Thesis (Ph.D.)--Ohio University, November, 2004. / Title from PDF t.p. Includes bibliographical references (p. 87-99 )

Indiana University-Purdue University Indianapolis (IUPUI) / In glaucoma, the connection between the eye and the brain is severed leading to the degeneration of retinal ganglion cells (RGCs) and eventual blindness. A need exists to better understand the maturation of human RGCs as well as their degeneration, with the goal of developing new therapeutics diseases like glaucoma. Human pluripotent stem cells (hPSCs) provide an advantageous model for the study of RGC development and disease as they can be differentiated into RGCs in large, reproducible quantities. Efforts of the current studies initially focused on the development and maturation of RGCs from hPSCs. RGCs derived from hPSCs were a diverse population of cells and matured in a temporal fashion, yielding morphological and functional characteristics similar to their in vivo counterpart. CRISPR/Cas9 gene editing was then utilized to insert the OPTN(E50K) glaucomatous mutation into hPSCs to model RGC degeneration. RGCs harboring this mutation exhibited numerous degenerative phenotypes including neurite retraction an autophagy dysfunction. Within the retina, many cell types contribute to the health and maturation of RGCs including astrocytes. As such, a co-culture system of hPSC-derived RGCs and astrocytes was developed to better understand the interaction between these two cell types. When grown in co-culture with astrocytes, hPSC-derived RGCs demonstrated significantly enhanced and accelerated morphological and functional maturation, indicating an important relationship between these cells in a healthy state. Astrocytes have also been shown to encompass neurodegenerative phenotypes in other diseases of the CNS, with these deficits profoundly effecting the health of surrounding neurons. hPSC-derived astrocytes grown from OPTN(E50K)-hPSCs demonstrated cell autonomous deficits and exhibited significant effects on the degeneration of RGCs. Taken together, results of this study demonstrated the utilization of hPSCs to model RGC maturation and degeneration in glaucoma. More so, these results are one of the first to characterize astrocyte deficits caused by the OPTN(E50K) mutation and could provide a new therapeutic target for pharmacological screenings and cell replacement therapies to reverse blindness in optic neuropathies.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is a genetic condition caused by the triplication of human chromosome 21 and presents with many phenotypes including decreased brain size, hypocellularity in the brain, and assorted ocular phenotypes. Some of the ocular phenotypes seen are increased risk of cataracts, accommodation difficulties, increased risk of refractive errors, and increased retinal thickness. The Ts65Dn mouse model of DS is a classically used mouse model as it presents a number of phenotypes also seen in those with DS. Some of these phenotypes include decreased brain volume, abnormal synaptic plasticity, and ocular phenotypes. These ocular phenotypes include decreased visual acuity, cataracts, and increased retinal thickness. The Ts65Dn mouse model is trisomic for Dyrk1a, a gene of interest in DS research. We hypothesize that there will be a genotypic and sex effect of retinal thickness and retinal ganglion cell (RGC) count at postnatal day 15 in the Ts65Dn mouse model of DS. Retinal slices were taken from male and female trisomic and euploid Ts65Dn mice at P15 and fluorescently labeled for RGCs and bipolar cells via immunohistochemistry. The retinas were measured for total retinal thickness and RNA-binding protein (RBPMS) positive cells in the RGC layer were counted. There was no genotypic or sex effect when comparing retinal thickness in trisomic mice as compared to euploid mice. There was a genotypic effect of RBPMS positive cell count in which the trisomic mice had a higher number of RBPMS positive cells than euploid mice. Increased retinal thickness along with increased RGC number have both been implicated with decreased apoptosis in the retina. In the Ts65Dn mouse model along with in individuals with DS, this could be due to an increase in DYRK1A protein levels reducing apoptosis. In future studies, determining DYRK1A’s influence in retinal thickness and RGC number could result in a treatment for overactive DYRK1A that could normalize retinal thickness and RGC number in those with DS.

No description available.

The superior cervical ganglion (SCG) is the largest of the sympathetic chain ganglia which control a number of autonomic cardiovascular reflexes via neural activity in the postganglionic nerve trunk. In addition to the large principal neurons, these ganglia contain a minority population of smaller cells, the small intensely fluorescent or SIF cells, so named because of their intense fluorescence following treatments which reveal the presence of endogenous catecholamines (mainly dopamine in the rat). The physiological functions of the SIF cells are largely unknown and various roles have been proposed including (i) dopaminergic interneuron, which modulates ganglionic transmission, (ii) endocrine function, since many of them have a close association with the vasculature and (iii) chemosensory function, similar to that of the arterial chemoreceptors which sense blood gases and pH. Understanding the physiological role of SIF cells has been hampered by their small size, sparse distribution and relative inaccessibility, all of which render microelectrode electrophysiological studies difficult. In this thesis these limitations were overcome by use of (i) dissociated cell cultures of the rat SCG, in which growth conditions favoured SIF cell survival but not that of the principal neurons, and (ii) the novel high resolution patch clamp/whole cell recording technique which is ideal for the study of the electrophysiology of small cells. The ionic currents, which underlie many basic electrophysiological processes, were characterized in 5-16 day old cultures of SIF cells obtained from the SCG of neonatal rats. The main methodology consisted of whole cell recording under voltage clamp conditions, which permit the study of membrane ionic currents. Five main ionic currents were identified in all of the SIF cells ( > 100) studied: (i) a fast transient inward Na+ current, sensitive to the well-known blocker of voltage-gated Na+ channels i.e. tetrodotoxin or TTX; (ii) the delayed rectifier outward K+ current that is found in a variety of cell types; (iii) a Ca2+- activated outward K+ current, sensitive to Ca2+ channel blockers; (iv) a transient inward Ca2+ current which appears to be carried by N-type Ca2+ channels and (v) a slower, sustained inward Ca2+ current which appears to be carried by L-type Ca2+ channels. In addition a third type of outward K+ current, the fast transient K+ current or lA, was found in SIF cells obtained from 3-7 day old rats, but not from 1 day old rats. It therefore appears that this lA current, which is known to modulate firing frequency in neurons develops rapidly in vivo during the first postnatal week. This broad repertoire of ion channels in SIF cells suggests several possible sites for modulation by various agents including neurotransmitters, neuromodulators, or other chemosensitive agents. Since SIF cells were recently proposed to have arterial chemoreceptor function similar to glomus cells, the effect of one such stimulus, i.e. an acidic (intracellular) pH, was tested. It has recently been suggested that a decrease in intracellular pH is part of the pathway responding to extracellular stimuli in the glomus cell (Stea, Alexander and Nurse, in press). Acidification of the SIF cell's cytoplasm with the K+/H+ ionophore nigericin resulted in a suppression of both the fast inward Na+ current as well as the outward K+ current. However, these effects do not appear to beunique to SIF cells and therefore the possibility of a chemoreceptor role m the cardiovascular system requires further study. In summary, the characterization of the various ionic currents in SIF cells resulting from this thesis provides the necessary background which should eventually resolve not only the question of the physiological role of SIF cells in autonomic ganglia, but also help to understand the underlying mechanisms responsible for SIF cell function. / Thesis / Master of Science (MS)

Ling Eva. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 175-202). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgement --- p.vi / Abbreviations Frequently Used --- p.vii / Table of Contents --- p.viii / Chapter Chapter One --- Introduction --- p.1 / Chapter Chapter Two --- Materials and Methods --- p.35 / Chapter Chapter Three --- Results --- p.64 / Chapter Chapter Four --- Discussion --- p.148 / References --- p.175 / Tables --- p.203

No description available.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 13, 2009). Includes bibliographical references.

Today, it is possible to restore hearing in congenitally deaf children and severely hearing-impaired adults through cochlear implants (CIs). A CI consists of an external sound processor that provides acoustically induced signals to an internal receiver. The receiver feeds information to an electrode array inserted into the fluid-filled cochlea, where it provides direct electrical stimulation to the auditory nerve. Despite its great success, there is still room for improvement, so as to provide the patient with better frequency resolution, pitch information for music and speech perception and overall improved quality of sound.  A better stimulation mode for the auditory nerves by increasing the number of stimulation points is believed to be a part of the solution. Current technology depends on strong electrical pulses to overcome the anatomical gap between neurons and the CI. The spreading of currents limits the number of stimulation points due to signal overlap and crosstalk. Closing the anatomical gap between spiral ganglion neurons and the CI could lower the stimulation thresholds, reduce current spread, and generate a more discrete stimulation of individual neurons. This strategy may depend on the regenerative capacity of auditory neurons, and the ability to attract and guide them to the electrode and bridge the gap. Here, we investigated the potential of cultured human and murine neurons from primary inner ear tissue and human neural progenitor cells to traverse this gap through an extracellular matrix gel. Furthermore, nanoparticles were used as reservoirs for neural attractants and applied to CI electrode surfaces. The nanoparticles retained growth factors, and inner ear neurons showed affinity for the reservoirs in vitro. The potential to obtain a more ordered neural growth on a patterned, electrically conducting nanocrystalline diamond surface was also examined. Successful growth of auditory neurons that attached and grew on the patterned substrate was observed. By combining the patterned diamond surfaces with nanoparticle-based reservoirs and nerve-stimulating gels, a novel, high resolution CI may be created. This strategy could potentially enable the use of hundreds of stimulation points compared to the 12 – 22 used today. This could greatly improve the hearing sensation for many CI recipients.

Neurotrophins BDNF and NT4 influence the development of the rodent gustatory system. Despite binding to the same receptor, TrkB, they have different roles. BDNF is chemo-attractive for gustatory neurons and regulates gustatory neuron targeting and number during development. NT4 regulates gustatory neuron number earlier in development than BDNF, but it is not chemo-attractive and does not regulate gustatory neuron targeting. To elucidate the mechanisms that regulate these processes we have examined which TrkB intracellular signalling pathways are required for specific aspects of gustatory development by studying the effect of specific point mutations in TrkB docking sites. We found that the TrkB/Shc docking site is involved in regulating the survival of geniculate ganglion neurons as a point mutation in this adaptor site (TrkbS/S) caused large losses of these neurons as early as E12.5. These losses were exacerbated throughout development until after birth. A point mutation in the TrkB/PLCγ (TrkbP/P) docking site did not cause loss of geniculate ganglion neurons at any point during development. Animals with a point mutation in both docking sites (TrkbD/D) caused a further decrease in neuron numbers compared to animals with a mutation in only one of the docking sites, similarly to what has previously been shown in Trkb null animals. We concluded that the TrkB/Shc docking site is crucial for determining the survival of geniculate ganglion neurons during mouse gustatory development, while the TrkB/PLCγ docking site does not affect the neuronal survival directly and likely plays a role in maintenance of these neurons. Examining the targeting of geniculate ganglion afferents into the tongue revealed large deficits in innervated neural bud and taste bud numbers in TrkbS/S animals both before and after birth. This was concluded to be reflecting the lack of neuronal survival in this ganglion, a result that was mirrored in TrkbD/D animals. TrkbP/P animals, on the other hand, exhibited a developmental delay in innervation. This was indicated by a low amount of innervated neural buds following the initial innervation period, which was compensated for by a large increase in the number of innervated taste buds by birth. By adulthood, the numbers of taste buds present on the tongues of TrkbP/P animals reached normal numbers compared to control animals. This suggested that the TrkB/PLCγ docking site is involved primarily in innervation. Finally, we examined the morphology of taste buds in newly born and adult animals. We found that the low amount of geniculate ganglion afferents innervating the tongue in TrkbS/S and TrkbD/D animals caused a decrease in size of taste buds. This effect was seen to be partially rescued by adulthood in TrkbS/S animals but not in TrkbD/D animals due to lack of viability. The morphology of taste buds was unaffected in TrkbP/P animals until adulthood, at which point the size of the taste buds was increased. These results are in agreement with previous findings showing dependency of taste bud morphology on the amount of innervation. Overall, our findings show a differential role of TrkB adaptor sites in gustatory development. Despite activated by the same ligands, the docking sites on this receptor are able to exert different influence on signalling pathways downstream of TrkB affecting neuronal survival, targeting and morphology of taste buds.