Scalable Optogenetic Modulation of Pain: A Novel Implant for Nociceptive Stimulation of the Sciatic Nerve

The study addresses how scalable nociceptive stimulation can be used to probe the relationship between pain severity and neuropsychiatric outcomes, including addiction. The context is the high prevalence of chronic pain and its association with negative reinforcement in substance use disorders, as well as anxiety and depression. Traditional pain models often induce constant, irreversible nerve injury (e.g., sciatic nerve cuffing), limiting control over pain dimensions. The authors hypothesize that an implantable, optogenetically driven sciatic nerve cuff can enable precise, reversible, and scalable nociceptive stimulation, where different LED light intensities elicit different pain responses, facilitating investigation of pain severity and its behavioral consequences.

The thesis situates chronic pain as multidimensional (sensory, affective-motivational, cognitive) and linked to psychiatric conditions (addiction, anxiety, depression). It reviews negative reinforcement’s role in addiction (Koob, 2013) and notes that chronic pain severity and disability correlate with drug dependence risk (Tetsunaga, 2018). Traditional neuropathic pain models (e.g., spared nerve injury, sciatic nerve cuffing; Yalcin, 2014) produce persistent allodynia but lack scalability and reversibility. Optogenetics enables precise control of nociceptors via channelrhodopsin-2 (ChR2) expressed through AAV vectors (Towne, 2013; Iyer, 2014), potentially activated transdermally or with implanted LEDs. Literature suggests ChR2 activation thresholds around 5–12 mW depending on preparation (Zhang, 2006; Boyden, 2015) and painful heat thresholds near 43°C in mice (Deuis & Vetter, 2016). The review motivates a scalable, reversible, and safe nociceptive device to study how pain severity modulates behavior.

Device design and fabrication: A 3D-printed mold (designed in TinkerCad) was created to encase up to five LEDs oriented toward a metal rod forming a 1 mm channel for the sciatic nerve. LED assembly involved soldering 36 AWG color-coded wires (9 cm, 1.55 mm strip for headcap end; 0.50 mm for LED end) to LED leads. Five such assemblies were placed in the 3D-printed mold. PDMS preparation used a 10:1 base-to-curing agent ratio, mixed 5 min, degassed repeatedly under vacuum/desiccation (including bubble popping by reintroducing air), then a 20 min vacuum step. PDMS was poured into the mold with LEDs, degassed 15 min, and cured at 50°C for 12–15 h to preserve mold integrity. Post-cure, cuffs were cut free, wires soldered to headcap connectors (MS303-120; Plastics1 Corp), insulated with non-conductive epoxy (15 min cure), cleaned with 70% ethanol, and autoclaved. Surgical implantation and viral transduction: Adult mice were anesthetized with isoflurane (1–4% induction; 1% maintenance) and placed in a stereotaxic frame. Scalp and right flank were sterilized; 0.4 mL of 0.25% bupivacaine was injected s.c. Incisions exposed the right sciatic nerve via blunt dissection between gluteus superficialis and biceps femoris. A 35G beveled needle connected via Tygon tubing (0.25 mm ID) to a 10 µL syringe was inserted under the epineurium to inject 1–2 µL AAV6-hSyn-ChR2-eYFP (2.4×10^13 vg/mL) at 1 µL/min. A subcutaneous tunnel was made between head and sciatic incisions; the head connector was temporarily mounted, and the cuff was tunneled to the sciatic nerve. Two configurations were tested: (1) above-nerve (PDMS cut flat at LED level); (2) cuff around nerve via slit accessing the circular channel; both secured with silk sutures. Wounds were closed with Vicryl and VetBond. The head connector was fixed to skull with #000 screws and dental cement. Mice recovered on warming pads, then were returned to home cages and allowed 3 weeks for recovery and viral expression. Paw withdrawal assay: Three weeks post-surgery, a button-activated circuit (Arduino 5V, button, series resistor) simultaneously drove the implanted LED and provided a TTL-like signal to TDT Synapse for synchronized video capture. A MATLAB program controlled LED illumination on button press and recorded press duration as paw withdrawal latency; release occurred upon observing nocifensive behaviors (paw flick/shake/stamp, sharp withdrawal, freezing with/without body shaking) or auto-termination at 20 s. Mice underwent 3 daily sessions, each with 3 trials separated by 5 min. LED intensity was varied via resistors corresponding to approximate powers: low 270 Ω (~1.4 mW, subthreshold control), medium 39 Ω (~8 mW), high 27 Ω (~12–15 mW). Three webcams recorded sessions; videos were also manually scored. Conditioned Place Aversion (CPA): Using Any-Maze in a 3-compartment chamber (Stoelting), the Any-Maze laser output triggered a relay to drive a 5V Arduino output to the cuff. Based on paw withdrawal results, a 27 Ω resistor (~12 mW) was used. Over 4 days: Day 1 pretest (baseline preference), Days 2–3 conditioning with a biased design pairing LED stimulation (1 Hz, 500 ms pulses) with each mouse’s initially preferred side, and Day 4 posttest. LED activated only while the mouse was in the paired chamber and ceased upon exit. Time in each chamber and freezing time were recorded. Parametric testing (bench characterization): To identify safe and effective operating points, the team varied series resistance and measured LED optical power (brightness) and temperature with and without PDMS encapsulation. Brightness incident on the nerve and temperature were recorded across resistances (22–470 Ω). Correlations among resistance, brightness, and temperature were computed to establish operating ranges that exceed ChR2 thresholds while keeping temperatures below nociceptive heat levels and below mouse core temperature (~36.7°C).

Bench parametrics: Increasing resistance decreased both brightness and temperature. Representative results: - 22 Ω: with PDMS 1.5±0.18 mW, 23.1±0.43°C; without PDMS 39±2 mW, 27.8±2.9°C. - 33 Ω: with PDMS 1.7±0.03 mW, 22.5±0.12°C; without PDMS 31±1 mW, 28.9±1.1°C. - 39 Ω: with PDMS 1.4±0.10 mW, 23.4±0.27°C; without PDMS 23.8±0.3 mW, 24.5±0.5°C. - 47 Ω: with PDMS 1.6±0.16 mW, 22.4±0.27°C; without PDMS 21±1 mW, 25.4±0.4°C. - 64 Ω: with PDMS 1.6±0.12 mW, 22.9±0.66°C; without PDMS 15.4±0.3 mW, 29.2±3.4°C. - 75 Ω: with PDMS 1.6±0.06 mW, 23.2±0.09°C; without PDMS 14.8±0.2 mW, 27.9±3.2°C. - 270 Ω: with PDMS 0.9±0.03 mW, 22.9±0.15°C; without PDMS 4.3±0.3 mW, 24.8±0.8°C. - 390 Ω: with PDMS 0.7±0.03 mW, 22.5±0.03°C; without PDMS 3.3±0.1 mW, 24.6±0.3°C. - 470 Ω: with PDMS 0.6±0.03 mW, 22.8±0.09°C; without PDMS 2.5±0.1 mW, 24.6±0.5°C. Averages across tested resistances (mean 156.6±57.7 Ω): brightness no PDMS 17.2±4.2 mW; brightness with PDMS 1.28±0.14 mW; temperature no PDMS 26.4±0.66°C; temperature with PDMS 22.8±0.11°C. Correlations: brightness vs resistance slopes m=-0.062 (no PDMS, r=-0.83) and m=-0.0024 (PDMS, r=-0.97); temperature vs resistance m=-0.0072 (no PDMS, r=-0.62) and m=-0.0054 (PDMS, r=-0.27); brightness vs temperature m=0.087 (no PDMS, r=0.56) and m=0.12 (PDMS, r=0.15). All temperatures were below mouse core temperature and below painful heat thresholds; resistances <~200 Ω yielded >5 mW (no PDMS), meeting/exceeding typical ChR2 activation thresholds. Paw withdrawal (limited N due to COVID-19): Latency inversely correlated with LED power (r=-0.46). Mean latencies: low ~1.4 mW: 12.0±1.748 s; medium ~8 mW: 11.4±1.451 s; high ~12–15 mW: 8.2±1.143 s. ANOVA across light levels: F(2,8)=4.152, p=0.058 (trend). Effects most pronounced at highest power, suggesting a critical activation threshold; ~8 mW resembled subthreshold behavior. Conditioned Place Aversion (N=5): Time in unpaired chamber increased from pretest 23.73%±3.512 to Day 1 59.93%±11.910, Day 2 75.597%±7.185, and posttest 58.980%±10.677. Time in paired chamber decreased from pretest 54.713%±9.124 to Day 1 12.095%±3.281, Day 2 5.768%±1.139, posttest 24.917%±4.864. Repeated measures ANOVA: main effect of Chamber F(2,8)=23.40, p<0.0001; Chamber×Session interaction F(6,24)=5.73, p=0.001; Session ns F(3,12)=0.99, p=0.43. Post-hoc paired vs unpaired: pretest ns t(24)=2.60, p=0.17; Day 1 trend t(24)=3.27, p=0.089; Day 2 significant t(24)=10.12, p=0.002; posttest ns t(24)=2.26, p=0.238. Within-chamber vs baseline: unpaired Day 2 increased significantly t(24)=6.89, p=0.014; paired Day 2 decreased significantly t(24)=5.35, p=0.035. These data indicate that pairing LED activation with a compartment produces aversion.

The work demonstrates feasibility of an open-source, implantable PDMS-embedded LED nerve cuff for optogenetic modulation of peripheral nociceptors. Bench tests established a controllable relationship between circuit resistance, emitted optical power, and LED temperature, identifying ranges that exceed ChR2 activation thresholds while remaining well below nociceptive heat and core body temperatures. In vivo, the cuff induced nocifensive behaviors with latencies decreasing at higher light intensities, consistent with scalable control of acute pain. Conditioned place aversion emerged when LED activation was paired with a compartment, indicating that the stimulation was aversive and behaviorally salient. These findings support the central hypothesis that light intensity can titrate nociceptive drive, enabling parametric control over pain severity. The device’s modularity and sterilizability make it compatible with common behavioral platforms, facilitating studies of how pain intensity contributes to negative reinforcement processes relevant to addiction and affective disorders. Observations suggest a practical activation threshold likely in the 8–12+ mW range for reliable peripheral activation, aligning with literature reporting higher power requirements in some preparations. Together, the device and preliminary data provide a pathway for dissecting how varying pain levels impact behavior and brain function, especially in models of the transition from acute to chronic pain and their interaction with reward/aversion systems.

This thesis introduces a medical-grade, autoclave-sterilizable, open-source implantable LED nerve cuff for optogenetic stimulation of the sciatic nerve, achieving scalable light delivery while maintaining safe temperatures. Bench characterization tied resistance to brightness and temperature, identifying operating points suitable for ChR2 activation. Preliminary in vivo experiments showed that higher light intensities shorten paw withdrawal latency and that pairing LED activation with a chamber induces place aversion, validating the device’s capacity to evoke and scale nociceptive responses. The platform enables controlled studies of pain severity and frequency, offering a tool to investigate how pain contributes to addiction and affective pathology. Future work should scale sample sizes, confirm ChR2 expression histologically, refine behavioral readouts (e.g., grimace scoring), and transition to more stable printed circuit boards with variable resistors for fine-grained intensity control, ultimately enabling studies of acute-to-chronic pain transitions and their neurobehavioral correlates.

In vivo data collection was curtailed by COVID-19-related lab closures, resulting in small sample sizes and limited statistical power. Video quality was insufficient for reliable facial grimace scoring, necessitating improved lighting and resolution. Histological verification of ChR2 expression (e.g., in dorsal root ganglia/sciatic nerve) was not performed, which could account for variability. The current prototype relied on static resistors limiting fine control; future versions will incorporate variable resistors on printed circuit boards for improved stability and scalability. Only initial cuff configurations (above-nerve vs around-nerve) were piloted; long-term stability and tissue responses were not systematically assessed.