AUTOMATED ANALYSIS OF NEURONAL MORPHOLOGY: DETECTION, MODELING AND RECONSTRUCTION

Ph.DDOCTOR OF PHILOSOPH

Segmentation strategies for polymerized volume data sets

A new technique, called the polymerization algorithm, is described for the hierarchical segmentation of polymerized volume data sets (PVDS) using the Lblock data structure. The Lblock data structure is defined as a 3dimensional isorectangular block of enhanced vertex information. Segmentation of the PVDS is attained by intersecting and merging Lblock coverings of the enhanced volumetric data. The data structure allows for easy compression, storage, segmentation, and reconstruction of volumetric data obtained from scanning a mammalian brain at submicron resolution, using threedimensional light microscopy (knifeedge scanning microscopy (KESM), confocal microscopy (CFM), and multiphoton microscopy (MPM)). A hybrid technique using the polymerization algorithm and an existing vectorbased tracing algorithm is developed. Both the polymerized and the hybrid algorithm have been tested and their analyzed results are presented

Miniaturized Fluorescence Biosensor for Studying Neuronal Events

When developing new techniques to analyze neuro-chemical microenvironments, it is important to realize the incredible variability in the cellular content and the response to stimulation between cells and within a single cell. Conventional analysis techniques yield an average result to describe the content and function of cells. This approach often misses important information since the onset of pathological conditions is always initiated in a small number of cells. New minimally invasive single cell analysis techniques are required for single cell studies in order to gain new insights and understanding of cells\u27 functions. The objective of my Ph.D. study was to fabricate, characterize, and apply submicrometric fluorescence sensors for the analysis of neuron cells. This dissertation will report the fabrication of miniaturized fluorescence sensors for Ca2+, pH and Zn2+ analysis. In the first approach, liposomes (phospholipid vesicles) were used as miniaturized containers for fluorescent sensing reagents. Liposome-based fluorescence sensing technology offers several advantages over commonly used fluorescence sensing techniques including high spatial resolution, protection of the sensing dye from quenchers and high biocompatibility. However, liposome based sensors were found to be unstable in the cellular environment. The second approach was to synthesize submicrometric particle-based fluorescence sensors named lipobeads to replace the fluorescent liposomes in cellular studies. Lipobeads are polystyrene particles that are coated with a phospholipid membrane. One unique advantage of fluorescent sensing lipobeads is the ability to immobilize hydrophobic indicator molecules in the phospholipid membrane. This enables the use of these indicators in aqueous media since the lipobeads are fully water miscible. The lipobeads also proved to be highly biocompatible in cellular studies. This is attributed to their phospholipid bilayer membrane, which is similar in structure to cell membranes. The dissertation will describe the analytical properties of fluorescence sensing lipobeads and their application in studying zinc ion release and pH changes near neuron cells under physiological conditions, conditions of neuronal injury and stress and acidic cortical spreading depression during stroke like conditions

Miniaturized Fluorescence Biosensor for Studying Neuronal Events

When developing new techniques to analyze neuro-chemical microenvironments, it is important to realize the incredible variability in the cellular content and the response to stimulation between cells and within a single cell. Conventional analysis techniques yield an average result to describe the content and function of cells. This approach often misses important information since the onset of pathological conditions is always initiated in a small number of cells. New minimally invasive single cell analysis techniques are required for single cell studies in order to gain new insights and understanding of cells\u27 functions. The objective of my Ph.D. study was to fabricate, characterize, and apply submicrometric fluorescence sensors for the analysis of neuron cells. This dissertation will report the fabrication of miniaturized fluorescence sensors for Ca2+, pH and Zn2+ analysis. In the first approach, liposomes (phospholipid vesicles) were used as miniaturized containers for fluorescent sensing reagents. Liposome-based fluorescence sensing technology offers several advantages over commonly used fluorescence sensing techniques including high spatial resolution, protection of the sensing dye from quenchers and high biocompatibility. However, liposome based sensors were found to be unstable in the cellular environment. The second approach was to synthesize submicrometric particle-based fluorescence sensors named lipobeads to replace the fluorescent liposomes in cellular studies. Lipobeads are polystyrene particles that are coated with a phospholipid membrane. One unique advantage of fluorescent sensing lipobeads is the ability to immobilize hydrophobic indicator molecules in the phospholipid membrane. This enables the use of these indicators in aqueous media since the lipobeads are fully water miscible. The lipobeads also proved to be highly biocompatible in cellular studies. This is attributed to their phospholipid bilayer membrane, which is similar in structure to cell membranes. The dissertation will describe the analytical properties of fluorescence sensing lipobeads and their application in studying zinc ion release and pH changes near neuron cells under physiological conditions, conditions of neuronal injury and stress and acidic cortical spreading depression during stroke like conditions

Synaptic patterns for reliable circuit function require postembryonic maintenance by Jeb-Alk and normal network activity during embryogenesis

A functioning nervous systems results from complex developmental processes. One requirement is that individual neurons need to form sufficient synaptic connections with adequate partners. Here, molecular signaling and neural activity control morphological development of axons and dendrites and synaptogenesis in order to establish and maintain stable networks. However, mechanisms maintaining stable postembryonic circuits are not well understood and the long-term effects of embryonic neural activity on neuronal morphology and connectivity are unkown. This thesis investigates trans-synaptic, anterograde Jelly-Belly-Anaplastic lymphoma kinase signaling in postembryonic circuit development and elucidates the establishment of synaptic patterns by embryonic neural activity in the motor circuit of Drosophila larva. I demonstrate that Alk activity inhibits the formation of postsynaptic specializations on motoneurons during postembryonic circuit growth by analyzing single cell connectivity. I employ a new Bxb1 integrase-based technique for targeted mutations to show that presynaptic release site number of an upstream interneuron is unchanged but Jeb-Alk seems to elicit a negative feedback that limits the formation of presynaptic filopodia. These Jeb-Alk devoid circuits with altered synaptic patterns produce epilepsy-like seizure behavior. Additionally, in vivo time lapse imaging of dendrites reveals that dendritic growth and postsynaptic synaptogenesis are regulated independently and presynaptic filopodia likely promote dendritic elaboration. During embryogenesis, neural activity adjusts the establishment of synaptic patterns in motoneurons. In a picrotoxin-induced epilepsy-like model, dendritic growth is unaffected, but synaptic input is increased. The number of release sites of an upstream interneuron is again unaffected. In summary, I identified cellular and molecular mechanisms required for the establishment and maintenance of synaptic patterns for reliable circuit function. With novel genetic and imaging techniques, I show embryonic neural activity is pivotal for the formation of functionally stable synaptic patterns, and establish Jeb-Alk signaling as a negative regulator of circuit expansion maintaining embryonically established connectivity. These developmental mechanisms highlight that balancing pre- and postsynaptic growth and synaptogenesis is central to stable network function

Microenvironmental Control in Microfluidic Bioreactors for Long Term Culture of Bone Marrow Cells.

The goal of this research is to create in vitro microenvironments for long term culture of hematopoeitic stem cell (HSC) in microfluidic bioreactors. In vivo, HSCs reside in the bone marrow osteoblastic and vascular niches in adult mammals. Defining features of their in vivo niche include: small number of HSCs, heterogeneous population of bone marrow cells that support HSCs, and low oxygen tension. We engineer niche elements in microfluidic bioreactors by modulating oxygen tension, optimizing attachment and growth of HSC-supporting bone marrow stromal cells, and culturing small numbers of HSCs in their physiologically relevant ratios between HSCs and supporting cells. By using a combination of a mathematical model and quantitative experiments, we have created a design tool to manipulate and control oxygen tension for cell culture inside the poly(dimethyl siloxane) (PDMS) microbioreactors. Dissolved oxygen concentrations in the microbioreactor are quantified in real time using fluorescence lifetime imaging of an oxygen sensitive dye. Experimental results are consistent with the mathematical model and give insight into operating conditions required for a desired oxygen tension in cell culture regions of the microbioreactor. We used microfluidic perfusion systems to develop nanocoatings made from electrostatic self assembly of PDDA (poly(diallyldimethyl ammonium chloride)), clay, type IV collagen and fibronectin to optimize attachment of primary murine bone marrow cells (support cells for HSCs) onto PDMS bioreactors. PDDA-topped coatings were found to be cytotoxic, while coatings with two or more bilayers of proteins collagen and fibronectin were found to optimize spreading, proliferation, and viability as compared to other surfaces. On-chip erythropoiesis was achieved with a 3-D co-culture of HSCs with supporting cells in PDMS bioreactors. In addition, an optimal ratio of support cells to HSCs was found to maximize self renewal potential of HSCs in vitro. By the combination of hypoxia (which simulates in vivo bone marrow oxygen tension), biofunctional surfaces, and 3-D co-cultures, we are moving towards a ‘microfluidic HSC niche’, in which hypothesis-driven studies about crosstalk between HSCs and stromal cells can be carried out.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/60852/1/mehtagee_1.pd

Dopamine and cAMP Regulated Phosphoprotein, 32 kDA: A Novel Therapeutic in Traumatic Brain Injury

Traumatic brain injury (TBI) represents a significant cause of death and disability in industrialized countries. Of particular importance to patients is the chronic effect that TBI has on cognitive function. Therapeutic strategies have been difficult to evaluate because of the complexity of injuries and variety of patient presentations within a TBI population. Experimental therapies based upon cortical and hippocampal neuroprotection have not translated clinically. However, pharmacotherapies targeting dopamine (DA) have consistently shown benefits in attention, behavioral outcome, executive function, and memory. Striatal damage causes deficits in executive function, learning, and memory. Dopamine and cAMP regulated phosphoprotein 32 (DARPP-32), expressed within striatal medium spiny neurons, is known to regulate several substrates of cognition. We found that controlled cortical impact injury in rats produces a chronic decrease in DARPP-32 threonine-34 phosphorylation and increase in protein phosphatase-1 activity. There is no effect of injury on threonine-75 phosphorylation or DARPP-32 protein. Amantadine has known benefits on post-TBI cognitive deficits and when given daily for two weeks reversed the DARPP-32 and protein phosphatase-1 changes. Amantadine also decreased the phosphorylation of threonine-75 consistent with activity as a partial N-methyl-D-aspartic acid receptor antagonist and partial dopamine agonist. FK-506, also known as tacrolimus, is a calcineurin inhibitor that has been shown to decrease cell death in the hippocampus following a fluid percussion experimental TBI. Calcineurin is also an important regulator of DARPP-32 phosphorylation in the striatum. We evaluated the effect of FK-506 on the hippocampus and DARPP-32 in the striatum to better detail its effects after a TBI. An acute administration of FK-506 following controlled cortical impact reversed the effects of TBI on DARPP-32 phosphorylation seen chronically. We then evaluated the effect of a combined drug therapy on cognitive deficits post TBI. An acute treatment with FK-506 post TBI followed by chronic Amantadine therapy demonstrated an improvement in both motor behavior and Morris water maze deficits seen following TBI. Neither drug produced benefit when given alone. These data demonstrate that DARPP-32 represents a promising new therapeutic target for TBI induced cognitive deficits