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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Exp Neurol. 2020 Sep 28;335:113480. doi: 10.1016/j.expneurol.2020.113480

A micro-LED implant and technique for optogenetic stimulation of the rat spinal cord

SE Mondello a,e, B Pedigo a, MD Sunshine a, AE Fischedick a, PJ Horner g, CT Moritz a,b,c,d,e,f
PMCID: PMC8284995  NIHMSID: NIHMS1720036  PMID: 32991934

Abstract

To date, relatively few studies have used optogenetic stimulation to address basic science and therapeutic questions within the spinal cord. Even less have reported optogenetic stimulation in the rat spinal cord. This is likely due to a lack of accessible optogenetic implants. The development of a device that can be fabricated and operated by most laboratories, requiring no special equipment, would allow investigators to begin dissecting the functions of specific neuronal cell-types and circuitry within the spinal cord, as well as investigate therapies for spinal ailments like spinal cord injury. Here, we describe a long-term implantable μLED device designed for optogenetic stimulation of the spinal cord in awake, freely moving rats that is simple enough to be fabricated, implanted and operated by most laboratories. This device, which sits above the dorsal cord, can induce robust movements for at least 6 weeks without causing physical or thermal damage to the underlying spinal cord. In this regard, the presented μLED device could help tease apart the complexities of the spinal cord and uncover potential future therapeutics.

Keywords: Optogenetic spinal stimulation, μLED implant, rat spinal cord, AAV transduction, neural devices

Introduction

Optogenetic stimulation a semi-noninvasive tool for both spatial and cell-specific stimulation of the nervous system. The capacity for multiple, wavelength-specific cell activation capabilities permits researchers to examine a range of basic science and therapeutic applications not possible with traditional, electrode-based stimulation techniques within the spinal cord. For example, identifying behavioral outcomes related to specific neuronal cells and circuitry, as well as uncovering spinal neuron populations that may have therapeutic potential for spinal cord disorders. To date, the majority of published works incorporating optogenetic spinal stimulation have been acute or short term studies of ≤ 2 weeks (Alilain et al., 2008; Bonin et al., 2016; Christensen et al., 2016; Lu et al., 2014, 2014; Mayer et al., 2019; Montgomery et al., 2016; Park et al., 2015). While short-term stimulation can be sufficient for circuit interrogation or even suggest therapeutic potential (Alilain et al., 2008), additional investigations are enabled by long-term optogenetic studies using a chronic implant in awake, freely moving animals (Montgomery et al., 2016; Rahman et al., 2018). However, long-term stimulation requires robust implantable devices that fit into small spaces without causing damage to the surrounding tissues. The majority of implants developed for long-term spinal optogenetic stimulation require specialized equipment and fabrication (Bonin et al., 2016; Lu et al., 2014; Montgomery et al., 2015; Park et al., 2015; Samineni et al., 2017). Commercial implants and associated equipment and software are expensive and the most widely available implants are designed for skull-based mounting, which is often not compatible with the spinal column.

Here we present a low-cost, microLED (μLED) implant for rat spinal stimulation that can be fabricated using standard tools and equipment. We also identify thermally safe optogenetic stimulation paradigms by testing 11 parameters and using a thermistor to measure real-time temperature changes in-vivo. Lastly, we describe a method of implantation between the spinal column and the dorsal surface of the cervical spinal cord that causes minimal damage to the spinal tissue and allows for at least 6 weeks of optogenetic spinal stimulation. This affordable spinal implant for optogenetic stimulation will accelerate research in spinal physiology and pathophysiology as well as provide a much needed tool to investigate therapeutic questions using optogenetic approaches in rodents.

Materials and Methods

Fabrication of μLED implant

Two silver plated soft copper core wires with PFA insulation (36 AWG, Micron Meters, Saint George, UT) were tightly twisted together and cut to 10 cm. Approximately 3-4 mm of insulation was then removed from both wires on each side. A high-power CREE μLED (Xlamp XB-D Blue, 475 nm, Cree Inc., Durham, NC; Figure 1A) was soldered to the copper wires such that the positive wire was soldered to the anode-soldering pad and the ground wire soldered to both the cathode and central heat sink pads. Epidural catheter tubing (19 ga.; Arrow International Inc., Reading, PA) was then cut to 2 mm, and slid over both wires adjacent to the LED (Figure 1B). A second piece of catheter was cut to 2 cm and positioned on the side furthest from the LED (Figure 1D, asterisk).

Figure 1: μLED implant.

Figure 1:

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A) Image of the Cree μLED used in our implants for optogenetic spinal stimulation. (B) The LED portion of the implant is coated in 2-ton epoxy with a small hole placed in the center to allow for anchoring with a microscrew. Asterisk identifies the small catheter above. (C) The total length of the LED portion of the implant is less than 1 cm. (D) The total implant length is approximately 10 cm. Asterisk identifies the long catheter above.

The connector end of the two de-insulated wires were then soldered to gold-plated pins (Plastics One, Roanoke, VA). A small, circular piece of laboratory tape was placed onto the underside of a 6-channel pedestal (Plastics One) to seal open holes from epoxy. The soldered pins were then pierced through the tape into the two central pinholes of the pedestal. The longer piece of catheter was then positioned immediately beside the connector pedestal for strain relief and 2-ton epoxy (Devcon, Danvers, MA) was poured onto the pedestal to completely cover all pins. Epoxy was allowed to cure overnight.

The following day, the smaller catheter was positioned 4-5 mm from the LED. The wires were then positioned at 45° relative to the LED. The wires between the catheter and LED were separated to allow space for a microscrew (5/32 inch #000 flat fillister, J.I. Morris Company, Oxford, MA) that is used to secure the implant to the vertebra (Figure 1B). The LED, wires, and small catheter were then dipped into 2-ton epoxy and allowed to cure overnight. This step was repeated several times until the LED and wires were fully insulated.

A small hole was then drilled into the epoxy between the two wires using a .0210”/.533 mm drill bit (Figure 1B, arrow; Gyros Precision Tools, Monsey, NY). The epoxy was then shaped into a smooth oval, approximately 8 mm long (Figure 1C) using a Dremel tool (407 1/2” Sanding Drum, 520-02 1/2” SIC Impregnated Wheels; Dremel, Racine, WI). A thin layer of epoxy was painted over the lens to reduce reflected light (Figure 1B,C). The entire length of the implant, including wiring and pedestal, was approximately 10 cm (Figure 1D). Lastly, complete insulation was confirmed by immersing the implant in 0.9% sodium chloride solution and testing the resistance of each wire against an independent wire via a multimeter to ensure there were no shorts. Implants were sterilized in an autoclave for 30 minutes.

Fabrication of μLED controller

To control the LED, we fabricated a simple LED driver circuit (Figure 2A). A 12V supply (3, 4V batteries in series) was used to power a constant current LED driver (1000 mA BuckBlock with DIM, LEDdynamics Inc., Randolph, VT), which is the dimmable power source for the implanted LED allowing for an adjustable light intensity. The timing of the implanted μLED (light on/light off) was controlled using an AtTiny85 microcontroller (Atmel). The “DIM” lead (Figure 2A) on the LED driver was connected to the collector of an NPN transistor and the emitter connected to the ground. The transistor base pin was switched ON (AtTiny GPIO pin 2 = high; Figure 2A) or OFF (GPIO pin 2 = low; Figure 2A), based on the information provided by the AtTiny. The AtTiny was programmed using a “Tiny AVR Programmer” (Sparkfun, PGM-11801 developed by David Mellis, MIT Media Lab), in Arduino (Arduino; Italy). The microcontroller (AtTiny85) gated the timing (pulse width and interpulse period) of the μLED, which was connected to the “LED+” and “LED−“ leads of the LED driver (Figure 2A). The current, which dictates the light intensity, was controlled using a 20 kOhm potentiometer (Figure 2A,B) attached to the LED driver between the “DIM GND” and “DIM” leads (Figure 2A). We added a connector to measure the resistance set by the potentiometer (Figure 2C) to standardize intensity. We included two indicator LEDs to monitor the two power sources (5V, 12V; orange; Figure 2A,B) and one indicator LED that mirrors the timing of the implanted μLED (blue; Figure 2A,B). Rats were tethered to the controller during periods of stimulation, which allowed them to walk freely around their cage while receiving stimulation (Figure 2D).

Figure 2: Implant controller.

Figure 2:

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(A) Schematic of μLED implant controller. (B,C) Images of the implant controller bud box. Orange LEDs indicate functional 5V and 12V power sources. Blue LED indicates the implant is properly powered. (D) Image of rat tethered to a functioning implant driver box.

Optional addition of thermistor to μLED implant

Construction of an implant that also contains a thermistor was similar to that of the μLED implant with a few additions. Four 30-gauge wires are wound together to form leads that connect to a pedestal connector for mounting on the animal’s headcap. On the opposite end, two of the four leads were soldered to a μLED as above, while the other two leads were soldered to a NTC thermistor 100 kOhm bead (Figure 3; Murata Electronics, Irvine, CA). The thermistor itself was positioned directly onto the back of the LED to ensure the temperature readouts were of the hottest point of the implant (Figure 3). The LED and thermistor were then epoxied together and tested to ensure no electrical shorts existed between channels. The entire device was then covered in 2-ton epoxy (Devcon) for insulation, with the epoxy shaped with a Dremel tool into a smooth oval (Figure 3). The other end of the leads were soldered to the pedestal and coated in epoxy to ensure insulation. A final check of the insulation was done by immersing the implant in 0.9% sodium chloride solution and testing the resistance of each channel via a multimeter.

Figure 3: Optional thermistor.

Figure 3:

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A thermistor (blue bead) was added to the μLED implant to allow for real-time temperature assessment.

Calibration of the μLED implant

The μLED implants were calibrated for light production as a function of current intensity. Current was adjusted using a potentiometer on the LED controller and resistance of the potentiometer was measured using a multimeter. Light intensity was measured using a ThorLabs light intensity meter paired with a standard photodiode power sensor attachment (Thorlabs, Newton, NJ). Light intensity and current intensity were measured at approximately 25 mW light intensity intervals, from the lowest to the highest light intensity produced by the μLED (~200 mW or 50 mW/mm2).

Calibration of the thermistor implant

Each thermistor implant was calibrated by comparison to a high-precision integrated temperature sensor (ITC; TSYS01, Elecrow, Shenzhen city, China). The ITC was connected to an Arduino Uno, and thermistor leads from the implant were directed to a voltage divider (100kOhm fixed resistance) that also connected to the Arduino. The ITC and implant were held together with a metal clip and submerged in a beaker containing water. The beaker was then set on a hot plate at a low setting. The water was heated from 25 to 50° C. Coefficients for the Steinhart-Hart equation were then calculated for each thermistor using MATLAB’s (Mathworks, Natick MA) linear regression function to fit parameters to the known temperature readings from the ITC.

Surgical procedures & animal care

Animals

All procedures were conducted in accordance with NIH guidelines and were approved by the University of Washington Institutional Animal Care and Use Committee. Long-Evans rats (250 – 350g; Harlan, Indianapolis, IN) were used in the implant longevity study (n=12) and in the thermal safety study (n=3). All rats received a cervical contusion injury since these implants will be used to test treatments for spinal cord injury in future studies. We therefore performed a C4 cervical contusion injury 2-5 weeks before LED implant (Mondello et al., 2015). In this regard, male rats were excluded from the study as they have a higher rate of urine retention and bladder infection after spinal injury.

Spinal cord injury and intraspinal injections of ChR2 AAV vector

During the same surgical procedure, rats received both a C4 hemicontusion (see below) and intraspinal injections of the optogenetic vector AAV2-hSyn-ChR2-YFP (UNC Vector Core, Chapel Hill, NC) at spinal segment C6 per our prior work (Mondello et al., 2018). Rats were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) with xylazine (10 mg/kg). Supplemental dosages of ketamine were supplied throughout the procedure, as needed. Rats were then shaved across the upper back, and eyes coated with a layer of ophthalmic ointment. Rats then received an intradermal injection of lidocaine (1 mg/kg) and bupivacaine (1 mg/kg) at the intended site of incision (C2-T2), as well as a subcutaneous injection of enrofloxacin (5 mg/kg) to reduce infection, and a subcutaneous injection of sterile saline (5 ml) to maintain hydration.

The surgery site was then prepared with betadine and alcohol. Rats were placed on a thermal pad and an incision was made in the skin and muscle layers over spinal segments C2-T2. A hemi-laminectomy was completed over C4 and a hemicontusion was produced using the Infinite Horizon Impactor (150 kdyne; Precision Systems and Instrumentation, LLC, Fairfax Station, VA).

Rats then received a partial laminectomy ipsilateral to injury, at the caudal aspect of C6 for spinal injections of the optogenetic AAV vector. Rats were then positioned in a spinal stereotaxic frame (Kopf, Tujunga, CA). Two injections were made using a Hamilton syringe (Hamilton Company, Reno, NV) with a glass-pulled pipette attached to a model 5000 microinjection unit (AgnTho’s AB, Lidingö, Sweden) into the dorsal root entry zone, and 1 mm lateral to the dorsal root entry zone, respectively.

A 1.5 μl deposit of AAV2-hSyn-ChR2-YFP (UNC Vector Core) was injected at a depth of 0.5 mm at both injection sites (3 μl total). The injection pipette was left in place for 10 minutes to permit complete dissipation of virus into the spinal tissue before withdrawing the syringe.

After the injections, gelfoam was placed over the exposed cord and the muscles and skin were sutured closed in layers. Rats were placed on a heating pad and provided intraperitoneal saline injections and a reapplication of ophthalmic ointment. Once the rats were awake, they received a subcutaneous injection of buprenorphine (0.05 mg/kg) for pain management, which continued 2x/day for the following two days. Weight and hydration were monitored daily for several days post-operatively.

μLED implant and thermistor implant procedure

Five weeks after receiving a spinal injury and injections, rats underwent a second surgery for μLED implant placement. The anesthesia and surgical preparations are the same as described above. A hemi-laminectomy was performed at C6, where the optogenetic AAV vector was previously injected, and a small hole was drilled into ipsilateral and contralateral C5 using a Dremel tool with a .0210”/.533 mm drill bit (Gyros Precision Tools). A 5/32 inch #000 flat fillister head screw (J.I. Morris) was then inserted into the contralateral hole at C5 (Figure 4A,B1) and a second screw was positioned in the hole of the μLED implant (Figure 1B). This second screw is included to increase anchoring of liquid dentin to the implanted region in one of the following steps. Liquid dentin adheres strongly to stainless steel and weakly to the bone and tissues surrounding the implant. The implant was then screwed into the hole at ipsilateral C5, positioning the LED over the laminectomy at C6 (Figure 4A,B2). Kwik-Sil (WPI, Sarasota, FL) was injected to fill the gap between the implant and top of the exposed spinal cord and dura. Next, UV-curable Fusio™ liquid dentin (Pentron, Orange, CA) was applied over the top of the implant and contralateral lamina and cured using an LED light cure lamp (NSKI dental, Hoffman Estates, IL; Figure 4A,B3). Importantly, liquid dentin provides an extra barrier to slow the infiltration of scar formation to the site of implantation, which eventually blocks the diffusion of light into the spinal cord for optogenetic stimulation.

Figure 4: μLED implant strategy.

Figure 4:

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(A) Schematic of the μLED spinal implant and current injury model. Rats received a spinal hemicontusion at C4 (purple oval) and the implant at C6. A microscrew is used to anchor the implant into ipsilateral C5. A second microscrew is positioned into contralateral C5 to help anchor liquid dentin around the implant as liquid dentin has a stronger adherence to stainless steel compared to bone/tissues. Kwik-sil is placed over the spinal cord dura and under the implant at C6 (green rectangle). Cyanoacrylate is painted onto the bony segments and liquid dentin is layered over the implant and surrounding vertebra, bilaterally. (B) Step-wise representative images of the implant procedure. (1) A hemi-laminectomy is performed at caudal C6 for optogenetic vector injection. Holes are drilled bilaterally at C5 and a microscrew is placed in the contralateral hole. * = hemi-laminectomy; # = microscrew; arrow = screw hole. (2) The implant is anchored into the ipsilateral hole at C5 using a second screw. # = microscrews; LED implant is located on the bottom half of the image (3) Liquid dentin is placed over both the implant and contralateral laminae. Liquid dentin = opaque white reagent covering the implant and screws.

The catheter was routed under the skin and the pedestal was fixed to the top of the skull using microscrews (5/32 inch #000 flat fillister head screw; J.I. Morris). A mixture of C & B Metabond Quick Adhesive System (Parkell, Edgewood, NY) and Ortho Jet Liquid Acrylic and Jet Denture Repair Powder (Lang, Wheeling, IL) created a skull cap that bonded the screws and connector together. The muscle and skin layers were sutured closed. Ophthalmic ointment was reapplied and buprenorphine was administered subcutaneously twice daily for the following two days. Enrofloxacin (25 mg/kg) was added to water bottles for 7 days to provide additional protection against infections.

Detecting in-vivo temperature changes

Rats implanted with the dual μLED and thermistor (n=3) were anesthetized with isoflurane to minimize variability in baseline temperature due to moving about their cages. Rats were placed in plexiglass induction chamber and anesthetized with 5% isoflurane and 1L/min oxygen. Once rats were sufficiently anesthetized, they were transferred to a heating pad and isoflurane was reduced to 1.5 – 2.5%. Before stimulation, the Arduino system and MATLAB software described above were used to estimate current amplitude as a function of potentiometer readings. Low (12.5 mW/mm2), medium (25 mW/mm2) and high (37.5 mW/mm2) current intensities were measured. Testing began when the temperature at the LED site was stable for 2 minutes. A total of 11 stimulation parameters were investigated (Table 1). Over the course of 2 – 3 hours, each animal received optical stimulation from several of the specified parameters, which were carried out in random order. An ATTiny85-20PU (Atmel, San Jose, CA) was programmed with the specified parameters. For each trial, a baseline temperature recording was measured over two minutes before initiating stimulation. If the temperature appeared stable by the end of this two-minute period, a trial began. Temperature changes were measured over a five-minute stimulation period.

Table 1: Parameters tested.

The last two parameters use continuous pulse trains.

Pulse width (ms) Intensity (mW/mm2) Frequency (Hz)
3 12.5 4
3 25 4
3 37.5 4
5 12.5 4
5 25 4
5 37.5 4
7 12.5 4
7 25 4
7 37.5 4
5 25 10
5 25 20
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Assessing in-vivo functionality of the μLED implant

To ensure the implant remained intact, we measured the current being passed across the LED once a week. To do this we placed a relatively small (22 ohms) resistor in series with the LED and observed the voltage drop across the resistor with an oscilloscope. If a voltage drop no longer occurred, paired with a lack of LED-induced movements, the implant was assumed to have shorted or otherwise failed.

Assessing in-vivo longevity of the μLED implant

The minimal light intensity to evoke a visible forelimb movement in rats implanted with a μLED implant (n=12) was determined at least 1x/ week for 6 weeks. For each testing period, a thermally safe protocol was used: 4 Hz, 5 ms pulse width, delivered using a stimulation paradigm delivering light pulses for 5 seconds followed by 15 seconds between stimulus trains. Rats were awake and freely-moving throughout testing. Light-evoked movements were discerned from spontaneous movements by first coaxing the rat to rear against their cage, leaving the optogenetically-stimulated limb with SCI-induced deficits dangling in the air, relatively still. The LED controller was then turned on containing an indicator LED that mirrors the in-vivo LED implant. Rhythmic movements (twitches) timed with the indicator LED on the LED controller box that also increased and decreased in magnitude with adjustments to the implant’s light intensity, were deemed true light-evoked movements.

Histology

Perfusion and tissue sectioning:

Rats received an intraperitoneal injection of Beuthanasia (200 mg/kg) and were transcardially perfused using 0.9% sodium chloride, followed by 10% formalin. The cervical spinal cord was dissected and placed in 10% formalin overnight for 24 hours at 4°C. Spinal cord tissue was cryopreserved in 30% sucrose for 24 hours at 4°C. Approximately 4 mm blocks of tissue were embedded in O.C.T. and then placed in a mixture of dry ice and acetone to rapidly freeze the tissue. Tissue was sectioned into 40 μm-thick crossections using a cryostat.

Assessment of AAV vector transduction

Tissue from the rats in the implant longevity study were checked to confirm transduction of the optogenetic AAV vector into the cervical spinal cord. Tissue sections from cervical segment C6 were mounted onto superfrost plus slides (Fisher Scientific, Hampton, NH) and covered with a coverslip using polyvinyl alcohol mounting media (PVA; Sigma-Aldrich, St. Louis, MO). Sections were then assessed using an SPX8 confocal microscope (Leica, Wetzlar, Germany) to confirm the presence of fluorescence indicating AAV transduction. Rats that did not have viral transduction were excluded from the study.

Cresyl violet and myelin dye

To determine if optogenetic stimulation using the μled implant induces tissue damage and/or demyelination, sections from spinal segment C6 were mounted onto subbed superfrost plus slides and stained with cresyl violet and myelin dyes as described in our previous work (Mondello et al., 2015). In short, tissue was fume fixed onto slides for 24 hours with 10% formalin. Tissue was then incubated for 5 minutes in water, followed by 70%, 95%, 100% Ethanol (EtOH), and Xylene. This process was repeated in the opposite order. Sections were then incubated for 10 minutes in myelin dye consisting of eriochrome cyanine R (Fluka, St. Louis, MO) and then differentiated in water mixed with ammonium hydroxide. Next, sections were incubated in cresyl violet dye consisting of cresyl violet with acetate (Sigma-Aldrich, St. Louis, MO) for 3 minutes. Afterwards, sections were dipped 10 times in 70%, and 95% EtOH then differentiated with glacial acetic acid mixed in 95% EtOH. Sections were then incubated for 10 mins in 100% EtOH followed by Xylene, then coverslipped with Eukitt (Sigma-Aldrich). Tissue was assessed for damage using a Zeiss Axio Zoom microscope (Oberkochen, Germany).

Immunohistochemistry

To identify if the μLED implant upregulates astroglioses, sections from spinal segment C6 were incubated in phosphate-buffered saline (PBS) mixed with 1% normal donkey serum (DS; Abcam, Cambridge, UK) and triton X-100 (T; 1% DS-PBS-T; Sigma-Aldrich) for 3 x 10 minutes on a shaker table at room temperature. Next, tissue was incubated in a blocking solution consisting of 5% DS-PBS-T for 1 hour at room temperature. Sections were transferred to a 1:750 GFAP antibody solution (AB5894; Millipore, Burlington, MA) for an overnight incubation at 4°C. The following day sections were washed with 1% DS-PBS-T for 3 x 10 minutes. Sections were then incubated in a 1:200 donkey anti-rabbit alexafluor 594 secondary antibody (Thermofisher, Waltham, MA) for 1 hour on a shaker table at room temperature. Afterwards, sections were washed 3 x 10 minutes with PBS and mounted onto superfrost plus slides (Fisher Scientific) and coverslipped with PVA mountant (Sigma-Aldrich). Sections were viewed with an SP8X confocal microscope (Leica).

Statistics

A one-way ANOVA test with Bonferroni corrections was used to compare changes in peak temperature during different stimulation paradigms. SPSS software (IBM, Armonk, NY) was used to complete this analysis. A significant p value was considered to be p < .05 for all tests.

Results

μLED device and implantation method

The μLED implant can provide up to 50 mW/mm2 of light for optogenetic spinal stimulation. This high light intensity is beneficial for epidural activation of transduced rodent spinal cords as there is often an accumulation of scar tissue between the implant and spinal cord, which impairs light penetration over time. The LED itself is 2 x 2 mm, which spatially allows for broad stimulation of the ~4 mm wide rat spinal cord. The small hole in the epoxy allows for secure tethering of the implant to the spinal column such that it “floats” closely over the spinal cord without spinal compression (Figure 1B,2). To minimize the accumulation of scar tissue under the implant, we applied silastic between the implant and spinal cord, which successfully elongated the length of time the implant produced neural activation (preliminary experiments; data not shown). We also applied UV-curable liquid dentin over the implant to further slow the infiltration of scar tissue (Figure 2).

Stimulation parameters affect thermal characteristics in-vivo

The FDA indicates that a temperature increase below 1°C for neural implants is considered safe. In order to identify the stimulation parameters that follow this criteria, we incorporated a thermistor into the μLED implant for real-time analysis of temperature changes in-vivo (Figure 5A). Due to the placement of the thermistor onto the back of the LED, we are likely obtaining thermal readings from the hottest point of the implant. A total of 11 different stimulation parameters were tested comparing varying pulse widths, light intensities, and frequencies (Table 1). These parameters were chosen to mimic those from a previous study that showed robust forelimb movements during acute optogenetic spinal stimulation in rats (Mondello et al., 2018). For comparisons between pulse widths and light intensities, the frequency was maintained at 4 Hz with a train duration of 5 seconds of stimulation, followed by 15 seconds between stimulus trains. For frequency comparisons, the pulse width was a constant 5 ms and the light intensity 25 mW/mm2. The change in temperature profile closely followed that of the stimulation paradigm, with temperatures increasing sharply while the LED delivered stimulus trains, and then decreasing towards baseline between stimulus trains (Figure 5A). The peak increase in temperature was first determined for each stimulation cycle (baseline + stimulation train), by subtracting the baseline from the peak temperature. This controls for changes in overall body temperature induced by anesthesia, which often led to reducing baseline temperatures during the earlier trials (Figure 5A). Then, the peak increase in temperature during every stimulus train was averaged across cycles for each parameter (Figure 5B).

Figure 5: In-vivo thermal changes during μLED activation.

Figure 5:

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(A) Representative profile of temperature changes with a 4 Hz frequency, 5 ms pulse width delivered for 5 seconds with 15 seconds between stimulus trains. Circles indicate peak increases in temperature for each cycle. (B) Average change in peak temperature (n=3) at varying pulse widths and current amplitudes. The frequency was held at 4 Hz. (C) For temperature assessments at different frequencies (orange bars), the parameters were held at a 5 ms pulse width and 25 mW/mm2 light intensity. Error bars = SEM.

All investigated parameters led to peak temperature increases that were less than 1°C. As expected, the lowest intensity (12.5 mW/mm2) and shortest pulse width (3 ms) resulted in the smallest increase in peak temperature (0.08°C, Figure 5B). Increasing the light intensity to 25 or 37.5 mW/mm2 at the same pulse width led to small but significant increases in peak temperatures (0.12°C and 0.17°C, respectively, p<0.05; Figure 5B). Increasing the pulse width from 3 ms to 5 ms also led to marginal but significant increases in peak temperature at 12.5, 25, and 37.5 mW/mm2 compared to similar light intensities at the 3 ms pulse width parameter (0.10°C, 0.18°C, 0.26°C, respectively, p<0.05; Figure 5B). The average peak temperatures at 12.5, 25, and 37.5 mW/mm2 at the 7 ms pulse width paradigm were significantly different from one another (0.14°C, 0.24°C, 0.36°C, respectively, p<0.05; Figure 5B) and also led to significant increases in peak temperatures compared to the 3 and 5 ms pulse width paradigms (p<0.05; Figure 5B). Notably, adjustments in frequency affected temperature the most, with 10 Hz frequency stimulation leading to a 0.40°C temperature increase, and 20 Hz frequency resulting in a 0.78°C temperature increase (p<0.05; Figure 5B). These results indicate that our implant is unlikely to cause excess heating or tissue damage to the spinal cord using a variety of stimulation parameters.

Long-term in-vivo functionality of the μLED implant

Implants were tested for light-induced movements beginning 1 or 2 weeks after implantation and continued for 6 weeks post-implant. All rats exhibited dense transduction of the optogenetic viral vector (e.g., Figure 6A) and were included in this analysis. For the first four weeks post-implant, all of the rats had light-induced movements in the ipsilateral forelimb (Figure 6B). This began to decline at 5 weeks post-implant when two rats no longer exhibited light-induced movements. This appeared to be due to increasing scar formation between the implant and spinal cord as determined post-mortem (n=1; Figure 6B) or a malfunction within the implant itself (n=1; Figure 6B). At 6 weeks post-implant, two more rats no longer exhibited light-induced effects for similar reasons (Figure 6B).

Figure 6: Implant longevity, in-vivo.

Figure 6:

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(A) Representative sections of spinal cord segment C6 (N=4) illustrating typical viral transduction (green). Scale bar = 1 mm. (B) Light intensity needed to evoke a movement was determined 1x/ week for 6 weeks using 4 Hz, 5 ms pulse width, 5 s trains with 15 s between trains. Each line represents an individual rat (N=12). The table above the line graph displays the number of rats at each timepoint with light-induced movements (“Mvmt”), a functional implant but no light-induced movements (“No Mvmt”), or a non-functional implant (“Shorted”). The asterisk indicates that while 3 of the 12 rats were not tested for movement thresholds at 1 week post-implant, their implants were likely functional based on their results the following week.

The greatest variability in movement thresholds occurred during the first and second week post-implant. For instance, during the first week, movement thresholds ranged from 4.5 to 31.5 mW/mm2 (Figure 6B) and from 4.5 to 24.8 mW/mm2 in the second week (Figure 6B). This variability was likely a result of continued progression of viral transduction during these earlier weeks. The thresholds were more consistent in the following weeks, generally between 4.5 to 12.5 mW/mm2 (Figure 6B). Overall, these findings suggest the implant is robust and viable for at least 4 - 6 weeks post-implantation.

Effects of μLED stimulation on tissue integrity:

Spinal cord tissue below the implant at C6 was stained with cresyl violet and myelin to search for gross tissue damage, such as cysts, demyelination, and other tissue disruptions resulting from excess heating or mechanical trauma from the μLED implant. Staining was compared in rats that did or did not receive optogenetic stimulation. Both groups received the implant. Our results indicate that neither the implant nor the heat/light produced by optogenetic stimulation had apparent effects on tissue integrity (Figure 7A,B).

Figure 7: Tissue integrity after spinal implant and stimulation.

Figure 7:

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Representative sections from spinal segment C6 below the site of implantation from rats that either received the inactive implant alone (A, B) or the implant receiving optogenetic stimulation (C,D). Tissue was stained with cresyl violet and myelin dyes. Tissue was also stained for GFAP (red) in rats that either did not (E,F) or did (G,H) receive optogenetic spinal stimulation. All rats received an implant. Insets display higher magnification view of the dorsal hemicord immediately under the implant for each type of stain and group (scale bar = 500 μm). Asterisks indicates the stimulated side of the spinal cord. Tissue was nicked on the left ventral side to identify orientation, though not all rats were stimulated on the left side. Scale bar = 1 mm for A,C,E,G.

We also investigated the effects of optogenetic stimulation via our implant on the expression of GFAP, a marker of glial astrocytes that upregulate in response to inflammation. Both groups receiving implants had a similarly minor increase in GFAP expression ipsilateral and ventral to the implant (Figure 7C,D). It did not appear that GFAP was increased by optogenetic stimulation. This indicates that modestly increased GFAP expression at C6 in optogentic and control tissues is likely a result of either the rostral, C4 hemicontusion, the viral vector injection at C6, or the presence of the implant regardless of stimulation.

Discussion

The current study describes a μLED implant for optogenetic spinal stimulation that can induce robust neuronal activation, while causing minimal tissue disruption under typical optogenetic stimulation parameters. This implant can provide optogenetic stimulation in awake, freely moving rats for up to 6 weeks. Choosing a thermally safe stimulation parameter is critical for preventing tissue disruptions for not only the currently discussed implant, but all implanted light sources. For our implant, we determined that pulse width and frequency have substantial impacts on heat production and should be carefully chosen. We found that pulse widths less than 7 ms paired with a 25% stimulation paradigm at 4 Hz produced minor temperature increases that were well within what is considered to be safe by the FDA. A similar finding occurred with 4 and 10 Hz frequencies when held at a 5 ms pulse width and 25% stimulation paradigm. This data are in line with other studies using optogenetic spinal stimulation with 5 or 10 ms pulse widths and 1-10 Hz frequencies (Caggiano et al., 2016; Chang et al., 2018; Hayashi et al., 2018; Lu et al., 2017). Studies that incorporate longer pulse widths and frequencies are at a higher risk of producing unsafe thermal outputs of > 1°C and would benefit from completing preliminary thermal tests. In these cases, a stimulation paradigm less than the currently tested 25% would be required.

The current device is one of the few spinal implants to be designed for optogenetic stimulation in rats, with the majority of approaches targeting mice (Lu et al., 2017, 2014; Montgomery et al., 2015; Park et al., 2015; Samineni et al., 2017). Rats are the preferred animal model when complex behavioral tasks must be trained to assess recovery from injuries (Aitman et al., 2016; Alaverdashvili et al., 2008; Ellenbroek and Youn, 2016; Hirst et al., 2003; Irvine et al., 2010; Jones and Schallert, 1992; Kjell and Olson, 2016; Schrimsher and Reier, 1993; Whishaw et al., 2008). Other implants targeting rat spinal stimulation (Montgomery et al., 2015) use a laser light source similar to others designed for mice (Bonin et al., 2016; Lu et al., 2017, 2014). The use of laser light sources comes with the need for laser coupling, which often leads to light loss and the need for larger, high-powered lasers. Further, laser-based implants likely limit the number of animals that can be treated simultaneously due to the high expense of a single laser. While lasers can be split to accommodate more than one animal, this decreases the light intensity delivered to each rat, limiting optogenetic excitation. For studies that require the ability to run multiple animals at once, optogenetic spinal implants with an LED light source may be a better option.

In addition to our implant, several other optogenetic spinal implants have been designed to use an LED light source, however these implants were designed for mice (Mayer et al., 2019; Montgomery et al., 2015; Park et al., 2015; Samineni et al., 2017). These elegantly designed implants are soft and flexible, cause minimal tissue disruption, and can be powered wirelessly. While it may be possible to upscale these devices for use in rats, many require the use of special equipment for fabrication whereas our μLED implant does not and is an accessible option for a large range of laboratories.

Therapeutic stimulation typically requires multiple weeks of activation. This can be challenging as implants can fall victim to the body’s harsh immune response leading to electrical shorts, or the build-up of physical scarring can block light penetration and disrupt activation. Our laboratory is one of the few to have shown long-term implant functionality, with others reporting implant longevity of either 1-2 days post-implant (Montgomery et al., 2015), 1 week post-implant (Mayer et al., 2019; Park et al., 2015), or 3 weeks post-implant (Samineni et al., 2017). Our implant can induce spinal activation for at least 6 weeks making it a promising option for studies requiring both acute and long-term stimulation.

The presented μLED implant is versatile with multiple applications as it can be modified to work with different opsins, like a red-shifted opsin (Govorunova et al., 2011; Lin et al., 2013; Zhang et al., 2008) by changing the LED wavelength. This change may even permit light penetration to more ventral aspects of the spinal cord with a lower light intensity, producing less heat and consuming less power. In this same vein, this implant could also be modified for neuronal inhibition by using a chloride pump like Halorhodopsin (Gradinaru et al., 2008; Han and Boyden, 2007), or a proton pump like Archaerhodopsin (Chow et al., 2010), and incorporating the appropriate wavelength LED into the implant.

Conclusions

The presented μLED implant boasts high accessibility, functional longevity, ease of fabrication and use, and a wide range of applications, making it possible for a variety of laboratories to incorporate optogenetic stimulation into spinal cord-focused investigations. This, in turn, will greatly expand the range of scientific questions that can be addressed and include both basic science and therapeutic applications.

Acknowledgements

This work was supported by the Craig H. Neilsen Foundation (476694), the Center for Neurotechnology, a National Science Foundation-Engineering Research Center (EEC-1028725), the Christopher and Dana Reeve Foundation, the Morton Cure Paralysis Fund, and the Levinson Foundation.

Footnotes

Declarations of interest

None

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