Ultrastructural changes in synaptic and mitochondrial structure throughout postnatal development and long-term potentiation in rat hippocampus




Smith, Heather Lynne

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Mitochondria, by providing the vast majority of ATP produced in neurons, fuel many steps of the synaptic vesicle cycle, but little is known about their role in synaptic vesicle clustering and mobilization during synaptic development and plasticity. Long-term potentiation (LTP), a cellular model for learning and memory, has a protein synthesis dependent late phase (L-LTP) that has been shown to recruit both pre- and postsynaptic mechanisms. In spite of their seeming importance in synaptic function, mitochondria are only found in roughly half of all mature presynaptic boutons, which implies that only a subset of boutons are capable of supporting increases in vesicle release. Preliminary exploration of CA1 boutons from perfusion- xed P15 rats, however, demonstrated that while smaller than boutons that contain mitochondria, boutons that are within three microns of mitochondria contain significantly more vesicles than those that are farther away from them, which raises the possibility that diffusion of ATP from nearby mitochondria could be sufficient to fuel LTP. To determine whether this was the case, I prepared 3D reconstructions from serial electron micrographs (3DEM) in order to quantify mitochondrial distance and vesicle counts in CA1 boutons following long-term potentiation in both P15 and adult Long-Evans rats. At both ages, mobilization of reserve pool vesicles following LTP required the presence of mitochondria. Mitochondria themselves also undergo structural changes following LTP. In adults, mitochondria become longer and less frequent along axons, implying that they undergo fusion. Although P15 mitochondria undergo no change in frequency, mitochondrial cristae become wider as mitochondrial matrix becomes more compact at both ages, which are both changes that correlate well with increased rates of respiration. To even further explore ultrastructural components required to support LTP, I used 3DEM to track changes in synaptic and subcellular structure from P8 to P12, during which animals undergo radical changes in their ability to support LTP. I found that spinogenesis may begin at P10 but increases sharply at P12, which coincides perfectly with the onset age of LTP. In conjunction with this increase in dendritic spines, axons built new single synaptic boutons from clusters of dense core and amorphous vesicles previously known to transport proteins and membrane to developing synapses. To fuel the creation of new boutons, mitochondrial division increased at P12, as evidenced by decreased mitochondrial size and increased mitochondrial frequency.




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