Table 1. Comparison table of optogenetic tools suitable for fast in vivo use in mammals and other animals.
| Opsin | Host organism | Wavelength sensitivity | Mode of control | Modulatory capabilities | Experimental systems tested |
|---|---|---|---|---|---|
| ChR2, ChR2(H134R), ChR1/2 chimeras, fast (ChETA) mutants | Chlamydomonas reinhardtii | 470 nm (maximum activation) | Depolarizing | Rapid on/off, best used for precise activation of neurons on the millisecond timescale. Can be used to evoke single spikes or defined trains of action potentials over a range of frequencies. The H134R mutation yields larger photocurrents relative to wild-type ChR2, but with slower koff kinetics. ChR1/ChR2 chimera mutants78, 79 reduce inactivation and ChETA opsins allow for spiking up to 200 Hz while also improving overall performance80 | In vitro: dissociated neuron culture1, 2, 4, acute mouse and rat brain sections2, 13, 18, 33, 62, 63, 64, 78, 79, 293HEK cells20 In vivo: C. elegans (requires supplementation of all-trans-retinal, ATR)23, D. melanogaster (requires supplementation of ATR)65, 66, 67, 77, zebrafish68, chicken69, mouse8, 9, 11, 12, 14, 15, 32, 49, rat49, primate70 |
| Step function opsin (SFO) genes: ChR2 (C128A), ChR2 (C128S), ChR2 (C128T) | Chlamydomonas reinhardtii | 470 nm (switching on) 546 nm (switching off for C128A and C128S mutants) |
Depolarizing | Point-mutants of ChR2 with slow or optically switchable deactivation. C128A and C128S mutants show the most prolonged activation and the highest light sensitivity, while C128T retains more temporal precision of activation SFOs can be switched on and off with blue and green light pulses, respectively | In vitro: dissociated neuron culture25 |
| VChR1 | Volvox carteri | 535 nm (maximum activation) 589 nm (completely separable activation from ChR2) |
Depolarizing | Red-shifted action spectrum relative to ChR2. Similar to ChR2, VChR1 can be used to drive reliable action potential firing over a range of frequencies. With 589 nm light, VChR1 can be activated independently of ChR2 | In vitro: dissociated neuron culture3 |
| NpHR, eNpHR | Natronomonas pharaonis | 589 nm (maximum activation) | Hyperpolarizing | Light-activated chloride pump. Can be used to hyperpolarize neurons with high temporal precision; capable of inhibiting single action potentials within high frequency spike trains (up to 30 Hz). Also can be used to mediate sustained inhibition of neurons over many minutes | In vitro: dissociated neuron culture4, 21, 22, 73 and mouse brain slice4, 22 In vivo: C. elegans (requires supplementation of ATR)4 and mouse21, 22 |
| opto-α1AR | Synthetic | 500 nm (maximum activation) | Biochemical | Light-activated GPCR, via the Gq pathway | In vitro: 293HEK cell line5 In vivo: mouse5 |
| opto-β2AR | Synthetic | 500 nm (maximum activation) | Biochemical | Light-activated GPCR, via the Gs pathway | In vitro: 293HEK cell line5In vivo: mouse5 |
Table 2. List of cell type-specific expression strategies proven compatible with optogenetics in mammalian tissues.
| Method | Cell-types targeted | Timing | Pros | Cons | Comments |
|---|---|---|---|---|---|
| Transgenic technology | In principle, any cell type identifiable via a molecular marker. A transgenic construct can be made by fusing the opsin gene to the promoter region of the cell type-specific marker | 6 months to 1 year for obtaining a stable transgenic (mouse or rat) line; marmoset transgenics also now possible | Consistent spatial distribution of targeted cells within each animal in a given transgenic line Higher specificity can be obtained by using bacterial artificial chromosomes (BACs) to introduce a large fragment of the promoter sequence Relatively uniform levels of opsin gene expression across targeted cells in the targeted population |
Endogenous promoters tend to drive low levels of transgene expression, often leading to insufficient levels of opsin gene expression to mediate robust optical control Generation of a transgenic line is slow and labor intensive Transgenic technology is more challenging in non-murine species |
Two transgenic mouse lines expressing a microbial opsin gene have been described thus far: Thy1 ChR2–EYFP32, 33 and Thy1NpHR–EYFP22 |
| Lentivirus | Cell type-specific promoters: CaMKIIα49, excitatory glutamatergic neurons SynapsinI4, neuron specific GFAP9, astrocytes ppHcrt8, hypocretin neurons |
2 weeks for construction and production of the recombinant viral vector, 2 weeks after injection to achieve a high level of opsin gene expression | Short testing cycle enables rapid screening of recombinant promoters for targeting specific cell types Can be pseudotyped with rabies glycoprotein to gain retrograde-transduction properties Can achieve high levels of opsin gene expression by increasing the copy number of transgene Expression persists for years |
Limited packaging capacity (< 10 kb total length between long terminal repeats, LTRs) prevents the use of large promoter fragments, therefore compromising specificity Precision of stereotactic injection is limited Variable levels of opsin gene expression across transduced cells Small volume of transduction Some cell type-specific promoters have weak expression levels |
Lentiviral vectors have been successfully applied in a range of mammalian hosts, ranging from mice and rats to birds and monkeys. When pseudotyped with VSVg, lentiviral vectors can effectively transduce all mammalian neural tissues |
| Adeno-associated virus (AAV) | Cell type-specific promoters: SynapsinI71, neuron specific ppSST56, SST neurons ppMCH72, melanin concentrating hormone neurons |
2 weeks for construction and production of the recombinant viral vector, 3 weeks after injection to achieve a high level of opsin gene expression | Short testing cycle enables rapid screen of recombinant promoters for targeting specific cell types Retrograde-transduction properties possible in certain serotypes Can achieve high levels of opsin gene expression by increasing the copy number of transgene Expression persists for years Low immunogenicity |
Limited packaging capacity (<5 kb total length between LTRs) prevents the use of large promoter fragments, therefore compromising specificity Precision of stereotactic injection is limited Variable levels of opsin gene expression across transduced cells Moderate volume of transduction Some cell type-specific promoters have weak expression levels |
Different AAV serotypes have slightly different tropism and transduction efficiency. For neural tissue, AAV1, 2, 5, 8, and 9 have been shown to transduce neurons in the brain, but with varying distribution and efficiency. The cell type-specific tropisms of each serotype remain to be fully explored and compared |
| Cre-dependent AAV expression system | Cell type specificity is determined by the choice of Cre transgenic lines. | 3 weeks for construction and production of the viral vector, 3 weeks after injection to achieve expression | As with conventional AAV vectors High level of cell type specificity when used with Cre drivers Overcomes the low transcriptional strength of some cell type-specific promoters by amplifying opsin gene expression via a Cre-dependent strong promoter74 Leak in absence of Cre prevented by use of the doubly floxed inverted opsin (DIO) system34, 10, 11, 36, 74 |
As with the AAV vector system | |
| Herpes simplex virus 1 (HSV-1) | Cells can be labeled based on their projection targets | ~2 months for the construction of the expression vector and production of recombinant viral vectors 1 to 2 weeks to achieve sufficient level of opsin gene expression | Strong and rapid expression Robust retrograde transporting property75 Can target specific cell populations based on projection target Can be applied in all mammalian models Larger (~150 kb) packaging capacity than lentivirus and AAV |
More difficult to produce than lentivirus More stringent biosafety precautions than AAV |
Commercial HSV vectors may be obtained from NeurovexOther retrograde possibilities include pseudorabies virus and rabies virus vectors |
| Selective control of light-sensitive neural afferents | Axonal projections coming from a brain region of interest can be targeted via focal injection of virus into the axon tract origin. Light-sensitive axon processes coming from the site of injection can be activated at the target region. | For virus-mediated expression, wait at least 4 weeks after injection to allow accumulation in the axonal membrane in the target region | Can used to study specific neural projections Not limited to transgenic mice |
Must wait >4 weeks after viral injection Cannot discriminate among fibers with different local cellular targets, which will require additional emerging targeting technologies |
Table 3. Troubleshooting table.
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| 22 | Incomplete anesthesia: persistent response to toe pinch | High tolerance or clearance | Administer additional 10% volume ketamine/xylazine (or corresponding isoflurane increment) |
| 31 | Poor cannula positioning observed during histological validation | Stereotactic coordinates based on brain atlas are not accurate for the experimental animal age or strain | Tune the co-ordinates for age and species |
| Excess bleeding after craniotomy | Vessel trauma | Place cotton swab over craniotomy site. When hemostasis is achieved, irrigate craniotomy site with sterile saline | |
| 33 | Excess bleeding after cannula placement | Vessel trauma | Place cotton swab over craniotomy site. When hemostasis is achieved, irrigate the site with sterile saline |
| 37 | No solution can be injected | Needles may clog after advancement through intact tissue | Replace the needle |
| 46 | No electrical signal is recorded | Sensitive electrode tip has been damaged by contact | After confirming integrity of electrical connections, replace electrode |
| Electrical noise observed during recording | Ground loop present in the setup | Check all converging grounds to the table and the table ground to the amplifier. Confirm that the ground wire is in contact with the tissue and provide ~30–50 μl saline to the ground wire to ensure electrical connectivity | |
| Optical stimulation does not modulate physiology | Opsin-expressing cells may be distant from recording electrode | Calculate relevant scattering parameters (accounting for light wavelength, intensity and source geometry) in the context of local neuroanatomy. Optrode may need to be redesigned accordingly or optrode positioning may need to be revised by complete retraction and replacement | |
| 56 | Fiber breakage during behavior | Rotational behavior results in fiber torsion | Employ fiberoptic commutator to relieve torsion; some light loss may result |
From the following article
Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures