Voltage imaging reveals the emergence of population activity in the spinal cord

Reviewer #1 (Public review):

Summary:

This paper demonstrates the first application of voltage imaging using a genetically encoded voltage indicator, ArcLight, for recording the spontaneous activity of the developing spinal cord in zebrafish. This technology enabled better temporal resolution compared to what has been demonstrated with calcium imaging in past studies (Muto et al., 2011; Warp et al., 2012; Wan et al., 2019 ), which led to the discovery of the maturation process of "firing" shapes in spinal neurons. This maturation process occurs simultaneously with axonal elongation and network integration. Thus, voltage imaging revealed new biological details of the development of the spinal circuits.

Strengths:

The use of voltage imaging instead of calcium imaging revealed biological details of the functional maturation of spinal cord neurons in developing zebrafish.

Weaknesses:

This manuscript lacks many basic components and explanations necessary for understanding the methodologies used in this study.

Reviewer #2 (Public review):

The authors present highly impressive in vivo voltage‐imaging data, demonstrating neuronal activity at subcellular, cellular, and population levels in a developing organism. The approach provides excellent spatial and temporal resolution, with sufficient signal-to-noise to detect hyperpolarizations and subthreshold events. The visualization of contralateral synchrony and its developmental loss over time is particularly compelling. The observation that ipsilateral synchrony persists despite contralateral desynchronization is a striking demonstration of the power of GEVIs in vivo. While I outline several points that should be addressed, I consider this among the strongest demonstrations of in vivo GEVI imaging to date.

Major points:

(1) Clarification of GEVI performance characteristics

There is a widespread misconception in the GEVI field that response speed is the dominant or primary determinant of sensor performance. Although fast kinetics are certainly desirable, they are not the only (or even necessarily the limiting) factor for effective imaging. Kinetic speed specifies the time to reach ~63% of the maximal ΔF/F for a given voltage step (typically 100 mV, approximating the amplitude of a neuronal action potential), but in practical imaging, a slower sensor with a large ΔF/F can outperform a faster sensor with a small ΔF/F. In this context, the authors' use of ArcLight is actually instructive. ArcLight is one of the slower GEVIs in common use, yet Figures S1a-b clearly show that it still reports voltage transients in vivo very well. I therefore strongly recommend moving these panels into the main text to emphasize that robust in vivo imaging can be achieved even with a relatively slow GEVI, provided the signal amplitude and SNR are adequate. This will help counteract the common misunderstanding in the field.

(2) ArcLight's voltage-response range

ArcLight is shifted toward more negative potentials (V₁/₂ ≈ −30 mV). This improves subthreshold detection but makes distinguishing action potentials from subthreshold transients more challenging. The comparison with GCaMP is helpful because the Ca²⁺ signal largely reflects action potentials. Panels S1c-f show similar onset kinetics but a longer decay for GCaMP. Surprisingly, the ΔF/F amplitudes are comparable; typically, GCaMP changes are larger. To support lines 193-194, the authors should include a table summarizing the onset/offset kinetics and ΔF/F ranges for neurons expressing ArcLight versus GCaMP.

Additionally, the expected action-potential amplitude in zebrafish neurons should be stated. In Figure S1b, a 40 mV change appears to produce ~0.5% ΔF/F, but this should be quantified and noted. Could this comparison to GCaMP help resolve action potentials from subthreshold bursts?

(3) Axonal versus somatic amplitudes (Line 203)

The manuscript states that voltage amplitudes are "slightly smaller" in axons than in somata; this requires quantitative values and statistical testing. More importantly, differences in optical amplitude reflect factors such as expression levels, background fluorescence, and optical geometry, not necessarily true differences in voltage amplitude. The axonal signals are clearly present, but their relative magnitude should not be interpreted without correction.

(4) Figure 4C: need for an off-ROI control

Figure 4C should include a control ROI located away from ROI3 to demonstrate that the axonal signal is not due to background fluctuations, similar to the control shown in Figure S3. Although the ΔF image suggests localization, showing the trace explicitly would strengthen the point. The fluorescence-change image in Figure 4c should also be fully explained in the legend.

(5) Figure 5: hyperpolarization signals

Figure 5 is particularly impressive. It appears that Cell 2 at 18.5 hpf and Cell 1 at 18 hpf exhibit hyperpolarizing events. The authors should confirm that these are true hyperpolarizations by giving some indication of how often they were observed.

(6) SNR comparison (Lines 300-302)

The claim that ArcLight and GCaMP exhibit comparable SNR requires statistical support across multiple cells.

Reviewer #3 (Public review):

Summary:

The authors aimed to establish a long-term voltage imaging platform to investigate how coordinated neuronal activity emerges during spinal cord development in zebrafish embryos. Using the genetically encoded voltage indicator ArcLight, they tracked membrane potential dynamics in motor neurons at population, single-cell, and subcellular levels from 18 to 23 hours post-fertilization (hpf), revealing relationships between firing maturation, waveform characteristics, and axonal outgrowth.

Strengths:

(1) Technical advancement in developmental voltage imaging:

This study demonstrates voltage imaging of motor neurons in the developing vertebrate spinal cord. The approach successfully captures voltage dynamics at multiple spatial scales-neuronal population, single-cell, and subcellular compartments.

(2) Insights into the relationship between morphological and functional maturation:

The work reveals important relationships between voltage dynamics maturation and morphological changes.

(3) Kinetics analysis of membrane potential waveform enabled by voltage imaging:

The characterization of "immature" versus "mature" firing based on quantitative waveform parameters provides insights into functional maturation that are inaccessible by calcium imaging. This analysis reveals a maturation process in the biophysical properties of developing neurons.

(4) Matching of voltage indicator kinetics to biological signal:

The authors' choice of ArcLight, despite its slow kinetics compared to newer GEVIs, proved well-suited to the low-frequency activity patterns in developing spinal neurons (frequency ~0.3 Hz).

Weaknesses:

(1) Insufficient comparison with prior calcium imaging studies:

While the authors state that voltage imaging provides superior temporal resolution compared to calcium imaging (lines 192-196, 301), and this is generally true, the current manuscript does not adequately cite or discuss previous calcium imaging studies. Since neural activity occurs at low frequency in the developing spinal cord, calcium imaging is adequate for characterizing the emergence of coordinated activity patterns in the developing zebrafish spinal cord. Notably, Wan et al. (2019, Cell) performed a comprehensive single-cell reconstruction of emerging population activity in the entire developing zebrafish spinal cord using calcium imaging. This work should be properly acknowledged and compared. The specific advantages of voltage imaging over these prior studies need to be more clearly articulated, e.g. detection of subthreshold events and membrane potential waveform kinetics.

(2) Considerations for generalizability of the ArcLight-based voltage imaging approach:

While this study successfully demonstrates voltage imaging using ArcLight in the developing spinal cord, the generalizability of this approach to later developmental stages and other neural systems warrants discussion. ArcLight exhibits relatively slow kinetics (rise time ~100-200 ms, decay τ ~200-300 ms). In the current study, these kinetics are well-suited to the developmental activity patterns observed (firing frequency ~0.3 Hz), representing appropriate matching of indicator properties to biological timescales. However, the same approach may be less suitable for later developmental stages when neural activity occurs at higher frequencies.

(3) Incomplete methodological descriptions:

As a paper establishing a new imaging approach, several critical details are missing or unclear.

(a) Imaging system specifications: The imaging setup description lacks essential information, including light source specifications, excitation wavelength/filter sets, and light power at the sample. The authors should also clarify whether wide-field optics was used rather than confocal or selective plane imaging.

(b) Long-term imaging protocol: Whether neurons were imaged continuously or with breaks between imaging sessions is not explicitly stated. The current phrasing could be interpreted as a continuous 4.5-hour recording, which would be technically impressive but may not be what was actually done.

(c) Image processing procedures: Denoising and bleach correction procedures are mentioned but not described, which is critical for a methods-focused paper.

(d) The waveform classification (Supplementary Figure S6) shows overlapping kinetics between "immature" and "mature" firing, yet the classification method is not adequately justified.

(e) Given that photostability and toxicity are critical considerations for long-term voltage imaging, these aspects warrant further clarification. While the figures suggest stable ArcLight fluorescence during the experiments, the manuscript lacks quantification of photobleaching, a discussion of potential toxicity concerns associated with the indicator, and information regarding the maximum duration over which the ArcLight signal can faithfully report physiological voltage dynamics.

(4) Incomplete data representation and quantification:

(a) The claim of "reduced variability" in calcium imaging (line 194) is not clearly demonstrated in Supplementary Figure S1.

(b) Amplitude distributions for cell/subcellular compartments are not systematically quantified. Figure S3 shows ~5% changes in some axons versus ~2% in others, but it remains unclear whether these variabilities reflect differences between axonal compartments within the same cell, between individual cells, or between individual fish.