Auditory Nerve Root Stimulation and Postsynaptic Current Recordings in a Mouse Brain Wedge Slice

Abstract

Source: Fischl, M. J., et al. In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity. J. Vis. Exp.(2020)

This video demonstrates a protocol for stimulating the auditory nerve root and recording postsynaptic currents in medial olivocochlear neurons in mouse brain wedge slices.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Electrophysiology set-up and recording

1. Place the wedge slice in the recording chamber and secure the slice with a harp or stabilizing system. Perfuse the tissue continuously at a rate of 7-10 mL/min with warm (35 °C) ACSF bubbled with carbogen.
2. Identify genetically labeled medial olivocochlear MOC neurons in the ventral nucleus of the trapezoid body VNTB using epifluorescence with 561 nm emission filters for patch-clamp recordings. Flip slice if there are no potentially patchable cells.
3. 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 down to the auditory nerve root and gently into the surface of the tissue.
   NOTE: Suction electrodes have been used in auditory nerve stimulation experiments in other labs. Theta glass electrodes or optical stimulation methods can be employed if applicable to other specific preparations.
4. Move the field of view back to the VNTB to choose a MOC neuron to target for patch clamp electrophysiology.
5. Fill a recording pipette with an appropriate internal solution for the proposed experiment.
6. Patch and record from the MOC neuron in the whole-cell configuration. Compensate membrane capacitance and series resistance if required.
7. Adjust the electrical stimulation amplitude of the auditory nerve root to obtain consistent postsynaptic events in the MOC neuron.
   NOTE: It may be necessary to move the stimulation electrode.
8. Run appropriate stimulation protocols to observe evoked synaptic currents in MOC (voltage clamp).
   NOTE: The wedge slice preparation can be used with any typical patch-clamp tools such as loose patch recordings, pharmacology, optogenetics, calcium imaging, neurotransmitter uncaging, etc.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
A1R Upright Confocal Microscope	Nikon Instruments		Electrophysiology and imaging microscope, can be any microscope compatible with electrophysiology
Electrode Borosilicate glass w/ Filament OD 1.5mm, ID 1.1mm, 10 cm long	Sutter Instrument	BF150-110-10	Patch clamping pipette glass
Electrode Filler MicroFil	WPI	CMF20G	Patch electrode pipette filler
In-line solution heater	Warner Instruments (GSAdvantage)	SH-27B	Slice perfusion system heater
Multi-Micromanipulator Systems	Sutter Intruments	MPC-200 with MP285	Micromanipulators for patch clamp and stimulation electrode placement
P-1000 horizontal pipette puller for glass micropipettes	Sutter instruments	FG-P1000	Patch clamp pipetter puller
Patch-clamp amplifier and Software	HEKA	EPC-10 / Patchmaster Next	Can be any amplifier/software
Recording Chamber	Warner Instruments	RC26G	Slice "bath" during recording
Recording Chamber Harp	Warner Instruments	640253	Stablizes slice during electrophysiology recording
Slice Incubation Chamber	Custom Build		Heated, oxygenated holding chamber for slices during recovery after slicing
Stimulus isolation unit	A.M.P.I.	Iso-Flex	Stimulus isolation unit for electrophysiology
Syringe 60CC	Fischer Scientific (Intramalls)	14-820-11	Electrophysiology perfusion fluid handling
Temperature controller	Warner Instruments (GSAdvantage)	TC-324C	Slice perfusion system temperature controller
Tubing 1/8 OD 1/16 ID	Fischer Scientific (Intramalls)	14-171-129	Electrophysiology perfusion fluid handling
Tugsten concentric bipolar microelectrode	WPI	TM33CCINS	Stimulating electrode for electrophysiology