Optogenetics Protocol Designer

Optogenetics Protocol Designer

Purpose

Optogenetic protocol design requires domain expertise that a general-purpose programmer would systematically get wrong. Selecting an opsin is not like selecting a software library — it requires understanding photocycle kinetics, ion selectivity, spectral overlap, expression toxicity, and the biophysics of light propagation through neural tissue. A naive approach risks tissue damage from heating, silencing neurons you intended to activate (depolarization block), or producing uninterpretable results from inadequate controls. This skill encodes the decision logic that bridges the gap between "I want to activate neurons" and a rigorous, publishable optogenetic protocol.

When to Use

• Designing a new optogenetic experiment from scratch
• Selecting an opsin for a specific excitation/inhibition application
• Determining light delivery parameters (power, wavelength, pulse protocol)
• Planning fiber optic implant specifications
• Designing proper control conditions for optogenetic experiments
• Troubleshooting failed or ambiguous optogenetic manipulations

Research Planning Protocol

Before executing the domain-specific steps below, you MUST:

1. State the research question — What neural circuit question is this optogenetic manipulation addressing?
2. Justify the method choice — Why optogenetics (not chemogenetics, lesion, pharmacology)? What alternatives were considered?
3. Declare expected outcomes — What behavioral/neural changes do you expect from activation/inhibition?
4. Note assumptions and limitations — What does this approach assume about the circuit? Where could it mislead?
5. Present the plan to the user and WAIT for confirmation before proceeding.

For detailed methodology guidance, see the research-literacy skill.

⚠️ Verification Notice

This skill was generated by AI from academic literature. All parameters, thresholds, and citations require independent verification before use in research. If you find errors, please open an issue.

Decision Tree: Research Question to Protocol

Step 1: Define the Manipulation Type

Goal	Category	Key Constraint
Drive action potentials with millisecond precision	Excitation (fast)	Need opsin with tau-off < 15 ms
Sustained depolarization / increased excitability	Excitation (tonic)	Step-function opsin or low-frequency pulsed
Silence neurons during a behavioral epoch	Inhibition (sustained)	Need potent inhibitory opsin, manage heating
Brief synaptic suppression	Inhibition (phasic)	Fast inhibitory opsin, short pulses
Bidirectional control in same animal	Dual manipulation	Spectrally separated opsins required

Step 2: Select Opsin Class

Excitatory Opsins (Cation Channels)

Opsin	Peak lambda	Tau-off	Photocurrent	Best For	Key Citation
ChR2 (H134R)	470 nm	~10 ms	Moderate	Standard activation, well-characterized	Boyden et al., 2005; Nagel et al., 2005
ChETA (E123T)	470 nm	~3 ms	Lower	High-frequency spiking (>40 Hz)	Gunaydin et al., 2010
ChrimsonR	630 nm	~15 ms	Moderate	Red-shifted, deep tissue, dual-color	Klapoetke et al., 2014
ChRmine	520-530 nm	~60 ms	Very high	Ultra-sensitive, large volume activation	Marshel et al., 2019
CheRiff	460 nm	~8 ms	~2x ChR2	All-optical electrophysiology	Hochbaum et al., 2014
C1V1(TT)	540 nm	~50 ms	Moderate	Red-shifted, combinatorial experiments	Yizhar et al., 2011

Inhibitory Opsins

Opsin	Peak lambda	Mechanism	Photocurrent	Best For	Key Citation
eNpHR3.0	590 nm	Cl- pump	Low (pump)	Established inhibition, yellow-light	Gradinaru et al., 2010
eArch3.0	520-550 nm	H+ pump	Moderate (pump)	Green-light inhibition	Chow et al., 2010; Mattis et al., 2012
stGtACR2	480 nm	Anion channel	Very high	Most potent somatic inhibition	Mahn et al., 2018
SwiChR++	480 nm	Anion channel (bistable)	Moderate	Sustained inhibition, low light	Berndt et al., 2016

Step-Function Opsins (Bistable)

Opsin	Activation	Deactivation	Tau-off (dark)	Best For	Key Citation
SSFO	Blue (~470 nm)	Yellow (~590 nm)	~29 min	Sustained excitability increase	Yizhar et al., 2011
SOUL	Blue (~470 nm)	Yellow (~590 nm)	~29 min	Transcranial, minimally invasive	Gong et al., 2020
SwiChR++	Blue (~480 nm)	Red (~600 nm)	~115 s	Bistable inhibition	Berndt et al., 2016

See references/opsin-catalog.md for the complete opsin reference with detailed kinetics.

Step 3: Determine Light Parameters

Power Density at Target Tissue

• ChR2 EPD50: ~1.3 mW/mm2 (Mattis et al., 2012)
• stGtACR2 EPD50: ~0.05 mW/mm2 (Mahn et al., 2018) — 100-200x more sensitive than NpHR
• eNpHR3.0 EPD50: ~5-10 mW/mm2 (Mattis et al., 2012)
• ChRmine: effective at sub-mW/mm2 levels (Marshel et al., 2019)
• Typical working range: 1-10 mW/mm2 for most excitatory opsins at the fiber tip (Aravanis et al., 2007)

CRITICAL — Tissue Heating Threshold:

• Temperature increase of ~0.1-0.25 deg C per mW at fiber tip for 473 nm light (Stujenske et al., 2015)
• Keep total tissue temperature rise below 1 deg C to avoid artifacts (Christie et al., 2013; Owen et al., 2019)
• At 20 mW/mm2, duty cycles above ~40% risk exceeding 1 deg C (Stujenske et al., 2015)
• Blue light at high power can alter firing rates even WITHOUT opsin expression (Owen et al., 2019)

Light Attenuation in Tissue

• 90% of 473 nm light is lost within 1 mm of brain tissue (Aravanis et al., 2007; Yizhar et al., 2011)
• At 500 um from fiber tip: ~3.2% of initial intensity remains
• At 1 mm from fiber tip: ~0.56% of initial intensity remains
• Red-shifted light (>600 nm) penetrates deeper due to lower scattering (Klapoetke et al., 2014)

Wavelength Selection

Match the laser/LED wavelength to the opsin's absorption peak:

Opsin Class	Recommended Wavelength	Common Laser Lines
ChR2 / CheRiff / stGtACR2	450-490 nm	473 nm
C1V1 / ChRmine / eArch3.0	520-560 nm	532 nm, 561 nm
eNpHR3.0	570-600 nm	594 nm
ChrimsonR	600-650 nm	638 nm

See references/stimulation-parameters.md for complete pulse protocol recipes.

Step 4: Design Pulse Protocol

General Principles

• Pulse width: 1-10 ms for fast excitatory opsins; 5-25 ms for slower or inhibitory opsins (Mattis et al., 2012)
• Frequency: Must not exceed the opsin's temporal fidelity limit
• Duty cycle: Balance activation efficacy against heating; keep below 40% for sustained protocols at moderate power (Stujenske et al., 2015)
• Ramp-down for inhibition: When ending sustained inhibitory light, ramp down over 500 ms to 1 s to avoid rebound excitation (Mahn et al., 2016)

Frequency Limits by Opsin

Opsin	Max Reliable Spike Rate	Notes
ChR2 (H134R)	~30-40 Hz sustained	Fails above gamma range in sustained trains (Mattis et al., 2012)
ChETA	~100-200 Hz	Reduced photocurrent trade-off (Gunaydin et al., 2010)
ChrimsonR	~20-30 Hz	Slower kinetics than ChR2 (Klapoetke et al., 2014)
ChRmine	~50 Hz (80 Hz with hsChRmine)	Large photocurrent compensates for slower kinetics (Marshel et al., 2019)
Chronos	~100 Hz	Fastest known excitatory opsin (Klapoetke et al., 2014)

Step 5: Fiber Optic Specifications

Parameter	Standard Value	Rationale
Core diameter	200 um (mice), 200-400 um (rats/primates)	Balances illumination volume vs. tissue damage (Aravanis et al., 2007)
Numerical aperture (NA)	0.22 or 0.39	0.22 for focused beam; 0.39 for wider illumination
Fiber type	Multimode step-index	Standard for optogenetics (Sparta et al., 2012)
Ferrule diameter	1.25 mm (standard) or 2.5 mm	Compatibility with patch cables and commutators

Placement rule: Position the fiber tip 200-500 um above the target region to allow light cone to cover the structure while avoiding mechanical damage to the target itself (Yizhar et al., 2011).

Step 6: Control Conditions

A rigorous optogenetic experiment requires AT MINIMUM three of the following controls (Fenno et al., 2011):

Control	What It Rules Out	Implementation
Opsin-negative + light	Heating, visual, auditory artifacts from light	Inject control virus (e.g., AAV-hSyn-eYFP), deliver identical light
Opsin-positive + no light	Effects of viral expression alone	Implant fiber, run behavioral protocol without laser
Wavelength control	Non-specific photic effects	Deliver light at a wavelength outside the opsin's activation spectrum
Fiber implant only	Mechanical damage effects	Implant fiber without virus injection
Within-subject light-off epochs	Temporal confounds	Interleave light-on and light-off trials within sessions

The single most common critique of optogenetic studies is inadequate controls. The opsin-negative + light control is non-negotiable (Fenno et al., 2011).

Common Pitfalls and Domain-Specific Warnings

1. Depolarization Block (Silencing When You Intend to Activate)

At high ChR2 expression levels or with prolonged/high-frequency stimulation, excessive cation influx causes sustained depolarization that inactivates sodium channels, STOPPING action potentials (Herman et al., 2014; Lin et al., 2009). This is especially dangerous with interneurons, which enter depolarization block more readily than pyramidal cells.

Signs: Loss of spiking after initial pulses in a train; behavioral effect opposite to prediction. Prevention: Limit pulse width to 1-5 ms; keep frequency at or below 40 Hz for ChR2; titrate expression levels; use ChETA for high-frequency applications.

2. Tissue Heating Artifacts

Continuous illumination at high power heats tissue, altering neuronal firing even without opsin expression (Owen et al., 2019; Christie et al., 2013). Blue light (473 nm) is worse than red (638 nm) for heating.

Prevention: Use pulsed (not continuous) light; keep duty cycle below 40% at moderate power; use temperature modeling (Stujenske et al., 2015); always include opsin-negative light controls.

3. Viral Expression Toxicity

High viral titers (>1e13 vg/mL) can cause cytotoxicity, especially with prolonged expression times (>8 weeks) (Miyashita et al., 2013). Overexpression of membrane proteins disrupts normal cell physiology.

Prevention: Use titers of 1e12 to 5e12 vg/mL for standard applications; check for cell health at the injection site post-mortem; limit expression time to 3-6 weeks for most applications.

4. Backpropagation of Light Along Fibers

Light can scatter back up the fiber and illuminate unintended brain regions above the target. This is especially problematic for superficial targets near the brain surface.

Prevention: Use opaque ferrule sleeves; verify illumination volume with computational modeling; consider tapered fibers for focal illumination.

5. Antidromic Activation with Axonal Opsins

When inhibitory opsins (especially GtACR2, not soma-targeted) are expressed in axons, blue light can cause depolarization at the axon initial segment, producing paradoxical excitation (Mahn et al., 2018).

Prevention: Use soma-targeted variants (stGtACR2) for inhibition; avoid illuminating axon terminals with anion channelrhodopsins; verify with electrophysiology.

6. Chloride Loading with Halorhodopsin

Prolonged eNpHR3.0 activation loads neurons with chloride, shifting the GABA-A reversal potential and causing rebound excitation upon light offset (Raimondo et al., 2012).

Prevention: Limit continuous NpHR activation to <15 seconds; use pulsed protocols for longer inhibition; consider anion channels (stGtACR2) for sustained inhibition.

Viral Vector Quick Reference

Serotype	Tropism	Onset	Spread	Use Case
AAV1	Broad neuronal	1-2 weeks	Large	General transduction (Aschauer et al., 2013)
AAV2	Neuronal (restricted)	2-4 weeks	Small	Precise local targeting
AAV5	Neurons + glia	2-4 weeks	Moderate	Use with neuron-specific promoter
AAV8	Broad neuronal	1-2 weeks	Large	Deep brain structures
AAV9	Broad, crosses BBB	1-2 weeks	Large	Systemic delivery, broad transduction
AAVrg	Retrograde neuronal	2-4 weeks	Projection-specific	Circuit-specific targeting (Tervo et al., 2016)

Always use a neuron-specific promoter (hSyn, CaMKII) with AAV1/5/8/9, as ubiquitous promoters (CMV, CAG) will also transduce glia (Aschauer et al., 2013).

Standard injection volume: 200-500 nL per site in mice; 1-2 uL per site in rats (Cetin et al., 2006). Standard titer: 1e12 to 5e12 vg/mL (Miyashita et al., 2013). Wait for expression: Minimum 2-3 weeks post-injection; optimal at 3-6 weeks for most AAVs.

Protocol Assembly Checklist

Before finalizing a protocol, verify:

• Opsin matches the manipulation type (excitation/inhibition/bistable)
• Wavelength matches the opsin's absorption spectrum (+/- 20 nm)
• Power density is within the opsin's effective range but below heating threshold
• Pulse frequency does not exceed the opsin's temporal fidelity
• Duty cycle is below 40% for sustained protocols at moderate-to-high power
• Fiber is positioned 200-500 um above target
• At least opsin-negative + light control is planned
• Viral titer is in the 1e12-5e12 vg/mL range
• Expression time is 3-6 weeks (not <2 weeks, not >8 weeks without toxicity check)
• Post-hoc histology is planned to verify expression and fiber placement

Key References

• Aravanis, A. M. et al. (2007). An optical neural interface: in vivo control of rodent motor cortex. J. Neural Eng., 4(3), S143-S156.
• Boyden, E. S. et al. (2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci., 8(9), 1263-1268.
• Chow, B. Y. et al. (2010). High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature, 463, 98-102.
• Deisseroth, K. (2015). Optogenetics: 10 years of microbial opsins in neuroscience. Nat. Neurosci., 18(9), 1213-1225.
• Fenno, L., Yizhar, O. & Deisseroth, K. (2011). The development and application of optogenetics. Annu. Rev. Neurosci., 34, 389-412.
• Gunaydin, L. A. et al. (2010). Ultrafast optogenetic control. Nat. Neurosci., 13(3), 387-392.
• Hochbaum, D. R. et al. (2014). All-optical electrophysiology in mammalian neurons. Nat. Methods, 11, 825-833.
• Klapoetke, N. C. et al. (2014). Independent optical excitation of distinct neural populations. Nat. Methods, 11, 338-346.
• Mahn, M. et al. (2018). High-efficiency optogenetic silencing with soma-targeted anion-conducting channelrhodopsins. Nat. Commun., 9, 4125.
• Marshel, J. H. et al. (2019). Cortical layer-specific critical dynamics triggering perception. Science, 365(6453), eaaw5202.
• Mattis, J. et al. (2012). Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nat. Methods, 9, 159-172.
• Stujenske, J. M. et al. (2015). Modeling the spatiotemporal dynamics of light and heat propagation for in vivo optogenetics. Cell Rep., 12(3), 525-534.
• Yizhar, O. et al. (2011). Optogenetics in neural systems. Neuron, 71(1), 9-34.