Human TRPV1 is an efficient thermogenetic actuator for chronic neuromodulation

Figures

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  Fig. 1. Quick heating of hTRPV1⁺ HEK293TN cells even below the activation threshold elicits elevations in cytosolic Ca2+. A Changes in the GCaMP6s calcium sensor fluorescence in hTRPV1⁺ cells (N = 45) in response to heating up to 39, 41, and 43 °C from the baseline temperature of 37 °C. B Absence of changes in the GCaMP6s calcium sensor fluorescence in control cells (N = 45) in response to heating up to 39, 41, and 43 °C from the baseline temperature of 37 °C. In A and B the upper graphs show time-courses of the averaged GCaMP6s signal (the Y-axis on the left) and temperature at the imaging site (the Y-axis on the right), and the lower heat-maps demonstrate respective individual cell responses. Yellow stripes indicate periods of time when temperature at the imaging site was above the baseline temperature of 37 °C. Quantitative data are shown as mean ± SD. See also Supplementary Video S1. The heating/temperature control setup and two independent replicates of the same experiment are shown in Suppl. Fig. S1

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  Fig. 2. Theromogenetic stimulation of hippocampal CA1 pyramidal cells in acute brain slices. A Viral vector delivery sites in hTRPV1⁺ and control mice. B Experimental setup for the IR-laser-induced heating of cells with concurrent patch-clamp recording and calcium imaging. This setup consists of heating and targeting lasers with the 1375 nm and 520 nm wavelengths, respectively, optics for combining both lasers into the same optical path, an optical fiber with a flat endface, an upright epi-fluorescence microscope, and the equipment for electrophysiological recordings. Mic Obj: microscope objective; PD: photodiode. C Representative images of the same field of view in various modes: fluorescence mode for calcium imaging (left image), transmitted light mode for electrophysiological recordings (middle image), and a mode for visualization of the reflected light from the 520 nm laser spot for targeting the heating 1375 nm laser to a desired site on an acute slice (right image). Scale bar: 50 µm. D and E Intraneuronal calcium changes (upper graphs) (see also Supplementary Video S2), electrophysiological traces (voltage) (middle graphs), and heating patterns (lower graphs) for pyramidal cells in…

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  Fig. 3. Theromogenetic stimulation of neuronal circuits in vivo. A Experimental design. B Targeting the cuneiform nucleus (CnF) with viral intracerebral injections followed by fiber-optic interface implantation. C Experimental setup for simultaneous thermogenetic stimulation using the IR laser and detection of the GCaMP6s calcium sensor signal. Arduino: Arduino controller; BP1 and BP2: bend-pass interference filters; C: multimode collimator; CMOS: videorecorder with CMOS sensor; DM1 and DM2: dichroic mirrors; Fiber: optical fiber; HWP: Zero-Order Half-Wave Plate; L1, L2, and L3: lenses; LED: light-emitting diode with the wavelength of 470 nm; M1 and M2: broadband dielectric mirrors; Obj: objective; PBSC: polarizing beamsplitter cube. D IR-laser stimulation patterns. Heating periods when the IR laser was on were commonly 20 s. If a mouse ran to too fast, the IR laser was switched off earlier than the 20-s period over. E The average speed of mice within the 30 s period prior to the first application of the heating IR laser on the first day of thermal stimulation. Quantitative data are shown as mean ± SEM. Statistics: Student’s t-test. p = 0.5198. t = 0.6616. F = 2.066. hTRPV1⁺: N = 7 mice.…

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  Fig. 4. Thermal stimulation increases running speed and elevates GCaMP6s signal in hTRPV1+ mice but not in control mice. A Average speed of hTRPV1+ and control mice during periods when the IR laser was on or off, on stimulation days 1, 2, and 3. B Average integral GCaMP6s signal in the CnF of hTRPV1+ and control mice during periods when the IR laser was on or off, on stimulation days 1, 2, and 3. Expression of hTRPV1 was confirmed by immunohistochemical staining (Suppl. Fig. S5) Quantitative data are shown as mean ± SEM. Statistics: paired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. hTRPV1⁺: N = 7 mice. Control: N = 8 mice. Details of statistical analysis are shown in Supplementary Tables S2 and S3

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  Fig. 5. Repeated theromogenetic neuromodulation is accompanied by activation of astrocytes and microglial cells. A GFAP immunopositive cells underneath the track (arrows) in hTRPV1+ and control mice (maximal projections). B GFAP positive area underneath the track in hTRPV1+ and control mice. C Iba-1 immunopositive cells underneath the track (arrows) in hTRPV1+ and control mice (maximal projections). D Iba-1 positive area underneath the track in hTRPV1+ and control mice. Quantitative data are shown as mean ± SEM. Statistics: Student’s t-test. B p = 0.0047. t = 3.407. F = 1.793. D p = 0.0008. t = 4.359. F = 1.705. *p < 0.05, **p < 0.01, ***p < 0.001. hTRPV1+: N = 7 mice. Control: N = 8 mice. Confocal images were acquired with the same settings for both hTRPV1+ and control mice

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  Fig. 6. Thermal stimulation increases the number of c-Fos positive cells in the CnF of hTRPV1+ mice. A Experimental design. B Average speed of hTRPV1+ and control mice during periods when the IR laser was on or off. Statistics: paired t-test. hTRPV1+ mice: p = 0.1698. t = 1.839. Control mice: p = 0.6462. t = 0.4955. C Average integral GCaMP6s signal in the CnF of hTRPV1+ and control mice during periods when the IR laser was on or off. Statistics: paired t-test. hTRPV1+ mice: p = 0.0395. t = 3.012. Control mice: p = 0.0236. t = 3.562. D Representative images of sites of thermal stimulation in the brains of hTRPV1+ and control mice. Brain slices were co-stained for revealing hTRPV1 (anti-FLAG antibody), GCaMP6s (anti-GFP antibody), and c-Fos (anti-c-Fos antibody). Scale bars: 1000 µm. E Representative images of c-Fos immunopositive cells underneath sites of fiber-optic implantation in the brains of hTRPV1+ and control mice. Scale bars: 100 µm. F Numbers of c-Fos immunopositive cells in the CnFs of ipsi- and contralateral hemispheres of hTRPV1+ and control mice. Statistics: Kruskal–Wallis test with Dunn’s post hoc test. p = 0.0154. KW = 10.406. *p < 0.05, **p < 0.01, ***p < 0.001. Confocal…

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  Fig. 7. Thermometry in vivo. A Experimental setup for simultaneous IR laser treatment and acquiring photoluminescence readout from negatively charged nitrogen vacancy (NV0) color centers in diamond. AOM: acousto-optic modulator; DM1 and DM2: dichroic mirrors; Fiber: dual cladding optical fiber; HWP: Zero-Order Half-Wave Plate; L1, L2, and L3: spherical lenses; LP: low pass filter; M1 and M2: broadband dielectric mirrors; Obj: objective; PBSC: polarizing beamsplitter cube; PG: pulse generator; SM: spectrometer. B A dual core optical fiber with an NV0 vacancy-containing diamond attached to its endface. IR: infrared irradiation, VIS: visible light. C A dual core optical fiber was inserted slightly above the CnF. D Experimental spectrum and its approximation for determining the position of the zero-phonon line ZPLNV⁰. E Temperature kinetics at the tip of the optical fiber upon IR laser treatments with laser power of 20 (green line), 40 (vermilion line), and 60 (blue line) mW over 20 s. Temperature kinetics curves given as ∆T (°C) were determined experimentally (N = 3 mice, solid lines) and theoretically (dashed black lines). Quantitative data are shown as mean ± SEM