Thread by @MichaelLinLab

Super-excited to reveal our latest fluorescent voltage indicator, ASAP5, now online @NeuroCellPress!

ASAP5 features higher responsivity and kinetics, allowing detection of single synaptic transmission events.

The link gives free access until 2024.11.09:
authors.elsevier.com/a/1joYd3BtfH9B…

To create ASAP5, amazing graduate student Alex Hao developed a biophysical model for how mutations on the voltage-sensing domain influence kinetics and shift voltage sensitivity, then rationally mutated 20 sites, finding an optimized combination of 5 mutants.

Previously the genetically encoded voltage indicators (GEVIs) with the largest response per mV were ASAP3, JEDI-1P (under 1-photon), and JEDI-2P (under 2-photon). ASAP5 has a steeper fluorescence-voltage relationship and improved kinetics over ASAP3, allowing larger responses.

A real-time movie of ASAP5 in an excitatory neuron in mouse motor cortex reveals single spikes with high clarity, individual spikes within bursts, and subthreshold deflections.

Have to use Youtube as framerate is too high for Twitter (but just 100fps):

In vivo comparisons done by awesome postdoc Sungmoo Lee confirmed improved SNR for spikes with ASAP5 vs other ASAP-family GEVIs under both 1p and 2p illumination.

Collaborators @JiannisTax @pgolshani @ZeguanWang @eboyden3 helped us extend these findings to interneurons and to zebrafish. Sparing you the stats, but they are there.

@JiannisTax @pgolshani @ZeguanWang @eboyden3 As with the recent JEDI-2P, photostability is excellent; after 50 minutes of 2p imaging, we observed only minor loss of SNR.

Using ASAP5 with random-access 2p microscopy, we detected the very short APs of fly neurons at >3kHz (thanks to @FemtonicsUSA and with Alex's coadvisor Tom Clandinin), and confirmed fast response kinetics in mice by imaging ASAP5 responses to APs at >7kHz (with @DieudonneLab)

Collaborators Laura Gomez, Na Ji, and @DanFeldmanPhD used ASAP5 to show that whisker-induced activation of neurons in the barrel cortex is followed by late hyperpolarization in the same neurons, demonstrating the ability of ASAP5 to detect inhibitory inputs as well.

Next, we asked "how low can we go?" with ASAP5. Could we detect spontaneous synaptic events on a single-trial basis? Alex and Sungmoo applied the voltage-gated sodium channel blocker TTX to neurons so that only spontaneous individual synaptic vesicle release events occurred.

Patch-clamping revealed miniature excitatory postsynaptic potentials (mEPSPs), as expected.

Excitingly, ASAP5 also detected these mEPSPs well, with a 0.83% response per 1mV.

We were curious how ASAP5 compared to Voltron2, a chemigenetic voltage indicator. Voltron2 is a non-conducting channelrhodopsin mutant fused to a Halotag domain that can be conjugated with various JaneliaFluor dyes, which are much brighter per molecule than a GFP fluorophore.

Voltron2's response relies on voltage-dependent protonation of the retinal, which is tuned over a very wide voltage range, creating a shallower response per mV. We tested Voltron2 with JF525, the dye yieldingthe steepest response. Responses were about 40% lower than ASAP5.

Since Voltron2+JF525 is brighter, it is actually similar to ASAP5 in SNR for mEPSP detection. (SNR is related linearly to relative response and to the square root of baseline brightness.) But with ASAP5, one does not need to apply a dye, so it's probably more convenient.

If we can detect spontaneous mEPSPs by ASAP5 in the cell body, then can we see them at their origin at the synapse, then follow their propagation to the cell body?

Yes, we can! And their amplitudes decay monoexponentially.

Jeff Magee, @nspruston, and others have observed that distal synapses elicit larger EPSPs than proximal synapses. Previous work used dendritic patching of neurons in brain slices, so we asked if we can see the same thing using ASAP5 and in the absence of tissue organization.

Indeed we found that the further away an EPSP originates, the larger its amplitude. Thus neuron-intrinsic mechanisms are enough to generate the scaling effect. Interestingly, it is not sufficient to make up for distance; distally generated EPSPs are still smaller at the cell body

Finally, we worked with @silvianatale92, Tom Sudhof, and @WernigLab to test ASAP5 in human neurons differentiated from ES cells

ASAP5 revealed mEPSPs in human neurons with high fidelity. Indeed ASAP5 seems to be expressed to higher levels than rat neurons, and SNR appears better

ASAP5 also revealed activity in networks of human neurons. Here, Sungmoo observed how 8 neurons exhibited synchronized EPSPs and spikes (no TTX here), and of course some asynchronous events too. Thus with ASAP5 one can assess excitability across multiple individual units.

To demonstrate the V and time resolution of ASAP5, here's a movie of two human neurons with ASAP5, with one neuron patch-clamped for ground truth (trace 1).

ASAP5 (trace 2) mirrors the ephys trace, and picks up coordinated nearby activity too (trace 3).
youtube.com/watch/LEFXmGpG…

Physiological phenotypes of neurogenetic diseases, such as altered mEPSP or mIPSP frequencies, have previously required patch-clamping to characterize.

We believe ASAP5 can now be used for high-throughput electrical phenotyping of human neurons with disease-associated mutations.

Tagging Laura Gomez @golauragomez. Thanks, Laura!

And the in vivo work was a close collaborative effort with Jun Ding's lab, with training and guidance from postdoc Richard Roth. Richard performed the non-AOD 2p imaging including the 1h recording. Thanks, Jun and Richard!

@golauragomez And finally a big thanks to @StanfordBrain and the BRAIN Initiative at @NIH, @NIH_NINDS, and @NIMHgov for their steadfast support for technologies to advance our understanding of the nervous system.

@golauragomez @StanfordBrain @NIH @NIH_NINDS @NIMHgov BTW from now on, odd-numbered ASAPs are negative, even-numbered ASAPs are positive.

Negative: ASAP3, ASAP5 (today)
Positive: ASAP4, ASAP6 (biorxiv)

And "-Kv" denotes somatic targeting by Kv2.1 PRC following the convention of PMID 30007418 (2018)
pubmed.ncbi.nlm.nih.gov/30007418/#&gid…

Packaged AAVs available now:
AAV9-EF1a-DIO-ASAP5-WPRE: Stanford GVVC AAV-301
With -Kv tag: AAV-302
Link:

Plasmids available soon:
pAAV.hSyn-ASAP5-Kv.WPRE: Addgene 225707
pAAV.hSyn-ASAP5.WPRE: Addgene 225709
pAAV.EF1a-DIO-ASAP5-Kv.WPRE: Addgene 225708neuroscience.stanford.edu/shared-resourc…

Alex presented the latest on ASAP5 and ASAP6 at SFN yesterday. Was great to see all the new enthusiasm for voltage imaging!

The final print version of our paper on ASAP5, "A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events", is now out in Neuron

cell.com/neuron/fulltex…

Thanks to @NIH_NINDS, @NIMHgov, the rest of the Brain Initiative, and the Stanford Wu Tsai Neuroscience Institute for their long-term vision in supporting technology development. I discuss this in the spiffy accompanying article

@threadreaderapp unroll so I can post a link from a place with 450nm atmosphere