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Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.
The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.
The bipolar neurons of the vestibulocochlear nerve connect the sensory hair cells of the inner ear to the brainstem. They are principal carriers of information about sound and head movements; damage to these important cells leads to deafness and balance disorders. The vestibular and auditory portions of the nerve are each comprised of distinct cell types that are morphologically and functionally diverse1,2. In the vestibular system, two afferent subpopulations fire spontaneously at intervals that are either regular or irregular2. Afferent spike timing is thought to reflect an underlying diversity in ion channel composition3,4. In the auditory system, there are two main subpopulations of spiral ganglion neurons (SGNs); whereas Type I SGNs contact individual inner hair cells5, Type II SGNs contact multiple outer hair cells5. In vitro recordings from semi-intact and organotypic cultures suggest differences in the membrane properties of Type I and Type II SGNs6,7.
Many ion channels and neurotransmitter receptors found at the terminals of these neurons are also found in their cell bodies. As such, cultures of the isolated vestibular and the spiral ganglion somata can be studied in vitro to understand how ion channels and neurotransmitter receptors contribute to the response of these neurons. The compact morphology of the isolated cell bodies allows for high-quality electrical recordings, suitable for detailed characterization of voltage-gated ion channels and neurotransmitter receptors. Easy access to a representative variety of neuron subtypes allows for high-throughput analysis of cell diversity.
This article presents a method for isolating and culturing dissociated ganglion cell bodies from the superior portion of the vestibular ganglion in rats at postnatal day (P)9 to P20. Suggestions are also provided for extending these methods to the spiral ganglion, in addition to the steps required for successfully extracting, dissociating, and plating the ganglion cells. These methods are an evolution of those devised in publications from various laboratories8,9,10. Also included in this paper is guidance for selecting healthy cells for patch-clamp recordings.
Finally, the protocol outlines the procedure for patch-clamp recording using the perforated-patch configuration11. Though the perforated-patch configuration is more time-consuming and more technically challenging than the more common ruptured-patch configuration, it is better for maintaining the cytoplasmic milieu that allows for long and stable recording sessions. The benefits of this recording configuration are illustrated here through the improved stability of hyperpolarization-activated cationic currents in perforated-patch relative to ruptured-patch recordings.
This protocol is organized into five sections. Sections 1-3 describe solutions and tools that can be prepared and stored ahead of time. Section 4 describes the steps for dissecting and plating the vestibular and SGNs. Section 5 describes the steps for recording from the neurons after a period in culture. In our hands, section 4 and section 5 are performed over a period of 2 consecutive days.
All animal use described here has been approved by the Institutional Animal Care and Use Committee at the University of Southern California. Animals in this protocol are P3- to P25-aged Long Evans rats of both sexes obtained from Charles River Laboratories, but these methods can be applied to other rodent strains. A laboratory coat and gloves must be worn during all procedures, as well as splash-protective goggles when making solutions.
1. Preparations
NOTE: The solutions and tools described in this section can be made well ahead of time to be used on the day of dissection and recording.
2. Fabricating trituration pipettes
NOTE: Trituration pipettes can be reused for multiple experiments. Flush the pipettes with ethanol, water, and L-15 solution before each use and clean them with water and ethanol after each use. Carefully store between uses.
3. Fabricating patch pipettes
NOTE: Prepare a set of patch pipettes before the recording session, but after ganglion dissection and culture. Although electrodes that are made one at a time during the recording session are optimal in performance, reasonable success has been achieved when these are made as a batch the night before. Store the pipettes in a covered glass container to protect the tips from dust.
4. Extraction of vestibular ganglion and plating of vestibular neurons
5. Recording
NOTE: In this procedure, patch-clamp recordings from the isolated ganglion are typically performed 12-24 h after the plating. Other labs have reported results from neurons after much longer periods in culture10.
Running voltage-clamp protocols by applying families of voltage steps reveals the voltage-dependent activation of a variety of different families of currents. Representative examples of whole-cell currents evoked from a VGN and adapted from published recordings13 are shown in Figure 1A,B. Applying depolarizing voltages (Figure 1B) activates an inward current (negative by convention) that activates and inactivates very rap...
The methods presented here are specific to recordings from isolated neurons; previous studies have focused on recordings from axon terminals in a semi-intact preparation. When compared to existing terminal recording techniques, isolated recordings offer superior space-clamp and iso-potential behavior. In addition, this protocol provides access to a broader sample of neurons, since only calyx-bearing subpopulations are accessible in semi-intact recordings of the vestibular epithelia. Finally, isolated recordings allow for...
The authors declare that there are no conflicts of interest.
We acknowledge Drs. Jing Bing Xue and Ruth Anne Eatock for their early contributions to these methods. This work was supported by NIH NIDCD R03 DC012652 and NIH NIDCD DC012653S, and R01 DC0155512 to RK and T32 DC009975 to DB, NN, and KR.
Name | Company | Catalog Number | Comments |
Amphotericin | Sigma-Aldrich | A4888-100MG | For perforated patch recordings. |
ATP di-sodium | Sigma-Aldrich | A7699 | Additive to internal solution |
B27 Supplement (50x), serum free | Thermo Fisher Scientific | 17504044 | additive to culture medium, for SGN |
Beakers (1000, 100, 10) milliliter | |||
bench-top centrifuge | USA Scientific | 2641-0016 | |
Bunsen burner | |||
CaCl2 | J.T. Baker | 1311-01 | Additive to internal solution |
Collagenase | Sigma-Aldrich | C5318 | one out of three enzyme to digest tissue |
Coverglass, rectangular, #1 thickness, 22x40 | Warner Instruments | 64-0707 | |
DMSO | Biotium | 90082 | |
Dnase I,from bovine pancreas | Sigma-Aldrich | 11284932001 | one out of three enzyme to digest tissue |
Dumont #3 Forceps (Blunt) | Fine Science Tools | 11231-30 | |
Dumont #5 Forceps (Fine) | Fine Science Tools | 11251-10 | |
Dumont #55 Forceps (Fine) | Fine Science Tools | 11255-20 | |
EGTA | Sigma-Aldrich | E0396 | Additive to internal solution |
Electrode Puller | Narashige | PC-10 | |
Epi-illumination light source | Zeiss | CL 1500 ECO | |
Ethanol | Decon Labs | 2716 | for cleaning head and around dissection bench |
Filamented Borosilicate Capillaries for electrodes | Sutter Instruments | BF140-117-10 | |
Fine-edged dissection blade | Fine Science Tools | 10010-00 | |
Glass Pasteur Pipettes | VWR | 14673-010 | to pull trituration pipettes |
Heat-inactivated Fetal Bovine Serum | Thermo Fisher Scientific | 16140063 | additive to culture medium |
HEPES | Sigma-Aldrich | H3375-100G | for pH buffering all solutions in protocol |
Hot plate / magnetic stirrers | VWR | 76549-914 | |
Insulated bucket filled with ice | to keep all samples and solutions cool | ||
K2SO4, Potassium Sulfate | Sigma Aldrich | P9458-250G | Additive to internal solution |
KCl | Sigma-Aldrich | P93333 | Additive to internal solution |
KOH (1 M) | Honeywell | 319376-500ML | To bring internal solution to desired pH. |
Large Spring Scissors | Fine Science Tools | 14133-13 | |
Leibovitz medium | Sigma Aldrich | L4386 | dissection and bath solutions |
Low-profile-bath recording chamber for culture dishes | Warner Instruments | 64-0236 | |
luer-lok syringes, 30 ml | BD | 302832 | for drawing L-15/HEPES/HEPES solution. |
MEM + Glutamax Supplement | Fisher Scientific | 41-090-101 | base of the culture medium |
MgCl2-Hexahydrate | Sigma-Aldrich | M1028 | Additive to internal solution |
microFil needle for filling micropipettes - 34 gauge | World Precision Instruments | MF34G | |
Microforge | Narashige | MF-90 | For electrode polishing. |
N2 Supplement (100x) | Thermo Fisher Scientific | 17502-048 | additiive to culture medium, for SGN |
NaCl | Sigma-Aldrich | S7653 | Additive to internal solution |
NaOH (1 M) | Thomas Scientific | 319511-500ML | for titration pH |
Osmometer | Advanced Instruments Inc. | 3320 | |
Oxygen, Medical grade, with adequate regulator and tubing | USC Material Management | MEDOX200 (Identifier: 00015) | for dissolving into dissection and bath solutions |
Parafilm | Bemis | PM992 | |
Pasteur pipette bulb (3 ml) | Fisher Scientific | 03-448-25 | bulb for trituration pipettes |
Penicillin/Streptomycin | Thermo Fisher Scientific | 15140122 | additive to prevent contamination of culture medium |
Pentobarbital based euthanasia solution (e.g., Fatal Plus. 50 – 60 mg/kg dosing) | MWI Animal Health | 15199 | for euthanasia |
PES membrane filters , 0.2 micrometer | Nalgene | 566-0020 | for filtering solutions |
PES membrane sterile syringe filters, 0.22 um, 30 mm | CELLTREAT | 229747 | for filtering solutions drawn into syringes |
Petri dishes, 35 x 10 mm | Genessee Scientific | 32-103 | for micro dissection and to hold Tip dip solution in perforated-patch configuration |
Petri Dishes, 60 x 15 mm | Midland Scientific | P7455 | for gross dissection |
pH Meter | Mettler Toledo | Model S20 | |
Pipettors (1000, 200, 10) microliter | USA Scientific | ||
Poly-d-lysine coated glass bottomed culture dish | Mattek | P35GC-0-10-C | to plate neurons for culture |
Quick change platform, heated base, for 35 mm culture dishes | Warner Instruments | 64-0375 | |
Reference Cell | World Precision Instruments | RC1T | |
Scalpel blade | Miltex | 4-315 | |
Scalpel Handle | Fine Science Tools | 10003-12 | |
Scientific Scale | Mettler Toledo | XS64 | |
Serological Pipettes (10, 25) milliliter | Fisher Scientific | ||
Silicone Grease Kit (for sealing coverglass and chamber) | Warner Instruments | 64-0378 | |
Small Animal Guillotine | Kent Scientific | DCAP | |
Small animal guillotine | Kent Scientific | DCAP | for decapitation if dissecting rats older than P15. |
Stereo Dissection Microscope | Zeiss | Stemi 2000 | |
Straight surgical scissors | Fine Science Tools | 14060-09 | |
Syringe (3, 10, 30) milliliter | |||
Trypsin | Sigma Aldrich | T1426 | one out of three enzyme to digest tissue |
Tuberculin syringe | Covidien | 8881500105 | for delivering euthanasia solution by intraperitoneal injection |
Vannas Spring Scissor, 2.5 mm Cutting Edge | Fine Science Tools | 15000-08 | |
Volumetric flask, 1000 milliliter | |||
Vortex | VWR | 945300 | |
Water, sterile u ltrapure, R>18.18 megaOhms cm (e.g., filtered by a Millipore-Sigma water purification system) | Millipore-Sigma | CDUFBI001 |
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