The Search for and Formation of Synapses on Zebrafish Motoneurons Across Natural Periods of Activity and Quiescence

Abstract

The work presented in this dissertation is an in-depth analysis of how a single neuron, the primary motoneuron of zebrafish, searches for and forms synapses across the circadian periods of activity and quiescence that occur from day to night. Dendrites repeatedly extend and retract filopodia in a search for synapses, the formation of which stabilizes the process, leading to growth of the arbor. I first focused my attention on zebrafish primary neurons during the day to explore, for the first time, the search process from the level of individual filopodial dynamics to the distribution of dynamics across an entire dendritic arbor. We found the magnitude of searching at individual locations varied tremendously across locations, with filopodia extending and retracting as much as 3 microns and averaging about 12 dynamics events per filopodium in a given 30-minute period. An analysis of the temporal sequence of these dynamic events showed the pattern of dynamics at individual locations also varied tremendously. Only retractions showed a consistent trend, tending to stabilize for some time and rarely being followed by an extension. This shows that dendrites are not just simply extending and stabilizing filopodia. Instead, filopodia at each individual location are engaging in highly dynamic periods of searching, with most extensions retracting within 5 minutes. Only about 4% of extensions survive for an hour or more, possibly representing the formation of synapses. We further find that these dynamic locations are equally distributed across the dendrite arbor independent of their individual dynamics and only the furthest extents of dendrite had higher than average numbers of motile locations, suggesting the search is largely unbiased except for those furthest regions. A comparison of how the search differs between day and night shows surprisingly, that the magnitude of this searching increases at night, a quiescent time, when motoneuron are much less active. While the extensions at night are less likely to stabilize, this increase in searching may partly account for these cells still forming synapses at a similar rate as during the day, with only the rate of synapse removal changing, increasing significantly at night. These results demonstrate that during times of behavioral and activity quiescence, neurons continue to search for and form synapses at the expense of losing others, providing potential insight into the role these quiescent states may play in network development and neuronal plasticity.