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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 under....
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
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.......
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, 22x40Â | 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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