Optogenetic and chemogenetic strategies for sustained inhibition of pain

Abstract

Spatially targeted, genetically-specific strategies for sustained inhibition of nociceptors may help transform pain science and clinical management. Previous optogenetic strategies to inhibit pain have required constant illumination, and chemogenetic approaches in the periphery have not been shown to inhibit pain. Here, we show that the step-function inhibitory channelrhodopsin, SwiChR, can be used to persistently inhibit pain for long periods of time through infrequent transdermally delivered light pulses, reducing required light exposure by >98% and resolving a long-standing limitation in optogenetic inhibition. We demonstrate that the viral expression of the hM4D receptor in small-diameter primary afferent nociceptor enables chemogenetic inhibition of mechanical and thermal nociception thresholds. Finally, we develop optoPAIN, an optogenetic platform to non-invasively assess changes in pain sensitivity, and use this technique to examine pharmacological and chemogenetic inhibition of pain.

Figure 1. Intraneural injection of AAV6-hSyn-SwiChR-eYFP enables optogenetic inhibition of nociceptors during illumination in vitro and in vivo.

(a) SwiChR-eYFP+ neurons project to i), v) lamina I/IIo in the spinal cord, are ii) unmyelinated, form free nerve endings in the iii) glabrous and iv) hairy paw and vi) are small in diameter. vi) Histogram based on 3 DRGs from 2 mice. n = 362 SwiChR+ neurons (green), n = 1078 SwiChR− neurons (grey). Scale bars: spinal cord (i): 250 μm, spinal cord (iv): 500 μm, nerve: 25 μm, paw (iii): 150 μm, paw (iv): 200 μm. Colors: i), ii), and v) magenta: myelin, green: SwiChR-eYFP. iii), iv) magenta: PGP9.5, green: SwiChR-eYFP. (b) i) Representative DRG sections showing overlap between SwiChR-eYFP and calcitonin gene-related peptide (CGRP), substance P (SP), isolectin B4 (IB4) binding neurons, and neurofilament-200 (NF200). Colors: green: SwiChR-eYFP, magenta: marker, white: overlap, arrowheads: co-expressing neurons. Scale bar: 100 μm. ii), and iii) Quantification, showing ii) percentage of SwiChR-eYFP+ neurons expressing a marker, and iii) percentage of neurons expressing a marker that co-express SwiChR-eYFP. Group data from >300 SwiChR-eYFP+ or marker + neurons from 3 different DRG sections from different mice. (c) i) Reversal potential of SwiChR relative to measured V_AP and V_rest. (P = 0.0002, pH = 7.4: n = 14 cells for V_rev , n = 15 cells for V_AP , n = 21 cells for V_rest ; pH = 6.0: n = 8 cells for V_rev , n = 7 cells for V_AP , n = 9 cells for V_rest). Gray zones: mean ± SEM. ii) Photocurrent amplitudes at V_AP . (P = 0.000174, pH = 7.4: n = 15 cells; pH = 6.0: n = 9). iii) Changes in cellular input resistance during and after blue light application normalized to pre-light value (pH = 7.4: P = 0.0018, n = 12 cells; pH = 6.0: P = 0.0050, n = 6 cells). (d) Mechanical and thermal thresholds and latencies increase significantly during blue-light illumination in SwiChR+ and iC1C2+ mice, but not YFP+ mice. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

SwiChR inhibits nociceptor-driven pain after illumination (a) i) Voltage traces from SwiChR+ neurons stimulated with pulsed current injection (480 pA, 10 Hz, 30 ms) ii) Probability that an action potential is inhibited at 60 seconds after blue light application in the protocol shown in (b) i), using 10 Hz current injection (pH = 7.3, 10 ms pulses: 297.1 ± 43.8 pA, n = 11 cells; 30 ms pulses: 256.2 ± 33.8 pA, n = 15 cells; pH = 6.0, 10 ms pulses: 374.4 ± 85.1 pA, n = 8 cells; 30 ms pulses: 507.1 ± 72.5 pA, n = 8 cells). (b) Mechanical thresholds measured in the absence of blue light and 10 seconds after light application. (c) Mechanical thresholds measured in SwiChR+ mice after a two-pulse sequence of different colors of light. (d) Mechanical thresholds measured in SwiChR+ mice at time-points after a single blue light pulse. (e) Mechanical thresholds measured in SwiChR+ mice after trains of blue light pulses (1 s pulses, 1/60 Hz). Response to a i) single pulse at t = 60 s, ii) 3 pulses at t = 180 s, and iii) 10 pulses at t = 600 s. (f) Mechanical thresholds measured before and after a one hour train of blue light pulses (1 s pulses, 1/60 Hz). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Chemogenetic inhibition of pain and the OptoPAIN assay (a) Representative DRG from mouse injected intraneurally with AAV6-hSyn-HA-hM4D(Gi)-IRES-mCitrine. Scale bar: 100 μm. (b) Histogram of size-distribution of hM4D+ (green) and hM4D− (gray) cells, expressed as relative percentage. (c) Mechanical thresholds in hM4D+ and YFP+ mice following CNO administration. (d) Thermal thresholds in hM4D+ and YFP+ mice following CNO administration. (e) Qualitative light-sensitivity scores showing differential response to various administered agents (saline: n = 5 mice, gabapentin: n = 3 mice, buprenorphine: n = 4 mice). (f) Light-intensity thresholds following intraperitoneal injection of buprenorphine (25 mg/kg) or saline (100 μl). (g) Light-intensity thresholds following intraplantar injection of lidocaine (20 μl of 2% lidocaine), or saline (20 μl). (h) Light-intensity thresholds in mice co-injected with AAV6::ChR2 and AAV6::hM4D following intraperitoneal injection of CNO or saline. *P < 0.05, **P < 0.01, ***P < 0.001. Bonferroni correction applied in Fig. 3d,f, with significance level 0.025.