Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2 methods
Aim. Evidence-backed execution summary for Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2 methods from Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2.
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This experiment, in seven questions
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Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
EQUIPMENT
reagent used in the protocol.
- Use
- SMA connectors (Fiber SMA Connectorization Kit, SMA 905 connectors, Thorlabs)
REAGENT SETUP
reagent used in the protocol.
- Use
- Add a small amount (~0.05 to 0.1 µl) of Fast Green to 1 µl of the virus solution.
Electrical artifacts (Step 16)
reagent used in the protocol.
- Use
- If field potential or intracellular signals are desired, one optimal approach is to use glass electrodes with a more distal metal wire insert that is not contacted by the light, a strategy typically used in slice or culture recordings. For field potential recordings made with glass electrodes, further adding a non-r...
EQUIPMENT
Hair trimmer (Harvard Apparatus, cat. no. 525204)
- Use
- Hair trimmer (Harvard Apparatus, cat. no. 525204)
EQUIPMENT
Pipette Puller (Sutter Instrument, Sutter Model P-97)
- Use
- Pipette Puller (Sutter Instrument, Sutter Model P-97)
EQUIPMENT
~473 nm laser (e.g., Opto Engine LLC or IkeCool)
- Use
- ~473 nm laser (e.g., Opto Engine LLC or IkeCool)
No light-evoked activity (Step 16)
ChR2-expressing cells may be located too deep to stimulate with an optical fiber placed at the surface of the brain. In this case, a small diameter optical fiber can be lowered into the brain tissue in the same manner as an electrode. Take care to minimize bleeding, as blood can reduce laser light transmission.
- Use
- ChR2-expressing cells may be located too deep to stimulate with an optical fiber placed at the surface of the brain. In this case, a small diameter optical fiber can be lowered into the brain tissue in the same manner as an electrode. Take care to minimize bleeding, as blood can reduce laser light transmission.
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...
- Use
- 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...
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,,. The...
- Use
- 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,,. The...
Cell-type-specific expression of light-activated channels
Targeted expression of genes to specific cell types can be robustly achieved with the Cre-loxP system. Recent studies have used 'double-floxed' inverted open reading frame (DIO) viral vectors to achieve high specificity and expression levels,,,. We have recently used the viral vector AAV DIO-Ch...
- Use
- Targeted expression of genes to specific cell types can be robustly achieved with the Cre-loxP system. Recent studies have used 'double-floxed' inverted open reading frame (DIO) viral vectors to achieve high specificity and expression levels,,,. We have recently used the viral vector AAV DIO-Ch...
Stimulation and recording of ChR2-expressing neurons ● TIMING 1-4 h
▲ CRITICAL STEP Disrupting the dura or causing direct brain tissue damage with the drill will result in bleeding, obscuring the placement of electrodes and reducing laser light transmission.
- Use
- ▲ CRITICAL STEP Disrupting the dura or causing direct brain tissue damage with the drill will result in bleeding, obscuring the placement of electrodes and reducing laser light transmission.
Light stimulation parameters
Software used for acquisition, scoring, statistics, or reporting.
- Use
- 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 p...
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Equipment is listed but no product mappings are linked.
Confirm before execution
This page is backed by a publishable Replication Data Ledger package with zero critical source-verification issues.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
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.
Measurement outputs
What raw and processed outputs should exist?
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 i...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The most commonly used strategy to date for the expression of ChR2 in brain tissue is through viral transduction. Viral vectors driving ChR2 expression can be delivered directly...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Targeted expression of genes to specific cell types can be robustly achieved with the Cre-loxP system. Recent studies have used 'double-floxed' inverted open r...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Successful use of optogenetic techniques relies on sufficient expression levels of the light-activated channels, which limits the use of small promoter elements. To circumvent...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
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.
from paperScoring or quantification
Quantify the primary readouts for this experiment: 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 i...; The most commonly used strategy to date for the expression of ChR2 in brain tissue is through viral transduction. Viral vectors driving ChR2 expression can be delivered directly...; Targeted expression of genes to specific cell types can be robustly achieved with the Cre-loxP system. Recent studies have used 'double-floxed' inverted open r...; Successful use of optogenetic techniques relies on sufficient expression levels of the light-activated channels, which limits the use of small promoter elements. To circumvent....
from paperStatistical comparison
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...; Several strategies can be used to minimize optical contamination of electrophysiological recording in vivo. The primary goal is to limit metal exposure to light by limiting the...
from paperReporting output
Report representative outputs alongside summary comparisons for 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 i..., The most commonly used strategy to date for the expression of ChR2 in brain tissue is through viral transduction. Viral vectors driving ChR2 expression can be delivered directly..., Targeted expression of genes to specific cell types can be robustly achieved with the Cre-loxP system. Recent studies have used 'double-floxed' inverted open r..., Successful use of optogenetic techniques relies on sufficient expression levels of the light-activated channels, which limits the use of small promoter elements. To circumvent....
inferred from protocolStructured statistical methods
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...; Several strategies can be used to minimize optical contamination of electrophysiological recording in vivo. The primary goal is to limit metal exposure to light by limiting the...
source structuredSource and audit
What supports the facts on this page?
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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