WO2021247623A1

WO2021247623A1 - Compositions and methods for optogenetic control

Info

Publication number: WO2021247623A1
Application number: PCT/US2021/035320
Authority: WO
Inventors: Paola Arlotta; James Harris
Original assignee: President And Fellows Of Harvard College
Priority date: 2020-06-01
Filing date: 2021-06-01
Publication date: 2021-12-09

Abstract

Disclosed are methods and apparatus for optogenetic control of neurite growth. In some aspects, the disclosed methods and apparatus can be used to create in vitro models of neurological conditions or diseases. In other aspects, the methods and apparatus can be used to treat or prevent neurological diseases or nerve injuries.

Description

COMPOSITIONS AND METHODS FOR OPTOGENETIC CONTROL

RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application No.

63/033,137, filed on June 1, 2020, the entire teachings of which are incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Axons wire neurons into networks capable of efficiently transmitting and processing information across enormous biological distances. During development, the general architecture of these neuronal networks is established in a complex, highly reproducible process of axon extension and guidance that allows axons to reliably find their appropriate targets. However, in most organisms, axons of the adult central nervous system have lost this ability, and are largely incapable of reestablishing appropriate axonal circuitry following injury. This lack of intrinsic axonal regeneration leads to permanent disability after injury, and methods to overcome this barrier will be required to recover functionality. Great progress has been made in understanding the molecular events in the growth cone, a specialized structure at the leading tip of the axon, that directs outgrowth in appropriate directions during development. This knowledge now offers the opportunity to manipulate the endogenous axon guidance machinery to construct novel neuronal circuit architectures and repair defective or injured circuits. If successful within the intact organism, such approaches would enable directed experimental manipulation of circuit wiring, a requisite for downstream understanding of the rules that wire neuronal networks and define their properties in vivo.

[0003] Previous work demonstrated that changes to the molecular composition of the microenvironment in which axons grow can successfully influence their growth trajectories, by ectopically providing guidance factors and permissive environments in vitro and in vivo. Since these methods rely on the creation of highly stereotyped environments to extrinsically influence axonal growth, by definition they lack spatial and temporal flexibility and precision, and require invasive procedures for in vivo application. Precise, non-invasive control of the intrinsic axon guidance machinery within the growth cone itself would alternatively provide unprecedented capacity and flexibility to direct novel connectivity.

SUMMARY OF THE INVENTION

[0004] Using zebrafish motor neurons engineered to express a photoactivatable Racl protein, the inventors have surprisingly found that the endogenous guidance machinery of the growth cone can be co-opted to precisely and noninvasively direct axon growth and guidance using light. Axons can be guided directionally, over large distances, and within the complex environment of the live, intact organism. Notably, competing endogenous signals can be overridden to redirect axon growth across typically repulsive barriers and construct novel circuitry. In addition, optogenetic Racl stimulation can rescue genetic axon guidance defects in mutant zebrafish and restore synaptic function. The results shown herein demonstrate that a ubiquitous central component of the intrinsic growth cone guidance machinery can be co opted to non-invasively shape connectivity within neuronal networks in intact living organisms. The invention described herein can be used to manipulate the axon guidance machinery to construct novel neuronal circuit architectures for, e.g., neuropsychiatric disease modeling, and repair defective or injured neuronal circuits.

[0005] Some aspects of the disclosure are related to a method for controlling neurite growth of a neuronal cell, comprising providing a neuronal cell comprising photoactivatable Racl (PA-Racl), and contacting a portion of the neuronal cell with radiation (i.e., light of a specific wavelength or range of wavelengths) that activates or increases activation of the photoactivatable Racl (e.g., causes or increases binding of the photoactivatable Racl with one or more downstream effector molecules, increases GTPase activity), thereby controlling neurite growth of the neuronal cell. In some embodiments, neuronal cell comprises a neurite. In some embodiments, the neurite or a portion thereof is contacted with the radiation. In some embodiments, the neurite comprises a growth cone. In some embodiments, the growth cone or a portion thereof is contacted with the radiation. In some embodiments, the neurite is an axon. In some embodiments, the neurite is a dendrite.

[0006] In some embodiments, the neuronal cell expresses PA-Racl (e.g, a transgenic cell expressing PA-Racl or a cell contacted with a nucleic acid coding for PA-Racl). In some embodiments, the expression of PA-Racl is under the control of an inducible or constitutive promoter. In some embodiments, the genome of the neuronal cell codes for PA-Racl. In some embodiments, the neuronal cell comprises an expression vector coding for PA-Racl. In some embodiments, the neuronal cell comprises an mRNA sequence coding for PA-Racl. In some embodiments, the neuronal cell further expresses channelrhodopsin (e.g., ReaChR (red-shifted channelrhodop sin) ) .

[0007] In some embodiments, the neuronal cell does not express PA-Racl. In some embodiments, PA-Racl is delivered to the cell. Any suitable vehicle for delivering PA-Racl to the cell may be used.

[0008] In some embodiments, the PA-Racl comprises a LOV (light oxygen voltage) domain from phototropin. In some embodiments, the PA-Racl comprises a Racl, a Racl mutant, or a functional fragment thereof. In some embodiments, the PA-Racl has reduced or no effector activity in the absence of the radiation. In some embodiments, contact of the portion of the neuronal cell with radiation increases the activation (e.g., binding with downstream effectors) of the PA-Racl by 2-fold or more.

[0009] In some embodiments, the radiation (i.e., light) has a wavelength of about 450 nm to 480 nm. In some embodiments, the radiation has a wavelength of 458 nm or 473 nm. In some embodiments, the radiation is produced from a light source selected from a laser, a light emitting diode (LED), and a digital micromirror device (DMD). In some embodiments, the radiation is contacted with the neuronal cell for at least 5 minutes.

[0010] In some embodiments, the methods described herein increase neurite length by at least 50 pm. In some embodiments, the contacted neurite crosses a repulsive boundary. In some embodiments, the contacted neurite forms a synapse (e.g., functional synapse, synapse capable of transducing a signal).

[0011] In some embodiments, the neuronal cell is a sensory neuron, motor neuron, intemeuron, projection neuron, or cortical neuron. In some embodiments, the neuronal cell is derived from an induced pluripotent stem cell or progenitor cell. In some embodiments, the neuronal cell is a human neuronal cell.

[0012] In some embodiments, the neuronal cell is contacted with the radiation in vivo, in vitro, or ex vivo. In some embodiments, a plurality of neuronal cells are contacted with the radiation. In some embodiments, the plurality of contacted neuronal cells form or comprise one or more neural circuits. In some embodiments, the one or more neural circuits provide a model of a neural structure (e.g., a neural structure found in an organism, an aberrant neural structure found in an organism) or aberrant neural condition. In some embodiments, the one or more neural circuits form a biological computer.

[0013] In some embodiments, the methods disclosed herein treat or prevent a nerve injury, neurological disease or neurological condition in a subject in need thereof. In some embodiments, the nerve injury comprises one or more severed neurons. [0014] Some aspects of the disclosure are directed to a biological computer comprising contacted neuronal cells of the methods described herein. Some aspects of the disclosure are directed to an in vitro neural circuit comprising contacted neuronal cells of the methods described herein. Some aspects of the disclosure are directed to a non-human animal comprising contacted neuronal cells of the methods described herein.

[0015] Some aspects of the disclosure are directed to a method of establishing or manipulating a neural connection in a subject in need thereof, comprising providing photoactivatable Racl (PA-Racl) in a neuronal cell in the subject and establishing or manipulating the neural connection by contacting a portion of the neuronal cell with radiation that activates or increases activation (e.g., downstream effector binding) of the photoactivatable Racl. In some embodiments, the neural connection is located in the central nervous system of the subject. In some embodiments, the neural connection is located in the spinal cord of the subject. In some embodiments, the neural connection is located in the peripheral nervous system of the subject. In some embodiments, the neural connection comprises or is partially or fully located in grafted tissue.

[0016] In some embodiments, a plurality of neuronal cells having PA-Racl are contacted with the radiation. In some embodiments, the method causes the formation of one or more synapses. In some embodiments, the neural cell comprises an exogenously added mRNA sequence or expression vector coding for PA-Racl. In some embodiments, the neural cell is a transgenic neural stem or progenitor cell expressing PA-Racl. In some embodiments, the PA- Racl was intracellularly delivered to an endogenous neural cell.

[0017] In some embodiments, the method repairs a severed nerve. In some embodiments, the method treats or prevents a neurological or neuropsychiatric disease or condition.

[0018] In some embodiments, the radiation is produced by one or more light sources selected from a laser and a light emitting diode (LED). In some embodiments, the one or more light sources are implanted. In some embodiments, the location of the radiation is controlled by a digital micromirror device (DMD). In some embodiments, the one or more light sources are external to the subject. In some embodiments, the subject is human. In some embodiments, the subject is an embryo.

[0019] Some aspects of the disclosure are directed to an apparatus for establishing or manipulating a neural connection in a subject, comprising one or more light sources, a power supply operably linked to the one or more light sources, and a processor configured to control the one or more light sources. In some embodiments, the one or more light sources are configured for implantation in a subject. In some embodiments, the one or more light sources comprise a plurality of light sources configured to be implanted at a nerve injury site having a first end and a second end, and direct regrowth and/or repair of the nerve by sequentially turning on and off each light source from the first end to the second end. In some embodiments, the plurality of light sources are a linear string of light sources that are configured to be secured across a nerve injury site, and wherein the light sources are configured to enable growth of one or more neurites along the linear string. In some embodiments, the apparatus comprises a DMD to control light source location and neurite growth.

[0020] Some aspects of the disclosure are related to a kit comprising an apparatus described herein and an agent capable of providing PA-Racl in a neuronal cell in a subject. In some embodiments, the agent comprises a nucleic acid coding for PA-Racl. In some embodiments, the agent is a transgenic stem cell or progenitor cell capable of expressing PA- Racl, and, optionally, ReaChR. In some embodiments, the kit further comprises an agent which comprises a nucleic acid coding for ReaChR.

[0021] Some aspects of the disclosure are related to an isolated transgenic stem cell or progenitor cell capable of expressing PA-Racl and, optionally, ReaChR.

[0022] Some aspects of the disclosure are related to a method of screening for therapeutic agents comprising, providing an in vitro neural circuit modeling a neurological disease or condition and comprising contacted neuronal cells of the method of claims 1-31, contacting the neural circuit with a test agent, and assessing whether the test agent improves one or more aspects of the neurological disease or condition present in the neural circuit. In some embodiments, the neural circuit comprises neuronal cells derived from a subject with the neurological disease or condition. In some embodiments, the neuronal cells are derived from induced pluripotent cells derived from one or more cells from the subject. In some embodiments, the one or more aspects of the neurological disease or condition comprises neuronal cell viability, neuronal cell proliferation, and synaptic signaling.

[0023] Some aspects of the disclosure are related to a method of treating a neuronal guidance defect in a subject in need thereof, comprising administering to the subject an agent that modulates the expression or activity of Racl in a neuronal cell. The agent is not limited and may be any agent described herein. In some embodiments, the agent comprises PA-Racl or a nucleic acid encoding for PA-Racl. In some embodiments, administration of the agent stabilizes the level of Racl in a neuronal cell of the subject. In some embodiments, administration of the agent increases the level of a Racl in the neuronal cell of the subject. [0024] In some embodiments, the method further comprises growth of neurites in the subject mediated by Racl. In some embodiments, the Racl is a PA-Racl described herein. In some embodiments, neurite growth is controlled in the subject via photo-activation of the PA- Racl (e.g., with an apparatus described herein).

[0025] The above discussed, and many other features and attendant advantages of the present inventions will become better understood by reference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0027] Figs. 1A-1C show optogenetic stimulation of PA-Racl results in directed axonal growth of cultured zebrafish spinal motor neuron axons. Fig. 1A shows a schematic of the expression construct and experimental design. Fig. IB shows the time series of cultured PA-Racl+ neurons, either unstimulated (top row) or illuminated in a region of interest focused on the leading edge of the growth cone (blue circle) (bottom row). White arrowheads indicate the initial position of the growth cone (scale bar lOpm). Fig. 1C shows stimulated axons (n=5) grew significantly greater distances over the trial period (left), resulting in a faster rate of growth (middle), and deviated significantly from the initial trajectory compared to unstimulated PA-Racl + axons or stimulated photo-insensitive Racl-i- (PI-Racl) axons (right, paired-end Student’s T-test, **p<0.01, *** p<0.001. Error bars are 95% confidence intervals). See also Fig. 6.

[0028] Figs. 2A-2E show optogenetic stimulation of PA-Racl directed zebrafish CaP axon growth across the repulsive somitic boundary into the neighboring somitic musculature. Fig. 2A shows a schematic of the experimental design; zebrafish expressing PA-Racl in CaP neurons were illuminated at the leading edge of the growth cone from 24 to 29hpf (right). Fig. 2B shows the position of axons prior to stimulation. All CaP neurons were within the somitic boundaries (dashed white chevrons). A single CaP axonal growth cone per fish (red box) was illuminated, while other CaP neurons, including the rostral neighbor (white box), remained unilluminated (scale bar 20pm). Fig. 2C shows the time series of CaP axons in an unstimulated rostral axon (upper series, corresponding to white dashed box in (Fig. 2B)) and illuminated CaP axon (lower series, corresponding to red box in (Fig. 2B)). The stimulated axon was guided across the rostral somitic boundary, while the unstimulated axon remained entirely within its native somite (arrowheads indicate the initial position of the axon; scale bar 20pm). Fig. 2D shows the distribution of outcomes of optogenetic stimulation. Fig. 2E shows PA-Racl stimulation of CaP axons in vivo resulted in significantly longer axons over the trial period, corresponding to faster axonal growth (top two graphs respectively, n=16 zebrafish). The angle of axon growth deviated significantly in both the rostral and caudal directions from the initial trajectory compared with unilluminated controls (middle). Stimulated axons were guided across the somitic boundary, while control axons never crossed [bottom, negative values indicate growth across the somitic boundary, (FDR adjusted, difference of least square means from mixed linear model, n=17 fish, * p<0.05, **P<0.01, *** p<0.001), Error bars are 95% confidence intervals]. R- Rostral; C-Caudal; D-Dorsal; V- Ventral; DM-dorsal myotome; SC-spinal cord; VM-ventral myotome; HMS -horizontal myoseptum; VMS-vertical myoseptum; CaP, MiP, and RoP-caudal, middle and rostral primary spinal motor neurons, respectively. See also Fig. 6.

[0029] Figs. 3A-3E show the molecular investigation of novel neuromuscular synaptic formation between ectopically-directed CaP neuron and foreign myotomes. Fig. 3A shows the Z-projection of a zebrafish CaP neuron (red) deviated across a somitic boundary (dashed white line) revealed by a-bungarotoxin staining (cyan; scale bar lOpm). Fig. 3B shows a 3D rendering from confocal stacks of the deviated (red) and neighboring control (green) axon in (Fig. 3A) with the somitic boundary (blue; scale bar 50pm). Fig. 3C shows the axonal segments in the white dashed box in (Fig. 3A), pseudocolored to reflect the Z-position of the deviated CaP axon (white arrowheads) which passed lateral to the neighboring rostral CaP neuron (scale bar 10pm). Fig. 3D shows the same region as (Fig. 3C), with two regions of interest from the unstimulated CaP axon (dashed white box, control) and the invading CaP axon (dashed white box, deviated; scale bar 10pm). Fig. 3E shows immunohistochemistry of regions of interest in (Fig. 3D) for mCherry (red), synaptic vesicle 2 (green), and a- bungarotoxin (cyan) for the unstimulated control axon (top) and stimulated axon deviated across the somitic boundary (bottom). White arrowheads indicate points of juxtaposition of pre- and post-synaptic markers, suggesting the presence of synapses (scale bar 1 pm). R- rostral, C-caudal, D-dorsal, V-ventral. See also Fig. 7.

[0030] Figs. 4A-4E show optogenetic stimulation of plod3 zebrafish CaP neurons rescues their axon guidance defect allowing for juxtaposition of pre- and postsynaptic machinery within the ventral myotome. (FIG. 4 A) PA-Racl expression (red) in spinal motor neurons of plod3⁺ zebrafish at 28hpf (scale bar 50pm). (FIG. 4B) Left: An age-matched plod3^A mutant sibling prior to stimulation fails to extend CaP axons into the ventral myotome past the horizontal myoseptum (dashed white line). Right: In plod3^A fish, a single PA-Racl⁺ CaP axon extended into the ventral myotome after illumination, while unilluminated axons remain arrested at the horizontal myoseptum (blue circle: region of illumination; scale bar 50mhi). (FIG. 4C) Enlarged image of plod3^A CaP axon following stimulation (solid white box in (B); scale bar 50 pm). (FIG. 4D) Illumination of PA-Racl⁺ CaP axons in plod3^A mutant fish induced growth significantly farther past the horizontal myoseptum than unilluminated axons. There was no significant difference in the distance grown past the horizontal myoseptum in illuminated plod3^A fish compared to plod⁺ fish (FDR adjusted, difference of least square means from mixed linear model, n=8 axons, one axon per fish, *** p<0.001, mean and 95% confidence intervals shown). (FIG. 4E) Immunohistochemistry for mCherry (red, left), SV2 (green, middle left), and aBT (cyan, middle right) showing colocalization of pre- and post-synaptic markers (merge, right) in the ventral myotome of the stimulated axon (bottom), but not in the unstimulated axon (top; scale bar 5pm). Stimulated and unstimulated regions correspond to solid white boxes in (C).

[0031] Figs. 5A-5F shows functional restoration of synaptic connectivity with ventral myotome following rescue of plod3^A zebrafish CaP axons. Fig. 5A shows before (top) and after (bottom) PA-Racl optogenetic mediated rescue of plod3^A mutant zebrafish expressing both the mCherry-PA-Racl (red, left) and the ReaChR-citrine (green, middle) transgenes (merge right, scale bar 50pm). Only the rescued axon extends into the dorsal myotome, past the horizontal myoseptum (dashed white line). Fig. 5B shows immunohistochemistry for mCherry (red, left) and aBT (cyan, middle) illustrates that only the rescued axon extends past the horizontal myoseptum (merge, right; scale bar 50pm). Fig. 5C shows immunohistochemistry for mCherry (red, left), SV2 (green, middle left), and aBT (cyan, middle right), showed colocalization of pre- and post-synaptic markers (merge, right) in the ventral myotome of the stimulated axon (solid white box in Fig. 5B; scale bar 5pm). Fig. 5D shows the time series of ventral myofibril contractions following ReaChR stimulation of a rescued CaP axon. The white arrows show the movement of the somatic boundaries (white lines) in the ventral, but not dorsal myotome compared with time 0 (grey dashed lines, scale bar 50pm). Fig. 5E shows particle image velocimetry using optical flow analysis was performed on bright field time series images to illustrate the motion of the zebrafish body wall following ReaChR channelrhodopsin stimulation after plod3^A axon guidance defect rescue. Color coded arrows represent the direction of optical flow and demonstrate contraction specifically in the ventral musculature following axonal rescue. Purple and green arrows show the direction of tissue motion that results from the myofibril contraction. Fig. 5F shows the number of contractions of ventral myofibrils were counted in response to focal neuronal illumination. Optogenetic depolarization of rescued plod3 via activation of ReaChR channelrhodopsin (Resc+ RChR+) resulted in significantly more contractions of ventral myofibrils compared with ReaChR activation of a neighboring axon that had not been rescued (Resc- RChR+) or illumination of the rescued axon stimulated with wavelengths of light outside of the ReaChR activation spectrum (Resc+ RChR-). (Pairwise comparison of estimated marginal means from a Poisson general linear model, n=3 fish, 5 trials per condition, * p<0.05, **p<0.01, Error bars are 95% confidence intervals.

[0032] Figs. 6A-6E show PA-Racl expression and activation in response to specific wavelengths of light. Fig. 6A shows mCherry-PA-Racl⁺ neurons were FACS sorted for gene expression analysis at 24hpf. Fig. 6B shows expression of transgene pa-racl compared with endogenous racla and raclb. Fig. 6C shows zebrafish spinal motor neuron axons cultured at 18hpf were illuminated in a small region of interest at the edge of the growth cone. The area of the illuminated region that overlapped with the axonal growth cone was measured before and after 5 minutes of exposure first to 514nm light, followed by 5 minutes of exposure to 458nm light. Only after activation with the 458nm light, which stimulates PA-Racl, did the axons extend into the region of illumination. Fig. 6D shows a comparison in the change in area within region of interest occupied by the illuminated growth cone from (Fig. 6C) after illumination with 514nm or 458nm light (paired T-test, n=6, * p<0.05; Error bars are 95% confidence intervals). Fig. 6E shows immunohistochemical detection of autophosphorylation of PAK1 (a downstream effector of Racl signaling) in cultured zebrafish neurons demonstrates light-induced activation of the Racl pathway. Top row: Immunohistochemistry for mCherry, a proxy for PA-Racl expression (a-d, red). Bottom row: Immunohistochemistry for phosphorylation at an activating autophosphorylation site of PAK1 (PAK1-P, bottom row, e-h, pseudocolored blue to yellow to indicate low to high signal, respectively). Panels present comparison of neurons without PA-Racl or stimulation (a,e); with PA-Racl, but no stimulation (b,f); or with PA-Racl and stimulation (c,d,g,h; scale bar lOpm). Enlarged image region of dashed white box in (c,g) shown in (d,h); scale bar 5pm). The blue circle represents the region of illumination and the white arrow indicates increased local PAK1 phosphorylation.

[0033] Figs. 7A-7F sho electron micrographs of an optogenetically-guided neuron confirms axon growth into ectopic tissue. Fig. 7A shows a confocal image of PA-Racl- CaP axon (red arrowheads) that was optogentically guided out of the ventral myotome (outlined by green dashed lines) and adjacent to the kidney duct (outlined by yellow dashed lines; scale bar 30mhi). Fig. 7B show's a schematic of a cross section of zebrafish tail. A stimulated CaP axon was guided out of the myotome to ectopic kidney tissue (red), while an unstimulated CaP axon remains closely associated with the myotome (blue). Fig. 7C shows an electron micrograph of a cross section of zebrafish tail shows an unstimulated CaP axon (blue) and a stimulated CaP axon (red, scale bar 20 pm). Fig. 7D shows the magnification of unstimulated CaP axon (blue arrows) from Fig. 7B. A migratory cell is highlighted in brown. Fig. 7E shows the magnification of stimulated CaP axon (red arrows) from Fig. 7B. Fig. 7F shows a boxed region in Fig. 7D which shows juxtaposition of stimulated CaP axon with kidney duct tissue. Migratory cells accompany this aberrantly guided axon. VM- ventral myotome; NT-neural tube; M- myotome; NC- notochord; DA- dorsal aorta; AV- axial vein; KD- kidney duct; MC- migratory cell.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Axons connect neurons together, establishing the wiring architecture of neuronal networks that transmit and process information. Axonal connectivity is largely built during embryonic development through highly constrained processes of axon guidance, for which the underlying mechanisms have been extensively studied. However, the inability to precisely control axon guidance, and thus the architecture of neuronal networks, has limited investigation of how axonal connections influence the subsequent development and functionality of neuronal networks. Using zebrafish motor neurons engineered to express a photoactivatable Racl protein, it is shown that the endogenous guidance machinery of the growth cone can be co-opted to precisely and noninvasively direct axon growth and guidance using light. Axons can be guided directionally, over large distances, and within the complex environment of the live, intact organism. Competing endogenous signals can be overridden to redirect axon growth across potent repulsive barriers to construct novel circuitry. Notably, optogenetic Racl stimulation can rescue genetic axon guidance defects in mutant zebrafish, restoring functional connectivity. These data demonstrate that a ubiquitous component of the intrinsic growth cone guidance machinery can be co-opted to non-invasively build new connectivity and allow investigation of neural network dynamics in the intact living organism.

[0035] Some aspects of the disclosure are directed to a method for controlling neurite growth of a neuronal cell, comprising providing a neuronal cell comprising photoactivatable Racl (PA-Racl), and contacting a portion of the neuronal cell with radiation that activates or increases activation (e.g., downstream effector binding, GTPase activity) of the photoactivatable Racl, thereby controlling neurite growth of the neuronal cell. In some embodiments, the radiation is contacted with a portion of the neuronal cell that is not a neurite and the neuronal cell is induced to form a new neurite. In some embodiments, the radiation is contacted with a neurite or portion thereof of the neuronal cell. In some embodiments, the neural cell comprises a neurite with a growth cone. In some embodiments, the radiation is contacted with the growth cone or portion thereof. In some embodiments, the neurite is an axon. In some embodiments, the neurite is a dendrite.

[0036] In some embodiments, the neuronal cell expresses PA-Racl. In some embodiments, the neuronal cell or neuronal cell progenitor may be transformed with an expression vector. In some embodiments, the neuronal cell further expresses a channelrhodopsin (e.g., ReaChR).

[0037] As used herein, the term “transform” means to introduce into a neuronal cell an exogenous polynucleotide (e.g., a nucleic acid or nucleic acid analog) which replicates within that neuronal cell, that encodes a gene product (e.g., an amino acid, polypeptide sequence, protein or enzyme) which is expressed in that neuronal cell, and/or that is integrated into the genome of that neuronal cell so as to affect the expression of a genetic locus within the genome. The term “transform” is used to embrace all of the various methods of introducing such polynucleotides (e.g., nucleic acids or nucleic acid analogs), including, but not limited to the methods referred to in the art as transformation, transfection, transduction, or gene transfer, and including techniques such as microinjection, DEAE-dextran-mediated endocytosis, calcium phosphate coprecipitation, electroporation, liposome-mediated transfection, ballistic injection, viral-mediated transfection, and the like.

[0038] In some embodiments, disclosed herein are methods of transforming a neuronal cell or neuronal cell progenitor, wherein such methods comprise a step of contacting the neuronal cell with an expression vector under conditions sufficient for the vector to integrate into the neuronal cell genome.

[0039] As used herein, the term “vector” means any genetic construct, such as for example, a plasmid, phage, transposon, cosmid, chromosome, vims and/or virion, which is capable transferring nucleic acids between cells. Vectors may be capable of one or more of replication, expression, and insertion or integration, but need not possess each of these capabilities. Thus, the term includes cloning, expression, homologous recombination, and knock-out vectors. [0040] In some embodiments, the expression of PA-Racl is under the control of an inducible or constitutive promoter. In some embodiments, the genome of the neuronal cell codes for PA-Racl. In some embodiments, the neuronal cell comprises an expression vector coding for PA-Racl.

[0041] In some embodiments, the neuronal cell comprises an mRNA sequence coding for PA-Racl. Standard methods such as plasmid DNA transfection, viral vector delivery, transfection with modified or synthetic mRNA (e.g., capped, polyadenylated mRNA), or microinjection can be used to introduce a nucleic acid (e.g., a ribonucleic acid) to the neuronal cell. In some embodiments, the modified or synthetic mRNA comprises one or more modifications that stabilize the mRNA or provide other improvements over naturally occurring mRNA (e.g., increased cellular uptake). Examples of modified or synthetic mRNA are described in Warren et al. (Cell Stem Cell 7(5):618-30, 2010, Mandal PK, Rossi DJ. Nat Protoc. 2013 8(3):568-82, US Pat. Pub. No. 20120046346 and/or PCT/US2011/032679 (WO/2011/130624). mRNA is also discussed in R.E. Rhoads (Ed.), “Synthetic mRNA: Production, Introduction Into Cells, and Physiological Consequences,” Series: Methods in Molecular Biology, Vol. 1428. Additional examples are found in numerous PCT and US applications and issued patents to Moderna Therapeutics, e.g., PCT/US2011/046861; PCT/US2011/054636, PCT/US2011/054617, USSN 14/390,100 (and additional patents and patent applications mentioned in these.) If DNA encoding the PA-Racl is introduced, the coding sequences should be operably linked to appropriate regulatory elements for expression, such as a promoter and termination signal.

[0042] In some embodiments, an induced pluripotent stem cell derived from a cell of a subject is modified with an expression vector to express PA-Racl. In some embodiments, the induced pluripotent stem cell or a progenitor cell thereof is modified using CRISPR to express PA-Racl. In some embodiments, a neural cell is modified using CRISPR to express PA-Racl. Methods of using CRISPR to modify the genome of a cell are known in the art. Any suitable CRISPR method may be used herein.

[0043] In some embodiments, the neuronal cell does not express PA-Racl but instead exogenous PA-Racl is delivered to the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, biopolymer nanoparticle, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer. [0044] Racl (Rac Family Small GTPase 1) is a key GTPase regulating actin cytoskeletal dynamics. In some embodiments, the PA-Racl described herein comprises Racl or a functional fragment thereof. In some embodiments, Racl refers to Racla or Raclb. In some embodiments, Racl refers to Racla. In some embodiments, Racl refers to Raclb. In some embodiments, the PA-Racl comprises a Racl mutant or functional fragment thereof. In some embodiments, the Racl mutant has L514K and L531E mutations in the J-oc helix (see, S. K. Yoo et ah, Dev Cell 18, 226-236 (2010) and A. Hayashi-Takagi et ah, Nature 525, 333-338 (2015)). In some embodiments, the Racl or mutant thereof is human Racl or a mutation thereof. In some embodiments, the PA-Racl is PA-Racl disclosed in S. K. Yoo et ah, Dev Cell 18, 226-236 (2010) or A. Hayashi-Takagi et ah, Nature 525, 333-338 (2015). In some embodiments, the PA-Racl is the PA-Racl disclosed in the Examples section herein.

[0045] ReaChR (red-shifted channelrhodopsin) is a channelrhodopsin (ChR), which are used to optogenetically depolarize neurons. ReaChR is an engineered variant of ChR, that is optimally excited with orange to red light (l -590-630 nm) and offers improved membrane trafficking, higher photocurrents, and faster kinetics compared to existing red-shifted ChRs (see, Lin et ah, Nat Neuroscience 16(10) 1499-1508 (2013). In some embodiments, polarization/depolarization of a neuronal cell comprising PA-Racl and a channelrhodopsin (e.g., ReaChR) may be controlled via excitation with an appropriate wavelength of light to activate the ChR. In some embodiments, functioning of neurites grown via PA-Racl activation are confirmed via photoactivation of ChR in the neurite.

[0046] The PA-Racl may contain any suitable photo-activation domain and is not limited as long as the PA-Racl has increased activity (e.g., effector binding, downstream effector binding, GTPase activity) (including increased from no activity or substantially no activity) when contacted with radiation (e.g., light). The term “light activation” (also referred to herein as “photoactivation”) is used herein to refer to control of a protein activity by application of light of selected wavelengths to a fusion protein as described herein. The fusion protein is “activated” when light applied to the photoreceptor causes a change in conformation of the output module of the fusion protein such that it changes the activity of the output module. This change is believed to be caused, at least in part, by rotation of the monomeric output modules with respect to each other such that a desired activity of the fusion protein is changed, e.g., started or enhanced. The term “light activated” (also called “photoactive” in reference to proteins hereof) means a protein capable of being controlled by light (i.e., radiation) to be active or more active. [0047] In some embodiments, the photo-activation domain activates (e.g., increases effector binding, increases GTPase activity) of a PA-Raclwhen contacted with NIR, far-red, or visible light. “Near infrared” (NIR) light is generally considered in the art to have a wavelength of between about 700-750 and about 3000 nm. “Far-red” light is generally defined as light having a wavelength at the long-wavelength red end of the visible (red) spectrum, from about 700 to about 750 nm. Visible light is generally defined as having a wavelength of about 390 to about 750 nm.

[0048] Types of photoactivatable domains that can be used in embodiments of the present invention include, by way of example but not limitation, phytochromes, light-oxygen- voltage (LOV) proteins, functional fragments thereof and variants thereof. In some embodiments, the photoactivatable domain is PhyB or a variant thereof. In certain embodiments, the photoactivatable domain is the LOV domain from Avena sativa phototropin 1 protein or a variant thereof. In some embodiments, the photoactivatable domain can be at least a portion or variant of PhyB the LOV domain from Avena sativa phototropin 1 protein, Dronpa, or Cry2. In some embodiments, the photoactivation domain is a bacteriophytochrome or functional fragment thereof. Bacteriophytochromes generally switch conformation in the presence of NIR or far-red light. Use of such Bacteriophytochromes as photoactivation domains can be advantageous as NIR or far-red light penetrates animal tissues to the depths of several centimeters.

[0049] In some embodiments, PA-Racl is activated (e.g., downstream effector binding is started or increased, GTPase activity is started or increased) by contact with radiation (i.e., light) having a wavelength of about 450 nm to 480 nm. In some embodiments, the radiation has a wavelength of 458 nm or 473 nm.

[0050] In some embodiments, the PA-Racl is disclosed in Wu YI et al., Nature. 2009; 461(7260): 104-8. In some embodiments, the PA-Racl is coded by a nucleic acid sequence comprising SEQ ID NO: 22. In some embodiments, the PA-Racl is coded by a nucleic acid sequence comprising a sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the PA-Racl is coded by a nucleic acid sequence comprising SEQ ID NO: 24. In some embodiments, the PA-Racl is coded by a nucleic acid sequence comprising a sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 24. In some embodiments, the PA-Racl comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the PA-Racl comprises an amino acid sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least

94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the PA-Racl comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the PA-Racl comprises an amino acid sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least

86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least

93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, pTriEx-mCherry-PA-Rac 1 (Addgene cat# 22027) comprises the PA-Racl nucleotide sequence.

[0051] In some embodiments, the PA-Racl sequence further comprises a detectable tag. In some embodiments, the detectable tag comprises a luciferase. As known in the art, “luciferase” refers to members of a class of enzymes that catalyze reactions that result in production of light. Luciferases are found in a variety of organisms including a variety of marine copepods, beetles, and others, and a number of these proteins have been cloned. Examples of luciferases include, e.g., luciferase from species of the genus Renilla (e.g., Renilla reniformis (Rluc), or Renilla mulleri luciferase), luciferase from species of the genus Gaussia (e.g., Gaussia princeps luciferase, Metridia luciferase from species of the marine copepod Metridia, e.g., Metridia longa, luciferase from species of the genus Pleuromamma, beetle luciferases (e.g. luciferase of the firefly Photinus pyralis or of the Brazilian click beetle Pyrearinus termitilluminans), etc. As known in the art, a number of luciferases contain signal sequences (sequences that direct secretion of the protein by cells that express it). For example, naturally occurring Gaussia princeps luciferase contains a signal sequence and is ordinarily secreted when expressed by mammalian cells. In certain embodiments of the invention in which a luciferase is used as a label for a bait or prey protein and the naturally occurring form of the luciferase contains a signal sequence effective to direct secretion of the luciferase when expressed in cells to be used in an inventive assay (e.g., mammalian cells), the signal sequence may be at least in part removed or modified so that is no longer functional in the cells to be used in the assay. “Luciferin” is used herein to refer to any substrate utilized by a luciferase or photoprotein in a light-emitting reaction. Examples include, e.g., firefly luciferin and coelenterazine. Coelenterazine is the substrate in many luciferases and photoproteins including Renilla, Gaussia, and Metridia luciferases, and aequorin.

[0052] In some embodiments, a fluorescent or luminescent protein or luciferase is an engineered variant of a naturally occurring protein. Such variants may, for example, have increased stability (e.g., increased photo stability, increased pH stability), increased fluorescence or light output, reduced tendency to dimerize, oligomerize, or aggregate, an altered absorption/emission spectrum (in the case of a fluorescent protein) and/or an altered substrate utilization. See, e.g., Chalfie, M. and Kain, SR (cited above) for examples. For example, the A. Victoria GFP variant known as enhanced GFP (eGFP) may be used. A variant of a naturally occurring luciferase that provides higher light output than the naturally occurring form and/or utilizes a coelentarazine analog as a substrate can be used. See, e.g., Foening, AM, et al., Protein Engineering, Design and Selection (2006) 19 (9): 391-400, for examples with respect to Renilla luciferase. In some embodiments, the detectable tag is mCherry.

[0053] In some embodiments, a nucleic acid sequence encoding a detectable protein (e.g., GFP, luciferase, etc.) is codon-optimized for expression in cells that are to be used in an assay. For example, the sequence may be codon-optimized for expression in mammalian cells, e.g., human cells. See, e.g., Tannous, BA, et al., Mol Ther. ll(3):435-43, 2005 for an example of Gaussia luciferase cDNA codon-optimized for expression in mammalian cells.

[0054] In some embodiments, the PA-Racl has reduced or no activation (e.g., effector binding, GTPase activity) in the absence of the radiation (i.e., light). In some embodiments, contact with light of a certain wavelength or range of wavelengths increases activation of the protein by at least about 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 1000% or more. In some embodiments, contact with light of a certain wavelength or range of wavelengths increases activation of the protein by at least about 0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 10,00- fold, or more. In some embodiments, contact with light of a certain wavelength or range of wavelengths starts activation of the protein. [0055] Methods of producing the radiation are not limited and may be any method known in the art. In some embodiments, the radiation is produced from a light source selected from a laser, a light emitting diode (LED), and a digital micromirror device (DMD). In some embodiments, the light source is a laser or LED emitting blue light.

[0056] The portion of the neuronal cell may be contacted with light of a certain wavelength or range of wavelengths for any suitable period causing a desirable extension or redirection of a neurite. In some embodiments, radiation is contacted with the neuronal cell for at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 3 hours, at least 5 hours, at least 10 hours, at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 1 month, or more.

[0057] The neurite may be increased in length by any suitable amount. In some embodiments, the neurite is sufficiently increased in length to establish (or enable establishment of) a synapse with another neuron. In some embodiments, the neurite increases in length by at least 20 pm, at least 30 pm, at least 40 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 100 pm, at least 150 pm, at least 200 pm, at least 500 pm, at least 1 mm, at least 2 mm, at least 5 mm, at least 10 mm, at least 100 mm, at least 500 mm, at least 1 cm, or more.

[0058] In some embodiments, the contacted neurite crosses an attractive/repulsive boundary. In some embodiments, the contacted neurite crosses a laminin/CSPG boundary.

[0059] One of the results of damage to the nervous system is the formation of glial scar tissue. Glial scar tissue is composed of astrocytes, oligodendrocytes and microglia, as well as a rich meshwork of extracellular matrix proteins including proteoglycans. Glial scar tissue formed in response to cellular damage presents a physical and/or a molecular barrier to regeneration. The content of glial scar tissue is complex, and thus the inhibitory effects of glial scar tissue may be due to multiple components of the scar. Proteoglycans are molecules consisting of one or more glycosaminoglycan (GAG) chains attached to a core protein. For example, it has been demonstrated that chondroitin sulfate proteoglycans (CSPGs) and chondroitin sulfate glycosaminoglycans (CS-GAGs), which are found in the glial scar, are inhibitory to regenerating neurons (reviewed in Silver (1994) J Neurol. 242: S22-4; Yu and Bellamkonda (2001) J. Neurosci. Res. 66: 303-310). These studies indicate that chondroitin sulfate GAGs play a role in the inhibitory effects of glial scar tissue, and additional studies suggest that the protein core of CSPGs may also play a role in inhibiting regeneration (Margolis and Margolis (1997) Cell Tissue Res. 290: 343-8; Friedlander et al. (1994) J. Cell Biol. 125: 669-680). In some embodiments, the method is performed in vivo and neurite growth across a repulsive boundary (e.g., a laminin/CSPG boundary) is obtained. In some embodiments, the method is performed on an embryo (e.g., blastocyst) and neurite growth across a somatic boundary is obtained. In some embodiments, the embryo has a neurological disease or condition that can be treated or prevented by the methods disclosed herein. In some embodiments, the neurological disease or condition is or is associated with a neuronal guidance defect.

[0060] In some embodiments, the contacted neurite forms a synapse (i.e., a signal- transmitting junction between two nerve cells). In some embodiments, the method is performed in a subject and the synapse forms between the contacted neurite and a neuronal cell endogenous to the subject. In some embodiments, the method is performed at the site of nerve damage and/or degradation and the method reestablishes nerve transmission through the site. In some embodiments, the site is in the central nervous system. In some embodiments, the site is in the peripheral nervous system. In some embodiments, the site is at a growth promoting peripheral nerve graft in the central nervous system that provide "bridges” to bypass inhibitory scars through which damaged axons can grow. The method may be used in such grafts to connect axons from the graft to the central nervous system or the peripheral nervous system.

[0061] The neuronal cells of the methods described herein are not limited. In some embodiments, the neuronal cell is a hippocampal neuronal cell, a cortical neuronal cell, a Purkinje neuronal cell, a basal ganglia neuronal cell, an olfactory neuronal cell, a dopaminergic neuronal cell, retinal neuronal cell, or a noradrenergic neuronal cell. According to another embodiment, the neuronal cell is a motor neuronal cell. According to another embodiment, the motor neuronal cell is a spinal motor neuronal cell. According to another embodiment, the neuronal cell is an interneuron neuronal cell. According to another embodiment, the neuronal cell population comprises a neuron population of the peripheral nervous system. In some embodiments, the neuronal cell is a sensory neuron, motor neuron, intemeuron, or cortical neuron.

[0062] In some embodiments, the neuronal cell is derived from a stem cell (e.g., an embryonic stem cell, a mammalian embryonic stem cell, a human embryonic stem cell, a murine embryonic stem cell). In some embodiments, the neuronal cell is derived from an embryonic stem cell. In some embodiments, the neuronal cell is derived from an induced pluripotent stem cell. In some embodiments, the induced pluripotent stem cell is derived from a subject having a disease or condition of interest. In some embodiments, the induced pluripotent stem cell is from a subject having a neurological condition or disease. In some embodiments, the induced pluripotent stem cell is from a subject having a nerve injury (e.g., spinal cord or peripheral nerve injury). In some embodiments, the neuronal cell is a human neuronal cell.

[0063] In some embodiments, the neuronal cell is contacted with the radiation in vivo, in vitro, or ex vivo. In some embodiments, a plurality of neuronal cells are contacted with the radiation. In some embodiments, the plurality of contacted neuronal cells form one or more neural circuits. In some embodiments, the one or more neural circuits provide a model of n neural structure or aberrant neurological disease or condition or a nerve injury.

[0064] As used herein, neurological disease or condition refers to neurodegenerative disorders, neuropsychiatric disorders and/or neurodevelopmental disorders. Neuro disorders may be any disease affecting neuronal network connectivity, synaptic function and activity. “Neurodegenerative disorder” refers to a disease condition involving neural loss mediated or characterized at least partially by at least one of deterioration of neural stem cells and/or progenitor cells. Non-limiting examples of neurodegenerative disorders include polyglutamine expansion disorders (e.g., HD, dentatorubropallidoluysian atrophy, Kennedy's disease (also referred to as spinobulbar muscular atrophy), and spinocerebellar ataxia (e.g., type 1, type 2, type 3 (also referred to as Machado-Joseph disease), type 6, type 7, and type 17)), other trinucleotide repeat expansion disorders (e.g., fragile X syndrome, fragile XE mental retardation, Friedreich's ataxia, myotonic dystrophy, spinocerebellar ataxia type 8, and spinocerebellar ataxia type 12), Alexander disease, Alper's disease, Alzheimer disease, amyotrophic lateral sclerosis (ALS), ataxia telangiectasia, Batten disease (also referred to as Spielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Guillain-Barre syndrome, ischemia stroke, Krabbe disease, kuru, Lewy body dementia, multiple sclerosis, multiple system atrophy, non- Huntingtonian type of Chorea, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum's disease, Sandhoff disease, Schilder's disease, spinal cord injury, spinal muscular atrophy (SMA), SteeleRichardson-Olszewski disease, and Tabes dorsalis.

[0065] In certain contexts, neurodegenerative disorders encompass neurological injuries or damages to the CNS or the PNS associated with physical injury (e.g., head trauma, mild to severe traumatic brain injury (TBI), spinal cord injury, diffuse axonal injury, craniocerebral trauma, cranial nerve injuries, cerebral contusion, intracerebral haemorrhage and acute brain swelling), ischemia (e.g., resulting from spinal cord infarction or ischemia, ischemic infarction, stroke, cardiac insufficiency or arrest, atherosclerotic thrombosis, ruptured aneurysm, embolism or haemorrhage), certain medical procedures or exposure to biological or chemic toxins or poisons (e.g., surgery, coronary artery bypass graft (CABG), electroconvulsive therapy, radiation therapy, chemotherapy, anti-neoplastic drugs, immunosuppressive agents, psychoactive, sedative or hypnotic drugs, alcohol, bacterial or industrial toxins, plant poisons, and venomous bites and stings), tumors (e.g., CNS metastasis, intraaxial tumors, primary CNS lymphomas, germ cell tumors, infiltrating and localized gliomas, fibrillary astrocytomas, oligodendrogliomas, ependymomas, pleomorphic xanthoastrocytomas, pilocytic astrocytomas, extraaxial brain tumors, meningiomas, schwannomas, neurofibromas, pituitary tumors, and mesenchymal tumors of the skull, spine and dura matter), infections (e.g., bacterial, viral, fungal, parasitic or other origin is selected from the group consisting of pyrogenic infections, meningitis, tuberculosis, syphilis, encephalomyelitis and leptomeningitis), metabolic or nutritional disorders (e.g., glycogen storage diseases, acid lipase diseases, Wernicke's or Marchiafava-Bignami's disease, Lesch- Nyhan syndrome, Farber's disease, gangliosidoses, vitamin B12 and folic acid deficiency), cognition or mood disorders (e.g., learning or memory disorder, bipolar disorders and depression), and various medical conditions associated with neural damage or destruction (e.g., asphyxia, prematurity in infants, perinatal distress, gaseous intoxication for instance from carbon monoxide or ammonia, coma, hypoglycaemia, dementia, epilepsy and hypertensive crises).

[0066] “Neuropsychiatric disorder” encompasses mental disorders attributable to diseases of the nervous system. Non-limiting examples of neuropsychiatric disorders include addictions, childhood developmental disorders, eating disorders, degenerative diseases, mood disorders, neurotic disorders, psychosis, sleep disorders, depression, obsessive-compulsive disorder, schizophrenia, visual hallucination, auditory hallucination, eating disorder, bipolar disorder, epilepsy, autism, and ALS.

[0067] In some embodiments, the neural circuit provides a model of a neurological disease or condition selected from epilepsy, schizophrenia, and autism. In some embodiments, the neural circuit comprises neural cells derived from induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs are derived from a subject (e.g., a subject having a neurological disease or condition described herein).

[0068] In some embodiments, the one or more neural circuits form a biological computer. [0069] In some embodiments, the methods disclosed herein are used to treat or prevent a neurological disease or condition or a nerve injury in a subject in need thereof. In some embodiments, the nerve injury comprises one or more severed neurons.

[0070] Some aspects of the present disclosure are directed to a biological computer comprising contacted neuronal cells of the methods described herein. Some aspects of the present disclosure are directed to an in vitro neural circuit comprising contacted neuronal cells of the methods described herein. Some aspects of the present disclosure are directed to a non human animal comprising contacted neuronal cells of the methods described herein.

[0071] Some aspects of the present disclosure are directed to a method of establishing or manipulating a neural connection in a subject in need thereof, comprising providing photoactivatable Racl (PA-Racl) in a neuronal cell in the subject and establishing or manipulating the neural connection by contacting a portion of the neuronal cell with radiation (i.e., light) that activates or increases the activation of the photoactivatable Racl. In some embodiments, the subject is further treated with a Rho inhibitor. In some embodiments, the Rho inhibitor is VX-210.

[0072] The PA-Racl is not limited and may be any PA-Racl described herein. In some embodiments, the PA-Racl is coded by a sequence comprising SEQ ID NO: 24 or comprises the amino acids of SEQ ID NO: 25. The neuronal cell is also not limited and may be any neuronal cell described herein. In some embodiments, the subject is administered a transgenic neuronal cell or neuronal stem cell expressing PA-Racl. In some embodiments, the subject is administered a nucleic acid (e.g., ribonucleic acid) coding for PA-Racl. In some embodiments, the subject is administered PA-Racl. The method of administration is not limited and may be any suitable administration known in the art. In some embodiments, administration is localized to a desired location (e.g., the site of a nerve injury or nerve deterioration or degradation). In some embodiments, administration immediately precedes contact of cells containing PA-Racl with radiation (i.e., light). In some embodiments, administration is at least about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, or longer prior to contact of cells containing PA-Racl with radiation (i.e., light).

[0073] The period of contact of cells containing PA-Racl with radiation (i.e., light) is not limited and may be any period disclosed herein.

[0074] The location of the neural connection in the subject is not limited. In some embodiments, the neural connection is located in the central nervous system (e.g., the brain or spinal cord) of the subject. In some embodiments, the neural connection is located in the spinal cord of the subject. In some embodiments, the neural connection is located in the peripheral nervous system of the subject. In some embodiments, the neural connection comprises grafted tissue. In some embodiments, the neural connections are located in the left frontal and/or temporal lobe.

[0075] As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient”, “individual” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In addition, the methods described herein can be used to treat domesticated animals and/or pets. A subject can be male or female. A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, but need not have already undergone treatment for a condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition in need of treatment or one or more complications related to such a condition. Rather, a subject can include one who exhibits one or more risk factors for a condition or one or more complications related to a condition. A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at increased risk of developing that condition relative to a given reference population. In some embodiments, the subject is an embryo. In some embodiments, the subject has amyotrophic lateral sclerosis, Parkinson’s, or schizophrenia.

[0076] In some embodiments, a plurality of neuronal cells having PA-Racl are contacted with the radiation. In some embodiments, a sufficient number of neuronal cells containing PA-Racl are contacted to treat or prevent a neurological disease or condition or a nerve injury. The neurological disease or condition or nerve injury is not limited and may be any neurological disease or condition or nerve injury described herein. [0077] As used herein, “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder, medical condition, or injury, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state as compared to that expected in the absence of treatment. In some embodiments, treatment of a nerve injury includes partial or full restoration of nerve functionality. In some embodiments, the method causes the formation of one or more synapses. In some embodiments, the method repairs a severed nerve. In some embodiments, the method treats or prevents a neurological or neuropsychiatric disease or condition.

[0078] The method of producing the radiation is not limited and may be any method known in the art or described herein. In some embodiments, the radiation is produced by one or more light sources selected from a laser and a light emitting diode (LED). The wavelength of light is not limited and may be any wavelength suitable to photoactivate the PA-Racl. In some embodiments, the radiation has a wavelength of 458 nm or 473 nm.

[0079] In some embodiments, the one or more light sources are external to the subject. In some embodiments, the one or more light sources are used to contact the PA-Racl with radiation during a surgical procedure. Contacting the PA-Racl with radiation during a surgical procedure is especially advantageous when sufficient growth of neurites to treat or prevent a neurological disease, condition or injury can be accomplished in an amount of time suitable for surgery (e.g., less than about a day, less than about 18 hours, less than about 12 hours, less than about 6 hours). Performing the method during surgery is also advantageous as the radiation may not have to penetrate intervening tissue (e.g., the site of nerve injury or degradation is exposed). Furthermore, performing the method during surgery may enable use of light sources, such as lasers, that can precisely guide one or more neurites to a desired location (e.g., to reconnect a severed nerve) via controlled movement in the site. In some embodiments, the subject is secured during surgery so that the site being treated by the methods disclosed herein does not move. [0080] In some embodiments, the one or more light sources are externally secured to a subject. In some embodiments, the one or more light sources are immobilized on the subject so they do not move in relation to the site being treated. In some embodiments, the PA-Racl is photoactivated by NIR or far-red light which is capable of penetrating tissue to a greater depth.

[0081] In some embodiments, the one or more light sources are implanted in the subject (e.g., surgically implanted). In some embodiments, the implanted light source comprises an apparatus described herein. In some embodiments, a digital micromirror device is implanted with the light source.

[0082] Apparatus

[0083] Some aspects of the disclosure are related to an apparatus for establishing or manipulating a neural connection in a subject, comprising one or more light sources, a power supply operably linked to the one or more light sources, and a processor configured to control the one or more light sources.

[0084] The light sources are not limited and may be any light source described herein. In some embodiments, the one or more light sources are LEDs emitting radiation of a suitable wavelength to photoactivate PA-Racl. In some embodiments, the light sources comprise biologically inert materials. In some embodiments, the one or more light sources are suitable for implantation. In some embodiments, the apparatus comprises a digital micromirror device.

[0085] The power supply is not limited and may be any suitable power supply known in the art. In some embodiments, the power supply comprises one or more batteries. In some embodiments, the power supply comprises a plug or other connection to external power. In some embodiments, the power supply is configured to be suitable for powering a light source implanted in a subject (e.g., meeting safety standards for implanted medical devices). In some embodiments, the power supply is configured for implantation in the subject. In some embodiments, the power supply is configured to be located external to a subject and deliver power to one or more implanted light sources. In some embodiments, the power supply provides power to the light source wirelessly.

[0086] In some embodiments, the one or more light sources are configured for implantation in a subject. In some embodiments, the one or more light sources comprise a plurality of light sources configured to be implanted at a nerve injury site having a first end and a second end, and direct regrowth and/or repair of the nerve by sequentially turning on and off each light source from the first end to the second end. In some embodiments, the plurality of light sources are a linear string of light sources that are configured to be secured across a nerve injury site, and wherein the light sources are configured to enable growth of one or more neurites along the linear string. In some embodiments, the plurality of light sources are a string of LEDs configured to be surgically secured at a first location comprising neuronal cells with PA-Racl at one end and a second location wherein a synaptic connection with the PA-Racl containing cells is desired (e.g., the LED string is secured across a nerve injury or nerve graft site as described herein). In some embodiments, the processor is configured to sequentially turn on and off the light sources (e.g., LEDs) in a timed manner so that neurites from the PA-Racl containing cells bridge the site of nerve injury, deterioration or damage. In some embodiments, the processor is configured to control a digital mirror device (DMD) and direct light from a light source to multiple desired locations. A DMD is a chip has on its surface several hundred thousand microscopic mirrors arranged in a rectangular array. The mirrors can be individually rotated and reflect a light source to desired locations, enabling complex and fine-tuned control of neurite growth either for single neurites or a plurality of neurites simultaneously.

[0087] Some aspects of the disclosure are related to a kit comprising the apparatus described herein and an agent capable of providing PA-Racl in a neuronal cell in a subject. In some embodiments, the agent comprises a nucleic acid coding for PA-Racl. In some embodiments, the agent is a transgenic stem cell or progenitor cell capable of expressing PA- Racl and, optionally, ChR (e.g., ReaChR). In some embodiments, the PA-Racl is coded by a sequence comprising SEQ ID NO: 24 or comprises the amino acids of SEQ ID NO: 25. In some embodiments, the kit further comprises an agent which comprises a nucleic acid coding for a ChR (e.g., ReaChR).

[0088] Some aspects of the disclosure are related to an isolated transgenic stem cell or progenitor cell capable of expressing PA-Racl. The PA-Racl is not limited and may be any PA-Racl described herein. In some embodiments, the PA-Racl is coded by a sequence comprising SEQ ID NO: 24 or comprises the amino acids of SEQ ID NO: 25. In some embodiments, the transgenic stem cell or progenitor cell further expresses a ChR (e.g., ReaChR).

[0089] Some aspects of the disclosure are related to a method of screening for therapeutic agents comprising, providing an in vitro neural circuit modeling a neurological disease or condition and comprising contacted neuronal cells of the disclosed herein, contacting the neural circuit with a test agent, and assessing whether the test agent improves one or more aspects of the neurological disease or condition present in the neural circuit. The neurological disease or condition may be any neurological disease or condition disclosed herein. In some embodiments, the neurological disease or condition is a central nervous system or peripheral nervous system injury. In some embodiments, the neurological disease or condition is Parkinson’s, ALS, or schizophrenia.

[0090] The test agent is not limited. “Test agent” is used herein to refer to any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. In some aspects, a test agent can be represented by a chemical formula, chemical structure, or sequence. Example of a test agent, include, e.g., small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids, polysaccharides, peptide mimetics, etc. In general, a test agent may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the test agent. A test agent may be at least partly purified. In some embodiments a test agent may be provided as a salt, ester, hydrate, or solvate. In some embodiments a test agent is cell-permeable, e.g., within the range of typical agents that are taken up by cells and acts intracellularly, e.g., within mammalian cells. Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z-isomers, R- and S -enantiomers, diastereomers, (D)-isomers, (L)- isomers, (-)- and (-i-)-isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated. Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates (e.g., with water (i.e. hydrates) or common solvents) and/or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable. In some embodiments, the test agent is a small molecule, a peptide, an RNA or a DNA.

[0091] In some embodiments, the test agent is a small molecule. The term “small molecule” refers to an organic molecule that is less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, the small molecule is less than about 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a saccharide. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and / or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups. Small molecules often comprise one or more cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups.

[0092] In some embodiments, the neural circuit comprises neuronal cells derived from a subject with the neurological disease or condition. In some embodiments, the neural circuit provides a model of a neurological disease or condition selected from epilepsy, schizophrenia, and autism. In some embodiments, the neural circuit comprises neural cells derived from induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs are derived from a subject (e.g., a subject having a neurological disease or condition described herein).

[0093] The “one or more aspects” of the neuronal circuit measured are not limited and may be any aspect relevant to the modeled neurological disease or condition. In some embodiments, the one or more aspects measured includes radical oxidative stress, mitochondrial biogenesis, intracellular protein aggregation les, cellular NAD or ATP levels, autophagy activity, protein aggregation, GSK3P or Tau protein phosphorylation, and neuronal plasticity. In some embodiments, the one or more aspects measured comprise one or more of neuronal cell viability, neuronal cell proliferation, and synaptic signaling.

[0094] Neuronal Guidance Defect

[0095] Some aspects of the disclosure are related to a method of treating a neuronal guidance defect in a subject in need thereof, comprising administering to the subject an agent that modulates the expression or activity of Racl in a neuronal cell. The agent is not limited and may be any agent described herein. In some embodiments, the agent comprises PA-Racl or a nucleic acid encoding for PA-Racl. In some embodiments, administration of the agent stabilizes the level of Racl in a neuronal cell of the subject. In some embodiments, administration of the agent increases the level of a Racl in the neuronal cell of the subject.

[0096] In some embodiments, the method further comprises growth of neurites in the subject mediated by Racl. In some embodiments, the Racl is a PA-Racl described herein. In some embodiments, neurite growth is controlled in the subject via photo-activation of the PA- Racl (e.g., with an apparatus described herein).

[0097] In some embodiments, the neuronal guidance defect is a plod3 mutation. In some embodiments, the neuronal guidance defect is associated with or caused by a disease or condition selected from corpus callosum agenesis, LI syndrome, Joubert syndrome, horizontal gaze palsy with progressive scoliosis, Kallmann syndrome, albinism, congenital fibrosis of the extraocular muscles type 1, Duane retraction syndrome, and pontine tegmental cap dysplasia. [0098] These defective axon guidance disorders are major human genetic disorders that result or have been proposed to result from defective axon guidance. In many of these disorders, there are symptoms and signs of aberrant axon connectivity in humans. Genes mutated in these disorders can encode axon growth cone ligands and receptors, downstream signaling molecules, and axon transport motors, as well as proteins without currently recognized roles in axon guidance.

[0099] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.

[0100] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0101] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or prior publication, or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. [0102] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0103] The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more active agents, additives, ingredients, optional agents, types of organism, disorders, subjects, or combinations thereof, can be excluded.

[0104] Where the claims or description relate to a composition of matter, it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0105] Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by “about” or “approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by “about” or “approximately”, the invention includes an embodiment in which the value is prefaced by “about” or “approximately”.

[0106] “Approximately” or “about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered “isolated”.

EXAMPLES

[0107] Axonal connections define how information flows through neuronal networks. As the nervous system develops, axonal innervation allows for the exchange of molecular and electrophysiological signals between projecting axons and their target tissues, with axonal input playing a key role in shaping the information processing functions of downstream neuronal networks. One of the most compelling demonstrations for the validity of this neurodevelopmental principle is the fact that sensory areas of the mammalian cerebral cortex switch to processing information of alternative sensory modalities (i.e. visual instead of auditory) when they receive rerouted afferent axonal connectivity from a different sensory organ (Pallas et ah, 1999; Pallas et ah, 1990; Pallas and Sur, 1993; Sur et ah, 1999). Additionally, axonal input plays a significant role in shaping postsynaptic neuronal identity by influencing key cellular properties including neurotransmitter selection, dendritic architecture, gene expression, and electrophysiological features (De Marco Garcia et ah, 2011; Dehorter et ah, 2015). And, conversely, retrograde signals from target tissue can shape neurotransmitter expression profiles (Kou et ah, 1995; Russ and Kaltschmidt, 2014) and cell body positioning (Haase et ah, 2002; Lin et ah, 1998) of the projecting neurons. How the molecular and electrophysiologic interaction of axons and their targets shape their mutually defined identities and processing functions remains largely unexplored. To pursue these foundational questions, approaches to precisely and noninvasively sculpt axonal connections within the intact organism are required, but are currently missing.

[0108] Example 1-Optogenetic stimulation of PA-Racl resulted in directed axonal growth of cultured zebrafish spinal motor neuron axons

[0109] In order to investigate whether axon guidance could be controlled by light activation of PA-Racl, spinal motor neurons were used in the developing zebrafish as a model system. Zebrafish embryos are optically transparent, develop rapidly, and have highly stereotyped programs of spinal motor axon guidance (Beattie, 2000). To express PA-Racl specifically in spinal motor neurons, transgenic zebrafish were generated with an optimized PA-Racl fused with the mCherry fluorescent reporter under a tissue specific mnxl minimal enhancer (Zelenchuk and Bruses, 2011) and a b-globin minimal promoter (Fig. 1A). Transgenic fish expressed PA-Racl RNA in spinal motor neurons approximately two-fold higher than the endogenous racl genes, racla and raclb, at 24 hours post fertilization (hpf) (Figs. 6A-6B), indicating robust expression of the exogenous construct. Given that the PA- Racl protein is only active upon illumination and that illumination was restricted to a small portion of the growth cone, it is likely that optogenetically driven Racl activity remained within the physiologic range of overall cellular Racl activity (Figs. 6A-6B).

To determine if optogenetic activation of Racl could co-opt the axon guidance machinery in the growth cone, a 2D in vitro culture of spinal motor neurons was first used. To clearly identify mCherry-positive spinal motor neurons, sparse monolayers of dissociated Tg(mnxl: mCherry -PA-Racl);mitfa^w2/w2;mpvl7^a9/a9 zebrafish tails at 18hpf were cultured, a time when motor neurons have differentiated and expressed PA-Racl, but before they have completed axonal pathfinding. mCherry-expressing neurons with projecting axons were visually identified, and the growth cone was asymmetrically illuminated with 458nm light in a small region of interest using a Zeiss 880 confocal microscope (see also Methods; Fig. 1A). Within 3 to 5 minutes from the start of illumination, growth cones responded by projecting membrane extensions in the direction of the illumination, and by 10 to 20 minutes most axons had visibly extended in the direction of the light stimulus (Fig. IB). The region of stimulation was progressively advanced to guide the growth of each axon perpendicular to its original trajectory (n=5 axons from separate cultures of independent fish, average illumination time of 83.2 minutes). As controls, the directed growth obtained by 458nm illumination of PA-Racl+ axons was compared to the growth of both unilluminated axons and 458nm illuminated axons from Tg(mnxl:mCherry-PI-Racl);mitfa^w2/w2;mpvl7^a9/a9 fish, which carry a mutated version of PA-Racl that renders this construct photo-insensitive (PI-Racl). Light- stimulated PA- Racl+ axons deviated from their initial trajectories at much greater angles compared to unilluminated controls and light stimulated PI-Racl+ axons (19.8° +/- 20.9°, 25.3° +/- 16.0 °, and 99.2° +/- 13.8° mean deviation angle +/- 95% confidence interval (Cl) in unilluminated, PI-Racl, and PA-Racl axons, respectively; Fig. 1C). In addition, stimulated growth cones extended axons over greater distances during the trial period (3.3pm +/- 6.1pm, 1.8pm +/- 3.4pm, 15.4pm +/- 6.0pm mean distance +/- 95% Cl in unilluminated, PI-Racl, and PA-Racl axons, respectively), resulting in faster rates of growth (2.1 pm/hr +/- 4.0 pm/hr, 2.0pm/hr +/- 3.6 m m/hr, and 11.4 mhi/hr +/- 5.2 m m/hr mean growth rate +/- 95% Cl in unilluminated, PI- Racl, and PA-Racl axons, respectively; Fig. IB, Fig. 1C.

[0110] To further control for possible physical effects of laser illumination, PA- Racl+ growth cones were exposed to 514nm light, outside of the spectrum of PA-Racl activation. As expected, exposure to 458nm light induced axonal extension in the direction of illumination, while exposure to 514nm light did not (Figs. 6C-6D). To assess if illumination of PA-Racl⁺ neurons activated Racl signaling, immunohistochemistry was performed for autophosphorylated Pakl, which is a major downstream signaling target of Racl. Increased autophosphorylation of Pakl was seen within a region of illumination compared with the rest of the cell (Fig. 6E). These results demonstrate that light activation of PA-Racl in growth cones allows targeted, directional control over intrinsic axonal extension programs.

[0111] Example 2-Optogenetic stimulation of PA-Racl directed zebrafish CaP axon growth across repulsive somitic boundaries into neighboring somitic musculature

[0112] While PA-Racl stimulation guided axonal growth in sparse neuronal cultures, in vivo applications present additional challenges for engineering axon guidance, including competition from endogenous axonal guidance signals, complex 3D tissue environments, and repulsive molecular and physical barriers. To test if optogenetic axon guidance could overcome these obstacles, intact embryonic zebrafish were used as an in vivo model of directed axon growth.

[0113] Within each somitic unit of the developing zebrafish, three bilaterally symmetric pairs of primary spinal motor neurons innervate nonoverlapping regions of the animal’s somatic musculature (Beattie, 2000; Eisen et al., 1986) (Fig. 2A). Of these neurons, only the caudal primary (CaP) spinal motor neuron extends an axon into the ventral myotome, in a stereotyped path halfway between the vertical myosepta that separate neighboring somitic myotomes (Beattie, 2000; Eisen et al., 1986) (Fig. 2A). In healthy animals, the path of CaP axons never deviates from this well-defined trajectory (Beattie, 2000; Myers et al., 1986). This strict pathfinding program is imposed via molecular instructions from several sources within the somite, including repulsive Sema3Ab expressed in the posterior somitic compartment and at the vertical myoseptal boundaries of the somites (Roos et al., 1999), repulsive chondroitin sulfate proteoglycans (CPSGs) at vertical myoseptal boundaries (Bernhardt et al., 1998), and initially attractive and subsequently repulsive Sema5a in the anterior portion of the ventral somite (Hilario et al., 2009).

[0114] Given that Racl molecularly links the transduction of a variety of extracellular signals to the cytoskeletal machinery that directs axon growth (Hall and Lalli, 2010; Hua et al., 2015b; Ng et al., 2002), it was reasoned that manipulation of Racl activity might allow us to override these endogenous axonal guidance signals to drive axonal growth in new directions, within the intact organism. To test this possibility, Tg(mnxl:mCherry-PA- Racl );mitfa^w2/w2 ;mpvl7^a9/a9 zebrafish were illuminated with 458nm light in a region of interest focused on the leading edge of the CaP growth cone, beginning between 23 and 25hpf, a time when only the CaP axon is extending into the ventral myotome. The region of illumination was periodically updated to attempt to deviate the growing axon in either the rostral or caudal directions over the course of 4 to 5 hours (Fig. 2A). Strikingly, in the majority of stimulated axons, growth was deflected in the direction of light application (n=17 axons/23 stimulated; Figs. 2B-E).

[0115] As the region of stimulation was updated, 3 of the 23 illuminated axons grew in the direction of light stimulation within their native somite and 14 of the 23 illuminated axons grew up to and eventually crossed the repulsive somitic boundary, extending into the neighboring somitic musculature (Figs. 2B-D), despite the presence of strong opposing endogenous signals at this boundary (Bernhardt et al., 1998; Roos et al., 1999). Axons could be guided across both rostral (Figs. 2B-2C) and caudal somitic boundaries. In contrast, unstimulated neighboring axons never crossed the somitic boundary (n=57 axons, Fig. 2E). Stimulated axons grew longer distances than unstimulated ones over the trial period, resulting in faster rates of growth (Fig. 2E), a finding that confirms the in vitro measurements. .In initial experiments, it was noted that the direction of axonal growth could be dynamically altered over the course of stimulation, as for the cultured neurons discussed earlier. In two instances, it appeared that the optogenetically guided axons could be induced to grow out of the myotome altogether, continuing their extension ventrally (Fig. 7), while all unstimulated axons remained within the musculature. To investigate these events in greater detail, serial EM reconstructions of one such deviated axon were performed, which confirmed that it grew past the edge of the myotome, extending ventrally to the kidney duct (Fig. 7). Interestingly, the EM suggests that migratory cells, possibly neural crest or sclerotomal cells, which normally migrate along the same path as the extending CaP axon (Melancon et al., 1997), also aberrantly accompanied the extending axon to its ectopic target in the kidney (Fig. 7).

[0116] In these experimental paradigms, optical activation of PA-Racl allowed direct axonal growth across somitic boundaries, such that the deviated axons became juxtaposed with the myotome of the neighboring somite, a target these axons do not typically innervate. It was unclear whether new connectivity could be established between the directed, aberrant axons and their new ectopic targets. To test whether new connectivity could be established between the directed, aberrant axons and their new ectopic targets, evidence of synapse formation was investigated. First, CaP axons were deviated across the rostral somitic boundary and into the neighboring somite as described above (Fig. 2A). To allow time for synapse formation, embryos were grown for an additional 1.5 hours past the end of stimulation before fixation. Immunohistochemical detection of mCherry-expressing CaP axons confirmed that the stimulated axon did indeed cross the somitic boundary (Fig. 3A), passing laterally to the CaP axon of the neighboring somite (Figs. 3B-3C). Within the target somitic myotome, both the control and deviated axon exhibited significant colocalization of the presynaptic marker synaptic vesicle protein 2 (SV2) with postsynaptic acetylcholine receptors (aBT), as quantified by volumetric signal correlation (mean Pearson’s correlation coefficient ± 95% confidence interval 0.347 ± 0.086 95%, n=3 fish, Costes p-value >0.95; Figs. 3D-3E), suggesting the presence of ectopic neuromuscular junctions between the deviated axon and the musculature of the neighboring, invaded somite.

[0117] Example 3-Optogenetic stimulation of plod3^~A zebrafish CaP neurons rescued their axon guidance defect allowing functional synaptic connectivity with the ventral myotome

[0118] In these experiments, optogenetic activation of Racl was sufficient to control the guidance of otherwise normally-developing CaP motor neurons in the context of a wild- type tissue environment. To test whether optogenetically directed axonal growth could also rescue CaP axon guidance defects in a pathological environment, a zebrafish mutant was employed in which CaP axon pathfinding and connectivity are disrupted. Specifically, a plod3^tv205a mutant zebrafish was used, (which is referred to as plod3 ), which carried a loss-of- function point mutation in the lh3 glycosyltransferase plod3 (Schneider and Granato, 2006). Animals homozygous for this mutation have disrupted myotomal type XVIII collagen, and consequently display premature cessation of CaP motor neuron growth cone pathfinding at or before the horizontal myoseptum, with associated failure to extend appropriately into the ventral myotome (Figs. 4A-4B) (Granato et al., 1996; Schneider and Granato, 2006; Zeller and Granato, 1999).

[0119] Transgenic plod3^A mutant embryos expressing PA-Racl in spinal motor neurons were generated (see also Methods) and rescue of the axon guidance defects in the CaP neurons was attempted using optogenetic stimulation. Light guidance was initiated in mutant growth cones at 28hpf, a point at which CaP axons of heterozygote plod3⁺ animals have projected well beyond the horizontal myoseptum (Fig. 4A), but growth cones of plod3^A fish have stalled at this boundary. The illuminated axons grew significantly farther past the horizontal myosepta compared with their neighboring, unilluminated axons (p<0.001, FDR adjusted, difference of least square means from mixed linear model), and extended distances that were not significantly different from wildtype fish (mean distance from CaP axon tip to the horizontal myoseptum ± 95% confidence interval: 0.2 ± 2.5mhi, 63.6 ± 9.2mhi, and 44.9 ± 16.5mhi in plod3^A fish, plod3⁺ fish, and optogenetically rescued plod3^A fish respectively, n=8 axons, one axon per fish; Figs. 4B-4D). Furthermore, immunohistochemical detection of pre- and post-synaptic proteins indicated that the rescued CaP axons could form synaptic connections with the surrounding myotome (Fig. 4E).

[0120] In order to determine whether functionals synapses were formed between optogenetically-rescued plod3^A CaP axons and the myotome, plod3^A zebrafish were generated that expressed both PA-Racl and ReaChR, a red-shifted channelrhodopsin (Lin et al., 2013) in spinal motor neurons (Fig. 5A). First, the axonal guidance defect in plod3^A mutants were rescued by growing axons into the ventral myotome (Fig. 5B). Following rescue, neuronal activity was induced in these neurons by stimulating the ReaChR channelrhodopsin using 594nm and 633nm light. ReaChR stimulation produced muscle contractions that in >85% of cases were specifically restricted to the ventral myofibrils innervated by the redirected axon (15/17 contractions, Fig. 5D), as assessed using motion tracking with particle image velocimetry analysis (see methods) (Fig. 5E). To control for axonal depolarization outside of the ventral myotome and the physical effects of light exposure, neighboring unrescued axons were illuminated with the same wavelengths of light (594nm and 633nm), and the rescued axon was illuminated with 405nm and 440nm light, which is outside the spectrum of ReaChR activation. ReaChR activation of rescued axons resulted in significantly more contractions of the ventral myotome than either control condition (n=3 fish, 5 trials per condition in each fish, Fig. 5F). In addition to ReaChR stimulation, immunohistochemistry was performed for synaptic markers (SV2 and aBT) on the same experimental animals after conclusion of the stimulation experiments. Presynaptic SV2 in the rescued plod3^A CaP axons and postsynaptic aBT in the ventral myofibrils were significantly colocalized, indicating that the recovery of synaptic function observed using optogenetic stimulation correlates with molecular evidence of synapse formation (mean Pearson’s correlation coefficient ± 95% confidence interval 0.367 ± 0.105, n=3 fish, Costes p-value>0.95; Fig. 5C). Thus, PA-Racl mediated axonal rescue resulted in formation of functional synaptic connectivity between the rescued axons and their new ventral myofibril targets, demonstrating that PA-Racl activation can be used to rescue defective axon guidance within the intact developing organism despite a pathologic environment that lacks type XVIII collagen, a necessary myotomal guidance signal. All together, these results show that PA-Racl provides a powerful means to direct axonal growth at the level of individual growth cones, irrespective of endogenous repulsive barriers or defective pathfinding signaling to create functional novel circuitry.

[0121] MATERIALS AND METHODS

[0122] STAR Methods:

[0123] Experimental Model and Subject Details

[0124] Zebrafish

[0125] All zebrafish ( Danio rerio ) lines were grown under standard conditions (16:8 hour light dark cycle at 28.5°C) approved by the Harvard Institutional Animal Care and Use Committee (IACUC). Transgenic lines were derived from two initial strains: mitfa^w2 ;mpvl7^a9 transparent fish (ZIRC ZL1714)(White, et ah, 2008), or plod3^tv205a mutant fish that have disrupted spinal motor neuron axon guidance in homozygotes (European Zebrafish Resource Center #941) (Schneider and Granato, 2006; Granato, et ah, 1996). From these two initial lines, transgenic animals were created to express an optimized photoactivatable Racl (PA- Racl) (Hayashi-Takagi, et ah, 2015; Lungu, et ah, 2012), a light insensitive mutant Racl (PI- Racl) (Wu, et ah, 2009), or a red-shifted channelrhodopsin (ReaChR) (Lin, et ah, 2013), in spinal motor neurons. Three stable transgenic lines were created: Tg(mnxl :mCherry-PA- Racl );mitfa^w2;mpvl 7^a9, Tg( mnxl :mCherry-PI-Racl );mitfa^w2;mpvl 7^a9, and Tg( mnxl :mCherry- PA-Racl );plod3^tv205a. Embryos were visually screened at 48hpf for mCherry expression in spinal motor neurons using a Zeiss fluorescent dissecting microscope; positive fish were grown to adulthood and outcrossed to identify founders with germline transmission. Male and female embryos from incrosses of founders and progeny were used for subsequent experiments between 18 and 32hpf. All control animals were clutchmates or age-matched if clutchmates were unavailable.

[0126] In addition to generating stable lines, transient transgenesis was performed to create Tg(mnxl:ReaChR-citrine);Tg(mnxl:mCherry-PA-Rcicl);plod3^{tv205a/tv205a} fish, by injecting Tg(mnxl:mCherry-PA-Rcicl);plod3^{tv205a/tv205a} embryos with the pDestTol2CG2- mnxl:ReaChR-citrine transgenesis construct. To determine experimental candidates, transgenic fish were screened for strong expression of mCherry-PA-Racl, mCherry-PI-Racl, or ReaChR-citrine transgenes by visualization of their respective fluorophores in CaP motor neurons.

[0127] Primary zebrafish motor neuron cultures [0128] Primary zebrafish motor neurons were cultured from pooled 18hpf male and female Tg(mnxl:mCherry-PA-Racl);mitfci^w2/w2;mpvl7^a9/a9 and Tg(mnxl:mCherry-PI- Racl);mitfa^w2/w2 ;mpvl7^a9/a9 fish at room temperature in Leibovitz’s L-15 media (Thermo Fischer 21083027) supplemented with 2% fetal bovine serum and lOOU/mL penicillin/streptomycin .

[0129] Method Details

[0130] Plod3 ^tv205a genotyping

[0131] DNA was extracted from tail clippings of adult zebrafish using the DNAeasy Blood and Tissue kit (Qiagen 69504). Plod3 ^tv205a fish were genotyped using a Custom TaqMan SNP genotyping assay (Thermo Fisher 4332077, Assay ID ANXGU9F) with an Applied Biosystems 7900HT RT-PCR machine, according to the manufacturer’s instructions.

[0132] Cloning of transgenesis constructs

[0133] To generate transgenic zebrafish lines, three transgenesis constructs were created: pDestTol2CG2-mnxl :mCherry-PA-Racl , pDestTol2CG2-mnxl :mCherry-PI-Racl , and pDestTol2CG2-mnxl:ReaChR-citrine. The Multisite Gateway technology (Invitrogen) and the Tol2 Kit (Kwan, et al., 2007) were used to generate pDestTol2CG2-mnxl :mCherry- PA-Racl, according to the manufacturer’s instructions. Three entry vectors were used: a 5’ entry vector (p5E ), a middle entry vector (pME ), and a 3’ entry vector (p3E ). The p5E entry vector contained three tandem repeats of a 125bp mnxl enhancer to drive transgene expression in zebrafish spinal motor neurons and was constructed as described previously (Zelenchuk and Bruses, 2011). Briefly, 3 copies of the 125bp mnxl enhancer were PCR amplified from a 3kb portion of the mnxl promoter with 3 sets of primers with unique restriction sites (primers and restriction enzymes listed in Table SI). PCR products were digested, gel purified, and sequentially cloned into the p5E vector using the pENTR 5’-TOPO TA Cloning kit (Invitrogen K59120, Tol2Kit plasmid #228)(Kwan, et al., 2007). The pME vector contained the mouse b-globin minimal promoter (Tamplin, et al., 2011) and mCherry-PA-Racl optimized to reduce background activity in the absence of light stimulation by introducing the previously described L514K and L531E mutations in the J-a helix (Hayashi-Takagi et al., 2015; Lungu et al., 2012). These mutations were introduced into the pTriEx-mCherry-PA- Racl plasmid (a gift from Klaus Hahn; Addgene plasmid #22027) (Wu et al., 2009) using the Q5 site-directed mutagenesis kit according to the manufacturer’s instructions (NEB E0554S, primers listed in Table 1). The L514K mutation altered two bases and required two sequential rounds of site directed mutagenesis, one for each base. This optimized PA-Racl was PCR amplified, gel purified, and TOPO cloned into the pENTR vector (Invitrogen K240020) using primers listed in Table 1. The mouse b-globin minimal promoter (Tamplin, et al., 2011) was PCR amplified and inserted upstream of optimized mCherry-PA-Racl in the pME-mCherry- PA-Racl vector linearized with Notl using Gibson Assembly (NEB E5510S, primers listed in Table 1) to allow robust gene expression. The p3E vector contained an SV40 poly-A signal (Tol2Kit plasmid #302)(Kwan, et al., 2007). The p5E-3xl25bp-mnxl, pME-B-globin- mCherry-PA-Racl, and p3E-SV40-poly-A entry vectors were recombined with the pDestTol2CG2 destination vector (Tol2Kit plasmid #395)(Kwan, et al., 2007) using LR Clonase II Plus according to the manufacturer’s instructions (Invitrogen 12538200) to produce the final transgenesis constructs (Fig. 1A).

[0134] To generate the pDestTol2CG2-mnxl:ReaChR-citrine plasmid, a pME vector containing the b-globin minimal promoter and ReaChR-citrine was constructed by swapping mCherry-PA-Racl for ReaChR-citrine in the pME-B-globin-mCherry-PA-Racl vector. Briefly, mCherry-PA-Racl was removed from pME-B-globin-mCherry-PA-Racl using BssHII and Hpal digestion and the backbone fragment was gel purified. A ReaChR-citrine PCR product amplified from Addgene plasmid #50956 (a gift from Roger Tsien) (Lin et al., 2013) and a PCR amplified fragment of the backbone lost in the restriction reaction were gel purified and inserted using the NEBuilder HiFi DNA Assembly kit according to the manufacturer’s instructions (NEB E2621, primers listed in Table SI). The final transgenesis construct was generated via recombination as described above.

[0135] To generate the light insensitive pDestTol2CG2-mnxl:mCherry-PI-Racl transgenesis construct, L514K and L531E mutations were introduced into the pTriEx- mCherry-PA-Racl -C450A plasmid (a gift from Klaus Hahn; Addgene plasmid #81031), which harbors a single amino acid substitution rendering PA-Racl insensitive to light (Wu, et al., 2009), and which is referred to here as PI-Racl. Thus, PA-Racl and PI-Racl were identical except for the C450A mutation rendering PI-Racl insensitive to light. The pDestTol2CG2- mnx l:mC he rry- PA-Racl and pTriEx-mCherry-PA-Racl-C450A plasmids were digested with Agel and BbvCI and the PI-Racl fragment and the destination vector backbone were gel purified and ligated together using Quick T4 DNA ligase (NEB M2200S) to create the final transgenesis construct. All cloning reactions were sequence verified.

[0136] Zebrafish transgenesis

[0137] Transgenic lines were derived by injecting mitfa^w2/w2;mpvl7^a9/a or plod3^tv205a/+ embryos at the one-cell stage with InL of 25ng/uL of pDestTol2CG2-mnxl:mCherry-PA- Racl, pDestTol2CG2-mnxl:mCherry-PI-Racl, or pDestTol2CG2-mnxl:ReaChR-citrine transgenesis constructs along with 35ng/uL Tol2 mRNA. Capped Tol2 mRNA was transcribed in vitro using the mMessage mMachine SP6 kit (Ambion AM1340) from a Notl linearized pCS2FA-transposase plasmid (Tol2Kit plasmid #396)(Kawakami and Shima, 1999) according to the manufacturer’s instructions. Tol2 RNA was purified with the RNAeasy Mini Kit (Qiagen 74104).

[0138] Zebrafish embryo dissociation and culture

[0139] Nitric acid etched glass coverslips (25mm No 1.5; neuVitro GG-25-1.5-Pre) were incubated with lOOug/mL poly-D-lysine (VWR 35210) in lOOmM borate buffer overnight at room temperature. The following day, coverslips were washed 5x with phosphate buffered saline (PBS) followed by incubation with 5ug/mL of laminin (VWR 354232) in water for one hour at 37°C. Coverslips were then washed twice with sterile water. Randomly selected embryonic embryonic zebrafish at 18hpf were dissociated as described previously (Andersen, 2001). Briefly, the outer chorionic surface was sterilized for 5 seconds in 70% ethanol. Embryos were then washed twice and then anesthetized in E3 (5mM NaCl, 0.17mM KC1, 0.33mM CaCl₂, 0.33mM MgS0₄) with 0.0016% tricaine (MS-222; Sigma-Aldrich E10505) for 10 minutes before manual removal of the chorion. Embryos were washed twice in MMR (lOOmM NaCl, 2mM KC1, ImM MgS0₄, 2mM CaCl₂ and 5mM Na-HEPES, adjusted to pH 7.8 with NaOH) and the skin and head were dissected away. The body was incubated in lOOuL of ATV solution (0.6mM EDTA, 5.5mM glucose, 5.4mM KC1, 136.8mM NaCl, 0.05% trypsin, 5.5mM Na₂C0₃) for 10 minutes at 30°C followed by trituration with a lOuL pipette tip 30 times. Culture media (2% fetal bovine serum, lOOU/mL penicillin/streptomycin in Liebowitz L-15 media [Thermo Fischer 21083027]) was then added. A pool of three embryos was plated on each coverslip and incubated at room temperature overnight before stimulation trials.

[0140] Imaging and optogenetic stimulation

[0141] Whole zebrafish embryos were mounted in 1% low melt agarose (Sigma A6877), while primary neurons grown on coverslips were mounted in Attofluor Cell Chambers (ThermoFischer A-7816) for imaging and stimulation experiments. Live embryos were anesthetized with 0.0016% tricaine (MS-222; Sigma-Aldrich E10505) in E3 solution prior to mounting in 1% low melt agarose in tricaine solution. Transmitted and fluorescent images of embryos and primary motor neurons were collected using a Zeiss 880 LSM confocal microscope with a GaSaP detector using the 20X objective (Plan-Apochromat 20x/0.8 NA M27) focused in the plane of the growth cone during stimulation experiments. These images were used to create time-lapse movies with a custom Jython script using the ImageJ API. For still “snapshots”, an AiryScan detector was used to acquire fluorescent Z- stacks for whole zebrafish embryos or a single Z plane for primary motor neuron cultures. Snapshots were acquired at 4-6 timepoints during PA-Racl stimulation experiments and following immunohistochemistry. For in vitro Pakl immunohistochemistry experiments, neurons were imaged using a Plan-Apochromat 63x/1.4 NA oil DIC M27 lens. AiryScan deconvolution was performed on images collected with the AiryScan detector using the AiryScan module in ZEN Black (Zeiss). Maximum intensity projections of Z stacks or three- dimensional representations are shown for whole embryos. 3D rendering of confocal Z-stacks was done using VAST (Berger, et ah, 2018).

[0142] For both in vitro and in vivo PA-Racl optogenetic stimulation experiments, a 3-10mhi diameter circle was raster scanned with 5% laser power of 458nm wavelength light (300-650nW, measured with a ThorLabs PM100D optical power meter) through the 20X objective in a region of interest (ROI) placed asymmetrically on the leading edge of the growth cones in the direction of desired outgrowth. Stimulation and imaging were alternated without delay with each stimulation lasting roughly 2 milliseconds and each stimulation/imaging cycle lasting 1-3 seconds. This cycle was repeated until the axon extended into the ROI, at which point the ROI was manually updated in the direction of desired growth. This process was repeated throughout the duration of the trial (up to 90 minutes for in vitro experiments and up to 4 hours for in vivo experiments). An identical illumination procedure was followed for in vitro control experiments of axons expressing the photo-insensitive Racl (PI-Racl).

[0143] To determine the effect of wavelength on axonal outgrowth, the growth cones of cultured zebrafish motor neurons expressing PA-Racl were exposed to 514nm light (outside the spectrum of PA-Racl activation) for 5 minutes, followed by exposure to 458nm light (within the spectrum of PA-Racl activation) within the same region of interest. For assessment of Racl signaling activity, PA-Racl expressing cultured zebrafish motor neurons were exposed to 458nm light in a region of interest as described above for 5 minutes and then immediately fixed for immunohistochemistry of downstream Pakl phosphorylation.

[0144] For in vivo deviation of axons across somite boundaries in Tg(mnxl:mCherry- PA-Rcicl);mitfa^w2/w2;mpvl7^a9/a9 fish, stimulation was initiated between 23 and 25hpf. For rescue experiments of Tg(mnxl:mCherry-PA-Rcicl);plod3^{tv205a/tv205a} fish, stimulation was initiated later at 28hpf to ensure that mutant CaP axons were stalled at the horizontal myoseptum prior to stimulation. All in vivo stimulation experiments were replicated in independent fish from distinct clutches on separate days and one neuron was optogenetically manipulated per fish. Candidate neurons for optogenetic stimulation were selected based on appropriate initial axonal morphology and strong transgene expression.

[0145] For functional interrogation of synapse formation with ReaChR optogenetic depolarization, mounting agarose was dissected away from the zebrafish embryos following PA-Racl mediated optogenetic rescue of plod3^{tv205a/tv205a} axons. Embryos were placed in room temperature E3 solution for 1.5 hours to ensure removal of tricaine anesthetic and to allow time for synapses to form. Embryos were then transferred to 4°C E3 solution for 1 minute to anesthetize them before remounting in 1% low melting point agarose in E3. ReaChR optogenetic depolarization trials were performed using a similar illumination strategy by raster scanning a region of interest surrounding the target neuron with 100% laser power of 594nm and 633nm light, within the ReaChR activation spectrum. Stimulation trials tested three conditions: ReaChR activation of the rescued neuron, ReaChR activation of a neighboring neuron whose axonal morphology was not rescued, and illumination of the rescued neuron with 100% laser power 405nm and 440nm light (which are outside of the ReaChR activation spectrum) in the same ROI, to control for light exposure. Each stimulation trial consisted of a time series of 1000 bright field imaging frames acquired at 9.5 Hz, with optogenetic stimulation occurring every 200 frames, for a total of 5 stimulations per trial. Five trials were performed per condition (n=3 fish, each tested for all conditions). Trials were initiated 1.5 hours after PA-Racl mediated rescue of the axonal guidance defect to allow for synapses to form. All stimulation experiments were performed at room temperature.

[0146] FACS purification and digital droplet PCR

[0147] Randomly sampled embryonic Tg(mnxl :mCherry-PA-

Racl );mitfa^w2/w2 ;mpvl 7^a9/a9 zebrafish were dissociated at 24hpf as described above. This age was chosen for gene expression analysis as it corresponded to the time of initiation of in vivo axon guidance experiments. mCherry-expressing spinal motor neurons were FACS-sorted into Trizol LS (Thermo Fisher 10296010) using a MoFlo XDP Cell Sorter (Beckman Coulter ML99030) with Summit 5.4 (Beckman Coulter) data collection software. Total RNA was purified from 10,000 sorted cells according to the Trizol LS manufacturer’s instructions (ThermoFisher). cDNA was synthesized using the iScript Select cDNA synthesis kit and random primers (BioRad 1708896). Digital droplet PCR was performed to quantify the expression levels of racla, raclb and mCherry expression in sorted spinal motor neurons was performed using the QX200 Evagreen ddPCR supermix (BioRad) (primers in Table 1). mCherry expression was used as a proxy for PA-Racl expression, since they originate from a single transcript.

[0148] Immunohistochemistry

[0149] For in vivo experiments, whole mount immunohistochemistry protocols were adapted from previous methods (Panzer et ah, 2005). To allow time for synapse formation, zebrafish embryos were fixed 1.5 hours following the termination of optogenetic stimulation experiments in 4% paraformaldehyde in PBS overnight at 4°C. Embryos were washed three times for five minutes in PBT (0.1% Tween20 and 1% DMSO in PBS), heads were removed, and the tails were incubated for 45 minutes in lmg/mL collagenase (Sigma-Aldrich C9891) in PBS. They were then washed three times in PBT for five minutes, and incubated in blocking solution (2% BSA, 0.5% TritonX in PBS) for several hours. They were then incubated with mouse anti-synaptic vesicle 2 (1:100 DSHB SV2) and rabbit anti-RFP (1:500, Rockland 600- 401-379S Lot 35868) primary antibodies in blocking solution overnight at 4°C. Embryo tails were washed 5X with PBT and incubated overnight at 4°C with AlexaFluor 488 conjugated goat anti-mouse (1:750, Invitrogen A11001) and AlexaFluor 546 conjugated goat anti-rabbit (1:750, Invitrogen A11035) secondary antibodies in blocking solution. AlexaFluor 647- conjugated a-bungarotoxin (Thermo B35450) was added during the last 45 minutes of secondary antibody incubation. The embryo tails were washed three times in PBT for five minutes each before mounting in 1% low melting point agarose (Sigma A6877) in PBS for confocal imaging.

[0150] For in vitro experiments, after 5 minutes of PA-Racl optogenetic stimulation as described above, cultured zebrafish motor neurons were immediately fixed in 4% paraformaldehyde in PBS overnight at 4°C, washed three times in PBT for five minutes each, and incubated in blocking solution as above. They were then incubated overnight with rabbit anti-Thr423 phospho-PAKl (1:100, Cell Signaling Technology 2601T) and rat anti-RFP (1:100, ChromoTek 5F8) primary antibody in blocking solution. Coverslips were washed three times in PBT for five minutes each, followed by incubation with AlexaFluor 488 conjugated goat anti-rabbit (1:750, Invitrogen A11008) and AlexaFluor 546 conjugated goat anti-rat (1:750, Invitrogen A11081) secondary antibody in blocking solution. Coverslips were washed three times with PBT for five minutes each and mounted with Fluoromount-G (SouthernBiotech 0100-01).

[0151] Dissection and tissue preparation for electron microscopy

[0152] Following induced axon guidance experiments (~30hpf), , Tg(mnxl:mCherry- PA-Racl);mitfa^w2/w2 ;mpvl7^a9/a9 embryos (~30hpf) still under anesthesia were placed in a dissection solution (64 mM NaCl, 2.9 mM KC1, 10 mM HEPES, 10 mM glucose, 164 mM sucrose, 1.2 mM MgCh, 2.1 mM CaCh, pH 7.5) (Hildebrand et al., 2017). Embryos were carefully removed from the agarose and grown for an additional 1.5 hours. The tail was then isolated using sharp scissors (WPI 501778) and immediately transferred to fixative at 4°C (2.5% glutaraldehyde, 2% PFA in 0.5x cacodylate buffer supplemented with 3.5% mannitol, pH 7.4, Cacodylate buffer: 0.3M sodium cacodylate, 6mM CaCh, pH7.4). To improve fixation, the tissue was rapidly microwaved (Ted Pella, cat. no. 36700, with power controller, steady-temperature water recirculator and cold spot) in the fixative solution at 10°C (<5 min after initial transfer into fixative). The following microwaving sequence was performed: at power level 1 (100 W) for 1 min on, 1 min off, 1 min on; then increased to power level 3 (300 W) and fixed for 20s on, 20s off, 20s on, three times (Tapia et al., 2012). Fixation was then continued overnight at 4°C in the same solution. The following day, samples were washed in 0.5X cacodylate buffer (3 exchanges, 30 min each at room temperature) and then reduced in freshly made 0.8% (w/v) sodium hydrosulfite in 60% (v/v) 0.1 M sodium bicarbonate, 40% (v/v) 0.1 M sodium carbonate buffer with 3 mM CaCh for 20 min at room temperature (Joesch et al., 2016). This step improves contrast-to-noise ratio between membrane and cytosol. Samples were then washed again in 0.5x cacodylate buffer (3 exchanges, 30 min each at room temperature) before osmication (2% OsCU in 0.5x cacodylate buffer, 4 h at room temperature), and then incubated overnight at 4°C. Samples were then reduced in 2.5% potassium ferrocyanide in 0.5x cacodylate buffer for 4 h at room temperature and then overnight at 4°C. The following day, samples were washed with filtered H2O (3 exchanges, 30 min each at room temperature) and incubated with 1% (w/v) thiocarbohydrazide (TCH) in H2O (filtered with a 0.20um syringe filter before use) at room temperature to enhance staining (Hua et al., 2015a). Due to poor dissolution of TCH in water, the solution was heated at 60°C for ~lh with occasional shaking before filtering. Samples were then washed with filtered H2O (3 exchanges, 30 min each at room temperature) before the second osmication (2% OsCU in filtered H2O, 4 h at room temperature) and then washed again (3 exchanges, 30 min each at room temperature). En bloc staining was performed using 1% uranyl acetate overnight in filtered water. The OsCU solution was sonicated for ~lh and then filtered with a 0.20um syringe filter before use. The following day, samples were washed with filtered H2O (3 exchanges, 30 min each at room temperature) and dehydrated in serial dilutions of ethanol (25%, 50%, 75%, 90%, 100%, 100% for 10 min each) followed by propylene oxide (PO) (100%, 100%, 30 min each). Infiltration was performed using LX112 epoxy resin with BDMA (21212, Ladd) in serial PO dilutions steps (25% resin/75% PO, 50% resin/50% PO, 75% resin/25% PO, 100% resin, 100% resin, 4h each). Samples were mounted using a mouse brain as support tissue (Hildebrand et al., 2017) in fresh resin to facilitate cutting. For this, mouse tissue was fixed using standard procedures, pierced using a puncher (EMS 57395) to insert the sample, and stained along with fish samples. The samples with support tissue were then cured for 3 days at 60°C. A rotator was used for all steps. Aqueous solutions were prepared with water passed through a purification system (Arium 611VF, Sartorius Stedim Biotech).

[0153] Electron microscopy

[0154] The cured blocks were trimmed as previously described (Hildebrand et al., 2017) and ~30nm sections were automatically collected using a custom tape collection device (ATUM) (Hayworth et al., 2014) mounted to a commercial ultramicrotome. Sections were collected and post-stained as published (Hildebrand et al., 2017). Images were acquired using back-scatter detection with a Sigma scanning electron microscope (Carl Zeiss) equipped with the ATLAS software (Fibics). Custom made algorithms were used for non-affine alignment, and volume annotation and segmentation were performed with VAST (Berger et al., 2018).

[0155] Quantification and Statistical Analysis

[0156] Analysis of PA-Racl axon guidance

[0157] For all experiments using transgenic zebrafish, candidate animals were first visually screened for high transgene expression. For in vitro optogenetic axon guidance experiments (Fig. 1), each replicate represents an axon from a separate culture with cells from independent Tg(mnxl :mCherry -PA-Racl );mitfa^w2/w2 ;mpvl7^a9/a9 or Tg(mnxl:mCherry-PI- Racl );mitfa^w2/w2 ;mpvl7^a9/a9 fish and cultures were randomly assigned to treatment conditions where applicable. Candidate axons were selected based on high expression of mCherry-PA- Racl or mCherry-PI-Racl and lack of contact of growth cones with any neighboring cells. To quantify in vitro optogenetic guidance (Fig. 1C), the distance grown by CaP axons over the course of the stimulation experiments and the deviation from their original growth trajectories were measured using the neurite tracing function of Imaris (Bitplane) and FIJI (ImageJ 2.0.0- rc-69/1.52p) respectively. The distance grown was quantified by taking the difference of the length of the axon before and after the stimulation trial. The speed of growth was calculated by dividing this distance by the time elapsed over the course of the stimulation trial. The angle of axonal trajectory deviation was measured using overlapped images of axons immediately prior to optogenetic stimulation with images of axons following stimulation. An angle of 0° reflected no change in trajectory, while any deviation was considered a positive angle of deflection, since there was no anatomic frame of reference in the monolayer cultures. Measurements were performed on stimulated PA-Racl expressing axons, as well as two controls: unstimulated PA-Racl expressing neurons and stimulated axons expressing photo insensitive PI-Racl. Independent samples Student’s T-tests were used to compare each control condition to PA-Racl stimulated axons.

[0158] For in vitro control experiments (Fig. 6C,D), candidate cultured Tg(mnxl:mCherry-PA-Racl);mitfci^w2/w2;mpvl7^a9/a9 zebrafish motor neurons were selected as described above and outgrowth was measured in response to different wavelengths of light. Specifically, the area occupied by the growth cone within the region of illumination was measured before and after 5 minutes of exposure first to 514nm light (outside the activation spectrum of PA-Racl) and then to 5 minutes of 458nm light (which activates PA-Racl). Since the experimental conditions were applied to the same growth cones sequentially, the response to different wavelengths was compared with a paired samples Student’s T-test.

[0159] For optogenetic guidance across somitic boundaries in vivo in Tg(mnxl:mCherry-PA-Racl);mitfci^w2/w2;mpvl7^a9/a9 zebrafish (Figs. 2,3), candidate axons for optogenetic manipulation were selected based on high transgene expression and having growth cones between the horizontal myoseptum and inferior ventral myotomal border. Neighboring axons within the same fish served as internal controls. For these in vivo optogenetic axon guidance experiments, the change in axon length, growth speed, and angle of growth trajectory deviation were measured as above. In this case, an angle of 0° reflected no change in trajectory, while positive angles represent deflections in the rostral direction and negative angles represent deflections in the caudal direction. Additionally, the distance between the CaP growth cone tip and the horizontal somitic boundary was measured using FIJI (ImageJ). These distances were arbitrarily assigned positive values if the axon had not crossed the boundary and negative values if the axon had crossed into the neighboring somite. The number of axons that responded to stimulation, crossed a horizontal somitic boundary or did not respond to stimulation was counted. In one fish, the initial axon lengths could not be determined, and this fish was excluded from subsequent analysis. Since somitic units develop asynchronously in a rostral-to-caudal gradient, rostral and caudal neighbors were treated separately. Furthermore, since neighboring axons reside in the same organism, and are thus not independent observations, these data were fit with a mixed linear model that included a random effect corresponding to the animal in which axonal measurements were made. Pairwise comparisons of the least square means were performed, and p-values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure for controlling the false discovery rate. [0160] For plod3^A rescue experiments, candidate fish were screened based the axonal guidance defect phenotype in which no CaP axons extended past the horizontal myoseptum (Figs. 4,5). To quantify the results of these experiments, the shortest distance from the horizontal myoseptum to the leading tip of the CaP axon was measured using FIJI (ImageJ)(Fig. 4D). A linear model analysis was similarly applied to measurements of the distance from the tip of the CaP axon to the horizontal myoseptum in plod3^/ mutant fish, plod3^+/ wildtype fish, and plod3 mutant fish that had been rescued with optogenetic PA- Racl activation. The linear model also included a random variable accounting for the fish in which the measurements were made. Measurements were made on rescued axons and their neighbors in the same fish and on axons from separate plod3^+/ fish identified phenotypically. Comparisons were performed as above.

[0161] Analysis of immunohistochemistry

[0162] To quantify immunohistochemical colocalization of pre- and postsynaptic proteins (SV2 and oc-BT), 3D regions of interest were extracted from Z-stacks and Pearson’s correlation coefficients between the SV2 and oc-BT were calculated from the raw data (Figs. 3E and 5C). These correlation coefficients were compared to a random distribution of correlations calculated by randomly shuffling blocks of the original images. Empiric p-values were obtained using the FIJI (ImageJ) Coloc 2 package in accordance with the Costes method (Costes, et ah, 2004).

[0163] ReaChR stimulation analysis

[0164] To analyze motion from muscle contraction after ReaChR channelrhodopsin stimulation, particle image velocimetry using optical flow (PIV) analysis was performed on bright field images using FIJI (ImageJ)(Fig. 5E). Specifically, 2D vector fields representing the translation of 32x32 pixel boxes were generated by comparing the bright field image immediately before the initiation of contraction and the subsequent frame in which motion was first detected. These vector fields representing the motion of the zebrafish body were calculated for every observed contraction. From these vector fields, foci of contraction were localized at the site where vector fields converged. Once these foci were identified, anatomical boundaries traced from the bright field images were superimposed on the vector fields. This allowed us to determine whether the focus of contraction was in the ventral or dorsal musculature and in which somite the contraction originated. The total number of stimulations that induced any muscle contraction and the number of stimulations that induced muscle contractions specifically in the ventral musculature of the somite targeted with optogenetic stimulation were counted for the following conditions: rescued axons receiving ReaChR stimulation, rescued axons not receiving ReaChR stimulation, and neighboring unrescued axons receiving ReaChR stimulation. These data were modeled using a Poisson general linear model and p-values were computed from pairwise comparisons of the estimated marginal means (Fig. 5F). Error bars represent 95% normal confidence intervals. All statistical analyses were performed in R studio 3.5.1 using the following packages: dplyr 0.7.6, reshape2 1.4.3, ggplot2 3.0.0, ggbeeswarm 0.6.0, ImerTest 3.0.1, stats 3.5.1, lubridate 1.7.4, multcomp 1.4-10 and emmeans 1.4.2.

[0165] Table 1: Primers used in this study.

bMutated bases indicated with bold and underline c Taken from Rosowski et al. 2016. dTaken from Jacobs, Badiee, and Lin. 2018.

[0166] PA-Racl DNA (SEQ ID NO: 22)

[0167] GGATCCTTGGCTACTACACTTGAACGTATTGAGAAGAACTTTGTCA TTACTGACCCAAGATTGCCAGATAATCCCATTATATTCGCGTCCGATAGTTTCTTG CAGTTGACAGAATATAGCCGTGAAGAAATTTTGGGAAGAAACTGCAGGTTTCTAC AAGGTCCTGAAACTGATCGCGCGACAGTGAGAAAAATTAGAGATGCCATAGATA ACCAAACAGAGGTCACTGTTCAGCTGATTAATTATACAAAGAGTGGTAAAAAGTT CTGGAACCTCTTTCACTTGCAGCCTATGCGAGATCAGAAGGGAGATGTCCAGTAC TTTATTGGGGTTCAGTTGGATGGAACTGAGCATGTCCGAGATGCTGCCGAGAGAG AGGG AGTC AT GCT GATT A AG A A A ACT GC AG A A A AT ATT GAT G AGGC GGC A A AAG AACTTATCAAGTGTGTGGTGGTGGGAGACGGAGCTGTAGGTAAAACTTGCCTACT GATCAGTTACACAACCAATGCATTTCCTGGAGAATATATCCCTACTGTCTTTGACA ATTATTCTGCCAATGTTATGGTAGATGGAAAACCGGTGAATCTGGGCTTATGGGA TACAGCTGGACTAGAAGATTATGACAGATTACGCCCCCTATCCTATCCGCAAACA GATGTGTTCTTAATTTGCTTTTCCCTTGTGAGTCCTGCATCATTCCACCACGTCCGT GCAAAGTGGTATCCTGAGGTGCGGCACCACTGTCCCAACACTCCCATCATCCTAG TGGGAACTAAACTTGATCTTAGGGATGATAAAGACACGATCGAGAAACTGAAGG AGAAGAAGCTGACTCCCATCACCTATCCGCAGGGTCTAGCCATGGCTAAGGAGAT TGGTGCTGTAAAATACCTGGAGTGCTCGGCGCTCACACAGCGAGGCCTCAAGACA GTGTTTGACGAAGCGATCCGAGCAGTCCTCTGCCCGCCTCCCGTGAAGAAGAGGA AG AG A A A AT GCCTGCT GTT GT A A [0168] PA-Racl Amino Acid sequence (SEQ ID NO: 23)

[0169] MRDQKGD V QYFIGV QLDGTEHVRD AAEREGVMLIKKT AENIDE A AK ELIKC V V V GDG A VGKT CLLIS YTTN AFPGE YIPT VFDN Y S AN VM VD GKP VNLGLWDT AGLEDYDRLRPLSYPQTDVFLICFSLVSPASFHHVRAKWYPEVRHHCPNTPIILVGTKL DLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQRGLKTVFDEAIRA VLCPPPVKKRKRKCLLL

[0170] PA-Racl with L514K and L53 IE mutations DNA (SEQ ID NO: 24)

[0171] GGATCCTTGGCTACTACACTTGAACGTATTGAGAAGAACTTTGTCA TTACTGACCCAAGATTGCCAGATAATCCCATTATATTCGCGTCCGATAGTTTCTTG CAGTTGACAGAATATAGCCGTGAAGAAATTTTGGGAAGAAACTGCAGGTTTCTAC AAGGTCCTGAAACTGATCGCGCGACAGTGAGAAAAATTAGAGATGCCATAGATA ACCAAACAGAGGTCACTGTTCAGCTGATTAATTATACAAAGAGTGGTAAAAAGTT CTGGAACCTCTTTCACTTGCAGCCTATGCGAGATCAGAAGGGAGATGTCCAGTAC TTTATTGGGGTTCAGAAGGATGGAACTGAGCATGTCCGAGATGCTGCCGAGAGAG AGGG AGTC AT GG AG ATT A AG A A A ACT GC AG A A A AT ATT GAT G AGGC GGC A A AAG AACTTATCAAGTGTGTGGTGGTGGGAGACGGAGCTGTAGGTAAAACTTGCCTACT GATCAGTTACACAACCAATGCATTTCCTGGAGAATATATCCCTACTGTCTTTGACA ATTATTCTGCCAATGTTATGGTAGATGGAAAACCGGTGAATCTGGGCTTATGGGA TACAGCTGGACTAGAAGATTATGACAGATTACGCCCCCTATCCTATCCGCAAACA GATGTGTTCTTAATTTGCTTTTCCCTTGTGAGTCCTGCATCATTCCACCACGTCCGT GCAAAGTGGTATCCTGAGGTGCGGCACCACTGTCCCAACACTCCCATCATCCTAG TGGGAACTAAACTTGATCTTAGGGATGATAAAGACACGATCGAGAAACTGAAGG AGAAGAAGCTGACTCCCATCACCTATCCGCAGGGTCTAGCCATGGCTAAGGAGAT TGGTGCTGTAAAATACCTGGAGTGCTCGGCGCTCACACAGCGAGGCCTCAAGACA GTGTTTGACGAAGCGATCCGAGCAGTCCTCTGCCCGCCTCCCGTGAAGAAGAGGA AG AG A A A AT GCCTGCT GTT GT A A

[0172] PA-Racl with L514K and L53 IE mutations DNA (SEQ ID NO: 25)

[0173] MRDQKGD V QYFIGV QKDGTEHVRDAAEREGVMEIKKT AENIDE AAK ELIKC VVV GDG A VGKT CLLIS YTTN AFPGE YIPT VFDN Y S AN VM VD GKP VNLGLWDT AGLEDYDRLRPLSYPQTDVFLICFSLVSPASFHHVRAKWYPEVRHHCPNTPIILVGTKL DLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQRGLKTVFDEAIRA VLCPPPVKKRKRKCLLL [0174] Example 4

[0175] I. Construction of artificial biological neural networks [0176] One aspect of this invention relates to the construction of standardized neural networks composed of neurons isogenic to patients to allow for screening of therapeutic compounds. Diseases of neural connection and activity including, but not limited to epilepsy, schizophrenia, and autism, currently lack robust preclinical models for drug development. By converting a patient’s own cells into neurons using standard stem cell techniques, i.e. differentiation of induced pluripotent stem cells (iPSCs), robust, reproducible neural networks can be constructed using light to pattern the axonal connections of neurons expressing PA- Racl. Since the cells comprising these networks share their genome and thus underlying molecular programs, with the patient from which they are derived, they provide tremendous potential for recapitulating the pathogenesis of disease. Such a model would provide a robust platform for screening pharmacologic agents to correct aberrant network connections for high personalized medical interventions.

[0177] Another aspect of this invention relates to the construction of biological computers. Much work has gone into developing artificial neural networks for machine learning and artificial intelligence applications in software implemented on silicon chips. However, the organic substrates that inspire these algorithms are vastly more resource efficient and flexible. This invention allows for precise wiring of individual neurons into highly stereotyped circuits that could comprise an in vitro biological computer. These biocomputers would have natural advantages in efficiency and parallel processing.

[0178] II. Therapeutic restoration of abnormal neural network topology

[0179] Neurological diseases ranging from degenerative aging to neurodevelopmental disease and to acute trauma can disrupt essential neural circuitry, leading to significant functional impairment. This damage is often irreversible, since the nervous system largely lacks the capacity to regenerate in response to these insults. A central clinical challenge is to restore lost function by reconnecting or replacing neurons damaged by these pathologies. Unlocking such therapeutic options will require reestablishment of highly stereotyped axonal wiring, which may be especially challenging in adults lacking developmental cues required for axon guidance. Current therapeutic approaches for functional restoration of axonal connections involve either surgical implantation of exogenous substrates for axon growth or treatment with axonal growth promoting drugs.

[0180] However, complications of invasive neurosurgical procedures, off-target side effects, and nonspecific axonal connections have limited their clinical use. These limitations may be circumvented by using light to noninvasively and specifically manipulate regenerating axons expressing PA-Racl, to guide them precisely to their appropriate destinations. [0181] Given Rad’s ubiquitous presence in shaping the circuits of the nervous system during development and the emergence of clinical methods for delivering light to the neural parenchyma of patients for optogenetic therapies, there is much promise in manipulating this system in flexible, reversible and noninvasive ways for therapeutic intervention in these diseases. Indeed, Racl is a promising therapeutic target given its direct involvement in the pathophysiology of these diseases. To demonstrate the broad applicability of our axonal guidance system for therapeutic use, here we outline the potential application of this tool in three diseases representing broad categories of CNS damage: 1) acute trauma resulting in spinal cord injury 2) neurodegeneration in amyotrophic lateral sclerosis and 3) neurodevelopmental disruptions in schizophrenia.

[0182] Upon acute injury to the spinal cord, inhibitory molecules such as myelin associated glycoprotein (MAG) and Nogo-A are expressed by glial scar tissue, inhibiting the natural regenerative response of axons. MAG and Nogo-A increase RhoA activity causing growth cone collapse. Rho and Racl act antagonistically in regulating growth cone dynamics and thus stimulating Racl activity along with Rho inhibition may prove synergistic in these patients. Since Racl has such pleotropic effects, limiting Racl activation to the site of injury with precise, laser illumination would greatly widen the therapeutic window. Furthermore, targeted light stimulation will allow for precise axon guidance, limiting the formation of spurious connections, which is a major limitation of nonspecific modulation of axonal growth with drug treatment.

[0183] Finally, patients with neurodevelopmental and psychiatric diseases also suffer from inappropriate axonal connections. For example, a meta- analysis of diffusion tensor imaging in schizophrenic patients revealed a loss of deep white matter and connections in the left frontal and temporal lobe. A highly penetrant genetic determinant of schizophrenia is the gene Disrupted-in-schizophrenia 1 (DISCI). Heterologous expression of DISCI in C. elegans demonstrates that DISCI accumulates in the growth cone, where it activates Racl, altering axon guidance. Given the known deficits in cortical white matter with Racl loss, schizophrenia could be therapeutically addressed by reconnecting areas that are disconnected by the disease process, using our light activated axonal guidance system.

[0184] DISCUSSION

[0185] The ability to manipulate axonal growth and guidance in living organisms would offer an opportunity for experimental investigation of fundamental neurodevelopmental questions regarding the relationship between axons and their targets and how network architecture shapes the development and function of the nervous system. [0186] New approaches are however needed to precisely and noninvasively sculpt axonal connections in the intact organism. Manipulating axonal growth trajectories in living organisms is particularly challenging, since growth cones are subjected to complex physical and molecular environments that have evolved to ensure axons adopt specific morphologies and only connect to stereotyped targets (Dickson, 2002; Tessier-Lavigne and Goodman, 1996). The ability to direct axons to new targets across these powerful endogenous repulsive barriers is necessary to wire novel circuitry in vivo , where a diversity of axon guidance signals would otherwise impede directed re-wiring.

[0187] In addition to guidance during development, axons can also encounter pathfinding barriers that impede regeneration following injury. Here it was shown that CaP axon growth could be directed across vertical myoseptal boundaries, which are typically never crossed (Beattie, 2000; Myers et al., 1986) due to potent molecular deterrents including chondroitin sulfate proteoglycans (CSPGs) and semaphorins (Bernhardt et al., 1998; Roos et al., 1999). Interestingly, a number of these same inhibitory molecules have homologues in glial scars, which form following injury in the mammalian CNS (Pasterkamp et al., 1999; Siebert et al., 2014). These scars become impenetrable barriers and are a major obstacle for subsequent axonal regeneration (Yiu and He, 2006). The ability to overcome such barriers in the zebrafish opens new doors to investigation of similar non-invasive guidance approaches to aid mammalian CNS axon regeneration.

[0188] In order to functionally repair and rewire circuitry, active synaptic connectivity must be established between the redirected axon and its new target. Optogenetic activation of PA-Racl in CaP neurons could not only overcome the axon guidance defects associated with pathological loss of plod3 activity, but also notably resulted in the formation of new functional synapses, capable of mediating ventral myofibril contraction following optogenetic depolarization of the rescued axons. These results serve as a proof of principle for the value of this approach to wire novel circuitry that can be used for downstream investigation of the role of new connectivity on target development and function.

[0189] In addition to enabling future investigation in the development, function, and possibly regeneration of neuronal networks, an intriguing application of this experimental paradigm relates to the engineering of neural networks in vitro. Human induced pluripotent and embryonic stem cells can be differentiated into neurons in a variety of configurations ranging from monolayers to 3D brain organoids (Quadrato and Arlotta, 2017; Velasco et al., 2019). Endogenous patterns of connectivity, however, are difficult to attain within these model systems, which sets practical limits to studies of human circuit formation and functionality. Although here single growth cones were manipulated, the use of light as a stimulus allows enormous flexibility in delivering patterned stimulation to these in vitro models. For instance, digital micromirror devices (DMDs) provide a scalable, parallel method for generating complex patterns of light (Avants et al., 2015; Brinks et al., 2016; de Beco et al., 2018; Hakim et al., 2018; Jung et al., 2017; Sakai et al., 2013). Future work can combine these devices with the optogenetic axon guidance approach demonstrated here, to guide many axons simultaneously and create precise, realistic, and reproducible axonal architectures in complex three-dimensional in vitro models, such as brain organoids.

[0190] Beyond its potential for powerful applications in the nervous system, this work also serves as an example of how optogenetic control of cellular morphology can reshape development to engineer novel tissue architectures, an approach that may carry particular value for regenerative biology and tissue engineering across a variety of tissue types.

[0191] REFERENCES

[0192] Airan, R.D., Thompson, K.R., Fenno, L.E., Bernstein, H., and Deisseroth, K. (2009). Temporally precise in vivo control of intracellular signalling. Nature 458, 1025.

[0193] Andersen, S.S. (2001). Preparation of dissociated zebrafish spinal neuron cultures. Methods Cell Sci 23, 205-209.

[0194] Avants, B.W., Murphy, D.B., Dapello, J.A., and Robinson, J.T. (2015). NeuroPG: open source software for optical pattern generation and data acquisition. Front Neuroeng 8, 1.

[0195] Beattie, C.E. (2000). Control of motor axon guidance in the zebrafish embryo. Brain Res Bull 53, 489-500.

[0196] Berger, D.R., Seung, H.S., and Lichtman, J.W. (2018). VAST (Volume Annotation and Segmentation Tool): Efficient Manual and Semi-Automatic Labeling of Large 3D Image Stacks. Front Neural Circuits 12, 88.

[0197] Bernhardt, R.R., Goerlinger, S., Roos, M., and Schachner, M. (1998). Anterior-posterior subdivision of the somite in embryonic zebrafish: implications for motor axon guidance. Dev Dyn 213, 334-347.

[0198] Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G., and Deisseroth, K. (2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8, 1263-1268.

[0199] Brinks, D., Adam, Y., Kheifets, S., and Cohen, A.E. (2016). Painting with Rainbows: Patterning Light in Space, Time, and Wavelength for Multiphoton Optogenetic Sensing and Control. Acc Chem Res 49, 2518-2526. [0200] Costes, S.V., Daelemans, D., Cho, E.H., Dobbin, Z., Pavlakis, G., and Lockett, S. (2004). Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys. J. 86, 3993-4003.

[0201] David, S., and Aguayo, A.J. (1981). Axonal elongation into peripheral nervous system "bridges" after central nervous system injury in adult rats. Science 214, 931-933.

[0202] de Beco, S., Vaidziulyte, K., Manzi, J., Dalier, F., di Federico, F., Comilleau, G., Dahan, M., and Coppey, M. (2018). Optogenetic dissection of Racl and Cdc42 gradient shaping. Nat Commun 9, 4816.

[0203] De Marco Garcia, N.V., Karayannis, T., and Fishell, G. (2011). Neuronal activity is required for the development of specific cortical interneuron subtypes. Nature 472, 351-355.

[0204] Dehorter, N., Ciceri, G., Bartolini, G., Lim, L., del Pino, L, and Marin, O. (2015). Tuning of fast-spiking intemeuron properties by an activity-dependent transcriptional switch. Science 349, 1216-1220.

[0205] Dickson, B.J. (2002). Molecular mechanisms of axon guidance. Science 298, 1959-1964.

[0206] Eisen, J.S., Myers, P.Z., and Westerfield, M. (1986). Pathway selection by growth cones of identified motoneurones in live zebra fish embryos. Nature 320, 269-271.

[0207] Endo, M., Hattori, M., Toriyabe, H., Ohno, H., Kamiguchi, H., lino, Y., and Ozawa, T. (2016). Optogenetic activation of axon guidance receptors controls direction of neurite outgrowth. Sci Rep 6, 23976.

[0208] Granato, M., van Eeden, F.J., Schach, U., Trowe, T., Brand, M., Fumtani- Seiki, M., Haffter, P., Hammerschmidt, M., Heisenberg, C.P., Jiang, Y.J., et al. (1996). Genes controlling and mediating locomotion behavior of the zebrafish embryo and larva. Development 123, 399-413.

[0209] Haase, G., Dessaud, E., Garces, A., de Bovis, B., Birling, M.-C., Filippi, P., Schmalbmch, H., Arber, S., and deLapeyriere, O. (2002). GDNF Acts through PEA3 to Regulate Cell Body Positioning and Muscle Innervation of Specific Motor Neuron Pools. Neuron 35, 893-905.

[0210] Hakim, R., Shamardani, K., and Adesnik, H. (2018). A neural circuit for gamma-band coherence across the retinotopic map in mouse visual cortex. Elife 7.

[0211] Hall, A., and Lalli, G. (2010). Rho and Ras GTPases in axon growth, guidance, and branching. Cold Spring Harb Perspect Biol 2, a001818. [0212] Hayashi-Takagi, A., Yagishita, S., Nakamura, M., Shirai, F., Wu, Y.I., Loshbaugh, A.L., Kuhlman, B., Hahn, K.M., and Kasai, H. (2015). Labelling and optical erasure of synaptic memory traces in the motor cortex. Nature 525, 333-338.

[0213] Hayworth, K.J., Morgan, J.L., Schalek, R., Berger, D.R., Hildebrand, D.G., and Lichtman, J.W. (2014). Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits. Front Neural Circuits 8, 68.

[0214] Hilario, J.D., Rodino-Klapac, L.R., Wang, C., and Beattie, C.E. (2009). Semaphorin 5A is a bifunctional axon guidance cue for axial motoneurons in vivo. Dev Biol 326, 190-200.

[0215] Hildebrand, D.G.C., Cicconet, M., Torres, R.M., Choi, W., Quan, T.M.,

Moon, J., Wetzel, A.W., Scott Champion, A., Graham, B.J., Randlett, O., et al. (2017). Whole- brain serial-section electron microscopy in larval zebrafish. Nature 545, 345-349.

[0216] Hua, Y., Laserstein, P., and Helmstaedter, M. (2015a). Large-volume en-bloc staining for electron microscopy-based connectomics. Nat Commun 6, 7923.

[0217] Hua, Z.L., Emiliani, F.E., and Nathans, J. (2015b). Racl plays an essential role in axon growth and guidance and in neuronal survival in the central and peripheral nervous systems. Neural Dev 10, 21.

[0218] Jacobs, C.L., Badiee, R.K., and Lin, M.Z. (2018). StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins. Nat. Methods 15, 523-526.

[0219] Joesch, M., Mankus, D., Yamagata, M., Shahbazi, A., Schalek, R., Suissa- Peleg, A., Meister, M., Lichtman, J.W., Scheirer, W.J., and Sanes, J.R. (2016). Reconstruction of genetically identified neurons imaged by serial- section electron microscopy. Elife 5.

[0220] Jung, H., Kang, H., and Nam, Y. (2017). Digital micromirror based near- infrared illumination system for plasmonic photothermal neuromodulation. Biomed Opt Express 8, 2866-2878.

[0221] Kawakami, K., and Shima, A. (1999). Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240, 239-44.

[0222] Kim, J.-M., Hwa, J., Garriga, P., Reeves, P.J., RajBhandary, U.L., and Khorana, H.G. (2005). Light-Driven Activation of b2- Adrenergic Receptor Signaling by a Chimeric Rhodopsin Containing the b2- Adrenergic Receptor Cytoplasmic Loops. Biochemistry 44, 2284-2292. [0223] Kou, S.Y., Chiu, A.Y., and Patterson, P.H. (1995). Differential regulation of motor neuron survival and choline acetyltransferase expression following axotomy. J Neurobiol 27, 561-572.

[0224] Kwan, K.M., Fujimoto, E., Grabher, C., Mangum, B.D., Hardy, M.E., Campbell, D.S., Parant, J.M., Yost, H.J., Kanki, J.P., and Chien, C.B. (2007). The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn 236, 3088-3099.

[0225] Lin, J.H., Saito, T., Anderson, D.J., Lance-Jones, C., Jessell, T.M., and Arber, S. (1998). Functionally Related Motor Neuron Pool and Muscle Sensory Afferent Subtypes Defined by Coordinate ETS Gene Expression. Cell 95, 393-407.

[0226] Lin, J.Y., Knutsen, P.M., Muller, A., Kleinfeld, D., and Tsien, R.Y. (2013). ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat Neurosci 16, 1499-1508.

[0227] Lohof, A.M., Quillan, M., Dan, Y., and Poo, M.M. (1992). Asymmetric modulation of cytosolic cAMP activity induces growth cone turning. J Neurosci 12, 1253- 1261.

[0228] Lungu, O.I., Hallett, R.A., Choi, E.J., Aiken, M.J., Hahn, K.M., and Kuhlman, B. (2012). Designing photoswitchable peptides using the AsLOV2 domain. Chem Biol 19, 507-517.

[0229] Melancon, E., Liu, D.W., Westerfield, M., and Eisen, J.S. (1997). Pathfinding by identified zebrafish motoneurons in the absence of muscle pioneers. J Neurosci 17, 7796- 7804.

[0230] Myers, P.Z., Eisen, J.S., and Westerfield, M. (1986). Development and axonal outgrowth of identified motoneurons in the zebrafish. J Neurosci 6, 2278-2289.

[0231] Nagel, G., Brauner, M., Liewald, J.F., Adeishvili, N., Bamberg, E., and Gottschalk, A. (2005). Light Activation of Channelrhodopsin-2 in Excitable Cells of Caenorhabditis elegans Triggers Rapid Behavioral Responses. Current Biology 15, 2279- 2284.

[0232] Ng, J., Nardine, T., Harms, M., Tzu, J., Goldstein, A., Sun, Y., Dietzl, G., Dickson, B.J., and Luo, L. (2002). Rac GTPases control axon growth, guidance and branching. Nature 416, 442-447.

[0233] Pallas, S.L., Littman, T., and Moore, D.R. (1999). Cross-modal reorganization of callosal connectivity without altering thalamocortical projections. Proc Natl Acad Sci USA 96, 8751-8756. [0234] Pallas, S.L., Roe, A.W., and Sur, M. (1990). Visual projections induced into the auditory pathway of ferrets. I. Novel inputs to primary auditory cortex (AI) from the LP/pulvinar complex and the topography of the MGN-AI projection. J Comp Neurol 298, 50- 68.

[0235] Pallas, S.L., and Sur, M. (1993). Visual projections induced into the auditory pathway of ferrets: II. Corticocortical connections of primary auditory cortex. J Comp Neurol 337, 317-333.

[0236] Panzer, J.A., Gibbs, S.M., Dosch, R., Wagner, D., Mullins, M.C., Granato, M., and Balice-Gordon, R.J. (2005). Neuromuscular synaptogenesis in wild-type and mutant zebrafish. Dev Biol 285, 340-357.

[0237] Pasterkamp, R.J., Giger, R.J., Ruitenberg, M.J., Holtmaat, A.J.G.D., De Wit,

J., De Winter, F., and Verhaagen, J. (1999). Expression of the Gene Encoding the Chemorepellent Semaphorin III Is Induced in the Fibroblast Component of Neural Scar Tissue Formed Following Injuries of Adult But Not Neonatal CNS. Molecular and Cellular Neuroscience 13, 143-166.

[0238] Piantino, J., Burdick, J.A., Goldberg, D., Langer, R., and Benowitz, L.I. (2006). An injectable, biodegradable hydrogel for trophic factor delivery enhances axonal rewiring and improves performance after spinal cord injury. Exp Neurol 201, 359-367.

[0239] Quadrato, G., and Arlotta, P. (2017). Present and future of modeling human brain development in 3D organoids. Curr Opin Cell Biol 49, 47-52.

[0240] Richardson, P.M., McGuinness, U.M., and Aguayo, A.J. (1980). Axons from CNS neurons regenerate into PNS grafts. Nature 284, 264-265.

[0241] Roos, M., Schachner, M., and Bernhardt, R.R. (1999). Zebrafish semaphorin Zlb inhibits growing motor axons in vivo. Mech Dev 87, 103-117.

[0242] Rosowski, E.E., Deng, Q., Keller, N.P., and Huttenlocher, A. (2016). Rac2 Functions in Both Neutrophils and Macrophages To Mediate Motility and Host Defense in Larval Zebrafish. J. Immunol. 197, 4780-4790.

[0243] Russ, J.B., and Kaltschmidt, J.A. (2014). From induction to conduction: how intrinsic transcriptional priming of extrinsic neuronal connectivity shapes neuronal identity. Open Biol 4, 140144.

[0244] Ryu, M.-H., Moskvin, O.V., Siltberg-Liberles, J., and Gomelsky, M. (2010). Natural and engineered photoactivated nucleotidyl cyclases for optogenetic applications. The Journal of biological chemistry 285, 41501-41508. [0245] Sakai, S., Ueno, K., Ishizuka, T., and Yawo, H. (2013). Parallel and patterned optogenetic manipulation of neurons in the brain slice using a DMD-based projector. Neurosci Res 75, 59-64.

[0246] Schneider, V.A., and Granato, M. (2006). The myotomal diwanka (lh3) glycosyltransferase and type XVIII collagen are critical for motor growth cone migration. Neuron 50, 683-695.

[0247] Schnell, L., Schneider, R., Kolbeck, R., Barde, Y.A., and Schwab, M.E.

(1994). Neurotrophin-3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesion. Nature 367, 170-173.

[0248] Siebert, J.R., Conta Steencken, A., and Osterhout, D.J. (2014). Chondroitin sulfate proteoglycans in the nervous system: inhibitors to repair. Biomed Res Int 2014,

845323.

[0249] Sur, M., Angelucci, A., and Sharma, J. (1999). Rewiring cortex: the role of patterned activity in development and plasticity of neocortical circuits. J Neurobiol 41, 33-43.

[0250] Tamplin, O.J., Cox, B.J., and Rossant, J. (2011). Integrated microarray and ChIP analysis identifies multiple Foxa2 dependent target genes in the notochord. Dev. Biol. 360, 415-25.

[0251] Tapia, J.C., Kasthuri, N., Hayworth, K.J., Schalek, R., Lichtman, J.W., Smith, S.J., and Buchanan, J. (2012). High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy. Nat Protoc 7, 193-206.

[0252] Tessier-Lavigne, M. (2002). Wiring the brain: the logic and molecular mechanisms of axon guidance and regeneration. Harvey Lect 98, 103-143.

[0253] Tessier-Lavigne, M., and Goodman, C.S. (1996). The molecular biology of axon guidance. Science 274, 1123-1133.

[0254] Velasco, S., Kedaigle, A.J., Simmons, S.K., Nash, A., Rocha, M., Quadrato, G., Paulsen, B., Nguyen, L., Adiconis, X., Regev, A., et al. (2019). Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature 570, 523-527.

[0255] Walter, J., Kem-Veits, B., Huf, J., Stolze, B., and Bonhoeffer, F. (1987). Recognition of position- specific properties of tectal cell membranes by retinal axons in vitro. Development 101, 685-696.

[0256] Wheeler, B.C., and Brewer, G.J. (2010). Designing Neural Networks in Culture: Experiments are described for controlled growth, of nerve cells taken from rats, in predesigned geometrical patterns on laboratory culture dishes. Proc IEEE Inst Electr Electron Eng 98, 398-406. [0257] White, R.M., Sessa, A., Burke, C., Bowman, T., LeBlanc, J., Ceol, C., Bourque, C., Dovey, M., Goessling, W., Burns, C.E., et al. (2008). Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2, 183-189.

[0258] Wu, Y.I., Frey, D., Lungu, O.I., Jaehrig, A., Schlichting, L, Kuhlman, B., and Hahn, K.M. (2009). A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104-108.

[0259] Yazawa, M., Sadaghiani, A.M., Hsueh, B., and Dolmetsch, R.E. (2009). Induction of protein-protein interactions in live cells using light. Nature Biotechnology 27, 941.

[0260] Yiu, G., and He, Z. (2006). Glial inhibition of CNS axon regeneration. Nat Rev Neurosci 7, 617-627.

[0261] Yoo, S.K., Deng, Q., Cavnar, P.J., Wu, Y.I., Hahn, K.M., and Huttenlocher,

A. (2010). Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev Cell 18, 226-236.

[0262] Zelenchuk, T.A., and Bruses, J.L. (2011). In vivo labeling of zebrafish motor neurons using an mnxl enhancer and Gal4/UAS. Genesis 49, 546-554.

[0263] Zeller, J., and Granato, M. (1999). The zebrafish diwanka gene controls an early step of motor growth cone migration. Development 126, 3461-3472.

[0264] Zhao, S., Cunha, C., Zhang, F., Liu, Q., Gloss, B., Deisseroth, K., Augustine, G.J., and Feng, G. (2008). Improved expression of halorhodopsin for light-induced silencing of neuronal activity. Brain cell biology 36, 141-154.

Claims

CLAIMS What is claimed is:

1. A method for controlling or directing neurite growth of a neuronal cell, comprising providing a neuronal cell comprising photoactivatable Racl (PA-Racl), and contacting a portion of the neuronal cell with radiation that activates or increases activity of the photoactivatable Racl, thereby controlling neurite growth of the neuronal cell.

2. The method of claim 1, wherein the neuronal cell comprises a neurite.

3. The method of claim 2, wherein the neurite or a portion thereof is contacted with the radiation.

4. The method of claims 2-3, wherein the neurite comprises a growth cone.

5. The method of claim 4, wherein the growth cone or a portion thereof is contacted with the radiation.

6. The method of claims 2-5, wherein the neurite is an axon.

7. The method of claims 2-5, wherein the neurite is a dendrite.

8. The method of claims 1-7, wherein the neuronal cell expresses PA-Racl.

9. The method of claim 8, wherein the expression of PA-Racl is under the control of an inducible or constitutive promoter.

10. The method of claims 8-9, wherein the genome of the neuronal cell codes for PA- Racl.

11. The method of claims 8-9, wherein the neuronal cell comprises an expression vector coding for PA-Racl.

12. The method of claim 8, wherein the neuronal cell comprises an mRNA sequence coding for PA-Racl.

13. The method of claims 1-7, wherein the neuronal cell does not express PA-Racl.

14. The method of claims 1-13, wherein the PA-Racl comprises a LOV (light oxygen voltage) domain from photo tropin.

15. The method of claims 1-14, wherein the PA-Racl comprises a Racl, a Racl mutant, or a functional fragment thereof.

16. The method of claims 1-15, wherein the PA-Racl has reduced or no activity in the absence of the radiation.

17. The method of claims 1-16, wherein contact of the portion of the neuronal cell with radiation increases the activity of the PA-Racl by 2-fold or more.

18. The methods of claims 1-17, wherein the radiation has a wavelength of about 450 nm to 480 nm.

19. The method of claim 18, wherein the radiation has a wavelength of 458 nm or 473 nm.

20. The method of claims 1-19, wherein the radiation is produced from a light source selected from a laser and a light emitting diode (LED).

21. The method of claims 1-20, wherein the radiation is contacted with the neuronal cell for at least 5 minutes.

22. The method of claims 1-21, wherein the neurite increases in length by at least 50 pm.

23. The method of claims 1-22, wherein the contacted neurite crosses a repulsive boundary.

24. The method of claims 1-23, wherein the contacted neurite forms a functional synapse.

25. The method of claims 1-24, wherein the neuronal cell is a sensory neuron, motor neuron, interneuron, or cortical neuron.

26. The method of claims 1-25, wherein the neuronal cell is derived from an induced pluripotent stem cell or progenitor cell.

27. The method of claims 1-26, wherein the neuronal cell is a human neuronal cell.

28. The method of claims 1-27, wherein the neuronal cell is contacted with the radiation in vivo, in vitro, or ex vivo.

29. The method of claims 1-28, wherein a plurality of neuronal cells are contacted with the radiation.

30. The method of claims 1-29, wherein the PA-Racl comprises an amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 90% identity to SEQ ID NO: 24.

31. The method of claim 30, wherein the plurality of contacted neuronal cells form one or more neural circuits.

32. The method of claim 31, wherein the one or more neural circuits provide a model of a neural structure or aberrant neural condition.

33. The method of claim 31 or 32, wherein the one or more neural circuits form a biological computer.

34. The method of claims 1-30, wherein the method treats or prevents a nerve injury, neurological disease or neurological condition in a subject in need thereof.

35. The method of claim 34, wherein the nerve injury comprises one or more severed neurons.

36. The method of claim 34, wherein the neurological disease or neurological condition is or is associated with a neuronal guidance defect.

37. A biological computer comprising contacted neuronal cells of the method of claims 1- 31.

38. An in vitro neural circuit comprising contacted neuronal cells of the method of claims 1-31.

39. A non-human animal comprising contacted neuronal cells of the method of claims 1- 31.

40. A method of establishing or manipulating a neural connection in a subject in need thereof, comprising providing photoactivatable Racl (PA-Racl) in a neuronal cell in the subject and establishing or manipulating the neural connection by contacting a portion of the neuronal cell with radiation that activates or increases activation of the photoactivatable Racl.

41. The method of claim 40, wherein the neural connection is located in the central nervous system of the subject.

42. The method of claim 41, wherein the neural connection is located in the spinal cord of the subject.

43. The method of claim 40, wherein the neural connection is located in the peripheral nervous system of the subject.

44. The method of claims 40-43, wherein the neural connection comprises grafted tissue.

45. The method of claims 40-44, wherein a plurality of neuronal cells having PA-Racl are contacted with the radiation.

46. The method of claims 40-45, wherein the method causes the formation of one or more synapses.

47. The method of claims 40-46, wherein the neural cell comprises an exogenously added mRNA sequence or expression vector coding for PA-Racl.

48. The method of claims 40-46, wherein the neural cell is a transgenic neural stem or progenitor cell expressing PA-Racl.

49. The method of claims 40-46, wherein the PA-Racl was intracellularly delivered to an endogenous neural cell.

50. The method of claims 40-49, wherein the neural cell further expresses ReaChR.

51. The method of claims 40-50, wherein the method repairs a severed nerve.

52. The method of claims 40-50, wherein the method treats or prevents a neurological disease or condition.

53. The method of claims 40-50, wherein the radiation is produced by one or more light sources selected from a laser and a light emitting diode (LED).

54. The method of claim 53, wherein the one or more light sources are implanted.

55. The method of claim 53, wherein the one or more light sources are external to the subject.

56. The method of claims 40-55, wherein the subject is human.

57. The method of claims 40-56, wherein the subject is an embryo.

58. The method of claims 40-57, wherein the PA-Racl comprises an amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 90% identity to SEQ ID NO: 24.

59. An apparatus for establishing or manipulating a neural connection in a subject, comprising: a. one or more light sources, b. a power supply operably linked to the one or more light sources, and c. a processor configured to control the one or more light sources.

60. The apparatus of claim 59, wherein the one or more light sources are configured for implantation in a subject.

61. The apparatus of claim 60, wherein the one or more light sources comprise a plurality of light sources configured to be implanted at a nerve injury site having a first end and a second end, and direct regrowth and/or repair of the nerve by sequentially turning on and off each light source from the first end to the second end.

62. The apparatus of claim 60, wherein the plurality of light sources are a linear string of light sources that are configured to be secured across a nerve injury site, and wherein the light sources are configured to enable growth of one or more neurites along the linear string.

63. The apparatus of claims 59-62, wherein the light sources comprise biologically inert materials.

64. The apparatus of claims 59-63, further comprising a digital micromirror device.

65. A kit comprising the apparatus of claims 59-64, and an agent capable of providing PA-Racl in a neuronal cell in a subject.

66. The kit of claim 65, wherein the agent comprises a nucleic acid coding for PA-Racl.

67. The kit of claim 65, wherein the agent is a transgenic stem cell or progenitor cell capable of expressing PA-Racl.

68. The kit of claims 65-67, wherein the PA-Racl comprises an amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 90% identity to SEQ ID NO: 24.

69. An isolated transgenic stem cell or progenitor cell capable of expressing PA-Racl.

70. The isolated transgenic stem cell or progenitor cell of claim 69, wherein the PA-Racl comprises an amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 90% identity to SEQ ID NO: 24.

71. A method of screening for therapeutic agents comprising, a. providing an in vitro neural circuit modeling a neurological disease or condition and comprising contacted neuronal cells of the method of claims 1-

31, b. contacting the neural circuit with a test agent, and c. assessing whether the test agent improves one or more aspects of the neurological disease or condition present in the neural circuit.

72. The method of claim 71, wherein the neural circuit comprises neuronal cells derived from a subject with the neurological disease or condition.

73. The method of claim 72, wherein the neuronal cells are derived from induced pluripotent cells derived from one or more cells from the subject.

74. The method of claims 71-73, wherein one or more aspects of the neurological disease or condition assessed is neuronal cell viability, neuronal cell proliferation, and/or synaptic signaling.

75. A method of treating a neuronal guidance defect in a subject in need thereof, comprising administering to the subject an agent that modulates the expression or activity of Racl in a neuronal cell.

76. The method of claim 75, wherein administration of the agent stabilizes a level of Racl in the neuronal cell of the subject.

77. The method of claim 75, wherein administration of the agent increases a level of Racl in the neuronal cell of the subject.

78. The method of any one of claims 75-77, wherein the agent is a photoactivatable Racl or a nucleic acid coding for the same.

79. The method of any one of claims 75-78, wherein the neuronal guidance defect is associated with a plod3 mutation.

80. The method of any one of claims 75-79, wherein the neuronal guidance defect is associated with or caused by a disease or condition selected from the group consisting of corpus callosum agenesis, LI syndrome, Joubert syndrome, horizontal gaze palsy with progressive scoliosis, Kallmann syndrome, albinism, congenital fibrosis of the extraocular muscles type 1, Duane retraction syndrome, and pontine tegmental cap dysplasia.