Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2 methods
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Stimulation and recording of ChR2-expressing neurons ● TIMING 1-4 h
Place the extracellular electrodes in the brain tissue at the desired angle ( and ) and depth by lowering slowly. If using an array of electrodes or a large single electrode, allow tissue to recover from electrode placement for up to 30 min before stimulating or recording. (If using intracellular electrodes, perform Step 15 first.)
Stimulation and recording of ChR2-expressing neurons ● TIMING 1-4 h
Give short light pulses (~1 to 10 ms duration at 20-70 mW mm -2 ) to evoke activity from ChR2-expressing cells ( and ).
Stimulation and recording of ChR2-expressing neurons ● TIMING 1-4 h
Steps 1-10, virus injection: ~30 to 45 min
Cell-type-specific expression of light-activated channels
A variety of approaches can be used to achieve insertion of light-activated elements into neurons that are otherwise not optically sensitive, including constitutive expression in transgenic mice, in utero electroporation and use of viral approaches both with and without associated use of a Cre-loxP system,,. These methods have strengths and weaknesses in generating cell-type-specific expression. Generation of transgenic mice expressing cell-type-specific ChR2 is effective, but costly and time-consuming. In utero electroporation can be targeted to general cell classes by varying the site and prenatal day of treatment -. This early surgery has the advantage of allowing maximal recovery of the pups across development and of providing at least some cell-type specificity in any given model species. However, this approach requires an invasive surgery in utero in which probes are...
Experimental design
The selection of a viral vector is important, as different viruses and virus serotypes exhibit different cellular tropisms -. Both AAV, - and lentivirus, - have been successfully used for expression of light-activated channels in several brain areas, including the cortex and hippocampus. In addition, the degree of viral spread from the injection site can vary widely with virus and tissue type transduced. One advantage of AAV is the broader spatial spread of ChR2 expression around the injection site. We routinely observe expression over a distance of up to 2 mm of cortical tissue ( ). The protocol outlined here specifies the use of an AAV vector, but may be successfully used with other virus types.
Light stimulation parameters
Light spreads in an approximate cone from the point source at the tip of the optical fiber, and light power from a fiber at the surface of the brain decreases with tissue depth. Light intensity is usually measured in mW mm - 2, and a safe range for in vivo experiments is up to ~75 mW mm - 2 for short pulses (~0.5 to 50 ms). We have observed significant tissue damage in the cortex immediately under the optical fiber from sustained stimulation (>500 ms duration) at levels above 100 mW mm - 2. Light can be applied through the mouse dura or thinned skull, and surface positioning of the optical fiber allows effective stimulation throughout supragranular and granular cortical layers in the mouse sensory cortex (~500 µm depth). Alternatively, optical fibers of smaller diameter (50-100 µm) can be placed in the brain for stimulation of deeper cells or small...
MATERIALS
Iodine-based wash (Betadine, Henry Schein, cat. no. 6906950)
Anesthesia setup
The animal should be anesthetized initially with a small dose of isoflurane administered in an induction chamber or small container, as determined by institutional policy. The animal should then be placed in the stereotaxic frame with the nose in the anesthesia head-holder with (at ~0.5 to 1 liter min - 1 ) running to the apparatus. Initial isoflurane in O 2 isoflurane levels after induction may be as high as 2% and may be lowered over the course of surgery to ~1% as the animal reaches a stable plane of anesthesia.
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Evidence quotes (8)
Place the extracellular electrodes in the brain tissue at the desired angle ( and ) and depth by lowering slowly. If using an array of electrodes or a large single electrode, allow tissue to recover from electrode placement for up to 30 min before stimulating or recording. (If using intracellular electrodes, perform Step 15 first.)
Give short light pulses (~1 to 10 ms duration at 20-70 mW mm -2 ) to evoke activity from ChR2-expressing cells ( and ).
Steps 1-10, virus injection: ~30 to 45 min
A variety of approaches can be used to achieve insertion of light-activated elements into neurons that are otherwise not optically sensitive, including constitutive expression in transgenic mice, in utero electroporation and use of viral approaches both with and without associated use of a Cre-loxP system,,. These methods have strengths and weaknesses in generating cell-type-specific expression. Generation of transgenic mice expressing cell-type-specific ChR2 is effective, but costly and time-consuming. In utero electroporation can be targeted to general cell classes by varying the site and prenatal day of treatment -. This early surgery has the advantage of allowing maximal recovery of the pups across development and of providing at least some cell-type specificity in any given model species. However, this approach requires an invasive surgery in utero in which probes are inserted into the fetal brain, which can have negative scientific and practical implications for a given experiment. Most importantly, cell-type specificity of expression is limited to the resolution provided by knowledge of cell birth date and relatively broad localization to the targeted germinal...
The selection of a viral vector is important, as different viruses and virus serotypes exhibit different cellular tropisms -. Both AAV, - and lentivirus, - have been successfully used for expression of light-activated channels in several brain areas, including the cortex and hippocampus. In addition, the degree of viral spread from the injection site can vary widely with virus and tissue type transduced. One advantage of AAV is the broader spatial spread of ChR2 expression around the injection site. We routinely observe expression over a distance of up to 2 mm of cortical tissue ( ). The protocol outlined here specifies the use of an AAV vector, but may be successfully used with other virus types.
Light spreads in an approximate cone from the point source at the tip of the optical fiber, and light power from a fiber at the surface of the brain decreases with tissue depth. Light intensity is usually measured in mW mm - 2, and a safe range for in vivo experiments is up to ~75 mW mm - 2 for short pulses (~0.5 to 50 ms). We have observed significant tissue damage in the cortex immediately under the optical fiber from sustained stimulation (>500 ms duration) at levels above 100 mW mm - 2. Light can be applied through the mouse dura or thinned skull, and surface positioning of the optical fiber allows effective stimulation throughout supragranular and granular cortical layers in the mouse sensory cortex (~500 µm depth). Alternatively, optical fibers of smaller diameter (50-100 µm) can be placed in the brain for stimulation of deeper cells or smaller volumes of tissue. It is important to consider the duration and strength of the light pulse, as continuous light application can cause abnormal levels of neural activity and excitotoxicity. Power transmitted by the fiber will vary with fiber type (multimode or single-mode) and the spread of light...
Iodine-based wash (Betadine, Henry Schein, cat. no. 6906950)
The animal should be anesthetized initially with a small dose of isoflurane administered in an induction chamber or small container, as determined by institutional policy. The animal should then be placed in the stereotaxic frame with the nose in the anesthesia head-holder with (at ~0.5 to 1 liter min - 1 ) running to the apparatus. Initial isoflurane in O 2 isoflurane levels after induction may be as high as 2% and may be lowered over the course of surgery to ~1% as the animal reaches a stable plane of anesthesia.
Machine-readable layer
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