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Title
Neuromuscular transmission in a naturally occurring mouse mutant of the ß subunit of the neuronal calcium channel
Creator
Molina Campos, Elizabeth
Date
2011
Collection
Electronic Theses & Dissertations
Description
Entry of Ca2+ through voltage gated calcium channels (VGCCs) into nerve terminals is a necessary step coupling the action potential to release of acetylcholine (ACh). VGCCs are heteromultimeric complexes of α1, α2δ, and β subunits, and sometimes γ subunits. The specific α1-β combination assembled determines the channel properties. The mouse mutant lethargic (lh) has severe neurological defects due to a...
Show moreEntry of Ca2+ through voltage gated calcium channels (VGCCs) into nerve terminals is a necessary step coupling the action potential to release of acetylcholine (ACh). VGCCs are heteromultimeric complexes of α1, α2δ, and β subunits, and sometimes γ subunits. The specific α1-β combination assembled determines the channel properties. The mouse mutant lethargic (lh) has severe neurological defects due to a mutation that deletes α1 subunit interaction domain of the β4 subunit. β4 normally associates with the α1A subunit of the P/Q-type VGCCs, and has a major role in stabilizing the final α1A subunit conformation and targeting it to the cell membrane. Loss of the β4 subunit could alter the channel characteristics and localization of α1A. The overall goal of this dissertation was to test the hypothesis that disruption of the β4 subunit affects the function of the α1A subunit of the P/Q-type VGCCs. Electrophysiological recordings were performed at neuromuscular junctions (NMJs) of adult lh and wild type (wt) mice. The quantal content and phrenic nerve evoked release showed a significant decrease in lh with respect to wt. The frequency of spontaneous release of ACh also decreased significantly, although the reduction was only evident when Ca2+ was replaced by Sr2+ or Ba2+ as charge carriers. The amplitude of spontaneous release was not affected by this mutation, implying that each vesicle contains approximately the same amount of ACh in wt and lh mice. These results are due to a significantly slower process of neurotransmitter vesicles release, as confirmed by FM1-43 staining method.There are specific VGCCs antagonists that can be used to determine the contribution of the different types of VGCCs in nerve-stimulated ACh release from motor nerve terminals. ω-agatoxin IVA and SNX-482, specific antagonists for P/Q- and R-type VGCCs respectively, significantly reduced the quantal content in adult lh mice. Immunolabeling of VGCC subunits revealed an increase in α1E, β1 and β3, but no apparent change in the levels of α1A at adult lh neuromuscular junctions. Therefore, lh animals control ACh release by P/Q- and R-type VGCCs. The studies of this dissertation provide evidence for: 1) decreased nerve-evoked ACh release in lh mice, 2) slowed vesicle release process in lh mice, 3) increased level of β1 and β3, compensating for the lack of β4 subunit, and 4) P/Q- and R-type VGCC involvement in release of ACh from motor nerve terminals.
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Title
Pathology at the neuromuscular junction in mouse models of spinal bulbar muscular atrophy
Creator
Poort, Jessica Erin
Date
2014
Collection
Electronic Theses & Dissertations
Description
Spinal bulbar muscular atrophy (SBMA) is a progressive, late onset neuromuscular disease that results in muscle weakness and atrophy, as well as motoneuron death in men. While pathology at the neuromuscular junction (NMJ) is noted in numerous neurodegenerative diseases, disease-related changes at the NMJ in SBMA have not been explored. Characterizing such changes is not only important for determining whether the NMJ has any role in the functional changes underlying motor dysfunction, but also...
Show moreSpinal bulbar muscular atrophy (SBMA) is a progressive, late onset neuromuscular disease that results in muscle weakness and atrophy, as well as motoneuron death in men. While pathology at the neuromuscular junction (NMJ) is noted in numerous neurodegenerative diseases, disease-related changes at the NMJ in SBMA have not been explored. Characterizing such changes is not only important for determining whether the NMJ has any role in the functional changes underlying motor dysfunction, but also in determining how such potential pathology at the NMJ develops as disease progresses. If for example, pathology emerges first at the NMJ followed by motoneuron death, then the NMJ offers future promise as a therapeutic target for preventing or reversing symptoms of SBMA before motoneurons are lost. We evaluated three different mouse models of SBMA, one overexpressing a wildtype androgen receptor (AR) exclusively in muscle fibers (so called "myogenic" model), a second which expressed the endogenous AR gene with the first exon of the human mutant AR gene "knocked in" (the so called "knock-in" model), and a final model that broadly expresses a full length human AR transgene harboring the SBMA mutation (the so called "97Q" model). Using both confocal microscopy and electron microscopy, I found that all three mouse models show a pathological fragmentation of the NMJ suggestive of functionally weakened synapses. Other changes at the neuromuscular synapse suggesting decreases in synaptic strength that were found in some but not all models include a decline in the number of docked vesicles ready for release in nerve terminals, a widening of synaptic clefts, simplified postsynaptic folds, and an abnormal accumulation of synaptic vesicle and neurofilament proteins. Retrograde axonal transport of endosomes was also characterized in the 97Q model using live imaging confocal microscopy. Despite previously published data, I found no evidence for a disease-related defect in retrograde transport in the 97Q model. The strikingly abnormal morphology of NMJs in all three models raises the possibility that synaptic function is impaired. Such synaptic dysfunction may contribute to or underlie the impairments in motor function associated with SBMA.
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