Photostimulation for optogenetics or uncaging

Obtain brain slices of region of interest according to existing protocols

Optogenetic experiments require the expression of genetically-encoded opsins in neurons projecting to the area of interest. Mice lines expressing opsins under cell-specific or inducible promoters are available. Alternatively, opsin expression can be induced via injection of appropriate viral vectors (see existing protocols for viral injections). For the specific case we are considering experiments on slices expressing the excitatory opsin Chronos, which is stimulated by blue light (~470nm). For slices expressing opsins, limit exposure to intense light during slice preparation and incubation steps.

Uncaging experiments can be performed on naive slices, and different caged compound can be applied to the slice of interest and photostimulated as needed. For this specific case, we are considering experiments using RUBI-GABA, which is stimulated by blue light (~470nm). If the experiments include imaging, plan experiments accordingly so that compatible excitation wavelengths and/or intensity probes/compounds are employed. Caged compounds can be bath applied or microinjected over the site of recording.Caged compounds are intrinsically light sensitive , so any light exposure should be strictly avoided during reconstitution, and particular attention should be paid to shielding all solutions, tubings, and equipment from light.

Turn on hardware (including the light source for the desired photostimulation approach) and software required for the electrophysiology/imaging experiment of choice.

Identify/patch neurons of interest and adjust settings according to the desired experimental protocol .

Once a recording on a cell of interest is established, set up the stimulation protocol.

When performing photostimulation combined with imaging experiments, if needed the photostimulation protocol should include a protective step for the light detectors (e.g., the closure of a shutter synchronized with the photostimulation pulse).

Either a wide-field LED stimulation or a target focal spot blue laser can be utilized, depending on the preferred experimental approach. Note that the excitation wavelength might vary with the opsin/compound utilized.

Wide-field LED stimulation is achieved with the CoolLED pE-100 blue LED (470nm) activated via the recording/controlling software. 

For target focal spot blue laser (473nm) stimulation, stimulating spots (sub-micron size) were positioned adjacent to individual spines. The laser power was calibrated to evoke a somatic postsynaptic potential of 2-5mV. For simultaneous stimulation of 5-10 spines, a larger blue spot (8 microns) is used.

Synchronize desired stimulation protocol with recording.

Start recording/imaging acquisition synchronized with a stimulation protocol.

For uncaging: if the caged compounds are bath applied, allow sufficient time for full replacement of the solution in the recording chamber and permeation in the slice before initiating the stimulation protocol (at least ~5minutes, but this might vary based on the length of tubing, flow rate, size of the chamber). If the caged compounds are microinjected over the site of recording, the stimulation protocol can be initiated shortly after the application of the compounds.

Adjust stimulation settings based on the desired experimental outcomes.

Terminate experiment, remove electrode and slices, and shut down software/hardware according to the experimental protocol used.

Discard waste according to institutional guidelines.

Clean up work station.