Fluorescent labeling of rat auditory brainstem circuits for synaptic and electrophysiological studies

We visualized neurons with fluorescent agents, both retrogradely and anterogradely, to identify the input and output neuronal pathways in the rat auditory system. Output neurons of dorsal cochlear nucleus (DCN) were labeled retrogradely by injecting fluorescent microspheres into the inferior colliculus. Electrical recordings were made from the labeled fusiform cells of DCN with the whole-cell patch-clamp recording technique in slice preparations. DiI and Sindbis virus expressing membrane-targeted green fluorescent protein (GFP) were adopted for anterograde labeling. Auditory nerve fibers (ANFs) were labeled by injecting DiI into the cochlea, and the contralateral projection to the medial nucleus of the trapezoid body (MNTB) by injecting DiI and GFP into the ventral cochlear nucleus. A single ANF projecting to a DCN fusiform cell was electrically stimulated by a glass electrode and EPSCs were recorded using whole-cell patch-clamp recording methods. EPSCs were sensitive to the positioning of the electrode, and the size of EPSCs was constant irrespective of stimulus intensity, indicating that a single fiber was stimulated. Large EPSCs were generated from MNTB principal cells by stimulating the labeled fiber with a Calyx of Held terminal. The membrane excitability and EPSCs recorded after fluorescence labeling were similar to those previously reported. We confirm that the fluorescence labeling is effective to visualize neural networks and is useful to investigate the electrophysiological properties of neurons and synapses.