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 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.
1. Culture of Spiral Ganglion Neurons
2. Preparation for Patch Clamp Recordings
3. Patch Clamp Recordings for Investigation of INS
4. INS Experiments
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.
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 illumination has ceased. The shape of the laser induced currents can be seen to vary as the membrane potential is changed, indicating that it may be important to conduct experiments at a range of holding potentials in order to gain a complete understanding of the processes underlying INS. These experiments can be carried out with minor modification of the current protocol and analyzed using established techniques such as charge-voltage (Q-V) analysis (see Reference 7 for examples of Q-V curves obtained from INS in vitro).
The data presented in Figure 3b are indicative of the change in membrane potential typically evoked by a 2.5 msec, 0.8 mJ laser pulse (initial membrane potential -73mV, averaged over 16 laser pulses delivered at a repetition rate of 4 Hz). Current-clamp recordings show a steady membrane depolarization over the course of the laser pulse followed by an approximately exponential decrease towards the resting membrane potential after the pulse. The example in Figure 3b also exhibits a small additional membrane depolarization following the laser pulse. Shapiro et al. 7 have demonstrated that laser-induced changes in membrane potential are closely related to local changes in temperature (i.e. the temperature in the direct vicinity of the cell). Further, the model described by Thompson et al. 27 has determined that under certain alignment conditions, diffusion resulting from axial and radial temperature gradients in the illuminated region can lead to local temperature variations closely resembling the changes in membrane potential shown in Figure 3b. As a result of these findings, it is thought that the position of the target cell relative to the end-face of the light delivery fiber plays a significant role in determining both the time course of laser-evoked variations in membrane potential and the maximum temperature in the region of the cell.
Illuminating with excessive energy or exposure to large increases in temperature may result in damage to the target neuron. This can often be observed through deterioration of cell electrical properties (e.g. an abrupt increase in current required to maintain the membrane potential at a steady level, and/or a large increase in noise and instability within the signal). In extreme cases cell death occurs almost instantaneously upon laser exposure. Figure 4 shows a voltage-clamp recording of the death of a spiral ganglion neuron resulting from exposure to a 25 msec, 8 mJ laser pulse.
Figure 1. Phase contrast images showing a typical spiral ganglion neuron and the relative positions of optical fiber and micropipette (as seen from above) during optical stimulation experiments. a) Typical spiral ganglion neuron. b) The optical fiber in position (image is focused on the top edge of the optical fiber). c) Overlaid images showing the fiber position relative to the cell (note: the top edge of the fiber is slightly overhanging the cell i.e. Δ is negative). As seen from above, δy and Δ are the radial displacement of the neuron center from the fiber axis and the distance from the top edge of the fiber to the center of the neuron respectively. Arrows indicate the position of the spiral ganglion neuron. Scale bars 20 μm.
Figure 2. Schematic of the experimental setup for optical stimulation experiments (not to scale). Inset: position of optical fiber and microelectrode relative to the target cell. θ, Δ and z are as defined in protocol steps 3.4.2 and 3.8.2.
Figure 3. a) Voltage-clamp (average response from 6 laser pulses delivered at a rate of 1 Hz) and b) current-clamp (average response from 16 laser pulses repeated at a rate of 4 Hz) recordings from spiral ganglion neurons showing laser induced changes in membrane current and membrane potential upon illumination by a 2.5 msec, 0.8 mJ laser pulse. Shaded regions indicate the timing of laser exposure. Insets show responses during laser illumination in more detail. Note: the inset in a) focuses on the trace obtained with a membrane potential of -60 mV. Click here to view larger figure.
Figure 4. Voltage-clamp recording showing cell death resulting from exposure to an excessively energetic (25 msec, 8 mJ) laser pulse. Note that the amplitude of the signal is shown in nA and is significantly larger than the pA currents shown in Figure 3.
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 addition, the environment in which the neurons are maintained (e.g. solution temperature, chemical factors) can be systematically varied, making it possible to study the membrane properties and hence the mechanisms underlying infrared neural stimulation. The interaction between electrical and optical stimulation modalities can also be investigated in a controlled manner. These fundamental studies can be further advanced by introducing fluorescent probes to monitor additional parameters such as ionic concentrations or the expression of heat shock proteins. A clear understanding of these various parameters is not only critical to achieve a complete understanding of the phenomenon, but also to achieve more efficient stimulation through process optimization.
Due to the important role played by temperature in the mechanism of INS 5-7, accurate measurement of the localized heating due to laser illumination is of paramount importance in defining this mechanism 27. A detailed method of obtaining calibrated temperature measurements by recording the current flowing through an open patch pipette has been described by Yao et al. 28 and employed by numerous authors, to determine the magnitude and time course of laser induced temperature changes in environments representative of those found in vitro (e.g. see References 7, 8). Provided the position of the light delivery fiber can be precisely determined (e.g. using the current protocol), this method of temperature measurement is likely to enable accurate mapping of the local change in temperature due to typical INS stimuli.
The wavelength of the stimulating laser is a parameter that should be considered in INS experiments, since laser induced temperature changes (and hence the underlying mechanisms of INS) are mediated by the wavelength dependent absorption characteristics of water 7 (see Thompson et al. 25 for a detailed discussion of expected wavelength effects). Aside from the 1,870 nm diode laser used in this protocol (Infrared Nerve Stimulator, Optotech P/L), a variety of wavelengths and laser packages have been used by other authors. Some common examples of the lasers used in existing INS publications are: diode lasers from Aculight with wavelengths ranging from 1,840-1,940 nm 6,7,10,13,16-18; Holmium:YAG lasers (2.12 μm) from Laser 1-2-3 6,11,12,14,15; and diode lasers operating at 1,875 nm5,8, 1,470 nm 8, and 1,535 nm 8 from Sheaumann laser.
A potential drawback of applying this technique to cultured spiral ganglion neurons is that owing to the relatively small size of the neurons (~10-15 μm diameter), the recording electrode is directly illuminated by the laser. It has been suggested that laser illumination beyond a certain threshold may change the properties of the recording circuit 7 (i.e. seal and pipette resistance). Shapiro et al. 7 measured this threshold by monitoring the change in reversal potential of Q-V curves as laser power was increased, finding a threshold pulse energy of 3 mJ. As an alternative approach, it may be possible to determine the magnitude of this effect by measuring the combined resistance of the seal and pipette in whole cell mode (e.g. by recording the current response to a 10 msec, +10 mV voltage pulse) while varying the temperature of the extracellular solution. In order to fully understand the mechanisms of INS, it is vital that laser induced resistance changes be measured and taken into account.
To date the majority of experimental work concerning infrared neural stimulation has been undertaken in vivo. While reported radiant exposure thresholds for infrared stimulation in vivo vary somewhat (e.g. 0.32 Jcm-2 at 2.12 μm for rat sciatic nerves 1, <0.1 Jcm-2 at 1.855 μm for gerbil auditory nerves 18), they are significantly lower than thresholds determined through in vitro studies (e.g. approximately 20 Jcm-2 at 1.875 μm for mouse retinal ganglion cells and rat vestibular ganglion cells 5,8, 8.3 J cm-2 for rat neonatal cardiomycytes 9). At this stage the details of the INS process are not sufficiently well understood to speculate about the cause of this difference; however, using in vitro models such as auditory neurons that closely resemble the targets of previous in vivo work may be advantageous in finding an explanation.
Some other in vitro models involve larger cells such as Xenopus oocytes (~1 mm diameter) used by Shapiro et al. 7 These may be useful for probing spatial variations in the effectiveness of INS across individual cells in order to investigate whether cellular components are influenced by stimulation. Simpler models such as lipid bilayer vesicles may allow even more precise control over experimental parameters than in vitro experiments, however such models are somewhat limited in scope and may not reveal the full complexity of INS.
The authors have nothing to disclose.
This work was supported by the Australian Research Council under Linkage Project grant LP120100264.
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. |