Claims 17
From the paper Excitation and polarization of isolated neurons by high-frequency sine waves for temporal interference stimulation · claims file · checked 2026-10-22: ready
- fig4-polarized-poles-face-electrodes
A 2-us pulse polarizes mainly the membrane facing the electrodes, depolarized at the cathode-facing pole and hyperpolarized at the anode-facing one, while even large neurites show little or no polarization.
In neurons of all shapes, the electric field primarily polarized membrane portions facing the electrodes ... Unexpectedly, even large neurites showed little to no polarization and did not seem to impact the charging of cell soma
Results, Kinetics of membrane charging by pulsed electric fields, the paragraph after the Figure 4 legend; Figures 4A to 4CHow it will be judged
Per experiment, the filtered RATIO image of the Methods (mean of the frames in the last 0.5 us of the pulse over the mean of the frames before the pulse and late after it, each low-pass filtered over 12 x 12 pixels). The field direction per experiment is taken from the dataset. The paper gives no measure; reproduced when, in more than half of the experiments of each shape group of the Figure 4D and 4E legend (or of all 19 when the labels are missing, O4), the extreme depolarized and extreme hyperpolarized pixels of the cell lie on opposite sides of its centroid along the field axis (the angle between their separation and the field axis below 45 degrees). The majority, the centroid and the 45 degrees are standard choices. The part on neurites needs a neurite mask that the paper does not give and is reported, not judged.
- fig4-orientation-time-constants
In elongated neurons, charging was about 2-fold faster with the field orthogonal to the long axis; aligning elongated cells with the field doubled the time constants compared with the same cells perpendicular to it or with randomly shaped cells.
Aligning elongated cells with the electric field doubled the time constants, as compared to either the same cells oriented perpendicular to the field ( p < 0.001, paired t test) or to randomly shaped cells ( p < 0.01, unpaired t test)
Results, Kinetics of membrane charging by pulsed electric fields, the paragraph after the Figure 4 legend; Figures 4A, 4D and 4EHow it will be judged
Fits as in the header (F1 to F4), groups from the dataset's labels (O4), as in the Figure 4D and 4E legend (parallel n = 3, perpendicular n = 6, random n = 10, random cells with both orientations pooled). The tau compared are charging and discharging, at each pole. The text names a paired t test (p < 0.001) for parallel against perpendicular; the legend marks p < 0.01 between orientations with an unpaired two-tailed t test (O5). Readings of that test (a) paired, on elongated cells imaged in both orientations, (b) unpaired, on the legend's groups. Which time constants "the time constants" means is not given, and the text's first sentence names charging only; readings of the subset (i) charging tau, (ii) charging and discharging tau, each at both poles. Reproduced when, under one subset reading, the parallel mean is within tolerance of twice the perpendicular mean and of twice the random mean, and parallel against random reaches p < 0.01 and the orientation test its stated p (0.001 paired, 0.01 unpaired) at one pole at least, under every reading of the header (O1 to O3, F2) and both readings of the test; blocked as methods_underspecified when they disagree. The ratios at each pole are reported.
- fig4-no-pole-difference
The maximum optical TMP and the charging and discharging time constants did not differ between the cathode- and anode-facing poles, indicating no rectification of the field.
Δ F max did not differ at the cathodic and anodic cell poles, indicating the lack of rectification of the applied electric field. Charging and discharging time constants did not differ between the two poles either
Results, Kinetics of membrane charging by pulsed electric fields, the paragraph after the Figure 4 legend; Figures 4D and 4EHow it will be judged
A claim of absence. The text names no test for it; the Methods' test is a two-sided Student's t test, paired or unpaired as appropriate, at p < 0.05, and the two poles are in the same experiment, so a two-sided paired t test over the 19 experiments of |dFmax|, charging tau and discharging tau, cathodic against anodic. Fits as in the header. Reproduced when none of the three is significant at p < 0.05 under every reading of the header; not reproduced when any is under every reading; blocked as methods_underspecified when the readings disagree. The mean differences and their 95% intervals are reported either way.
- fig4-discharging-slower
Discharging time constants were larger than charging ones in the same cell, except in elongated cells aligned with the field.
discharging time constants were always larger than the charging ones in the same cell ( p < 0.001, paired t test), with the exception of elongated cells aligned with the field
Results, Kinetics of membrane charging by pulsed electric fields, the paragraph after the Figure 4 legend; Figures 4D and 4EHow it will be judged
Fits as in the header. Two-sided paired t test of discharging against charging tau. The pooling behind the text's p < 0.001 is not given; readings (a) all 19 experiments, both poles, (b) the experiments other than elongated parallel, both poles, (c) as (b), each pole alone, and per group and pole for the legend's marks. Reproduced when, under one of (a) to (c), discharging tau is larger with p < 0.001, and the elongated parallel group shows no difference at p < 0.05 at either pole (which needs the labels, O4), with every reading of the header giving that outcome; blocked as methods_underspecified when the header's readings disagree.
- fig4-pooled-time-constants
Pooled over all 19 experiments, charging and discharging time constants averaged 0.7 and 0.91 us at the cathodic pole and 0.65 and 0.8 us at the anodic pole.
The time constants pooled together for all cells averaged 0.7 ± 0.08 (charging) and 0.91 ± 0.08 (discharging) μs for the cathodic cell pole and 0.65 ± 0.05 and 0.8 ± 0.07 μs for the anodic pole, respectively (mean ± SEM, n = 19)
Results, Kinetics of membrane charging by pulsed electric fields, the paragraph after the Figure 4 legend; Figures 4D and 4E (ALL)How it will be judged
Fits as in the header. The mean over all 19 experiment fits, as the text (n = 19) and the Figure 4D and 4E legend (ALL, n = 19, all cells and all orientations) give it. Tolerances are the SEMs the paper gives. Reproduced when all four are within tolerance, with every reading of the header (O1 to O3, F2) giving that outcome; blocked as methods_underspecified when the header's readings disagree. The SEMs are reported.
- fig4-full-charge-within-3us
Membranes exceed 95% of full charge in under 3 us, even in the largest neurons and with the least efficient alignment with the field, so each cycle of a sine wave fully polarizes the membrane even at 100 to 200 kHz.
It will take <3 μs to exceed 95% of the full charge, even in the largest neurons and with the least efficient cell alignment with the field
Results, Kinetics of membrane charging by pulsed electric fields, the paragraph after the Figure 4 legendHow it will be judged
Under the paper's exponential model (Methods), 95% of full charge is reached at 3 tau (ln 20 = 3.0). Charging tau from the fits of the header. The paper does not say whether the bound holds for every cell or for the least efficient group; readings (a) 3 times the mean charging tau of the elongated parallel group (the least efficient alignment, Figure 4D and 4E) is below 3 us at both poles, (b) 3 times the largest charging tau of any experiment is below 3 us. Reproduced when (a) holds under every reading of the header; partially reproduced when (a) holds and (b) does not. The 100 to 200 kHz part is an inference and is not tested.
- fig1-pure-sine-excites-all
Unmodulated 250-ms sine waves at 2 and 20 kHz excited every neuron tested, with APs 100 to 200 ms after onset near threshold, firing rate rising to a ceiling of 60 to 90 Hz, and an excitation block with sustained depolarization at the highest fields.
Pure sine waves at 2 and even 20 kHz excited 100% of neurons ... The maximum firing rate increased linearly until reaching a ceiling of 60-90 Hz
Results, Stimulation by 2- and 20-kHz unmodulated sine waves, paragraph 1; Figures 1A to 1C and insetHow it will be judged
AP detection is given only as "brief emission upticks" in neurons whose APs peak above 5% dF and last about 10 ms (Methods, Recording of the optical APs); readings (a) a peak above 5% dF over F0, (b) a rise of at least 5% dF within 5 ms. The maximum firing rate of a session is the inverse of the interval between its first two AP peaks (Methods). Reproduced when, under both readings, at least 90% of neurons are excited at each frequency and the median ceiling lies within 54 to 99 Hz (the reported 60 to 90 Hz widened by 10% of each bound, written above as its midpoint and half-width), and the median first-AP latency at threshold lies within 90 to 220 ms (the reported 100 to 200 ms widened the same way); blocked as methods_underspecified when the readings disagree. The median over neurons is a standard choice. The block is reported, not judged.
- fig2d-modulated-thresholds-higher
Thresholds for 1 and 5 APs at 20 kHz were about 2-fold those at 2 kHz, and thresholds for sine waves modulated at 20 Hz were significantly higher than for pure sine waves for 5 APs at 2 kHz and for 1 and 5 APs at 20 kHz.
All thresholds at 20 kHz were about 2-fold higher than at 2 kHz ( p < 0.0001, two-tailed unpaired t test) ... thresholds for modulated sine waves, which emulate the TI stimulus at the target, were significantly higher than those for pure sine waves at the same frequency
Results, Excitation thresholds are similar or lower for pure 2- and 20-kHz sine waves than those for modulated sine waves, paragraph 1; Figure 2DHow it will be judged
The threshold of a neuron for a stimulus is the lowest field of a session with at least 1 (or 5) APs during the 250-ms stimulus (Figure 2D legend), AP detection as in fig1-pure-sine-excites-all, both readings. Two-tailed unpaired t tests as the text names them. The paper gives one p for 20 against 2 kHz over "all thresholds"; readings (a) one test per stimulus and AP count, each p < 0.0001, (b) one test pooling the four. Reproduced when each ratio is within tolerance, 20 against 2 kHz reaches p < 0.0001 under both readings of that test, and the three modulated pairs are higher with p < 0.05 (Methods), under both AP readings; blocked as methods_underspecified when the readings disagree.
- fig2-modulated-locks-to-beats
With 20-Hz modulation, firing above threshold locked to the beats, one AP near the peak of each of the five beats and at most two per beat (ten in all), and modulated sine waves triggered no sustained firing or sustained depolarization.
single APs emerged near the peak of each beat, so the firing rate equaled the modulation rate ... the maximum number of APs evoked by five beats was limited to ten. Unlike pure sine waves, modulated stimuli did not trigger sustained firing or sustained depolarization
Results, Stimulation by 2- and 20-kHz sinusoids modulated at 20 Hz, paragraph 1; Figures 2A and 2BHow it will be judged
AP detection as in fig1-pure-sine-excites-all, both readings. Modulated sessions only. Reproduced when no session has more than 10 APs, in sessions with 5 APs or more most APs fall in the half of each 50-ms beat around its peak (12.5 to 37.5 ms into the beat), and no session has, over the last 100 ms of the stimulus, no APs with mean dF above 5% (the block test of fig3c-sustained-firing-2s applied to that window). The paper does not quantify "near the peak"; the window, the majority and the block test are standard choices, and the AP phase histogram is reported.
- fig2c-pure-more-aps
In the same neuron at the same field strength, pure sine waves triggered more APs than modulated ones.
At the same field strengths, pure sine waves triggered a larger number of APs
Results, Stimulation by 2- and 20-kHz sinusoids modulated at 20 Hz, paragraph 2; Figure 2CHow it will be judged
AP detection as in fig1-pure-sine-excites-all, both readings. Pairs are a neuron's modulated and unmodulated sessions at the same carrier and the same field. The paper reports no test; reproduced when the unmodulated count is larger in more pairs than the reverse under both readings. A two-sided paired t test of the counts (the Methods' general test) is reported.
- fig3b-low-conductivity-halves-threshold
Lowering solution conductivity from 16.4 to 8.4 mS/cm with sodium gluconate reversibly lowered excitation thresholds about 2-fold at all sine wave frequencies from 2 to 20 kHz.
Switching to the NaGlu-based solution reversibly and reproducibly decreased excitation thresholds ~2-fold across all sine wave frequencies tested
Results, Decreased extracellular conductivity lowers excitation thresholds, paragraph 1; Figures 3A and 3BHow it will be judged
1-AP thresholds for 250-ms unmodulated sine waves (Figure 3B legend) as in fig2d-modulated-thresholds-higher, per frequency and solution, 4 to 8 neurons per point. Reproduced when the ratio is within tolerance at every frequency under both AP readings.
- fig3c-sustained-firing-2s
In the low-conductivity solution, a 2-s unmodulated 2-kHz sine wave ramped over 250 ms evoked firing for its whole duration, rising with field until replaced by a firing block with sustained depolarization.
we observed sustained firing for the entire duration of the sine wave ( Figure 3C ). The rate of firing increased with the electric field strength until it was replaced by a firing block and sustained depolarization
Results, Decreased extracellular conductivity lowers excitation thresholds, paragraph 3; Figure 3CHow it will be judged
Traces corrected for bleaching by subtracting a trace without APs (Methods); AP detection as in fig1-pure-sine-excites-all, both readings. Reproduced when an intermediate field has APs in the last 250 ms of the stimulus, the AP count rises with field up to some field, and the highest field has no APs after 250 ms with mean dF above F0 by more than 5% over the rest of the stimulus. The 5% is the Methods' AP selection level, used here as a standard choice.
- figs1-di8-anepps-control
With Di-8-ANEPPS instead of FluoVolt, in eight neurons, near-threshold stimulation elicited one or a few delayed APs and stronger fields induced sustained firing ending in sustained depolarization, as with FluoVolt.
Stimulation near the threshold elicited one or a few delayed APs. Increasing the electric field strength reduced the delay and induced sustained firing that culminated in sustained depolarization
Results, Sensitivity to unmodulated sine waves was not caused by FluoVolt dye, paragraph 1; Figure S1How it will be judged
Traces inverted, as Di-8-ANEPPS emission falls with depolarization (Methods). AP detection as in fig1-pure-sine-excites-all, with a third reading of 5 SD of the pre-stimulus trace, as the paper notes the dye is less sensitive. Reproduced when, under every reading, every neuron fires, first-AP latency falls with field in more than half, and the highest field ends in depolarization as in fig3c-sustained-firing-2s in more than half.
- figs2-ttx-blocks-depolarization
1 uM tetrodotoxin abolished both the APs and the sustained depolarization caused by intense unmodulated 2-kHz sine waves.
the inhibition of sodium channels with 1 μM tetrodotoxin not only abolished the APs but also fully blocked the sustained depolarization
Results, Tetrodotoxin blocks sustained depolarization, paragraph 1; Figure S2How it will be judged
Per neuron, sessions before and about 5 min after TTX or vehicle at the same field (Figure S2 legend). Reproduced when, in every TTX neuron, APs (as in fig1-pure-sine-excites-all) present before are absent after and the mean dF over the last 100 ms of the stimulus is above 5% before and below it after, while vehicle neurons keep their response. The window and level are standard choices.
- fig6-sine-less-than-pulse
FluoVolt sensitivity to 2-us pulses averaged 28% plus or minus 2.5% per 1 kV/cm (15% to 47%), and to 2-kHz sine waves 11.8% plus or minus 1.7% per 1 kV/cm, 42% of the pulse value, smaller in every ROI.
All numbers were invariably smaller than those measured for the same ROIs with the pulse ( Figure 6D ). The electric field sensitivity averaged across all ROIs in all the cells was 11.8% ± 1.7% per 1 kV/cm, just 42% of the value for the 2-μs pulses
Results, Asymmetric and less-efficient membrane charging by sine waves compared to pulses, paragraphs 4 and 5; Figures 6B to 6DHow it will be judged
Pulse sensitivity is |dF| averaged from 1 to 2 us into the 257 V/cm pulse, per kV/cm (Results; Figure 6B legend). Sine sensitivity is the regression coefficient of the emission change on the electric field after subtracting the LOESS fit of the whole-cell response (Figure 5H and 6C legends); the LOESS span and the form of the regression are not given, and their readings are those of the claims file for DANDI:001274, which tests this claim on its own data. Reproduced when the ratio is below 1 in all 18 ROIs and the expected values are within tolerance under every reading.
- fig5-sine-asymmetry
In single cells, sine waves polarized the two poles more asymmetrically than a 2-us pulse (ROI 2 up to 90% smaller than ROI 1 with sine waves against 30% with the pulse in Figure 5; 40% against 20% in Figure S3), with carrier oscillations antiphasic in the two ROIs on a gradual whole-cell depolarization.
Unexpectedly, in this cell, the oscillations in ROI 2 were up to 90% smaller than those in ROI 1 (compared to just 30% smaller when the same cell was polarized with the 2-μs pulse)
Results, Asymmetric and less-efficient membrane charging by sine waves compared to pulses, paragraphs 2 and 3; Figures 5B to 5D and S3How it will be judged
Measures and readings as in the claims file for DANDI:001274 (fig5-sine-asymmetry-c7, figs3-sine-asymmetry-c8, fig5-carrier-antiphasic and fig5-whole-cell-depolarization), which tests this finding on its own data. Reproduced when all four values are within tolerance and the antiphasic oscillations and whole-cell depolarization are found, under the readings that file names.
- threshold-depolarization-estimate
The 2-kHz AP threshold of 57.7 V/cm times the sine wave sensitivity gives a 0.68% emission change, about 6.8 mV, so the threshold depolarization is 5 to 15 mV, as with intracellular current injection.
This threshold corresponds to a 0.68% ± 0.11% FluoVolt emission change (11.8 × 57.7/1,000) ... a 0.68% emission change corresponds to a 6.8-mV depolarization at the AP threshold
Results, Asymmetric and less-efficient membrane charging by sine waves compared to pulses, paragraph 6How it will be judged
Mean sine sensitivity of fig6-sine-less-than-pulse times the mean unmodulated 2-kHz 1-AP threshold of fig2d-modulated-thresholds-higher, divided by 1,000, and that times 10 mV per 1% (the calibration of reference 47 as the paper uses it). Tolerances are the SEMs the paper gives, the last being the 0.11% times 10 mV per 1%. Reproduced when all three are within tolerance.
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