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Ubiquitin Expression in the Lumbar Spinal Cord Motoneurons of Postnatal Mice-- an Immunohistochemical StudyChaube, Sanjay 12 1900 (has links)
Maturation of spinal motoneurons in rodents is characterized by a period of cell loss in the embryo, but researchers have claimed that some cell death occurs postnatally. This form of cell death is called apoptosis and involves active participation of the cell. Apoptotic cells have certain recognizable morphological and molecular features. I have used a monoclonal antibody against ubiquitin, (a putative marker of apoptotic cells), to do immunochemistry on mouse spinal cords at various postnatal ages till early adulthood. Staining is seen in large amotoneurons in the ventral horn. Staining is intense till P28, and faint thereafter. Substantial proportions of motoneurons stain till P21, followed by a sharp decline in the number of immunopositive cells. None of the cells exhibit signs of apoptosis.
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Investigations of neuronal network responses to electrical stimulation in murine spinal cultures.Sparks, Christopher A. 12 1900 (has links)
Spontaneous activity in neuronal networks in vitro is common and has been well documented. However, alteration of spontaneous activity in such networks via conditioning electrical stimulation has received much less experimental attention. Two different patterns of electrical stimulation were used to enhance or depress the level of spontaneous activity in spinal cord cultures. High-frequency stimulation (HFS), a method routinely shown to increase the efficacy of synaptic transmission, was employed to augment spontaneous activity. Low-frequency stimulation (LFS), the technique often applied to depress synaptic efficacy, was employed to decrease spontaneous activity. In addition, LFS was used to reverse the effect of HFS on spontaneous activity. Likewise, HFS was applied to counter the effect of LFS. Because these networks were grown on multi-microelectrode plates (MMEPs), this allowed the simultaneous stimulation of any combination of the 64 electrodes in the array. Thus, the possible differences in response to single versus multi-electrode stimulation were also addressed. Finally, test-pulses were delivered before and after the conditioning stimulation on the same stimulation electrode(s) in order to assess the change in mean evoked action potentials (MEAPs). Dissociated spinal tissue from embryonic mice was allowed to mature into self-organized networks that exhibited spontaneous bursting activity after two weeks of incubation. Spontaneous activity was monitored from up to 14 recording channels simultaneously. Although uniform responses to stimulation across all recording electrodes were rarely observed, a large majority of the recording channels had similar responses. Spontaneous activity was increased in 52% of 89 HFS trials, whereas activity was decreased in 35% of 75 LFS trials. The duration of most of these increases was less than 5 minutes. When there were substantial and long-term (> 15 min) changes in spontaneous activity, the opposing stimulation pattern successfully reversed the effect of the previous stimulation. The percent change in MEAPs following conditioning stimulation suggested that synaptic modification had taken place in 75% of all test-pulse stimulation trials.
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Mutation of Polaris, an Intraflagellar Transport Protein, Shortens Neuronal CiliaMahato, Deependra 08 1900 (has links)
Primary cilia are non-motile organelles having 9+0 microtubules that project from the basal body of the cell. While the main purpose of motile cilia in mammalian cells is to move fluid or mucus over the cell surface, the purpose of primary cilia has remained elusive for the most part. Primary cilia are shortened in the kidney tubules of Tg737orpk mice, which have polycystic kidney disease due to ciliary defects. The product of the Tg737 gene is polaris, which is directly involved in a microtubule-dependent transport process called intraflagellar transport (IFT). In order to determine the importance of polaris in the development of neuronal cilia, cilium length and numerical density of cilia were quantitatively assessed in six different brain regions on postnatal days 14 and 31 in Tg737orpk mutant and wildtype mice. Our results indicate that the polaris mutation leads to shortening of cilia as well as decreased percentage of ciliated neurons in all brain regions that were quantitatively assessed. Maintainance of cilia was especially affected in the ventromedial nucleus of the hypothalamus. Furthermore, the polaris mutation curtailed cilium length more severely on postnatal day 31 than postnatal day 14. These data suggests that even after ciliogenesis, intraflagellar transport is necessary in order to maintain neuronal cilia. Regional heterogeneity in the effect of this gene mutation on neuronal cilia suggests that the functions of some brain regions might be more compromised than others.
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Optimization of Cell Culture Procedures for Growing Neural Networks on Microelectrode ArraysSanta Maria, Cara L. 12 1900 (has links)
This thesis describes the development of an optimized method for culturing dissociated, monolayer neuronal networks from murine frontal cortex and midbrain. It is presented as a guidebook for use by cell culture specialists and laboratory personnel who require updated and complete procedures for use with microelectrode array (MEA) recording technology. Specific cell culture protocols, contamination prevention and control, as well common problems encountered within the cell culture facility, are discussed. This volume offers value and utility to the rapidly expanding fields of MEA recording and neuronal cell culture. Due to increasing interest in determining the mechanisms underlying Parkinson's disease, the newly developed procedures for mesencephalon isolation and culture on MEAs are an important research contribution.
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Roles of Primary Cilia in the Oligodendrocyte LineageSubedi, Ashok 12 1900 (has links)
Primary cilia are nonmotile, hair-shaped organelles that extend from the basal body in the centrosome. The present study is the first investigation of this organelle in the oligodendrocyte lineage in vivo. I used immunohistochemical approaches in normal and cilia-deficient mutant mice to study cilia in relation to oligodendrogenesis and myelination. Primary cilia immunoreactive for Arl13b and ACIII were commonly present in NG2+ oligodendrocyte progenitor cells (OPCs), in which cilia-associated pathways control proliferation, differentiation, and migration. The loss of primary cilia is generally associated with enhanced Wnt/β-catenin signaling, and Wnt/β-catenin signaling has been shown to promote myelin gene expression. I examined whether the lack of cilia in the oligodendrocyte lineage is associated with elevated Wnt/β-catenin activity. I found that absence of a primary cilium was associated with with higher levels of TCF3, and with β-galactosidase in Axin2-lacZ Wnt reporter mice. This evidence supports the proposal that cilia loss in oligodendrocytes leads to enhanced Wnt/β-catenin activity, which promotes myelination. Cilia are dependent on the centrosome, which assembles microtubules for the cilium, the cytoskeleton, and the mitotic spindle. Centrosomes are the organizing center for microtubule assembly in OPCs, but this function is decentralized in oligodendrocytes. I found that the intensity of centrosomal pericentrin was reduced in oligodendrocytes relative to OPCs, and γ-tubulin was evident in centrosomes of OPCs but not in mature oligodendrocytes. These decreases in centrosomal proteins might contribute to functional differences between OPCs and oligodendrocytes. The importance of cilia in the oligodendrocyte lineage was examined in Tg737orpk mice, which have a hypomorphic IFT88 mutation resulting in decreased cilia numbers and lengths. These mice showed marked, differential decreases in numbers of oligodendrocytes and myelin, yet little or no change in OPC populations. It appears that sufficient cells were available for maturation, but lineage progression was stalled. There were no evident effects of the mutation on Wnt/β-catenin. Factors that might contribute to the abnormalities in the oligodendrocyte lineage of Tg737orpk mice include decreased cilia-dependent Shh mitogenic signaling and dysregulation in cilia-associated pathways such as Notch and Wnt/β-catenin.
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The origin and early development of the intrinsic innervation in the foetal mouse lungTollet, Cecilia Jenny January 2003 (has links)
In this study, the origin and development of the intrinsic innervation in the foetal mouse lung is described and experimental evidence is provided to support the involvement of glial cell line-derived neurotrophic factor (GDNF) in the guidance of nerves and neuronal precursors in the developing lung. Antibodies were used to stain for neuronal precursors, neurones, nerve fibres, primordial epithelium and smooth muscle. These structures were revealed in whole mounts of foetal mouse lungs by immunofluorescence and confocal microscopy, and their spatial and temporal distribution was mapped from the onset of lung development and through the pseudoglandular period. The results showed that neuronal precursors, positive for neural crest cell markers, were present in the vagal tract of the foregut at embryonic day 10 (E10), the time of the evagination of the lung buds. These neural crest-derived cells (NCC) migrated into the lung at E11, along nerve processes directed from the vagus to the smooth musclecovered trachea and emerging lobar bronchi. During E11-E14, a network of nerves and ganglia became established along the dorsal trachea, and large ganglia formed a plexus at the ventral hilum. Nerve trunks issued from these ganglia, travelled along the smooth muscle-covered bronchi, providing a pathway for migrating NCC. To investigate the role of GDNF in the innervation of the lung, an in vitro model of left lung lobes was established. Lung growth and tubule branching was comparable to that in vivo, and neural tissue and smooth muscle continued to grow and thrive. A significant increase in nerve growth occurred when explants were cultured with GDNF compared to controls. Nerves extended, and NCC migrated towards GDNF-impregnated beads suggesting that GDNF may be the molecule guiding nerve fibres and NCC in the lung. The migrating NCC were negative for GDNF-family receptor α1 (GFRα1) during their migration into the lung while the nerves were positive. Since GDNF needs to be associated with its binding receptor, GFRα1, for cellular signalling, GDNF may induce the migration of the NCC if they migrate along the GFRα1-positive nerve fibres. It is concluded that neural tissue and smooth muscle become integral components of the lung shortly after the onset of lung development. The results show that the migration of neural crest-derived cells into the lung and the establishment of the innervation requires coordinated cross-talk between NCC, nerves and smooth muscle throughout development.
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