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 addition of a particular molecule (e.g. caged molecules) ³ or some form of genetic manipulation (e.g. optogenetics) ⁴, neither of which are easy to apply outside of a research setting. Of particular interest therefore is infrared neural stimulation (INS), whereby neural tissue is excited by pulsed infrared laser light. INS has the potential to overcome many of the shortcomings of electrical stimulation by enabling highly specific, non-contact stimulation of neural tissue ². However, while INS has been successfully demonstrated in a variety of settings in vivo, the precise mechanism of excitation remains uncertain.

Some recent publications have shown progress towards uncovering the mechanism behind INS ^5-7. Rapid heating due to absorption of the laser light by water appears to play a key role. However, beyond this a consensus is yet to be reached. Shapiro et al. ⁷ propose a highly general mechanism whereby rapid heating causes a perturbation in the distribution of charged particles adjacent to the cell membrane, leading to a change in the capacitance of the cell membrane and subsequent depolarization. In addition, Albert et al. ⁵ assert that laser induced heating activates a specific class of temperature sensitive ion channels (transient receptor potential vanilloid channels), allowing ions to pass through the cell membrane. At this stage it is unclear how these mechanisms combine, or indeed whether there are further factors that are yet to be identified.

Although a small number of publications (references ^5,7-9) have investigated INS in vitro, the vast majority of work published in this field has been carried out in vivo (e.g. references ^1,6,10-18). Infrared stimulation of auditory neurons has been an area of particular interest, owing to the potential applications in cochlear implants ^10,14-18. While in vivo experiments are important to verify the effectiveness of the technique in various settings, the increased level of control afforded by in vitro studies is expected to lead to a more detailed understanding of the mechanism responsible for INS. This report describes the preparation of cultured spiral ganglion neurons for patch clamp investigations, as these can be used to study fundamental mechanisms while also linking to the large body of existing data from the auditory system.

The patch clamp technique is an excellent tool for investigations of electrophysiological phenomena, providing a means of recording electrical activity in single cells and studying the contribution of the individual underlying currents¹⁹. When this technique is applied to a stable in vitro preparation of primary neurons, such as cultured spiral ganglion neurons, it offers the opportunity to study in depth the mechanisms by which neural activity is controlled and manipulated.

The protocols specified in this work outline methods for investigating the effect of laser stimulation on the electrical properties of spiral ganglion neurons through patch clamp recordings. The approach is based on a fiber-coupled laser rather than a free-space laser, allowing safer operation as well as easier and more repeatable alignment without the need to modify the standard microscope configuration. On the basis of these protocols, it should be possible to conduct a wide range of experiments in order to more clearly determine the mechanism or mechanisms behind INS.

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 supplement, 1 ml B27 supplement, 0.5 ml L-glutamine, and 0.5 ml penicillin-streptomycin. Note: Supplements can be frozen, stored at -20 °C and added to the media on the day required.
3. Dissociate spiral ganglion neurons from post-natal day 4-7 rat pups as previously described ^20,21, using both enzymatic (0.025% trypsin and 0.001% DNase I) and mechanical techniques. Refer to Whitlon et al. ²² and Vieira et al. ²³ for detailed procedures of spiral ganglion neuron culture, or Parker et al. ²⁴ for a demonstration of modiolus isolation.
4. Aspirate any remaining poly-L-ornithine/laminin solution from the coverslips and wash briefly with NBM.
5. Add 150-200 μl of the dissociated spiral ganglion neuron suspension to the coverslips and place into an incubator (37 °C, 10% CO₂). Note: up to 20 coverslips can be prepared from an average litter of 8 rat pups.
6. Four hours after plating neurons, aspirate the solution to remove cell debris and replace with 150-200 μl warmed fresh NBM. Note: media may require daily replenishment to avoid dehydration.
7. Return coverslips to the incubator until required for electrophysiological recordings. Note: dissociated spiral ganglion neuron cultures can be used for electrophysiological experiments four hours after dissociation and for up to two days thereafter. Time in vitro should be taken into account during analysis of results. Replenish NBM every 24-48 hr.

2. Preparation for Patch Clamp Recordings

1. Prepare solutions
   1. Intracellular (micropipette) solution: 115 mM K-gluconate, 7 mM KCl, 10 mM HEPES, 0.05 mM EGTA, 2 mM Na₂ATP, 2 mM MgATP, 0.5 mM Na₂GTP (adjust to pH 7.3 with KOH; adjust to 295 mOsmol/kg with sucrose). Pass the solution through a sterile filter (0.2 μm) and divide into 200 μl aliquots to be stored at -20 °C until the day of recording.
   2. Extracellular (bath) solution: 137 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 10 mM glucose (adjust to pH 7.4 with NaOH; adjust to 300-310 mOsmol/kg with sucrose). This solution is made on the day of recording.
2. Prepare recording micropipettes with a resistance of 2-6 MΩ. We use a CO₂ laser puller (P-2000; Sutter Instruments) and borosilicate glass (1.0 mm outer diameter; 0.58 mm inner diameter; 75 mm length).
3. Prepare the laser. This protocol is intended for use with a fiber-coupled laser, such as the 1,870 nm Infrared Nerve Stimulator from OptoTech P/L. The optical fiber used for light delivery in our experiments is a 200/220 μm core/cladding diameter silica fiber with a numerical aperture of 0.22 and FC-PC connectors at both ends (AFW Technologies MM1-FC2-200/220-5-C-0.22). The patch cords were cut in half to produce two fiber pigtails (i.e. connectorized at one end and cleaved at the other). The effects of fiber core diameter and numerical aperture on laser induced temperature changes have been discussed in detail by Thompson et al. ²⁵
   1. Cleave the tip of the light delivery fiber using standard techniques and ensure that the resulting tip is of high quality by observation with an optical microscope (i.e. the tip should be perpendicular to the fiber axis and appear flat upon visual inspection). Connect the light delivery fiber to the fiber-coupled output of the stimulation laser using an appropriate through connector (e.g. Thorlabs ADAFC2).
   2. Measure the output laser power from the cleaved tip of the light delivery fiber using an appropriate instrument (e.g. Coherent FieldMate with LM-3 detector head). It is recommended to check the laser power every time the light delivery fiber is cleaved or a significant adjustment is made to the laser (e.g. transportation from one lab to another).
   3. Insert light delivery fiber into a fiber chuck or equivalent device and affix the chuck to the appropriate micropositioner. It is important to be able to accurately determine the angle θ that the optical fiber makes with the coverslip. This angle can be measured by taking a photograph of the experimental arrangement and using image processing software (e.g. ImageJ) to obtain the angle. The angle θ (or range of possible angles) is primarily constrained by spatial limitations of the experimental arrangement - in particular the microscope. Typical values of θ in our experiments (based on an upright microscope) are around 36°, however the optimal angle may differ significantly for alternative setups (e.g. those using an inverted microscope).
   4. Make connections between the laser and the patch clamp data acquisition system (e.g. Digidata 1440A, Molecular Devices) as shown in Figure 2. The digital output from the patch clamp data acquisition system should be connected to the laser via an external function generator, making it possible to specify laser pulse parameters independent of the data acquisition system. Alternatively this output can be connected directly to the laser driver (requiring the pulse length and repetition rate to be set by the data acquisition software). In either case, the signal used to trigger the laser should be connected back to an input of the data acquisition system to ensure that the timing and length of the laser pulses can be recorded concurrently with the electrophysiological signal.

3. Patch Clamp Recordings for Investigation of INS

1. Fill the appropriate container of the perfusion system with extracellular solution and adjust the flow rate to provide perfusion of the bath at a rate of 1-2 ml/min. We use a gravity-fed system (aspirator bottle, pinch valve, and PE tubing) with an in-line heater to enable rapid heating of the solution, and a peristaltic pump to remove spent solution by suction.
2. Place a coverslip with cultured cells into the recording chamber (bath) of an upright microscope. Using a high magnification water-immersion objective (e.g. 40X) and phase-contrast (e.g. differential interference contrast or Dodt gradient contrast), visually locate the spiral ganglion neurons within the culture. A typical spiral ganglion neuron is phase-bright, round and approximately 15 μm in diameter with a prominent nucleus, as shown in Figure 1a.
3. Once a neuron has been located, switch to a lower magnification objective (e.g. 10X) and locate the target neuron within the visual field.
4. Move the light delivery optical fiber into position using the following procedure (or equivalent):
   1. Use the micropositioner to move the output fiber until the tip is close to the target neuron in both the horizontal and vertical planes. The vertical position of the fiber tip can be verified by moving the objective up and down (scanning the focus).
   2. Switch back to the high magnification objective and position the tip of the optical fiber in its intended position next to the neuron (see Figure 2 inset). When adjusting the vertical position of the fiber, it is important that the bottom edge of the fiber is resting on (i.e. just touching) the coverslip, to minimize uncertainty in the location of the fiber. Optimal alignment in the horizontal plane will depend on the angle θ that the fiber makes with the coverslip and the radius of the fiber r. In order to position the fiber so that the center of the target neuron lies along the fiber axis, the horizontal distance between the upper edge of the fiber and the target cell should be
      Δ_target = r (cosec θ - 2 sin θ),
      where negative values would indicate that the optical fiber overhangs the cell. When aligning the fiber, a distance of Δ_target between the top edge of the fiber and the center of the neuron can be approximately achieved by visual inspection. However Δ_target is designed to serve as a rough guideline only. Positioning the fiber such that the actual distance Δ between the upper edge of the fiber and the center of the neuron differs measurably from Δ_target (as in Figure 1) can provide insight into the effect of both radial displacement (i.e. from the center of the beam) and axial displacement (i.e. along the fiber axis) of the beam relative to the target neuron. For example, setting Δ ≈ 0 would be expected to increase the local temperature in the vicinity of the cell - i.e. since the beam profile for typical multimode fibers is well approximated by a top hat distribution ²⁶, the decrease in locally absorbed energy (temperature) due to displacement from the center of the beam is minimal compared to the increase in absorbed energy from moving the fiber end face closer to the cell.
      While care should be taken to position the fiber as accurately and repeatably as possible using visual cues, precise measurement of the fiber location relative to the target neuron (e.g. Δ) is of greater importance than positioning it a predetermined distance away. Δ can be determined (with an estimated maximum uncertainty of ±3 μm) by image analysis as described in step 3.8.2 of the protocol. In some experiments ^5,8, white light sources have been coupled through the fiber in order to target the desired cell. This approach was found to be ineffective in the upright microscope setup, as the intensity of light scattered from the target region was relatively low at these shallow angles (θ).
   3. Once the fiber is in position, move it out by a known amount to enable straightforward positioning of the micropipette. The fiber can then be returned to the original position when a neural recording is achieved.
5. Fill the micropipette with intracellular solution and securely fit into place on the headstage of the amplifier (e.g. Multiclamp 700B, Molecular Devices). Using tubing attached to the side of the microelectrode holder, apply a small amount of positive pressure to prevent clogging of the micropipette.
6. Using a micromanipulator, move the micropipette into position just above the target neuron.
7. Protocol for whole cell recordings:
   1. Apply a 10 msec, +10 mV square wave pulse (e.g. Seal Test) in voltage-clamp mode of the amplifier and adjust the micropipette potential (pipette offset) of the baseline signal to 0 nA. The seal test should also be used to determine whether the resistance of the microelectrode is within the desired range (e.g. 2 - 6 MΩ).
   2. While monitoring the resistance, use fine adjustments of the micromanipulator to gently position the micropipette onto the surface of the target neuron. When the micropipette is in contact with the neuron, the resistance will increase (by ~ 0.5 to 1 MΩ). Immediately following the resistance increase, remove the positive fluid pressure and apply a small negative pressure. Once resistance has passed 10 - 20 MΩ, fix the membrane potential to a holding level (e.g. around -60 mV).
   3. The electrode resistance should continue to increase until it exceeds 1 GΩ, signifying that an effective seal (termed 'gigaseal') has been formed between the cell membrane and micropipette. At this point, remove all fluid pressure from the micropipette.
   4. Once the gigaseal has been formed, use short current pulses (25 to 100 μsec; +1 V) or brief pulses of negative fluid pressure to rupture the cell membrane and achieve whole cell configuration.
   5. Record the membrane capacitance, series resistance and input resistance as determined from the exponential curve fitted to the current during the seal test pulse. Minimize the capacitance transients by adjusting the CpFast and CpSlow controls on the amplifier, then switch the amplifier into whole cell mode, and compensate capacitance and resistance until a flat current is observed during the seal test. Apply series resistance compensation (~70% correction; 70% prediction), adjusting capacitance and resistance controls to maintain a flat test seal response.
   6. Switch to current-clamp mode in the amplifier. Take note of the resting membrane potential (in the absence of current injection). Set a holding current to stabilize the membrane potential at the desired level (e.g. -60 mV). Neutralize the pipette capacitance and adjust the bridge balance to balance the voltage drop.
   7. Check the firing properties of the neuron by stimulating with depolarizing current (+10 to +200 pA in +10 pA steps; 300 msec duration).
8. 1. Move the optical fiber back into position next to the neuron. Using imaging software coupled to a CCD camera, capture images of the position of the fiber with respect to the target neuron, focusing on the plane of the neuron initially, and then on the top edge of the optical fiber (see Figure 1).
   2. By subsequent analysis of the resulting images (e.g. Figures 1b and 1c) it is possible to precisely determine Δ (the position of the upper edge of the optical fiber relative to the center of the target neuron). Once Δ is known, parameters such as the distance from the fiber end face to the target neuron (along the fiber axis) z and the radial displacement of the neuron from the center of the beam δr can be calculated using simple trigonometric relations:
      z = r sin 2θ + Δ cos θ
      δr = ((r cos 2θ - Δ sin θ)² + δy²)^1/2,
      where δy is the distance between the fiber axis and the center of the neuron as seen from above (e.g. see Figure 1).
      Analysis should take into account positional variations from cell to cell, as accurate knowledge of these location parameters may be required to resolve possible differences between stimulation processes ²⁵.

4. INS Experiments

1. While recording electrophysiological data in either current clamp or voltage clamp configurations, run the stimulation laser at the desired parameters (e.g. power, pulse length, repetition rate etc.). With our laser, the optical power is controlled via a direct input to the laser driver and is specified manually before each recording. Pulse length and repetition rate can be controlled by either an external signal generator or the data acquisition software (as described in step 2.3.4). Ensure that data are recorded from both the patch clamp channel and the laser trigger channel.

Laser pulses with lengths ranging from around 500 μsec to 15 msec and energies of ~0.25-5 mJ per pulse typically yield measurable electrical responses. Setting the repetition rate of laser pulses to be 1 Hz or less may be useful for initial experiments, since it will minimize the effects of this parameter. Typical results showing the change in the recorded signal are presented in the following Section.

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

Name	Company	Catalog Number	Comments
Name of Reagent/Material	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.