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

Podsumowanie

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.

Streszczenie

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).

Wprowadzenie

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.

In many species including rodents, the hearing function is immature in early postnatal epoch^4,5. No action potential in response to sound signals could be detected in the auditory cortex before the hearing onset^6,7. Development of morphology and function of the cochlea has been widely studied in mouse, gerbil, and rat^4,5,8. The mechanotransduction and electromotility of hair cells are also developed in the early life epoch⁵.

In order to evaluate the hearing sensitivity of rats at different postnatal ages, we have developed a method for auditory brainstem response (ABR) recording in rat pups. Whole-cell patch clamp is an ideal technology to investigate the OHCs electrophysiologically. However, compared with the patch clamp performed in neurons and other epithelial cells, the low rate of whole-cell sealing limited the investigation of electromotility of isolated OHCs.

Here we describe a procedure to investigate the OHCs morphologically and electrophysiologically in acutely dissociated cochlea from postnatal rats. This method can be modified to study the molecular mechanisms that regulate inner hair cell development and function.

Protokół

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 Materials) in dissection solution prepared in step 1.2.
4. Prepare the immunostaining solutions (see Table of Materials): Dissolve the 4% paraformaldehyde in phosphate buffered saline (PBS).
   Caution: Paraformaldehyde is toxic; wear appropriate protection.
5. Dilute the 0.3% permeability agent in PBS. Dilute the 10% normal goat serum in PBS as the blocking buffer. Dilute the prestin antibody 1:200 in blocking buffer. Dilute the Alexa488-conjugated antibody 1:600 in blocking buffer. Dilute the Phalloidin-Tetramethylrhodamine B isothiocyanate 1:200 in PBS.
6. Prepare the extracellular solution (see Table of Materials): Prepare the Leiboviz's L-15 medium as described above (step 1.2).
7. Prepare the intracellular solution (see Table of Materials).

2. ABR Recording

NOTE: ABR recording has been previously described in detail in our previous study⁹.

1. Anesthetize Sprague Dawley (SD) rats (1-14 day old pups and 2-month old adults, both sexes) with a single injection of 1.5% sodium pentobarbital (22 mg/kg for pups and 30 mg/kg for adults, intraperitoneal (i.p.)).
2. Place the anesthetized animal in a polyethylene-foam mold to immobilize the animal's body. Place the animal on an anti-vibration table in a sound-attenuating room. Keep the animal's body temperature at 37.5 °C with a heating pad.
3. Wipe the head area with 70% (vol/vol) ethanol. By using a fine scissors, make a 1-2 mm incision ventrolateral to the external pinna to place the reference electrode or ground. A subdermal needle electrode (recording electrode) is located over the skull vertex (Figure 1A).
4. Using the function generator, generate calibrated tone bursts (1 ms rise/fall, 3 ms plateau) of various frequencies (2, 4, 8, 16, 24, and 32 kHz) and intensities (decreased from 85 to 10 dB SPL in 5 dB step). Vary the frequency and amplitude manually or using a computer. Deliver sound stimuli through an electrostatic speaker located 10 cm away toward the head of animal.
5. Filter (100-1,000 Hz), amplify and average (256 times) the sound elicited potential using a multi-function processor to get the ABRs. The ABR traces is monitored online and stored for offline analysis. It takes about 40 min to complete the ABR recording from one animal.
   NOTE: Usually, three to seven peaks (wave I to wave VII) with latencies less than 15 ms can be identified (Figure 1B). The ABR threshold is defined as the minimum sound level at which wave I and II could be detected.
6. Euthanize before the animals awake from anesthesia (with an intraperitoneal injection of 0.3 mL 1.5% sodium pentobarbital).

3. The Organ of Corti (OC) Dissection

1. Anesthetize rat pups. For rats less than 6 days old (<P6), keep the animal in a 100 mm culture dish, and cover the dish with ice for 10 min. For rats >P6, anesthetize the animal with a single injection of 1.5% sodium pentobarbital (22 mg/kg, i.p.). Decapitate the rat pups after anesthesia.
2. Sterilize the head by spraying with 70% (vol/vol) ethanol.
3. Open the skull along the sagittal midline with scissors; remove the brain to expose the inner ear.
4. Transfer the inner ear into a 35-mm Petri dish filled with 3 mL of ice-cold L-15 (Figure 2A).
5. Under the dissection microscope, use fine forceps to remove the bony capsule of the cochlea (Figure 2B).
6. Unwrap the OC and associated stria vascularis (SV) from the modiolus.
7. By holding the basal portion of the SV with forceps, separate the OC from the SV completely by unwinding slowly from base-to-apex (Figure 2C).
8. Cut the OC evenly into three pieces by using fine scissors (Figure 2D).

4. Immunofluorescence Staining

1. By using a 200 µL pipette tip, transfer the segments of OC (step 3.8) onto a glass slide, and fix with 100 µL of 4% paraformaldehyde for at least 4 h at 4 °C.
   Caution: Paraformaldehyde is toxic; wear appropriate protection.
2. Wash the tissue by displacing the paraformaldehyde with 100 µL of fresh PBS. Wash the tissue three times for 10 min.
3. Incubate the tissue with 0.3% permeability agent in PBS for 30 min at room temperature.
4. Discard the permeability agent in PBS and wash the tissue three times with PBS.
5. Block with 10% normal goat serum in PBS for 1 h at room temperature.
6. Incubate with anti-prestin-C-terminus antibody (1:200) in blocking solution for 2 h at room temperature or at 4 °C overnight.
7. Wash three times with PBS for 5 min.
8. Incubate with Alexa488-conjugated secondary antibodies (1:600) in blocking solution for 1 h at room temperature in the dark.
9. Wash three times with PBS for 5 min in the dark.
10. Incubate with rhodamine-phalloidin (1:200) for 10 min at room temperature in the dark.
11. Wash three times with PBS for 5 min in the dark.
12. Mount on a glass slide with mounting medium (containing DAPI). Image with confocal microscopy by using 405 nm, 488 nm, and 594 nm lasers. Set the laser power at 0.1-0.5 mW and the scan speed at 0.5 frame/s.

5. Patch clamp Recording of Isolated OHCs

1. Use the pipette puller and micro-forger to make patch pipettes with a tip diameter 2-3 µm. Back-fill the pipettes with intracellular solution. Usually, the initial resistance of patch pipette was 2.5-3.5 MΩ in bath solution.
2. By using a 200 µL pipette tip, transfer a piece of the OC (from step 3.8) into a 35 mm Petri dish. Digest the tissue with 100 µL of enzymatic digestion medium (collagenase IV, see Table of Materials) for 5 min at room temperature.
3. Displace the enzymatic digestion medium with 100 µL of L-15. Cut the tissue into small pieces using a micro scalpel to isolate the hair cells.
4. After gentle pipetting, transfer the cells into a homemade small plastic chamber (diameter ~ 1.5 cm) filled with enzyme-free bath solution (~ 1.5 mL, see Table of Materials).
5. Place the chamber on the stage of an inverted microscope. Find the healthy-appearing solitary OHCs.
6. Load the patch pipette into the head stage of a 700B amplifier. Move the patch pipette carefully by a micromanipulator and position it around the bottom of the outer hair cell (Figure 3A).
7. Whole-cell patch clamp the OHC by sealing the lateral wall of the cell body. Apply light suction until the cell membrane is ruptured. Set the holding potential at -70 mV. Cells with access resistance ranged from 10 to 17 MΩ and membrane resistance ranged from 100 to 500 MΩ, and are considered a successful whole-cell configuration.
8. Using computer control, apply hyperpolarizing and depolarizing voltage (250 ms duration and ranging from −140 to +94 mV in 13 mV steps) to the cell to elicit whole-cell currents.
9. Amplify the whole-cell currents, filter (corner frequency of 5 kHz) by a 700B amplifier. Convert the data by a 1440A converter and store for offline analysis.
10. Measure the membrane capacitance of OHCs using a two sine-wave voltage stimulus protocol controlled by a patch clamp software. Set the stimulate voltage range from -140 to 110 mV. Store the data for offline analysis.

Wyniki

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).

For the ...

Dyskusje

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...

Materiały

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