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 and/or imaging configuration (cell attached, whole cell, perforated patch) 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 compoound (e.g., RUBI-GABA, RUBI-Glutamate, MNI-glutamate...).

[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
2. Identify and patch neurons of interest according to the desired experimental protocol.
SECTION: Preliminary steps
3. Once a recording on a cell of interest is establish, set up the stimulation protocol.
SECTION: Stimulation protocol
4. Either a wide-field LED stimulation or a target focal spot blue laser can be utilized, depending on the preferred experimental approach.
SECTION: Preliminary steps
1.1. Optogenetic experiments require the expression of genetically-encoded opsins in neurons projecting to the area of interest. Mice line expressing opsins under cell-specific or inducible promoters re 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
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). 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
4.1. Wide-field LED stimulation is achieved with the CoolLED pE-100 blue LED (470nm) activated via the  recording/controlling software. 

SECTION: Stimulation protocol
4.2. 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
5. Synchronize desired stimulation protocol with recording.
SECTION: Stimulation protocol
6. Start recording.
SECTION: After the experiment
7. Terminate experiment, remove electrode and slices, and shut down software/hardware according to the experimental protocol used.
SECTION: After the experiment
8. Discard waste according to institutional guidelines.
SECTION: After the experiment
9. Clean up work station.