Optogenetic Stimulation of the Auditory Nerve

The overall goal of this procedure is to optically stimulate the auditory nerve of animals with optogenetically modified spiral ganglion neurons. This is accomplished by first preparing either a blue micro LED stimulator or an optical fiber coupled to a blue laser. The next step of the procedure is to assess the cochlea using a retro auricular microsurgical approach.

The third step is to position the optical stimulator to record optically evoked brainstem responses. The final step is to access the inferior colus to record optically evoked activity. Ultimately, results can show successful stimulation of the auditory system through recordings of auditory brainstem responses and local field potentials.

In the inferior colliculus Optogenetic stimulation of the auditor nerve. As an alternative to electric, cochlear implants promises better frequency and intensity resolution. This is of great interest for auditor research, but the implications of this technique extend toward improved restoration of hearing in the deaf.

This method can help answer key questions in auditory research, such as the role of pattern activity for the refinement of to orthopedic development, the requirement for spectral integration in cell localization, and the extent of interaction between frequency specific frame projections. In the central auditory system, Visual demonstration of the procedure is critical as the appropriate positioning and orientation of the micro LED or optical fiber are difficult to learn. The micro LED stimulator uses a blue LED with a 200 square micron active surface first solder wires to the micro LED.

Next, using an epoxy encapsulate the LED and its connection, allow it to dry overnight. Once cured, feed the wires through a one millimeter outer diameter. Borrow silicate glass capillary.

The glass provides mechanical stability and electrical insulation. Seal the junction of the capillary and the epoxy coated LED with more epoxy position the capillary so that it can be mounted to a manipulator. Now check the electrical connections.

First, prepare three copper wires. Dip one end of each into a 0.9%sodium chloride bath. Then connect their other ends to an A BR amplifier.

Monitor the signal with an oscilloscope. Next, connect the micro LED to the custom made stimulator for the test. Immerse the stimulator connected micro LED in the bath and power the LED for at least 20 minutes.

While stimulating the LED monitor the oscilloscope for artifacts. Discard any faulty LEDs. Anesthetize a four to 10 week old mouse.

This mouse expresses channel opsin two under the thigh 1.2 promoter 10 minutes. After injecting the anesthetics, check the animals withdrawal reflex to confirm a surgical plane of anesthesia. Then apply ointment to the eyes and start the surgery.

First, carefully shave the retro auricular area to make an approach to the bullah, the air-filled middle ear cavity. Set the mouse on a heating pad at the surgical side using micro forceps. Carefully dissect the neck muscles, namely the platysma, sternocleidomastoid, and scalenes.

Now expose the bulla. It is a spherical, bony structure with a ring on the surface where the tympanic membrane is located. Using an insulin needle, poke a hole just below the ring from the hole.

Remove the bone that covers the bulla. Use fine Rons and micro forceps exposing the pro auditorium of the cochlea in the process. Next, start the cochlea.Ostomy.

First shave off the bony capsule without disrupting the membranous labyrinth. Now open a small 500 to 800 micron window on the second cochlear turn far from the staed artery and the apex opening. The apex could rupture the cochlea and fracture the modis.

Prepare to measure optogenetic auditory brainstem responses or OABR. First, insert needle electrodes at the following locations underneath the pinna, on the vertex, and on the back near the legs with the electrodes connected to an amplifier. Amplify the difference potential between the vertex and mastoid subdermal needles.

Sample at 50 kilohertz and filter the responses between one and 10, 000 hertz for trans cochlear stimulation. Mount a micro LED or fiber, coupled with the micro LED gripped by a mechanical manipulator. Carefully position it into place.

Visualize the difference potential on an oscilloscope. Place it near the recording setup so that it is easy to optimize the position of the optical stimulator. Use signal averaging and store the data on a computer for offline analysis.

Now optimize the position and orientation of the micro LED using responses to three to 10 millisecond pulses of current at one to five hertz. Another option is laser stimulation by a 473 nanometer continuous wave laser attached to a 250 micrometer optical fiber. This requires fast analog control of the laser power or an al optical device.

With a fast shutter position the fiber with a micro manipulator directly onto the cochlea ostomy and fix it with dental cement for intra cochlear stimulation. Insert the fiber through the round window into the s Scala.Tempe. Optimize the position and orientation of the fiber by eliciting responses with three to 10 millisecond pulses of 10 to 30 milliwatts at one to five hertz.

Positioning of the stimulator is the most critical step to the protocol. The micro ED or the optical fiber should be patiently relocated until the OABR is evident. Once good optogenetic, auditory brainstem responses are made, fix the fiber with cyanoacrylate glue or by dental cement.

For this protocol, use a stereotaxic frame and prepare a mouse by opening the skull over the inferior colus. Using a microscope, position a recording probe over the inferior colus and insert it so that the topmost channel is just visible at the surface. Now, amplify the difference potential between the individual channels of the recording array and the reference screw and the parietal bone.

Use a sample rate of 32 kilohertz, a 300 hertz low pass filter and save the data. An optimal cochlea ostomy is critical and increases the probability of a successful experiment. This means the window is regular small and there is no injury of the internal cochlear structures.

For example, bleeding indicates damage of the STR ssis using channel Rodin two transgenic mice. Channel Rodin two is expressed in the spiral gang neurons within the cochlea blue light illumination either by micro LED or by laser elicits large optogenetic A BR, which differ from acoustic A BR in amplitude and waveform optogenetic. A BR has larger amplitudes than acoustic A BR optogenetic.

A BR is thus more comparable to electrical A BR.They may both recruit more spiral ganglion neurons or elicit a more synchronized activation of the spiral ganglion neurons. Propagation of the activity through the auditory pathway was verified by extracellular recordings in the central auditory system. For example, optogenetic stimulation of the cochlea elicited neural activity in the auditory midbrain.

Mechanical stability and safe electrical isolations of micro LED stimulators, as well as avoidance of excessive carbon flow are all critical factor to successful use of cochlea optogenetics. While attempting this pro, it is important to remember to avoid injuring distro artery in Ebola. Once mastered recordings from the inferior colliculus can be done in about three hours if performed properly.

Following this procedure a, a methods like single unit recordings in the inferior colliculus can be performed. Optogenetic stimulation of the auditory pathway will pave the way for researches in auditory neuroscience to ask questions that could not previously be addressed with acoustic or electrical stimulation. We also hope that this technique will be translated into clinical rehabilitation of hearing impairment.