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.
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).
Two distinct functions of cochlear sensory hair cells are essential for mammalian hearing: mechanoelectric transduction (MET) and electromotility1,2. 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 epoch4,5. No action potential in response to sound signals could be detected in the auditory cortex before the hearing onset6,7. Development of morphology and function of the cochlea has been widely studied in mouse, gerbil, and rat4,5,8. 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.
All experimental protocols involving animal subjects were approved by the Animal Ethics Committee of the Southern Medical University.
1. Prepare Solutions for Experiments
2. ABR Recording
NOTE: ABR recording has been previously described in detail in our previous study9.
3. The Organ of Corti (OC) Dissection
4. Immunofluorescence Staining
5. Patch clamp Recording of Isolated OHCs
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 following immunostaining and electrophysiological measurement, rat inner ear was freshly dissected from the temporal bone. We show the consecutive steps of tissue dissection to isolate the OC from SV and the modiolus (Figure 2A–2C). The OC was cut into three pieces evenly with micro-scissors (Figure 2D). To show the morphology of the hair cells, the segments were stained with phalloidin, prestin antibody, and DAPI (Figure 2E). The hair bundles (in red) indicate the apical surface of hair cells, whereas the cell body of the OHCs was marked by prestin (in green). The nucleus was labeled by DAPI (in blue) located in the bottom region of the IHCs and OHCs. As shown in Figure 2E, no prestin was detected in the OHCs of cochlea at P5. Prestin was first observed at P7 in OHCs located in the basal cochlea.
After gentle digestion, the OHCs were isolated from the OC (Figure 3A). Whole-cell voltage-clamp recordings were performed from OHCs acutely isolated from the rat cochlea. A representative example of the whole-cell current recorded from an isolated P9 OHC in response to membrane potential changed from −140 to +107 mV is shown in Figure 3B. Please note the N-shape region of the I-V curve, indicating the presence of KCa current which is a unique response of OHCs. Driven by the membrane voltage, the intracellular Cl– combined with the prestin, appearing as a nonlinear membrane capacitance (NLC) changes under whole-cell patch clamp mode. The typical NLC of a mature OHC is characterized by bell-shaped dependence on membrane potential. The NLC can be fitted with a two-state Boltzmann function and can reflect the electromotility function of OHCs. The Boltzmann function for capacitance fitting is described as:
The equation and the parameters were described in our previous paper9. Two representative NLCs are shown in Figure 3C.
Figure 1. Auditory brainstem response recording. (A) The recording electrode was inserted into the scalp between two ears. Ground and reference electrodes were placed under the left and right pinna, respectively. An open field speaker was placed 10 cm away from the nasal tip of the rat. (B) Two representative ABR waveforms in response to 16 kHz, 80 dB SPL tone bursts. Upper trace recorded from a P8 pup. Bottom trace recorded from an adult rat. The numbers indicate the different peaks of waveforms. Please click here to view a larger version of this figure.
Figure 2. The organ of Corti dissection. (A) The inner ear dissected from a P9 rat pup. The cochlea and vestibular region are shown. (B) The structure of cochlea was exposed after removal of the bony wall. (C) The organ of Corti (OC) and stria vascularis (SV) were isolated from the modiolus. (D) The organ of Corti was cut evenly into three pieces. Scale bars in A-D represent 1,000 µm. (E) Confocal images show the hair cells located in different segments (basal, middle, and apical) along the cochlea. Rhodamine-phalloidin (red) represents the hair bundles located on the apical surface of the hair cells. DAPI (blue) represents the nuclei located in the middle-bottom portion of hair cells. The prestin (labeled in green) was only expressed on the lateral wall of the outer hair cells. For middle and basal segments at P7, only the green channel is shown to illustrate the expression of prestin in OHCs. Scale bar represents 20 µm. Please click here to view a larger version of this figure.
Figure 3. Whole-cell patch clamp recording of isolated outer hair cells. (A) An isolated outer hair cell was sealed in whole-cell mode. Scale bar represents 10 µm. (B) A representative I-V curve recorded from a P9 outer hair cell. (C) The nonlinear membrane capacitance (NLC) obtained from two outer hair cells at different ages. The capacitance-voltage responses (open circles) were fitted to the Boltzmann function (shown as the thick lines). The NLC were normalized to the corresponding linear capacitance (Clin). Please click here to view a larger version of this figure.
In rats younger than day 11, no action potential in response to sound stimulation could be observed in auditory cortex6,7. 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 (Figure 1). However, the ABR waveforms obtained from rat pups showed some different features from those of adult animals. The ABR responses are small in amplitude with relatively high thresholds. This result implies that the developing auditory system has low sensitivity to sound signals. Significant difference was observed in the ABR waveforms recorded from rat pups. Usually, up to seven peaks were observed in adult ABR waveforms9. These peaks with different latency are generated by different brainstem nuclei along the auditory pathway11. Our results indicate that only the I-IV waves were detectable in ABR from rat pups (Figure 1B). The waves I and II represent the responses from the cochlea and the auditory nerve11. Therefore, the results indicate that the higher level auditory nuclei failed to respond to acoustic stimuli during development. These data suggest that cochlea reach functional maturity earlier than does the central auditory system. The method we describe here is important for the study of molecular mechanisms that regulate cochlea development and hair cell function.
High quality dissection of the OC is critical for further experiments including immunostaining, Western blotting, and electrophysiological measurement. These experiments have been performed using adult SD rats and rat pups between 1 and 14 days old. Rat pups are ideal for fine dissection as the cochlear bone is soft and can be easily removed by forceps without damaging the OC. For adult rats, particular caution is required to avoid rupturing the OC while removing the hard-bony capsule. By using fine forceps, the OC and associated SV can be lifted off from the modiolus. The separated OC and SV have different texture (Figure 2C). For rat pups, the OC could be cut into three segments with a length of 800-1200 µm. Please note, all steps should be performed in ice-cold L-15 as fast as possible in order to minimize degradation and tissue deterioration.
Isolated segments of the OC are good preparations for immunostaining, Western blotting, and RNA analysis. Here we show the morphology of sensory hair cells in OC by immunostaining. OHCs have hair bundles on their apical surface whereas the nucleus has them located basally12. The motor protein prestin is expressed on the lateral membrane of OHCs. The confocal imaging data indicated that the prestin was first expressed at P6-P7 and showed a continued increase over the following week (Figure 2E). In further experiments, Western blotting and q-PCR were performed to quantify the observed expression level changes of prestin during development (data not shown). Because the hair cells are the minority in the OC, 6 to 10 cochleae are recommended in a single experiment to achieve robust signals.
For patch clamp recordings of OHCs, mild enzymatic digestion was performed to separate the OHCs. The hair cells are fragile, and special caution is required in this step. Use a 200 µL pipette tip to transfer the tissue and separated cells. The isolated segment of OC was transferred to a drop of collagenase solution (~ 100 µL) in a Petri dish for about 5 min at room temperature. Avoid long digestion times, which will damage the cell membrane of OHCs. Under the microscope, healthy looking OHCs were selected for the patch clamp recording. Cells showing any signs of shrinkage, swelling, damage, or translocation of the nucleus were excluded from next experiment. Healthy appearing solitary OHCs and IHCs are easy to identify by their morphological difference. OHCs show a cylindrical shape and a larger axis-diameter ratio, whereas IHCs are flask-shaped with a tight neck12. Whole-cell patch clamp in OHCs is somewhat difficult from those in neurons and other epithelial cells. For successful whole-cell configuration, the patch pipette was usually located near the nucleus, kept away from the upper lateral membrane containing a high density of prestin particles (Figure 3A). After the membrane was ruptured, a two sine-wave voltage stimulus protocol was applied to the cell in order to obtain the membrane capacitance of OHCs. With the same technique, by using voltage steps, it is possible to elicit whole-cell current.
This method is a good tool to investigate the electromotility function of OHCs in vitro9,13. This method could also be used to investigate the function of ion channels, as well as the pharmacological properties of receptors in OHCs and IHCs14.
The authors have nothing to disclose.
This work was supported by grants from the 973 Program (2014CB943002) and the National Natural Science Foundation of China (11534013, 31500841).
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 |