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1 Orderly recruitment of motor units under opticl control in vivo Michel E Llewellyn 1, Kimerly R Thompson 1, Krl Deisseroth 1, & Scott L Delp 1,3 1 Nture Americ, Inc. All rights reserved. A drwck of electricl stimultion for muscle control is tht lrge, ftigle motor units re preferentilly recruited efore smller motor units y the lowest-intensity electricl cuff stimultion. This phenomenon limits therpeutic pplictions ecuse it is precisely the opposite of the norml physiologicl (orderly) recruitment pttern; therefore, mechnism to chieve orderly recruitment hs een long-sought gol in physiology, medicine nd engineering. Here we demonstrte technology for relile orderly recruitment in vivo. We find tht under opticl control with microil opsins, recruitment of motor units proceeds in the physiologicl recruitment sequence, s indicted y multiple independent mesures of motor unit recruitment including conduction ltency, contrction nd relxtion times, stimultion threshold nd ftigue. As result, we oserved enhnced performnce nd reduced ftigue in vivo. These findings point to n unnticipted new modlity of neurl control with rod implictions for nervous system nd neuromusculr physiology, disese reserch nd therpeutic innovtion. A typicl muscle is comprised of mny thousnds of fiers tht contin its contrctile mchinery. Rther thn controlling ech fier individully, the nervous system controls motor units, which re groups of fiers innervted y single motor neuron. These motor units vry in size, from severl fiers to severl thousnd fiers, nd in the type of constituent muscle fiers. Smll motor units hve smll-dimeter xons typiclly innervting slow muscle fiers tht re ftigue resistnt, wheres lrge motor units hve lrge-dimeter xons tht typiclly innervte fster muscle fiers tht re more ftigle 1,. In 197, Elwood Hennemn et l.,3 discovered tht motor units re recruited in order, from smll to lrge, with incresing levels of motor ctivtion. Hennemn s size principle hs ecome one the mjor tenets of neurophysiology nd provides the iophysicl sis for the recruitment of the smllest, most ftigue-resistnt fiers for tsks tht require fine motor control over long periods of time while reserving lrger motor units for rief ursts of high force production,. Smll motor neurons hve smll cell odies with fewer prllel ion chnnels nd tend to hve higher input resistnce thn lrge motor neurons 6. Smll motor neurons therefore respond to given synptic current with greter chnge in memrne potentil, cusing them to rech the threshold for initition of n ction potentil with less synptic current thn lrge motor neurons 6. Under electricl stimultion of motor nerve y cuff, however, lrger myelinted xons re typiclly recruited t lower pplied voltges thn smller myelinted xons, lthough sometimes this order is more rndom (Supplementry Methods) 7 9. This reverse or rndom recruitment order of motor units y electricl stimultion mkes it difficult to modulte nd mintin muscle force 1. Recent discoveries hve mde it possile to stimulte mmmlin neurons with pulses of light 11,1. This is done y geneticlly inserting photo-ctivtle protein into the desired neuron. For exmple, the memrne-trgeted photoctivted ction chnnel chnnelrhodopsin- (ChR) from the lg Chlmydomons reinhrdtii hs enled millisecond-precision control of centrl nervous system neurons in response to rief pulses of light 11,1. We explored the possiility of recruiting motor neurons y photoctivtion, resoning tht if photoctive chnnel density were similr in lrge nd smll xons, the higher input resistnce of smller motor neurons 6 would result in greter memrne potentil chnges in response to fixed conductnce chnges. Smller motor units would then e ctivted efore lrger motor units, chieving the gol of orderly recruitment. RESULTS Chrcteriztion of Thy1::ChR expression in mouse motor neurons To chieve microil opsin expression in peripherl nerves, we used trnsgenic mouse line expressing the ChR-YFP fusion protein under the Thy1 promoter, which drives roust expression in neurons of oth the centrl nd peripherl nervous systems 13, including lower motor nd dorsl root gnglion neurons (Fig. 1,). We quntified the distriution of the YFP-tgged chnnels within motor xons of Thy1::ChR mouse y exmining cross-sections of the scitic nerve oth prllel nd perpendiculr to the long xis of the motor xons (Fig. 1,). Nodl nd internodl regionl ntomy nd morphology ppered norml, indictive of suitle preprtion for explortion of opticl recruitment (Fig. 1). Using confocl microscopy, we found no correltion etween xon size nd fluorescent intensity in the trnsverse sections (Fig. 1c, R =.1, P =.88). The oserved ChR-YFP fusion protein undnce ws found to systemticlly 1 Deprtment of Bioengineering, Stnford University, Stnford, Cliforni, USA. Deprtment of Psychitry nd Behviorl Sciences, Stnford University, Stnford, Cliforni, USA. 3 Deprtment of Mechnicl Engineering, Stnford University, Stnford, Cliforni, USA. Correspondence should e ddressed to S.L.D. ([email protected]) or K.D. ([email protected]). Received 11 Jnury; ccepted 3 June; pulished online 6 Septemer 1; doi:1.138/nm.8 nture medicine VOLUME 16 NUMBER 1 OCTOBER

2 Figure 1 ChR in mouse scitic nerve. () Confocl imge of scitic nerve in cross-section. Red, fluorescently leled lminin of the sl lmin of the peripherl nerve. Green, YFP fluorescence expressed from ChR- YFP fusion protein expressed under control of the Thy1 promoter. Scle r, μm. () Confocl imge of scitic nerve in longitudinl section; stining s in, illustrting severl nodes of Rnvier. Scle r, μm. (c) YFP fluorescence intensity versus motor xon size in cross-section (n = ). (d) Averge YFP fluorescence intensity prllel to the long xis of smpled xons, where the origin indictes the center of the node of Rnvier (n = 1, shded region represents s.d.). 1 Nture Americ, Inc. All rights reserved. c YFP fluorescence intensity (AU) Motor xon dimeter (µm) vry long the xolemm (Fig. 1d), with distriution well suited to the prospect of opticl control; the fluorescence intensity t the center of the nodl region represented minimum long the intermodl distnce (proly owing to locl pcking of N + chnnels) 1, wheres the fluorescence intensity in the perinodl region represented mximum. Force nd electromyogrphic response to opticl stimultion We next chrcterized motor unit recruitment in these mice using opticl stimultion y mesuring whole-muscle prmeters in vivo. In lrge nimls such s cts, motor unit recruitment cn e chrcterized y first isolting individul motor units t the ventrl root to determine motor unit type y their contrctile properties. Once the motor units hve een typed, their recruitment order cn e determined pirwise y stimultion known to elicit orderly recruitment, such s homonymous muscle stretch reflex 1,1. In smll nimls such s mice, however, this technique is imprcticl. We therefore used opticl or electricl cuffs round the scitic nerve in Thy1::ChR or control mice (Fig. ). Stimuli provided vi the cuffs evoked electricl nd contrctile responses of the muscle. We mesured muscle electricl response (M wve) y fine-wire electromyogrphic (EMG) electrodes plced in elly-tendon configurtion, wheres we mesured contrctile force output y force trnsducer ttched to the Achilles tendon (Fig. nd Supplementry Methods). The medil gstrocnemius, the lterl gstrocnemius nd the soleus hve free d YFP fluorescence intensity (AU) Nodl region Distnce from node (xon di.) tendons tht ttch to the distl end of the Achilles tendon. To mesure medil gstrocnemius muscle forces (Figs. nd 3), we detched the free tendons of the other muscles not eing mesured from the Achilles tendon nd mnully seprted the medil gstrocnemius from the djcent muscles without interrupting the neurovsculr supply to the medil gstrocnemius. The tetnus response generted y opticl stimultion (Fig. nd Supplementry Fig. 1) differs from typicl electriclly stimulted tetnus response. The stedy-stte force vlue during tetnus response from opticl stimultion pproched pproximtely one-hlf of its initil pek vlue (Fig. ). This chrcteristic mirrors the pek stedy-stte reltionship of ChR chnnels expressed in neurons when exposed to light 1 nd proly does not ffect twitch mesurements due to the longer time course of this property. The twitch responses generted y opticl stimultion from the medil gstrocnemius (Fig. ) differ from twitches evoked y electricl stimultion in terms of contrction nd relxtion times (Fig. 3c,d). stimultion produced no stimultion rtifct in the EMG response. There ws no response to opticl stimultion in control mice (Fig. ), indicting tht opticl stimultion occurs y photostimultion of the ChR chnnels nd not y het or electricl mens. To compre electricl nd opticl stimultion cross rnge of intensities, we clculted the rectified integrted EMG (iemg) from cuff.1 N 1 ms 1 Hz 8 Hz 6 Hz Hz Hz EMG 1 mv 1 ms 6 Hz Figure Optogenetic control of peripherl nerve. () An opticl or electricl cuff is plced round the scitic nerve of n nesthetized Thy1:: ChR mouse. Inset photomicrogrph: custom-designed light-emitting diode sed opticl cuff. Fine-wire EMG leds re plced in the muscle of interest; EMG plot shows typicl response from opticl stimultion. The Achilles tendon is fixed to force trnsducer; force trces show typicl rw dt of contrctions t vrious frequencies of opticl stimultion. The sg in force fter initil stimultion seen in opticl stimultion proly rises from the iophysicl properties of the ChR chnnel itself. An exmple EMG trce is shown for ms t 6 Hz stimultion. () Typicl rw EMG nd force trces from twitches elicited y opticl nd electricl stimultions in Thy1::ChR mice nd control C7BL/6 mice. The colored rs ner ech trce indicte the durtion of stimultion. C7BL/6 control Thy1::Chr Force trnsducer EMG 1 mv 1 ms Force.1 N 1 ms 116 VOLUME 16 NUMBER 1 OCTOBER 1 nture medicine

3 1 Nture Americ, Inc. All rights reserved. Figure 3 Orderly recruitment nd ftigue resistnce with opticl stimultion. Ech point represents the men ± s.e.m. (opticl, n = mice, 6 trils; electricl, n = mice, 73 trils; P <.1). Error rs re present t ll points nd my e smller thn the dt-point mrkers throughout the figure. () Pek force during single twitch versus rectified iemg for oth electricl nd opticl stimultion. () Averge ltency mesured from initition of stimuli to detection of EMG. (c) Averge contrction time mesured from 1% of pek force to pek force. (d) Averge relxtion time mesured from pek force to 1% of pek force. (e) Averge tetnic tension over min in muscle eing stimulted with -ms trins t 1 Hz using electricl nd opticl stimultion (n = 7, shded region is s.e.m., verge ody weight (BW) =.8 ±.1 N, BW is pproximtely % of mximl isometric tension, Supplementry Methods). (f) Averge ftigue index for electricl nd opticl stimultion, mesured s decline in tetnic tension over min (n = 7). (g) Exemplr tetnic tension from single mouse using oth opticl nd electricl stimultion in hindlims over min. rw EMG dt over the time intervl required for ctivity to return to within. mv of zero in ech tril nd compred verge pek force during twitch to the iemg response (Fig. 3). The shpe of the force reltionship under voluntry conditions is thought to e recruitment order dependent 16 ; however, we found tht overll response chrcteristics were similr etween electricl nd opticl stimultion. Averge pek twitch forces chieved y electricl stimultion were slightly higher thn verge pek twitch forces with opticl stimultion (1. ±.19 ody weights versus 1.1 ±. ody weights; P <.1; verge ody weight =.8 ±.1 N). Twitch forces for oth clsses of stimultion were lso consistent with previous mesurements 17,18. Mesures of orderly recruitment using opticl stimultion Motor xon conduction ltency is widely used mesure of motor unit recruitment, given tht smller xons hve slower conduction speeds nd longer ltencies for given distnce 1,19. Typicl mesurements of ltency record conduction long nerve only; however, we mesured ltency (Fig. 3) s the time etween the nerve stimulus nd the depolriztion mesured on EMG (M wve), ecuse the physicl size of our opertive field did not llow us to record from two plces long the cut scitic nerve without introducing error. Ltencies mesured under opticl stimultion for ll intensities (rnging from.18 ±. ms to 1.7 ±.13 ms) were significntly longer thn ltencies under electricl stimultion (rnging from 1.1 ±. ms to.99 ±.1 ms, P <.1 in ll cses). At the lowest levels of ctivity (1 mv ms), the ltency decresed under opticl stimultion (P <.1) ut not under electricl stimultion (P =.11). This suggested tht smller xons re recruited preferentilly t the lowest levels of opticl stimultion ut not under electricl stimultion. Ltency did not decrese with opticl stimultion t ctivities greter thn mv ms, presumly ecuse the medil gstrocnemius is innervted y only smll frction of smll motor units. Other mesures of motor unit recruitment, the contrction nd relxtion times 1,, lso suggested orderly recruitment with opticl stimultion. Contrction time (Fig. 3c), the time required for twitch tension to increse from 1% of pek force to pek force, ws significntly longer using opticl stimultion (11.1 ±.1 ms) t the lowest levels of muscle ctivity when compred to electricl stimultion (8.8 ±.1 ms, P <.1). In contrst, t the highest levels of muscle ctivity, contrction time using opticl nd electricl stimultion ws not significntly different (8.3 ±.1 ms, P =.6) (Fig. 3c). Furthermore, relxtion time (time required from twitch tension to Pek tension (BW) c Contrction time (ms) e Avg. tension (BW) g Muscle tension (BW) NS Time (s) 1 NS 6 8 fll from pek force to 1% of pek force; Fig. 3d) ws significntly longer t the lowest level of muscle ctivity using opticl stimultion (1.7 ±. ms) thn electricl stimultion (17. ±.7 ms, P <.1), wheres relxtion time t the highest levels of muscle ctivity were not significntly different etween the different types of stimultion (1. ±.1 ms, P =.1). The mesurements of contrction nd relxtion times, which re consistent with other dt 17,3, oth imply tht t the lowest levels of muscle ctivity opticl stimultion preferentilly recruits slower motor units. To test whether opticl stimultion of muscle elicits less ftigue thn electricl stimultion, we mesured tetnic tension generted y the plntrflexor group (lterl gstrocnemius, medil gstrocnemius, soleus nd plntris) of mice using opticl nd electricl stimultion to ctivte motor units regrdless of their origin in prticulr muscle. The ftigility of muscle fiers is ffected y multiple fctors, including the frequency of stimultion, nd is known to e highly plstic under chronic electricl stimultion ; however, ll prmeters were held constnt etween the electricl nd opticl stimultion, nd we ssumed no plstic chnges during the short time of our trils. At stimultion intensities in ech modlity tht initilly elicited force of two times the ody weight for ech unftigued mouse (Supplementry Methods), the force generted y the muscle declined much more rpidly with electricl stimultion thn with opticl stimultion (Fig. 3e). Indeed, the ftigue index, defined s the verge tetnic tension of the lst trin divided y the verge tetnic tension in the first trin, ws strikingly smller in trils using electricl stimultion (.11 ±.9) thn in those using opticl stimultion (.6 ±.9, P <.1; Fig. 3f). When we extended the ftigue protocol Ltency (ms) d Relxtion time (ms) Avg. ftigue index NS Time (min) f nture medicine VOLUME 16 NUMBER 1 OCTOBER

4 1 Nture Americ, Inc. All rights reserved. Figure Differentil recruitment of soleus nd lterl gstrocnemius with electricl nd opticl stimultion. Ech point represents men ± s.e.m. (opticl, n = mice, 1,99 trils; electricl, n = mice, 88 trils; P <.1). Error rs re present t ll points nd my e smller thn the dtpoint mrkers throughout the figure. () Rectified iemg versus estimted opticl intensity t surfce of the scitic nerve for soleus () nd lterl gstrocnemius (). () Rectified iemg versus electricl stimultion voltge pplied to scitic nerve. (c) intensity required to chieve mximum iemg in soleus nd lterl gstrocnemius. (d) stimultion to chieve 9% of mximum iemg in soleus nd lterl gstrocnemius. (e) Distriution of motor xon dimeters for soleus nd lterl gstrocnemius found in cross-section of the scitic nerve. (f) Distriution of soleus nd lterl gstrocnemius motor xon depths from the surfce of the scitic nerve. (g) Exmple cross-section of the scitic nerve where retrogrde dye ws injected into the lterl gstrocnemius only. Scle r, 1 μm. to min in n individul mouse in the contrlterl hindlims, electricl stimultion diminished tetnic tension completely fter min, wheres opticl stimultion continued to elicit 3% of the initil tension fter the -min tril (Fig. 3g). These dt revel roustly enhnced performnce of the system in the cse of opticl stimultion, which would e expected from orderly recruitment. Recruitment of smll motor units with opticl stimultion Differentil motor unit recruitment my lso enle differentil control of distinct muscles tht re controlled y the sme peripherl nerve. To explore this possiility, we compred the recruitment of two muscles, the soleus nd the lterl gstrocnemius (Fig. ). The soleus contins 8 ± % slow oxidtive fiers nd % fst glycolytic fiers, wheres the lterl gstrocnemius hs 1 ± 3% slow oxidtive fiers nd 69 ± 13% fst glycolytic fiers. Smller motor units hve smller xons nd tend to hve higher compositions of slow oxidtive fiers, nd thus we expected tht soleus motor units would e recruited efore the fster motor units of the lterl gstrocnemius with lrger motor units nd lrger xons, s reported in other recruitment studies 6. We found under opticl stimultion tht soleus (1.9 ± 1.9 mw mm ) reched 9% pek ctivity t significntly lower opticl intensity thn lterl gstrocnemius (Fig.,c,. ± 1.9 mw mm, P <.1). We lso found tht the electricl stimultion used to evoke 9% of pek ctivity in soleus (.6 ±.1 V) nd lterl gstrocnemius (.6 ±.9 V) ws not significntly different (Fig.,d, P =.98). These findings suggest tht slower muscle fiers re preferentilly recruited y opticl stimultion efore fster fiers; however, it is impossile to know the order of motor unit recruitment without knowing the size distriution of the motor xons innervting ech muscle. Therefore, to nlyze xon size distriution nd to identify ny is in the loction of the xons innervting ech muscle within the cross-section of the peripherl nerve, we injected retrogrde dye (Fst Blue) intrmusculrly into the muscles of interest to ckfill only the xons innervting the muscle in which it ws injected 7. We found in cross-sections of the scitic nerve (Fig. ) tht soleus nd lterl gstrocnemius do not contin significntly different numers of motor xons (Fig. e, 3. ±.9 xons for the solueus nd. ± 3.7 xons for the lterl gstrocnemius, P =.71). However, the verge motor xon innervting the lterl gstrocnemius hd significntly lrger dimeter thn those stimultion (V) e f 18 g Averge numer power (mw mm ) Motor xon dimeter (µm) Averge numer Depth in peripherl nerve (µm) innervting the soleus (6.7 ±.16 μm for the solueus nd. ±.17 μm for the lterl gstrocnemius, P <.1). Quntittive histology reveled tht soleus xons tended to e t greter distnce from the outer nerve sheth thn lterl gstrocnemius xons (Fig. f), indicting tht soleus xons were not exposed to higher intensities of light thn lterl gstrocnemius xons (Supplementry Methods). These oservtions support the premise tht smll motor units re preferentilly recruited with opticl stimultion nd tht the oserved difference in opticl intensity required for pek muscle ctivity in soleus nd lterl gstrocnemius is not influenced y either the numer of xons or the position of those xons within the peripherl nerve. DISCUSSION Together, these dt demonstrte tht opticl stimultion chieves the elusive gol of orderly recruitment nd cn mrkedly enhnce functionl performnce. The ility to preferentilly recruit slower motor units with opticl stimultion hs potentilly enormous functionl significnce. Functionl electricl stimultion systems hve een developed to serve s neuroprosthetics for individuls with prlysis ut hve not een dopted widely in prt ecuse of erly onset ftigue due to reversed or rndom motor unit recruitment 7,8. A numer of complex electricl stimultion strtegies hve een developed tht replicte orderly recruitment under controlled circumstnces 9,3. However, opticl stimultion of motor neurons is fundmentlly different pproch tht my solve the motor recruitment prolem y stimulting motor units closer to their physiologicl recruitment pttern without hving to suppress lrge motor units typiclly required with electricl stimultion. This unique pproch is comptile with other forms of optogenetics 31,3, pointing to unprecedented levels of control over motor function. These techniques re currently limited to reserch in nimls; however, recent dvnces in humn gene therpy 33,3 point the wy towrd eventul clinicl pplictions of this technology. For exmple, mny motor neuron diseses result from overctivity (such s spsticity resulting from cererl plsy, stroke nd trumtic rin injury) 3 ; in these cses the light-sensitive Cl pump (hlorhodopsin) could e employed s n inhiitory gte to control when motor units re ctivted 36. Additionlly, individul motor units or individul muscles could e essentilly color-coded with the devices nd principles outlined c stim. power (mw mm ) stim. d stim. (V) ns stim. 116 VOLUME 16 NUMBER 1 OCTOBER 1 nture medicine

5 1 Nture Americ, Inc. All rights reserved. here to control individul elements within muscle volume. This technique opens the door to vst rry of new studies involved in mpping nd control of the peripherl nervous system for clinicl nd ioengineering pplictions. Methods Methods nd ny ssocited references re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Medicine wesite. Acknowledgments We thnk R. Brretto nd D. Wetmore for technicl ssistnce nd K. McGill, Z. Ltev, R. Lieer, D. Lin nd F. Zjc for fruitful discussions. This work ws supported y Stnford Bio-X Interdisciplinry Inititives wrd (S.L.D. nd K.D.), the Ntionl Allince for Reserch on Schizophreni nd Depression (K.R.T.), nd the Stnford US Ntionl Institutes of Helth Medicl Scientist Trining Progrm (M.E.L.). AUTHOR CONTRIBUTIONS M.E.L. conducted the experiments, performed the nlysis nd wrote the mnuscript. K.R.T. performed the imging experiments nd wrote the mnuscript. S.L.D. nd K.D. supervised the project nd wrote the mnuscript. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Burke, R.E., Levine, D.N., Tsiris, P. & Zjc, F.E. III. Physiologicl types nd histochemicl profiles in motor units of the ct gstrocnemius. J. Physiol. (Lond.) 3, (1973).. Hennemn, E. Reltion etween size of neurons nd their susceptiility to dischrge. Science 16, (197). 3. Hennemn, E., Somjen, G. & Crpenter, D.O. Functionl significnce of cell size in spinl motoneurons. J. Neurophysiol. 8, 6 8 (196).. Gordon, T., Thoms, C.K., Munson, J.B. & Stein, R.B. The resilience of the size principle in the orgniztion of motor unit properties in norml nd reinnervted dult skeletl muscles. Cn. J. Physiol. Phrmcol. 8, ().. Mendell, L.M. The size principle: rule descriing the recruitment of motoneurons. J. Neurophysiol. 93, 3 36 (). 6. Bkels, R. & Kernell, D. 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Mnipultion of muscle force with vrious firing rte nd recruitment control strtegies. IEEE Trns. Biomed. Eng. 3, (1987). 31. Zhng, F. et l. Red-shifted optogenetic excittion: tool for fst neurl control derived from Volvox crteri. Nt. Neurosci. 11, (8). 3. Berndt, A., Yizhr, O., Gunydin, L.A., Hegemnn, P. & Deisseroth, K. Bi-stle neurl stte switches. Nt. Neurosci. 1, 9 3 (9). 33. Binridge, J.W. et l. Effect of gene therpy on visul function in Leer s congenitl murosis. N. Engl. J. Med. 38, (8). 3. Mguire, A.M. et l. Sfety nd efficcy of gene trnsfer for Leer s congenitl murosis. N. Engl. J. Med. 38, 8 (8). 3. McClellnd, S., Teng, Q., Benson, L.S. & Boulis, N.M. Motor neuron inhiition sed gene therpy for spsticity. Am. J. Phys. Med. Rehil. 86, 1 1 (7). 36. Grdinru, V. et l. Moleculr nd cellulr pproches for diversifying nd extending optogenetics. Cell 11, 1 16 (1). nture medicine VOLUME 16 NUMBER 1 OCTOBER 1 116

6 1 Nture Americ, Inc. All rights reserved. ONLINE METHODS Nerve microscopy. Fresh mouse scitic nerve ws fixed in % prformldehyde for 3 min nd wshed in PBS. The smples were then emedded in % low melting-point grose nd cut (into -μm slices) with virtome. The sections were leled with ntiodies to tu (Acm) nd lmin (Dko). The sections were imged on confocl microscope (Leic, DM6). The numer, size nd fluorescence intensity of the motor xons ( 3 μm nd G rtio.) 37,38 were determined y mnul nlysis in ImgeJ (US Ntionl Institutes of Helth). In vivo studies. All studies were pproved y Stnford University Institutionl Animl Cre nd Use Committee. Norml-ppering, l-red 9-1-week-old Thy1-ChR or C7BL/6 control mice were nesthetized, nd the hindlim ws shved nd fixed in frme. The Achilles tendon ws freed y cutting the distl end of the clcneous to force trnsducer (Auror Scientific, 3C-LR) y thin steel wire. An opticl cuff, mde of 16 lightemission diodes (Rohm, SMLP1BC7T, 6 nm) rrnged in concentric perimeter fcing the peripherl nerve center, or ipolr hook electricl cuff ws inserted round the exposed scitic nerve tht ws cut proximl to the site of stimultion (Supplementry Fig. 1). In most cses, opticl nd electricl stimultion were conducted in the sme leg t different times. Stinless steel hook electrodes were inserted for differentil EMG recordings. EMG recordings were filtered in hrdwre only (BP 33 Hz). All force, EMG nd stimuli dt were smpled t 1 khz. All other dt nlysis ws conducted in Mtl. All dt reported for the medil gstrocnemius ws roken in ritrrily defined ins on the sis of iemg vlue. To determine stimuli needed for 9% mximum iemg in soleus nd lterl gstrocnemius, Weiull cumultive distriution function ws fit to dt points. The confidence intervl generted y the curve fit ws used to define the 99% confidence intervl of the required stimuli. Smples tested for sttisticlly significnt differences were first tested for normlity with the Lilliefors test (α =.) nd then tested with the unpired two-tiled Student s t test (α =.). All smple groups tested were found to e of norml distriution, except for the xon size dt, which ws tested with the Mnn-Whitney U test. All dt points listed re men ± s.e.m. or dt ± 99% confidence intervl (Fig. c,d). 37. Irintchev, A., Drguhn, A. & Wernig, A. Reinnervtion nd recovery of mouse soleus muscle fter long-term denervtion. Neuroscience 39, 31 3 (199). 38. McHnwell, S. & Biscoe, T.J. The sizes of motoneurons supplying hindlim muscles in the mouse. Proc. R. Soc. Lond. B Biol. Sci. 13, 1 16 (1981). nture medicine doi:1.138/nm.8