Optimizing dye microinjection for labeling single cells in the lateral superior olive of rats

Abstract

A healthy nervous system relies on the balance between excitation and inhibition. Much of what we know about the early development of circuits comes from excitatory projections. The development of inhibitory projections has been especially challenging to study due to their diversity and abundance. The lateral superior olive (LSO), a nucleus responsible for sound localization, is an established model for studying how inhibitory inputs develop because the inhibitory projections to LSO cells are of a similar type and can be easily manipulated. The excitatory and inhibitory inputs to the LSO go through substantial structural and functional changes before and after hearing onset. Previous research has shown that after hearing the inhibitory inputs of the medial superior olive (MSO), a neighboring nucleus also responsible for sound localization, increase in number and density on the soma while decreasing on the distal dendrites. In the MSO, the redistribution of inhibitory synapses has been shown to be activity-dependent, controlled by acoustically-driven activity after hearing onset. However, throughout the superior olive, many developmental events occur both before and after hearing onset, and in the LSO, significant functional reorganization has been demonstrated before hearing onset. In order to determine whether inhibitory or excitatory synapses show redistribution before or after hearing onset in the LSO, fluorescent dye was microinjected into the principal cells of the rat LSO. Excitatory and inhibitory synapses were identified using immunolabeling. The dataset is small, as significant troubleshooting was needed to label cells in older tissue. Here, I report on optimized parameters for labeling individual cells, and I show pilot data that support a pattern of synaptic redistribution that starts before hearing onset and continues after, with inhibitory synapses aggregating on the soma and proximal dendrites as the excitatory synapses decrease in number on the soma and proximal dendrites.