The overall goal of this procedure is to prepare chicken, auditory brain stem slices to be used for in vitro whole cell physiology or long-term organotypic cultures. This is accomplished by first removing the embryo from the egg. The second step of the procedure is to dissect the brain from the skull and to isolate the auditory brainstem region.
The third step of the procedure is to prepare slices of the brainstem region on a viome. The final step of the procedure is to place brainstem slices in a chamber for physiology recordings, or onto Millipore plates for culturing. Ultimately, results can be obtained that show healthy auditory brainstem slices from chicken embryos through physiological recordings or long-term organotypic cultures.
Hi, my name is Armand Sidle. Both this method can provide insight into avian auditory development. It can also be used to study topographic exon regeneration, dendrite dynamics, as well as intrinsic and synaptic properties due to the unique structure and function of this binaural auditory circuit.
Generally, individuals new to this technique will struggle. Tissue needs to be rapidly dissected, sliced and stored in order to have viable samples for physiological and culturing approaches. First, make a 4%agar solution by adding 40 milligrams of agar per milliliter of distilled water and heat in the microwave until boiling.
Pour the hot agar solution into a Petri dish and let it cool until firm once solidified. Cut out a small block of the agar and glue it to the vibrato stage. Next, prepare 500 to 1000 milliliters of artificial cerebral spinal fluid or A CSF for later.
Use in the vibrato stage chamber by bubbling continuously with a mixture of 95%oxygen, 5%carbon dioxide pH range, 7.2 to 7.4, and an osmolarity of 295 to 310 milli osmoles per liter. While the A CSF is bubbling, clean the dissecting into culture areas, Vibram and blade with 70%ethanol. Rinse the vibram blade with distilled water and let dry place a clean fluid absorption pad on the dissecting area and lay out the appropriate dissecting tools, usually consisting of a scalpel, scissors, tweezers, razor blades, pins, petri dish, and a spatula under a sterile tissue culture hood fill one well per chicken embryo of a six.
Well plate with one milliliter of culture, medium and incubate at 35 degrees Celsius and 5%carbon dioxide. Prepare as many culture membrane inserts as needed and store them in the hood for later. Use on the dissection pad.
Place an egg under a light source to locate the air-filled space void of the embryo, which is typically the large side of the egg. Break open the large side of the egg using the flat end of the spatula, exposing the membrane sack. Make a slice in the membrane sac with the scalpel or scissors, and gently extract the head of the chicken embryo with the spatula.
Using scissors. Quickly decapitate the head of the embryo. Place the tip of a razor blade slightly posterior to the middle of the eyes and make a rostral to coddle midline incision through the skull.
Applying gentle pressure for younger embryos. Use less pressure for older embryos. Use more now that the incision has been made.
Gently push aside the skin and feathers for older animals to expose the skull and verify the midline.Cut. Using strong pressure, block the rostral portion of the skull by slicing with a razor blade. Position the blade posterior to the eyes and rapidly cut down through the entire skull and brain tissue.
Place the blocked tissue onto a Petri dish with the coddle side facing up. Pin the tissue through the lateral muscle region and completely submerge with bubbled. A CSF.
Make midline to lateral incisions with small scissors in the caudal region of the skull, slightly anterior to the visible neck muscles on both sides of the head. Pull away the skull and tissue to expose the cerebellum and brain. Now that the brain tissue is exposed, locate the cerebellum and brainstem to remove the tissue from the skull.
Use a pair of small scissors to gently cut away from the skull base the attached tissue that surrounds the brainstem on the ventral side. Once the brainstem has been removed from the skull, pin it down onto a petri dish filled with cigar through the optica. Next, completely remove the cerebellum by cutting the peduncles with small scissors, exposing the floor of the fourth ventricle.
Use tweezers to remove any membrane is tissue and blood vessels from the surface of the brainstem and make lateral cuts through the optic teta. To free the brain stem, place a small amount of superglue on the Vibram stage in front of the agar block using angled tweezers gently but swiftly. Lift the brainstem at the spinal cord and place the tissue onto the superglue with the rostral side down and dorsal side towards the vibram blade.
Take up any excess glue with a paper tissue or filter paper. Gently fill the vibram stage with approximately 100 milliliters of oxygenated A CSF. If the slicing is performed rapidly, it is okay to use the prepared oxygenated A CSF.
From the first section. Position the vibram blade so that it is at a 20 to 22 degree angle with respect to the Vibram stage. Start the vibram at maximum oscillation amplitude.
Then move the stage up the blade so that the top of the tissue is parallel with the blade. Before slicing. Quickly move the blade towards the brainstem tissue, but slow down considerably just prior to the blade making contact with the tissue.
Now that the blade is in position, begin slicing the tissue with the slowest possible forward motion. Once through the entire coronal section of tissue, gently move the slices away from the blade with a paintbrush and discard. Lower the vibrato stage in 300 to 500 micrometer steps and slice.
Again, repeat until anatomical signatures are visible in the brainstem tissue, discarding all previous slices. Now slice and collect brainstem tissue containing auditory structures at 200 to 1000. Micrometer thicknesses carefully collecting usable slices in the order of slicing.
If the slices are to be used for in vitro physiology, which will be shown in a later section of this video, they should at this point be individually stored in custom chambers made of hollow plastic tubes open at the top and nylon mesh glued to the bottom. Each individual chamber is attached to a larger storage chamber in which approximately 350 milliliters of A CSF is continuously bubbled. If slices are to be used for organic typic cultures, see the next section immediately following slice collection transfer slices with approximately 500 milliliters of A CSF using a fire polished glass pipette fitted at one end with a rubber bulb to a 48 well plate on ice.
Placing one slice per well and maintaining the order of slicing. When finished, collecting all the slices of one brain, bring the 48 well plate to the tissue culture hood. Retrieve the six well plate that was prepared earlier from the incubator and bring it to the hood as well.
Place a membrane insert into a well with culture medium, making sure no air bubbles are trapped underneath. Using a glass pipette transfer one slice in a drop of A CSF onto the moist membrane. Insert and remove any excess A CSF that is still on top with a micro pipette.
Repeat for a maximum of four slices per membrane and position the slices so that they do not touch each other or the rim of the membrane. Store the cultures in a 35 degree Celsius incubator and maintain them by changing the culture medium three times per week under a tissue culture hood. Place each slice in an individual holding chamber using a fire polished glass pipette fitted at one end with the rubber bulb.
Continuing to maintain the order of slicing. The holding chamber should be filled with the A CSF prepared earlier and continuously bubbled with a mixture of 95%oxygen, 5%carbon dioxide the entire time during the physiology experiments. Allow the slices to equilibrate by placing the holding chamber into a 35 degree Celsius water bath for approximately one hour, and then allow it to rest at room temperature for 30 minutes.
Slices can be used for up to six hours after removal from the warm bath. After 30 minutes at room temperature, the slices can be transferred from the holding chamber to a 0.5 milliliter recording chamber mounted on a microscope for electrophysiological experiments. A representative schematic and in vitro slice images of the chicken auditory circuit are shown here.
The slice images are typical of the auditory brainstem region and with practice can be easily identified. The ENT excitatory inputs from the auditory nerve or a N project to both the nucleus Magno cellis or NM and nucleus angularis or N na. The NM projects bilateral excitatory inputs to the nucleus, lamins or NL on both sides of the brainstem.
An inhibitory input from the superior olivar nucleus or SON projects to the na, nm and nl. Here, a coronal slice of auditory brainstem from an E 11 chicken is shown after seven days. In culture.
Neurons were labeled using immunohistochemical methods, an antibody against the microtubule associated protein two, which occurs in the soma and dendrites labels neurons in green. A cluster of NM neurons and axons is labeled in red via electroporation of an rin dye. The ipsilateral nm axon terminals indicated by the white arrowheads can be seen projecting to the dorsal NL dendrites.
Shown here are superimposed membrane voltage changes in response to hyperpolarizing and depolarizing current steps from acute and cultured tissue at four and seven days in culture. Note the changes in hyperpolarizing voltage sag, strong reduction in outward current and single action potential firing that develops similarly in the slice culture approach. Compared to age equivalent acute tissue resting membrane potentials were between minus 55 and minus 60 millivolts Once mastered, this technique can be performed in less than 15 minutes if done Correctly.
While attempting this procedure, it's important to remember to use appropriate sterile techniques, And after watching this video, you should have a good understanding of how to prepare chicken auditory braid stem slices to be used for physiological recordings or to be used for culturing.
