Development of a topographically organized auditory network in slice culture is calcium dependent

Inhibitory and excitatory connections of remarkably precise topographic order are characteristic features of the mammalian auditory system, particularly within the superior olivary complex (SOC). Little is known about the requirements for the correct development of these specific connections. Previous in vivo experiments have demonstrated a high expression of calcium-binding proteins in this system during development, pointing to the need for precise calcium regulation. Here, we have employed an organotypic slice culture from the above neuronal network and analyzed the requirements for the maintenance and development of this system in vitro. When slices from neonatal rats were incubated in standard culture medium for up to 7 days, we found no organotypic features. Only if 25 mM KCl was added to the culture medium, the cytoarchitecture of the nuclei, the neuronal morphology, and the specificity and topography of internuclear connections were indistinguishable from that in vivo. The addition of calcium channel blockers (MgCl2 and nifedipine) to the high-KCl medium reduced organotypicity drastically, indicating that a depolarization-induced increase of intracellular calcium is indispensable. Furthermore, the temporal course of the expression of the calcium-binding protein parvalbumin in culture under high KCl mimics that in vivo, demonstrating developmental processes during incubation. The need for calcium influx into neurons of this auditory network in vitro (which is not seen in other slice culture systems) strengthens the hypothesis that an optimal calcium concentration is exceptionally important in auditory neurons. The effect of KCl in the slice cultures may substitute for input activity regulating intracellular calcium in auditory neurons in vivo.