Voltage indicator polypeptides and methods of use for recording electrical activity of neurons

Abstract

Provided are nucleic acids encoding voltage indicator polypeptides. In some instances, the voltage indicator polypeptide comprises a voltage-sensing domain (VSD) comprising four transmembrane segments, and a circularly permuted fluorescent protein inserted into an extracellular loop between the third transmembrane segment (S3) and the fourth transmembrane segment (S4) of the VSD. The voltage indicator polypeptide may comprise one or more amino acid substitutions and / or deletions, non-limiting examples of which include one or more substitutions at I412, F413 and / or Q414, wherein numbering of positions is according to SEQ ID NO: 1. Cells comprising the nucleic acids are also provided. In some embodiments, the cells are neurons that express the voltage indicator polypeptide on the plasma membrane of the neuron in a pan-membrane or targeted manner. Methods of recording the electrical activity of neurons are also provided, as are methods of assessing an effect of an agent on the electrical activity of neurons.

Description

[0001] Atty. Docket: STAN-2221 WO (S24-333)

[0002] VOLTAGE INDICATOR POLYPEPTIDES AND METHODS OF USE FOR RECORDING ELECTRICAL ACTIVITY OF NEURONS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 696,775, filed September 19, 2024, which application is incorporated herein by reference in its entirety.

[0005] STATEMENT OF GOVERNMENT SUPPORT

[0006] This invention was made with Government support under contracts MH136462 and NS123681 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0007] INTRODUCTION

[0008] Neurons process and transmit information by responding to and generating a rich repertoire of transmembrane voltage dynamics. These membrane potential changes are diverse in amplitude and timescale, differ by cell type, and propagate across sub-cellular compartments. For instance, action potentials (APs) are -100 mV in amplitude and persist for less than 5 ms1. In contrast, the effect of a unitary single synaptic transmission event, which in mammalian excitatory neurons usually entails the release of a single synaptic vesicle, is usually less than 5 mV in amplitude when measured at the cell body but can persist over tens of milliseconds2.

[0009] In addition to their importance in information processing in the healthy brain, unitary events are also a sensitive sign of synaptic dysfunction in disease3 4. When released spontaneously in the absence of action potentials, unitary synaptic events are termed miniature excitatory postsynaptic potentials (mEPSPs). Measurement of the frequency of mEPSPs, or the corresponding miniature excitatory postsynaptic currents (mEPSCs), can reveal defects in pre- or postsynaptic function in cultured neurons, including neurons derived from human stem cells5-8. However, mEPSP or mEPSC measurements have so far only been possible with patch-clamp electrophysiology, which is inherently low-throughput in nature, creating a rate-limiting step in characterizing synaptic effects of disease mutations or in screening for corrective drugs. Thus, recording neuronal populations non-invasively with millivolt resolution and millisecond temporal resolution has been a longstanding goal. Atty. Docket: STAN-2221 WO (S24-333)

[0010] SUMMARY

[0011] Provided are nucleic acids encoding voltage indicator polypeptides. In some instances, the voltage indicator polypeptide comprises a voltage-sensing domain (VSD) comprising four transmembrane segments, and a circularly permuted fluorescent protein inserted into an extracellular loop between the third transmembrane segment (S3) and the fourth transmembrane segment (S4) of the VSD. The voltage indicator polypeptide may comprise one or more amino acid substitutions and / or deletions, non-limiting examples of which include one or more substitutions at 1412, F413 and / or Q414, wherein numbering of positions is according to SEQ ID NO: 1. Cells comprising the nucleic acids are also provided. In some embodiments, the cells are neurons that express the voltage indicator polypeptide on the plasma membrane of the neuron in a pan-membrane or targeted manner. Methods of recording the electrical activity of neurons are also provided, as are methods of assessing an effect of an agent on the electrical activity of neurons.

[0012] BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1A-1G: Protein engineering and biophysical properties of ASAP5. (A) A structural model of ASAP3 based on homology modeling. The engineered sites are highlighted by sticks representation. (B) 3 rounds of screening and the engineering pathway from ASAP3 to ASAP5. (C) Scatter plot for normalized kinetics index and normalized responsivity of variants in the first round of screening. ASAP5 is added for comparison. Colored lines represent the product of the two parameters, ranging from 0.2 to 2.0 with equal distance.(D) Steady-state fluorescencevoltage (F-V) function normalized to -70 mV (left), and the normalized slope of this function (right). Error bars, standard deviation (SD) of 5, 5 HEK293A cells for ASAP3 and ASAP5, respectively.

[0014] (E) Example traces of action potentials induced by current injection in cultured hippocampal neurons expressing ASAP3 (left) or ASAP5 (right), recorded by electrode and camera (800 fps).

[0015] (F) Left: averaged voltage and fluorescence waveforms of current-induced spikes in cultured rat hippocampal neurons at room temperature. N = 10 cells, shaded area: ± 1 SEM. Right: Statistical comparison of the peak amplitudes of the voltage and optical spikes. Optical recording was at 800 fps. Welch's t test was used for the four comparisons. (G) Relative brightness of GEVI to fused mCyRFP3 in cultured hippocampal neurons. N = 21 and 27 neurons for ASAP3 and ASAP5, respectively. Welch’s t test was used for the comparison.

[0016] FIG. 2A-2D: Steepening and accelerating ASAP3. (A) Responsivity and kinetics index of ASAP variants from screening. Top, normalized fluorescence of individual HEK293-Kir cells aligned to the onset of electrophoresis (gray) and the averaged trace (black). Responsivity, i.e. Atty. Docket: STAN-2221 WO (S24-333) the dynamic range, is the response after it reached the steady state. Bottom, the kinetics index was defined by the percentage of steady-state response reached at round 6.7 ms. Left panels are the results of ASAP3 and right panels are the results of ASAP3 412V 413V 414R, the top performer from the first round of screening. Images were recorded at 300 Hz. (B) Performance of variants in screening rounds 1 and 2 normalized to each parent. The top two performers are highlighted. Response to AP is computed as the product of the responsivity and the kinetics index. (C) Fluorescence-voltage curves of GEVIs, normalized to resting membrane potential (left) and Sigmoidal-curve-fitted maximum (middle). Physiological voltage range is indicated by the shadow. Error bars: SD of 4~5 HEK293 cells. Right: statistical comparison of the responsivity around -70 mV. Bonferroni's multiple comparisons test was used for the comparison. (D) Response of GEVIs to voltage-clamp-commanded action potentials with 2 ms FWHM (up panel) and 4 ms FWHM (down panel). Shaded area: SEM from 4~6 HEK293 cells. Bonferroni's multiple comparisons test was used for the comparison.

[0017] FIG. 3A-3D: Comparative performance of ASAP5 in vivo under 1 -photon illumination. (A) Left, schematic drawing of a head-fixed mouse running on a wheel during in vivo voltage imaging. Right, Representative post-hoc histology images of sparsely labelled CaMKIIch neurons expressing soma-targeted versions of ASAP3, JEDI-2P, JEDI-1 P, and ASAP5. The sparse expression was achieved by injecting mixed AAVs of double-floxed GEVI and diluted CaMKIIa-Cre. The brain slices were imaged under a confocal microscope. (B) Left, representative baseline-corrected 2-min 500 fps GEVI recordings. Middle, single-AP examples. Right, average waveform of all detected APs for each GEVI. Error bars, SEM. N = 17 cells from 6 mice for ASAP3- Kv, 16 cells from 4 mice for JEDI-1 P-Kv, 11 cells for 5 mice for JEDI-2P-Kv, and 15 cells from 4 mice for ASAP5-Kv. (C) Statistical analyses for AF / F0amplitude, cellular brightness averaged from the first second, SNR for detected optical spikes, and detected spike numbers (mean ± SEM). Outlined dots correspond with the displayed traces in (B). (D) An ASAP5-Kv expressing cortical neuron showing spikes time-locked to the running periods. The pseudo-colored bar below the fluorescence trace shows the running speed. Red ticks in (B) and (D) indicate spikes detected by VolPy.

[0018] FIG. 4A-4C: Single-trial imaging of graded and action potentials in fruit flies. (A) Comparison of ASAP5 and JEDI-2P in Mil neurons. Left: 2-photon voltage imaging of Mil neurites in the M10 layer of the medulla in awake flies. Middle: Stimulus-triggered average of fluorescence trace, peak response, SNR at peak, and time of detected peak response, in response to a 24-ms contrast increment or decrement (gray box). Right: photostability and baseline brightness of ASAP5 and JEDI-2P. Shadow represents SEM. N = 59 ASAP5 neurons Atty. Docket: STAN-2221 WO (S24-333) from 5 flies and 60 JEDI-2P neurons from 5 flies. Each dot represents one neuron. Welch’s t test was used for all the comparisons. (B) Left: Example single trials of JEDI-2P or ASAP5-expressing Mil neurons responding to visual stimuli. Red ticks denote the onset of a 24 ms contrast increment; magenta ticks denote the onset of a 24 ms contrast decrement. Traces were binned to 70 fps. Right: Stimulus-triggered average of the example traces. (C) Left: The scanned image of the fly brain with two ASAP5-expressing TPN-II neurons and a template fly brain showing R60H12-Gal4 expression pattern. Middle: example traces of two ASAP5-expressing TPN-II neurons firing spikes recorded using a 3D AOD random-access scope (Femtonics) at 3288 fps. Gaussian filter (sigma=1.5 frame) was applied to the trace. Red ticks denote the peaks of each spike. Right: Averaged waveform of all spikes in each cell, as well as the spike-triggered average of the response of the other neuron. Dots represent the measurement points.

[0019] FIG. 5A-5C: Single-trial imaging of graded potentials in L2 and action potentials in TPN-II with different imaging modalities. (A) Left: example of the terminals of L2 neuron. Middle: Averaged fluorescence traces of L2 neurons expressing JEDI-2P or ASAP5. Shade: SEM. Number of cells and flies: 56 (ASAP5, 4 flies), 55 (JEDI-2P, 4 flies). Right: statistical comparison of the peak responses for both contrast decrement (depolarization) and increment (hyperpolarization). Welch’s t test was used. (B) Example trace of an ASAP5-expressing TPN-II neuron spiking spontaneously, recorded using a resonant galvanometric scanner (Broker) at 779 fps. From left to right: expression pattern of R60H12-GAL4 in virtual adult fly brain, the scanned ROI, Gaussian-filter smoothed trace (sigma=1 frame), and averaged waveform of spikes. Red ticks denote spike peaks. Dots represent the measurement points. (C) Example trace of an ASAP5-expressing TPN-II neuron spiking spontaneously, recorded using a random-access scanner (Karthala) at 3333 fps. From left to right: expression pattern of R60H12-GAL4 in virtual adult fly brain, the scanned ROI, Gaussian-filter smoothed trace (sigma=1 .5 frame), and averaged waveform of spikes. Ticks above the traces denote spike peaks. Dots represent the measurement points.

[0020] FIG. 6: Comparative performance of ASAP5 in vivo under 2-photon illumination. Comparison of ASAP5-Kv and JEDI-2P-Kv 2-photon performance. Left: representative layer-2 motor cortex neurons expressing JEDI-2P-Kv or ASAP5-Kv. Middle: Corresponding baseline- corrected fluorescence traces, with shaded intervals enlarged at the right. Right: Comparison of SNR and brightness corrected for post-objective power (mW) used for each neuron, spike amplitudes in AF / F0, and depth of each neuron from dura (mean ± SEM). N = 12 JEDI-2P-Kv cells from 3 mice, and 7 ASAP5-Kv cells from 4 mice. Outlined dots correspond with the displayed traces. Atty. Docket: STAN-2221 WO (S24-333)

[0021] FIG. 7A-7C: Sustained and multi-unit 2-photon voltage imaging of ASAP5-Kv in mice. (A) Photostability comparison of ASAP5-Kv and JEDI-2P-Kv in mouse cortex in vivo. Left, normalized raw traces from the 2-min recordings shown in Figure 6A (N = 7 for ASAP5-Kv, and N = 12 for JEDI-2P-Kv). Right, normalized raw traces from long-term recordings (N = 3 for each indicator). Shaded area, SEM. (B) One-hour recording of ASAP5-Kv in mouse cortex in vivo. Left, image of the neuron. Bright spots are processes perpendicular to the image plane. Top, baseline- corrected trace extracted using masks generated by VolPy. Red ticks indicate spikes. Bottom, zoomed-in views of the trace, indicated by the thin red box in the upper trace. The raw movie was recorded at 500 fps and binned to 250 fps for visualization. The mouse was awake, head-fixed, and behaving on a running wheel. The neuron was 100-pm deep from dura and imaged with 37- mW post-objective power tuned to 925 nm. (C) Three-dimensional ASAP5-Kv imaging of population dynamics of mouse cortical neurons during running. Left, coordinates of the imaged neurons. Middle, images of the 9 sampled neurons. Right, baseline-corrected traces with running speed indicated above. Segments highlighted by magenta windows were enlarged to the right.

[0022] FIG. 8A-8F: Optical detection of mEPSPs in cultured rat hippocampal neurons. (A) Left: a neuron expressing ASAP5-Kv. Middle: simultaneous voltage and optical recording of the neuron. The voltage recording was downsampled to match the optical recording. Red circles denote mEPSPs detected in the voltage recording. Green circles denote mEPSPs detected in the optical recording. Right: The amplitude of mEPSP voltage signals and the corresponding optical signals from the example neuron. Black line is the linear fitting of the data. (B) A neuron expressing Voltron2525-Kv and the recording of mEPSPs. Panels are the same with (A). (C) Above, SNR of the peak amplitude of mEPSPs. Below, responsivity of the indicators for reporting mEPSPs. Each dot is a neuron. Welch’s t test was used for statistical comparison of the mean. (D) Above, mean electrical waveforms (left) and optical waveforms (right) of detected mEPSPs of the two sensors. Each optical waveform was normalized to its peak value for comparing kinetics. Below, time-lagged cross-correlograms of simultaneous voltage and optical recordings. Shaded area: SEM. Number of neurons: 6 (ASAP5-Kv) and 7 (Voltron2525-Kv). Welch’s t test was used to compare the mean of the peak cross-correlations. (E) Fidelity of optical detection of mEPSPs using deconvolution-based method. Top: Averaged false positive (FP) rate as a function of mEPSP amplitude for all events from all neurons. Bottom: Averaged false negative (FN) rate as a function of mEPSP amplitude for all events from all neurons. Shaded area: SEM. Number of neurons and events detected per neuron: 6, 89 ± 31 (ASAP5-Kv; mean ± SD) and 7, 61 ± 29 (Voltron2525-Kv; mean ± SD). (F) Fidelity of optical detection of mEPSPs using miniML trained on paired electrophysiological and optical recordings. Top: Averaged FP rate as a function of mEPSP Atty. Docket: STAN-2221 WO (S24-333) amplitude for all events from all neurons. Bottom: Averaged FN rate as a function of mEPSP amplitude for all events from all neurons. Shaded area: SEM. Number of neurons and events detected per neuron: 6, 144 ± 97 (ASAP5-Kv; mean ± SD) and 7, 97 ± 99 (Voltron2525-Kv; mean ± SD).

[0023] FIG. 9A-9F: Optical recording and deconvolution-based detection of mEPSPs in cultured rat neurons. (A) Construction of the deconvolution divisor. Left: individual mEPSPs from voltage recordings with highest amplitudes aligned by the peaks (colored traces) and the averaged template (black trace). Right: The repolarizing phase of the averaged template normalized to its peak, as the deconvolution divisor. (B) Deconvolution-based detection of mEPSPs of an ASAP5-Kv expressing neuron. Top: Example voltage recording and the deconvolved trace. Bottom: Optical recording and the deconvolved trace. Red dashed line indicates the threshold of z-score=2.5. Red and green circles denote the location of detected mEPSPs in the voltage trace and the fluorescence trace, respectively. (C) Deconvolution-based detection of mEPSPs of an Voltron2525-Kv expressing neuron. Layout is the same as (B). (D) Brightness comparison of ASAP5-Kv (N = 6) and Voltron2525-Kv (N = 7) mEPSP recordings (mean ± SEM). Unpaired t-test was used for statistical comparison. (E) Photostability of the two sensor systems during mEPSP recordings under conditions that produce the same SNR. The normalized fluorescence traces were averaged from 6 (ASAP5-Kv) and 7 (Voltron2525-Kv) neurons. Excitation Bleaching rate was adjusted to match SNR (See Methods). Shared area: SEM. (F) Fidelity of optical detection of mEPSPs using miniML trained on the electrophysiological recordings and applied to both modalities. Top: Averaged FP rate as a function of mEPSP amplitude for all events from all neurons. Bottom: Averaged FN rate as a function of mEPSP amplitude for all events from all neurons. Shaded area: SEM. Number of neurons and events detected per neuron: 6, 144 ± 97 (ASAP5-Kv; mean ± SD) and 7, 97 ± 99 (Voltron2525-Kv; mean ± SD).

[0024] FIG. 10A-10E: Voltage imaging of dendrosomatic propagation of mEPSPs. (A) ASAP5 reveals EPSPs originating from distal dendrites in a rat hippocampal neuron (DIV 26). Top, whole-cell current clamp recording of spontaneous activity. The red boxes indicate two time periods shown below. Bottom-left, a time-period where the dendritic ROIs 2, 3, and 4 (from proximal to distal) show subthreshold events with decreasing AF / F0amplitudes as it propagates from distal to the soma (marked by a black arrow). Note that these dendritic EPSPs are not present in ROIs 5, 6, and 7 which are on another dendrite. Bottom-right, another time-period where ROIs 5, 6, and 7, but not ROIs 2, 3 and 4, are showing a dendritic EPSP with larger amplitudes in distal part than in near-soma ROIs. (B) An example neuron and an observation of the spatial propagation of mEPSP. Top: An example neuron expressing ASAP5. The blue solid Atty. Docket: STAN-2221 WO (S24-333) disk marked the initiation site of the propagation, and the blue circles marked the pixels with coincident optical signals. The yellow line marked the path along with the distance to soma was calculated. Middle: The optical traces of the propagation event at the initiation site and the soma. Black arrows indicate the time of the peak signals of the propagation. Bottom: The amplitudes of the optical signals in the propagation event as a function of the distances between the pixels and the soma, do marked the distance of the initiation site to the soma. Only the signals from pixels closer to the soma than the initiation site were included. The red dashed line is the exponential function that fits the data. (C) Top: amplitude of signals at the initiation sites correlates with the distance between the initiation site and the soma. Middle: amplitude of signals at the soma correlates with the distance between the initiation site and the soma. Bottom: dendrosomatic attenuation ratio correlates with the distance between the initiation site and the soma. In the three plots, each dot is a propagation event, and each color is a neuron. Two example neurons are shown here, and the black lines are the linear fit of the data. (D) Top: The Pearson correlation coefficients of signal amplitude at the initiation sites and the distance between initiation site and soma. Middle: The Pearson correlation coefficients of signal amplitude at the soma and the distance between initiation site and soma. Bottom: The Pearson correlation coefficients of dendrosomatic attenuation ratio and the distance between the initiation site and the soma. In the three plots, each dot is a neuron. Number of neurons: 21 . (E) Histogram of the length constant L fit from the exponential function in (C) for all propagation events from all neurons. Welch’s t test was used to compare the data and the shuffled control in D, E and F. Number of neurons and propagation events: 21 , 309.

[0025] FIG. 11A-11C: Pharmacological conditions change the optical signals. (A) A cultured cortical neuron showed optical mEPSPs at the presence of TTX and PTX. With additional CNQX, the optical signals corresponding to millivolt-scale transients vanished. After washing out CNQX for 10 min, the optical signals reappeared. (B) Two rat cortical neurons expressing nonsoma targeted ASAP5 also show excitatory and inhibitory postsynaptic potentials as well as APs. Top, the two neurons show time-correlated activity. Red boxes indicate the time periods shown in the insets below. Inset-left, this panel shows a time-period where both neurons show subthreshold activity. Note the pronounced hyperpolarizing activity in cell 2 (magenta). Insetmiddle, coincident firing of APs from both neurons. Inset-right, the 3rdred-box shows an AP from cell 1 (blue) only. Images were acquired at 400 fps at 25 DIV. (C) ASAP5-Kv expressed in rat hippocampal neurons reveal coincident inputs to the two neurons. Suprathreshold events in cell 1 (blue) are often coincident with subthreshold events in cell 2 (magenta). Inset-left, the 1stred- box shows uncorrelated subthreshold activities from the two neurons. Inset-middle, this time Atty. Docket: STAN-2221 WO (S24-333) window shows an AP from cell 1 but subthreshold activity from the other. Inset-right, this panel shows coincident APs from both neurons. Images were acquired at 400 fps at 29 DIV.

[0026] FIG. 12A-12C: Characterization of electrical activity in induced human neurons by ASAP5-Kv. (A) Two human iNs expressing ASAP5-Kv under the Ubiquitin-C (UbC) promoter imaged at 40 DIV. Celli was patched in whole-cell mode for simultaneous recording of its spontaneous activity in electrophysiology (black) and in ASAP5-Kv fluorescence (dark blue). Cell2 was only imaged for its fluorescence change (blue). The asterisks indicate three exemplary states of the two cells and their zoomed-in traces are shown on the lower panel. Frame rate was 400 fps. (B) Eight ASAP5-Kv expressing human iNs network (hSyn promoter; 41 DIV) imaged at 100 fps revealing their supra- and sub-threshold activity in cellular resolution. (C) Left, a human iN expressing virally introduced ASAP5-Kv (hSyn promoter; 80 DIV). Middle, simultaneous recording of spontaneous activity in electrophysiology (black) and ASAP5-Kv fluorescence (blue) with TTX (1 pM) and PTX (50 pM) in the bath solution to block AP-generated synaptic activity and inhibitory transmission. The red circles indicate miniature events detected by using a template search algorithm. Right, averages of electrical (black) and fluorescent signal (blue) for 42 mEPSPs recorded from the same neuron during 1 min.

[0027] FIG. 13A-13B: Recording conditions for miniature events in human iNs. (A) The effect of different sampling rates on detection of APs and subthreshold potentials in ASAP5-Kv. Top, whole-cell current clamp recording of a 40-DIV human iN expressing ASAP5-Kv. Middle, 400 fps unfiltered ASAP5-Kv trace. Bottom, 100 fps timebinned ASAP5-Kv trace. Inset, enlarged traces showing similar subthreshold activity waveforms. (B) Validation of mEPSPs by whole-cell patch clamp recording of an untransduced human iN. i) voltage-clamp recording with TTX (1 pM) and PTX (50 pM) in the bath solution showing mEPSCs. ii) current clamp recording with the same condition to measure mEPSPs. iii) CNQX was added to the bath solution to remove GluA- mediated miniature signal, iv) The neuron recovered both APs and EPSPs after 25 min of washing with normal extracellular solution without TTX, PTX, and CNQX.

[0028] FIG. 14: Fluorescence excitation spectra demonstrating that ASAP7y and ASAP8y are 1030nm-excitable in two-photon microscopy at resting membrane potential.

[0029] FIG. 15: Fluorescence excitation spectra demonstrating that ASAP7y and ASAP8y are 1030nm-excitable in two-photon microscopy at depolarized potential.

[0030] FIG. 16: Plot illustrating that the fluorescence of ASAP5, ASAP7y, and ASAP8y is modulated by transmembrane voltage in mammalian cells to a larger extent than ASAP3.

[0031] FIG. 17: Plot illustrating the signal readout of ASAP7y as a function of membrane voltage and that ASAP7y has a steeper modulation (higher sensitivity) to voltage than ASAP5. Atty. Docket: STAN-2221 WO (S24-333)

[0032] FIG. 18: Plot illustrating the signal readout of ASAP8y as a function of membrane voltage and that ASAP8y becomes brighter when the voltage increases. In contrast, ASAP5 and ASAP7y become dimmer.

[0033] FIG. 19: Plots showing the signal readout of ASAP molecules when expressed in primary (rat) neuron culture. As shown, ASAP7y and ASAP8y exhibit higher signals than ASAP5.

[0034] DETAILED DESCRIPTION

[0035] Before the nucleic acids, voltage indicator polypeptides and methods of the present disclosure are described in greater detail, it is to be understood that the nucleic acids, voltage indicator polypeptides and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the nucleic acids, voltage indicator polypeptides and methods will be limited only by the appended claims.

[0036] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the nucleic acids, voltage indicator polypeptides and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the nucleic acids, voltage indicator polypeptides and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the nucleic acids, voltage indicator polypeptides and methods.

[0037] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the nucleic acids, voltage indicator polypeptides and methods belong. Although any nucleic acids, voltage indicator polypeptides and methods similar or equivalent to those described herein can also be used in the Atty. Docket: STAN-2221 WO (S24-333) practice or testing of the nucleic acids, voltage indicator polypeptides and methods, representative illustrative nucleic acids, voltage indicator polypeptides and methods are now described.

[0039] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present nucleic acids, voltage indicator polypeptides and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0040] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0041] It is appreciated that certain features of the nucleic acids, voltage indicator polypeptides and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the nucleic acids, voltage indicator polypeptides and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present nucleic acids, voltage indicator polypeptides and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0042] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Atty. Docket: STAN-2221 WO (S24-333)

[0043] NUCLEIC ACIDS AND VOLTAGE INDICATOR POLYPEPTIDES

[0044] Aspects of the present disclosure include nucleic acids that encode voltage indicator polypeptides. In certain embodiments, the voltage indicator polypeptide comprises a voltagesensing domain (VSD) comprising four transmembrane segments, a circularly permuted fluorescent protein inserted into an extracellular loop between the third transmembrane segment (S3) and the fourth transmembrane segment (S4) of the VSD. According to some embodiments, the voltage indicator polypeptide comprises an amino acid substitution at position 1412, F413, Q414, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1 . In some instances, the voltage indicator polypeptide comprises an amino acid substitution at position D145, R149, or both.

[0045] Voltage indicator polypeptides (also referred to as genetically encoded voltage indicators (GEVIs)) have improved dramatically in the last decade, such that single-trial imaging of spikes and subthreshold events is now routine in the mouse brain9-14. However reliable single-trial detection of electrical events has not yet been achieved for mEPSPs. The voltage indicator polypeptides of the present disclosure (a non-limiting embodiment of which is sometimes referred to herein as ASAP5) exhibit faster and steeper fluorescence responses to changes in membrane potential than their predecessors ASAP3, JEDI-1 P, and JEDI-2P. As demonstrated herein, the voltage indicator polypeptides of the present disclosure allow for single-trial 2-photon detection of graded and subthreshold events in flies and mice, and outperformed ASAP3, JEDI-1 P, and JEDI- 2P in response amplitude and signal-to-noise ratio (SNR) in action potential (AP) detection under 1 -photon or 2-photon illumination. In cultured rodent neurons, the voltage indicator polypeptides detected single-mV mEPSPs at the cell body with higher responsivity and similar accuracy when compared to Voltron2, a leading opsin-based sensor that requires loading of an exogenous chemical dye15. Taking advantage of the superior responsivity of the voltage indicator polypeptides of the present invention for detecting subthreshold changes in membrane potential, the dendrosomatic propagation of mEPSPs were measurable in cultured rodent neurons. These studies revealed that mEPSP amplitude at the synapse scales with distance from the soma, and that these distal signals are attenuated more strongly than proximal signals, results that extend previous measurements of dendritic properties using electrophysiological approaches. Thus, the voltage indicator polypeptides of the present disclosure improve AP and subthreshold detection in a variety of illumination regimes in vivo and expands the scope of voltage imaging to detect quantal synaptic events in culture, including in human stem cell-derived neurons. Details regarding embodiments of the nucleic acids and voltage indicator polypeptides of the present disclosure will now be provided. Atty. Docket: STAN-2221 WO (S24-333)

[0046] The present disclosure provides nucleic acids encoding voltage indicator polypeptides. The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length composed of nucleotides, e.g., deoxyribonucleotides, and may be produced enzymatically or synthetically. The term “nucleotide” is intended to include those moieties that contain not only the naturally occurring purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like. The terms “polypeptide”, “peptide”, or “protein” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acids may include the 20 “standard” genetically encodable amino acids, amino acid analogs, or a combination thereof.

[0047] In certain embodiments, a nucleic acid of the present disclosure encodes a voltage indicator polypeptide comprising a voltage-sensing domain (VSD) comprising four transmembrane segments, and a circularly permuted fluorescent protein inserted into an extracellular loop between the third transmembrane segment (S3) and the fourth transmembrane segment (S4) of the VSD. In some instances, the circularly permuted fluorescent protein is inserted at a position 4, 5, or 6 amino acids downstream of the C-terminal end of the S3.

[0048] The VSD of the voltage indicator polypeptide may be derived from a voltage sensitive phosphatase or a voltage-gated ion channel or transporter. In certain embodiments, the fluorescent protein voltage sensor comprises a VSD from a mammal, bird, fish, amphibian, reptile, or insect. Exemplary fluorescent protein voltage sensors comprising voltage sensitive phosphatase VSDs from Gallus gallus, Xenopus laevis, Danio rerio, Alligator mississippiensis, and Metaseiulus occidentalis.

[0049] The voltage indicator polypeptide can be constructed with any circularly permuted fluorescent protein, wherein the fluorescence intensity of the fluorescent protein voltage sensor is voltage dependent. The choice of a particular circularly permuted fluorescent protein for use in a fluorescent protein voltage sensor may depend on the desired emission spectrum for detection, and includes, but is not limited to, circularly permuted fluorescent proteins with green, blue, cyan, yellow, orange, red, or far-red emissions. Exemplary circularly permuted fluorescent proteins that can be used in the practice of the invention include circularly permuted green fluorescent protein (cpGFP), circularly permuted superfolder GFP (cpsfGFP), circularly permuted mApple Atty. Docket: STAN-2221 WO (S24-333)

[0050] (cpmApple), circularly permuted mCherry (cpmCherry), circularly permuted mKate (cpmKate), circularly permuted enhanced green fluorescent protein (cpEGFP), circularly permuted Venus (cpVenus), circularly permuted Citrine (cpCitrine), and circularly permuted enhanced yellow fluorescent protein (cpEYFP).

[0051] In certain embodiments, the voltage indicator polypeptide is a variant of a predecessor voltage indicator polypeptide. Non-limiting examples of such predecessors include ASAP3 (the amino acid sequence of which is set forth in SEQ ID NO: 1 ), JEDI-1 P, and JEDI-2P. For example, in certain embodiments, a nucleic acid of the present disclosure encodes a voltage indicator polypeptide comprising an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, or 98% or greater amino acid sequence identity to SEQ ID NO: 1. In some instances, such a voltage indicator polypeptide comprises an amino acid substitution at position 1412, F413, Q414, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1 . Non-limiting examples of such substitutions include 1412V, F413I, Q414R, or any combination thereof. Non-limiting examples of such substitutions also include 1412V, F413V, Q414R, or any combination thereof. According to some embodiments, the voltage indicator polypeptide comprises an amino acid substitution at each of 1412, F413, and Q414. In certain embodiments, the voltage indicator polypeptide comprises an amino acid substitution at position D145, R149, or both (e.g., alternatively or in addition to a substitution at 1412, F413, and / or Q414). Non-limiting examples of such substitutions include D145A, R149K, or both. Non-limiting examples of such substitutions also include D145C, R149K, or both. According to some embodiments, the voltage indicator polypeptide comprises an amino acid substitution at each of D145 and R149. In certain embodiments, the voltage indicator polypeptide comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% amino acid sequence identity to SEQ ID NO: 3 or SEQ ID NO: 5.

[0052] In certain embodiments, the voltage indicator polypeptide is a variant of ASAP5 (SEQ ID NO: 3) or ASAP5-Kv (SEQ ID NO: 5), wherein relative to the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 5, the voltage indicator polypeptide comprises 146L(insert), T 148A, T207Y, or any combination thereof. In certain embodiments, such a voltage indicator polypeptide comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91 % or greater, 92% or greater, 93% or greater, 94% or greater, or 95% or greater amino acid sequence identity to SEQ ID NO: 3 or SEQ ID NO: 5. Atty. Docket: STAN-2221 WO (S24-333)

[0053] In some instances, the voltage indicator polypeptide is a variant of ASAP5 (SEQ ID NO: 3) or ASAP5-Kv (SEQ ID NO: 5), wherein relative to the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 5, the voltage indicator polypeptide comprises one or any combination (e.g., each) of N138A, 146L(insert), G147A, T148A, G151 S, D152E, R172I, T207Y, F227D, and T392I. In certain embodiments, such a voltage indicator polypeptide comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, or 95% or greater amino acid sequence identity to SEQ ID NO: 3 or SEQ ID NO: 5.

[0054] According to some embodiments, the voltage indicator polypeptide is a variant of ASAP5 (SEQ ID NO: 3) or ASAP5-Kv (SEQ ID NO: 5), wherein relative to the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 5, the voltage indicator polypeptide comprises one or any combination (e.g., each) of 146L(insert), G147E, T148A, G151 D, D152T, R172F, T207Y, F227N, and Q316A. In certain embodiments, such a voltage indicator polypeptide comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91 % or greater, 92% or greater, 93% or greater, 94% or greater, or 95% or greater amino acid sequence identity to SEQ ID NO: 3 or SEQ ID NO: 5.

[0055] In general, “identity” refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353 358, National biomedical Research Foundation, Washington, D.C., which adapts the local homology algorithm of Smith and Waterman Advances in AppL Math. 2:482 489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions. Atty. Docket: STAN-2221 WO (S24-333)

[0056] Another method of establishing percent identity in the context of the present disclosure is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity.” Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by =HIGH SCORE; Databases=non redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs are readily available.

[0057] The amino acid sequences of voltage indicator polypeptides and nucleic acids encoding the same according to embodiments of the present disclosure are provided in Table 1 .

[0058] Table 1 Atty. Docket: STAN-2221 WO (S24-333) Atty. Docket: STAN-2221 WO (S24-333) Atty. Docket: STAN-2221 WO (S24-333) Atty. Docket: STAN-2221 WO (S24-333)

[0059] Because of the knowledge of the codons corresponding to the various amino acids, availability of an amino acid sequence of a voltage indicator polypeptide of the present disclosure provides a description of all the polynucleotides capable of encoding the voltage indicator polypeptide. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the voltage indicator polypeptides and domains disclosed herein. Thus, having identified a particular amino acid sequence, those of ordinary skill in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the voltage indicator polypeptide of interest. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based upon the possible codon choices, and all such variations are to be considered specifically disclosed for any voltage indicator polypeptide disclosed herein, including the amino acid sequences set forth in Table 1 . Accordingly, the nucleotide sequences in Table 1 are only by way of example. The nucleotide sequences of the nucleic acids of the present disclosure may be codon- optimized. “Codon-optimized” refers to changes in the codons of the polynucleotide encoding a Atty. Docket: STAN-2221 WO (S24-333) polypeptide to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, a nucleic acid of the present disclosure encoding a voltage indicator polypeptide may be codon-optimized for optimal production from the host organism selected for expression, e.g., human cells, such as human neurons.

[0060] Also provided are expression constructs comprising any of the nucleic acids of the present disclosure. As used herein, an “expression construct” is a circular or linear polynucleotide (a polymer composed of naturally occurring and / or non-naturally occurring nucleotides) comprising a region that encodes a voltage indicator polypeptide of the present disclosure, operably linked to a suitable promoter, e.g., a constitutive or inducible promoter. In some embodiments, expression of the voltage indicator polypeptide is under the control of one or more exogenous (including heterologous) regulatory elements, e.g., promoter, enhancer, etc., present in the expression construct. In some embodiments, expression of the voltage indicator polypeptide may be controlled by one or more endogenous regulatory elements, e.g., promoter, enhancer, etc., at or near a genomic locus into which the expression construct is inserted.

[0061] The expression constructs (e.g., vectors) can be suitable for replication and integration in prokaryotes, eukaryotes, or both. The expression constructs may contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the voltage indicator polypeptide. The expression constructs optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0062] To obtain high levels of expression of a cloned nucleic acid it is common to construct expression constructs which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription / translation terminator, each in functional orientation to each other and to the protein-encoding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (PL), and the L-arabinose (araBAD) Atty. Docket: STAN-2221 WO (S24-333) operon. The inclusion of selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing the polypeptide are available using, for example, E. coli, Bacillus sp. and Salmonella. E. co / / systems may also be used. Transducing cells with nucleic acids (e.g., expression constructs) can involve, for example, incubating lipidic microparticles containing nucleic acids with cells or incubating viral vectors containing nucleic acids with cells within the host range of the vector.

[0063] In certain embodiments, upon delivery of an expression construct to cells, the expression constructs is episomal (e.g., extra-chromosomal), where by “episome” or “episomal” is meant a polynucleotide that replicates independently of the cell’s chromosomal DNA. A non-limiting example of an episome that may be employed is a plasmid.

[0064] According to some embodiments, upon delivery of an expression construct to cells, the expression construct integrates into the genome of the cell. In certain embodiments, the expression construct is adapted for site-specific integration into the genome. For example, an expression construct may be adapted for site-specific integration into the genome, where the sitespecific integration inactivates a target gene within the genome of the cell. Functional integration of an expression construct may be achieved through various means, including through the use of integrating vectors, including viral and non-viral vectors. In some instances, a retroviral vector, e.g., a lentiviral vector, may be employed. In some instances, a non-retroviral integrating vector may be employed. An integrating vector may be contacted with the cells in a suitable transduction medium, at a suitable concentration (or multiplicity of infection), and for a suitable time for the vector to infect the target cells, facilitating functional integration of the expression construct. Nonlimiting examples of useful viral vectors include retroviral vectors, lentiviral vectors, adenoviral (Ad) vectors, adeno-associated virus (AAV) vectors, hybrid Ad-AAV vector systems, and the like.

[0065] Also provided by the present disclosure are messenger RNAs (mRNAs) encoding any of the polypeptides of the present disclosure. In some instances, the mRNAs are encapsulated within particles, e.g., lipid nanoparticles. Such mRNAs and particles find use, e.g., for expression of an indicator polypeptide in vivo upon delivery of the mRNA to cells of a subject, e.g., an insect, a non-human animal (e.g., a rodent such as a mouse), or the like.

[0066] CELLS

[0067] Aspects of the present disclosure further include cells comprising a nucleic acid of the present disclosure, as well as cells comprising an expression construct of the present disclosure. In certain embodiments, the cells are insect (e.g., drosophila) cells, amphibian (e.g., frog, e.g., Atty. Docket: STAN-2221 WO (S24-333)

[0068] Xenopus) cells, or mammalian cells. Mammalian cells of interest include rodent cells (e.g., mouse cells), non-human primate cells, and human cells.

[0069] According to some embodiments, the cells are stem cells, e.g., mammalian (e.g., human) stem cells. Non-limiting examples of stem cells include embryonic stem (ES) cells, neural stem cells (NSCs), and induced pluripotent stem cells (iPSCs).

[0070] In certain embodiments, the cells are neurons. Neurons of the present disclosure may be induced neurons (iNs), primary neurons (e.g., neurons obtained from a healthy human subject or a human subject with a neurological disease or condition), neurons from a neuronal cell line, or the like.

[0071] Approaches for introducing the nucleic acid or expression construct into cells of interest are known and may include contacting a population of cells with the nucleic acid or expression construct under conditions in which the nucleic acid or expression construct is delivered to cells of the population of cells. The contacting step may comprise contacting the population of cells with the nucleic acid or expression construct, e.g., by combining the cells and the nucleic acid or expression construct in a single mixture under conditions suitable for delivery (e.g., transfection, transduction, etc.) of the nucleic acid or expression construct into cells of the population of cells.

[0072] A variety of suitable approaches and conditions for the delivery of nucleic acids and expression constructs to cells are known. According to some embodiments, delivery is carried out by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, DEAE-dextran-mediated transfer, and / or the like. In certain embodiments, the nucleic acid or expression construct is introduced into cells of the population of cells by AAV transduction. The AAV vector may comprise ITRs from AAV2, and a serotype from any one of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV 10. According to some embodiments, the AAV vector comprises ITRs from AAV2 and a serotype from AAV6. In some instances, the nucleic acid or expression construct is introduced into the cells (e.g., human ES cells, human iPSCs, human neurons, or the like) by lentiviral or retroviral transduction. The lentiviral vector backbone may be derived from HIV-1 , HIV-2, visna-maedi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (Fl V) , bovine immune deficiency virus (Bl V), or simian immunodeficiency virus (SIV). The lentiviral vector may be integration competent or an integrase deficient lentiviral vector (TDLV). In some embodiments, IDLV vectors including an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) are employed. Atty. Docket: STAN-2221 WO (S24-333)

[0073] Example lentiviral-mediated approaches for transducing cells with nucleic acids and expression constructs for pan-membrane or localized expression of a voltage indicator polypeptide are described in detail in the Experimental section below.

[0074] The nucleic acid or expression construct of interest can also be delivered without a viral vector. For example, the nucleic acid or expression construct can be packaged as DNA or RNA in liposomes for delivery to cells. Lipid encapsulation is generally accomplished using liposomes which can stably bind or entrap and retain nucleic acid.

[0075] Liposomal preparations for delivery of the nucleic acid or expression construct may include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA and mRNA in functional form.

[0076] Cationic liposomes are readily available. For example, N[1 -2,3-dioleyloxy)propyl]-N,N,N- triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413- 7416). Other commercially available lipids include (DDAB / DOPE) and DOTAP / DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., PCT Publication No. WO 1990 / 011092 for a description of the synthesis of DOTAP (1 ,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

[0077] Similarly, anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0078] The liposomes may comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs)

[0079] The nucleic acid or expression construct of interest may also be encapsulated, adsorbed to, or associated with, particulate carriers. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. Atty. Docket: STAN-2221 WO (S24-333)

[0080] Moreover, other particulate systems and polymers can be used for the in vivo or ex vivo delivery of the nucleic acid or expression construct of interest. For example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest. Similarly, DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, may be employed to deliver a nucleic acid or expression construct to a cell of interest.

[0081] Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten, are useful for delivering the nucleic acids or expression constructs of the present disclosure. The particles may be coated with the nucleic acids or expression constructs to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a “gene gun.” For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371 ,015; and 5,478,744. Also, needle-less injection systems can be used (Davis, H. L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

[0082] In certain embodiments, a cell of the present disclosure expresses the polypeptide. For example, the present disclosure provides neurons that express the voltage indicator polypeptide on the plasma membrane of the neuron. In some instances, such neurons of the present disclosure express the voltage indicator polypeptide in a pan-membrane fashion - that is, not localized to the plasma membrane of a particular neuronal compartment. In other embodiments, neurons of the present disclosure may exhibit localized (or “targeted”) expression of the voltage indicator polypeptide. For example, expression of the voltage indicator polypeptide may be localized to the plasma membrane of a neuronal compartment such as the cell body (soma), dendrites, or axons of the neuron. Localization tags for achieving localized neuronal expression are known. For example, when localized expression on the cell body is desired, the voltage indicator polypeptide may be fused to a Kv tag, e.g., Kv2.1 tag. A non-limiting example of a Kv tag amino acid sequence is set forth in SEQ ID NO: 7.

[0083] Aspects of the present disclosure further include methods comprising or consisting of differentiating (or “inducing”) a stem cell of the present disclosure (e.g., a human stem cell comprising a nucleic acid or expression construct of the present disclosure) into a neuron. Protocols that find use in practicing such methods are known and include those for differentiating iPSCs into neurons (as described, e.g., in Bell et al. (2019) Bio Protoc. 9(5):e3188; and Ju et al. (2021 ) Neural Regen Res. 16(4):653-658) and for differentiating ES cells into neurons (as Atty. Docket: STAN-2221 WO (S24-333) described, e.g., in the Experimental section below as well as Zhang et al. (2013) Neuron 78(5):785-798), the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0084] METHODS

[0085] Aspects of the present disclosure further include methods of recording electrical activity of a neuron. In some embodiments, such methods comprise irradiating a neuron of the present disclosure (e.g., a neuron that expresses a voltage indicator polypeptide of the present disclosure on the plasma membrane of the neuron) with light at an excitation wavelength of the voltage indicator polypeptide, and recording fluorescence emission from the voltage indicator polypeptide over time, wherein the fluorescence emission is correlated with the electrical activity of the neuron. Such methods may be performed in vitro, ex vivo, or in vivo (e.g., in insects, non-human animals (e.g., rodents or non-human primates), or the like).

[0086] The methods find use in recording a variety of types of electrical activity. Non-limiting examples of electrical activity that may be recorded according to the methods of the present disclosure include excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs), or both. EPSPs and IPSPs are voltage events induced by excitatory or inhibitory synaptic activity, respectively. EPSPs and IPSPs occur to induce or inhibit action potentials in response to excitatory or inhibitory neurotransmitter release, but they can be detected in isolation by blocking action potential generation, e.g., using a voltage-gated sodium channel blocker, a non-limiting example of which is tetrodotoxin (TTX). The EPSP and I PSP frequency produces a measure of relative function of excitatory vs. inhibitory synapse function, which is commonly affected in neurological disease. In some instances, the electrical activity recorded by the methods of the present disclosure comprises spontaneous EPSPs and IPSPs.

[0087] According to some embodiments, the methods comprise recording electrical activity in response to a stimulus. Non-limiting examples of stimuli include an electrical current, a drug, a ligand for a receptor, a ligand for an ion channel, a ligand for an ion transporter, a hormone, a second messenger, photostimulation of a photogated channel, or any combination thereof.

[0088] In some instances, the neuron comprises a mutation associated with neuronal dysfunction, non-limiting examples of which include mutations underlying neurological disorders in humans (e.g., neuropsychiatric diseases such as autism, schizophrenia, and neurodegenerative disorders), mutations that result in the neuron resembling / mimicking a neuron characteristic of a neurological disorder in humans. In other embodiments, the neurons are primary neurons transfected / transduced with a nucleic acid or expression construct of the present Atty. Docket: STAN-2221 WO (S24-333) disclosure, where the primary neurons were obtained from one or more subjects having a neurological disorder. Neurological disorders of interest include, but are not limited to, acute spinal cord injury, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), ataxia, autism, Bell’s Palsy, epilepsy, Guillain-Barre syndrome (GBS), migraine headaches, multiple sclerosis, muscular dystrophy, neurodegenerative disorders, Parkinson's Disease, and schizophrenia, among others.

[0089] Non-limiting examples of equipment (e.g., excitation sources, lenses, filters, cameras), settings and conditions for imaging and recording electrical activity of neurons expressing voltage indicator polypeptides according to embodiments of the present disclosure are described in detail in the Experimental section below

[0090] Aspects of the present disclosure further include methods of assessing an effect of an agent on the electrical activity of a neuron. In certain embodiments, such methods comprise contacting a neuron of the present disclosure (e.g., a neuron that expresses a voltage indicator polypeptide of the present disclosure on the plasma membrane of the neuron) with the agent, irradiating the contacted neuron with light at an excitation wavelength of the voltage indicator polypeptide, and recording fluorescence emission from the voltage indicator polypeptide over time, wherein the fluorescence emission is correlated with the electrical activity of the neuron. Such methods further comprise assessing an effect of an agent on the electrical activity of a neuron.

[0091] In some embodiments, assessing an effect of the agent on the electrical activity of the neuron comprises comparing the electrical activity of the neuron contacted with the agent to the electrical activity of the neuron in the absence of the agent. Alternatively, or additionally, assessing an effect of the agent on the electrical activity of the neuron may comprise comparing the electrical activity of the neuron contacted with the agent to the electrical activity of a control neuron not contacted with the agent.

[0092] The agent may be assessed for its ability to affect / alter a variety of types of electrical activity. In certain embodiments, the electrical activity comprises one or more of: the frequency of action potentials, the frequency of EPSPs and / or IPSPs, the frequency of EPSPs and / or IPSPs when action potential generation is blocked, the extent of EPSP and / or IPSP decay during propagation along dendrites, baseline voltage, and synchrony of electrical activity with one or more surrounding neurons.

[0093] The neuron may be any neuron of interest including any of the neurons described elsewhere herein, e.g., neurons comprising a mutation of interest, primary neurons from a human Atty. Docket: STAN-2221 WO (S24-333) subject having a neurological disorder, and the like. As will be appreciated with the benefit of the present disclosure, the methods of assessing an effect of an agent on the electrical activity of a neuron find use, inter alia, in identifying therapeutic agents or candidate therapeutic agents for the treatment of neurological disorders characterized by abnormal electrical activity of neurons. For example, an agent that restores (partially or completely) normal electrical activity of a neuron that models abnormal electrical activity associated with a neurological disorder identifies the agent as a therapeutic agent or candidate therapeutic agent for that neurological disorder.

[0094] In some embodiments, provided are methods of identifying chemical compounds that alter electrical activities recorded according to the methods of the present disclosure, including frequency of action potentials, frequency of excitatory and / or inhibitory postsynaptic potentials (EPSPs and / or IPSPs), synchrony of these events between neurons, the extent of EPSP or IPSP decay during propagation along dendrites, and / or baseline voltage. In some embodiments, provided are methods of identifying chemical compounds that alter frequency of excitatory and inhibitory postsynaptic potentials (EPSPs or IPSPs) or the extent of EPSP or IPSP decay during propagation along dendrites, in which the electrical activity comprises or consists of spontaneous excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) while action potential generation is pharmacologically blocked. In some embodiments, provided are methods of identifying chemical compounds that alter electrical events in stem cell-derived neurons by performing quantitation of electrical events, including frequency of action potentials, frequency of excitatory and inhibitory postsynaptic potentials (EPSPs or IPSPs), or synchrony of these events between neurons, or the extent of EPSP or IPSP decay during propagation along dendrites, or baseline voltage, in electrical activity recordings obtained from neurons that have been treated with individual compounds from a compound collection, compared to neurons that have not been treated. In some embodiments, provided are methods of identifying chemical compounds that alter frequency of excitatory and inhibitory postsynaptic potentials (EPSPs or IPSPs) or the extent of EPSP or IPSP decay during propagation along dendrites, in electrical activity recordings obtained while action potential generation is pharmacologically blocked from neurons that have been treated with individual compounds from a compound collection, compared to neurons that have not been treated.

[0095] As will be appreciated with the benefit of the present disclosure, the neurons and methods of the present disclosure find use in screening libraries of agents for candidate therapeutic agents for neurological disorders of interest, e.g., any of the neurological disorders described elsewhere herein. The methods of the present disclosure are compatible for integration into a variety of existing platforms for high-throughput screening of drug libraries. Such platforms in the context Atty. Docket: STAN-2221 WO (S24-333) of neuronal function include those described in Gohel et al. (2023) Journal of Pharmacology and Experimental Therapeutics 385 (S3) 11 1 ; Ahfeldt et al. (2016) Brain Res. 1656:40-48; Sridharan et al. (2019) Scientific Reports volume 9, Article number: 9000 (“A Simple Procedure for Creating Scalable Phenotypic Screening Assays in Human Neurons”); Little et al. (2019) Neurobiology of Aging 78:130-141 (“Using stem cell-derived neurons in drug screening for neurological diseases”); and Zhou et al. (2024) Neuro-Oncology 26(9):1685-1699; and Sterin et al. (2022) Micromachines (Basel) 13(9):1500; the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0096] In some embodiments, the agent is a small molecule. By “small molecule” is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 900 amu or less, 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In some instances, the small molecule is not made of repeating molecular units such as are present in a polymer.

[0097] Compound libraries for high-throughput screening are readily available and include Bioactive Libraries, Natural Product Libraries, Fragment Libraries, and Diversity Sets. In some instances, the methods are used to screen a library of FDA approved small molecule drugs, a non-limiting examples of which include SCREEN-WELL® FDA approved drug library V2 (Enzo Life Sciences), DiscoveryProbe™ FDA-approved Drug Library (APExBIO), Tocriscreen FDA- Approved Drug Library (Tocris Bioscience), and the like.

[0098] KITS

[0099] Aspects of the present disclosure further include kits. In certain embodiments, a kit of the present disclosure includes a nucleic acid or expression construct of the present disclosure. Such a kit may further include one or more reagents that find use in transfecting or transducing a cell (e.g., an ES cell, an iPSC, or a neuron such as an induced neuron or primary neuron) with the nucleic acid or expression construct.

[0100] Components of the subject kits may be present in separate containers, or multiple components may be present in a single container.

[0101] In addition to the above-mentioned components, a subject kit may further include instructions for using the components of the kit, e.g., to transfect or transduce a cell with the nucleic acid or expression construct. The instructions are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling Atty. Docket: STAN-2221 WO (S24-333) of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, Hard Disk Drive (HDD) etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0102] Non-limiting aspects and embodiments of the present disclosure are also disclosed in the following numbered clauses.

[0103] 1 . A nucleic acid encoding a voltage indicator polypeptide, wherein the voltage indicator polypeptide comprises:

[0104] (a) a voltage-sensing domain (VSD) comprising four transmembrane segments; and

[0105] (b) a circularly permuted fluorescent protein inserted into an extracellular loop between the third transmembrane segment (S3) and the fourth transmembrane segment (S4) of the VSD, wherein the voltage indicator polypeptide comprises an amino acid substitution at position 1412, F413, Q414, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1.

[0106] 2. The nucleic acid of clause 1 , wherein the voltage indicator polypeptide comprises an amino acid substitution chosen from 1412V, F413I, Q414R, or any combination thereof.

[0107] 3. The nucleic acid of clause 1 , wherein the voltage indicator polypeptide comprises an amino acid substitution chosen from 1412V, F413V, Q414R, or any combination thereof.

[0108] 4. The nucleic acid of any one of clauses 1-3, wherein the voltage indicator polypeptide comprises an amino acid substitution at each of 1412, F413, and Q414.

[0109] 5. The nucleic acid of any one of clauses 1-4, wherein the voltage indicator polypeptide further comprises an amino acid substitution at position D145, R149, or both.

[0110] 6. The nucleic acid of clause 5, wherein the voltage indicator polypeptide comprises an amino acid substitution chosen from D145A, R149K, or both.

[0111] 7. The nucleic acid of clause 5, wherein the voltage indicator polypeptide comprises an amino acid substitution chosen from D145C, R149K, or both. Atty. Docket: STAN-2221 WO (S24-333)

[0112] 8. The nucleic acid of any one of clauses 5-7, wherein the voltage indicator polypeptide comprises an amino acid substitution at each of D145 and R149.

[0113] 9. The nucleic acid of any one of clauses 1-8, wherein the voltage indicator polypeptide comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% amino acid sequence identity to SEQ ID NO: 3 or SEQ ID NO: 5.

[0114] 10. The nucleic acid of any one of clauses 1-8, wherein the voltage indicator polypeptide comprises 146L(insert), T148A, T207Y, or any combination thereof, relative to the amino acid sequence set forth in SEQ ID NO: 3, and wherein the voltage indicator polypeptide comprises 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, or 95% or greater amino acid sequence identity to SEQ ID NO: 3.

[0115] 11 . The nucleic acid of any one of clauses 1-8, wherein the voltage indicator polypeptide comprises N138A, 146L(insert), G147A, T148A, G151 S, D152E, R172I, T207Y, F227D, T392I, or any combination thereof, relative to the amino acid sequence set forth in SEQ ID NO: 3, and wherein the voltage indicator polypeptide comprises 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, or 95% or greater amino acid sequence identity to SEQ ID NO: 3.

[0116] 12. The nucleic acid of any one of clauses 1-8, wherein the voltage indicator polypeptide comprises 146L(insert), G147E, T148A, G151 D, D152T, R172F, T207Y, F227N, Q316A, or any combination thereof, relative to the amino acid sequence set forth in SEQ ID NO: 3, and wherein the voltage indicator polypeptide comprises 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, or 95% or greater amino acid sequence identity to SEQ ID NO: 3.

[0117] 13. The nucleic acid of any one of clauses 1-12 operably linked to a promoter.

[0118] 14. A cell comprising the nucleic acid of clause 13.

[0119] 15. The cell of clause 14, wherein the cell is a human cell.

[0120] 16. The cell of clause 14 or 15, wherein the cell is a stem cell.

[0121] 17. The cell of clause 16, wherein the stem cell is an embryonic stem cell.

[0122] 18. The cell of clause 16, wherein the stem cell is an induced pluripotent stem cell. Atty. Docket: STAN-2221 WO (S24-333)

[0123] 19. The cell of clause 14 or 15, wherein the cell is a neuron that expresses the voltage indicator polypeptide on the plasma membrane of the neuron.

[0124] 20. The cell of clause 19, wherein expression of the voltage indicator polypeptide is targeted to the cell body of the neuron.

[0125] 21 . The cell of clause 19 or 20, wherein the neuron is an induced neuron (iN) differentiated from the stem cell of any one of clauses 16-18.

[0126] 22. A method of recording electrical activity of a neuron, the method comprising: irradiating the neuron of any one of clauses 19-21 with light at an excitation wavelength of the voltage indicator polypeptide; recording fluorescence emission from the voltage indicator polypeptide over time, wherein the fluorescence emission is correlated with the electrical activity of the neuron.

[0127] 23. The method of clause 22, wherein the electrical activity comprises excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs), or both.

[0128] 24. The method of clause 23, wherein the EPSPs and IPSPs are spontaneous EPSPs and IPSPs.

[0129] 25. The method of any one of clauses 22-24, wherein action potential generation is blocked for a duration of the recording, optionally wherein action potential generation is blocked during the entire duration of the recording.

[0130] 26. The method of clause 25, comprising contacting the neuron with a voltage-gated sodium channel blocker to block action potential generation.

[0131] 27. The method of any one of clauses 22-24, wherein the electrical activity comprises action potentials.

[0132] 28. The method of any one of clauses 22-27, wherein the electrical activity is in response to a stimulus.

[0133] 29. The method of clause 28, wherein the stimulus is an electrical current, a drug, a ligand for a receptor, a ligand for an ion channel, a ligand for an ion transporter, a hormone, a second messenger, photostimulation of a photogated channel, or any combination thereof.

[0134] 30. The method of any one of clauses 22-29, wherein the neuron comprises a mutation associated with neuronal dysfunction.

[0135] 31 . The method of any one of clauses 22-30, wherein the method is performed in vitro. Atty. Docket: STAN-2221 WO (S24-333)

[0136] 32. A method of assessing an effect of an agent on the electrical activity of a neuron, the method comprising: contacting the neuron of any one of clauses 19-21 with the agent; irradiating the contacted neuron with light at an excitation wavelength of the voltage indicator polypeptide; recording fluorescence emission from the voltage indicator polypeptide over time, wherein the fluorescence emission is correlated with the electrical activity of the neuron; assessing an effect of an agent on the electrical activity of a neuron.

[0137] 33. The method of clause 32, wherein the assessing comprises comparing the electrical activity of the neuron contacted with the agent to the electrical activity of the neuron in the absence of the agent.

[0138] 34. The method of clause 32, wherein the assessing comprises comparing the electrical activity of the neuron contacted with the agent to the electrical activity of a control neuron not contacted with the agent.

[0139] 35. The method of any one of clauses 32-34, wherein the electrical activity comprises one or more of: the frequency of action potentials, the frequency of EPSPs and / or IPSPs, the frequency of EPSPs and / or IPSPs when action potential generation is blocked, the extent of EPSP and / or IPSP decay during propagation along dendrites, baseline voltage, and synchrony of electrical activity with one or more surrounding neurons.

[0140] 36. The method of any one of clauses 32-35, wherein the neuron comprises a mutation associated with neuronal dysfunction.

[0141] 37. The method of any one of clauses 32-36, wherein the agent is from a library of agents, and wherein the method comprises screening the library for agents that alter the electrical activity of neurons.

[0142] 38. The method of any one of clauses 32-37, wherein the agent is a small molecule.

[0143] Atty. Docket: STAN-2221 WO (S24-333)

[0144] The following examples are offered by way of illustration and not by way of limitation.

[0145] EXPERIMENTAL

[0146] Example 1 - Improving ASAP for Larger Responsivity and Faster Activation Kinetics

[0147] A remaining challenge for genetically encoded voltage indicators (GEVIs) is the reliable detection of excitatory post-synaptic potentials (EPSPs). Described herein is the development of a GEVI (referred to herein as Allosteric Sensor for Action Potentials 5 (or “ASAP5”) with enhanced activation kinetics and responsivity near resting membrane potentials for improved detection of both spiking and subthreshold activity. ASAP5 reports action potentials (APs) in vivo with higher signal-to-noise ratios than previous GEVIs, and successfully detects graded and subthreshold responses to sensory stimuli in single 2-photon trials. In cultured rat or human neurons, somatic ASAP5 reported synaptic events propagating centripetally, and could detect ~1-mV EPSPs. By imaging spontaneous EPSPs throughout dendrites, it was found that EPSP amplitudes decay exponentially during propagation, and that amplitude at the initiation site generally increases with distance from the soma. These results extend the applications of voltage imaging to the quantal response domain including in human neurons, opening up the possibility of high-throughput high- content characterization of neuronal dysfunction in disease as well as high-throughput screening for therapeutic agents for diseases characterized by neuronal dysfunction.

[0148] ASAP3 was selected as a template for making a GEVI with high responsivity around -70 mV and fast activation kinetics. The fluorescence-voltage conversion of ASAP3 is largest around -88 mV, making it more promising for detecting millivolt-scale events around -70 mV (resting membrane potential) than ASAP4b and ASAP4e, whose most sensitive voltage values are 0 mV and 30 mV respectively. Since the response to voltage transients is a function of both activation kinetics and steady-state response, designed was a multi-parametric screening protocol intended to improve both activation kinetics and steady-state response.

[0149] To screen for improved sensors, a platform was deployed that can measure changes in sensor fluorescence across a near-instantaneous voltage transition between -70 mV and 0 mV with high temporal resolution as previously described9. For fast electrical events such as APs (which have a time to peak of ~ 2 ms) and EPSPs (which have a time to peak of ~ 5 ms), the peak fluorescence change (AFevent / F0) can be related to the screen parameters by the equation Where AFsteady / F0is the steady-state fluorescence change to a step voltage change of the event amplitude, cfastis the fractional contribution of the fastest-rising component and Tfastthe fastest time constant in the case of Atty. Docket: STAN-2221 WO (S24-333) multiexponential activation kinetics, and t is the time to peak of the voltage transition. For reference, ASAP3 exhibits AFsteady / F0of -51% for 100-mV depolarizations, Tfast of 3.7 ms, and cfast of 81%. ASAP3 responses to AP waveforms in vitro are about 20% at peak, consistent with continued kinetic limitation9. To improve responses to fast events, it was therefore sought to increase steady-state responsivity, enlarge the contribution of the fast-rising component, and decrease the fast time constant.

[0150] It was hypothesized that both responsivity and kinetics could be improved by optimizing the interactions between ASAP3 and the surrounding lipid milieu to reduce energetic barriers to moving the S4 voltage-sensing helix (Fig. 1A). In previous mutagenesis screens of ASAP3 and ASAP4, conservative mutations at T399, 1412, F413, and Q414 were found to modulate voltage tuning and activation kinetics. As these sites are predicted to contact the lipid bilayer, a 324- member library comprising 9x9 amino acid (aa) choices at positions F413 and Q414 in combination with T399T / R and 14121 / V was designed (Fig. 1 B). This library was expressed in HEK 293T cells expressing the inwardly rectifying potassium channel Kir2.1 , which have a resting membrane potential of -70 mV, and implemented software and hardware improvements to enable imaging of electroporation-induced depolarization at 300 frames per s (fps, Fig. 2A). ASAP3 variants were then ranked by the product of AFsteady / F0to capture responsivity, and by a kinetics index that captured the fraction of the full response that was reached in 6.66 ms (corresponding to 2 video frames) as an estimation of relative responses to APs (Fig. 1 C). This process identified ASAP3 1412V F413V Q414R (ASAP3 VVR), as well as ASAP3 1412V F413I Q414R (ASAP3 VIR) (Fig. 2B).

[0151] In a second round of screening, 16 sites that contacted the voltage-sensing S4 helix in ASAP3 VVR were separately mutated to all possible amino acids, comprising a second library of 320 variants (Fig. 1A, B). This screen identified D145A and D145C as improved variants (Fig. 2B). Finally, recorded from single cells using patch-clamp electrophysiological were recordings to test 8 combinations of D145A / C and nearby linker mutations in ASAP3 VVR and ASAP3 VIR. From this final round of screening, the mutant with the largest responses to AP-like waveforms proved to be ASAP3 VIR D145A R149K (Fig. 1C; Table 2), which was designated and referred to herein as ASAP5. Atty. Docket: STAN-2221 WO (S24-333)

[0152] Table 2 - Kinetics of different GEVIs.

[0153] GEVI-expressing HEK293A cells were imaged at 1888 fps with simultaneous voltage clamping. Reported values are mean + SEMs, measured from 6 to 9 cells. ND, not determined. NR, not relevant.

[0154] Table 3 - Comparing different combinations of beneficial mutations 4-ms AP 4-ms AP 2-ms AP 2-ms AP

[0155] -AF / F SNR -AF / F SNR

[0156] ASAP3-VVR 0.52 0.37 0.27 0 24 0.17

[0157] ASAP3-VVR-145A 0.53 0.4 0.29 0 27 0.20

[0158] ASAP3-VVR-145C 0.59 0.4 0.31 0 26 0.20

[0159] ASAP3-VVR-145C-149K 0.53 0.39 0.28 0 28 0.20

[0160] ASAP3-VIR 0.71 0.34 0.29 0 22 0.19

[0161] ASAP3-VIR-145A 0.50 0.42 0.30 0 29 0.21

[0162] ASAP3-VIR-145C 0.65 0.37 0.30 0 25 0.20

[0163] ASAP3-VIR-145A-149K (ASAP5) 0.54 0.43 0.32 0 32 0.24

[0164] GEVI-expressing HEK293A cells were imaged at 800 fps with simultaneous voltage clamping. Baseline fluorescence BF = F-zo / Fmax was obtained from the Fluorescence-Voltage curve. SNR was computed by SNR = -AF / FAPx VBF. Reported values were mean values from 4~6 cells.

[0165] Example 2 - Characterizing ASAP5 Performance for Small- and Large-Amplitude Events

[0166] ASAP5 was compared in the steepness of the response to the earlier ASAP-family GEVIs ASAP314, JEDI-1 P16, and JEDI-2P10. When depolarized from -70 mV to 30 mV, ASAP5 displayed a 59% decrease in fluorescence, representing a 20% improvement in responsivity compared to ASAP3 (Fig. 1 D). The F-V curve of ASAP5 exhibited a steeper slope at the resting membrane potential of -70 mV than the F-V curve of ASAP3 (Fig. 1 D), such that the fluorescence-per-voltage conversion ratio was 1.3% per mV, compared to 0.9% for ASAP3, 0.9% for JEDI-1 P, and 0.7% for JEDI-2P (Fig. 2C). This higher slope is expected to improve detection of sub-threshold events such as EPSPs. Atty. Docket: STAN-2221 WO (S24-333)

[0167] Characterized next were activation and deactivation kinetics. ASAP5 displayed an activation time constant of 2.61 ms at room temperature with a fractional fast component of 92%, and an activation time constant of 0.78 ms with a fractional fast component of 95% at 37eC. Similarly, ASAP5 displayed a deactivation time constant of 4.30 ms with a fractional fast component of 75% at room temperature and 1 .12 ms with a fractional fast component of 63% at 37eC (Table 2). ASAP3, JEDI-1 P and JEDI-2P kinetic measurements using the same method revealed that ASAP5 is the fastest member of the ASAP family in both activation and deactivation (Table 2).

[0168] Compared next were responses of ASAP5 and other ASAP-family GEVIs to neuronal activity waveforms. In response to commanded 2-ms AP waveforms, ASAP5 showed approximately 40% larger responses than JEDI-1 P or JEDI-2P (Fig. 2D). In rat hippocampal neurons injected with current, ASAP5 traces corresponded well to electrical responses for both sub- and supra-threshold components (Fig. 1 E). Fluorescent responses to current-evoked APs were 2-fold larger in amplitude with ASAP5 than with ASAP3 (Fig. 1 F). As expected from their similar F / Fmax ratios at -70 mV (Fig. 2C), ASAP5 and ASAP3 exhibited similar baseline brightness when expressed in cultured neurons (Fig. 1G).

[0169] Example 3 - Comparing ASAP5 Performance In Vivo Under 1 -Photon Illumination

[0170] In this example, ASAP5 was tested in awake behaving mice (Fig. 3A), comparing it to other GEVIs under 1 -photon illumination. To achieve sparse labeling of neurons in motor cortex, CaMKIIa-cre AAV and cre-dependent AAV expressing ASAP3-Kv, JEDI-1 P-Kv, JEDI-2P-Kv, or ASAP5-Kv were co-injected, where Kv denotes the proximal retention and clustering signal of Kv2.1 for somatic enrichment (Fig. 3A). Imaging through an optical window at 500 fps with a sCMOS camera detected optical spikes with the amplitude and duration expected for APs (Fig. 3B), with ASAP5-Kv exhibiting significantly higher SNR for AP detection than the other GEVIs (Fig. 3B, C). In some neurons, ASAP5-Kv optical spikes occurred preferentially with running (Fig. 3D), as expected for APs in pyramidal neurons of the motor cortex.

[0171] The higher single-AP SNR of ASAP5-Kv versus ASAP3-Kv and JEDI-2P-Kv derived from a larger relative fluorescence change (AF / F0) without improved brightness. In contrast, the higher SNR versus JEDI-1 P was due to only higher basal brightness (Fo) (Fig. 3C). That boosting either parameter can improve SNR is expected, as SNR in shot noise-limited conditions scales with and thereby relates to both relative fluorescence change Atty. Docket: STAN-2221 WO (S24-333) and basal brightness. Essentially, ASAP5 combines the larger AF / F0of JEDI-1 P with the higher brightness of ASAP3 and JEDI-2P to improve 1 -photon SNR beyond earlier GEVIs.

[0172] Example 4 - Single-Trial Imaging of Graded and Action Potentials in Flies

[0173] Compared next were ASAP5 and JEDI-2P responses in fruit flies under 2-photon illumination. In Drosophila, Mil neurons depolarize when the fly is presented with a contrast increment on a gray background and hyperpolarize when contrast decrement is presented17 18. When expressed in Mil , ASAP5 responses were approximately 100% larger in amplitude and 60% higher in SNR than JEDI-2P to both hyperpolarization and depolarization, while JEDI-2P displayed a 20 % brighter baseline and slightly better photostability (Fig. 4A). ASAP5 response amplitudes were also significantly larger than JEDI-2P when tested in L2 neurons, which hyperpolarize to contrast increment and depolarize to contrast decrement (Fig. 5A).

[0174] Single-trial 2-photon imaging of both graded responses and action potentials have not previously been reported in the fly. ASAP5 reported individual visually evoked excitatory and inhibitory graded potentials in both Mil ; by comparison, visually evoked excitatory and inhibitory graded potentials were challenging to discern using JEDI-2P, given its lower SNR (Fig. 4B). Also tested was the ability of ASAP5 to detect APs in single trials, using the bilaterally symmetric pair of temperature projection neurons, TPN-II19'20. TPN-II neurons receive inputs from temperature receptor neurons and respond to changes in absolute temperature by modulating their spike rate. ASAP5 reported APs in TPN-II neurons using either resonant galvanometric scanning at 779 fps or Acousto-Optic Deflector (AOD)-based random-access scanning at 3.3 kHz, with similar SNR and multiple measurements per spike (Fig. 5B, C). The ability of AODs to rapidly excite arbitrary locations allowed recording of both TPN-II neurons simultaneously at 3.3 kHz and it was found the two TPN-II neurons fired APs in an uncorrelated manner, consistent with receiving independent inputs (Fig. 4C). Taken together, these experiments demonstrate that ASAP5 can detect both graded and action potentials in single trials in the fly.

[0175] Example 5 - Single-Trial Voltage Imaging by 2-Photon Excitation in Mice

[0176] Next, ASAP5-Kv and JEDI-2P-Kv were compared in pyramidal neurons of the mouse cortex under 2-photon illumination. Cell bodies of the neurons in the two groups were located at similar depths between 100 pm and 200 pm below the cortical surface (Fig. 6). ASAP5-Kvand JEDI-2P-Kv optical spikes recorded at 500 fps were similar in response amplitude, but ASAP5- Kv exhibited higher brightness and higher SNR (Fig. 6). This differs from the observations with ASAP5 and JEDI-2P in fly Mil visual neurons, so it is speculated that the relative brightness of Atty. Docket: STAN-2221 WO (S24-333) the two indicators may depend on the presence of the soma-localization tag or on the experimental system. Remarkably, both ASAP5-Kv and JEDI-2P-Kv demonstrated exceptional photostability under 2-photon illumination (Fig. 7A). In one case ASAP5-Kv reported voltage continuously at 500 fps for over 1 hour without discernable reduction in SNR (Fig. 7B).

[0177] Multi-unit voltage imaging is challenging with 2-photon excitation because photons collected per voxel will drop with more locations or higher sampling rates. With higher labeling densities, raster scanning is economical9’21, whereas random-access strategies are better suited for more sparsely labeled specimens14. ASAP5-Kv was expressed sparsely in mouse motor cortex and recorded from multiple three-dimensional locations using a random-access 2-photon microscope during locomotion. In one example, recordings were obtained from 9 neuronal cell bodies distributed across -85 pm to -278 pm in depth at 450 fps, and contrasting relationships were observed between locomotion and firing rates in individual neurons across long time scales (Fig. 7C). These results demonstrate the feasibility of expanding random-access 2-photon voltage imaging from two to three dimensions while increasing the number of recorded cells compared to previous work10’14.

[0178] Example 6 - Optical Detection of Miniature EPSP in Cultured Rat Neurons

[0179] Spontaneous exocytosis of single synaptic vesicles continues when APs are blocked by tetrodotoxin (TTX) to produce miniature EPSPs (mEPSPs), whose frequency and amplitude reflect synaptic number and function26. To determine if ASAP5-Kv can detect mEPSPs at the soma, simultaneous fluorescence imaging and whole-cell electrophysiological recording of cultured neurons was performed in the presence of TTX and picrotoxin (PTX) to remove APs and inhibitory potentials (Fig. 8A), and mEPSPs were identified in both traces by a deconvolutionbased algorithm27(Fig. 9A-C). As a comparison, Voltron2-Kv labelled with the JaneliaFluor525 fluorophore (Voltron2525-Kv), the combination of protein and chemical dye with the largest response per mV in the Voltron chemigenetic voltage indicator system15, was also imaged (Fig. 8B). One filter set was used to excite both ASAP5-Kv and Voltron2-Kv, but as transmission wavelengths were better aligned to the excitation and emission wavelengths of ASAP5-Kv, calculated was the relative advantage of ASAP5-Kv in fluorescence collection compared to Voltron2-Kv, then shot noise was added to the ASAP5-Kv trace to compensate for its better collection so that SNR would mimic the situation where ASAP5-Kv had similar collection efficiency to Voltron2-Kv. Although brightness per cell of Voltron2s25-Kv was 4.1 -fold higher than that of ASAP5-Kv (after adjusting for filter efficiency, Fig. 9D), SNRs for mEPSPs were similar (Fig. 8C). The ability of ASAP5-Kv to match Voltron2525-Kv in SNR was due to its larger responsivity of - Atty. Docket: STAN-2221 WO (S24-333)

[0180] 0.83% / mV compared to -0.48% / mV (Fig. 8C), and the fact that SNR scales linearly with response size but only with the square root of brightness28. Optical waveforms of detected mEPSPs were similar in shape between ASAP5-Kv and Voltron2525-Kv (Fig. 8D), and cross-correlograms between optical and electrophysiological recordings were also similar (Fig. 8D), implying no substantial differences in kinetics between the two systems. At excitation rates that produce similar SNR, ASAP5Kv and Voltron2s25-Kv exhibited similar photostability (Fig. 9E).

[0181] Next, the false positive (FP) rate was quantified and compared, where the FP rate was defined by the proportion of optically detected events without a corresponding mEPSP in the voltage trace. The false negative (FN) rate was also quantified and compared, where the FN rate was defined by the proportion of electrically detected events without a corresponding mEPSP event in the optical trace. The two systems exhibited similar FP and FN rates across the 0-3 mV range of mEPSP amplitudes (Fig. 8E). For mEPSPs with amplitudes >1 mV, both sensors reported mEPSPs with an FP rate of <20%. To cross-validate the comparison results, the machine-learning algorithm miniML29was also used to detect optical mEPSPs after training on paired segments of electrical and optical recordings, or after training on electrical recordings only. In these analyses, ASAP5-Kv and Voltron2525-Kv again produced similar FP and FN rates (Fig. 8F, Fig. 9F).

[0182] Taken together, these results demonstrate that both ASAP5-Kv and Voltron2525-Kv can be used in cultured neurons to detect spontaneous synaptic events at the cell body in the single mV range using standard illumination conditions (~46 mW / mm2). Between these systems, ASAP5-Kv has the advantage of being completely genetically encoded without requiring the addition of a chemical dye.

[0183] Example 7 - Recording Propagations of Synaptic Inputs Along Dendrites

[0184] Constructing accurate models of neuronal computation requires understanding how synapses in dendritic trees signal to the site of AP generation in the cell body. Voltage signals originating at synapses attenuate as they propagate passively along dendrites, with the distance of the synaptic input to the soma influencing the degree of attenuation30-32. Previous studies have demonstrated the imaging of EPSPs in the dendritic spines in vitro using ASAP133or in vivo using postASAP12. However, single-trial visualization of EPSP propagation from dendrites to soma have not yet been demonstrated. Pan-membrane ASAP5 was therefore imaged in cultured neurons (Fig. 10A). Low-amplitude ASAP5 transients were observed in the cell body that correlated with low-amplitude EPSPs recorded by simultaneous whole-cell electrophysiology, and were clearly distinct from APs (ROI 1 ). These somatic ASAP5 signals correlated with larger fluorescence Atty. Docket: STAN-2221 WO (S24-333) transients in one primary dendrite but not another primary dendrite (ROI 4 or 7), further confirming they are not back-propagating APs. Centripetal decay of these transients was apparent along the dendrite toward the cell body.

[0185] Investigated next was the relationship between initial locations of mEPSPs and their initial and final amplitudes, imaging pan-membrane ASAP5 in cortical neurons with TTX and PTX. For each mEPSP, the location with the highest signal amplitude was defined as the initiation site of the event, and the corresponding somatic signal (Fig. 10B). Queried was how the amplitude of the same mEPSP event at the initiation point ( b) or at the soma ( Vs) correlates with the distance between the two locations (cbs). It was found that V positively correlated with dos in individual examples (Fig. 10C) and across most neurons (Fig. 10D), while Vs negatively correlated with dns (Fig. 10C,D). As a result, the attenuation coefficient ADS = Vb / Vs positively correlated with cfas (Fig. 10C,D). Overall these results suggest that a greater distance between the dendritic input site and the soma correlates with a larger initial voltage signal, a smaller somatic voltage signal, and higher attenuation during the propagation. These observations are consistent with previous electrophysiological measurements in brain slices32, 34.

[0186] Cable theory predicts exponential decay of mEPSP amplitude as signals propagate passively along the dendrite35. At the same time, precise quantification of spatial attenuation and determination of the length constant is challenging using electrophysiological approaches in many neurons, as these measurements require multiple patch recordings at different locations along the same dendrite. Asked therefore was whether voltage changes along multiple points of each dendrite could be directly measured to map spatial attenuation using voltage imaging. For each propagation event, correlated was the distance between the peak ASAP5 signal at each point along the dendrite and the soma, and it was fit with an exponential function (Fig. 10B) to estimate the length constant. These studies revealed that the measured length constants varied from 25 pm to 240 pm, with an average of 90 pm (Fig. 10E). These signals were inhibited by CNQX, identifying them as mediated by GluA receptors (Fig. 11 A).

[0187] In addition, network effects were observed in cultured rat cortical neurons without channel blockers, with either pan-membrane ASAP5 or soma-localized ASAP5-Kv (Fig. 11 B, C), with neuron pairs displaying shared activating inputs. Inhibitory post-synaptic potential (IPSP)-like events could also be detected (Fig. 11 B). Taken together, these results suggest that ASAP5 can be used to study the passive and active propagation of EPSPs and the effects of I PSPs. These excitatory and inhibitory PSPs and synchronized spiking activities from two neurons are examples Atty. Docket: STAN-2221 WO (S24-333) of mixed sub- and suprathreshold network-dependent activity that would be difficult to observe by multi-electrode arrays (MEAs) or calcium imaging.

[0188] Example 8 - Reoortinq Network Activity and Unitary Synaptic Events in Human Neurons

[0189] Patch-clamp electrophysiology, multi-electrode arrays (MEAs), and calcium imaging have been widely used to study the effects of disease-associated mutations in human stem cell-derived neuronal cultures. Patch-clamp electrophysiology remains the gold standard in its ability to detect both spiking and subthreshold activity, but can only be performed on a handful of neurons at a time. As large samples sizes are required to detect small differences in mEPSP frequency due to the intrinsic variability in mEPSP rate between neurons, the limited throughput of patch-clamp electrophysiology limits assessment of synaptic function in neuronal populations using this approach. In contrast, MEAs and calcium imaging allow parallel detection of spiking activity in multiple neurons, allowing assessment of changes in overall network excitability by mutations found in neurological disease such as autism and schizophrenia36. However, neither MEAs nor calcium can directly detect subthreshold or inhibitory activity, and MEAs do not allow genetic specification of recorded cell types.

[0190] Previously, an opsin-based GEVI, a two-component chemigenetic voltage indicator, and a small-molecule voltage-sensitive dye have been used to measure neuronal excitability of human stem cell-derived neurons by AP detection37-39. Asked was whether ASAP5 could extend the uses of voltage imaging in human neurons to EPSP detection. Human embryonic stem (ES) cells were differentiated into neurons using an established protocol40. ASAP5-Kv was introduced by lentiviral transduction, and one-photon imaging was performed at 400 fps. In one example, activity between two human induced neurons (human iNs) was correlated in multiple ways (Fig. 12A): EPSPs in both neurons, an AP in one neuron with a corresponding EPSP in the other, or APs in both neurons (Fig. 12A, bottom). Because simulations demonstrated that 100 fps was as effective as faster imaging framerates in detecting subthreshold activity, while preserving distinctly sharper optical responses to APs (Fig. 13A), human iNs were imaged at this lower speed to acquire a larger FOV. This allowed the observation of correlated APs and EPSPs in up to 8 human iNs simultaneously (Fig. 12B).

[0191] It was hypothesized that ASAP5-Kv could also be used to detect mEPSPs in human iNs. Abnormalities in synapse development or vesicular release probability caused by disease- associated mutations are manifested as altered mEPSP or mEPSC frequencies, but phenotypes can differ between human iNs and rodent neurons, underscoring the importance of studying human iNs41. As most synaptic activity studies in human iNs used somatic voltage-clamp Atty. Docket: STAN-2221 WO (S24-333) recordings to detect mEPSCs rather than mEPSPs, compared first were somatic current-clamp and voltage-clamp recordings with TTX and PTX to allow only spontaneous quantal glutamate release. Both mEPSCs and mEPSPs were detected with similar frequency (Fig. 13B), and did not occur in the presence of the GluA channel blocker CNQX, indicating these were excitatory synaptic events40. Finally, to assess the ability to image mEPSPs, simultaneous current-clamp recording and ASAP5-Kv imaging was conducted in human iNs in the presence of TTX and PTX. ASAP5-Kv detected mEPSPs with amplitudes as low as 1 mV, with a mean AF / F0of -0.67% per mV depolarization (Fig. 12C). These results demonstrate that mEPSPs, representing synaptic quantal events, can be easily detected in cultured human iNs.

[0192] Summary

[0193] To summarize, ASAP5 is an improved voltage indicator polypeptide with 0.78 ms onset kinetics at physiological temperature, larger responses to APs and subthreshold events, achieving single-trial EPSP and IPSP detection at cell bodies in vivo and 1 -mV responsivity in cultured neurons. ASAP5 exhibits higher sensitivity and SNR than JEDI-2P and JEDI-1 P for detecting spikes in vivo, while achieving similar SNR to Voltron2 for detecting mEPSPs in cultured neurons. With its superior SNR for both sub- and supra-threshold activities, ASAP5 enabled single-trial two-photon recordings of graded potentials and APs in fly neurons, of APs at multiple 3D locations in motor cortex, and of EPSP and IPSPs in barrel cortex. Most importantly, with its higher gain near resting membrane potentials, ASAP5 enabled optical recording of unitary synaptic events in human neurons, previously only achievable with patch-clamp electrophysiology. ASAP5 detected coordinated APs and EPSPs in multiple human neurons within a network, a feat not possible with multi-electrode arrays (MEA) and challenging for conventional patch-clamping.

[0194] ASAP5 conveniently maintains large responses across imaging modalities. Under 1 - photon excitation of sparsely-labeled neurons in vivo, ASAP5 showed higher SNR than previous ASAP-family GEVIs. When used in cultured neurons, where the specimen is nearly 2- dimensional, out-of-focus cells are not a problem and thus ASAP5 provides excellent sensitivity even from very densely labelled specimens. So far, one-photon imaging of soma-targeted GEVIs has been demonstrated through 300 pm of tissue using a combination of targeted illumination and confocal gating47, while two-photon voltage imaging with subcellular resolution has been performed through 600 pm of tissue10. Two-photon excitation is therefore still essential when imaging deeper below the surface, and also useful for reducing background in densely labelled specimens as out-of-focus cells are not excited. Under 2-photon excitation by resonant galvanometric scanning or random-access scanning, ASAP5 was superior in SNR to previous Atty. Docket: STAN-2221 WO (S24-333)

[0195] GEVIs. Thus, ASAP5 is widely useful in vivo under 1 -photon excitation with sparse labeling, in vivo under 2-photon excitation at any labeling density, or in vitro under any illumination.

[0196] The high responsivity of ASAP5 near the resting membrane potential, which enhances the detection of mEPSPs at soma and along the dendrites in vitro, is expected to aid the detection of EPSPs and their spatial propagation in vivo.

[0197] With its enhanced SNR for electrical events from 1 to 100 mV, ASAP5 is expected to permit high-throughput characterization of synaptic and network function in human neurons in culture. Specifically, ASAP5 can report all levels of electrical activity — APs, EPSPs and IPSPs, and mEPSPs — in multiple neurons in parallel. ASAP5 is thus expected to allow high-throughput functional characterization of physiological phenotypes of mutations associated with neuropsychiatric diseases such as autism, schizophrenia, and neurodegenerative disorders, substituting for low-throughput patch-clamp electrophysiology for many tasks. Finally, the improved throughput of voltage imaging is expected to enable direct screening of drug libraries for improved synaptic function in human neurons.

[0198] Example 9 - ASAP5 Variants: ASAP7v and ASAP8v

[0199] Described herein is the development of two ASAP5 variants (designated ASAP7y and ASAP8y) with mutations relative to ASAP5 that cause a shift of their excitation and emission spectra to redder wavelengths. The shift permits the use of ASAP7y and ASAP8y in 2-photon excitation with common 1030-nm lasers. ASAP7y and ASAP8y exhibit higher contrast as compared to ASAP5. Relative to ASAP5, ASAP7y exhibits higher sensitivity to voltage at the subthreshold range which is useful for revealing EPSPs and IPSPs. Relative to ASAP5, ASAP8y has higher sensitivity to voltage at the supra-threshold range or for action potentials. Also demonstrated in this example is that all three sensors work in neurons.

[0200] Relative to ASAP5, ASAP7y comprises the following mutations: N138A, 146L(insert), G147A, T148A, G151 S, D152E, R172I, T207Y, F227D, and T392L Relative to ASAP5, ASAP8y comprises the following mutations: 146L(insert), G147E, T148A, G151 D, D152T, R172F, T207Y, F227N, and Q316A.

[0201] Data demonstrating that ASAP7y and ASAP8y are 1030nm-excitable in two-photon microscopy at resting membrane potential is provided in FIG. 14. The plot shows the fluorescence excitation spectra of ASAP5, ASAP7y, and ASAP8y, each normalized to its own peak. The spectra reveal a right-shift in ASAP7y and ASAP8y that allows more efficient excitation by 1030- Atty. Docket: STAN-2221 WO (S24-333) nm two-photon lasers (dashed line). Data demonstrating that ASAP7y and ASAP8y are 1030nm- excitable in two-photon microscopy at depolarized potential is provided in FIG. 15.

[0202] Provided in FIG. 16 is data demonstrating that the total contrast of ASAP7y and ASAP8y is increased relative to ASAP5. The plot illustrates how fluorescence of each ASAP protein is modulated by membrane voltage relative to its minimum value across all voltages when expressed in mammalian cells.

[0203] FIG. 17 is a plot showing how the change of fluorescence normalized to a baseline at -70mV is modulated by membrane voltage. The change of fluorescence is AF, and the baseline is Fo. The plot illustrates the signal readout of ASAP7y as a function of membrane voltage and that ASAP7y has a steeper modulation (higher sensitivity) to voltage than ASAP5.

[0204] FIG. 18 is a plot showing how the change of fluorescence normalized to a baseline at -70mV is modulated by membrane voltage. The change of fluorescence is AF, and the baseline is Fo. The plot illustrates the signal readout of ASAP8y as a function of membrane voltage and that ASAP8y becomes brighter when the voltage increases. In contrast, ASAP5 and ASAP7y become dimmer.

[0205] FIG. 19 includes plots showing the signal readout of ASAP molecules when expressed in primary (rat) neuron culture. The spikes represent action potentials of neurons. As shown, ASAP7y and ASAP8y exhibit higher signals than ASAP5.

[0206] Methods

[0207] Plasmid construction

[0208] For electroporation-based screening in HEK293-Kir2.1 cells, the ASAP3 genes with designed mutations were cloned into pcDNA3.1 with a CMV enhancer and promoter and bGH poly(A) signal. For patch-clamp characterization in HEK293A cells, all voltage indicators were subcloned into a pcDNA3.1 / Puro- GAG vector between Nhel and Hindlll sites.

[0209] For in vitro characterization in cultured neurons, ASAP5 was subcloned into pAAV-hSyn- WPRE. For making transgenic fly lines, ASAP5 was cloned into pJFRC7-20XUAS vector. For in vivo mouse voltage imaging, ASAP5 was cloned into pAAV-hSyn-WPRE or pAAV-EF1 a-Dio- WPRE, and then packaged into AAV9 capsids by the Neuroscience Gene Vector and Virus Core (GVVC) at Stanford University. For somatic targeting expression in vivo, a 65-amino-acid cytoplasmic segment of Kv2.1 potassium channel was added to the C terminus of ASAP5 followed by an LKGSSGSSGSSTR linker. All plasmids were made by standard molecular biology Atty. Docket: STAN-2221 WO (S24-333) techniques with all cloned fragments or whole plasmids confirmed by sequencing (Sequetech and Primordium labs).

[0210] For the comparison with Voltron2 in cultured neurons, pAAV-hSyn-Voltron2-Kv-WPRE was cloned by taking the Voltron2, the Golgi export trafficking sequence, and the endoplasmic reticulum export sequence (ER) from pGP-pcDNA3.1 Puro-CAG-Voltron2-ST plasmid. Then, the fragment was inserted into a pAAV-hSyn-WPRE backbone vector together with the somatargeting tag from Kv2.1 by using In-Fusion HD cloning kit (Takara). pGP-pcDNA3.1 Puro-CAG- Voltron2-ST was a gift from GENIE Project (RRID: Addgene_172910).

[0211] Lentivirus plasmid construction and packaging for human iNs experiments

[0212] Lentivirus constructs for induction of human neurons from H1 ESCs were prepared as previously described40. Briefly, Rev, RRE, and VSV helper plasmids were used for lentiviral packaging of Ngn2 and rtTA. Additionally, ASAP5-Kv was packaged with either hSyn or Ubiquitin-C promoter to express the GEVI into human iNs.

[0213] All lentiviruses were packaged using HEK293T cells (ATCC) as previously described48. Briefly, lentiviral vector plasmid (12 pg) and helper plasmids (4 pg Rev, 8 pg RRE, and 6 pg VSVG) were co-transfected in T75 flask by the calcium phosphate method. Lentivirus particles were harvested from the medium 48 h after transfection. To concentrate viral particles, supernatant was pelleted by centrifugation at 19,000 g for 2 h, resuspended in DMEM, and frozen in aliquots at -80 °C.

[0214] Cultured cell lines

[0215] For electroporation-based screening, the previously described HEK293-Kir2.1 cell line was maintained in high-glucose DMEM (Thermo Fisher Scientific, 31053036), 5% FBS (Gemini Bio), 2 mM L-glutamine (Gemini Bio) and 500 pg / mL geneticin (Thermo Fisher Scientific, 10131035). For electrophysiological recordings to measure the responsivity and kinetics of voltage indicators, HEK293A cells (Thermo Fisher Scientific, R70507) were cultured in high- glucose DMEM with 5% FBS and 2 mM L-glutamine. All cell lines were maintained in a humidified incubator at 37 °C with 5% CO2.

[0216] Primary neuronal culture and transfection

[0217] Primary culture of rat neurons and transfection were conducted as previously described9. Briefly, hippocampus and cortex from embryonic day 18 Sprague Dawley rat’s brain were dissected separately and dissociated in TrypLE Select enzyme (Gibco) mixed with 0.005% DNasel. The dissociated neurons were seeded at 70,000 hippocampal cells / well or 100,000 Atty. Docket: STAN-2221 WO (S24-333) cortical cells / well (500-pL working volume per well) on poly-D-lysine (0.1 mg / mL; Gibco) coated round coverslips (12-mm diameter and 0.13-0.17-mm thick coverslips; Carolina biological). At 9- 12 days in vitro (DIV), the neurons were transfected with 100-200 ng of GEVI plasmid under the hSyn promoter using Lipofectamine 2000 (Invitrogen). For the dendritic mEPSP mapping experiment (see below), the rat cortical neurons were cultured on 24-well glass-bottom plates (0.170 ± 0.005mm thick cover-glass; CellVis) at the same density as described above. The transfection was done at 16 DIV. All the neurons were maintained until the day of imaging by performing a half-media change per well on every 3rdday, for example, on 1 , 4, 7, 10, 13, 16, 19, 22, and 25 DIV with fresh Neurobasal media supplemented with 2 % v / v B-27 and 2 mM GlutaMax (all from Gibco).

[0218] Preparation of ESC-derived human iNs and transduction of ASAP5-Kv

[0219] H1 ESCs were maintained in mTeSRI medium (Stem Cell Technologies). Human induced neurons were generated from H1 ESCs as previously described40with a few modifications. On the day of induction, ES cells were treated with Accutase, plated as dissociated cells on matrigel- coated wells in mTeSRI medium containing 2 pM thiazovivin, and infected with TetO-Puro-Ngn2 and rtTA (3.5 pL for each well in a 6-well plate) lentivirus particles prepared as described above. On day 1 , doxycycline (2 mg / L, Sigma) was added to the induction media (Non-Essential Amino Acids, BDNF (10 ng / ml, PeproTech), human NT3 (10 ng / ml, PeproTech) and mouse Laminin-1 (0.2 pg / mL, PeproTech)) to induce TetO gene expression. On days 2 and 3, puromycin (1 mg / L) was added to the induction media to select only previously infected cells. On day 4, IN cells were dissociated with Accutase and plated 100,000 cells / well on a 24-well plate in Neurobasal media with Gem21 (Gemini bio), doxycycline, BDNF, NT3, Laminin-1 , and Glutamax. On day 5, 2.3 pL of ASAP5-Kv lentivirus was added to each well of a 24-well plate and on day-6, glial cells (cultured from forebrain of newborn wildtype CD1 mice) were added. On days 7 and 9, 4Ara-C (2 pM, Sigma) was added to the medium to inhibit astrocyte proliferation. From day 10, 5% FBS was added to the medium. Since then, a half of the medium in each well was changed weekly. The human iNs were then used for experiments on days 30-80.

[0220] Animals

[0221] For in vivo 2-photon imaging in fruit flies, ASAP5 was inserted into the attP40 phiC31 landing site by injection of a pJFRC7-20XUAS-ASAP5 plasmid (BestGene). The cell-type-specific driver GMR19F01 -Gal4 was used to express GEVIs in Mil , 53G02AD-29G11 DBD split-Gal4 to express GEVIs in L2 neurons, and R60H12-Gal4 to express ASAP5 in TPN-II neurons. The genotypes of the imaged flies in Fig. 4 and Fig. 5 were: Atty. Docket: STAN-2221 WO (S24-333)

[0222] Mi1 »JEDI-2P: + / +; UAS-JEDI-2P / +; UAS-jRGECO1 a / GMR19F01 -Gal4

[0223] Mil »ASAP5: + / +; UAS-ASAP5 / +; UAS-jRGECO1 a / GMR19F01-Gal4

[0224] L2»JEDI-2P: + / +; UAS-JEDI-2P / 53G02-AD; UAS-jRGECO1a / 29G1 1-DBD

[0225] L2»ASAP5: + / +; UAS-ASAP5 / 53G02-AD; UAS-JRGECO1 a / 29G11 -DBD

[0226] TPN-II»ASAP5: + / +; UAS-ASAP5 / R60H12-Gal4; +

[0227] For rat hippocampal and cortical neuronal culture, Sprague Dawley rat embryos at embryonic day 18 were obtained with approval by the Stanford Institutional Animal Use and Care Committee. For in vivo experiments in mouse motor cortex, wild-type C57BL / 6 mice (P51 -101 ) were used with procedures approved by the Stanford Institutional Animal Use and Care Committee. For in vivo experiments in mouse barrel cortex, all animal procedures were approved by the UC Berkeley Animal Care and Use Committee and followed NIH guidelines. Prior to surgery, mice were housed in cohorts of 3-4 in reverse 12 / 12-hour light-dark cycles with humidity 30-70% and temperature 20-26 °C. All behavioral training and imaging experiments were conducted in the dark cycle. Mice were singly housed in separate cages with running wheels after performing cranial window surgery and mounting head fixation gear. Drd3-Cre mice were used with viral expression of ASAP5-Kv under the hSyn promoter, which achieved neuron specific expression of ASAP5-Kv in S1. For in vivo experiments in mouse visual cortex, all protocols adhered to the guidelines of the French National Ethics Committee for Sciences and Health report on Ethical Principles for Animal Experimentation in agreement with the European Community Directive 86 / 609 / EEC under agreement #29791. 6 male wildtype C57BL / 6J adult mice (>P40 - body weight 20-24 g) were housed in standard conditions (12-hour light / dark cycles, light on at 7 a.m., with water and food ad libitum). A preoperative analgesic was used (buprenorphine, 0.1 mg / kg), and Ketamine- Xylazine were used as anesthetic (Centravet).

[0228] Cell screening

[0229] HEK293-Kir2.1 cells were screened in 384-well plates (Grace Bio-Labs) on conductive glass slides (Sigma-Aldrich) with customized electrophoresis-based screening system. 2 days before imaging, cells were transfected with PCR-generated libraries with Lipofectamine 3000 followed by a media change after 6 h. Cells were imaged at room temperature on an 1X81 inverted microscope (Olympus). A blue LED (Prizmatix UHP-Mic-LED-460) passed through a 480 / 40-nm excitation filter and focused on the sample through a x20 0.75-NA objective (Olympus). Emitted fluorescence was passed through a 503-nm long-pass emission filter. A single FOV was imaged for a total of 3 s with a 10-ps 150-V electrical square pulse applied. Images was recorded at 300 Atty. Docket: STAN-2221 WO (S24-333) fps by an ORCA Flash4.0 V2 CMOS camera (Hamamatsu, C11440-22CA) with pixel binning set to 4 x 4. All operations were controlled by a customized software in MATLAB (MathWorks).

[0230] Whole-cell patch-clamping and imaging of HEK293A cells

[0231] Cells were transfected with pcDNA3.1 / Puro-CAG-based plasmids expressing each GEVI using Lipofectamine 3000 in 24-well plates following the manufacturer’s recommended instructions. 30 h after being plated on 12-mm glass coverslips (Carolina Biological), the cells were voltage-clamped (Multiclamp 700B amplifier with pCIamp software, Molecular Devices) and recorded by an iXon 860 EMCCD camera (Oxford Instruments) with a x40 1.3-NA oil-immersion objective (Zeiss). Blue light from a UHP-F-455 LED (Prizmatix) served as the excitation light source. The filter cube set consisted of a 484 / 15-nm excitation filter and a 525 / 50-nm emission filter. The power density at the sample was 20-41 mW / mm2.

[0232] T o characterize the fluorescence-voltage curve of the indicators, cells were clamped at a baseline potential of -70 mV, then 400 ms of voltage steps at -200, -160, -140, -120, -100, -80, -70, - 60, —40, -20, 0, 30, 50, 80 mV followed by 400 ms of -70 mV baseline. To characterize the response of voltage indicators to action potentials, a scaled AP waveform (FWHM 2.0 ms or 4.0 ms, -70 mV to +30 mV) recorded from a cultured hippocampal neuron was applied to HEK293A cells.

[0233] To characterize kinetics of ASAP indicators at both 22 °C and 37 °C, a 30-mV voltage step was applied to cells from -70 mV baseline and images were acquired at 1888 fps. Time constants were obtained using MATLAB curve fitting (MathWorks).

[0234] Electrophysiology and voltage imaging from cultured neurons

[0235] All electrophysiological recordings were conducted with Multiclamp 700B amplifier, Digidata 1440A digitizer, and pCIamp software (all from Molecular devices). Glass capillaries with filament having 1.5 mm outer diameter, 1.0 mm inner diameter and 75 mm axial length (King Precision Glass) were used for whole-cell voltage and current clamp recordings. Recordings from cultured rat neurons and human iNs were conducted at room temperature under similar conditions described in a previously reported paper41. The pipette solution contained 123 mM K-gluconate, 10 mM KCI, 8 mM NaCI, 1 mM MgCL, 10 mM HEPES, 1 mM EGTA, 0.1 mM CaCL, 1.5 mM MgATP, 0.2 mM Na4GTP, and 4 mM glucose, and the pH was adjusted to 7.2 with KOH. Osmolarity was 295-300 mOsm / kg. The resistance of intracellular solution filled pipettes was 3- 5 MOhm. The extracellular solution contained 145 mM NaCI, 3 mM KCI, 2 mM CaCL, 2 mM MgCh, 10 mM HEPES, and 10 mM glucose, and the pH was adjusted to 7.4 with NaOH. Osmolarity was Atty. Docket: STAN-2221 WO (S24-333)

[0236] 310 mOsm / kg. Whole-cell current clamp recordings requiring more than ± 100 pA current injection to maintain resting membrane potential near -70 mV were excluded from analysis. Tetrodotoxin (TTX; 1 pM) and picrotoxin (PTX; 50 pM) were added to the bath solution to isolate miniature excitatory postsynaptic activity. To remove AMPA-receptor dependent miniature events, 6-cyano- 7-nitroquinoxaline-2, 3-dione (CNQX; 20 pM) was added to the bath solution. For the miniature EPSP recording from rat hippocampal neurons (Fig. 8B), the extracellular solution containing (in mM) 110 NaCI, 26 sucrose, 23 glucose, 5 HEPES-Na, 5 KCI, 2.5 CaCh, 1.3 MgSC , and pH adjusted to 7.4 was used. The patch pipette was filled with 115 mM K-gluconate, 10 mM HEPES- Na, 10 mM EGTA, 10 mM glucose, 8 mM KCI, 5 mM MgCl2, and 1 mM CaCh, and pH was adjusted to 7.4. Electrophysiological recordings were acquired at 10 kHz sampling rate, but they were resampled to 0.4 or 1 kHz for visualization in the figures. Clampfit 11 (Molecular devices) was used for post-processing and analyses of all recordings. For detection of miniature EPSPs in Fig. 12C, the template search function in Clampfit 11 was first used, and then detected events were further sorted with a minimum threshold of 0.5 mV followed by a visual inspection of each event.

[0237] For voltage imaging of cultured neurons, the blue light from UHP-F-455 LED (Prizmatix) filtered through a 482 / 18-nm bandpass filter (Semrock) and focused by a x40 1.3-NA oilimmersion objective lens (Zeiss) was used for excitation. The power density at the sample ranged from 56 to 92 mW / mm2. The resulting fluorescence emission filtered by a 525 / 50 nm bandpass filter (Semrock) was imaged on a sCMOS camera (Flash4-V2 C11440-22CU, Hamamatsu) with various sub-array sizes to achieve suitable framerate for each experiment. To acquire optical miniature EPSP signal from the ASAP5-Kv transfected rat neuron in Fig. 8B, 484 / 15 nm excitation filter and iXon 860 EMCCD camera (Andor - Oxford instruments) were used. The power density at the sample was 40 mW / mm2. For voltage imaging of spontaneous activity from the two rat hippocampal neurons shown in Fig. 11 A, HBSS solution containing 10 mM HEPES, 2mM GlutaMax, and 1 mM sodium pyruvate was used. The excitation was delivered by a 20x objective lens (NA 1.0, Olympus) and the power density at the sample was 70 mW / mm2. Photobleaching in resulting fluorescence traces was corrected by normalizing with exponential decay function using MATLAB. Fluorescence traces in figures 11 B, and 12C were lowpass filtered with passband frequency of 80 Hz in MATLAB. The sCMOS camera (Flash4-V2 C1 1440-22CU, Hamamatsu) used for the dendritic EPSPs voltage imaging result in Fig. 10A acquires signal with rolling shutter, which causes time delay in y-axis of the image. Since the result compared time-series signal from multiple ROIs, digital interpolation was used to correct for the delay in different rows. Atty. Docket: STAN-2221 WO (S24-333)

[0238] For Voltron2-Kv expressing neurons, JaneliaFluor 525 (JF525) - HaloTag ligands (Item No.: CS315102; Promega) were added prior to experiments following the procedures described in the original literature15and by the manufacturer. Briefly, the neurons were gently rinsed with the extracellular bath solution. Then, JF525-HaloTag ligands were added to each well at 100 nM concentration. After 10 min of incubation, the neurons were washed twice with the extracellular solution.

[0239] To compare ASAP5-Kv and Voltron2-Kv for optical detection of mEPSPs, cultured rat hippocampal neurons at 14-21 DIV were patched and imaged simultaneously. For electrophysiological recordings, extracellular solution containing 145 mM NaCI, 3 mM KCI, 2 mM CaC , 2 mM MgCk, 10 mM HEPES, and 10 mM glucose (pH adjusted to 7.4) supplemented with TTX (1 pM) and PTX (50 pM), and the pipette solution containing 123 mM K-gluconate, 10 mM KCI, 8 mM NaCI, 1 mM MgCh, 10 mM HEPES, 1 mM EGTA, 0.1 mM CaCL, 1 .5 mM MgATP, 0.2 mM Na4GTP, and 4 mM glucose (pH adjusted to 7.2) were used. The excitation for ASAP5-Kv was provided by SOLIS-470C LED (Thorlabs), filtered by a 482 / 18-nm bandpass filter (Semrock), and the emission was acquired after 525 / 50-nm bandpass filter (Semrock). For Voltron2525-Kv, M530L4 LED (Thorlabs) was used as the light source, and a filter cube consisting of 520 / 35-nm bandpass filter (Semrock), 552nm long-pass dichroic mirror (Semrock), and 572 / 35-nm bandpass filter (Chroma) was used to collect the fluorescence. For both sensors, the excitation light was set to provide the irradiance of 46 mW / mm2. All the images were acquired at 400 fps by using an iXon 860 EMCCD camera (Andor - Oxford instruments). Based on the overlap of the spectra of normalized LED power, excitation filter transmission, and fluorophore excitation, and the overlap of the spectra of fluorophore emission, emission filter transmission, and camera sensitivity, the conditions provided 2.3-fold less efficient excitation (relative to peak excitation) and collection (relative to total emission) of Voltron2525-Kv fluorescence compared to ASAP5-Kv at the same irradiance. For the brightness comparison, used were mean intensities in the 0.5-1 .5 s time frame due to the rapid decay of fluorescence signals in the first 0.5 s; by bypassing the brighter but highly transient state, the practically usable brightness is measured. Likewise, photostability curves were normalized to values at 0.5 s. To compare the photostability of the two different sensor systems, time-lapse images acquired as described above at an irradiance of 46 mW / mm2were used. But as a 2.3-fold lower irradiance of ASAP5-Kv was found to yield the same SNR for mEPSPs as Voltron2525-Kv irradiated at 46 mW / mm2, the time-axis of ASAP5-Kv was multiplied by 2.3 to produce photobleaching curves at irradiances that provide the same SNR. The difference was also used as correction factor to either add corresponding Gaussian noise to the Atty. Docket: STAN-2221 WO (S24-333)

[0240] ASAP5-Kv fluorescence traces or to improve the SNR of Voltron2-Kv detected events by multiplying the square root of 2.3 assuming photon shot noise limited condition.

[0241] Detecting mEPSPs at the soma in cultured neurons

[0242] A deconvolution-based method was applied to detect mEPSPs in both electrophysiological and optical recordings of cultured neurons. The electrophysiological recording was first downsampled from 10 kHz to 400 Hz to match the sampling rate of the optical recordings, from which 30 mEPSPs with the highest peak amplitude were collected and the waveforms aligned by their peaks were averaged to construct a template. The deconvolution divisor was the repolarizing phase of the template normalized to its peak value. Then the electrophysiological and optical recordings were deconvolved with the divisor, applying a Gaussian filter (sigma=3) to remove the high-frequency noise and a 2 Hz high-pass filter to remove the baseline fluctuation. The peaks in the deconvolved traces were identified with a z- score higher than 2.5 as the detected events. For SNR comparison, the noise was estimated by the standard deviation of the 20 Hz-high-pass-filtered trace to exclude sub-threshold activities in the recording, and the signal was defined by the optical signal corresponding to the detected mEPSPs in the electrophysiological recording.

[0243] To detect electrical and optical mEPSPs using miniML, events and event-free segments from electrophysiological data were extracted and labeled using a template-matching algorithm. These annotated data (total segments, 900; label ratio 1 :1) were used to train a machine-learning model for event detection using transfer learning29. Corresponding imaging data from both sensors (Voltron2-Kv and ASAP5-Kv; total segments, 800; label ratio 1 :1) were used to train a separate model to detect events in the fluorescence traces. mEPSPs in electrophysiological recordings were detected using the electrophysiology-trained miniML model. Fluorescence recordings were filtered using an 8-sample Hann window and then resampled to 4 kHz. Events in the imaging data were detected using the distinct optical model (Fig. 8) or the electrophysiology- only trained model (Fig. 9).

[0244] Mapping the propagation of mEPSPs in cultured neurons

[0245] Cultured rat cortical neurons at 23 and 24 DIV in extracellular solution containing 145 mM NaCI, 3 mM KCI, 2 mM CaCh, 2 mM MgCh, 10 mM HEPES, and 10 mM glucose (pH adjusted to 7.4) with TTX (1 pM) and PTX (50 pM) were imaged with a sCMOS camera (Flash4-V2 C11440- 22CU, Hamamatsu). Excitation light from UHP-F-455 LED (Prizmatix) passed through a 482 / 18- nm bandpass filter (Semrock) and was focused by a x40 1 ,2-NA water-immersion objective lens (Zeiss), and the emission light was collected after a 525 / 50-nm bandpass filter (Semrock). Atty. Docket: STAN-2221 WO (S24-333)

[0246] Neurons were imaged for 13000 frames at 200 fps, with the irradiance of 114 mW / mm2. To analyze the images, the rolling-shutter delay of the camera was first corrected for. Then the image was binned by 8x8 to enhance the SNR of single pixels. The masks of neurons were generated by Otsu-thresholding, from which fluorescence traces from each pixel were extracted. A 0.5 Hz high-pass filter was applied to detrend the traces and peaks with z-scores higher than 3.5 were identified as signals. All peaks were sorted in time, and consecutive peaks within 15 ms were collected and defined as a propagation event. Propagation events that contained less than 5 pixels were removed due to less reliability. For each propagation event, the pixel with the highest peak amplitude was identified as the initiation site of the propagation, and the distance to soma was measured along the dendrite. The somatic trace was pooled from a manually-drew somatic mask and the signal that corresponds to a propagation was identified according to the averaged peak time of all the signals in a propagation event.

[0247] Brightness comparison in cultured neurons

[0248] Hippocampal neurons were transfected at 9-1 1 DIV with 200 ng of indicator DNA (pAAV- hSyn-ASAP-Kv-mCyRFP3) and 1 pL of Lipofectamine 2000 (Thermo Fisher Scientific) in 200 pL of Neurobasal with 2 mM GlutaMAX and imaged 2 d after transfection. Neurons were imaged on an inverted microscope (Zeiss Axiovert 200M) with a x40 1 ,2-NA objective (Zeiss). Excitation light was 488 / 30-nm filtered from a 120-W mercury vapor short arc lamp (X-Cite 120PC, Exfo). Fluorescence was collected using a 531 / 40-nm filter (for green channel) and a 625 / 60-nm filter (for red channel). Images were taken using an Orca Flash4.0LT+ C11440-42U CMOS camera (Hamamatsu) with pManager software.

[0249] One-photon voltage imaging in mouse and sensor comparison

[0250] Surgery and viral injection

[0251] To prepare mice for both one and two-photon in vivo imaging from primary motor cortex, a procedure previously described was followed9 49with a few modifications. Briefly, 8-14 weeks- old wild-type C57BL / 6J mice (Jackson Laboratories, No. 000664) of both genders were injected with relevant GEVI viruses (floxed versions of ASAP3-Kv, JEDI-1 P-Kv, JEDI-2P-Kv, and ASAP5- Kv) mixed with diluted CaMKIla Cre virus (2.8x1013GC / mL, Addgene #105558). All four GEVI viruses were produced by the Stanford GVVC in the same batch, but for a better comparison, higher titer viruses were further diluted to match with the lowest titer one which had the titer of 4.59x1012vg / mL. For each mouse, 450 nL of the GEVI virus was mixed with 50 nL of CaMKIla Cre virus pre-diluted for 100 times in volume to make the GEVI virus (floxed) to diluted CaMKIla Atty. Docket: STAN-2221 WO (S24-333)

[0252] Cre virus ratio to be 148:1 in terms of virus particles. The intracranial virus injection was done on the left hemisphere at the coordinate; ML -1.5 mm, AP 1.0 mm, and DV 1.2 -1.0 mm (all measured from bregma), for sparse labeling of layer 2 / 3 pyramidal neurons in motor cortex. Cranial window surgery was done either directly following the injection or 3-4 weeks after the injection. No noticeable difference was observed in expression or cranial window quality between the two groups. In brief, a 3x3-mm craniotomy was cut using #11 scalpel blades (Fine Science Tools) and a square coverslip (#1 coverslip glass, Warner Instruments) was implanted on top of the dura within the craniotomy with mild compression of the brain. The window was centered at ML -2.0 mm, AP 0.5 mm. The window was sealed to the skull using dental cement (C&B Metabond, Parkell) and at the end of surgery, a custom titanium headplate was attached to the skull to fixate the head to the microscope stage during subsequent imaging. Once the animals recovered, and the window and head-plate stayed in good quality, all the mice were made blind to the experimenter by another researcher. The experimenters were left blind for the identity of each animal until all one-photon in vivo imaging and analyses were done. The identity was only revealed for the statistical comparison between GEVI groups.

[0253] One-photon in vivo imaging and analysis

[0254] The 1 -photon wide-field voltage imaging setup was similar to what was previously described9. Briefly, a BX-51 microscope (Olympus) equipped with a 470-nm LED (SOLIS-470C, Thorlabs), a long-working distance x20 objective lens (NA 1 .0, Olympus), a Flash4.0 V2 scientific CMOS (sCMOS) camera (C11440-22CA, Hamamatsu), and a FITC-5050A filter set (Semrock) was used. High-speed acquisition of fluorescence images at 500 fps was achieved by reading 64 vertical lines from the 4x4-binned sCMOS sensor. Light intensity was 50 mW / mm2for all animals. Resulting images were converted to TIF files and corrected for lateral motion using turboReg50(imagej.net / plugins / turboregj. The ROIs for neurons and background were selected using a customized program in MATLAB (Mathworks). Then the fluorescence trace was extracted from the TIF files, background-subtracted, and photobleaching-corrected for spike detection and quantification in customized Python program. To correct for photobleaching, a piece-wise step function with 0.5-s window size was used to fit the fluorescence trace to estimate the baseline. In each 0.5-s window, the baseline was the average of the 30-80 percentile of the fluorescence values, to exclude extreme values and signals. Spike location was determined using the spikepursuit algorithm in Volpy51. For brightness comparison, background subtracted fluorescence intensity during the first second of each cellular ROI was averaged over time and compared. For SNR comparison, the noise was estimated by the standard deviation of the 20 Hz- Atty. Docket: STAN-2221 WO (S24-333) high-pass-filtered and spike-removed trace, to exclude supra- and sub-threshold activities in the recording, and the signal was defined by the peak value of the spikes. The Python programs for data processing can be find in a GitHub repository (github.com / AlexYkHao / spikenotes.git).

[0255] Confocal imaging of brain slices

[0256] To evaluate sparse GEVI expression in motor cortex after one-photon in vivo voltage imaging, the brain was sliced and imaged as previously described49. Briefly, mice were anesthetized with isoflurane and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) by transcardiac perfusion. After craniotomy, the brain was dissected out and then further fixed in 4% PFA / PBS in room temperature for 1-2 h, and then further incubated in 4 °C for 24 h. The brain was transferred to 30% sucrose / PBS solution for at least 3 days. The fixed brain was frozen and sliced into 50-pm thick coronal sections using a sliding microtome (Leica). The brain slices were mounted on a slide glass in DAPI containing mounting media (VectaShield H-1500, Vector Laboratories). The slide was imaged using a confocal microscope (LSM900, Zeiss).

[0257] Two-photon in vivo imaging of Drosophila

[0258] Female flies, 5 days post eclosion, were mounted, dissected to expose the brain, and perfused with an oxygenated saline-sugar solution during imaging, following standard methods18. For Mil and L2, a 920 nm laser beam with 25 mW of post-objective power excited the fluorophores, and photons were collected with a 525 / 50-nm filter. Data was collected at a framerate of 354 fps and a resolution of 0.265 pm in X and Y using a resonant-galvanometric scanning microscope (Broker) with a x20 1 .0-NA objective (Leica).

[0259] Visual stimuli were generated as previously described52. Briefly, two projectors (Lightcrafter 4500, Texas Instruments) with a 482 / 18-nm filter generated visual stimuli at 120 Hz. A photodiode (Thorlabs, SM05PD1A) was used to align the timing of the stimulus to image acquisition. Visual stimuli were full-field 24-ms contrast increments and decrements (50 presentations each, in random order), interleaved in time by 500 ms presentations of gray. Images were motion-corrected using turboReg50. Background was subtracted for each resonant- galvanometric scanning line before analysis. To do this, 20 contingent pixels with the lowest brightness value were averaged to calculate the background value for each scanning line. The extracted fluorescence traces were filtered by 120Hz-band-pass filter to further remove bleed- through photons from the projectors with 120 Hz refresh rate. The peak response (peak AF / F) was computed by identifying the AF / F value farthest from zero in the expected direction (depending on the stimulus), and the time to peak (tpeak) was the time of this response relative to Atty. Docket: STAN-2221 WO (S24-333) the start of the contrast change. For calculating SNR, the noise was estimated by the standard deviation of the signal in the 100 ms window before the onset of the visual stimulus, and the signal was defined by the peak value of the stimulus-triggered optical signal.

[0260] For the TPN-II neuron, when imaged using the resonant scanning microscope (Broker), a 920 nm laser beam with 25 mW of post-objective power was used and data was collected at 779 fps and 0.38 pm x-y resolution using a resonant-galvanometric scanning microscope (Broker) with a x20 1 .0-NA objective (Leica). On one AOD microscope (Karthala) a 920 nm laser beam with 25 mW of post-objective power was used and data was collected at 3333 fps using a x25 1.1-NA objective (Nikon). To simultaneously image two TPN-II neurons in the same brain, a second 3D AOD microscope (Femtonics) with 25 mW of post-objective power was used and data was collected at 3288 fps using a x25 1.1 -NA objective (Nikon). For calculating SNR, the noise was estimated by the standard deviation of the 20 Hz-high-pass-filtered and spike-removed trace, to exclude supra- and sub-threshold activities in the recording, and the signal was defined by the peak value of the spikes.

[0261] Two-photon in vivo voltage imaging in mouse motor cortex

[0262] Resonant-galvanometric two-photon voltage imaging

[0263] Two-photon imaging in motor cortex was performed in the same mice used for one-photon in vivo imaging in mouse motor cortex. Following surgery and one-photon imaging (described under “One-photon voltage imaging in mouse and sensor comparison”), images were acquired using a custom-built two-photon microscope system with a resonant scanner (LotosScan, Suzhou Institute of Biomedical Engineering and Technology) and a x25 / 1.0 NA water immersion objective lens (Olympus). A mode-locked tunable ultrafast laser (lnSightX3, Spectra-Physics) at 925 nm was used for imaging with a post-objective power of 30-160 mW. A 240x48-pixel field of view was imaged at 500 fps using a pixel size of 0.6 pm in X and 1.0 pm in Y. Resulting images were exported in TIF files and analyzed in the same pipeline as described in the one-photon imaging method session.

[0264] Three-dimensional in vivo random access two-photon voltage imaging

[0265] For the 3D random-access two-photon voltage imaging, used was a Femto 3D Atlas Plug & Play microscope which was made portable for demonstration purposes by Femtonics (Hungary). This portable stand-alone rig was equipped with a 920nm fiber-laser and acousto-optic Atty. Docket: STAN-2221 WO (S24-333) crystals for random-access two-photon volumetric excitation through a x160.80-NA long-working distance objective lens (Nikon). The post-objective laser power was 25 mW.

[0266] During imaging, a mouse sparsely expressing ASAP5-Kv in motor cortex under the CaMKIla promoter was head-fixed to the custom-made head-post and run on a running wheel device modified to fit onto the demo scope’s specimen stage. The running was recorded by a running encoder and the mouse motion was imaged using an infrared-camera for the duration of imaging. Resulting images were exported in TIP files and fluorescence traces were extracted using a customized Python program.

[0267] Two-photon in vivo voltage imaging in mouse visual cortex in behaving mice

[0268] Viral injections, and surgeries

[0269] AAV9-EF1 -DIO-ASAP5-Kv viruses were injected at a titer of 3x1012vg / ml and AAV2 / 1 hSyn-Cre (Addgene, AV-1 -PV2676) was co-injected at a final titer of 2.109GC / ml). Viruses were combined in a saline solution containing 0.001% of pluronic acid (ThermoFischer 24040032), 300 nl of which was injected at a flow rate of 75 nl / min into the visual cortex (V1 coordinates from bregma: anteroposterior -31-3.5 mm, mediolateral —2.5 / — 3 mm, and dorsoventral -0.3 mm from brain surface). A custom-designed aluminum head-plate was fixed on the skull with layers of dental cement (Metabond). A 5-mm diameter #1 coverslip was placed on top of the visual cortex and secured with dental cement (Tetric evoflow). Mice were allowed to recover for at least 15 days before recording sessions and housed at least 2 mice per cage. Behavioral habituation was adopted, involving progressive handling by the experimenter with gradual increases in head fixation duration53. Mice were handled before recording sessions to limit restraint-associated stress, and experiments were performed during the light cycle.

[0270] Signal analyses, spikes extraction and waveform analyses

[0271] Photobleaching was assessed by bi-exponential fitting and corrected by division of the raw trace by the normalized fit function. After removing the remaining low frequency drift using a zero-phase distortion filter (high pass: 0.5 Hz), the trace was converted to % AF / F0taking the mean signal as Fo. Spikes were detected with a custom designed algorithm, which utilizes three metrics to sort aspects of spike shape and threshold, all of them with zscores above 3. The first metric is to high pass filter the trace (second order Butterworth filter with lower limit set at 40Hz). The second metric is a cumulative probability transformation of the signal using the standard erf function for a duration of 1 .6 ms. The third metric takes the cumulative product of the 2ndto the Atty. Docket: STAN-2221 WO (S24-333)

[0272] 5thscale of the coif 1 discrete wavelet transform and applies a global realignment to retrieve energy in various frequency bands in one peak. To extract the spike waveform metrics, an average spike waveform was aligned on the spike onset. Then spike amplitude was taken as the peak value of the spike waveform average, measured from the onset point. FWHM corresponds to the extent in time at half maximum amplitude, taken from the average spike waveform after 20KHz linear interpolation. To quantify subthreshold fluctuations, traces were filtered using a bidirectional Butterworth bandpass filter between 0.1 and 30Hz. The cell up state corresponds to the mean signal within 5ms surrounding detected spikes, and the cell down state corresponds the 1stpercentile of that filtered trace. The subthreshold fluctuation is taken as the difference between the cell up and the cell down states.

[0273] Statistics

[0274] Statistical analyses were performed in Prism 10 (GraphPad). Specifically, distributions were first checked for normality by the Kolmogorov-Smirnov test. For comparisons of two groups, unpaired t-tests were performed for normally distributed data, and Mann-Whitney tests for nonparametric data. For comparisons of more than two groups, one-way analysis-of-variance (ANOVA) followed by pairwise t-tests with Bonferroni corrections was performed for normally distributed data. For nonparametric data, Kruskal-Wallis tests followed by Dunn's multiplecomparisons tests was used.

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[0333] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents Atty. Docket: STAN-2221 WO (S24-333) thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

Atty. Docket: STAN-2221 WO (S24-333)WHAT IS CLAIMED IS:1 . A nucleic acid encoding a voltage indicator polypeptide, wherein the voltage indicator polypeptide comprises:(c) a voltage-sensing domain (VSD) comprising four transmembrane segments; and(d) a circularly permuted fluorescent protein inserted into an extracellular loop between the third transmembrane segment (S3) and the fourth transmembrane segment (S4) of the VSD, wherein the voltage indicator polypeptide comprises an amino acid substitution at position 1412, F413, Q414, or any combination thereof, wherein numbering of positions is according to SEQ ID NO: 1.

2. The nucleic acid of claim 1 , wherein the voltage indicator polypeptide comprises an amino acid substitution chosen from 1412V, F413I, Q414R, or any combination thereof.

3. The nucleic acid of claim 1 , wherein the voltage indicator polypeptide comprises an amino acid substitution chosen from 1412V, F413V, Q414R, or any combination thereof.

4. The nucleic acid of any one of claims 1 -3, wherein the voltage indicator polypeptide comprises an amino acid substitution at each of 1412, F413, and Q414.

5. The nucleic acid of any one of claims 1 -4, wherein the voltage indicator polypeptide further comprises an amino acid substitution at position D145, R149, or both.

6. The nucleic acid of claim 5, wherein the voltage indicator polypeptide comprises an amino acid substitution chosen from D145A, R149K, or both.

7. The nucleic acid of claim 5, wherein the voltage indicator polypeptide comprises an amino acid substitution chosen from D145C, R149K, or both.

8. The nucleic acid of any one of claims 5-7, wherein the voltage indicator polypeptide comprises an amino acid substitution at each of D145 and R149.Atty. Docket: STAN-2221 WO (S24-333)9. The nucleic acid of any one of claims 1 -8, wherein the voltage indicator polypeptide comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% amino acid sequence identity to SEQ ID NO: 3 or SEQ ID NO: 5.

10. The nucleic acid of any one of claims 1 -8, wherein the voltage indicator polypeptide comprises 146L(insert), T148A, T207Y, or any combination thereof, relative to the amino acid sequence set forth in SEQ ID NO: 3, and wherein the voltage indicator polypeptide comprises 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, or 95% or greater amino acid sequence identity to SEQ ID NO: 3.11 . The nucleic acid of any one of claims 1 -8, wherein the voltage indicator polypeptide comprises N138A, 146L(insert), G147A, T148A, G151 S, D152E, R172I, T207Y, F227D, T392I, or any combination thereof, relative to the amino acid sequence set forth in SEQ ID NO: 3, and wherein the voltage indicator polypeptide comprises 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, or 95% or greater amino acid sequence identity to SEQ ID NO: 3.

12. The nucleic acid of any one of claims 1 -8, wherein the voltage indicator polypeptide comprises 146L(insert), G147E, T148A, G151 D, D152T, R172F, T207Y, F227N, Q316A, or any combination thereof, relative to the amino acid sequence set forth in SEQ ID NO: 3, and wherein the voltage indicator polypeptide comprises 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, or 95% or greater amino acid sequence identity to SEQ ID NO: 3.

13. The nucleic acid of any one of claims 1 -12 operably linked to a promoter.

14. A cell comprising the nucleic acid of claim 13.

15. The cell of claim 14, wherein the cell is a human cell.

16. The cell of claim 14 or 15, wherein the cell is a stem cell.Atty. Docket: STAN-2221 WO (S24-333)17. The cell of claim 16, wherein the stem cell is an embryonic stem cell.

18. The cell of claim 16, wherein the stem cell is an induced pluripotent stem cell.

19. The cell of claim 14 or 15, wherein the cell is a neuron that expresses the voltage indicator polypeptide on the plasma membrane of the neuron.

20. The cell of claim 19, wherein expression of the voltage indicator polypeptide is targeted to the cell body of the neuron.21 . The cell of claim 19 or 20, wherein the neuron is an induced neuron (iN) differentiated from the stem cell of any one of claims 16-18.

22. A method of recording electrical activity of a neuron, the method comprising: irradiating the neuron of any one of claims 19-21 with light at an excitation wavelength of the voltage indicator polypeptide; recording fluorescence emission from the voltage indicator polypeptide over time, wherein the fluorescence emission is correlated with the electrical activity of the neuron.

23. The method of claim 22, wherein the electrical activity comprises excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs), or both.

24. The method of claim 23, wherein the EPSPs and IPSPs are spontaneous EPSPs and IPSPs.

25. The method of any one of claims 22-24, wherein action potential generation is blocked for a duration of the recording, optionally wherein action potential generation is blocked during the entire duration of the recording.

26. The method of claim 25, comprising contacting the neuron with a voltage-gated sodium channel blocker to block action potential generation.Atty. Docket: STAN-2221 WO (S24-333)27. The method of any one of claims 22-24, wherein the electrical activity comprises action potentials.

28. The method of any one of claims 22-27, wherein the electrical activity is in response to a stimulus.

29. The method of claim 28, wherein the stimulus is an electrical current, a drug, a ligand for a receptor, a ligand for an ion channel, a ligand for an ion transporter, a hormone, a second messenger, photostimulation of a photogated channel, or any combination thereof.

30. The method of any one of claims 22-29, wherein the neuron comprises a mutation associated with neuronal dysfunction.31 . The method of any one of claims 22-30, wherein the method is performed in vitro.

32. A method of assessing an effect of an agent on the electrical activity of a neuron, the method comprising: contacting the neuron of any one of claims 19-21 with the agent; irradiating the contacted neuron with light at an excitation wavelength of the voltage indicator polypeptide; recording fluorescence emission from the voltage indicator polypeptide over time, wherein the fluorescence emission is correlated with the electrical activity of the neuron; assessing an effect of an agent on the electrical activity of a neuron.

33. The method of claim 32, wherein the assessing comprises comparing the electrical activity of the neuron contacted with the agent to the electrical activity of the neuron in the absence of the agent.

34. The method of claim 32, wherein the assessing comprises comparing the electrical activity of the neuron contacted with the agent to the electrical activity of a control neuron not contacted with the agent.Atty. Docket: STAN-2221 WO (S24-333)35. The method of any one of claims 32-34, wherein the electrical activity comprises one or more of: the frequency of action potentials, the frequency of EPSPs and / or IPSPs, the frequency of EPSPs and / or IPSPs when action potential generation is blocked, the extent of EPSP and / or IPSP decay during propagation along dendrites, baseline voltage, and synchrony of electrical activity with one or more surrounding neurons.

36. The method of any one of claims 32-35, wherein the neuron comprises a mutation associated with neuronal dysfunction.

37. The method of any one of claims 32-36, wherein the agent is from a library of agents, and wherein the method comprises screening the library for agents that alter the electrical activity of neurons.

38. The method of any one of claims 32-37, wherein the agent is a small molecule.

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