Lights, motor neurons, and muscle action!

Mice motor neurons

Editor's Introduction

Optical control of muscle function by transplantation of stem cell-derived motor neurons in mice

annotated by
Polly Huang

After nerve injury or neurodegenerative disease, patients suffer paralysis and loss of muscle mass due to limited neuron regeneration. Major obstacles to restoring muscle function include the ability to effectively replace damaged motor axons, make connections to muscles, and to generate normal muscle activity. In this report, scientists engineer light-sensitive motor neurons derived from embryonic stem cells and transplant these motor neurons into mice with nerve injury. By switching on a light, scientists can control these light-sensing motor neurons and restore muscle activity.

Paper Details

Original title
Optical control of muscle function by transplantation of stem cell-derived motor neurons in mice
Authors
J. Barney Bryson
Original publication date
Reference
Vol. 344 no. 6179 pp. 94-97
Issue name
Science
DOI
10.1126/science.1248523
Topics
Neuroscience and behavior
Technology and engineering
Cell and molecular biology

Abstract

Damage to the central nervous system caused by traumatic injury or neurological disorders can lead to permanent loss of voluntary motor function and muscle paralysis. Here, we describe an approach that circumvents central motor circuit pathology to restore specific skeletal muscle function. We generated murine embryonic stem cell–derived motor neurons that express the light-sensitive ion channel channelrhodopsin-2, which we then engrafted into partially denervated branches of the sciatic nerve of adult mice. These engrafted motor neurons not only reinnervated lower hind-limb muscles but also enabled their function to be restored in a controllable manner using optogenetic stimulation. This synthesis of regenerative medicine and optogenetics may be a successful strategy to restore muscle function after traumatic injury or disease.
 

Report

Electrical stimulation of motor axons within peripheral nerves has been known to induce muscle contraction since Luigi Galvani’s early experiments. In more recent times, phrenic nerve pacing has been used clinically to control the function of the diaphragm, the major muscle involved in respiration, in some patients with high-level spinal cord injury (1) or amyotrophic lateral sclerosis (ALS) (2). However, peripheral nerves are composed of efferent motor axons as well as afferent sensory axons (which are unaffected in ALS). Functional electrical stimulation, which stimulates nerves indiscriminately, can thus cause considerable discomfort (3). Furthermore, functional electrical stimulation is ineffective if axon integrity is compromised because of injury or degenerative disease. Other strategies to replace lost motor neurons within the central nervous system include the use of embryonic stem cells (ESCs), but ESC-derived neurons do not always integrate into adult brain and spinal cord circuitry (4) and have difficulty overcoming molecular inhibitors of neuronal outgrowth (5) and extending axons across the barrier between the central and peripheral nervous system to reach the appropriate muscles (6).

It has previously been shown that motor neurons derived from ESCs can be engrafted into a peripheral nerve environment and successfully reinnervate denervated muscle (7). However, these engrafted cells are not connected to the descending inputs within the central nervous system that normally control motor function; therefore, their neural activity must be regulated by an artificial control system. Such engrafted ESC-derived motor neurons can be electrically stimulated (7), but this approach stimulates endogenous as well as engrafted neurons. In transgenic mice that express the light-sensitive ion channel channelrhodopsin-2 (ChR2) (8, 9) in endogenous motor neurons, it has been shown that the axons of these ChR2 motor neurons can be recruited by optical stimulation in a physiological and graded fashion, resulting in optogenetic control of muscle function (10). It has also been shown that viral expression of ChR2 in motor neurons of adult rats can enable optical stimulation of muscle function (11). In this study, we tested whether expression of ChR2 in ESC-derived motor neurons engrafted into a denervated peripheral nerve (i) confers optically regulated control of muscle function, without interfering with endogenous motor signals or afferent sensory axons, and (ii) enables physiological recruitment of motor units.

We generated genetically modified ESC-derived motor neurons that express both ChR2, to enable optical stimulation, as well as glial-derived neurotrophic factor (Gdnf), a neurotrophic factor that promotes long-term motor neuron survival (see supplementary materials and methods). To develop such ESCs suitable for in vivo engraftment of ESC motor neurons, we stably transfected an established mouse ESC clonal cell line that already carried the motor neuron–specific reporter Hb9::CD14-IRES-GFP (GFP, green fluorescent protein) (12) with a photoreceptor transgene that is expressed regardless of cell type, CAG::ChR2-YFP (YFP, yellow fluorescent protein), and with the neurotrophin-expressing CAG::Gdnf transgene. Embryoid bodies derived from CAG:ChR2-YFP/Gdnf ESCs, which express ChR2-YFP in all cells including motor neurons (Fig. 1A), were differentiated in vitro using an established protocol (13, 14). CAG::ChR2-YFP/Gdnf motor neurons also produce Gdnf (Fig. 1B), which improves their long-term survival in vitro (Fig. 1C). When cultured on an ESC-derived astrocyte feeder layer (fig. S1), ChR2 motor neurons mature electrically over a period of 35 days, until they fire trains of action potentials in response to optical stimulation and closely resemble adult motor neuron activity patterns induced by electrical stimulation (15) (Fig. 1D and fig. S2).

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Fig. 1. Expression of Gdnf in ChR2 motor neurons enhances survival and enables them to mature electrically in vitro. (A) Embryoid bodies derived from CAG::ChR2-YFP/Gdnftransgenic ESCs and parental controls stained for the pan-motor neuron marker Isl1/2. GFP and YFP signals were detected by direct fluorescence. (B) Confocal images of MACS-sorted ESC motor neurons derived from CAG::ChR2-YFP (MACS, magnetic-activated cell sorting) and CAG::ChR2-YFP/Gdnf ESCs immunostained for Gdnf and GFP/YFP. (C) Survival analysis of sorted CAG::ChR2-YFP and CAG::ChR2-YFP/Gdnf ESC motor neurons (MNs) (200 cells per well) plated on ESC astrocytes at indicated time points. CAG::ChR2-YFP motor neurons were cultured with (10 ng/ml) or without recombinant Gdnf (two replicates, analysis of variance with Bonferroni correction, *P < 0.25). Error bars indicate SEM. One representative of three separate experiments is shown. Wt, wild type. (D) Optogenetic stimulation (blue bars) of CAG::ChR2-YFP/Gdnf ESC motor neurons cultured on ESC astrocytes. Scale bars in (A) and (B), 50 μm.

Rationale 

Generate ESC-derived ChR2 motor neurons first by transfection of DNA containing ChR2, followed by selection of ChR2-expressing ESC clones for transfection of Gdnf transgene. Then, ESC clones that stably express ChR2 and Gdnf transgene were selected, cultured in astrocyte feeder layer, and allowed to differentiate into motor neurons suitable for engraftment and in vivo experiments

  1. Mouse embryonic stem cells are isolated from mouse embryo carrying the transgene Hb9::CD14-IRES-GFP, where green fluorescent protein (GFP) expression is controlled by Hb9 specifically in motor neurons. These ESCs were subsequently directed to differentiate into motor neurons. Therefore, motor neurons that express GFP indicate that they originate from ESCs and not any other origin.

  2. DNA containing CAG::ChR2-YFP is introduced into ESCs by transfection, followed by expression of the light-sensitive ChR2 and yellow fluorescent protein (YFP). Therefore, ESC-derived motor neurons that express the light-sensitive protein ChR2 can be selected by their GFP and YFP expression.

  3. DNA carrying CAG::Gdnf is transfected into cells expressing CAG::ChR2-YFP. Stable expression of Gdnf improves motor-neuron survival.

As a result, motor neurons suitable for engraftment express the following:

GFP: specific for ESC-derived motor neurons

ChR2-YFP: allows for light stimulation

Gdnf: improves neuron survival

Objectives

Show that ESC-derived motor neurons stably express light-sensitive ChR2 on the membrane and that the expression of Gdnf improves survival of motor neurons, and determine whether and when ESC-derived ChR2 motor neurons become functionally mature. These observations would establish the criteria for generating motor neurons suitable for engraftment for other experiments from this point forward.

1A Experiment 

Analysis of ESC-derived embryoid bodies containing ChR2 motor neurons using confocal scanning microscope. Embryoid bodies are immunostained for the motor neuron marker Isl 1/2. Expression of the following proteins is analyzed:

1) Isl1/2 (red) is a protein important for motor neuron growth and maintenance, and is expressed in all motor neurons (i.e. pan-motor neuron marker).

2) GFP (green) is expressed in ESCs used to generate motor neurons and indicates ESC origin.

3) YFP (blue) expression from ChR2-YFP transgenes indicates ESC-derived motor neurons successfully expressing ChR2 transgenes.

For more about immunostaining, some basic principles can be found here:

https://www.youtube.com/watch?v=HdBgTAAi3rU.

Images were obtained through a camera (A), light microscope (B), and scanning electron microscope (C) and (D). The images obtained through microscopy were used to make the idealized drawings in (E) and (F).
1A Results

Representative images show parental embryoid bodies (top) before transfection of ChR2 and Gdnf transgenes; embryoid bodies are positive for GFP (green) and Isl 1/2 (red), which indicate ESC origin and motor neuron cell identity, respectively. Transfected embryoid bodies (bottom) show expression of GFP (green), YFP (cyan) and Isl 1/2 (red), which indicate ESC-derived ChR2 motor neurons in embryoid bodies.

1B Experiment

Differentiated motor neurons are isolated using magnetic-activated cell sorting (MACS) and grown in culture. ESC-derived motor neurons are immunostained for Gdnf and stained with a dye for DNA, followed by analysis using fluorescent confocal microscopy:

1)      GFP and YFP (green) indicates ChR2-YFP transgene expression and ESC origin of the cells in the image.

2)      Gdnf (red) expression following transfection of CAG::Gdnf.

3)      DNA (blue) indicates nucleus of cells on the image and indicates that protein and transgene expression is localized with cells.

For more about MACS: https://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/macs-technology/microbeads_dp.aspx

For more about confocal microscopy: http://www.microscopyu.com/articles/confocal/confocalintrobasics.html.

1B Results

Left panel – Appearance of GFP/YFP (green) indicates ChR2 expression in motor neurons of ESC origin. Right panel – ESC-derived ChR2 motor neurons (green) also localize with Gdnf (red), indicating Gdnf-protein expression. DNA (blue) is present in both left and right images, which indicates GFP, YFP, and Gdnf are localized with cells and do not appear to be the result of non-specific immunostaining or fluorescence.

1C Results

 ESC-derived motor neurons transfected with Gdnf transgene (triangle, red line) are grown in culture in parallel with wild-type motor neurons with (open circles, dark blue line) or without (closed circles, light blue line) Gdnf supplement. Surviving cells per well are counted (y-axis) over 35 days in culture (x-axis) and plotted here. Surviving cells per well in CAG::Gdnf motor neurons culture remain significantly greater than wild-type motor neurons with or without Gdnf supplement over 35 days. This supports the hypothesis that stable Gdnf expression in motor neurons improves survival.

1D Experiment

In order to test functional maturation of ESC-derived motor neurons in culture, motor neurons expressing light-sensitive ChR2-YFP and Gdnf are optically stimulated at days 3, 7, and 35. Neuron action potentials are recorded and presented as spikes on the graph. Scale bar indicates duration of action potentials in milliseconds (ms) and voltage of action potentials in millivolts (mV).

For more about electrophysiology (recording action potentials): http://www.leica-microsystems.com/science-lab/the-patch-clamp-technique/.

1D Results

At day 3, ChR2 motor neurons did not generate action potentials following optical stimulation. At day 17, ChR2 motor neurons show action potentials in response to optical stimulation. At day 35, mature ChR2 motor neurons generate rapid action potentials following optical stimulation. Supplementary Figure 1A (below) shows representative action potential recordings of ESC-derived ChR2 motor neurons in response to electrical stimulation (top panel) and optical stimulation (bottom panel), up to 35 days in culture. The rapid action potentials induced by optical stimulation at 35 days in culture are similar to those induced by electrical stimulation. Together, the data indicate functional maturation of ChR2 motor neurons at 35 days.

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Conclusions

ESC-derived ChR2 motor neurons are generated by the stable transfection of embryonic stem cells with ChR2-YFP and Gdnf, followed by stable expression of ChR2 and Gdnf. Motor-neuron survival is improved with Gdnf protein expression. ESC-derived ChR2 motor neurons generate action potentials in response to optical stimulation at 35 days in culture, which indicates functional maturity. Together, these results establish the criteria for generating ESC-derived ChR2 motor neurons suitable to engraftment for the remainder of the study. The authors also concluded that ChR2 motor neurons mature after 35 days, when functional testing should be performed. 

After developing these ChR2 motor neurons, we next established an in vivo model to assess the feasibility of restoring muscle function with optical control of the engrafted cells, using the sciatic nerve. Muscle denervation was induced by sciatic nerve ligation in adult mice. This procedure results in a complete initial denervation, followed by limited regeneration of endogenous axons through the ligation site, thereby creating a partially denervated environment resembling the partial muscle denervation of early-stage ALS (fig. S3). Three days postligation, embryoid bodies containing ChR2 motor neurons were engrafted distal to the ligation into the tibial and common peroneal branches of the sciatic nerve. Histological analysis revealed that the engrafted ChR2 motor neurons not only survive for at least 35 days in the peripheral nerve environment (Fig. 2A), but also mature morphologically to resemble adult spinal motor neurons and express the mature motor neuron marker choline acetyltransferase (Fig. 2B). Immunodetection of ChR2-YFP, using an antibody to GFP, demonstrates that ChR2 is localized to the membrane of motor neurons, whereas direct detection of GFP versus YFP fluorescent signals reveals that Hb9-driven GFP expression is virtually absent in ChR2 motor neurons that have matured in vivo for 35 days (figs. S4 and S5). Additionally, ChR2 motor neurons extended large numbers of axons (Fig. 2C) distally toward both anterior [tibialis anterior (TA) and extensor digitorum longus (EDL)] and posterior [triceps surae (TS)] lower hind-limb muscles when grafted into the specific branches of the sciatic nerve that innervate these muscles. Engrafted ChR2 motor neuron axons, which grow alongside regenerating endogenous (YFP-negative) motor axons, are mostly myelinated (Fig. 2D). Histological analysis also revealed robust reinnervation of muscle fibers by ChR2 motor neurons, although the neuromuscular junctions exhibited hallmarks of inactivity, including poly-innervation as well as collateral and terminal axonal sprouting (16) (Fig. 2E), most likely because these motor neurons were inactive in vivo until stimulated by the external optical signal. Nevertheless, quantification of all end plates within a whole TS muscle revealed that 14.7% were innervated by YFP-positive ChR2 motor neurons axons after 35 days (Fig. 2F). Moreover, we observed YFP-positive neuromuscular junctions in both fast-twitch and slow-twitch regions of the TS, indicating that ChR2 motor neurons can innervate different muscle types. Therefore, this time point (35 days) was used in subsequent experiments to establish whether the transplanted ChR2 motor neurons were indeed functional and responsive to optical stimuli in vivo.

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Fig. 2. Robust axonal growth and reinnervation of distal muscles after engraftment of ChR2 motor neurons. (A) Image montage of a whole nerve and muscle section showing ChR2 motor neuron cell bodies at the graft site and axon projection (dashed lines indicate approximate trajectory). Scale bar, 500 μm. (B) Confocal image of engrafted ChR2 motor neurons immunolabeled for choline acetyltransferase (ChAT; left image) and GFP and/or YFP (merged image at right). Scale bar, 50 μm. (C) Confocal image of longitudinal and transverse common peroneal nerve sections showing both ChR2 motor neurons and endogenous axons. Scale bar, 50 μm. (D) Confocal images of engrafted ChR2 motor neuron axons showing myelination. Scale bar, 50 μm. (E) Confocal z-stack of ChR2 motor neuron axon terminals innervating multiple neuromuscular junctions within the TS muscle. Arrows indicate preterminal collateral sprouting, arrowheads denote terminal sprouting, and the asterisk indicates an endogenous motor axon. Scale bar, 50 μm. (F) Two-dimensional projection image of a TS muscle showing proportion of neuromuscular junctions (NMJs) innervated by engrafted ChR2 motor neurons, relative to the total number of end plates present [labeled with α-bungarotoxin (αBTx)]. Quantification is shown below. Representative images shown here are compiled from n = 4 engrafted nerves from three separate experiments.

Objectives

Present data to show that engrafted ESC-derived ChR2 motor neurons can survive and grow in the peripheral environment. The authors test the hypothesis that engrafted motor neurons resemble normal motor neurons in appearance, extend axons to distal muscles, and form neuromuscular junctions with muscle fibers.

Experiments

The authors used sciatic nerve ligation to induce peripheral nerve injury, a mouse model of muscle paralysis resulting from denervation. A suture is tied around the sciatic nerve of anesthetized mice, and animals were allowed to recover from the surgery. Sciatic nerve ligation induced nerve damage, axon degeneration and muscle denervation, followed by regeneration of a small population of endogenous motor neurons. This partial denervation resembles certain aspects of early-stage ALS. Three days after the ligation surgery, ESC-derived ChR2 motor-neuron embryoid bodies were engrafted into the injured nerve below the ligation site (i.e. closer to the target muscle and away from the spinal cord). Supplementary Figure 3 shows a schematic illustration of sciatic nerve ligation (below): The ligation site and ChR2 motor-neuron graft sites are indicated. Engrafted ChR2 motor neurons are predicted to extend axons along the indicated nerves in the lower leg (green). 

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At 35 days after engraftment, immunohistochemistry and confocal microscopy were used to analyze motor neuron growth. The following motor -neuron markers are examined in Figure 2A:

1)      Acetylcholine transferase (ChAT, red): immunostained; motor neuron marker; an enzyme involved in synthesizing the neurotransmitter acetylcholine.

2)      Myelin (red): stained with a fluorescent dye that binds to myelin; indicates myelin sheath which is characteristic of neurons in the peripheral nervous system.

3)      α-Bungarotoxin (αBtx, red): fluorophor-conjugated αBtx binds to nicotinic acetylcholine receptors on the axon terminal, and direct fluorescence is used to visualize fluorophor-stained axon terminals.

4)      GFP/YFP (green): indicates engrafted ESC-derived ChR2 motor neurons, which were immunostained with an antibody that binds to both GFP and YFP.

For more information about sciatic nerve ligation as a model of nerve damage: http://www.jove.com/video/3393/chronic-constriction-sciatic-nerve-pain-hypersensitivity-testing

Learn the basic principles of confocal microscopy here: http://www.microscopyu.com/articles/confocal/confocalintrobasics.html

Learn the basic principles of immunohistochemistry: https://www.youtube.com/watch?v=HdBgTAAi3rU

2A Results

Image shows dissected nerve and muscle after engraftment of ChR2 motor neurons. The tissue is immunostained for ChAT (red), which identifies both endogenous and engrafted motor neurons. The tissue is stained with a dye for DNA (blue), which helps visualize the outline of the tissue. GFP/YFP (green) appears at the ChR2 motor neuron graft site and along the nerve, which indicates healthy engrafted ESC-derived ChR2motor neurons with axon extensions along the nerve; endogenous motor neurons do not express GFP/YFP. Motor-neuron axon projections (green and red overlay) extend from the graft site toward target muscle. Together, the data suggest robust axon growth of engrafted ESC-derived ChR2 motor neurons from the graft site into tibial and common peroneal nerves. 

2B Results

Dissected and sectioned nerve tissue shows presence of GFP/YFP (green) and ChAT (red), which indicates ESC-derived ChR2 motor neurons. 

2C Results

Longitudinal (left) and transverse (right) sections of nerve tissue show GFP/YFP (green) and ChAT (red), and both co-localized (yellow). These images further provide evidence of ESC-derived ChR2 motor-neuron survival and axon extensions along the sciatic nerve and common peroneal nerve in the leg.

2D Results

Longitudinal (left) and transverse (right) sections of the peroneal nerve show GFP/YFP (green) ChR2 motor neurons and myelin (red), which indicate that ChR2 motor axons are insulated by myelin sheath along the peroneal nerve. Myelin sheath is a characteristic of peripheral motor axons. These data implicate healthy ESC-derived ChR2 motor neurons resembling normal motor neurons in the peripheral nerve environment. 

2E Results 

The image shows dissected posterior triceps surae (TS) muscle tissue. GFP/YFP (green) indicates axon terminals of engrafted ESC-derived ChR2 motor neurons. αBtx (red) indicates axon terminals in the muscle. Co-localization of GFP/YFP and αBtx (yellow) indicates the formation of neuromuscular junctions consisting of ChR2 motor neurons and TS muscle fibers. The asterisk indicates an endogenous motor neuron (red) alongside GFP/YFP-positive ChR2 motor axons. Notably, neuromuscular junctions show collateral sprouting and terminal sprouting, indicated by arrows and arrowheads, respectively, which are hallmarks of inactivity.  

2F Results

A representative image (top) of dissected TS muscle tissue shows GFP/YFP (green) for ChR2 motor neurons and αBtx (red) for axon terminals. Neuromuscular junctions (yellow) formed by co-localization of ChR2 and αBtx are also observed. The number of terminals innervated by engrafted ChR2 motor neurons (GFP, green) is counted and plotted (bottom panel), relative to the total number of neuromuscular junctions (end-plates) innervated by any motor neuron (red). The whole TS muscle shows 14.7% of end-plates are innervated by engrafted ChR2 motor neurons. 

Conclusions

After never injury in otherwise healthy adult mice, engraftment of ESC-derived ChR2 motor neurons can survive in the peripheral nerve environment. At 35 days after engraftment, ChR2 motor neurons are shown to extend axons along the sciatic nerve and common peroneal nerve to target muscles in the lower leg, where neuromuscular junctions are observed. The data suggest that engrafted ESC-derived ChR2 motor neurons appear to reennervate target muscles in the lower leg after sciatic nerve injury. 

In anesthetized animals, we used isometric muscle tension physiology to examine the contractile responses elicited from TA, EDL, and TS (data summarized in table S1) muscles after optical stimulation of the exposed sciatic nerve using finely controlled pulses of 470-nm blue light generated by a light-emitting diode (LED) unit and delivered via a light-guide to the graft site (Fig. 3A and movie S1). Short-duration (14-ms) light pulses were able to induce submaximal twitch contractions in muscles innervated by transplanted ChR2 motor neurons (Fig. 3B), whereas high-frequency illumination (40 to 80 Hz) induced tetanic muscle contraction (Fig. 3C) that can be repeated in a highly reproducible manner (Fig. 3D). Quantification of these contractile responses demonstrated that the ratio of tetanic to twitch force was 3.09 ± 0.52 and 2.34 ± 0.33 for TA and EDL muscles, respectively (Fig. 3E), similar to normal values in uninjured animals.

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Fig. 3. Restoration of muscle function, in a controlled manner, using optical stimulation of engrafted ChR2 motor neurons in vivo. (A) Schematic showing optical stimulation and isometric muscle tension recordings. EB, embryoid body. Representative twitch (B), tetanic (C), and repetitive tetanic (D) contraction traces obtained from the TA muscle, induced by optical stimulation. Blue line, muscle force; red line, electrical trigger signals sent to the LED unit. (E) Quantification of twitch and tetanic contraction of TA and EDL muscles. Time to peak contractile force, from initiation of the electrical trigger to the LED unit (F) or from the initiation of muscle contraction (G), is shown alongside direct electrical nerve stimulation (n values represent optical/electrical stimulation, respectively, compiled from four separate experiments). (H) Representative fatigue traces from TA muscles (different animals) produced by optical (top) or electrical (bottom) stimulation for 180 s. (I) Representative TA muscle optical stimulation motor-unit number estimate trace. The asterisk indicates square-wave trigger voltage to the LED unit and oscilloscope trigger. (J) Motor-unit number quantification of TA and EDL muscles after optical versus electrical stimulation. (K) Analysis of average motor-unit force. The dashed line indicates the normal EDL value. All error bars indicate SEM.

Rationale 

Engrafted ESC-derived ChR2 motor neurons appear to form neuromuscular junctions with distal muscle fibers, which suggest muscles reinnervate  after engraftment at the site of sciatic nerve injuries. The authors test the hypothesis that engrafted ChR2 motor neurons reinnervate distal leg muscles and restore muscle function. Optical stimulation of light-sensitive ChR2 motor neurons is hypothesized to activate this specific population of motor neurons, and consequently stimulate distal leg muscle fibers to generate muscle contraction. Further muscle-contraction characteristics in response to optical stimulation should be comparable to normal muscle physiology. 

Panel A 

Diagram of experimental set-up to test muscle function after ChR2 motor neuron engraftment and reinnervation of distal leg muscles. A Digitimer provides finely controlled pulses with duration and frequency determined by the experimenter. The LED light source provides a flashing blue light of 470 nm wavelength through the light guide and directed at the ChR2 motor neurons in the graft site (green). A force transducer attached to the tendon of individual leg muscles detects the contractile force of the muscle when light stimulates ChR2 motor neurons. Contractile force detected by the force transducer in response to different frequencies of light stimulation is recorded. A movie of experimental set-up is part of supplemental materials: http://www.sciencemag.org/content/344/6179/94/suppl/DC1

3B, C and D Experiments 

Engrafted ChR2 motor neurons at the site of engraftment were stimulated with either a single or repeated light pulses (red) at various frequencies. Force of muscle contraction (blue) is measured in response to optical stimulation. The scale bars indicate amplitude of muscle contractile force in grams and duration of muscle contraction in milliseconds or seconds.

3B, C and D Results

Increased contractile force following a single light pulse indicates muscle contraction in response to light stimulation of ChR2 motor neurons (B). High frequency (40 to 80 Hz) light stimulation on ChR2 motor neurons generated tetanic (sustained, uninterrupted) muscle contraction (C). Muscle contraction recordings show tetanic-muscle contractions in response to repeated bouts of high-frequency optical stimulation, which suggests that ChR2 motor neurons can be repeatedly stimulated and innervate leg muscles (D). 

2E Experiment 

Contractile force in grams is measured and quantified from two muscles in the leg, the tibialis anterior (TA) and extensor digitorum longus (EDL),following either twitch (short-duration, light blue) or tetanic (high-frequency, dark blue) optical stimulation. The histogram shows the average twitch and tetanic muscle contractile force in grams (y-axis) from repeated experiments. 

3E Results

In both muscles examined, high-frequency tetanic optical stimulation generated significantly greater force of muscle contraction compared to the contractile force generated by single, short-duration twitch stimulus. The authors note that these muscle contractile characteristics were expected of normal muscles in response to electrical stimulation. 

3F, G and H Rationale 

After nerve injury, a small number of endogenous motor axons regenerate and limited innervation of muscle fibers by endogenous motor neurons is maintained. Optical stimulation specifically activates light-sensitive, engrafted ChR2 motor neurons, whereas electrical stimulation activates both endogenous and engrafted motor neurons. Both populations of motor neurons innervate muscle fibers in the lower leg to generate contraction. In the following experiments, the authors compare muscle contraction generated by electrical stimulation of endogenous motor neurons vs. optical stimulation of engrafted ChR2 motor neurons. 

3F and G Experiments

Muscle contraction was examined in two leg muscles, tibialis anterior (TA) and extensor digitorum longus (EDL), following either optical stimulation (blue) or electrical stimulation (black). Latency to the start muscle contraction response in milliseconds (Figure 3F) and time to peak, contractile force (Figure 3G) were quantified. These experiments determine the conduction velocity of engrafted ChR2 motor neurons, compared to conduction velocity of endogenous motor neurons. Average values from repeated experiments are plotted on histograms.

3F and G Results

The histograms show that in TA and EDL muscles, optical and electrical stimulation of the sciatic nerve generated comparable characteristics of muscle contraction. Latency to response (F) was not different after optical and electrical stimulation. Time to peak muscle contractile force (G) was also similar when sciatic nerve was optically and electrically stimulated. These results suggest that the conduction velocity of engrafted ChR2 motor neurons is comparable to that of endogenous motor neurons.

3H Experiment

Sciatic nerves in different animals were optically and electrically stimulated for 180 seconds. TA muscle contraction in response to sustained stimulation was recorded and plotted in grams to compare fatigue characteristics after optical stimulation and electrical stimulation.

3H Results

Optical stimulation (top) specifically activates ChR2 motor neurons and generates repetitive muscle contraction. The maximal force of muscle contraction (amplitude of blue spikes) remains relatively consistent over 180 seconds of stimulation, which suggests that ChR2 motor neurons are resistant to fatigue. In contrast, electrical stimulation (bottom) generated repetitive muscle contraction, where the maximal force of muscle contraction (amplitude of black spikes) decreased over 180 seconds of stimulation, which indicates muscle fatigue.

3I, J, and K Rationale and Experiments 

The authors examine motor-unit recruitment, an important characteristic of normal muscle function, in response to optical and electrical stimulation. A motor unit consists of a motor neuron and the muscle fibers it innervates. Large motor neurons with high activation thresholds are part of stronger motor units and include more muscle fibers. Smaller motor units with lower activation thresholds innervate fewer muscle fibers and generate lower contractile force. Motor-unit recruitment in normal physiological order consists of increasingly higher intensity of the stimulus, and activation of smaller, low-threshold motor units followed by the activation of increasingly greater, higher-threshold motor units. This recruitment of small to large motor units results in a gradual increase in total force of muscle contraction as additional motor units are recruited in the whole muscle.

Optical stimulation activates engrafted ChR2 motor neurons in a neuron population-specific manner. As intensity of optical stimulus increases, motor-unit recruitment occurs in physiological order—from small, low-threshold motor units to large, high-threshold motor units. In contrast, electrical stimulation activates motor units in reverse order, where high-threshold motor units are recruited first, followed by lower-threshold motor units. To test whether engrafted ChR2 motor neurons can function in motor-unit recruitment, the authors stimulated engrafted ChR2 motor neurons with increasing intensity of light followed by measurement of muscle contractile force. Muscle contraction induced by optical stimulation vs. electrical stimulation was examined.

3I Results

Representative motor unit number estimate trace shows force generated from low- to high-threshold motor units. The asterisk indicates optical stimulation. Black waves indicate an increase in muscle contractile force, where low-amplitude waves represent small, low-threshold motor units activated by low intensity of light, and high-amplitude waves represent large, high-threshold motor-unit recruitment with increasing light intensity. Each wave represents increases in muscle contractile force over time. Each stochastic jump in force represents recruitment of additional motor units as intensity of the stimulus is increased.  The data support the hypothesis that engrafted ChR2 motor neurons reinnervate muscles and are able to participate in motor-unit recruitment—a normal muscle contraction characteristic.

3J Results

Motor-unit number estimate traces were recorded from TA and EDL muscles in response to optical (blue) and electrical (black) stimulation. The number of motor units was counted on the motor number estimate trace as each stochastic jump in contractile force represents recruitment of an additional motor unit (Figure 3I). Average values from different experiments were plotted on a histogram. In both muscles, optical stimulation recruits half of the total number of motor units recruited by electrical stimulation. The authors conclude that optical stimulation accounts for 50% of recruited motor units, and that regenerated endogenous axons account for the remainder of the motor units recruited.

3K Results

The average force per motor unit is quantified in TA and EDL muscles in response to either optical (blue) or electrical stimulation (black). In order to calculate the average force per motor unit, the maximal tetanic force is divided by the number of motor units recruited, represented by the number of stochastic jumps in contractile force (Figure 3I). The dotted line represents the average force per motor unit in the EDL muscle of normal, uninjured animals. Average force per motor unit in response to electrical stimulation is comparable to normal EDL values. However, optical stimulation, in which only ChR2 motor neurons are activated, generates a significantly lower average force per motor unit. The authors explain ChR2 motor neuron-associated motor units are likely slow-twitch in nature, resulting in lower average force per motor unit. In addition, inactivity of ChR2 motor neurons for 35 days after engraftment may have contributed to the lower motor unit strength. 

Conclusions

Engrafted ESC-derived ChR2 motor neurons respond to single twitch, high-frequency tetanic and repeated optical stimulation, and generate muscle contraction with characteristics similar to normal muscle physiology. ChR2 motor neurons, compared to endogenous motor neurons, are fatigue-resistant. Optical stimulation of ChR2 motor neurons also participated in motor-unit recruitment—an essential characteristic of normal muscle physiology. Importantly, optical stimulation activates ChR2 motor neurons and generates muscle contraction in a neuron population-specific manner, which can be distinguished from electrical stimulation of endogenous regenerated axons. Together, these data support the hypothesis that optical stimulation of engrafted ESC-derived ChR2 motor neurons can restore muscle function after nerve injury. 

Because nerve ligation enables regeneration of some endogenous motor axons, it was possible to directly compare the properties of these endogenous axons with those of the grafted ChR2 motor neurons by electrical nerve stimulation, which activates both populations of axons. This comparison demonstrated that the proportionate increase between twitch and tetanic stimuli after electrical stimulation was similar to that of optical stimulation [3.07 ± 0.5 and 2.96 ± 0.3 for TA and EDL muscles, respectively) (table S1)], although maximal force generation induced by optical stimulation of ChR2 motor neurons was weaker in comparison with electrical recruitment of both ChR2-expressing and endogenous motor neurons (12.2% of electrically induced force for TA and 12.3% for EDL). It is likely that this reduced force output of muscle fibers innervated by ChR2 motor neurons reflects the 35-day period of inactivity preceding optical stimulation, as indicated by the findings of the histological analysis of these muscles, which showed the presence of axonal sprouting and poly-innervated end plates, characteristic features of inactive muscles (16). Sustained optical stimulation in vivo would probably lead to reinforcement of these neuromuscular junctions and a corresponding increase in force output. Analysis of muscle contractile characteristics also revealed that the latency to peak twitch contraction from initiation of the electrical trigger to the LED unit, or direct stimulation of the nerve, was identical for both optical and electrical stimulation (Fig. 3F). This finding indicates that the nerve conduction velocities were similar for both types of stimulation and supports the histological findings showing that axons of ChR2 motor neurons are myelinated (Fig. 2D). Additionally, the contraction rate—that is, the time from initiation of muscle contraction to peak contraction—was also very similar (Fig. 3G) for both forms of stimulation. Repetitive trains of optical or electrical stimuli (40 Hz, 250-ms duration, every 1 s) were delivered for a period of 180 s to investigate the fatigue characteristics of the reinnervated muscles, which normally have a fast-twitch, fatigable phenotype. The results showed that muscle fibers innervated by grafted ChR2 motor neurons were fatigue-resistant, in contrast to muscle fibers activated by electrical stimulation (Fig. 3H). Again, this probably reflects the prolonged period of inactivity of ChR2 motor neurons before optical stimulation.

Simultaneous activation of all motor neurons innervating a specific muscle would result in inefficient spasmodic contraction and muscle fatigue. It is therefore important that motor neurons are recruited physiologically, according to their activation threshold, to generate a graded muscle contraction that is proportionate to the intended force output. Activation threshold is normally determined by motor neuron soma size (17), but in the case of optogenetic activation of motor neurons, axonal diameter and intermodal distance are also important factors (10, 18). With this in mind, it was important to determine whether grafted ChR2 motor neurons could also be recruited in a graded manner by optical stimulation to induce physiological motor-unit recruitment, where smaller motor units are recruited before larger motor units. To test this, the illumination intensity of the LED was varied from 0.8 to 8 mW/mm2, which resulted in stochastic increases in muscle contractile force, demonstrating that different motor units could be recruited according to their optical activation threshold (Fig. 3I). This technique also enabled us to count the number of individual motor units innervating a given muscle, which for the TA muscle was 15 ± 3.03. Furthermore, by comparing optical and electrical nerve stimulation, we found that grafted ChR2 motor neurons accounted for ~50% of all motor units (Fig. 3J). Moreover, the motor-unit counts enabled us to calculate the average motor-unit force (Fig. 3K), which, after optical stimulation, was found to be 0.21 ± 0.04 g and 0.16 ± 0.05 g for TA and EDL muscles, respectively. Combined recruitment of endogenous and ChR2 motor neuron axons by electrical stimulation resulted in average motor-unit force values of 0.67 ± 0.04 g and 0.59 ± 0.34 g for TA and EDL muscles, respectively, consistent with normal motor-unit force (0.62 g).

In this study, we show that ChR2 motor neurons can be successfully transplanted into a peripheral nerve, where they can survive and extend axons that not only replace lost endogenous motor axons but also reinnervate denervated muscle fibers. Moreover, these transplanted ChR2 motor neurons can be selectively activated by 470-nm light, in a controlled manner to produce graded muscle contractions. Major challenges still remain before this approach can be established as an effective clinical intervention: These obstacles include the development of an implantable optical stimulator, such as that shown by Towne et al. (11), and a means to encapsulate the grafted cells. Additionally, incorporation of sensitive, red-shifted channelrhodopsin variants, such as ReaChR (19), rather than ChR2 would abrogate potential cellular toxicity associated with short-wavelength light (20).

These results show that through the use of a synthesis of regenerative medicine and optogenetics, it is possible to restore specific motor nerve functions. Although this study is largely a proof-of-principle study, it is possible that with further development this strategy may be of use in conditions where muscle function is lost—for example, after traumatic injury or neurodegenerative disease.

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Acknowledgments: We thank T. Keck, D. Kullman, and G. Schiavo for constructive feedback on the manuscript and the Thierry Latran Foundation for supporting this study. I.L. is funded by the Medical Research Council (G0900585), the Biotechnology and Biological Sciences Research Council (G1001234), King’s Health Partners, and the Association Française contre les Myopathies. L.G. is the Graham Watts Senior Research Fellow, funded by The Brain Research Trust, and is supported by the European Community’s Seventh Framework Programme (FP7/2007-2013). J.B. is a Wellcome Trust Investigator. The data reported in this paper are tabulated in the supplementary materials. We declare no conflicts of interest. I.L., C.B.M., M.C., and D.S. developed and characterized ESCs, prepared EBs, and purified motor neurons; M.C. and J.B. performed in vitro physiology; J.B.B. performed surgery, in vivo physiology, and histology and drafted the manuscript; V.B.-F. and D.S. assisted with surgery and histology; and L.G. and I.L. developed the original concept, designed and oversaw the study, and revised the manuscript.