In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

Brain slice experiments allow interrogation of neuronal function with high electrical and temporal resolution, but these slices typically sever many presynaptic connections. A wedge-shaped slice maintains a more intact presynaptic circuitry. This modified brain slice maintains a more complete in vivo-like neuronal circuit while providing the benefits of in vitro experimentation, such as visually guided patch clamp recordings, pharmacology, and activity imaging.

Accurate wedge sliced geometry can be estimated based on the atlas locations of the neurons within the circuit, but the integrity of the presynaptic schemata and axons should be determined using histology. Begin by using a razor blade to cut the skin at the midline of the skull from the nose to the back of the neck. Peel back the skin to expose the skull and starting at the base of the skull and continuing towards the nose, use small scissors to make an incision in the skull through the midline.

At the lambda suture, make cuts in the skull from the midline laterally towards the ear on both sides to peel back the skull to expose the brain. Starting at the rostral end, use a small lab spatula to gently lift the brain from the skull to allow the optic nerves to be severed. Continue to gently work the brain backwards, exposing the ventral surface and use fine forceps to carefully pinch the trigeminal nerves near the ventral surface of the brainstem to cut them.

Place the preparation in a glass Petri dish filled with cold slicing solution and place the dish under a dissecting microscope. Trim the facial nerves close to the brainstem to expose the vestibulocochlear nerves. Use fine forceps to push the tips into the foramina where the vestibulocochlear nerve exits the skull as far as possible.

Pinch to sever the nerves on both sides, leaving the nerve root attached to the brainstem. Remove the meninges and vasculature from the ventral surface of the brainstem near the trapezoid body. Then pinch the remaining cranial nerves and connective tissue to free the brain completely from the skull, taking care to preserve the remaining spinal cord, if possible.

To prepare the surface of the brain for fixture to the stage place the brain ventral side up and use a blunt tool to gently immobilize the spinal cord to stabilize the brain tissue. Insert open forceps at an approximately 20 degree angle through the brain to the bottom of the dish so that the tips exit the dorsal surface of the brain caudal to the optic chiasm and use a razor blade to cut along the forceps. Next, prepare a one cubic centimeter block of 4%agar and spread a small drop of glue into a rectangle on the stage.

Use forceps to carefully lift the brain and gently dab the excess liquid with the edge of a paper towel. Then place the blocked surface onto the glue so that the ventral surface will be facing the direction of the blade during slicing and push the agar block gently against the dorsal surface as a support. To acquire wedge slices with the cochlear nerve root on the thick side and the medial olivocochlear neurons and the medial nucleus of the trapezoid body on the thin side, place the magnetic disc with the attached brain onto the stage holder and place the holder in the slicing chamber of a vibratome with the ventral surface of the brain oriented toward the blade.

Fill the chamber with ice cold slicing solution and lower the blade into the carbogen bubbled solution. Cut slices caudal to the region of interest to make sure the slices are symmetrical. Then shift the stage approximately 15 degrees to one side.

Continue slicing carefully until the auditory nerve root is close to the surface on one side and the facial nerve can be seen at the surface of the other side of the slice. Shift the stage 15 degrees back to the original position and move the blade away from the tissue. Spin the stage base 90 degrees so that the lateral edge of the thin side is facing the blade and lower the blade several hundred microns before slowly bringing the blade close to the edge of the tissue.

With the blade retracted slightly lower the blade to the desired thickness of the thin edge of the slice. Move the blade back from the tissue and spin the stage base back so that the ventral surface faces the stage base. Make a cut to designate the rostral surface of the wedge slice and transfer the slice to a one square centimeter piece of interface paper caudle surface down.

The facial nerve should be visible on both hemispheres of the slice on the rostral surface. Then move the slice to a 35 degrees Celsius incubation chamber to recover for 30 minutes. To set up the wedge slice for electrophysiology analysis place the sample into a recording chamber that is being continuously perfused with 35 degrees Celsius ACSF and stabilize the slice.

Using DIC optics, focus on the auditory nerve root on the thick side of the slice and use a micromanipulator to move the bipolar tungsten stimulating electrode to the auditory nerve root and gently into the surface of the tissue. Move the field of view to the ventral nucleus of the trapezoid body on the thin side and select a medial olivocochlear neuron as the target for patch clamp electrophysiology under epifluorescence using a 561 nanometer emission filter. Fill a recording pipette with the appropriate internal solution for the proposed experiment and under DIC optics patch and record from the medial olivocochlear neuron in the whole-cell configuration.

Adjust the electrical stimulation amplitude of the auditory nerve root to obtain consistent postsynaptic events in the medial olivocochlear neuron. Then run appropriate stimulation protocols to observe evoked synaptic currents in medial olivocochlear neurons. This wedge slice preparation is designed to contain the auditory nerve root and the cochlear nucleus contralateral to the medial olivocochlear neurons targeted for recordings.

In this cresyl violet stained resectioned wedge slice the cochlear nucleus is present in nearly its full rostral-caudal extent. And the auditory nerve root is observed entering the cochlear nucleus. In addition, the wedge slice contains neurons of the medial nucleus of the trapezoid body ipsilateral to the medial olivocochlear neurons from which recordings are performed.

To confirm the neuronal connectivity within a wedge slice presynaptic inputs are stimulated in two ways. First, the ventral acoustic stria at the midline is electrically stimulated, activating T-stellate axons directly and the medial nucleus of the trapezoid body neurons via globular bushy cell axon stimulation and resulting in postsynaptic currents that are measured at the medial olivocochlear neuron. The auditory nerve root is then stimulated to activate the entire monaural ascending brainstem circuitry and to evoke postsynaptic responses.

Comparison of the onset latency measures of the first postsynaptic current evoked with direct stimulation of the ventral acoustic stria with those evoked with auditory nerve stimulation reveals a significantly longer latency in the auditory nerve stimulation event, indicative of the synaptic delay resulting from the additional cochlear nucleus synapse activated during auditory nerve stimulation. The use of anatomical landmarks and careful maneuvering of the magnetic disc stage are critical for creating wedge slices with intact neuronal circuitry and reliable evoked post synaptic responses. This slicing technique provides a platform for using additional in vitro electrophysiology tools including calcium or voltage imaging, optogenetics, neurotransmitter uncaging, and both intracellular and extracellular pharmacology.