Precise spatiotemporal control of voltage-gated sodium channels by photocaged saxitoxin

Here we report the pharmacologic blockade of voltage-gated sodium ion channels (NaVs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of NaVs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of NaVs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to NaV-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial NaV block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.


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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Methods n/a Involved in the study

ChIP-seq
Flow cytometry MRI-based neuroimaging during this study are included in the supplementary information files. Source data are provided with this paper.
No sample size calculation was performed. For data collected on dissociated cells, sample sizes were chosen based on prior experiments with neurotoxins within the lab. Measurements on five or more cells typically yielded SEMs of less than 5%, consistent with literature precedent.
For slice experiments, at least three mice per group were used in order to assess reproducibility. Sample sizes were chosen based on established standards in the lab. At least eight brain slices were used in each condition, which is sufficient to reliably detect changes of the magnitudes shown in the paper, i.e. 10-50% saxitoxin dependent reduction.
For data collected on dissociated cells, cells were chosen as described in the Methods section of the manuscript. For voltage-clamp data on dissociated cells, data was included if the cell had >1 nA of sodium current and the leak current remained less than -0.3 nA. These cutoffs were predetermined, are consistent with literature precedent, and ensure that (a) the signal:noise ratio is low and (b) cell quality is maintained throughout the experiment.
Specifically for IC50 data, cells that recovered current to within +/-10% after initial toxin application and wash-off were used. These exclusion criteria were also pre-established-the rationale being that IC50 data should reflect the effects of the applied toxin and not inherent changes in the sodium current of a cell.
For current-clamp experiments, all cells firing action potential trains with frequencies greater than 5 Hz were taken, provided a seal was maintained for the duration of the experiment. Cells were chosen in this way to ensure precise reporting of action potential block.
Slice experiments were conducted as described in the manuscript; when measurements were not possible data was included in a failure graph.
For data collected on dissociated cells, all results were replicated against at least three distinct cells (most often five or more). Data were reproducible (see error measurements in the manuscript and supporting information). For slice experiments, each condition was tested on a least three mice with two slices taken per mouse. We confirmed reproducibility by showing that effects were consistently found in at least ten slices total from at least five mice. See manuscript and supporting information for exact replicate numbers and associated error measurements.
Allocation was random.
Blinding was not possible due to the requisite presence or absence of saxitoxin photocage in the test and control groups, respectively. Likewise, laser application requires manual input and cannot be blinded.