Orderly recruitment of motor units under optical control in vivo

Abstract

A drawback of electrical stimulation for muscle control is that large, fatigable motor units are preferentially recruited before smaller motor units by the lowest-intensity electrical cuff stimulation. This phenomenon limits therapeutic applications because it is precisely the opposite of the normal physiological (orderly) recruitment pattern; therefore, a mechanism to achieve orderly recruitment has been a long-sought goal in physiology, medicine and engineering. Here we demonstrate a technology for reliable orderly recruitment in vivo. We find that under optical control with microbial opsins, recruitment of motor units proceeds in the physiological recruitment sequence, as indicated by multiple independent measures of motor unit recruitment including conduction latency, contraction and relaxation times, stimulation threshold and fatigue. As a result, we observed enhanced performance and reduced fatigue in vivo. These findings point to an unanticipated new modality of neural control with broad implications for nervous system and neuromuscular physiology, disease research and therapeutic innovation.

Figure 1

ChR2 in mouse sciatic nerve. (a) Confocal image of sciatic nerve in cross-section. Red, fluorescently labeled laminin of the basal lamina of the peripheral nerve. Green, YFP fluorescence expressed from ChR2-YFP fusion protein expressed under control of the Thy1 promoter. Scale bar, 50 µm. (b) Confocal image of sciatic nerve in longitudinal section; staining as in a, illustrating several nodes of Ranvier. Scale bar, 50 µm. (c) YFP fluorescence intensity versus motor axon size in cross-section (n = 4). (d) Average YFP fluorescence intensity parallel to the long axis of sampled axons, where the origin indicates the center of the node of Ranvier (n = 15, shaded region represents s.d.).

Figure 2

Optogenetic control of peripheral nerve. (a) An optical or electrical cuff is placed around the sciatic nerve of an anesthetized Thy1::ChR2 mouse. Inset photomicrograph: custom-designed light-emitting diode–based optical cuff. Fine-wire EMG leads are placed in the muscle of interest; EMG plot shows a typical response from optical stimulation. The Achilles tendon is fixed to a force transducer; force traces show typical raw data of contractions at various frequencies of optical stimulation. The sag in force after initial stimulation seen in optical stimulation probably arises from the biophysical properties of the ChR2 channel itself. An example EMG trace is shown for 500 ms at 60 Hz stimulation. (b) Typical raw EMG and force traces from twitches elicited by optical and electrical stimulations in Thy1::ChR2 mice and control C57BL/6 mice. The colored bars near each trace indicate the duration of stimulation.

Figure 3

Orderly recruitment and fatigue resistance with optical stimulation. Each point represents the mean ± s.e.m. (optical, n = 5 mice, 625 trials; electrical, n = 5 mice, 573 trials; *P < 0.01). Error bars are present at all points and may be smaller than the data-point markers throughout the figure. (a) Peak force during a single twitch versus rectified iEMG for both electrical and optical stimulation. (b) Average latency measured from initiation of stimuli to detection of EMG. (c) Average contraction time measured from 10% of peak force to peak force. (d) Average relaxation time measured from peak force to 10% of peak force. (e) Average tetanic tension over 2 min in muscle being stimulated with 250-ms trains at 1 Hz using electrical and optical stimulation (n = 7, shaded region is s.e.m., average body weight (BW) = 0.258 ± 0.01 N, 2 BW is approximately 20% of maximal isometric tension, Supplementary Methods). (f) Average fatigue index for electrical and optical stimulation, measured as decline in tetanic tension over 2 min (n = 7). (g) Exemplar tetanic tension from a single mouse using both optical and electrical stimulation in hindlimbs over 20 min.

Figure 4

Differential recruitment of soleus and lateral gastrocnemius with electrical and optical stimulation. Each point represents mean ± s.e.m. (optical, n = 5 mice, 1,099 trials; electrical, n = 5 mice, 885 trials; *P < 0.01). Error bars are present at all points and may be smaller than the datapoint markers throughout the figure. (a) Rectified iEMG versus estimated optical intensity at surface of the sciatic nerve for soleus (SOL) and lateral gastrocnemius (LG). (b) Rectified iEMG versus electrical stimulation voltage applied to sciatic nerve. (c) Optical intensity required to achieve maximum iEMG in soleus and lateral gastrocnemius. (d) Electrical stimulation to achieve 95% of maximum iEMG in soleus and lateral gastrocnemius. (e) Distribution of motor axon diameters for soleus and lateral gastrocnemius found in cross-section of the sciatic nerve. (f) Distribution of soleus and lateral gastrocnemius motor axon depths from the surface of the sciatic nerve. (g) Example cross-section of the sciatic nerve where retrograde dye was injected into the lateral gastrocnemius only. Scale bar, 100 µm.