Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

Summary

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

Abstract

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

Introduction

Two distinct functions of cochlear sensory hair cells are essential for mammalian hearing: mechanoelectric transduction (MET) and electromotility^1,2. By MET channels located in the hair bundle, IHCs (IHCs) and OHCs convert sound vibration into membrane potential changes, as well as the electrical signals of innervated spiral ganglion neurons. OHCs change their cell length with the membrane potential and amplify the vibration of low-level sound. This activity termed electromotility is derived by the motor protein prestin located in the lateral wall of OHCs³.

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Protocol

All experimental protocols involving animal subjects were approved by the Animal Ethics Committee of the Southern Medical University.

1. Prepare Solutions for Experiments

1. Prepare the anesthetic (see Table of Materials): 1.5% pentobarbital sodium dissolved in ddH₂O.
2. Prepare the dissection solution (see Table of Materials): Dissolve one bag of Leiboviz's L-15 powder in 1 L of ddH₂O. Adjust pH to 7.3 with 1 M NaOH. Adjust osmolarity to 300-330 mOsm by using an osmometer and 10 mM HEPES before use.
3. Dissolve 2 mg/mL collagenase IV (see Table of Ma....

Results

ABR can be elicited from anesthetized rat pups older than postnatal day 7 (P7) using pure tone bursts (Figure 1A). As shown in Figure 1B, the ABR waveforms obtained from rat pups showed only three to four distinct waves with small amplitude. Usually, up to seven peaks were observed in the ABR waveforms of adult animals (Figure 1B).

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Discussion

In rats younger than day 11, no action potential in response to sound stimulation could be observed in auditory cortex^6,7. Therefore, postnatal day 11 is described as "hearing onset"¹⁰. The development of hearing function before hearing onset was not well studied yet. Using the similar method for adult ABR recording, we demonstrate that ABRs could be elicited by pure tone burst from rat pups younger than P11 (F.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anesthetic
Pentobarbital sodium	Sigma	P3761	1.5% in water
Name	Company	Catalog Number	Comments
Dissection solution
Leiboviz's L-15 Medium	Life Technologies	41300-039	1 pack in 1 L water
Collagenase IV	Sigma	C5138	2 mg/mL in L-15
HEPES	Sigma	7365-45-9	10 mM
Name	Company	Catalog Number	Comments
Immunostaining solutions
PBS	Thermo Fisher Scientific	10010023	PH 7.3
Paraformaldehyde	Sigma	158127	4% in PBS
Triton X-100	Amresco	ZS-0694	0.3% in PBS
Normal goat serum	Thermo Fisher Scientific	10000C	10% in PBS
prestin antibody	Santa Cruz	SC-22694	dil 1:200
Alexa Fluor 488-conjugated antibody	Thermo Fisher Scientific	A-11055	dil 1:600
Phalloidin-Tetramethylrhodamine B isothiocyanate	Sigma	P1951	dil 1:200
DAPI	Solarbio	C0060	dil 1:20
Name	Company	Catalog Number	Comments
Extracellular solution
Leiboviz's L-15 Medium	Life Technologies	41300-039	1 pack in 1 L water
HEPES	Sigma	7365-45-9	10 mM
Name	Company	Catalog Number	Comments
Intracellular solution
CsCl	Sigma	7647-17-8	140 mM
MgCl2	Sigma	7791-18-6	2 mM
EGTA	Sigma	67-42-5	10 mM
HEPES	Sigma	7365-45-9	10 mM
Name	Company	Catalog Number	Comments
Equipment
Osmometer	Gonotec	OSMOMAT 3000 basic
Forcep	WPI	14095	Tweezers dumont
Micropipette puller	Sutter Instrument	MODLE-P97
Micro Forge	Narishigen	MF-830
Mini Operating System	Sutter Instrument	MP-285
MultiClamp	Axon	700B
Low-Noise Data Acquisition System	Axon	1440A
ES1 speaker	Tucker-Davis Technologies
TDT system 3	Tucker-Davis Technologies
Name	Company	Catalog Number	Comments
Software
SigGenRP software	Tucker-Davis Technologies
BioSigRP software	Tucker-Davis Technologies
jClamp	Scientific Solutions
Name	Company	Catalog Number	Comments
Animal
SD rat	Experimental Animal Center of Southern Medical University