Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

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 diverse^1,2. 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

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.

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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.......

Materials

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