Developmental effects of nonuniform acetylcholine receptor expression on motor neuron and synapse morphology in larval zebrafish Public Deposited

It is well established that altered postsynaptic receptor activity leads to changes in presynaptic transmission and presynaptic connectivity throughout the nervous system (Turrigiano 2012; Davis and Muller 2015). Receptor-activity-dependent modulation of presynaptic transmission, termed “synaptic plasticity”, was first highlighted from studies at neuromuscular synapses and later recapitulated at central synapses (Robbins and Fishbach 1971, Turrigiano 2004, Slater 2015). The receptor-activity-dependent process responsible for reducing the number of presynaptic inputs from multiple neurons onto a target cell throughout synapse development, a form of synaptic competition, was also first demonstrated at the neuromuscular junction (NMJ) (Wyatt and Balice-Gordon 2003) and later observed at central synapses (Kano and Hashimoto 2009). This study, however, probes for evidence of receptor-activity-dependent intra-neuronal competition between axon terminals from the same motor neuron. In this study, I exploit the advantages provided by zebrafish including viable acetylcholine receptor (AChR) null juvenile fish and transparent bodies allowing for direct imaging of spinal motor neurons and neuromuscular synapses. By injecting AChR-δ subunit plasmid cDNA into single cell AChR-null fish embryos, I was able to stochastically rescue AChR expression in a subset of muscle cells (referred to as “partial rescue”) to create a unique environment whereby the axon terminals of a single motor neuron will be subjected to nonuniform AChR expression and amounts of AChR activity during development. Using partially rescued AChR-null zebrafish, I tested my hypothesis that primary motor axon terminal morphology and synapse number are dependent on the uniformity of AChR expression and AChR activity. Changes in axon terminal morphology and synapse number were measured from three-dimensional reconstructions of confocal images. I used this data to determine if there was evidence of AChR-activity-dependent, intra-neuronal competition. AChR-activity-dependent intra-neuronal competition at the developing zebrafish NMJ would provide evidence of a new, intracellular component of receptor-activity-dependent synaptic competition. The findings from this study, however, demonstrate that axon terminal morphology and synapse number are not dependent on either AChR receptor expression or activity. Despite these morphological results, the tools and techniques developed throughout this study provide the possibility to test whether nonuniform AChR receptor expression or activity impact synaptic transmission using patch clamp electrophysiology.

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