The overall goal of this procedure is to study desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time, using intercellular electrical recordings of photoreceptor cells in isolated eye stocks, employing the discontinuous single electrode switched voltage clamp configuration. This method can help answer key questions in the neurobiology field, such as the circadian time dependency of so many electro-physiological properties of photoreceptors in inactivation and recovery. The main advantage of this technique, is that it allows the measurement of photoreceptors'electrical activity.
In spite of the difficulty to isolate cells and without disturbing the intracellular milieu. Demonstrating this procedure will be Doctors Carolina Barriga-Montoya and Araceli de la O-Maritnez, coworkers of our laboratory. To prepare the intracellular electrode, pull the glass capillary tube with a micropipette puller to obtain a thin tip with a small opening.
Next, fill the pulled capillary glass with 2.7-molar potassium chloride solution by capillarity and then, fill half of the pipette with a fine injection needle. If necessary, tap the electrode pipette to eliminate air bubbles. After that, place the electrode on its holder.
Connect the holder to amplifier head stage with the amplifier. Following that, position the electrode holder head stage with a stable 3-D micromanipulator. Lower the electrode until bath solution covers its tip.
Select the bridge mode of the amplifier and measure the electrode resistance. Make sure that the resistance is about 50 megaohms. Null the offset current and compensate capacitive transients using the holding position button in the section voltage clamp of the amplifier and the capacitance neutralization button in the Microelectrode 1 section of the amplifier.
To construct the super fusion system, pour the bath solution into a suitable receptacle and connect it to an irrigation tubing set, thus connecting the recording chamber. Use a gravity-driven superfusion system. Regulate the flow rate to 0.5 milliliters per second.
Connect the chamber to a suction device. Regulate the solution suction system with a vacuum pump in such a way that the total volume of recording chamber does not vary. To isolate the eye stock of an adult crayfish, detach it from the base with a pair of fine scissors.
Make an opening of one square millimeter in the dorsal cornea using a razor blade to access the retina. Next, place the eye stock in the center of the recording chamber with the opening to the retina facing up. Keep the eye stock in darkness for 20 minutes.
Place the microelectrode parallel to the eye stocks longitudinal axis in such a way that the microelectrode is centered to access the retina. Use a stereoscopic microscope to place the devices in the correct configuration. Next, monitor the voltage difference between the reference and recording electrodes by selecting the bridge mode of the amplifier.
Lower the electrode into the bath and place it right over the retina. Remove the microscope and position the photostimulator lamp parallel to the eye stocks longitudinal axis. Then, slowly lower the microelectrode until a sudden voltage drop is detected.
Subsequently, deliver a test light flash to record the photoreceptor's receptor potential. To obtain appropriate recordings of light-elicited currents, it is critical to make a good impairment of a healthy photoreceptor cell. In this procedure, clamp the voltage at the measured resting membrane potential of the cell by selecting holding amplitude in the data acquisition software.
Then, select the DSEVC mode of the amplifier. In the mode section of the amplifier, select the SEVC button and switch the lever to the Discont. SEVC position.
Set the switching rate to 500 to 1, 000 hertz, as determined by the speed of electrode. After that, deliver a light flash and observe the evoked ion flux. Then, deliver a pair of light pulses and apply the second flash after a desired time interval.
Digitize the currents at 10 kilohertz sampling with the data acquisition software and save the data for offline analysis. Shown here is a two-pulse protocol. Light stimuli were applied at zero milliseconds and 700 milliseconds.
This figure shows the recovery from desensitization. Here is latency recovery, here is peak current recovery, and here is the desensitization time constant tau recovery. Experiments were performed at zero hours circadian time.
This figure shows the recovery from desensitization as a function of circadian time. This is iP recovery, this is L recovery, and this is tau recovery, and this graph shows the weighted time constants. Biphasic processes are marked with an arrow.
Once mastered, this technique can be done in two hours, if it's done properly. While attempting this procedure, it is important to remember to aim for a low noise and low leakage current, photoreceptor impairment. Following this procedure, other methods, like pharmacological assays, can be done in order to answer additional questions, like the involvement of particular channels, receptors, or signaling pathways.
After its development, this technique paved the way for researchers in the field of neurobiology to explore the circadian time dependence of electrophysiological properties of photoreceptor cells. After watching this video, you should have a good understanding of how to study desensitization and recovery of light-activated transduction current by using the intracellular recording technique.
