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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
AmphotericinSigma-AldrichA4888-100MGFor perforated patch recordings.
ATP di-sodiumSigma-AldrichA7699Additive to internal solution
B27 Supplement (50x), serum freeThermo Fisher Scientific17504044additive to culture medium, for SGN
Beakers (1000, 100, 10) milliliter
bench-top centrifugeUSA Scientific2641-0016
Bunsen burner
CaCl2J.T. Baker1311-01Additive to internal solution
CollagenaseSigma-AldrichC5318one out of three enzyme to digest tissue
Coverglass, rectangular, #1 thickness, 22x40 Warner Instruments64-0707
DMSOBiotium90082
Dnase I,from bovine pancreasSigma-Aldrich11284932001one out of three enzyme to digest tissue
Dumont #3 Forceps (Blunt)Fine Science Tools11231-30
Dumont #5 Forceps (Fine)Fine Science Tools11251-10
Dumont #55 Forceps (Fine)Fine Science Tools11255-20
EGTASigma-AldrichE0396Additive to internal solution
Electrode PullerNarashigePC-10
Epi-illumination light source Zeiss CL 1500 ECO
EthanolDecon Labs2716for cleaning head and around dissection bench
Filamented Borosilicate Capillaries for electrodesSutter InstrumentsBF140-117-10
Fine-edged dissection bladeFine Science Tools10010-00
Glass Pasteur PipettesVWR14673-010to pull trituration pipettes
Heat-inactivated Fetal Bovine SerumThermo Fisher Scientific16140063additive to culture medium
HEPESSigma-AldrichH3375-100Gfor pH buffering all solutions in protocol
Hot plate / magnetic stirrers VWR76549-914
Insulated bucket filled with iceto keep all samples and solutions cool
K2SO4, Potassium SulfateSigma AldrichP9458-250GAdditive to internal solution
KClSigma-AldrichP93333Additive to internal solution
KOH (1 M)Honeywell319376-500MLTo bring internal solution to desired pH.
Large Spring ScissorsFine Science Tools14133-13
Leibovitz medium Sigma AldrichL4386dissection and bath solutions 
Low-profile-bath recording chamber for culture dishesWarner Instruments64-0236
luer-lok syringes, 30 mlBD302832for drawing L-15/HEPES/HEPES solution.
MEM + Glutamax SupplementFisher Scientific41-090-101base of the culture medium
MgCl2-HexahydrateSigma-AldrichM1028Additive to internal solution
microFil needle for filling micropipettes - 34 gauge World Precision InstrumentsMF34G
MicroforgeNarashigeMF-90For electrode polishing.
N2 Supplement (100x)Thermo Fisher Scientific17502-048additiive to culture medium, for SGN
NaClSigma-AldrichS7653Additive to internal solution
NaOH (1 M)Thomas Scientific319511-500MLfor titration pH
OsmometerAdvanced Instruments Inc.3320
Oxygen, Medical grade, with adequate regulator and tubingUSC Material ManagementMEDOX200 (Identifier: 00015)for dissolving into dissection and bath solutions
ParafilmBemisPM992
Pasteur pipette bulb (3 ml)Fisher Scientific03-448-25bulb for trituration pipettes
Penicillin/StreptomycinThermo Fisher Scientific15140122additive to prevent contamination of culture medium
Pentobarbital based euthanasia solution (e.g., Fatal Plus. 50 – 60 mg/kg dosing) MWI Animal Health15199for euthanasia
PES membrane filters ,  0.2 micrometer Nalgene566-0020for filtering solutions
PES membrane sterile syringe filters, 0.22 um, 30 mm CELLTREAT229747for filtering solutions drawn into syringes
Petri dishes, 35 x 10 mmGenessee Scientific32-103for micro dissection and to hold Tip dip solution in perforated-patch configuration
Petri Dishes, 60 x 15 mmMidland ScientificP7455for gross dissection
pH MeterMettler ToledoModel S20
Pipettors (1000, 200, 10) microliterUSA Scientific
Poly-d-lysine coated glass bottomed culture dishMattekP35GC-0-10-Cto plate neurons for culture
Quick change platform, heated base, for 35 mm culture dishesWarner Instruments64-0375
Reference CellWorld Precision InstrumentsRC1T
Scalpel bladeMiltex4-315
Scalpel HandleFine Science Tools10003-12
Scientific ScaleMettler ToledoXS64
Serological Pipettes (10, 25) milliliterFisher Scientific
Silicone Grease Kit (for sealing coverglass and chamber)Warner Instruments64-0378
Small Animal GuillotineKent ScientificDCAP
Small animal guillotineKent ScientificDCAPfor decapitation if dissecting rats older than P15.
Stereo Dissection Microscope ZeissStemi 2000
Straight surgical scissorsFine Science Tools14060-09
Syringe (3, 10, 30) milliliter
TrypsinSigma AldrichT1426one out of three enzyme to digest tissue
Tuberculin syringe Covidien8881500105for delivering euthanasia solution by intraperitoneal injection
Vannas Spring Scissor, 2.5 mm Cutting EdgeFine Science Tools15000-08
Volumetric flask, 1000 milliliter
VortexVWR945300
Water, sterile u ltrapure, R>18.18 megaOhms cm (e.g., filtered by a Millipore-Sigma water purification system)Millipore-SigmaCDUFBI001

References

  1. Liberman, M. C. Single-neuron labeling in the cat auditory nerve. Science. 216 (4551), 1239-1241 (1982).
  2. Goldberg, J. M. Afferent diversity and the organization of central vestibular pathways. Exper....

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