Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

The overall goal of this procedure is to produce slice cultures with clear hippocampal topography and evaluate neurogenesis in the dentate gyrus of the hippocampus. This is accomplished by first culturing hippocampal slices in a culture dish fitted with a special membrane. Next, the dividing cells are labeled with a mitotic marker.

The labeled tissue is then cut into sections. Finally, the sections are stained using standard immunohistochemical procedures to identify newborn neurons. Ultimately, immunohistochemistry and cell counting is used to measure cell density and cell properties.

Generally, individuals new to this technique may find it challenging because it requires a lot of practice. Visual demonstration is critical since the dissection steps and the process of transferring tissue from the culture plate to the vibram stage can be difficult to learn. Demonstrating the procedure will be Adam Moza, a graduate student in our laboratory, using a clean scalpel blade, cut the two hemispheres of an isolated wrap brain apart.

Transfer the left hemisphere back to the large Petri dish, and place the other PS side down in ice. Cold dissecting solution. View the medial face of the right hemisphere and identify the edge fornix.

A prominent band of white matter along the medial edge of the hippocampus once identified, cut through the fornix using only about 0.5 centimeters of the scalpel blade. Using two micros spatulas. Remove the hippocampus by placing the right hand spatula on the brainstem and lifting the overlying cortex.

With the left hand spatula. Gently lift the cortex to reveal the lateral ventricle and medial surface of the hippocampus. A white curved line.

The FIA should now be visible. Align the curvature of the spatula with the curvature of the fia, and gently press the spatula under the FIA slide the spatula left and ride along the rostral coddle axis before lifting in the dorsal direction. To remove the hippocampus, transfer the hippocampus to a separate small Petri dish with ice cold dissecting solution.

Repeat this procedure on the left hemisphere to remove the left hippocampus. Once isolated, carefully transfer the hippo campi to the tissue chopper stage. Arrange them adjacent and parallel to each other and perpendicular to the axis of the chopper blade.

Use a paintbrush to position the tissue and add a few drops of the dissecting solution on top of the hippo.Campi. Cut the tissue in 400 micrometer slices without pausing to remove individual slices After the whole hippocampus has been cut, use a paintbrush to gently transfer the sections to a small Petri dish with dissection solution. Under a dissecting microscope, carefully separate the floating slices using a micros, spatula and paintbrush.

Next, remove a culture plate previously prepared with a culture insert from the incubator and place it into the laminar flow hood. Using a fire polished PEs or pipette, draw up four to five slices and place them onto the apical surface of the culture. Insert membrane.

Use a paintbrush to adjust their spacing, leaving space between individual sections and the border of the culture. Insert once in position, remove the excess dissecting solution from the apical surface of the membrane. Place the culture plate with serum containing culture medium and the hippocampal slices back into the incubator at 35 degrees Celsius and 5%carbon dioxide.

Feed the cultures in a sterile laminar flow hood, two days post dissection. First aspirate out the old culture medium, and then add one milliliter of fresh serum containing medium to the wells. Using a sterilized glass pipette, gently replace the culture insert and take care to remove any air bubbles that may have formed underneath the membrane surface.

After the first feeding, change the medium every other day. To study the maturation and integration of dentate granule cells, change the feeding medium to one with a thymidine analog and incubate for two hours at 35 degrees Celsius. Following the incubation, remove the analog containing medium and replace with regular feeding medium.

To resume the normal feeding schedule at the desired time point. Retrieve the culture plate and remove the culture medium. Next, add one milliliter of 4%PFA to each culture.

Well seal with paraform and store at four degrees Celsius for 24 hours the following day. Remove the fixative and add one milliliter of PBS with sodium azide seal and store the plates as shown previously. When ready for sectioning, use a scalpel to carefully cut along the perimeter of the circular insert and transfer the detached membrane to a Petri dish containing PBS.

After rinsing, use forceps to transfer the membrane to the vibram mounting stage. Next, use a scalpel to eliminate excess membrane surrounding two to three slices selected for sectioning clean edges. Help with sectioning ensures the membrane is flat, and that it can easily adhere to the cutting surface.

Place one to two drops of adhesive on the vibram cutting stage and spread it in an even layer. Using a 22 gauge needle, quickly spread the adhesive into a rectangular shape with a long edge parallel to the cutting blade. Use forceps to transfer the trimmed membrane to the glue on the cutting stage and ensure that there are no air bubbles as the super glue dries.

Transfer the Vibram stage back to the PBS containing Petri dish. Prepare the Vibram blade and a 48 well plate containing sodium azide to store the tissue sections. Use the vibrato to generate 30 micrometer sections of organ typic sliced tissue.

Transfer the sections to a 48 well plate containing PBS with sodium azide for storage and subsequent immunohistochemical staining. Perform standard immunohistochemical staining procedures by sequentially replacing PBS with solutions containing primary and secondary antibodies, and incubating the sections in these solutions for the desired periods of time. These fluorescence microscope photographs highlight the preserved hippocampal morphology in culture.

Here, organotypic slices were immuno labeled for chloro, deoxy, uridine, and calbindin. A sample micrograph of a slice immuno labeled for CLDU and double cottin is shown here. The left side image shows a neuron labeled with double cottin.

The middle image shows a chloro deoxy uridine labeled nucleus. The right side image shows both markers superimposed to illustrate an mature neuron. The left side image shows a chloro deoxy uridine labeled nucleus.

The middle image shows a neuron labeled with cow binding. The right side image shows both markers superimposed to illustrate a mature neuron Following this procedure. Other techniques, like in vivo immunohistochemistry can be used to ask additional questions such as, how does neuronal development in culture compare to neuronal development in vivo, especially in the adult brain?