This protocol facilitates preparation of horizontal hippocampal brain slices for their use in various scientific applications. This technique preserves the integrity of all the hippocampal fiber pathways within a single slice of hemisphere. This slice protocol is ideally suited to assess neurological changes that occur on brain pathologies that develop and manifest in the dentate gyrus.
The most important aspect of this protocol is to find the balance of a fast as possible execution and the gentle handling of the brain tissue. To prepare one liter of ACSF, slowly mix all of the solid chemicals as indicated in 800 milliliters of water under constant stirring with a magnetic stir bar before slowly dripping in the appropriate volumes of magnesium sulfate and calcium chloride. Then use a vapor pressure osmometer to validate the osmolarity between 305 to 315 milliosmole and continuously bubble the ACSF solution at room temperature with carbogen to set the pH between 7.3 and 7.4.
To prepare 250 milliliters of high-sucrose slice solution, add the compounds to 25 milliliters of pre-sliced solution as indicated in the table and verify that the osmolarity is between 320 to 325 milliosmole. Then bubble the high-sucrose slice solution for 10 to 15 minutes with carbogen to set the pH between 7.3 and 7.4 and store the high-sucrose slice solution for 20 to 30 minutes at minus 80 degrees Celsius until it is partially frozen. To prepare a workspace for the dissection, fill a recovery chamber with carbogenated ACSF solution and place the chamber in a 32 degree Celsius water bath.
Set the vibratome to the appropriate cutting program and fill the holder with ice, mount it on the vibratome, and attach a blade to the vibratome arm. Use a spatula to crush and mix the partially frozen high-sucrose slice solution until a heterogeneous slush is obtained. Then bubble the solution with carbogen and use the solution to hydrate a piece of filter paper on top of a chilled 90 millimeter culture dish and fill a 35 millimeter culture dish on ice.
To harvest the brain, use dissection scissors to cut open the scalp of a two to six-week-old male mouse and to open the calvaria along the sagittal suture. Use curved forceps to remove the skull until the whole brain including the olfactory bulbs is visible and use a spatula to carefully scoop out the intact brain tissue. Place the brain into the 35 millimeter culture dish and use a Pasteur pipette filled with high-sucrose slice solution to gently remove any hair or blood particles from the tissue.
Use the spatula to transfer the cleaned brain onto the piece of soaked filter paper and use a blade to cut the brain longitudinally in half. Place both hemispheres on the freshly cut medial side and use the blade to make a parallel cut on the dorsal top of each hemisphere to remove the dorsal region of each piece of brain. Place both hemispheres on the freshly cut dorsal side with the ventral part of the brain facing up and place a drop of super glue onto a specimen plate.
Use a pipette tip to spread the glue into a big area to accommodate both pieces of tissue and touch a piece of filter paper strap to the ventral side of one hemisphere. Use another filter paper strap to carefully semi-dry the dorsal side of the brain and position the hemisphere dorsal side down onto the glue on the specimen plate. Use two additional filter paper straps to position the second hemisphere onto the glue as just demonstrated and place the specimen plate into the slicing chamber.
Then quickly but carefully cover the plate with ice cold high-sucrose slice solution slush. To acquire sections of the brain tissue, position the vibratome blade in front of the medial side of the hemispheres and lift the vibratome table so that the blade is on the same height as the ventral sides of the hemispheres, which are now facing up. Use the vibratome control to lower the blade 600 microns further in the dorsal direction and slice the tissue until the first two slices are completely separated from the two hemispheres.
When the first two tissue slices have been obtained, reverse the cutting direction and lower the blade another 300 microns before slicing again. When the hippocampus becomes visible, use a widened plastic Pasteur pipette to collect and transfer the slices into the recovery chamber in the water bath. Leave the slices in the ACSF filled recovery chamber for one hour, then place the recovery chamber at room temperature for 30 minutes before performing electrophysiological recordings of the tissue samples.
Here, representative negative and positive field excitatory post-synaptic potential recordings from low and high-quality slices respectively can be observed. The negative example trace exhibits a large fiber volley amplitude upon depolarization of the stimulated neuronal fibers that is even higher than the actual fEPSP amplitude while the high-quality trace demonstrates a small fiber volley to fEPSP ratio and a high fEPSP amplitude. The viability of a brain slice can also be analyzed by plotting field excitatory post-synaptic potential slopes versus the fiber volley amplitudes.
Input/output curves which are typically used to determine the slice quality can be obtained by applying increasing current stimuli to the brain slice and monitoring the subsequent fEPSP responses. Due to the suboptimal conduction properties of poorly preserved brain tissue, low-quality brain slices demonstrate reduced input/output curves. Viable brain slices have stable synaptic transmission baselines while brain slices with an unstable baseline cannot be used for further conditioning protocols to study the synaptic plasticity of brain circuits.
fEPSP baseline recordings can also be useful for monitoring drug effects on synaptic transmission itself. In addition, the brain slices can be used in combination with a calcium indicator to acquire fluorescence imaging recordings and to study calcium influxes under different slice conditions or treatments. Ensure that the brain dissection is performed within no more than 1.5 minutes of sacrifice and this is still guarantee that the brain tissue is only briefly without cooling and oxygen supply.
Hippocampal brain slices have many different applications from molecular biological techniques to electrophysiology. This procedure facilitates the intense investigations of hippocampal anatomy and neurological processes.
