neuroscience, nanoscience, tissue engineering, stretch reflex arc, myelination
The overall focus of this research project was to develop an in vitro tissue-engineered system that accurately reproduced the physiology of the sensory elements of the stretch reflex arc as well as engineer the myelination of neurons in the systems. In order to achieve this goal we hypothesized that myelinating culture systems, intrafusal muscle fibers and the sensory circuit of the stretch reflex arc could be bioengineered using serum-free medium formulations, growth substrate interface design and microfabrication technology. The monosynaptic stretch reflex arc is formed by a direct synapse between motoneurons and sensory neurons and is one of the fundamental circuits involved in motor control. The circuit serves as a proprioceptive feedback system, relaying information about muscle length and stretch to the central nervous system (CNS). It is composed of four elements, which are split into two circuits. The efferent or motor circuit is composed of an [alpha]-motoneuron and the extrafusal skeletal muscle fibers it innervates, while the afferent or sensory circuit is composed of a Ia sensory neuron and a muscle spindle. Structurally, the two muscular units are aligned in parallel, which plays a critical role modulating the system's performance. Functionally, the circuit acts to maintain appropriate muscle length during activities as diverse as eye movement, respiration, locomotion, fine motor control and posture maintenance. Myelination of the axons of the neuronal system is a vertebrate adaptation that enables rapid conduction of action potentials without a commensurate increase in axon diameter. In vitro neuronal systems that reproduce these effects would provide a unique modality to study factors influencing sensory neuronal deficits, neuropathic pain, myelination and diseases associated with myelination. In this dissertation, results for defined in vitro culture conditions resulting in myelination of motoneurons by Schwann cells, pattern controlled myelination of sensory neurons, intrafusal fiber formation, patterned assembly of the mechanosensory complex and integration of the complex on bio-MEMS cantilever devices. Using these systems the stretch sensitive sodium channel BNaC1 and the structural protein PICK1 localized at the sensory neuron terminals associated with the intrafusal fibers was identified as well as the Ca2+ waves associated with sensory neuron electrical activity upon intrafusal fiber stretch on MEMS cantilevers. The knowledge gained through these multi-disciplinary approaches could lead to insights for spasticity inducing diseases like Parkinson's, demyelinating diseases and spinal cord injury repair. These engineered systems also have application in high-throughput drug discovery. Furthermore, the use of biomechanical systems could lead to improved fine motor control for tissue-engineered prosthetic devices.
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Doctor of Philosophy (Ph.D.)
College of Sciences
Length of Campus-only Access
Doctoral Dissertation (Open Access)
Rumsey, John, "Tissue Engineered Myelination And The Stretch Reflex Arc Sensory Circuit: Defined Medium Formulation, Interface Design And Microfabrication" (2009). Electronic Theses and Dissertations. 3826.
Video 1: Ca2+ flux in a type Ia sensory neuron generated after field stimulation of intrafusal myotubes on cantilevers (B & W)
Chapter 9 - Video 1 - ca11_pseudo_color.avi (10421 kB)
Video 1: Ca2+ flux in a type Ia sensory neuron generated after field stimulation of intrafusal myotubes on cantilevers (Color)
Chapter 9 - Video 2 - Ca flux blocked by amiloride -4.avi (81989 kB)
Video 2: Ca2+ flux attenuation after amiloride blockage of mechanically sensitive ion channels in DRG+ESM co-cultures on cantilevers