Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

Summary

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

Abstract

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Introduction

The fields of neurophysiology and medical bionics rely heavily on techniques that allow controllable stimulation of electrical responses in neural tissue. While electrical stimulation remains the gold standard in neural excitation, it suffers from a number of drawbacks such as the presence of stimulation artifacts when recording neural responses, and a lack of stimulation specificity due to the spread of current into surrounding tissue ¹.

The last two decades have seen the development of optically mediated stimulation techniques ². Several of these techniques require modification of the target tissue, either via the ad....

Protocol

1. Culture of Spiral Ganglion Neurons

1. Sterilize small round (e.g. 10 mm diameter) glass coverslips and curved forceps in an autoclave. Transfer the sterilized coverslips into individual wells of a sterile 4-ring 35 mm petri dish or 4-well plate, using the sterilized forceps. Apply 150 μl of poly-L-ornithine (500 μg/ml) and mouse laminin (0.01 mg/ml) to the top surface of the coverslip and place in an incubator (37 °C) for up to 48 hr. Ensure that the coverslips do not float away from the bottom of the well.
2. Prepare 50 ml sterile Neurobasal media (NBM) for each neural culture: 47.5 ml neurobasal A, 0.5 ml N₂ supplemen....

Results

Spiral ganglion neurons respond to laser illumination with repeatable waveforms in both voltage-clamp and current-clamp recording configurations. Figure 3a shows typical changes in current flow across a cell membrane in response to a 2.5 msec, 0.8 mJ laser pulse (average response from 6 laser pulses, repeated at 1 sec intervals) with the membrane potential held at -70 mV, -60 mV and -50 mV. Net inward currents are consistently evoked in response to laser pulses, returning to initial values after illumina.......

Discussion

Using the protocols outlined in this paper it is possible to extract and culture spiral ganglion neurons and to investigate laser-evoked electrical activity by performing whole cell patch clamp experiments. When used in vitro, the patch clamp technique provides a level of control over experimental parameters that is not achievable in vivo. Laser stimulation parameters such as wavelength, pulse energy, pulse length, pulse shape, and pulse repetition sequences can be studied in a reproducible setting. In .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell culture materials and equipment
Glass coverslips	Lomb Scientific	CSC 10 1 GP
4-ring cell culture dish	VWR International	82050-542
Poly-L-ornithine solution	Sigma-Aldrich	P4957
Laminin	Invitrogen	23017-015
Curved forceps	WPI	14101	Dumont #5 tweezers (45° angle tip)
CO2 Incubator	ThermoScientific	Heracell 150i
Table 1. Cell culture materials and equipment.
Neurobasal media
Neurobasal A	Gibco	10888-022
N-2 supplement	Invitrogen	17502-048
B27 serum-free supplement	Invitrogen	17504-044
Penicillin-Streptomycin	Invitrogen	15140-148
L-Glutamine	Invitrogen	25030-149
Intracellular solution
Potassium chloride	Sigma-Aldrich	P4504
HEPES	Sigma-Aldrich	H4034
Potassium D-gluconate	Sigma-Aldrich	G4500
EGTA	Sigma-Aldrich	E3889
Na2ATP	Sigma-Aldrich	A2383
MgATP	Sigma-Aldrich	A9187
NaGTP	Sigma-Aldrich	G8877
Potassium hydroxide	LabServ	BSPPL738.500
Sucrose	Sigma-Aldrich	S8501
Extracellular solution
Sodium chloride	Sigma-Aldrich	310166
Potassium chloride	Sigma-Aldrich	P4504
HEPES	Sigma-Aldrich	H4034
Calcium chloride	Sigma-Aldrich	383147
Magnesium chloride	Sigma-Aldrich	M8266
D-Glucose	Sigma-Aldrich	G8270
Sodium hydroxide	LabServ	BSPSL740.500
Sucrose	Sigma-Aldrich	S8501
Table 2. Solutions for cell culture and patch clamp. a) Neurobasal media. b) Intracellular solution. c) Extracellular solution.
Upright microscope	Zeiss	AxioExaminerD1	Equipped with Dodt contrast
Water-immersion objective	Zeiss	W Plan-APOCHROMAT 40x/0.75
Platform and X-Y stage	ThorLabs	Burleigh Gibraltar
Recording chamber	Warner Instruments	RC-26G
Vibration isolation table	TMC	Micro-g 63-532
CCD Camera	Diagnostic Instruments	RT1200
Camera software	Diagnostic Instruments	SPOT Basic
In-line solution heater	Warner	SH-27B
Temperature controller	Warner	TC-324B
Patch clamp amplifier	Molecular Devices	Multiclamp 700B
Patch clamp data acquisition system	Molecular Devices	Digidata 1440A
Micromanipulator	Sutter Instruments	MPC-325
Micropipette glass	Sutter Instruments	GBF100-58-15	Borosilicate glass with filament
Micropipette Puller	Sutter Instruments	P2000
Recording Software	AxoGraph		Lab pack and electrophysiology tools
Aspirator bottle	Sigma-Aldrich	CLS12201L	1 L Pyrex aspirator bottle, with outlet for tubing
PE Tubing	Harvard	PolyE #340
Masterflex peristaltic pump	Cole-Parmer	HV-07554-85
Table 3.Patch clamp equipment.
1,870 nm laser diode	Optotech
200/220 μm diameter multimode optical fiber patch cord (FC/PC)	AFW Technologies	MM1-FC2-200/220-5-C-0.22	Light delivery optical fiber, silica core and cladding, 0.22 NA
Optical fiber through connector (FC/PC)	Thorlabs	ADAFC2
Optical fiber cleaver	EREM	FO1
Optical fiber stripping tool (0.25 - 0.6 mm)	Siemens		For removing optical fiber jacket
Optical fiber stripping tool (0.6 - 1.0 mm)	Siemens		For removing outer coating of patch cord
Signal generator			Any signal generator that can output the necessary pulse shapes and is capable of being externally triggered
Optical fiber positioner			Custom made positioner. Could substitute with standard micropositioner used for patch clamp experiments
Optical fiber chuck	Newport	FPH-DJ
Laser power meter and detector head	Coherent	FieldMate (power meter) with LM-3 (detector head)
Table 4. Laser equipment.