The overall goal of the following experiment is to produce a model system to study the effects of infrared laser illumination on auditory neurons in vitro. This is achieved by patch clamping, cultured auditory neurons in the whole cell configuration in order to explore their electrical characteristics. Subsequently, exposing the neurons to laser irradiation can elicit electrical responses in the exposed cell, which can be measured with a patch clamp setup.
Illumination parameters and environmental variables can then be varied in order to observe their effects on laser induced electrical responses. The auditory neurons exposed to the laser light will exhibit repeatable electrical activity in response to each laser pulse for later analysis. This method can help answer key questions about the mechanisms underlying infrared stimulation of spiral ganglion neurons.
Though this method can provide insight into infrared stimulation of spiral ganglion cells specifically, it can also be extended to other cell types or simplified models such as lipid bilayers to further elucidate the physical processes that are involved. Visual demonstration of this method is important to ensure accurate positioning of the light delivery optical fiber, because the resulting radiance exposure is a critical parameter, Have prepared recording micro pipettes with a resistance of two to six mega ohms. They can be pulled from borrow silicate glass with a CO2 laser puller.
Prepare the fiber coupled laser. A wide array of optical fiber will work. Cutting a fiber in a patch cord configuration with FCPC connectors in half produces two links of fiber with a connector at one end and exposed fiber at the other end.
These are the pigtails. Prepare the bear tip of one pigtail by removing the fiber jacket, cleaning with ethanol and cleaving with an appropriate tool under a microscope. Check that the tip is perpendicular to the fiber axis and that it appears flat.
Connect the other end of the fiber pigtail to the output of the stimulation laser using an appropriate through connector if necessary. At this point, always be sure to measure the output laser power from the fiber. Do this after any further tip manipulations as well.
Now insert the fiber into a chuck and to fix the chuck to the appropriate micro positioner. Next, determine the angle that the optical fiber makes with a cover slip. Take a photograph of the arrangement and calculate the angle using image J.Now, secure the connections that synchronize the laser with the patch clamp data acquisition system.
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, 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, set the flow rate of the perfusion system to between one and two milliliters per minute. A gravity fed system with an inline heater for rapid heating of the solution and a peristaltic pump to remove spent solution by suction will work.Well. Place a cover slip with cultured cells into the recording chamber of an upright microscope using a high magnification water immersion objective and phase contrast, locate a spiral ganglion neuron.
A typical spiral ganglion neuron is phased bright round and approximately 15 microns in diameter with a prominent nucleus. Once a suitable neuron has been located, switch to a lower magnification objective and locate the target neuron. Then use the micro positioner to move the optical fiber until the tip is close to the target neuron in both the horizontal and vertical planes.
Switch back to the high magnification objective and position the tip of the optical fiber in its intended position next to the neuron. When adjusting the vertical position of the fiber, it's important for the bottom edge of the fiber to rest on the cover lip. To minimize uncertainty in the fiber's location, the point of contact can be identified from visual cues in the microscope image.
Once the fiber is in position, move it out by known amount along its longitudinal axis so as not to affect the positioning of the micro pipette. The micro pipette should be filled with intracellular solution and securely fitted into place on the head stage of the amplifier. Using tubing attached to the side of the micro electrode holder, apply a small amount of positive pressure to prevent clogging of the micro pipette.
Using a microm manipulator, move the micro pipette into position just above the target neuron and proceed with making a giga ohm seal. Make a record of 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 transient by adjusting the CP fast and CP slow controls on the amplifier. Then switch the amplifier into whole cell mode and modify capacitance and resistance compensation until a flat current is observed during the SEAL test. Next, apply series resistance compensation with about 70%correction, 70%prediction, adjusting the capacitance and resistance compensation controls to maintain a flat test seal response.
Then switch the amplifier to current clamp mode. Take note of the resting membrane potential in the absence of current injection. Now set a holding current to stabilize the membrane potential at the desired level.
Neutralize the pipette capacitance and adjust the bridge balance to balance the voltage drop. Check the firing properties of the neuron by stimulating with depolarizing current. At this point, move the optical fiber back into position next to the neuron using imaging software coupled to a CCD camera capture images focused on the plane of the neuron initially and then focused on the top edge of the optical fiber.
Analyze the images to determine the delta value, which is the position of the upper edge of the optical fiber relative to the center of the target neuron. It's very important to accurately know the position of the optical fiber. This is because the energy per unit, area, or radiance exposure delivered to the target cell is a critical parameter for infrared neural stimulation, and this can be significantly affected by the position of the fiber relative to the cell.
This laser is optical power is controlled by computer via a direct input to the laser and can be specified manually before each recording. Pulse length and repetition rate can be controlled via the function generator as described before, ensure that the data are recorded from both the patch clamp channel and the laser trigger channel one half to 15 millisecond laser pulses at 0.25 to five. Millijoules typically yield measurable electrical responses.Initially.
It may be useful to set the repetition rate of laser pulses to one hertz or less to minimize undesirable effects. Once all of the laser parameters have been set. Proceed with data collection.
Spiral ganglion neurons respond to laser illumination with repeatable wave forms in both voltage clamp and current clamp Recording configurations in response to 2.5 millisecond 0.8 millijoules laser pulses. A typical cell produces a net inward current at various holding potentials. Current clamp recordings show a steady membrane depolarization over the course of these laser pulses, followed by an approximately exponential decrease towards the resting membrane potential after the pulse.
In some cases, there is also a small additional membrane depolarization following the laser pulse. Illuminating with excessive energy or exposure to large increases in temperature may result in cell damage observed through deterioration of cell electrical properties or instantaneous cell death. By following this procedure, a range of environmental parameters such as solution temperature or chemical factors can be varied to test the effect of these on laser induced electrical activity.
Don't forget that working with lasers can be hazardous and standard safety precautions should be put in place. These include using laser safety goggles, warning signs, and making sure that beam does not unintentionally intersect with any highly reflective surfaces.
