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Nat Methods Tian He et al. reported the optimization of far-infrared membrane potential probes based on optical hybrid screening technology

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Membrane potential imaging is one of
the rapidly developing frontier technologies in neuroscience in recent years.
Compared with the classic patch-clamp technology, fluorescent membrane potential imaging has the characteristics of simple operation, high throughput, and genetic coding, which has unique advantages
for studying in vivo electrophysiological phenomena.
Among the genetically encoded voltage indicators (GEVIs), the Archaerhodopsin3-based membrane potential probe developed by Adam Cohen's group at Harvard University has the redtest spectrum 【1】
。 In 2014, Cohen's lab proposed the concept of "all-optical electrophysiology" [2].

Cohen's lab constructed and expressed them in neurons with red-shifted membrane potential fluorescent probes and blue-shifted channelrhodopsin, and then excited the action potentials of neurons with blue light, while recording membrane potential changes
in far-infrared fluorescence channels.
In 2019, the Nature paper published in Cohen's lab first reported membrane potential imaging recordings of the CA1 region of the mouse hippocampus [3].

Compared to the widely used calcium probe GCaMPs, genetically encoded membrane potential probes have several natural "disadvantages"
.
First, membrane potential probes are necessarily based on membrane proteins, and a larger number
of probe molecules can be accommodated in the cytoplasm than on the cell membrane.
Secondly, since the duration of the action potential is in the millisecond level, and the change time of the calcium signal is in the order of 100 milliseconds, the exposure time of membrane potential imaging is much lower than that of calcium imaging
.
In other words, the photon budget for membrane potential imaging is more constrained
.
The fluorescence of the Archaerhodopsin3-based membrane potential probe comes from a retinal group inside the protein
.
Although this probe has high membrane potential sensitivity, its low quantum yield affects the number of photons that can be collected in time, which in turn limits the signal-to-noise ratio of the probe
.
In the in vivo full optical electrophysiological recording experiment, the signal-to-noise ratio of the probe can be described as "the more baht must be more" and "the more, the better"
.
Therefore, after Cohen's lab reported the membrane potential probe based on Archaerhodopsin3, the research group of Frances Arnold and Viviana Gradinaru of Caltech [4] and the Ed Boyden group of MIT [5].
Attempts have been made to improve the performance of
such membrane potential probes by using directed evolution.

The directed evolution of biological probes is an important means to improve the properties of probes, and it is also one of the
main experimental bottlenecks.
According to past experience, probes modified from membrane proteins often need to be expressed on mammalian cells to function normally
.
Even if it can be expressed in prokaryotic cells, after several rounds of directed evolution, the resulting product may not fold properly in mammalian cells—such probes are meaningless
for neurological research.
In in vivo imaging, the performance of fluorescent probes is determined
by multiple indicators such as brightness, sensitivity, and response speed.
Therefore, the screening of probe mutants must be based on dynamic optical signals and consider multiple indicators
.
At present, most of the research groups working on the directed evolution of fluorescent probes have adopted the method
of microfluorescence imaging combined with array screening.
In general, experimenters construct thousands of mutant plasmids and express them one by one in
96- or 384-well plates.
This approach requires a lot of repetitive operations, is time-consuming, labor-intensive, and inefficient
.

On January 9, 2023, Professor Adam Cohen from Harvard University and Postdoctoral Fellow Tian He published a report in Nature Methods entitled Video-based pooled screening yields improved far-red genetically encoded voltage Research papers
by indicators.
This paper reports the screening, characterization and demonstration of the latest generation of Archaerhodpsin3-based membrane potential probes and their application in living mouse
brains.

To solve the flux bottleneck of fluorescent probe screening, the authors first developed a hybrid screening system based on dynamic optical signals (Figure 1).

Compared with array filtering, the construction and presentation of hybrid screening text libraries is far simpler and cheaper
.
The system consists of two elements: 1) a fluorescence microscopy imaging system with a very large field of view that can simultaneously characterize thousands of text library cells; 2) Photopick
, a new method for optical marking under a microscope.
To screen membrane potential probes, the authors genetically modified the wild-type HEK293 cell line to obtain an electrophysiological excitatory spiking HEK cell by co-expressing the voltage-gated sodium channel Na_V1.
5 and the inwardly rectified potassium channel_Kir2.
1 [6].

The authors then used lentiviral delivery systems to stably express 1) a single membrane potential probe mutant and 2) a fluorescent protein mEOS4a that can change color under violet (405 nm) light in spiking HEK cells
.
In the optical screening process, the authors first measured the membrane potential probe signals of thousands of mutant library cells in the imaging field of view, and used a digital micromirror array device (DMD) to accurately project violet light onto cells with both brightness and responsiveness
.
After exposure to violet light, mEOS4a changes from green to red
.
The authors then recovered the red cells with flow cytometry and repeated the screening process
after expanding the cells in a dish.
In this process, favorable mutations are continuously enriched
.
Finally, the authors used Illumina sequencing to analyze the change in abundance of the point mutations to identify the point mutations
that can enhance the membrane potential probe.

Based on the point mutations obtained from the mixed screening, the authors obtained the latest generation of far-infrared membrane potential probes QuasAr6a and QuasAr6b
.
Among them, QuasAr6a has higher sensitivity to changes in membrane potential, while QuasAr6b has faster response speed
.
The authors collaborated with Q-State, a biotech startup based in Cambridge, Massachusetts (Adam Cohen is one of the founders of Q-State), to conduct a high-throughput, head-to-head comparison of the new probe QuasAr6a/b with the previous generation probe Archon1 [5] in rat hippocampal primary neurons in vitro
。 The results show that in the neuronal cells cultured in vitro, the brightness, signal-to-noise ratio and response speed of QuasAr6a/b have been improved
.

The authors further compared the performance
of QuasAr6a/b with Archon1 in mouse in vivo neuronal membrane potential imaging.
Since the action potentials of different types of neurons tend to have different characteristics (such as threshold, amplitude, half-peak width), and the difference in these electrophysiological signals affects the intensity
of optical signals.
The authors utilized the Cre tool mouse strain, Neuron Derived Neurotrophic on the surface of the cerebral cortex QuasAr6a, QuasAr6b, and Archon1 were compared in Factor (NDNF) cells, while QuasAr6b and Archon1 were compared in Parvalbumin (PV) cells in the CA1
region of the hippocampus.
The results showed that QuasAr6a expressed in NDNF cells had a higher signal-to-noise ratio
than Archon1.
In PV cells, QuasAr6b surpassed Archon1
in signal-to-noise ratio and response speed.
Since the action potential half-peak width (<0.
5 ms) of PV cells is significantly narrower than that of NDNF cells, it has more advantages
for capturing fast electrophysiological signals and faster response speed of QuasAr6b.

The improved signal-to-noise ratio of membrane potential probes means that it is easier
to record electrical signals from multiple neurons in vivo.
Using the combination of QuasAr6a/b and Optopatch, a light-sensitive channel excited by blue light, the authors developed an all-optical electrophysiological method
for detecting neuronal connections in the brains of living mice.
Neurons can be connected
by both chemical synapse and electrical synapse.
Chemical synapses require neurotransmitter transduction, while electrical synapses rely on connexins to form ion channels that directly diffuse electrical signals
.
If classical electrophysiological techniques are used to study neuronal connections in living organisms, researchers need to record two neurons simultaneously with patch-clamps, which requires considerable craftsmanship and experience, is difficult, and has a high
failure rate.
The authors demonstrated that with the new generation of Optopatch combinations, it was possible to quantitatively analyze the strength of inhibitory synaptic connections between NDNF cells and between PV cells in the hippocampus
.
Notably, the authors found that electrical synapses between PV cells tend to be
asymmetrical.
In both experiments, the authors recorded more than twenty pairs of NDNF and PV neurons, demonstrating the flux advantage
of optical recording.

In addition, in a 2022 eLife paper published in Bernardo Sabatini's lab at Harvard Medical School [7], QuasAr6a was used to track potential changes
in the distal dendrites of neurons in brain slices.

In summary, this study developed a simpler and easier high-throughput optical screening platform and used this platform to optimize
membrane potential probes.
The latest generation of membrane potential probes can be used to quantitatively analyze neuronal connections in the brains of living mice, advancing the development of
all-optical electrophysiology.

Original link:

https://doi.
org/10.
1038/s41592-022-01743-5

Platemaker: Eleven

References

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