NEUR 327  ·  Group 6  ·  2024
BiPOLES
Bidirectional Dual-Color
Optogenetic Control of Neurons
⬤  470 nm  Inhibition ⬤  595–635 nm  Excitation
Vierock, Rodriguez-Rozada et al.
Nature Communications (2021)  |  12:4527
GtACR2 anion channel
Chrimson cation channel
02Background

What Is Optogenetics?

[Click to edit — Add your content here]

  • Brief definition of optogenetics
  • Explanation of opsins (light-sensitive proteins derived from microbes)
  • How light can activate (depolarize) or inhibit (hyperpolarize) neurons
  • Why this technique is revolutionary for neuroscience research
[Key takeaway — e.g., "Optogenetics gives researchers millisecond-precision control of specific neurons using light."]
Figure

[Insert diagram: Neuron responding to light stimulation — show opsin in membrane, light source, and resulting ion flow]

03Motivation

The Problem: Why Bidirectional Control?

To prove a neuron population is necessary and sufficient, you need both excitation and inhibition.

Necessity & Sufficiency

[Click to edit — Explain why proving a neural circuit's role requires both activating and silencing the same population]

Current Approach Limitations

[Click to edit — Separate experiments, co-expression of two opsins with mismatched trafficking, variable ratios, spectral overlap]

The Core Challenge

[Click to edit — All opsins have some blue-light sensitivity, making truly independent dual-color control very difficult]

04Background

Previous Tools & Their Limitations

🔌
Gene Fusion

[Click to edit]

  • ChR2 + NpHR/Arch fused together
  • Poor membrane trafficking
  • Limited potency for reliable neuronal control
⚒
Bicistronic (eNPAC)

[Click to edit]

  • 2A ribosomal skip sequence
  • Variable expression ratios
  • No guaranteed co-localization
⚡
Ion Pumps

[Click to edit]

  • NpHR, Arch — one charge per photon
  • Weak at negative voltages
  • Don't work in invertebrates (Drosophila)
05Design

The BiPOLES Strategy: Design Logic

Inverting the color scheme — red excitation + blue inhibition in a single fusion protein.

[Click to edit — Explain the conceptual innovation]

  • Red-light cation channel (Chrimson) for excitation
  • Blue-light anion channel (GtACR2) for inhibition
  • Single open reading frame ensures 1:1 stoichiometry
  • Inverted color scheme solves blue-light cross-talk
[Key insight — The inverted spectrum means Chrimson's blue-light sensitivity is cancelled by GtACR2's inhibitory current]

Fusion Construct Architecture

GtACR2
—
TS + mCerulean + βHK
—
Chrimson
Diagram

[Insert diagram of the fusion construct showing membrane topology, linker components, and both opsin domains]

06Engineering

Systematic Screen of Opsin Combinations

[Click to edit — Describe the screening approach]

  • ACRs tested: GtACR1, GtACR2, iC++, Aurora
  • CCRs tested: Chrimson, ChRmine, bReaChES, f-Chrimson, vf-Chrimson
  • Linkers tested: L1, L2, L3, L4

Key metrics evaluated:

  • Photocurrent density
  • Reversal potential
  • Spectral separation (λrev)
[Result — GtACR2-L2-Chrimson (BiPOLES) emerged as the best: largest photocurrents, ~150 nm spectral separation, reversal potentials matching individually expressed channels]
Figure

[Insert figure from paper: Screening results showing photocurrent densities and spectral separation for all tested combinations]

07Results

HEK Cell Characterization

Biophysical validation of BiPOLES in vitro.
490 nm → Cl− outward 600 nm → cation inward

[Click to edit — Describe the HEK cell data]

  • Representative photocurrent traces at 490 nm vs. 600 nm
  • Current-voltage relationship: outward (Cl−) currents with blue, inward (cation) currents with red
  • BiPOLES vs. eNPAC2.0: 2 orders of magnitude more light-sensitive for inhibition
  • Reversal potentials close to individually expressed channels
Figure 1

[Insert simplified Figure 1 — photocurrent traces, I-V curves, comparison with eNPAC2.0]

08Optimization

somBiPOLES: Improved Membrane Trafficking

[Click to edit — Explain the optimized variant]

  • Addition of Kv2.1 soma-targeting sequence
  • Improved membrane localization: soma + proximal dendrites
  • No intracellular accumulation
  • Absent from axon terminals (critical: Cl− reversal potential in axons could make GtACR2 excitatory)
[Key point — Soma targeting avoids confounding effects from axonal chloride gradients where GtACR2 could be depolarizing instead of inhibitory]
SOMA
Kv2.1 targeted
↑ Pulsing glow = membrane localization
Confocal

[Insert confocal images showing improved membrane localization vs. non-targeted variant]

09Characterization

Neuronal Excitation with somBiPOLES

Red-light spiking performance matches Chrimson alone.
595 nm → Excitation

[Click to edit — Present the excitation data]

  • AP probability in hippocampal CA1 neurons: 100% at 0.5 mW/mm² (595 nm)
  • Comparable performance to Chrimson alone
  • Blue light never triggered APs in somBiPOLES neurons (unlike Chrimson alone, which spikes at 470 nm)
  • Light-ramp threshold data confirms similar sensitivity at orange wavelengths
Figure

[Insert figure: AP probability curves, comparison with Chrimson alone, blue-light test showing no spiking]

10Characterization

Neuronal Inhibition with somBiPOLES

Blue-light silencing matches GtACR2 alone.
490 nm → Inhibition

[Click to edit — Present the inhibition data]

  • Rheobase shift experiments in CA1 neurons
  • Blue light (490 nm) blocked spiking starting at 0.1 mW/mm²
  • Comparable to somGtACR2 alone
  • Silencing effective even at 100 mW/mm² — Chrimson cross-activation does not compromise inhibition
Figure

[Insert figure: Rheobase shift data, AP blocking at various light intensities, comparison with somGtACR2]

11Application 1

Bidirectional Control & Optical Voltage Tuning

[Click to edit — Describe bidirectional control results]

  • Red light pulses reliably trigger APs
  • Concomitant blue light blocks them
  • Optical tuning of membrane voltage by varying blue/red ratio
  • Wavelength sweep: membrane potential smoothly transitions from Cl− Nernst potential to AP threshold
[Key result — A single wavelength sweep across the spectrum can smoothly control membrane potential between inhibition and excitation]
470 nm
Inhibit
↔
635 nm
Excite
Figure

[Insert figure: Voltage tuning traces, spectral sweep data]

12Application 2

Dual-Population Control with a Second ChR

[Click to edit — Describe the dual-population experiment]

  • somBiPOLES in VIP interneurons
  • CheRiff (blue-light ChR) in pyramidal neurons
  • Red light spikes only VIP cells
  • Blue light spikes only pyramidal cells
  • Postsynaptic recordings in OLM neurons confirm distinct EPSCs and IPSCs
Figure

[Insert figure: Circuit diagram showing VIP + pyramidal cell populations, color-coded stimulation, and postsynaptic recordings in OLM neurons]

13Application 3

Two-Photon Holographic Control

First demonstration of two-photon bidirectional control in the same neuron.
920 nm → 2P Inhibition 1100 nm → 2P Excitation

[Click to edit — Describe the two-photon results]

  • 920 nm two-photon for GtACR2 inhibition
  • 1100 nm two-photon for Chrimson excitation
  • AP trains at 1100 nm shunted by co-incident 920 nm
  • Enabled by the 1:1 stoichiometry of the fusion protein
  • Single-cell resolution bidirectional control
Figure

[Insert figure: Two-photon holographic stimulation data — AP trains with and without co-incident inhibition]

14In Vivo

In Vivo: C. elegans

Cross-species utility demonstrated in invertebrates.

[Click to edit — Describe the C. elegans experiments]

  • BiPOLES expressed in cholinergic motor neurons
  • Red light → body contraction (excitation)
  • Blue light → body elongation (inhibition)
  • Previous tools (ChR2 + NpHR) could not achieve both in the same animal
[Key point — BiPOLES achieves bidirectional motor control in worms, something no prior tool could accomplish]
C. elegans — animated worm body
Figure

[Insert figure: Body length quantification — contraction (red) vs. elongation (blue)]

15In Vivo

In Vivo: Drosophila

First bidirectional optogenetic tool that works in flies.

[Click to edit — Describe the Drosophila experiments]

  • Motor neuron control: body length changes with red/blue light
  • Nociceptive circuit (Dp7 neurons):
  • Blue light reduced rolling escape response
  • Red light enhanced rolling escape response
  • Rhodopsin pumps don't work in flies — BiPOLES provides the first bidirectional tool for Drosophila
Figure

[Insert figure: Drosophila body length and nociceptive behavior data — blue vs. red light conditions]

16In Vivo

In Vivo: Mouse & Ferret

Mouse — Locus Coeruleus

[Click to edit]

  • somBiPOLES in locus coeruleus (TH-Cre)
  • Orange light triggered pupil dilation (arousal)
  • Blue light blocked or reversed dilation
Mouse Figure

[Insert figure: Pupil dilation traces — orange vs. blue light]

Ferret — Visual Cortex

[Click to edit]

  • BiPOLES in GABAergic neurons (mDlx promoter)
  • Blue light increased baseline activity (disinhibition)
  • Red light suppressed visually evoked responses
Ferret Figure

[Insert figure: Visual cortex neural activity modulation data]

17Summary

Advantages Over Previous Tools

✓ Channels, not pumps — Higher sensitivity, no disruption of ion gradients, moves multiple ions per photon
✓ Inverted color scheme — Exclusive red-light excitation, fully compatible with blue-light ChRs for dual-population experiments
✓ Fixed 1:1 stoichiometry — Single ORF ensures reliable and predictable expression ratio across all cells
✓ Soma targeting (somBiPOLES) — Avoids axonal artifacts where chloride gradients could invert GtACR2 function
✓ Cross-species utility — Validated in C. elegans, Drosophila, mice, and ferrets
✓ Single- and two-photon compatible — Works with standard widefield and advanced holographic stimulation
18Discussion

Limitations & Considerations

⚠ Chloride-dependent — Won't work where Cl− reversal potential is depolarized (immature neurons, axon terminals)
⚠ Not for presynaptic control — Soma-targeting excludes the tool from axon terminals by design
⚠ Limited spike frequency — Max reliable spiking ~10–20 Hz, not ideal for fast-spiking interneuron studies
⚠ Large construct size — May limit AAV packaging flexibility for certain viral delivery strategies

[Click to edit — Add additional critical evaluation notes here]

19Future

Future Directions & Broader Impact

[Click to edit — Discuss future applications]

  • Optotagging + silencing — Tag and silence the same neurons during electrophysiology recordings
  • Engram studies — Test necessity and sufficiency of memory engrams in a single experiment
  • Multiplexing — Combine with fluorescent sensors or optogenetic cyclases
  • Modular architecture — Swap channel components for tailored spectral or kinetic properties
  • Clinical relevance — Understanding circuit-level dysfunction in neurological disorders
Summary Graphic

[Insert graphic showing future applications branching from BiPOLES — or a timeline of expected developments]

Questions?
Thank you for your attention
⬤  GtACR2 — Blue Inhibition ⬤  Chrimson — Red Excitation

[Click to edit — Add summary graphic, key references, or group member credits]

Vierock, Rodriguez-Rozada et al. Nature Communications (2021) 12:4527

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