Examining the emergent properties of neuronal networks towards the creation of oscillatory biological machines

The field of neuroscience may be one of the last great frontiers of human medicine. Much of the rest of mammalian physiology has been understood at the macromolecular level with regards to anatomical form and function as well as how organ systems functions. Although gross anatomical form and function of the nervous system is understood, much still needs to be learned about neural development and how cognition and memory function and integrate through neurons. A key part of this will be understanding the emergent properties and development of neuronal circuits and networks. Research has found that neurons respond to a variety of endogenous and extracellular cues in order to develop their specific and unique morphologies. Additionally, many extracellular cues have been found to guide the development of neuronal circuits through neurite differentiation, guidance, and branch and the subsequent formation of functional synapses. We know many of the proteins and other small molecules that play a role in neuronal function, but what has yet to be understood is how all these work in synchrony to create a thinking a processing unit like the central nervous system. The thesis work presented here attempts to fill this gap in our knowledge by using a variety of engineering tools to analyze and guide the development of active neuronal networks. First, glia are analyzed for their role in how functioning neuronal networks develop. Using novel imaging techniques, overall network connectivity is examined to show that glia plays an important role in the development of active networks. Additionally, the role of tension in the developing neural network is examined. Clustering of neuronal bodies has been observed but underreported in many studies. We have found that neurons generate tension upon the substrate and between cells to actively migrate in vitro. These findings may help to explain brain development and structural organization of the nervous system. Beyond understanding self-guided behaviors of neurons, we looked at the role of the extracellular microenvironment in determining the properties of developing neuronal networks. Using a novel 3D engineering scaffold constructed of alginate hydrogels with directed porous alignments, we cultured central and peripheral nervous system neurons. We found that the physical microenvironment plays an important role in the emergent morphological features of neural tissue. By directing neurite outgrowths using an aligned porous hydrogel, we were able to take a step towards creating biomimetic neural tissue that was physiological active. This is a major step towards engineered nervous system tissue that can be used to study neural degenerative diseases, development of neurons, and possible therapies for treatment of diseases. Using extracellular chemoattractants, we also engineered 2D microenvironments for guiding neurite development. We found that a variety of shapes and chemical cues were able to guide axons and dendrites into specific orientations. Neurons followed along vertices of protein-patterned polygons, and along stamped lines of a variety of proteins. Previously, a variety of extracellular cues have been found to either repel or attract axons and dendrites. Using signaling molecules such as semaphorin 3a, a strong repellent of axons and attractant of dendrites, we showed that when given no choice, axons will still grow these proteins as opposed to a generally inhospitable glass surface that has been traditionally used for cell culture. We found that these cultured neuronal networks were still physiologically active through synaptic signal transduction. This work illustrates the complexity of neuronal development and neurite differentiation. No single factor has been shown to account for the bipolar morphologies of neurons and disruption of a single factor, where it is intracellular of extracellular, is not enough to prevent neuronal network formation and function. The end-product of understanding emergent properties of neuronal network formation is to develop in vitro platforms for engineered biological machines capable of integrating sensory information towards creating a programmed response. Previously studies have engineered integrated machines with a soft polymer skeleton and either cardiomyocytes or skeletal muscles for actuation. These machines used optogenetics as a means of external control of force generation but have lacked neuronal control. Towards this goal, this research has taken a significant step in the development of “smart” biological machines containing a neuronal oscillator. After extracting the lumbar region of a spinal cord, we have combined it with a muscle-containing biobot and formed active neuromuscular junctions. We found that stimulation of the spinal cord neurons either chemically or electrically is enough to generate force and movement of the machine. This biobot is a novel development in the area of tissue engineering and has potential to be used as a device in health, security, and medicine. The thesis work presented here has made significant advancements in understand the emergent properties of developing neuronal networks. It has examined the role of glia and tension in network formation. The extracellular microenvironment has also been found to play a significant role in the development of neuronal networks to create functional biomimetic tissue and guidance of neurites in creating active neuronal networks. We have incorporated this understanding of the properties of network development in engineering a novel biological machine that is neuronally mediated and capable of generating force and move in response to electrochemical stimulation of its spinal cord. This work helps to advance neuroscience towards a greater understanding of nervous system development and utilizing this knowledge for a variety of engineering purposes to further human technological advances