Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits

This method acquires live slices of the chicken auditory brainstem which encompasses a larger gradient of frequency region in the same slice. These slices are suitable for electrophysiological and anatomical experiments with a larger tonotopic axis. This methodology will help to understand microcircuit development in the auditory brainstem.

While the anatomy is specific to the chicken, other avian species like quails and songbirds have similar anatomy. To begin, sterilize the eggs with 70%ethanol and incubate them at 38 degrees Celsius and 50%humidity. Next, set the bubbling rate for the ACSF such that the pH is 7.2 to 7.4 with an osmolality between 300 and 310 milliosmolar per liter.

Then mix five grams of agarose in 100 milliliters of dACSF. Dissolve the agarose completely using a water bath or microwave until the agarose starts bubbling. Pour the melted agarose into a Petri dish up to a thickness of five millimeters and keep at room temperature to set.

After setting, seal the Petri dish and store at four degrees Celsius. Cut the agarose into cubicle blocks for use at the time of dissection. First, clean the dissection area using 70%ethanol.

Glue the supporting or angled agarose block onto the vibratome tray. Place the egg under a bright light and locate the air-filled space on the larger or rounder side of the egg, then crack the shell over the air-filled space and expose the membrane sac. Make a gentle incision in the sac to expose the beak.

With a scalpel, gently pull the neck and head out of the egg. After decapitation, clean the head with chilled dACSF. Hold the head steady in chilled dACSF and make a rostrocaudal incision.

Start the incision behind and between the eyes and follow the length of the harvested neck. Separate the skin to expose the skull. Cut the skull behind the eye in midline to lateral direction.

Repeat for both hemispheres. Slice the rostral portion of the skull by placing the blade behind the eyes and making a quick cut. Then immerse the head in a dish of chilled dACSF.

Using a pair of small scissors, make midline to lateral incisions in the caudal region of the skull to separate the brain without causing tissue damage. Gently expose the brainstem and cerebellum. Retract the dorsal area of the entire skull.

Remove the brainstem and expose it by mild brushing using a fine paint brush. Use curved forceps to clean the brainstem from connecting tissue and blood vessels. Separate the brainstem from the cerebellum by cutting the peduncles and removing the blood vessels carefully.

Trim the brainstem of additional blood vessels. First, place the vibratome blade along the horizontal axis. For coronal slices, glue the brainstem on a slicing tray by the rostral side, keeping the rostrocaudal axis vertical.

For sagittal slices, keep the lateral medial axis vertical. For horizontal slices, glue the ventral side keeping the dorsal ventral axis vertical. To achieve the acute angular sagittal horizontal plane, glue the ventral side of the brainstem such that the ventral dorsal axis is vertical on the hypotenuse surface agarose block.

Glue the opposite surface of the agarose block facing the slicing tray and keep the rostrocaudal axis parallel to the blade edge. As an alternative to vibratome slicing, immerse the isolated brainstem in 4%low melting point agarose at 40 degrees Celsius in a 35 by 10 millimeter Petri dish. After the agarose solidifies, cut the cubicle agarose block using a sharp razor blade.

Glue the agarose block on its rostral side keeping the rostrocaudal axis of the brainstem vertical. Take coronal slices until the NM region is seen. Remove the agarose block from the glue with a sharp blade.

With a fine needle, gently place 0.5 microliters of toluidine blue dye on the NM to spot the nuclei. Remount this block on the slicing tray for sagittal or horizontal slices. Identify the nuclei with respect to the stained region and section the brainstem.

After sectioning, place the 200 to 300 microns sequentially collected slices in a sliced chamber containing ACSF to equilibrate for one hour at room temperature. Coronal sections of brainstem tissue from an E19 to 21 chicken embryo represent the relative isofrequency regions of the auditory brainstem nuclei from the lowest to the highest characteristic frequency auditory region. The afferent auditory nerve fibers and the bifurcation of NM axons were visible.

Nucleus angularis was also observed. In rostral sections, the superior olivary nucleus is located along the ventral lateral region of the coronal slice. In sagittal sections, NM and nucleus laminaris or NL were identified where the auditory nerve fibers entered the cluster of neurons.

The superior olivary nucleus was identified in the rostrolateral region. Low and high characteristic frequency auditory regions from NM and NL along the rostrocaudal axis were seen. The most medial slice showed the ipsilateral and contralateral axonal tufts and the end point of the auditory region.

In the horizontal slices, NM and NL were identified toward the midline, and neurons were spread along the lateral medial axis. Slices of the brainstem tissue at an acute angle from a horizontal plane showed the auditory brainstem nuclei across a large diagonal spread. Magnified images showed the tonotopic axis of the auditory nuclei along the rostromedial to caudolateral axis.

Ensure that the whole process is performed in ice chilled dACSF continuously bubbled with carb oxygen. This protocol is used for researchers investigating anatomical and biophysical property of developing brainstem microcircuits, specifically the relationship between the major auditory nuclei.