Published: July 31st, 2013
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.
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.
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....
1. Culture of Spiral Ganglion Neurons
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.......
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 .......
|Name of Reagent/Material
|Cell culture materials and equipment
|CSC 10 1 GP
|4-ring cell culture dish
|Dumont #5 tweezers (45° angle tip)
|Table 1. Cell culture materials and equipment.
|B27 serum-free supplement
|Table 2. Solutions for cell culture and patch clamp. a) Neurobasal media. b) Intracellular solution. c) Extracellular solution.
|Equipped with Dodt contrast
|W Plan-APOCHROMAT 40x/0.75
|Platform and X-Y stage
|Vibration isolation table
|In-line solution heater
|Patch clamp amplifier
|Patch clamp data acquisition system
|Borosilicate glass with filament
|Lab pack and electrophysiology tools
|1 L Pyrex aspirator bottle, with outlet for tubing
|Masterflex peristaltic pump
|Table 3.Patch clamp equipment.
|1,870 nm laser diode
|200/220 μm diameter multimode optical fiber patch cord (FC/PC)
|Light delivery optical fiber, silica core and cladding, 0.22 NA
|Optical fiber through connector (FC/PC)
|Optical fiber cleaver
|Optical fiber stripping tool (0.25 - 0.6 mm)
|For removing optical fiber jacket
|Optical fiber stripping tool (0.6 - 1.0 mm)
|For removing outer coating of patch cord
|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
|Laser power meter and detector head
|FieldMate (power meter) with LM-3 (detector head)
|Table 4. Laser equipment.
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