, fine tip tweezers, a thin metal spatula, a large metal spatula, and transfer pipettes. Peel the Petri dishes with a cutting solution.
One dish to perform the dissection of the head and the other for the dissection of the brain. In the latter, also place a piece of filter paper. Also fill the beaker with 30 milliliter cutting solution.
This will be needed for Transcardial perfusion. Fill the vibratome tray with ice cold cutting solution and keep all solution oxygenated with a carbogen bubbling device. Add the crushed ice made from the frozen batch of cutting solution to the cold fresh cut-in solution to keep the temperature down.
Pay attention, to keep the ice from the blade while slicing. Nearly all steps after the capitation are performed within the solutions. This helps to keep metabolism oxygen deprivation to a minimum.
Prepare an incubation chamber filled with a cutting solution under constant oxygenation. Place the chamber in a pre-warmed water bath at 35 degree Celsius. Prepare a slice storage chamber with several independent wells and fill it with storage solution.
Keep it under constant oxygenation at room temperature. In case the tissue expresses a fluorescent protein or a light activated opsonin. It is recommendable to keep the chamber in the dark.
For example, inside the box. After performing Transcardial perfusion, place the head of the mouse into the Petri dish with the ice cold cutting solution. Open the skin with a fine scissors to expose the skull.
Starting on the foramen magnum, cut along the sagittal suture until reaching the nasofrontal suture. Be careful to slightly pull up the scissors to avoid damage of the brain. Make two lateral incisions into the base of the skull.
Use the rounded tip tweezers to pull up the parietal bones. Be careful to also remove the meninges. If left they can damage the brain during extraction.
Use the small spatula to gently loosen the brain from the base of the skull. With a small spatula, detach the brain from the cranial nerves and transfer the brain onto a small piece of filter paper in the second Petri dish. Cut the brain in halves along the longitudinal fissure using a pre-chilled blade.
Separate the two hemispheres using the smallest spatula. Use the largest spatula to transfer one hemisphere to the plastic Petri dish with its medial surface facing down. Align the parietal cortex with one of the parallel lines to have a reference for the second cut.
Use a piece of filter paper to dry excess solution. Position the blade in parallel to the lines and cut the ventral part of the hemisphere. This flat surface will be later glued onto the specimen holder.
Return the hemisphere into the oxygenated cotton solution of the glass Petri dish and perform the same procedure on the second hemisphere. Place a drop of cyanoacrylate glue onto the vibratome specimen holder and spread it with a spatula to create a thin layer. Transfer the hemisphere onto the vibratome holder with the ventral side down using the rounded side of the spatula.
Starting this slicing from the parietal cortex would reduce the time required to collect the dorsal region of the hippocampus. Add a few drops of cold cutting solution to solidify the glue. Move the specimen holder into the vibratome tray and with appropriate settings, slice the brain until the region of dorsal hippocampus is visible.
Then, collect the slices with the use of a plastic transfer pipette. Transfer each slice into the incubation chamber and let it rest for 12 minutes. In the meantime, proceed with cutting the next slices.
After the 12 minutes of incubation, transfer the slices into the storage chamber. And let it rest until the start of the experiment. To demonstrate the purity of our preparation, we compare it to a coronal preparation which is commonly used to record from the dorsal hippocampus.
Shown here are, differential interference contrast micrograph acquired from the upper blade of the dentate gyrus in acute slices from two month old mice. In the transversal slice the neurons mostly showed smooth surfaces and only likely contrasted borders, indicated by black arrows. Neurons in Coronal slices however often appeared course and displayed strongly contrasted outlines, indicated by white arrows.
This suggests better viability of neurons in the transversal slice. In accordance with this impression, the average time to seal formation during patching in our transversal slices was significantly more rapid than in the coronal slices. As a general proxy for cell integrity, we record that the resting membrane potentials of granule cells and parvalbumin positive inter neurons which where significantly more depolarize in both cell types in coronal versus transversal slices.
Which in the end resulted in significantly more cells that had to be excluded in the coronal preparation. As granule cell axons run in the transversal plane, reconstruction of recorded cells resulted in retrieval of complete axonal arborization in our preparation. This was not the case in coronal slices where the axons may easily have been severed.
In addition, the morphological reconstruction of parvalbumin positive inter neurons in transversal slices allowed the depiction of extensive axonal and dendritic arborization, including the visualization of a small details, such as dendritic spines. Over all, we have presented a slicing methods to obtain transversal hippocampus slices with high neuronal viability for adult mice. This method can be used to measure electrophysiological in vitro investigation with anatomical and behavioral studies focusing on the intermediate dorsal part of the hippocampus.
