Optogenetic Control of Targeted Peripheral Axons in Freely Moving Animals methods
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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.
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Gait recovery and locomotor function
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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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