Development of 3D neuromuscular bioactuators.

Neuronal control of skeletal muscle bioactuators represents a critical milestone toward the realization of future biohybrid machines that may generate complex motor patterns and autonomously navigate through their environment. Animals achieve these feats using neural networks that generate robust firing patterns and coordinate muscle activity through neuromuscular units. Here, we designed a versatile 3D neuron-muscle co-culture platform to serve as a test-bed for neuromuscular bioactuators. We used our platform in conjunction with microelectrode array electrophysiology to study the roles of synergistic interactions in the co-development of neural networks and muscle tissues. Our platform design enables co-culture of a neuronal cluster with up to four target muscle actuators, as well as quantification of muscle contraction forces. Using engineered muscle tissue targets, we first demonstrated the formation of functional neuromuscular bioactuators. We then investigated possible roles of long-range interactions in neuronal outgrowth patterns and observed preferential outgrowth toward muscles compared to the acellular matrix or fibroblasts, indicating muscle-specific chemotactic cues acting on motor neurons. Next, we showed that co-cultured muscle strips exhibited significantly higher spontaneous contractility as well as improved sarcomere assembly compared to muscles cultured alone. Finally, we performed microelectrode array measurements on neuronal cultures, which revealed that muscle-conditioned medium enhances overall neural firing rates and the emergence of synchronous bursting patterns. Overall, our study illustrates the significance of neuron-muscle cross talk for the in vitro development of neuromuscular bioactuators.

A connected cytoskeleton network generates axonal tension in embryonic Drosophila.

Axons of neurons are contractile, i.e., they actively maintain a rest tension. However, the spatial origin of this contractility along the axon and the role of the cytoskeleton in generating tension and sustaining rigidity are unknown. Here, using a microfluidic platform, we exposed a small segment of the axons of embryonic Drosophila motor neurons to specific cytoskeletal disruption drugs. We observed that a local actomyosin disruption led to a total loss in axonal tension, with the stiffness of the axon remaining unchanged. A local disruption of microtubules led to a local reduction in bending stiffness, while tension remained unchanged. These observations demonstrated that contractile forces are generated and transferred along the entire length of the axon in a serial fashion. Thus, a local force disruption results in a collapse of tension of the entire axon. This mechanism potentially provides a pathway for rapid tension regulation to facilitate physiological processes that are influenced by axonal tension.

Cancer Cells Invade Confined Microchannels via a Self-Directed Mesenchymal-to-Amoeboid Transition.

Cancer cell invasion through physical barriers in the extracellular matrix (ECM) requires a complex synergy of traction force against the ECM, mechanosensitive feedback, and subsequent cytoskeletal rearrangement. PDMS microchannels were used to investigate the transition from mesenchymal to amoeboid invasion in cancer cells. Migration was faster in narrow 3 µm-wide channels than in wider 10 µm channels, even in the absence of cell-binding ECM proteins. Cells permeating narrow channels exhibited blebbing and had smooth leading edge profiles, suggesting an ECM-induced transition from mesenchymal invasion to amoeboid invasion. Live cell labeling revealed a mechanosensing period in which the cell attempts mesenchymal-based migration, reorganizes its cytoskeleton, and proceeds using an amoeboid phenotype. Rho/ROCK (amoeboid) and Rac (mesenchymal) pathway inhibition revealed that amoeboid invasion through confined environments relies on both pathways in a time- and ECM-dependent manner. This demonstrates that cancer cells can dynamically modify their invasion programming to navigate physically confining matrix conditions.

A simple microfluidic platform for the partial treatment of insuspendable tissue samples with orientation control.

Microfluidic devices have extensively been applied to study biological samples, including single cells. Exploiting laminar flows on a small scale, microfluidics allow for the selective and partial exposure of samples to various chemical treatments. Traditionally, suspendable samples are first flowed into formed microchannels and are allowed to adhere to the channel floor randomly with no control over sample placement or orientation, before being subjected to partial treatment. This severely limits the choice of samples and the extent of sample preparations. Here, we overcame this limit by reversing the sequence. We prepared the samples first on glass substrates. A patterned silicone slab was then placed on the substrate to form channels at an appropriate orientation with respect to the sample. We used liquid silicone rubber (LSR) as the base material. Its compliance (low elastic modulus) and its adhesion to glass offer the necessary seal to form the microchannels naturally. The applicability of the device was demonstrated by testing single axons of embryonic Drosophila motor neurons in vivo. A segment of the axons was subjected to drugs that inhibit myosin activities or block voltage-gated sodium ion channels. In response, the axons reduced the clustering of neuro-transmitter vesicles at the presynaptic terminal of neuromuscular junctions, or increased the calcium intake and underwent membrane hyperpolarization, respectively. Such fundamental studies cannot be carried out using conventional microfluidics.

Coupled circumferential and axial tension driven by actin and myosin influences in vivo axon diameter.

It has long been known that neuronal axons are contractile. They actively maintain rest tension along the longitudinal direction both in vitro and in vivo. Here we show evidence that embryonic drosophila axons also actively maintain contractility/tension along the circumferential direction. We used confocal microscopy and spatial light interference microscopy to monitor axonal diameter along their length. We observed a decrease in diameter when microtubules are disrupted and an increase in diameter when actin filaments or myosin II are disrupted. Interestingly, active diameter reduction occurred consistently when axons were subjected to manipulations known to increase axial tension, suggesting that tension can be coupled in the axial and circumferential direction. This is further supported by the remarkably similar time constants for diameter reduction and rest tension increase of slackened axons. We infer that the actomyosin-driven circumferential contraction/hoop tension applies a squeezing force on the microtubule bundle of the axons. This hoop tension is balanced by the restoring force of the microtubule bundle. Therefore, axonal diameter increased when actin/myosin disrupting drugs relaxed the hoop tension and decreased when microtubule disrupting drug relaxed the restoring force. Circumferential tension thus can regulate axonal diameter and volume, as well as potentially microtubules alignment, inter-tubular spacing, and, by extension, axonal transport.

Mechanism of Axonal Contractility in Embryonic Drosophila Motor Neurons In Vivo.

Several in vitro and limited in vivo experiments have shown that neurons maintain a rest tension along their axons intrinsically. They grow in response to stretch but contract in response to loss of tension. This contraction eventually leads to the restoration of the rest tension in axons. However, the mechanism by which axons maintain tension in vivo remains elusive. The objective of this work is to elucidate the key cytoskeletal components responsible for generating tension in axons. Toward this goal, in vivo experiments were conducted on single axons of embryonic Drosophila motor neurons in the presence of various drugs. Each axon was slackened mechanically by bringing the neuromuscular junction toward the central nervous system multiple times. In the absence of any drug, axons shortened and restored the straight configuration within 2-4 min of slackening. The total shortening was ∼40% of the original length. The recovery rate in each cycle, but not the recovery magnitude, was dependent on the axon's prior contraction history. For example, the contraction time of a previously slackened axon may be twice its first-time contraction. This recovery was significantly hampered with the depletion of ATP, inhibition of myosin motors, and disruption of actin filaments. The disruption of microtubules did not affect the recovery magnitude, but, on the contrary, led to an enhanced recovery rate compared to control cases. These results suggest that the actomyosin machinery is the major active element in axonal contraction, whereas microtubules contribute as resistive/dissipative elements.

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging.

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice(1), we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Stretch induced hyperexcitability of mice callosal pathway.

Memory and learning are thought to result from changes in synaptic strength. Previous studies on synaptic physiology in brain slices have traditionally been focused on biochemical processes. Here, we demonstrate with experiments on mouse brain slices that central nervous system plasticity is also sensitive to mechanical stretch. This is important, given the host of clinical conditions involving changes in mechanical tension on the brain, and the normal role that mechanical tension plays in brain development. A novel platform is developed to investigate neural responses to mechanical stretching. Flavoprotein autofluoresence (FA) imaging was employed for measuring neural activity. We observed that synaptic excitability substantially increases after a small (2.5%) stretch was held for 10 min and released. The increase is accumulative, i.e., multiple stretch cycles further increase the excitability. We also developed analytical tools to quantify the spatial spread and response strength. Results show that the spatial spread is less stable in slices undergoing the stretch-unstretch cycle. FA amplitude and activation rate decrease as excitability increases in stretch cases but not in electrically enhanced cases. These results collectively demonstrate that a small stretch in physiological range can modulate neural activities significantly, suggesting that mechanical events can be employed as a novel tool for the modulation of neural plasticity.