Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The overall goal of this procedure is to record post-synaptic activity at inner hair cell afferent synapses. This is accomplished by first exposing the sensory epithelium of a cochlea recently removed from a decapitated rat. The second step of the procedure is to position the excised apical, turn on a modified cover slip, and transfer it to the recording chamber.

The third step of the procedure is to locate and gain access to an afferent baton with a recording electrode. The final step of the procedure is to form a tight seal onto and then rupture the afferent fiber membrane establishing the whole cell configuration of the patch clamp technique. Ultimately, results can be obtained that show post-synaptic occurrence or potentials from a single ribbon synapse through whole cell patch clamp electrophysiology.

Visual demonstration of this method is critical as the dissection and preparation of cochlear tissue is difficult to learn and the small afferent fibers are hard to recognize and access. Prior to beginning the dissection, prepare a modified cover slip that will hold the preparation in place During recordings. Using an eight to 12 millimeter circular glass cover slip, apply a drop of half cured syl guard towards the edge of the cover slip.

Place the thick end of a fine insect pin on the cover slip. Hold the insect pin tightly against the glass. With forceps, heat the sill guard against a heated coil to set.

Prepare 10 to 20 micro electrodes as described in the accompanying text. After fire polishing with a micro forge, the micro electrode should have an outer tip diameter of approximately one micron. The final thickness of the pipette wall equals about one third of a micrometer following anesthetization and decapitation of a three week old rat.

Begin the dissection by removing the skin bi dissecting the head and removing the brain To expose the temporal bone, remove both temporal bones. Place each temporal bone in a clean dissecting dish containing standard extracellular solution. The next step is to remove the bone, encapsulating the temporal bone, so as to expose the cochlea.

Hold the temporal bone securely at the base with a pair of forceps. Identify the round and oval window and the cochlea. Orient the cochlea so that the oval window and spiraling side of the cochlea face upwards.

Remove excess bone around the cochlea. Use a second pair of fine forceps to chip the bone directly off the cochlea, beginning with the area that is more transparent than the rest of the bone. Here the bone is thinner and easier to remove.

Continue removing the bone from around the apical coil. Use micros dissecting scissors to cut through the modis beneath the apical turn. Then disconnect the apical turn from the lower turns of the cochlea.

Take care to protect the apical coil. It should not be pulled or stretched. Finally, remove the apical turn from the cochlea without tearing the tissue too much.

Use fine forceps to remove the rest of the bone. The next step is to remove the STR of oculis and the tial membrane. Carefully remove the STR of oculis, the shiny strip of tissue situated outside the hair cell region.

Make sure to avoid removing the sensory hair cells, which can easily become detached along with the STR of oculis. Next, use fine forceps to detach the tial membrane. The shiny semitransparent membrane that sits above the sensory hair cells now trim excess tissue and bone and flatten the preparation with the forceps.

This is necessary so that the tissue can be placed evenly under a pin. The dissection is now finished. Place the preparation under the pin on the prepared cover slip taking care to position the pin away from the hair cells once the dissection is complete.

Use forceps to transfer the preparation to the recording chamber while keeping a drop of extracellular fluid on the cover slip that holds the cochlear tissue immediately begin profusion with extracellular solution. To ensure better survival of the preparation, use the microscope's 10 x objective or the 40 x water immersion DIC objective. To locate the preparation, orient the preparation so that the recording electrodes can approach the inner hair cells at a right angle to their lateral walls.

If the visibility of the basal region of the inner hair cells is limited because the preparation is curled over, use a recording electrode to push the outer edge of the preparation down against the glass cover slip. Taking care to avoid pressing the inner hair cells themselves. In this preparation, the upper electrode is holding the outer edge of the preparation down the lower electrode marks where the afferent batons are located.

For reference, the NC 70 Nuv Con camera displays the Forex magnified image on the monitor to assess whether the tissue in the inner hair cell region is healthy. Find the hair bundles as a point of reference and focus down to find the nuclei. Focus further down towards the base of the inner hair cells with its afferent endings and back up until reaching the level of the nuclei on the monitor.

Now locate the afferent batons around the base of the inner hair cells. Batons are spherical or ellipsoidal, approximately one micron in diameter and are light in color with a shiny appearance. You will find them by focusing up and down.

The next step is to clear a path to the afferent fibers using a recording electrode with a one micron tip filled with intracellular solution. Apply positive pressure and use the micro manipulator to maneuver the electrode to the preparation. Use this electrode to make an incision in the preparation at the level of the inner hair cell base.

Push the electrode between the thin layer of supporting cells and the inner hair cells using positive pressure to forge an access route to the area at the base of the inner hair cells. Carefully remove the electrode and replace it with a fresh electrode with a one micron diameter tip filled with intracellular solution. Using positive pressure to maintain a clean electrode tip, move the recording electrode through the access hole around adjacent supporting cells towards the afferent baton.

Ensure that the electrode tip is directly in front of the afferent baton batons offer more resistance to movement of the pipette than the supporting cells and the inner hair cell membrane. This difference can be felt as the pipette is moved, move the pipette up and down and push to make sure that the bhuton moves. This indicates that the pipette tip and bhuton are in the same plane of focus with the electrode pressed against the afferent bhuton Simultaneously release positive pressure and apply suction to form a giga ohm seal.

Apply gentle bursts of suction to rupture the membrane within the electrode to obtain a whole cell recording. If the cell patched is an afferent baton, small capacitive transients are observed in the whole cell recording mode. This figure shows typical transient recorded from an afferent fiber.

Begin recording synaptic activity. If the afferent fiber does not exhibit spontaneous activity, depolarize the hair cell by applying an extracellular solution with a higher potassium concentration. This figure shows typical current voltage relations for AFF fibers on the left and inner hair cells on the right.

The current voltage relations were recorded a holding potential of minus 84 millivolts with voltage steps from minus 124 millivolts to positive 36 millivolts in 10 millivolt increments. Voltages are shown to the right of some traces focusing on the current voltage relations from an afferent fiber. Note that the excitatory post-synaptic occurrence or EPCs are present during the majority of the voltage steps.

The EPCs reversed a positive at about positive six millivolts. This recording was carried out in the presence of tetra to toxin to block voltage-gated sodium currents without the blocker Fast activating sodium inward currents would've additionally appeared in the recording and identified the recording as an afferent fiber recording. Note, the slowly activating inward current at hyperpolarizing voltages.

This current is not present in inner hair cells or supporting cells and provides a good indication that the recording is from an afferent fiber. These recordings show the current voltage relations for an inner hair cell. The arrow points to the fast activating outward potassium currents at positive potentials.

These are followed by delayed rectifier potassium currents due to a narrow pipette diameter and high access resistance. The inner hair cell currents shown here are smaller than expected to obtain accurate recordings of inner hair cell currents. Pipettes with lower access resistances should be used.

However, the recordings presented here demonstrate that the current voltage relations of inner hair cells and afferent fibers can be clearly distinguished. This figure shows synaptic currents recorded from an afferent fiber. Note the variable size and shape of the EPCs.

The recordings were done at room temperature with tetra toin applied to block the voltage gated sodium currents. This figure shows simultaneous recordings from an inner hair cell and a contacting afferent baton. A voltage step was used to depolarize the inner hair cell and in response inward calcium currents were recorded from the inner hair cell calcium influx results in neurotransmitter release and activated EPCs in the afferent baton.

Note, the synaptic depression appearing during a 50 millisecond inner hair cell depolarization Whilst attempting this procedure. It's important to remember to take care dissecting and preparing cochlear tissue and to remain patient and optimistic when attempting to record from afferent terminals.