This study details the electrophysiological analysis of medial olivocochlear neurons in a transgenic mouse brain wedge slice. The methodology includes patch-clamp techniques to record neuronal activity in response to auditory nerve stimulation.
Secure a transgenic mouse brain wedge slice in a recording chamber perfused with oxygenated aCSF.
The thicker wedge side contains the auditory nerve root and cochlear nucleus, including T-stellate and globular bushy cells.
The thinner side contains the medial and ventral nucleus of the trapezoid body with fluorescent medial olivocochlear neurons.
Microscopically locate the auditory nerve root and position a stimulating electrode on it.
Identify a fluorescent medial olivocochlear neuron and advance a recording pipette toward it.
Patch the neuron to achieve a whole-cell configuration and record its electrical activity.
Apply electrical pulses to the auditory nerve root, activating cochlear nucleus cells.
Activated T-stellate cells release excitatory neurotransmitters to the medial olivocochlear neurons.
However, globular bushy cells activate inhibitory pathways, releasing inhibitory neurotransmitters to the medial olivocochlear neurons.
The integration of the excitatory and inhibitory signals results in postsynaptic currents, confirming neuronal activity within the slice.
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.
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