Anthony Zorzos

Three-dimensional multiwaveguide probe array for light delivery to distributed brain circuits

To deliver light to the brain for neuroscientific and neuroengineering applications like optogenetics, in which light is used to activate or silence neurons expressing specific photosensitive proteins, optical fibers are commonly used. However, an optical fiber is limited to delivering light to a single target within the 3D structure of the brain. Here, we describe the design and fabrication of an array of thin microwaveguides, which terminates at a three-dimensionally distributed set of points, appropriate for delivering light to targets distributed in a 3D pattern throughout the brain.

Multiwaveguide implantable probe for light delivery to sets of distributed brain targets

Optical fibers are commonly inserted into living tissues such as the brain in order to deliver light to deep targets for neuroscientific and neuroengineering applications such as optogenetics, in which light is used to activate or silence neurons expressing specific photosensitive proteins. However, an optical fiber is limited to delivering light to a single target within the three-dimensional structure of the brain. We here demonstrate a multiwaveguide probe capable of independently delivering light to multiple targets along the probe axis, thus enabling versatile optical control of sets of distributed brain targets. The 1.45-cm-long probe is microfabricated in the form of a 360-μm-wide array of 12 parallel silicon oxynitride (SiON) multimode waveguides clad with SiO(2) and coated with aluminum; probes of custom dimensions are easily created as well. The waveguide array accepts light from a set of sources at the input end and guides the light down each waveguide to an aluminum corner mirror that efficiently deflects light away from the probe axis. Light losses at each stage are small (input coupling loss, 0.4 ± 0.3 dB; bend loss, negligible; propagation loss, 3.1 ± 1 dB/cm using the outscattering method and 3.2 ± 0.4 dB/cm using the cutback method; corner mirror loss, 1.5 ± 0.4 dB); a waveguide coupled, for example, to a 5 mW source will deliver over 1.5 mW to a target at a depth of 1 cm.

A murine model of a novel surgical architecture for proprioceptive muscle feedback and its potential application to control of advanced limb prostheses

OBJECTIVE Proprioceptive mechanisms play a critical role in both reflexive and volitional lower extremity control. Significant strides have been made in the development of bionic limbs that are capable of bi-directional communication with the peripheral nervous system, but none of these systems have been capable of providing physiologically-relevant muscle-based proprioceptive feedback through natural neural pathways. In this study, we present the agonist-antagonist myoneural interface (AMI), a surgical approach with the capacity to provide graded kinesthetic feedback from a prosthesis through mechanical activation of native mechanoreceptors within residual agonist-antagonist muscle pairs. APPROACH (1) Sonomicrometery and electroneurography measurement systems were validated using a servo-based muscle tensioning system. (2) A heuristic controller was implemented to modulate functional electrical stimulation of an agonist muscle, using sonomicrometric measurements of stretch from a mechanically-coupled antagonist muscle as feedback. (3) One AMI was surgically constructed in the hindlimb of each rat. (4) The gastrocnemius-soleus complex of the rat was cycled through a series of ramp-and-hold stretches in two different muscle architectures: native (physiologically-intact) and AMI (modified). Integrated electroneurography from the tibial nerve was compared across the two architectures. MAIN RESULTS Correlation between stretch and afferent signal demonstrated that the AMI is capable of provoking graded afferent signals in response to ramp-and-hold stretches, in a manner similar to the native muscle architecture. The response magnitude in the AMI was reduced when compared to the native architecture, likely due to lower stretch amplitudes. The closed-loop control system showed robustness at high stretch magnitudes, with some oscillation at low stretch magnitudes. SIGNIFICANCE These results indicate that the AMI has the potential to communicate meaningful kinesthetic feedback from a prosthetic limb by replicating the agonist-antagonist relationships that are fundamental to physiological proprioception.

Transdermal optogenetic peripheral nerve stimulation

OBJECTIVE A fundamental limitation in both the scientific utility and clinical translation of peripheral nerve optogenetic technologies is the optical inaccessibility of the target nerve due to the significant scattering and absorption of light in biological tissues. To date, illuminating deep nerve targets has required implantable optical sources, including fiber-optic and LED-based systems, both of which have significant drawbacks. APPROACH Here we report an alternative approach involving transdermal illumination. Utilizing an intramuscular injection of ultra-high concentration AAV6-hSyn-ChR2-EYFP in rats. MAIN RESULTS We demonstrate transdermal stimulation of motor nerves at 4.4 mm and 1.9 mm depth with an incident laser power of 160 mW and 10 mW, respectively. Furthermore, we employ this technique to accurately control ankle position by modulating laser power or position on the skin surface. SIGNIFICANCE These results have the potential to enable future scientific optogenetic studies of pathologies implicated in the peripheral nervous system for awake, freely-moving animals, as well as a basis for future clinical studies.

Towards optogenetic sensory replacement

Over the last several years we have developed a rapidly-expanding suite of genetically-encoded reagents (e.g., ChR2, Halo, Arch, Mac, and others) that, when expressed in specific neuron types in the nervous system, enable their activities to be powerfully and precisely activated and silenced in response to light. If the genes that encode for these reagents can be delivered to cells in the body using gene therapy methods, and if the resultant protein payloads operate safely and effectively over therapeutically important periods of time, these molecules could subserve a set of precise prosthetics that use light as the trigger of information entry into the nervous system, e.g. for sensory replacement. Here we discuss the use of ChR2 to make the photoreceptor-deprived retina, as found in diseases such as retinitis pigmentosa, sensitive to light, enabling restoration of functional vision in a mouse model of blindness. We also discuss arrays of light sources that could be useful for delivering patterned sensory information into the nervous system.

On prosthetic control: A regenerative agonist-antagonist myoneural interface

The agonist-antagonist myoneural interface enables bidirectional signaling for enhanced prosthetic control and sensation. Prosthetic limb control is fundamentally constrained by the current amputation procedure. Since the U.S. Civil War, the external prosthesis has benefited from a pronounced level of innovation, but amputation technique has not significantly changed. During a standard amputation, nerves are transected without the reintroduction of proper neural targets, causing painful neuromas and rendering efferent recordings infeasible. Furthermore, the physiological agonist-antagonist muscle relationships are severed, precluding the generation of musculotendinous proprioception, an afferent feedback modality critical for joint stability, trajectory planning, and fine motor control. We establish an agonist-antagonist myoneural interface (AMI), a unique surgical paradigm for amputation. Regenerated free muscle grafts innervated with transected nerves are linked in agonist-antagonist relationships, emulating the dynamic interactions found within an intact limb. Using biomechanical, electrophysiological, and histological evaluations, we demonstrate a viable architecture for bidirectional signaling with transected motor nerves. Upon neural activation, the agonist muscle contracts, generating electromyographic signal. This contraction in the agonist creates a stretch in the mechanically linked antagonist muscle, producing afferent feedback, which is transmitted through its motor nerve. Histological results demonstrate regeneration and the presence of the spindle fibers responsible for afferent signal generation. These results suggest that the AMI will not only produce robust signals for the efferent control of an external prosthesis but also provide an amputee’s central nervous system with critical musculotendinous proprioception, offering the potential for an enhanced prosthetic controllability and sensation.

Spectrally distinct channelrhodopsins for two-colour optogenetic peripheral nerve stimulation

Technologies for peripheral nerve stimulation have conventionally relied on the anatomic placement of electrodes adjacent to subsets of sensory fibres or motor fibres that selectively target an end effector. Here, we demonstrate the use of optogenetics to directly target the innervating fibres of an end effector by relying on retrograde transfection of adeno-associated virus serotype 6 to restrict axonal opsin expression to the desired fibre targets. By using an in vivo screen in rats, we identify the first channelrhodopsins as well as a halorhodopsin that respond to red light in the peripheral nerve. Combining two channelrhodopsins with spectrally distinct activation profiles allowed us to drive opposing muscle activity via two-colour illumination of the same mixed nerve. We also show halorhodopsin-mediated reductions in electrically evoked muscle tremor spectrally optimized for deep peripheral nerves. Our non-invasive peripheral neurostimulator with targeted multi-fascicle resolution enables scientific and clinical exploration, such as motor control in paralysis, biomimetic sensation feedback for amputees and targeted inhibition of muscle tremor.Viral transfection of a mixed nerve with two channelrhodopsins with spectrally distinct activation sensitivities enables, via two-colour stimulation of the nerve, optogenetic control over the activity of opposing muscle pairs in a rat hindlimb.

Assessment of Nerve Regeneration through a Novel Microchannel Array

Advancements in robotic technologies have enabled a significant improvement in the clinical efficacy of prosthetic limbs for persons with upper and lower-extremity amputations. However, significant challenges still remain in establishing a biomimetic bidirectional neural communication between amputees and their external powered prostheses. Regenerative peripheral nerve interfaces may offer a high-resolution alternative to conventional nerve interface technologies for their unique potential to provide increased biospatial resolution for both the control of and feedback from an external powered prosthesis. Here, we present three active 3-D microchannel arrays, having 16-20 channels and each 200 μm by 200 μm: one passive (without integrated electrodes), one active (with integrated electrodes), and one active with a porous collagen scaffold. With the array positioned between proximal and distal nerve stumps, we evaluate their effectiveness in tibial n. regeneration in vivo in both rats (N=4) and ferrets (N=4). Using immunofluorescence, we report robust mixed sensory and motor nerve regeneration through the microchannels in all rats, and weak regeneration in 2 of 4 ferrets, suggesting both interspecies regeneration variability and the lack of benefit of axially oriented collagen in improving ferret nerve regeneration through microchannels.

Scalable, Modular Three-Dimensional Silicon Microelectrode Assembly via Electroless Plating

We devised a scalable, modular strategy for microfabricated 3-D neural probe synthesis. We constructed a 3-D probe out of individual 2-D components (arrays of shanks bearing close-packed electrodes) using mechanical self-locking and self-aligning techniques, followed by electroless nickel plating to establish electrical contact between the individual parts. We detail the fabrication and assembly process and demonstrate different 3-D probe designs bearing thousands of electrode sites. We find typical self-alignment accuracy between shanks of <0.2° and demonstrate orthogonal electrical connections of 40 µm pitch, with thousands of connections formed electrochemically in parallel. The fabrication methods introduced allow the design of scalable, modular electrodes for high-density 3-D neural recording. The combination of scalable 3-D design and close-packed recording sites may support a variety of large-scale neural recording strategies for the mammalian brain.