Optogenetic neuromuscular actuation of a miniature electronic biohybrid robot

Abstract

Neuronal control of skeletal muscle function is ubiquitous across species for locomotion and doing work. In particular, emergent behaviors of neurons in biohybrid neuromuscular systems can advance bioinspired locomotion research. Although recent studies have demonstrated that chemical or optogenetic stimulation of neurons can control muscular actuation through the neuromuscular junction (NMJ), the correlation between neuronal activities and resulting modulation in the muscle responses is less understood, hindering the engineering of high-level functional biohybrid systems. Here, we developed NMJ-based biohybrid crawling robots with optogenetic mouse motor neurons, skeletal muscles, 3D-printed hydrogel scaffolds, and integrated onboard wireless micro-light-emitting diode (μLED)-based optoelectronics. We investigated the coupling of the light stimulation and neuromuscular actuation through power spectral density (PSD) analysis. We verified the modulation of the mechanical functionality of the robot depending on the frequency of the optical stimulation to the neural tissue. We demonstrated continued muscle contraction up to 20 minutes after a 1-minute-long pulsed 2-hertz optical stimulation of the neural tissue. Furthermore, the robots were shown to maintain their mechanical functionality for more than 2 weeks. This study provides insights into reliable neuronal control with optoelectronics, supporting advancements in neuronal modulation, biohybrid intelligence, and automation.

Fig. 1.. Fabrication of NMJ-driven biohybrid crawler with μLED optoelectronics.

(A) Brief illustration of key components of the NMJ crawler. (B) Schematics of the experimental procedure for NMJ tissue preparation. Depending on where neural chambers exist, NTs are located at the center of or beside the muscle tissue. Inset shows the two versions of NMJ models, single-NT and dual-NT. Red dotted circle denotes boundary of NTs that form the NMJ in a lateral direction. (C) Pictures of single-and dual-NT NMJ crawlers. Scale bars, 5 mm. (D) Fluorescence expression of optogenetic mESCs transduced by HB9-GFP and ChR2-tdTomato. Scale bars, 200 and 100 μm. (E) Fluorescence mapping of a whole NMJ crawler showing the localization of neurospheres in the tissue. Scale bars, 3 mm. (F) Representative image of the completely integrated NMJ crawler. Scale bar, 5 mm.

Fig. 2.. Characterization of NMJs.

(A) Immunostaining images of an NMJ tissue, showing myosin (MF-20, red), neuronal units (Tuj1, green), AChRs (α-BTX, orange), and cell nucleus (DAPI, blue). Scale bars, 100 and 20 μm. White arrows denote the representative location of synaptic junctions. (B) Relative quantification (RQ) of NMJ-related genes functioning on development and stabilization of AChRs. In comparison with a muscle-only control group, neuromuscular tissues showed up-regulation of MuSK, DOK-7, Rapsyn, and CHRNA. Data represent means ± SD (N = 3 biological replicates; *P < 0.05). (C) Analysis of the fold change of RNA expression between muscle-only and neuromuscular tissues. Inset shows the heatmap of up/down-regulated genes under a q value of 0.05 (N = 3 biological replicates). (D) Biological gene ontology analysis based on differentially expressed genes (FDR < 0.05).

Fig. 3.. Analyzing locomotion of dual-NT NMJ crawlers driven by spontaneous neuronal activity.

(A) Brief illustration of a two-chamber dual-NT NMJ crawler. (B) FEA of the crawling behavior of the asymmetric scaffold. (C) Leg deflections of the long and short leg obtained from simulation (blue line) and experimental recording (black line with red circle). Data represent means ± SD (pink-shaded area) (N = 6 independent trajectories). The 2.2-Hz twitching frequency was applied to the simulation. (D) Snapshots of the NMJ crawler during locomotion. Scale bars, 3 mm. (E) Comparison of crawling trajectories extracted from simulation (red) and experiment (black). (F) Chemical inhibition of crawling by adding the inhibitory molecule curare. Scale bars, 3 mm. (G) Recorded crawling trajectories in the x-y plane and (H) corresponding net displacement with increasing concentration of the curare. (I) Effect of curare and l-glutamic acid on leg deflections. (J) Statistics of contraction forces in the presence of chemicals. Data represent means ± SD (N = 38, 71, and 44 for initial status, l-glutamic acid, and curare treatment, respectively; ***P < 0.001).

Fig. 4.. Optogenetic response of single-NT NMJ crawler controlled by an optical fiber.

(A) Illustration of the experimental setup consisting of single NT with an optical fiber. (B) Snapshots of the NMJ crawler and acceleration of crawling behavior by using 2-Hz optical stimulation (scale bars, 3 mm). (C) Crawling trajectory of the crawler over time. (D) Synchronized twitching of both legs under stimulation. (E) Observation of long leg twitching under 2- and 4-Hz frequencies of optical stimulation. (F) Representative deflection traces obtained from each section in (E). (G) PSDs of muscle twitching modes with respect to stimulus conditions. (H) Power densities obtained from continuous recording of muscle twitching after the 2-Hz stimulation.

Fig. 5.. Optogenetic control of single-NT NMJ crawler with onboard electronics.

(A) Structure of the one-chamber, single-NT NMJ crawler with onboard optoelectronics. (B) Images of the optoelectronics composed of a power-harvesting antenna with five μLEDs (fig. S10). (C) Comparison of crawling behaviors between nonoptogenetic pristine muscle and NMJ crawlers. Insets show the location of the crawlers after 5 min. Scale bars, 1 cm. (D) Optimization of the number of neural cells via measuring autonomous crawling of NMJ crawlers. (i) Optical images with different numbers of spheroids in the NT (scale bars, 1 mm) and (ii) the average velocities of autonomous crawling and yield (in parentheses) of autonomously crawling robots, both as a function of cell seeding density. (E) A case study of an optoelectronics-driven NMJ crawler that did not crawl before stimulation but showed boosted crawling after stimulation. (i) Images of the crawler for 5 min (scale bars, 5 mm). (ii) Recorded traces and velocity of the robot during the measurement. (F) A crawler showing high speed of autonomous crawling before stimulation but reduced its velocity after stimulation. (i) Actual displacement of the robot for 25 min. Scale bar, 5 mm. (ii) Measured traces and velocities under various stimulation conditions from 1 to 4 Hz.

Fig. 6.. Modulating neuromuscular actuation of the biohybrid crawler across samples and across time.

(A) Representative example of altering muscular actuation depending on neurostimulation frequency (1 to 4 Hz). Long-term performance of an NMJ crawler was verified by measuring the mechanical responses of the robot for 21 days. The mechanical output to 2-Hz optical stimulation was converted to normalized PSDs, and data for 2 days are shown at (B) coculture day 6 and (C) coculture day 12. (D) Measured muscle contraction force in these long-term measurement experiments of an NMJ biohybrid crawler showing functionality lasting more than 2 weeks. Data represent means ± SD (N > 25 independent actuation events).

Movie 1.

Overview of the NMJ-based biohybrid crawling robot.