DOI: 10.5061/dryad.mgqnk996r
title: Data from: Remote neurostimulation through an endogenous ion channel using a near infrared light-activatable nanoagonist

description: The development of noninvasive approaches to precisely control neural
 activity in mammals is highly desirable. Here we utilized the ion channel
 TRPA1 as a proof of principle, demonstrating remote near-infrared (NIR)
 activation of endogenous channels in the neural structures of living mice
 through an engineered nanoagonist. This achievement enables specific
 neurostimulation in wild-type, non-genetically modified mice. Initially,
 target-based screening identified flavins as photopharmacological
 agonists, allowing for the photoactivation of TRPA1 in sensory neurons
 upon UVA/blue light illumination. Subsequently, upconversion nanoparticles
 (UCNPs) were customized with an emission spectrum aligned to flavin
 absorption and conjugated with flavin adenine dinucleotide, creating a
 nanoagonist capable of NIR activation of TRPA1. Following the intrathecal
 injection of the nanoagonist, noninvasive NIR stimulation allows precise
 bidirectional control of nociception in mice through the remote activation
 of spinal TRPA1. This study demonstrates a noninvasive NIR
 neurostimulation method with the potential for adaptation to various
 endogenous ion channels and neural processes by combining photochemical
 toolboxes with customized UCNPs.
description: Remote neurostimulation through an endogenous ion channel using a near
 infrared light-activatable nanoagonist
 [https://doi.org/10.5061/dryad.mgqnk996r](https://doi.org/10.5061/dryad.mgqnk996r) Our manuscript demonstrates the specific and noninvasive stimulation of neural activity in mice using a NIR-activated agonist for an endogenous ion channel, resulting in the remote control of pain. The files are available in OriginPro or GraphPad Prism formats and are organized by figure panels corresponding to those in the original manuscript. Each file contains raw data used to produce the figure panels. An exception is made for Figure 4 panels, where the raw data sets are organized in Excel spreadsheets or text files, and the graphs generated from the raw data are provided in PDF format. Each file includes a legend describing the original experiment. Fig. 1A shows the whole-cell currents recorded at +80 and -80 mV in a human TRPA1-expressing HEK 293 cell in response to sequential applications of FAD (100 μM), the combination of FAD (100 μM) and blue light (460-490 nm, 6 mW/cm2), and the TRPA1-specific antagonist HC030031 (HC, 30 μM). Fig. 1B shows the power-density dependence of blue light-induced TRPA1 currents at +80 mV in the presence or absence of FAD (100 μM) (n = 6). Data are normalized to TRPA1 basal currents. Fig. 1C shows concentration dependence of FAD in blue light (6 mW/cm2)-induced TRPA1 currents at +80 mV (n = 6). Data are normalized to TRPA1 basal currents. The smooth curve was fit to the Hill equation, with a half maximal effective concentration (EC50) of 55 μM and a Hill coefficient of 1. Fig. 1D shows whole-cell currents in a TRPA1-expressing HEK 293 cell in response to sequential applications of FAD (100 μM), FAD (100 μM) plus UVA light (330-385 nm, 6 mW/cm2), and HC (30 μM) (n = 6). Fig. 1E shows whole-cell currents in a TRPA1-expressing HEK 293 cell in response to sequential applications of green light (510-550 nm, 6 mW/cm2) and blue light (6 mW/cm2) in the presence of FAD (100 μM) (n = 6). Fig. 1F shows the absorption spectrum of FAD in saline. Fig. 2A shows whole-cell currents in a TRPA1-expressing HEK 293 cell in response to sequential applications of a mixture of FAD (200 μM) and ascorbic acid (10 mM), the mixture with blue light (460-490 nm, 6 mW/cm²), FAD with blue light, and HC030031 (HC, 30 μM) (n = 6). Fig. 2B Left: Whole-cell currents in a TRPA1-expressing cell in response to sequential applications of a mixture of FAD (200 μM) and sodium azide (NaN3, 10 mM), the mixture with blue light (6 mW/cm²), FAD with blue light, and HC. Right: Quantification of the time required for blue light to induce steady-state photoactivation of TRPA1 with or without NaN3. Statistical significance is indicated by asterisks, evaluated using a two-tailed *t*-test (n = 6). Fig. 2D-G shows whole-cell currents in response to sequential applications of 2-APB (2-aminoethoxydiphenyl borate, 200 μM), FAD (300 μM) plus blue light (6 mW/cm2), and HC recorded in cells expressing WT or mutant human TRPA1 (mutated residues are indicated on top of each graph, n = 5). Fig. 2H shows ratios of photoactivation-induced currents to 2-APB-induced currents for wild-type (WT) or mutant TRPA1 as shown in (D–G) (n = 5). The statistical analysis was conducted using one-way ANOVA with post hoc Tukey test. Asterisks denote statistical significance between the WT and mutant channels. Fig. 3A shows whole-cell currents recorded at -70 mV in a mouse dorsal root ganglia (DRG) neuron in response to sequential applications of FAD (50 μM), FAD (50 μM) plus blue light (460-490 nm, 5 mW/cm2), and the TRPA1-specific antagonist HC030031 (HC, 30 μM) (n = 5). Fig. 3D shows quantifications of scratching behavior of *TRPA1+/+* and *TRPA1-/-* mice in response to UVA/blue light (358-381 and 445-488 nm, 30 mW/cm2 in total) after intradermal injection of FAD (150 μM, 50 μl volume) into the nape skin at 15 min before light stimulation. The light exposure protocol using optoelectronic devices in mice consisted of four phases: Activation of the devices for 40 minutes (Phase I), a 60-minute deactivation period (Phase II), an additional 40-minute deactivation (Phase III), and a final 40-minute reactivation (Phase IV). Statistical analysis was performed using two-way ANOVA with post hoc LSD test (n = 9). Asterisks denote statistical significance between the *TRPA1+/+* and *TRPA1-/-* mice. Fig. 3E shows quantifications of scratching behavior of *TRPA1*+/+ mice in response to UVA/blue light irradiation (30 mW/cm2, 40 min) or control white light (76 μW/cm2), along with intradermally injected FAD (150 μM, 50 μl) or saline (50 μl) in the nape skin (n = 9). Statistical analysis was conducted using one-way ANOVA with post hoc Tukey test. Asterisks denote statistical significance between the UVA/blue light + FAD group and other groups. Fig. 4E shows x-ray diffraction (XRD) patterns of core, CS and CSS nanocrystals. Fig. 4F shows** **fourier transform infrared (FTIR) spectra of UCNP-OA, DSPE-PEG-COOH, UCNP-PEG, FAD, and UCNP-FAD. Fig. 4G shows** **upconversion emission spectrum of upconversion nanoparticles (UCNPs)/UCNP-FAD with luminescence photograph (inset) under 808 nm NIR laser excitation, and the absorbance spectrum of FAD. Fig. 5B shows whole-cell currents recorded in a TRPA1-expressing HEK 293 cell in response to sequential applications of UCNP-FAD (4 mg/ml), UCNP-FAD (4 mg/ml) plus NIR light (808 nm, 200 mW/cm2), and the TRPA1-specific antagonist HC030031 (HC, 30 μM) (n = 6). Fig. 5C shows** **power-density dependence of 808 nm NIR light-induced TRPA1 currents at +80 mV in the presence of UCNP-FAD (4 mg/ml) (n = 6). Data are normalized to TRPA1 basal currents. Fig. 5D shows** **concentration dependence of UCNP-FAD in 808 nm NIR light (200 mW/cm2)-induced TRPA1 currents at +80 mV (n = 6). Data are normalized to TRPA1 basal currents. Fig. 5E shows** **the pulsed irradiation regime of NIR light enables TRPA1 photoactivation at much lower excitation powers in the presence of UCNP-FAD. Left, whole-cell currents in a TRPA1-expressing HEK 293 cell in response to pulsed 808 nm NIR light (peak power density at 200 mW/cm2, 15 ms pulse width, 20 Hz) in the presence of UCNP-FAD (4 mg/ml) (n = 6). Right, quantification of TRPA1 currents induced by pulsed (15 ms pulse width, 20 Hz) or continuous NIR light at the same peak power density of 200 mW/cm2 in the presence of 4 mg/ml UCNP-FAD. Data are normalized to TRPA1 basal currents. Statistical significance was evaluated using a two-tailed *t* test (n = 6). Fig. 6A Left: Averaged intracellular Ca2+ increases in cultured dorsal root ganglia (DRG) neurons from *TRPA1+/*+ or *TRPA1-/-* mice in response to various treatments for 5 minutes. Treatments included NIR irradiation (808 nm, 75 mW/cm², 15 ms pulse width, 20 Hz) with and without UCNP-FAD (4 mg/ml), and UCNP-FAD alone. The TRPA1 agonist allyl-isothiocyanate (AITC) and capsaicin (Cap), a TRPV1 channel agonist, served as positive controls. 216 AITC-responsive neurons from 6 *TRPA1+/+* mice and 72 Cap-responsive neurons from 3 *TRPA1-/-* mice were included. Right: Bar graph highlighting the Ca2+ increases induced by different treatments on the left, color-coded accordingly. Ca2+ responses were normalized to AITC or Cap-induced Ca2+ increases. Statistical analysis utilized one-way ANOVA with post hoc Tukey test. Sterisks denote statistical significance between the NIR + UCNP-FAD neuron group from *TRPA1+/+* mice and other groups. Fig. 6D shows quantification of NIR-induced scratching behavior in mice. After intradermal injection of UCNP-FAD (200 μg), UCNPs (200 μg) or control saline (50 μl) into the nape skin of *TRPA1+/+ *or *TRPA1-/-* mice, the injection site was illuminated by 808 nm NIR light (200 mW/cm2, 15 ms pulse width, 20 Hz) or control white light (76 μW/cm2) for 20 min. After NIR stimulation ceased, the scratching behavior of mice was recorded for 60 min. Statistical analysis was conducted using one-way ANOVA with post hoc Tukey test (n = 6). Asterisks denote statistical significance between the NIR + UCNP-FAD group in *TRPA1+/+* mice and other groups. Fig. 7B, C shows heat hyperalgesia time course in mice measured at 35 minutes, 1, 1.5, 2, and 2.5 hours post-intrathecal injection (B). Bar graph showing heat hyperalgesia in mice at 35 minutes post-intrathecal injection (C). Mice received intrathecal injections of 30 μg UCNP-FAD, UCNPs, or control saline (5 μl) in *TRPA1+/+* or *TRPA1-/-* mice. The injection site was illuminated by pulsed 808 nm NIR laser (750, 350, or 100 mW/cm², 15 ms pulse width, 20 Hz) or white light (76 μW/cm²) for 15 minutes. Statistical analysis was performed using two-way ANOVA with post hoc LSD test (B) or one-way ANOVA with post hoc Tukey test (C) (n = 9). Statistical significance is indicated by asterisks for comparisons between the Saline + White light group and other groups, and by pound signs for comparisons between the UCNP-FAD + NIR 750 mW/cm² group in *TRPA1+/+* mice and other groups. Fig. 7D shows c-fiber-evoked field potentials during 808 nm NIR irradiation (500 mW/cm², 15 ms pulse width, 20 Hz) with saline or UCNP-FAD (30 μg) at the spinal recording site in anesthetized mice. Top traces depict typical field potential recordings evoked by 15 V electrical stimulation during NIR irradiation, with either control saline or UCNP-FAD. Statistical analysis used a two-tailed *t*-test comparing control saline and UCNP-FAD groups at each stimulus voltage, with significance denoted by asterisks (n = 6). Fig. 7E shows quantification of c-fos-expressing neurons in the lumbar spinal cord following hot plate test under the same treatment conditions as (B, C) with 750 mW/cm² NIR light. Statistical analysis was conducted as in (C), with significance denoted by asterisks (n = 9). Fig. 8A, B shows** **time course of nociceptive responses to noxious heat in mice measured at 35 minutes, 1, 1.5, 2, and 2.5 hours post-intrathecal injection (A). Bar graph showing nociceptive responses in mice at 35 minutes post-intrathecal injection (B). Mice received intrathecal injections of 40 μg UCNP-FAD, upconversion nanoparticles (UCNPs), or control saline (5 μl) in *TRPA1+/+* or *TRPA1-/-* mice. The injection site was illuminated by 808 nm NIR laser (1.5, 1.25, or 1 W/cm2, 15 ms pulse width, 20 Hz) or white light (76 μW/cm²) for 15 minutes. Statistical analysis was performed using two-way ANOVA with post hoc LSD test or one-way ANOVA with post hoc Tukey test (n = 9). Statistical significance is indicated by asterisks for comparisons between the Saline + White light group and other groups, and by pound signs for comparisons between the UCNP-FAD + NIR 1.5 W/cm2 group in *TRPA1*+/+ mice and other groups. Fig. 8C shows** **quantification of c-fos-expressing neurons in the lumbar spinal cord following the hot plate test under the same treatment conditions as (A, B) with 1.5 W/cm² NIR light. Statistical analysis was performed between the two groups using a two-tailed *t* test. Fig. 8D shows time course of nociceptive responses to noxious heat in mice measured at 35 minutes, 1, 1.5, 2, and 2.5 hours after intrathecal injection of either control saline (5 μl), UCNP-FAD (30 μg) with or without the TRPA1 agonist cinnamaldehyde (CIN, 4 μg). Following intrathecal injection, mice were exposed to either NIR irradiation (750 mW/cm²) or white light (76 μW/cm2) for 15 minutes. Statistical analysis was performed using two-way ANOVA with post hoc LSD test (n = 9). Asterisks denote statistical significance between the Saline + White light group and other groups. Abbreviation FAD: flavin adenine dinucleotide UVA: ultraviolet A TRPA1: transient receptor potential ankyrin-repeat 1 UCNPs: upconversion nanoparticles HC: HC030031 2-APB: 2-aminoethoxydiphenyl borate WT: wild-type NIR: near-infrared AITC: allyl-isothiocyanate Cap: capsaicin CIN: cinnamaldehyde HEK: human embryonic kidney All data was generated from experiments in our laboratory and available in the paper and/or this repository. No new code or software in this research.