This technique leverages multi-photon laser-scanning microscopy in conjunction with laser-flash photolysis of a photoactivatable nicotine molecule. Allowing precise activation of nicotinic cholinergic receptors. This technique allows for fine spatiotemporal control over nicotinic receptor agonist application, providing a powerful tool for the study of receptor functional expression patterns and cholinergic synaptic or volume transmission.
Before beginning the procedure, use a programmable pipette puller to pull a glass micropipette with a 20-to 40-micrometer opening diameter. Strain approximately one milliliter of recording solution through a 0.22-micron filter. And resuspend enough lyophilized photoactivatable nicotine in the filtered recording solution to yield a two-millimolar final concentration.
Backfill the pulled local application micropipette with 50 microliters of the photoactivatable drug, and secure the pipette in a pipette holder mounted on a micromanipulator. Connect the pipette holder via an appropriate length of tubing to a pressure ejection system capable of sustained low-pressure application. And use the micromanipulator to maneuver the local application pipette into the extracellular recording solution.
Position the pipette tip slightly above a mouse brain tissue sample, approximately 50 micrometers from the cell of interest. And briefly apply one to two pounds per square inch of pressure to the pipette. There should be minimal to no displacement of the cell of interest.
After achieving a stable whole-cell patch clamp, turn on the low application on the pressure ejection device, and saturate the tissue surrounding the cell with photoactivatable nicotine for one to two minutes. Local application introduces additional complexity to the experiment. But it spares compound and allows higher concentrations of PA-Nic to be utilized.
For bath application of the photoactivatable nicotine, first dissolve enough of the lyophilized drug in a volume of recording solution appropriate for continuous recirculation to a 100-micromolar final concentration. And load the resulting mixture into a perfusion system. Then begin recirculation of the photoactivatable nicotine solution at a 1.5-to 2-millimeter-per-minute rate, using tubing with a minimal inner diameter and overall length to minimize the required recirculation volume.
During recirculation, continuously bubble the solution with carbogen, and maintain the bath temperature at 32 degrees Celsius under low-light conditions. Bath application benefits from the simplicity and uniformity of PA-Nic permeation of the tissue. But it necessarily utilizes lower micromolar PA-Nic concentrations, and expends higher total amounts of compound.
For live visualization of a medial habenula neuron, use transmitted light or infrared differential interference contrast optics and a video camera to establish a stable, whole-cell patch clamp recording. After establishing the high-resistance cell-attached configuration, but before break-in, switch the setup and software to laser scanning mode. After break-in, use laser scanning to verify that the imaging dye of interest is passively filling the neuron by diffusion.
Allow the dye to fill the cellular compartments for at least 20 to 30 minutes before using the live scan function to visualize the neuron and subcellular compartment of interest. Select imaging parameters that allow an accurate live visualization of the neuronal features, manipulating the appropriate settings to affect or alter the display visualization, resolution, signal-to-noise ratio, and image frame acquisition time as necessary. To enhance the signal contrast, open the lookup table window and adjust the lookup table floor and ceiling settings.
To locate an area of interest within the tissue, select the 1X optical zoom and use the panning controls. Use the Z-series tool to select a start and stop position that contains the cell of interest. Set a step size of one micrometer, and select an image size that will yield a highest-resolution image.
Often 1, 024 by 1, 024 pixels per line. Then consecutively image the neuron in every Z-plane that contains the cell. To calibrate the laser stimulation, start the system scanning with an imaging laser power greater than minimum, and fine-tune the objective focus onto the thin red marker fluorescence layer.
Select an area within the fluorescence field clear of debris and evenly coated with marker, and open the tools, calibration, and alignment menu to select the uncaging galvocalibration function. Follow the burn spots tutorial for the spatial calibration of the second galvanometer mirror pair, selecting the 405-nanometer laser, a laser stimulation power of 400, and a stimulation duration of 20 milliseconds. To yield one-to five-micrometer diameter holes in the red marker.
Select update to stimulate and refresh the image after the center spot burn, and move the round red indicator to the actual spot location of the center, right-center, and lower-center spots to obtain a grid of nine spots. To test the calibration, open the mark points window and manually activate the stimulation parameters of the defined spots in a new area of the sample, taking care that the correct latest calibration file is loaded into the mark points window. Then apply a test pulse to verify the correct calibration.
To briefly image and locate the subcellular area of interest, select live scan and use increase the optical zoom to visualize any small structures as necessary. Place the mark point single-spot crosshairs immediately adjacent to the cell membrane, and set the photostimulation parameters to a one-to 50-millisecond duration, a one-to four-milliwatt laser power, and at least one trial. Then select run mark points to initiate the mark points protocol, and observe the electrophysiology data acquisition in real time.
Photoactivatable nicotine 2PE fluorescence of one-millimolar PA-Nic is easily detected during pressure ejection from a local application pipette as demonstrated. Whereas the delivery of the main photochemical products of the photoactivatable nicotine photolysis reaction generate no fluorescent signal at the same concentration and excitation power and wavelength. Demonstrating the specificity of the photoactivatable nicotine results.
Imaging of the photoactivatable nicotine applied to the brain tissue as demonstrated reveals the presence of the drug within 100 to 200 micrometers of the local application pipette. Confirming that photoactivatable nicotine can be effectively delivered to the brain tissue via local application. Here, high-quality images within which the neuronal morphology appears to be complete, the noise is minimized, and the debris does not interfere with interpretation of the cellular morphology, are shown.
These images, however, are of a lower quality. Owing to a lower signal-to-background ratio and substantial debris. Pairing two-photon laser scanning microscopy of medial habenula neurons in brain slices with laser flash photolysis of PA-Nic as demonstrated, allows for the reconciliation of electrophysiological responses with cellular morphology.
Single-spot photolysis performed at 10-second intervals allows a sufficient recovery time for the baseline holding current. While a shorter one-second interval leads to a gradual increase in the holding current as the protocol proceeds. Suggesting that the nicotine does not have enough time to diffuse away from the system with the shorter intervals.
In designing experiments that utilize photoactivatable molecules, it's important to remember to select fluorescent indicators and report fluorophores with compatible excitation emission spectra. PA-Nic is compatible with most common fluorophores due to its short wavelength photolysis peak. When choosing the most appropriate PA-Nic application technique, it is important to carefully consider the functional expression, physiological activity of the nicotinic receptors in the neurons of interest.
Skilled utilization of this technique allows for fine spatiotemporal control of nicotine application, which enables exciting new possibilities for studying the kinetics of native nicotinic receptor engagement, subcellular localization, and modulation of neuronal activity by nicotinic receptor activation.
