TITLE: Photostimulation for optogenetics or uncaging
AUTHORS: enrico.zampese

[DESCRIPTION]
Protocol for photostimulation, to be combined with any other electrophysiology/imaging recording approach.
Once the desired patch clamp recording (e.g., cell attached, whole cell, perforated patch) and/or imaging (e.g., genetically encoded sensor or synthetic dye) configuration is achieved and a stable recording is established, photostimulation can be used to stimulate presynaptic terminals/cell expressing an opsin of interest (e.g., Chronos or Channelrhodopsin) or to release a photo-sensitive caged compound (e.g., RUBI-GABA, RUBI-Glutamate, MNI-glutamate...), and test the effect on the experimental outcome of interest (e.g., excitatory/inhibitory response).

[BEFORE_START]
This protocol assumes that slices have already been obtained (see for example dx.doi.org/10.17504/protocols.io.dm6gp328jvzp/v2) and that an experimental recording approach has already been established (see for example dx.doi.org/10.17504/protocols.io.36wgq3y9olk5/v1 or dx.doi.org/10.17504/protocols.io.eq2lyj8zrlx9/v1), and it is meant only as an add-on to those protocols.


[GUIDELINES]
Follow institutional guidelines.

[STEPS]
SECTION: Preliminary steps
1. Obtain brain slices of region of interest according to existing protocols
SECTION: Preliminary steps
3. Identify/patch neurons of interest and adjust settings according to the desired experimental protocol .
SECTION: Preliminary steps
4. Once a recording on a cell of interest is established, set up the stimulation protocol. 

SECTION: Stimulation protocol
5. 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.
SECTION: Preliminary steps
1.2. 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.
SECTION: Stimulation protocol
5.1. Wide-field LED stimulation is achieved with the CoolLED pE-100 blue LED (470nm) activated via the  recording/controlling software. 

SECTION: Stimulation protocol
5.2. 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.
SECTION: Stimulation protocol
6. Synchronize desired stimulation protocol with recording.
SECTION: Stimulation protocol
7. Start recording/imaging acquisition synchronized with a stimulation protocol.
SECTION: After the experiment
9. Terminate experiment, remove electrode and slices, and shut down software/hardware according to the experimental protocol used.
SECTION: After the experiment
10. Discard waste according to institutional guidelines.
SECTION: After the experiment
11. Clean up work station.
SECTION: Preliminary steps
2. Turn on hardware (including the light source for the desired photostimulation approach) and software required for the electrophysiology/imaging experiment of choice.
SECTION: Stimulation protocol
8. Adjust stimulation settings based on the desired experimental outcomes.
SECTION: Preliminary steps
1.1. 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. 
SECTION: Preliminary steps
4.1. 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).
SECTION: Stimulation protocol
7.1. 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.