Claims 19
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
- figs1-eight-neurons
The Di-8-ANEPPS control experiments were made on eight neurons, at field strengths tested in a random sequence.
Experiments were performed on eight neurons using different electric field strengths tested in a random sequence
Results, Sensitivity to unmodulated sine waves was not caused by FluoVolt dye, paragraph 1; Figure S1How it will be judged
Neurons are counted from the dataset's subjects, sessions or cell identifiers, each recording counted once (author, step 1). The tolerance is 0, as a count of neurons is whole and 10% of 8 admits no other value. The order of fields over each neuron's sessions is reported (the number of neurons whose fields rise monotonically with session order), not judged, since the Methods also allow escalating sequences.
- figs1-aps-as-emission-decrease
With Di-8-ANEPPS, APs appear as brief reductions of emission, opposite in sign to FluoVolt.
APs were identified by the reduction instead of the increase of emission
Results, Sensitivity to unmodulated sine waves was not caused by FluoVolt dye, paragraph 1; Methods, Recording of the optical APs and data analyses; Figure S1 legendHow it will be judged
F1 and F2, under the readings O1 and O2. Per neuron, over the sessions at fields above its threshold (F4, under O3), the largest brief deflections of dF during the stimulus are negative. Detection under O3 is run on the trace as recorded (not inverted) and on the inverted trace; reproduced when in every neuron with APs the inverted trace yields more APs than the trace as recorded, in more than 75% of its sessions with APs (a standard choice). The counts each way are reported.
- figs1-near-threshold-delayed-aps
Near threshold, 2-kHz sine waves elicited one or a few delayed APs in Di-8-ANEPPS neurons.
Stimulation near the threshold elicited one or a few delayed APs
Results, Sensitivity to unmodulated sine waves was not caused by FluoVolt dye, paragraph 1; Figure S1How it will be judged
F1 to F4, under O1 to O5. Per neuron, the near-threshold sessions (O4); their AP count and first-AP latency from the onset (F3). The paper gives no count for "a few"; at most 3 APs is a standard choice. Reproduced when, in more than half of the neurons with APs (a standard choice), the median near-threshold session has 1 to 3 APs and its first AP is delayed (O5). Counts and latencies per neuron are reported.
- figs1-stronger-field-shorter-delay-sustained-firing
In Di-8-ANEPPS neurons, increasing the field strength reduced the delay to the first AP and induced sustained firing.
Increasing the electric field strength reduced the delay and induced sustained firing
Results, Sensitivity to unmodulated sine waves was not caused by FluoVolt dye, paragraph 1; Figure S1How it will be judged
F1 to F4, under O1 to O3 and O6. Per neuron with sessions with APs at 3 or more fields, the Spearman correlation of first-AP latency with field over those sessions. The paper reports no test; reproduced when the correlation is negative in more than half of those neurons and more than half of all neurons show sustained firing (O6) in some session. The majority and the Spearman correlation are standard choices. The maximum firing rate (F5) against field is reported per neuron.
- figs1-culminates-in-sustained-depolarization
In Di-8-ANEPPS neurons, the strongest fields replaced firing with sustained depolarization (excitation followed by inhibition of firing).
induced sustained firing that culminated in sustained depolarization
Results, Sensitivity to unmodulated sine waves was not caused by FluoVolt dye, paragraph 1; Figure S1 legendHow it will be judged
F1 to F4, under O1 to O3 and O6. Per neuron, its sessions at its highest field. The paper gives no fraction of neurons; reproduced when more than half of the neurons (a standard choice) show sustained depolarization (O6) at their highest field and sustained firing or more APs at some lower field. The Figure S1 legend's "Excitation and inhibition of firing" describes one neuron; the number of neurons with each is reported.
- figs1-noisier-than-fluovolt
Di-8-ANEPPS traces were noisier than FluoVolt traces, as the dye has a lower voltage sensitivity.
Compared to FluoVolt recordings, the traces were expectedly noisier (Di-8-ANEPSS has a lower voltage sensitivity)
Results, Sensitivity to unmodulated sine waves was not caused by FluoVolt dye, paragraph 1How it will be judged
F1 to F3, under O1 to O3, for the Di-8-ANEPPS neurons of this dataset and for the FluoVolt neurons stimulated with 250-ms unmodulated 2-kHz sine waves in NaCl solution in DANDI:001030 and 001032 to 001035 (assigned from their titles; FluoVolt traces not inverted). Per neuron, the signal to noise ratio is the median AP height over the SD of dF before the onset. "Noisier" is not defined; readings (a) that ratio, (b) the SD of dF before the onset. Reproduced when the Di-8-ANEPPS median is worse (lower ratio, larger SD) than the FluoVolt median under both. A two-sided unpaired t test (F6) is reported. As a variant on this dataset alone, the median Di-8-ANEPPS AP height is compared with the 11.3% typical of FluoVolt (Methods).
- figs1-effects-same-as-fluovolt
Overall, the effects of 2-kHz sine waves in Di-8-ANEPPS neurons did not differ from those with FluoVolt, so the excitation by unmodulated sine waves is not a sensitization by FluoVolt.
Overall, stimulation effects were not different from those observed with FluoVolt dye
Results, Sensitivity to unmodulated sine waves was not caused by FluoVolt dye, paragraph 1; Discussion, paragraph 2How it will be judged
A claim of absence, for which the paper names no test, and the paper does not say which effects were compared; the threshold comparison below is a reading of "not different", not a measure the paper names. The response pattern holds when figs1-near-threshold-delayed-aps, figs1-stronger-field-shorter-delay-sustained-firing and figs1-culminates-in-sustained-depolarization are reproduced. The paper's own concern is a sensitization by FluoVolt (Discussion, "we ruled out possible sensitization"), which would show as lower thresholds with FluoVolt; so the 1-AP threshold (F4) of the Di-8-ANEPPS neurons is compared with that of the FluoVolt neurons of figs1-noisier-than-fluovolt by a two-sided unpaired t test at p < 0.05 (F6). Reproduced when the pattern holds and the test finds no difference; not reproduced when the test finds a difference in either direction. The mean FluoVolt threshold of the neurons used is reported beside the paper's 57.7 ± 3.6 V/cm (Results, Asymmetric and less-efficient membrane charging, last paragraph, from Figure 2D) as a check on the reference group, not judged. A one-sample t test of the Di-8-ANEPPS thresholds against 57.7 V/cm is shown as a variant on this dataset alone. The difference of means and its 95% interval are reported. The Di-8-ANEPPS sessions may not have been a threshold search (fields in a random sequence), so the threshold is the lowest exciting field tested, which is an upper bound; the notebook reports the gap to the next lower field 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
F1 to F5 and O1 to O3 and O6 of the header, applied to FluoVolt traces without inversion. Reproduced when, under every reading, at least 90% of neurons are excited at each frequency, the median ceiling lies within 54 to 99 Hz (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 (100 to 200 ms widened the same way); blocked as methods_underspecified when the readings disagree. Whether the ceiling is meant for the median neuron or every neuron is open (the inset shows three); the median is a standard choice and the range over neurons is reported. The block is reported under O6, not judged.
- fig1-20khz-needs-twice-the-field
Responses to 20 kHz needed about 2-fold stronger fields than at 2 kHz, and all thresholds at 20 kHz were about 2-fold higher than at 2 kHz.
All thresholds at 20 kHz were about 2-fold higher than at 2 kHz ( p < 0.0001, two-tailed unpaired t test)
Results, Stimulation by 2- and 20-kHz unmodulated sine waves, paragraph 1; Results, Excitation thresholds are similar or lower, paragraph 1; Figure 2DHow it will be judged
Thresholds for at least 1 and at least 5 APs (Figure 2D legend), as F4, with the AP readings of the header. The paper gives one p over "all thresholds"; readings (a) one test per stimulus (modulated or not) and AP count, each p < 0.0001, (b) one test pooling the four. Reproduced when each ratio is within tolerance and 20 against 2 kHz reaches p < 0.0001 under every reading; blocked as methods_underspecified when they disagree.
- fig2d-modulated-thresholds-higher
Thresholds for sine waves modulated at 20 Hz were significantly higher than for pure sine waves at the same carrier, for 5 APs at 2 kHz and for 1 and 5 APs at 20 kHz, so modulation did not lower excitation thresholds.
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 ... p = 0.0079 ... p = 0.0142 and p = 0.0037
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
Thresholds as in fig1-20khz-needs-twice-the-field. Two-tailed unpaired t tests, as the Figure 2D legend names them. Reproduced when the three modulated thresholds are higher with p < 0.05 (Methods) and the 1-AP threshold at 2 kHz is not lower for modulated waves at p < 0.05, under every AP reading; blocked as methods_underspecified when they 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 readings of the header, modulated sessions only. The 250-ms wave holds five 50-ms beats (Results), and modulated waves begin and end at minimal amplitude (Methods, Pulsed laser fluorescence microscopy, F0 for sine waves), so each beat peaks 25 ms into it; the stimulus record of the dataset is used instead when it gives the envelope. The paper does not quantify "near the peak"; as standard choices, reproduced when no session has more than 10 APs, in sessions with 5 APs or more most APs fall within 12.5 to 37.5 ms of each 50-ms beat, and no session shows sustained depolarization (O6). 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, and the threshold was typically lower for unmodulated signals.
the threshold was typically lower for unmodulated signals ( Figures 2A - 2C ). 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; Figures 2A to 2CHow it will be judged
AP readings of the header. Pairs are a neuron's modulated and unmodulated sessions at the same carrier and field. The paper reports no test; reproduced when the unmodulated count is larger in more pairs than the reverse, and the unmodulated threshold lower in more neurons than the reverse, under every reading. A two-sided paired t test (F6) of the counts 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 (F4) for 250-ms unmodulated sine waves per frequency and solution, 4 to 8 neurons per point (Figure 3B legend), AP readings of the header. Reproduced when the ratio is within tolerance at every frequency under every reading.
- 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 readings of the header. 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 shows sustained depolarization (O6, over the last 250 ms of the stimulus). The windows are standard choices.
- figs2-ttx-blocks-depolarization
1 uM tetrodotoxin abolished both the APs and the sustained depolarization caused by intense unmodulated 2-kHz sine waves, while the vehicle did not.
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 S2 legendHow it will be judged
Per neuron, sessions before and about 5 min after TTX or vehicle at the same field (Figure S2 legend), AP readings of the header. Reproduced when, in every TTX neuron, APs present before are absent after and sustained depolarization (O6) present before is absent after, while vehicle neurons keep their response.
- fig4-pooled-time-constants
Membrane charging by 2-us pulses is fast; pooled over 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, so membranes charge fully within each sine wave cycle up to 100 to 200 kHz.
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 4EHow it will be judged
Fits and readings as in the claims file for DANDI:001273 (fig4-pooled-time-constants), which tests this claim on its own data; tolerances are the paper's SEMs.
- fig4-orientation-and-poles
Aligning elongated cells with the field doubled the time constants; the maximum optical TMP and the time constants did not differ between the cathode- and anode-facing poles; and discharging was slower than charging except in elongated cells aligned with 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 ... Aligning elongated cells with the electric field doubled the time constants ... discharging time constants were always larger than the charging ones in the same cell ( p < 0.001, paired t test)
Results, Kinetics of membrane charging by pulsed electric fields, the paragraph after the Figure 4 legend; Figures 4D and 4EHow it will be judged
As in the claims file for DANDI:001273 (fig4-orientation-time-constants, fig4-no-pole-difference and fig4-discharging-slower), which tests each part on its own data; reproduced when all three are reproduced there. The Figure 4 legend reports these tests differently from the Results (p < 0.01, paired two-tailed, charging against discharging; p < 0.01, unpaired two-tailed, between the orthogonal orientations, with n = 3 parallel and n = 6 perpendicular); the statistics above are the Results', and the legend's are a reading the paper also allows.
- fig6-sine-less-than-pulse
FluoVolt sensitivity to 2-kHz sine waves (11.8% per 1 kV/cm) was 42% of that to 2-us pulses (28% per 1 kV/cm) and smaller in every ROI, with sine waves polarizing the two poles more asymmetrically than pulses.
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 2 to 5; Figures 5, 6B to 6D and S3How it will be judged
Measures and readings as in the claims file for DANDI:001274 (fig6b-pulse-sensitivity, fig6c-sine-sensitivity, fig6d-sine-less-than-pulse, fig5-sine-asymmetry-c7 and figs3-sine-asymmetry-c8), which tests this finding on its own data; tolerances are the paper's SEMs, and 10% for the ratio.
- 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, last paragraphHow it will be judged
Mean sine sensitivity of fig6-sine-less-than-pulse times the mean unmodulated 2-kHz 1-AP threshold (F4), divided by 1,000, and that times 10 mV per 1% (the calibration of reference 47 as the paper uses it). Tolerances are the paper's SEMs, the last being 0.11% times 10 mV per 1%. Reproduced when all three are within tolerance.
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