Claims 20
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
- figs2-neurons-tested
The tetrodotoxin experiment was replicated in several neurons, with vehicle controls; the representative traces of Figure S2 come from at least 4 independent replications in different cells.
Representative traces of the optical membrane potentials were selected from at least 4 and usually 8-15 independent replications in different cells
Figure S2 legend; Methods, Quantification and statistical analysisHow it will be judged
Neurons are counted from the dataset's subjects, sessions or cell identifiers, each recording counted once (author, step 1), and split into TTX and vehicle by the dataset's session metadata. The paper does not say whether the 4 replications count TTX neurons alone; readings (a) at least 4 TTX neurons, (b) at least 4 neurons over TTX and vehicle together. As a reported number read two ways, reproduced when either reading holds (protocol, step 3). The tolerance is 0 on a lower bound. The number of vehicle neurons is reported; the legend gives no count and the figure shows one, so at least one vehicle neuron is expected and its absence is reported as a finding.
- figs2-before-firing-then-depolarization
Before the drug, unmodulated 2-kHz sine waves evoked repetitive APs at lower fields and, at the most intense fields, a sustained depolarization in which APs merged into small oscillations.
How it will be judged
F1 to F3, under O1 to O4, sessions before the change only. Per neuron, the session at its highest field and its sessions at lower fields. Reproduced when every neuron (TTX and vehicle) has APs in some session, and more than half of them (a standard choice; the paper gives no fraction) show sustained depolarization (O4) at their highest field and APs without sustained depolarization at a lower field. The AP count and the maximum firing rate (F5) per session are reported against field.
- figs2-depolarization-reversible
The sustained depolarization induced by intense unmodulated sine waves reversed immediately when the stimulus ended, so it was not membrane electroporation, which would take seconds or longer to repair.
The sustained depolarization induced by intense unmodulated sine waves ( Figures 1 and 3 ) was immediately reversible upon stimulus cessation. It was not related to membrane electroporation, which would take seconds or longer to repair
Results, Tetrodotoxin blocks sustained depolarization, paragraph 1How it will be judged
F1 to F3, under O1 to O4, all sessions with sustained depolarization (O4), before the change and in vehicle neurons. "Immediately" is not quantified; readings of the window after the stimulus (a) 10 to 40 ms after its end, (b) the last 40 ms of the recording (the Methods give about 90 ms after the stimulus). Reproduced when, in every such session, the mean dF in the window is below the O4 level of that reading. A further check, from the paper's "seconds or longer", is that F0 of the next session of the same neuron (20 to 60 s later) is within 10% (a standard choice) of the F0 of the session before; it is reported, not judged. Windows are standard choices; the claim is tested on the Figure S2 neurons, while the paper's sentence cites Figures 1 and 3.
- figs2-ttx-abolishes-aps
1 uM tetrodotoxin abolished the APs evoked by unmodulated 250-ms, 2-kHz sine waves.
the inhibition of sodium channels with 1 μM tetrodotoxin not only abolished the APs
Results, Tetrodotoxin blocks sustained depolarization, paragraph 1; Figure S2 legendHow it will be judged
F1 to F4, under O1 to O3 and O5. Per TTX neuron, session pairs at the same field before and after TTX. Reproduced when, in every TTX neuron, no AP is detected in any session after TTX at a field where APs were detected before; not reproduced when any such session has an AP. The AP counts before and after per pair are reported.
- figs2-ttx-blocks-sustained-depolarization
Tetrodotoxin also fully blocked the sustained depolarization caused by intense sine waves, consistent with the depolarization arising from repeated activation of voltage-gated sodium channels by the cycling de- and hyperpolarization of the membrane.
Consistent with this hypothesis, 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
F1 to F4, under O1, O2, O4 and O5. Per TTX neuron, the session pairs at the same field where sustained depolarization (O4) was present before TTX. Reproduced when, in every such pair, sustained depolarization is absent after TTX, and the mean dF over the last 100 ms of the stimulus after TTX is below the O4 level. "Fully" is read as that level; the mean dF over the window before and after is reported per pair, with a two-sided paired t test (F6) over pairs shown as a variant, not judged. Blocked (other) when no TTX neuron has sustained depolarization before TTX.
- figs2-vehicle-keeps-response
In vehicle controls, recorded about 5 min after the solution was replaced by the same solution without TTX, the firing and sustained depolarization persisted.
How it will be judged
F1 to F4, under O1 to O5. Per vehicle neuron, session pairs at the same field before and after the change. Reproduced when, in every vehicle neuron, every pair with APs before has APs after, and every pair with sustained depolarization (O4) before has it after. AP counts and the maximum firing rate (F5) per pair are reported; the paper does not say whether the response must be unchanged, so a decline is reported, not judged. Blocked (other) when the dataset holds no vehicle neuron.
- ap-amplitude-duration
Neurons were selected for APs peaking above 5% dF and lasting about 10 ms; typical APs were 11.3% dF high and 8 ms long, excluding the afterhyperpolarization.
we selected neurons whose APs peaked at a more than 5% Δ F and lasted about 10 ms (typical values ... 11.3% ± 0.9% Δ F and 8 ± 0.7 ms duration, excluding afterhyperpolarization ... mean ± SEM, n = 22)
Methods, Recording of the optical APs and data analysesHow it will be judged
F1 and F2, under O1 to O3, sessions before the change. The paper does not say which 22 neurons these are or which experiments they come from; the neurons of this dataset are tested as neurons the same selection was applied to, and a mismatch may come from that. Per neuron, the mean peak dF and duration of its APs. Which APs are measured is not given; readings (a) all APs before the change, (b) the first AP of each session, which is not merged with others. The duration measure is not given; readings (a) width at half height, (b) time from the rise to the return to F0 on the decay, ending at the first crossing of F0 (so the afterhyperpolarization is left out). These readings are method details that can change the outcome, so the header's rule applies. Reproduced when the means over neurons are within the paper's SEM under every reading; partially reproduced when, under every reading, every neuron has APs above 5% dF and lasting 2 to 20 ms (O3) but a mean falls outside; blocked as methods_underspecified when the readings give different outcomes. The number of neurons with APs above 5% is reported.
- 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 sustained depolarization at the highest fields.
Pure sine waves at 2 and even 20 kHz excited 100% of neurons ... at near-threshold electric field strengths, isolated APs emerged with a delay of 100-200 ms after the sine wave onset ... 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 F3, F5, O1 to O4 of the header, for FluoVolt sessions of unmodulated sine waves. A neuron's threshold is the lowest field of a session with at least one AP during the stimulus (Figure 2D legend). Reproduced when, under every reading, at least 90% of neurons are excited at each frequency, the median over neurons of the highest maximum firing rate lies within 54 to 99 Hz (60 to 90 Hz widened by 10% of each bound) and the median first-AP latency at threshold lies within 90 to 220 ms (100 to 200 ms widened the same way). Whether the ceiling is meant for the median neuron or every neuron is open (the inset shows three); both are reported and the median decides. Sustained depolarization (O4) at the highest field is reported, not judged, as the paper gives no fraction of neurons.
- fig1-20khz-needs-twice-the-field
All excitation 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, 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 for at least 1 and at least 5 APs during the 250-ms stimulus (Figure 2D legend), from the threshold search of the Methods (5% to 10% steps), 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; the 2-kHz unmodulated 1-AP threshold was 57.7 V/cm.
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 2D; Results, Asymmetric and less-efficient membrane charging, last paragraphHow 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), the 1-AP threshold at 2 kHz is not lower for modulated waves at p < 0.05, and the mean 2-kHz unmodulated 1-AP threshold is within the paper's SEM, under every AP reading. The paper calls 57.7 V/cm "the AP threshold" for unmodulated 2-kHz sine waves (Figure 2D) without saying 1 or 5 APs; it is read as the 1-AP threshold, the excitation threshold of Figures 2D and 3B, and the 5-AP mean is reported beside it; 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), so each beat peaks 25 ms into it; the dataset's stimulus record 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 firing or sustained depolarization (O4).
- 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 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.
- fig3-not-heating
Excitation by pure sine waves was not caused by heating; the adiabatic temperature rise was 2.5 C after 250 ms at 63 V/cm in NaCl solution, at most 1.24 C for the same stimulus in the low-conductivity solution, and 4.1 C at 81 V/cm in NaCl.
the maximum theoretical temperature rise (assuming no heat dissipation 43 ) was 2.5°C after 250 ms of near-threshold stimulation at 2 kHz, 63 V/cm, in the NaCl-based solution ... These observations prove that excitation by pure sine waves was not caused by heating
Results, Decreased extracellular conductivity lowers excitation thresholds, paragraph 2; Figure 3AHow it will be judged
The rise is sigma E^2 t over the volumetric heat capacity (reference 43, cited for the adiabatic assumption). The paper does not give the heat capacity or whether E is the peak or the RMS field; readings (a) peak, (b) RMS, each with the heat capacity of water (4.18 J per cm3 per K, a standard choice). Reproduced when all three are within tolerance under a reading; since these are calculated numbers read two ways, that reading is reported. Computed from the paper's own values (1.64 S/m, 63 V/cm, 0.25 s), neither reading gives 2.5 C (peak about 3.9 C, RMS about 1.95 C), while the ratios 4.1/2.5 and 1.24/2.5 follow the formula's scaling with E^2 and with conductivity. The heat capacity or field definition the paper used is therefore not known; when no reading matches the absolute values but the two ratios are within 10%, the claim is blocked as methods_underspecified, and it is not reproduced only when the ratios fail as well. That the Figure 3A neuron fired at 63 V/cm in NaGlu as at 81 V/cm in NaCl is tested as in fig3b-low-conductivity-halves-threshold.
- 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 (O4, over the last 250 ms of the stimulus). The windows are standard choices.
- figs1-di8-same-as-fluovolt
With Di-8-ANEPPS in place of FluoVolt, in eight neurons, the effects of 2-kHz sine waves were not different, so the excitation by unmodulated sine waves is not a sensitization by FluoVolt.
Experiments were performed on eight neurons using different electric field strengths tested in a random sequence ... 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; Figure S1How it will be judged
As in the claims file for DANDI:001272 (figs1-eight-neurons and figs1-effects-same-as-fluovolt), which tests this finding on its own data.
- 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 gives other bounds and test types for these comparisons (p < 0.01, paired two-tailed, charging against discharging; p < 0.01, unpaired two-tailed, between orthogonal orientations); the statistics above are the Results'.
- 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 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 paper's, the last being 0.11% times 10 mV per 1%. Reproduced when both are within tolerance.
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