Optogenetic Control of Targeted Peripheral Axons in Freely Moving Animals methods
Aim. Evidence-backed execution summary for Optogenetic Control of Targeted Peripheral Axons in Freely Moving Animals methods from Optogenetic Control of Targeted Peripheral Axons in Freely Moving Animals.
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rat
Subject model for the experiment.
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- confirm full cohort details in the source paper
In vivo animal preparation and acute stimulation and recording
reagent used in the protocol.
- Use
- Animals expressing ChR2 were anesthetized using isoflurane and warmed with a heating pad. The hindlimb and back of the animal was shaved. The bone attached to the proximal tendon of the targeted muscle was immobilized. The muscle was exposed and kept moist with Ringer's solution. The distal tendon of the muscle was...
Histology
reagent used in the protocol.
- Use
- Rats were anesthetized and perfused with 4% paraformaldehyde (PFA) in PBS. Spinal cord, muscle, sciatic nerve, and dorsal root ganglia were dissected, postfixed (4°C overnight), and transferred to 30% sucrose in PBS (4°C until sectioning). Tissues were embedded in Tissue-Tek OCT compound (Sakura, Holland)...
Implantable optical nerve cuffs are tolerated and activate motor neurons expressing ChR2
( A ) Biocompatible spiral cuffs were constructed from polydimethylsiloxane (PDMS) and covalently bound to a silicon-based optical fiber that was terminated with a stainless steel ferrule. ( B ) Optical nerve cuffs were implanted into rats around the sciatic nerve 4 weeks following AAV6:ChR2 delivery. ( C ) Typical...
- Use
- ( A ) Biocompatible spiral cuffs were constructed from polydimethylsiloxane (PDMS) and covalently bound to a silicon-based optical fiber that was terminated with a stainless steel ferrule. ( B ) Optical nerve cuffs were implanted into rats around the sciatic nerve 4 weeks following AAV6:ChR2 delivery. ( C ) Typical...
In vivo animal preparation and acute stimulation and recording
Animals expressing ChR2 were anesthetized using isoflurane and warmed with a heating pad. The hindlimb and back of the animal was shaved. The bone attached to the proximal tendon of the targeted muscle was immobilized. The muscle was exposed and kept moist with Ringer's solution. The distal tendon of the muscle was...
- Use
- Animals expressing ChR2 were anesthetized using isoflurane and warmed with a heating pad. The hindlimb and back of the animal was shaved. The bone attached to the proximal tendon of the targeted muscle was immobilized. The muscle was exposed and kept moist with Ringer's solution. The distal tendon of the muscle was...
Implantation of cuffs and EMG electrodes
In some animals, implantable stainless steel electrodes (Plastics One, Cat: E363/76H, Roanoke, VA) were implanted directly after implantation of the cuff. The electrodes were sutured to the surface of the target muscle and the contralateral muscle on the opposite leg. A ground electrode was sutured to the base of th...
- Use
- In some animals, implantable stainless steel electrodes (Plastics One, Cat: E363/76H, Roanoke, VA) were implanted directly after implantation of the cuff. The electrodes were sutured to the surface of the target muscle and the contralateral muscle on the opposite leg. A ground electrode was sutured to the base of th...
Stimulation in awake and moving rats
Rats were trained to walk on a treadmill (Harvard Apparatus, Holliston, MA, 760303) at 25 cm/s for 10 minutes per day, 2 days per week for 2 weeks before implantation of the nerve cuff. At time of testing, a fiber optic cable and ceramic sleeve were used to connect the laser to the nerve cuff ferrule at the skull. C...
- Use
- Rats were trained to walk on a treadmill (Harvard Apparatus, Holliston, MA, 760303) at 25 cm/s for 10 minutes per day, 2 days per week for 2 weeks before implantation of the nerve cuff. At time of testing, a fiber optic cable and ceramic sleeve were used to connect the laser to the nerve cuff ferrule at the skull. C...
Gait testing for tolerability
Gait was examined one week following implantation using the Catwalk XT gait analysis system (Noldus Information Technology, Asheville, NC). Rats ambulated on an illuminated glass surface within a narrow corridor. Footprints were recorded with a high-speed camera. Five trials were acquired per animal and analyzed usi...
- Use
- Gait was examined one week following implantation using the Catwalk XT gait analysis system (Noldus Information Technology, Asheville, NC). Rats ambulated on an illuminated glass surface within a narrow corridor. Footprints were recorded with a high-speed camera. Five trials were acquired per animal and analyzed usi...
Histology
Rats were anesthetized and perfused with 4% paraformaldehyde (PFA) in PBS. Spinal cord, muscle, sciatic nerve, and dorsal root ganglia were dissected, postfixed (4°C overnight), and transferred to 30% sucrose in PBS (4°C until sectioning). Tissues were embedded in Tissue-Tek OCT compound (Sakura, Holland)...
- Use
- Rats were anesthetized and perfused with 4% paraformaldehyde (PFA) in PBS. Spinal cord, muscle, sciatic nerve, and dorsal root ganglia were dissected, postfixed (4°C overnight), and transferred to 30% sucrose in PBS (4°C until sectioning). Tissues were embedded in Tissue-Tek OCT compound (Sakura, Holland)...
Supporting Information
Experimental setup for treadmill experiment. AAV6:ChR2 was injected into the tibialis anterior or gastrocnemius, and four to six weeks later, an optical nerve cuff was implanted around the sciatic nerve. Surface EMG electrodes were also implanted on the targeted muscle and the non-injected contralateral muscle on th...
- Use
- Experimental setup for treadmill experiment. AAV6:ChR2 was injected into the tibialis anterior or gastrocnemius, and four to six weeks later, an optical nerve cuff was implanted around the sciatic nerve. Surface EMG electrodes were also implanted on the targeted muscle and the non-injected contralateral muscle on th...
Optical nerve cuffs modulate motor neuron activity in freely moving animals
Software used for acquisition, scoring, statistics, or reporting.
- Use
- ( A ) Optical nerve cuffs and EMG electrodes were implanted into rats 4 weeks following AAV6:ChR2 delivery. EMG electrodes were implanted onto the surface of the AAV6:ChR2-injected muscle (targeted) and onto the uninjected muscle on the opposite leg (contralateral). Non-anesthetized rats were tested for EMG activity...
Gait testing for tolerability
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Gait was examined one week following implantation using the Catwalk XT gait analysis system (Noldus Information Technology, Asheville, NC). Rats ambulated on an illuminated glass surface within a narrow corridor. Footprints were recorded with a high-speed camera. Five trials were acquired per animal and analyzed usi...
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Supporting Information
The effect of optical train frequency on ChR2 muscle activation. Typical EMG and force traces following pulses of blue light (20 mW, 2.5 ms) at 24, 36, 50, and 100 Hz.
Supporting Information
The effect of implanted nerve cuffs on gait. Coupling symmetry of paws in age-matched wild-type littermates ( n = 4) and rats 1 week post-implantation of optical nerve cuffs ( n = 5). HL, hind left, FL, front left, HR, hind right, FR, front right. There is no significant difference between wild-type and implanted animals. P values, HL-HR = 0.30, FL-FR = 0.97, FL-HL = 0.19, HR-FR = 0.26.
AAV6:ChR2 delivery enables optogenetic muscle-specific control of the sciatic nerve
We next examined light-mediated activation of virally expressed ChR2 in motor axons of the sciatic nerve. Four to six weeks after vector delivery, we anesthetized rats and exposed their sciatic nerves. To characterize contractile and electrical responses to optogenetic activation, we attached the distal tendon of the AAV6:ChR2-targeted muscle to a force transducer and placed fine wire electromyographic (EMG) electrodes in both the targeted and non-targeted muscles ( ). Pulses of blue light (473 nm) applied to the sciatic nerve using an optical fiber were sufficient to evoke robust muscle twitches ( ) and EMG activity in the targeted muscles. High frequency trains of blue light were capable of generating tetanic muscle contractions and corresponding EMG activity. The shape of the force traces for the tetanus trials was dependent on the frequency of the light pulses ( ). Optical activat...
AAV6:ChR2 delivery enables optogenetic muscle-specific control of the sciatic nerve
( A ) Blue light (473 nm) was applied to the sciatic nerve of anesthetized rats 4 weeks following injection of AAV6:ChR2 in the GN or TA. EMG plots show typical responses from optical stimulation taken with fine wire electrodes in the AAV6:ChR2 targeted-muscle (twitch trial: 20 mW, 5 ms, 1 Hz) (tetanus trial: 20 mW, 2.5 ms, 36 Hz). The distal tendon of the muscle was fixed to a transducer to measure force. Representative force traces are shown for corresponding optical activation and are scaled using supramaximal twitch force ( smf ). ( B ) Representative force traces in response to varying pulse widths of 20 mW blue light. ( C ) Percentage of smf versus pulse width (20 mW light power) for AAV6:ChR2 ( n = 7, GN and TA animals combined) or wild-type ( n = 3) rats. ( D ) Percentage of smf versus light power (5 ms pulse width). ( E ) Fine wire electrodes were pl...
Optical nerve cuffs modulate motor neuron activity in freely moving animals
( A ) Optical nerve cuffs and EMG electrodes were implanted into rats 4 weeks following AAV6:ChR2 delivery. EMG electrodes were implanted onto the surface of the AAV6:ChR2-injected muscle (targeted) and onto the uninjected muscle on the opposite leg (contralateral). Non-anesthetized rats were tested for EMG activity on a treadmill 3 days following the surgery. ( B ) EMG activity in response to a pulse of blue light (20 mW, 5 ms) in the targeted and contralateral muscles in awake, non-moving rats. ( C ) EMG activity in response to pulses of light (20 mW, 5 ms, 1 Hz) in awake rats walking on a treadmill at constant speed (20 cm/s). ( D ) EMG activity in response to 150 ms trains of light (20 mW, 5 ms, 36 Hz) in awake rats walking on a treadmill. ( E ) Integrated EMG in the targeted muscle in response to optogenetic or physiological activation ( n = 3, animals matched) for t...
Implantable optical nerve cuffs are tolerated and activate motor neurons expressing ChR2
( A ) Biocompatible spiral cuffs were constructed from polydimethylsiloxane (PDMS) and covalently bound to a silicon-based optical fiber that was terminated with a stainless steel ferrule. ( B ) Optical nerve cuffs were implanted into rats around the sciatic nerve 4 weeks following AAV6:ChR2 delivery. ( C ) Typical traces of EMG (targeted and non-targeted muscles) and force (targeted muscle only) following illumination using the optical nerve cuff (20 mW, 5 ms, 1 Hz) in anesthetized rats 1 week following cuff implantation. ( D ) Representative force trace following a train of light pulses (20 mW, 2.5 ms, 36 Hz) using the nerve cuff. ( E ) Percentage of smf versus pulse width (20 mW light power) using direct laser light application ( n = 7) or light transmitted through the implanted cuff ( n = 3). ( F ) Percentage of smf versus light power (5 ms pulse width) u...
Implantable optical nerve cuffs are tolerated and activate motor neurons expressing ChR2
We injected the GN or TA muscles of rats with AAV6:ChR2 and once ChR2 expression was established, three to five weeks later, implanted these rats with the optical nerve cuff ( ). We wrapped the cuff around the sciatic nerve and tunneled the optical fiber under the skin up to the head, cementing the ferrule to the skull. One week after implantation, we measured muscle force and EMG signals of the rats under anesthesia to determine if light delivery through the optical nerve cuff was possible following a week of free animal movement. We observed consistent light-mediated generation of twitch ( ) and tetanic force ( ) through the optical nerve cuff. Force and EMG activity was similar to that achieved with direct light application and was tunable by modulating light pulse width ( ) and light power ( ). EMG recordings demonstrated that the optical nerve cuff preferentially activated the ta...
Optical nerve cuffs modulate motor neuron activity in freely moving animals
We assessed whether the optical nerve cuffs could modulate PNS activity in freely moving rats using implanted EMG electrodes to record muscle activity. We sutured electrodes to the surface of the AAV6:ChR2-injected muscle and non-injected contralateral muscle (immediately following cuff implantation) and tunneled wires subcutaneously to a pedestal mounted at the skull ( ). We observed consistent muscle twitches ( ) and EMG activity in response to blue light (20 mW light power, 5 ms pulse width) in non-moving, but awake rats ( ). In wild-type animals implanted with the optical nerve cuff and electrodes, muscles were not activated by illumination of the sciatic nerve with blue light.
Measurement outputs
What raw and processed outputs should exist?
We injected the GN or TA muscles of rats with AAV6:ChR2 and once ChR2 expression was established, three to five weeks later, implanted these rats with the optical nerve cuff ( )...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
To test whether the optical nerve cuff was tolerated by rats, we performed automated gait analysis of the rats at one week post-implantation. Gait is sensitive to motor and sens...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
We assessed whether the optical nerve cuffs could modulate PNS activity in freely moving rats using implanted EMG electrodes to record muscle activity. We sutured electrodes to...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
AAV6 expressing ChR2 (AAV6:ChR2) was produced at the University of North Carolina Vector Core Facility. The expression cassette comprised ChR2 (H134R mutant) fused to yellow flu...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
The effect of implanted nerve cuffs on gait.
from paperScoring or quantification
Quantify the primary readouts for this experiment: We injected the GN or TA muscles of rats with AAV6:ChR2 and once ChR2 expression was established, three to five weeks later, implanted these rats with the optical nerve cuff ( )...; To test whether the optical nerve cuff was tolerated by rats, we performed automated gait analysis of the rats at one week post-implantation. Gait is sensitive to motor and sens...; We assessed whether the optical nerve cuffs could modulate PNS activity in freely moving rats using implanted EMG electrodes to record muscle activity. We sutured electrodes to...; AAV6 expressing ChR2 (AAV6:ChR2) was produced at the University of North Carolina Vector Core Facility. The expression cassette comprised ChR2 (H134R mutant) fused to yellow flu....
from paperStatistical comparison
The effect of implanted nerve cuffs on gait. Coupling symmetry of paws in age-matched wild-type littermates ( n = 4) and rats 1 week post-implantation of optical ne...; ( A ) Optical nerve cuffs and EMG electrodes were implanted into rats 4 weeks following AAV6:ChR2 delivery. EMG electrodes were implanted onto the surface of the AAV6:ChR2-injec...; To compare optogenetic and physiological activation, we calculated integrated EMG (iEMG) values for the various trials. We observed that light-mediated activation (20 mW light p...; Rats were trained to walk on a treadmill (Harvard Apparatus, Holliston, MA, 760303) at 25 cm/s for 10 minutes per day, 2 days per week for 2 weeks before implantation of the ner...
from paperReporting output
Report representative outputs alongside summary comparisons for We injected the GN or TA muscles of rats with AAV6:ChR2 and once ChR2 expression was established, three to five weeks later, implanted these rats with the optical nerve cuff ( )..., To test whether the optical nerve cuff was tolerated by rats, we performed automated gait analysis of the rats at one week post-implantation. Gait is sensitive to motor and sens..., We assessed whether the optical nerve cuffs could modulate PNS activity in freely moving rats using implanted EMG electrodes to record muscle activity. We sutured electrodes to..., AAV6 expressing ChR2 (AAV6:ChR2) was produced at the University of North Carolina Vector Core Facility. The expression cassette comprised ChR2 (H134R mutant) fused to yellow flu....
inferred from protocolStructured statistical methods
The effect of implanted nerve cuffs on gait. Coupling symmetry of paws in age-matched wild-type littermates ( n = 4) and rats 1 week post-implantation of optical ne...; ( A ) Optical nerve cuffs and EMG electrodes were implanted into rats 4 weeks following AAV6:ChR2 delivery. EMG electrodes were implanted onto the surface of the AAV6:ChR2-injec...; To compare optogenetic and physiological activation, we calculated integrated EMG (iEMG) values for the various trials. We observed that light-mediated activation (20 mW light p...; Rats were trained to walk on a treadmill (Harvard Apparatus, Holliston, MA, 760303) at 25 cm/s for 10 minutes per day, 2 days per week for 2 weeks before implantation of the ner...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
The effect of optical train frequency on ChR2 muscle activation. Typical EMG and force traces following pulses of blue light (20 mW, 2.5 ms) at 24, 36, 50, and 100 Hz.
The effect of implanted nerve cuffs on gait. Coupling symmetry of paws in age-matched wild-type littermates ( n = 4) and rats 1 week post-implantation of optical nerve cuffs ( n = 5). HL, hind left, FL, front left, HR, hind right, FR, front right. There is no significant difference between wild-type and implanted animals. P values, HL-HR = 0.30, FL-FR = 0.97, FL-HL = 0.19, HR-FR = 0.26.
We next examined light-mediated activation of virally expressed ChR2 in motor axons of the sciatic nerve. Four to six weeks after vector delivery, we anesthetized rats and exposed their sciatic nerves. To characterize contractile and electrical responses to optogenetic activation, we attached the distal tendon of the AAV6:ChR2-targeted muscle to a force transducer and placed fine wire electromyographic (EMG) electrodes in both the targeted and non-targeted muscles ( ). Pulses of blue light (473 nm) applied to the sciatic nerve using an optical fiber were sufficient to evoke robust muscle twitches ( ) and EMG activity in the targeted muscles. High frequency trains of blue light were capable of generating tetanic muscle contractions and corresponding EMG activity. The shape of the force traces for the tetanus trials was dependent on the frequency of the light pulses ( ). Optical activation at greater than 36 Hz resulted in a reduction in force through time and likely reflects a desensitization of ChR2. We speculate that excitatory opsins with faster kinetics, such as ChETA, may be used in the future to overcome this.
( A ) Blue light (473 nm) was applied to the sciatic nerve of anesthetized rats 4 weeks following injection of AAV6:ChR2 in the GN or TA. EMG plots show typical responses from optical stimulation taken with fine wire electrodes in the AAV6:ChR2 targeted-muscle (twitch trial: 20 mW, 5 ms, 1 Hz) (tetanus trial: 20 mW, 2.5 ms, 36 Hz). The distal tendon of the muscle was fixed to a transducer to measure force. Representative force traces are shown for corresponding optical activation and are scaled using supramaximal twitch force ( smf ). ( B ) Representative force traces in response to varying pulse widths of 20 mW blue light. ( C ) Percentage of smf versus pulse width (20 mW light power) for AAV6:ChR2 ( n = 7, GN and TA animals combined) or wild-type ( n = 3) rats. ( D ) Percentage of smf versus light power (5 ms pulse width). ( E ) Fine wire electrodes were placed in the targeted and non-targeted muscles of the sciatic nerve. Representative EMG traces are shown following electrical or optogenetic stimulation. ( F ) Integrated EMG versus pulse width (20 mW light power) in the targeted and non-targeted muscles following optical activation ( n =̴...
( A ) Optical nerve cuffs and EMG electrodes were implanted into rats 4 weeks following AAV6:ChR2 delivery. EMG electrodes were implanted onto the surface of the AAV6:ChR2-injected muscle (targeted) and onto the uninjected muscle on the opposite leg (contralateral). Non-anesthetized rats were tested for EMG activity on a treadmill 3 days following the surgery. ( B ) EMG activity in response to a pulse of blue light (20 mW, 5 ms) in the targeted and contralateral muscles in awake, non-moving rats. ( C ) EMG activity in response to pulses of light (20 mW, 5 ms, 1 Hz) in awake rats walking on a treadmill at constant speed (20 cm/s). ( D ) EMG activity in response to 150 ms trains of light (20 mW, 5 ms, 36 Hz) in awake rats walking on a treadmill. ( E ) Integrated EMG in the targeted muscle in response to optogenetic or physiological activation ( n = 3, animals matched) for twitch and tetanus contractions. Integrated EMG responses following optogenetic activation are greater or equal to physiological activity (* P <0.05; 2 tailed paired T-test). ( F ) Integrated EMG versus gait cycle demonstrating that activity was independent of the position of the legs ( n =...
( A ) Biocompatible spiral cuffs were constructed from polydimethylsiloxane (PDMS) and covalently bound to a silicon-based optical fiber that was terminated with a stainless steel ferrule. ( B ) Optical nerve cuffs were implanted into rats around the sciatic nerve 4 weeks following AAV6:ChR2 delivery. ( C ) Typical traces of EMG (targeted and non-targeted muscles) and force (targeted muscle only) following illumination using the optical nerve cuff (20 mW, 5 ms, 1 Hz) in anesthetized rats 1 week following cuff implantation. ( D ) Representative force trace following a train of light pulses (20 mW, 2.5 ms, 36 Hz) using the nerve cuff. ( E ) Percentage of smf versus pulse width (20 mW light power) using direct laser light application ( n = 7) or light transmitted through the implanted cuff ( n = 3). ( F ) Percentage of smf versus light power (5 ms pulse width) using direct laser light application ( n = 7) or light transmitted through the implanted cuff ( n = 3). ( G ) Integrated EMG in targeted and non-targeted muscles following light delivery using the optical nerve cuff (20 mW, 5 ms) ( n = 3). ( H ) Integrated EMG i...
We injected the GN or TA muscles of rats with AAV6:ChR2 and once ChR2 expression was established, three to five weeks later, implanted these rats with the optical nerve cuff ( ). We wrapped the cuff around the sciatic nerve and tunneled the optical fiber under the skin up to the head, cementing the ferrule to the skull. One week after implantation, we measured muscle force and EMG signals of the rats under anesthesia to determine if light delivery through the optical nerve cuff was possible following a week of free animal movement. We observed consistent light-mediated generation of twitch ( ) and tetanic force ( ) through the optical nerve cuff. Force and EMG activity was similar to that achieved with direct light application and was tunable by modulating light pulse width ( ) and light power ( ). EMG recordings demonstrated that the optical nerve cuff preferentially activated the targeted muscle over the non-targeted muscle ( ). We also observed that the optical nerve cuff could elicit muscle activation for at least 4 weeks following implantation ( ).
We assessed whether the optical nerve cuffs could modulate PNS activity in freely moving rats using implanted EMG electrodes to record muscle activity. We sutured electrodes to the surface of the AAV6:ChR2-injected muscle and non-injected contralateral muscle (immediately following cuff implantation) and tunneled wires subcutaneously to a pedestal mounted at the skull ( ). We observed consistent muscle twitches ( ) and EMG activity in response to blue light (20 mW light power, 5 ms pulse width) in non-moving, but awake rats ( ). In wild-type animals implanted with the optical nerve cuff and electrodes, muscles were not activated by illumination of the sciatic nerve with blue light.
Machine-readable layer
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"text": "The effect of optical train frequency on ChR2 muscle activation. Typical EMG and force traces following pulses of blue light (20 mW, 2.5 ms) at 24, 36, 50, and 100 Hz."
},
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"text": "The effect of implanted nerve cuffs on gait. Coupling symmetry of paws in age-matched wild-type littermates ( n = 4) and rats 1 week post-implantation of optical nerve cuffs ( n = 5). HL, hind left, FL, front left, HR, hind right, FR, front right. There is no significant difference between wild-type and implanted animals. P values, HL-HR = 0.30, FL-FR = 0.97, FL-HL = 0.19, HR-FR = 0.26."
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"name": "AAV6:ChR2 delivery enables optogenetic muscle-specific control of the sciatic nerve",
"text": "We next examined light-mediated activation of virally expressed ChR2 in motor axons of the sciatic nerve. Four to six weeks after vector delivery, we anesthetized rats and exposed their sciatic nerves. To characterize contractile and electrical responses to optogenetic activation, we attached the distal tendon of the AAV6:ChR2-targeted muscle to a force transducer and placed fine wire electromyographic (EMG) electrodes in both the targeted and non-targeted muscles ( ). Pulses of blue light (473 nm) applied to the sciatic nerve using an optical fiber were sufficient to evoke robust muscle twitches ( ) and EMG activity in the targeted muscles. High frequency trains of blue light were capable of generating tetanic muscle contractions and corresponding EMG activity. The shape of the force traces for the tetanus trials was dependent on the frequency of the light pulses ( ). Optical activat..."
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"name": "AAV6:ChR2 delivery enables optogenetic muscle-specific control of the sciatic nerve",
"text": "( A ) Blue light (473 nm) was applied to the sciatic nerve of anesthetized rats 4 weeks following injection of AAV6:ChR2 in the GN or TA. EMG plots show typical responses from optical stimulation taken with fine wire electrodes in the AAV6:ChR2 targeted-muscle (twitch trial: 20 mW, 5 ms, 1 Hz) (tetanus trial: 20 mW, 2.5 ms, 36 Hz). The distal tendon of the muscle was fixed to a transducer to measure force. Representative force traces are shown for corresponding optical activation and are scaled using supramaximal twitch force ( smf ). ( B ) Representative force traces in response to varying pulse widths of 20 mW blue light. ( C ) Percentage of smf versus pulse width (20 mW light power) for AAV6:ChR2 ( n = 7, GN and TA animals combined) or wild-type ( n = 3) rats. ( D ) Percentage of smf versus light power (5 ms pulse width). ( E ) Fine wire electrodes were pl..."
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"name": "Optical nerve cuffs modulate motor neuron activity in freely moving animals",
"text": "( A ) Optical nerve cuffs and EMG electrodes were implanted into rats 4 weeks following AAV6:ChR2 delivery. EMG electrodes were implanted onto the surface of the AAV6:ChR2-injected muscle (targeted) and onto the uninjected muscle on the opposite leg (contralateral). Non-anesthetized rats were tested for EMG activity on a treadmill 3 days following the surgery. ( B ) EMG activity in response to a pulse of blue light (20 mW, 5 ms) in the targeted and contralateral muscles in awake, non-moving rats. ( C ) EMG activity in response to pulses of light (20 mW, 5 ms, 1 Hz) in awake rats walking on a treadmill at constant speed (20 cm/s). ( D ) EMG activity in response to 150 ms trains of light (20 mW, 5 ms, 36 Hz) in awake rats walking on a treadmill. ( E ) Integrated EMG in the targeted muscle in response to optogenetic or physiological activation ( n = 3, animals matched) for t..."
},
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"name": "Implantable optical nerve cuffs are tolerated and activate motor neurons expressing ChR2",
"text": "( A ) Biocompatible spiral cuffs were constructed from polydimethylsiloxane (PDMS) and covalently bound to a silicon-based optical fiber that was terminated with a stainless steel ferrule. ( B ) Optical nerve cuffs were implanted into rats around the sciatic nerve 4 weeks following AAV6:ChR2 delivery. ( C ) Typical traces of EMG (targeted and non-targeted muscles) and force (targeted muscle only) following illumination using the optical nerve cuff (20 mW, 5 ms, 1 Hz) in anesthetized rats 1 week following cuff implantation. ( D ) Representative force trace following a train of light pulses (20 mW, 2.5 ms, 36 Hz) using the nerve cuff. ( E ) Percentage of smf versus pulse width (20 mW light power) using direct laser light application ( n = 7) or light transmitted through the implanted cuff ( n = 3). ( F ) Percentage of smf versus light power (5 ms pulse width) u..."
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"name": "Implantable optical nerve cuffs are tolerated and activate motor neurons expressing ChR2",
"text": "We injected the GN or TA muscles of rats with AAV6:ChR2 and once ChR2 expression was established, three to five weeks later, implanted these rats with the optical nerve cuff ( ). We wrapped the cuff around the sciatic nerve and tunneled the optical fiber under the skin up to the head, cementing the ferrule to the skull. One week after implantation, we measured muscle force and EMG signals of the rats under anesthesia to determine if light delivery through the optical nerve cuff was possible following a week of free animal movement. We observed consistent light-mediated generation of twitch ( ) and tetanic force ( ) through the optical nerve cuff. Force and EMG activity was similar to that achieved with direct light application and was tunable by modulating light pulse width ( ) and light power ( ). EMG recordings demonstrated that the optical nerve cuff preferentially activated the ta..."
},
{
"@type": "HowToStep",
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"name": "Optical nerve cuffs modulate motor neuron activity in freely moving animals",
"text": "We assessed whether the optical nerve cuffs could modulate PNS activity in freely moving rats using implanted EMG electrodes to record muscle activity. We sutured electrodes to the surface of the AAV6:ChR2-injected muscle and non-injected contralateral muscle (immediately following cuff implantation) and tunneled wires subcutaneously to a pedestal mounted at the skull ( ). We observed consistent muscle twitches ( ) and EMG activity in response to blue light (20 mW light power, 5 ms pulse width) in non-moving, but awake rats ( ). In wild-type animals implanted with the optical nerve cuff and electrodes, muscles were not activated by illumination of the sciatic nerve with blue light."
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"name": "In vivo animal preparation and acute stimulation and recording"
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"name": "Implantation of cuffs and EMG electrodes"
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"name": "Stimulation in awake and moving rats"
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