Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 158-196).

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--Indiana University, 2006. / Title from screen (viewed on Apr. 27, 2007) Department of Pharmacology & Toxicology, Indiana University-Purdue University Indianapolis (IUPUI) Includes vita. Includes bibliographical references (leaves 181-239)

by Vernon Rowland and Robert Blumenthal ; Yvonne M. Homsy, writer-editor. / "First published as volume 12, no. 1, March 1974, of the Neurosciences Research Program bulletin." Includes index. / Bibliography: p. 154-187.

The striatum is the major input structure of the basal ganglia, and is composed of two major populations of spiny projection neurons (MSNs), which give rise to the socalled direct and indirect pathways, and several types of interneuron. Dopaminergic inputs to striatum are critical for its proper function. Indeed, loss of dopaminergic neurons in Parkinsonism leads to motor disturbances, grossly disturbs striatal activity, and is associated with the emergence of excessively-synchronized network oscillations at beta frequencies (15-30 Hz) throughout the basal ganglia. How the distinct structural, neurochemical and other properties of striatal neurons are reflected in their firing rates and patterns in vivo is poorly defined, as are their possible cell-type-selective contributions to the aberrant oscillations arising in the Parkinsonian brain. To address these issues, I first used multi-electrode arrays to record the spontaneous firing of ensembles of neurons in dorsal striatum in both anaesthetised dopamine-intact and Parkinsonian (6-hydroxydopamine-lesioned) rats during two well-defined brain states, slow-wave activity (SWA) and spontaneous activation. The chronic loss of dopamine led to an overall increase in the average firing rates of striatal neurons, irrespective of brain state. However, many neurons in the Parkinsonian striatum still exhibited the low firing rates and irregular firing patterns typical of neurons in the dopamine-intact striatum. During SWA in Parkinsonian rats, the firing of striatal neurons was more strongly synchronized at low frequencies, in time with cortical slow (~1 Hz) oscillations. During spontaneous cortical activation in Parkinsonian rats, more striatal neurons engaged in synchronized firing in time with cortical beta oscillations. Under the same experimental conditions, I then recorded the spontaneous firing of individual striatal neurons and juxtacellularly labelled the same neurons to verify their cell types, and locations; indirect pathway and direct pathway MSNs were distinguished by the expression (and lack of expression respectively), of the neuropeptide precursor preproenkephalin (PPE). After chronic dopamine loss, and on average, only indirect pathway (PPE+) MSNs significantly increased their firing rates during both brain states, and engaged in widespread, synchronized firing in the beta-frequency range. This did not hold true for all PPE+ MSNs; the Parkinsonian striatum contained many MSNs that were virtually quiescent, which were just as likely to belong to the indirect pathway as the direct pathway. Direct pathway (PPE-) MSNs increased their firing only during SWA after chronic dopamine loss and rarely engaged in aberrant beta oscillations. Taken together, these data suggest that (1) the firing patterns, as well as the firing rates of many striatal neurons are grossly disturbed by chronic loss of dopamine and (2) that the pathological synchronization of the rhythmic firing of a subpopulation of indirect pathway MSNs could contribute to the propagation of aberrant beta-frequency oscillations to downstream basal ganglia nuclei in Parkinsonism.

In the case of Parkinson’s disease, a common neurodegenerative disorder, the loss of motor function results from the selective degeneration of dopaminergic neurons (DNs) in the brain. Current treatments focus on pharmacological approaches that lose effectiveness over time and produce unwanted side effects. A more complete concept of rehabilitation to improve on current treatments requires the production of DNs to replace those that have been lost. Although pluripotent stem cells (PSCs) are a promising candidate for the source of these replacement neurons, current protocols for the terminal differentiation of DNs require a complicated cocktail of factors. Recently, a naturally occurring steroid called guggulsterone has been shown to be an effective terminal differentiator of DNs and can simplify the method for the production of such neurons. I therefore investigated the potential of long-term guggulsterone release from drug delivery particles in order to provide a proof of concept for producing DNs in a more economical and effective way. Throughout my study I was able to successfully encapsulate guggulsterone in Poly-ε-caprolactone (PCL)-based microspheres and I showed that the drug was capable of being released over 44 days in vitro. These guggulsterone-releasing microspheres were also successfully incorporated in human induced pluripotent stem cell (hiPSC)-derived neural aggregates (NAs), providing the foundation to continue investigating their effectiveness in producing functional and mature DNs. Together, these data suggest that guggulsterone delivery from microspheres may be a promising approach for improving the production of implantable DNs from hiPSCs. / Graduate

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No description available.

Dopamine exerts an important role in the regulation of motor activity in humans. During the progression of Parkinson’s disease, patients are faced with the progressive neurodegeneration of nigro-striatal dopamine neurons resulting in an array of pathological symptoms characteristic of the disease. Current treatment relies on targeting symptomatic aspects of the disease but currently Parkinson’s disease is incurable. Targeting the regeneration of DA neurons in PD patients could offer an alternative therapeutic approach that could stall and perhaps even revert the progression of the disease and improve the quality of life for patients. Here, I describe the generation of a transgenic zebrafish line for the non-invasive, conditional and specific ablation of dopaminergic neurons in both larval and adult zebrafish. Understanding the endogenous regenerative ability of the zebrafish may in the future contribute to the development of novel therapeutic approaches targeting DA neuron regeneration in humans. The Tg(dat:CFP-NTR) line efficiently labels and ablates most clusters of DA neurons in both the larval and the adult zebrafish brain. Neuronal ablation is followed by a locomotor and tail bend phenotype as well as by an increase in exploratory behavior. Using double transgenic larvae, we showed through live imaging that loss of DA neurons induces an increase in nestin expression; in addition we show an increase in the number of proliferating cells and an up regulation of genes involved in neurogenesis and tissue repair. Adult zebrafish were able to fully recover their DA neuronal population in the olfactory bulb within 45 days post ablation. Overall the Tg(dat:CFP-NTR) zebrafish offers a novel tool for the study of the molecular and cellular mechanisms driving the regeneration of DA neurons in the zebrafish brain and will be a useful tool for the field of regenerative medicine.

The Distal-less homeobox (Dlx) genes encode homeodomain transcription factors found in all animals of the phylum Chordata. These genes are involved in early vertebrate development of limbs, sensory organs, branchial arches and the forebrain (telencephalon and diencephalon). The mouse and human genomes each have six Dlx genes organized into convergently transcribed bigene clusters (Dlx1/2, Dlx3/4 and Dlx5/6). In the forebrain, Dlx1/2 and Dlx5/6 genes play essential roles in GABAergic neuron proliferation, migration and survival. Each bigene cluster includes a short intergenic region (~3.5-16kb) harboring cis-regulatory elements (CREs) that control expression of the Dlx genes. The Dlx1/2 intergenic region harbors the I12b/I12a CREs, while Dlx5/6 includes I56i/I56ii. In determining the regulatory roles of the CREs on Dlx activity and forebrain development, I have characterized the phenotypic changes that occur in mice that have an I56i enhancer deletion. I have also characterized mice with double deletions of I56i and I12b as well as mice that harbored an I12b deletion and have a SNP in the I56i enhancer (vI56i). Mutant mice with a single targeted deletion of I56i are viable, fertile and do not show obvious developmental defects. These mice have significant decreases in Dlx5/6, Gad1/Gad2 and Evf-2 expression in the forebrain and have defects related to GABAergic neuron development. The ΔI56i mutants demonstrate a behavioral phenotype related to anxiety and learning deficits. Mice that lack the I12b enhancer and have the vI56i do not show morphological abnormalities but have severely disrupted Dlx expression. When mice are homozygous for the I56i and I12b enhancer deletion, they do not survive past post natal day 5 and exhibit a dwarfed body size. These mice look weak and seem to have limited motor ability. In characterizing mice with targeted deletions of highly conserved Dlx enhancers, we will have a better understanding of forebrain development.