Globular bushy cells in the cochlear nucleus of the auditory brainstem circuit are responsible for the fast relay of signals to the superior olivary complex. Their axons possess a low internode length/axon diameter ratio (L/D) of ~60 that allow action potentials to travel along their axons with exceptional speed and precision. We investigate how the unusual myelination pattern develops. Recently, we showed that the internode length is determined early in development followed – after hearing onset - by an increase in axon diameter, which causes the L/D ratio to decrease. Additionally, we found that the unmyelinated regions of the axon, the Nodes of Ranvier, develop sequentially along the axon [1]. However, it remains unclear whether patterned spontaneous neuronal activity is crucial for this process. This patterned network activity is present well before hearing onset and has been reported to influence other properties of the circuit’s development [2]. Therefore, the aim of this study is to determine how spontaneous neuronal activity before hearing onset affects myelination. We therefore established organotypic slice cultures of the gerbil auditory brainstem. Cultures could be maintained over a period of two weeks, allowing to study the development of myelin ex vivo. Additionally, we are using a viral vector encoding a light sensitive ion channel to manipulate the bushy cell activity at early developmental timepoints.[1] Nabel, Alisha L., et al. “Development of Myelination and Axon Diameter for Fast and Precise Action Potential Conductance.” GLIA, Jan. 2024, https://doi.org/10.1002/glia.24504.[2] Clause, Amanda, et al. “The Precise Temporal Pattern of Prehearing Spontaneous Activity Is Necessary for Tonotopic Map Refinement.” Neuron, vol. 82, no. 4, May 2014, pp. 822–35. https://doi.org/10.1016/j.neuron.2014.04.001.