Identification of auditory neurons by retrograde labelling for patch-clamp recordings in a mixed culture of chick brainstem

We present a method to identify specific sub-populations of auditory neurons in a mixed primary cell culture of the chicken brainstem, allowing the study of individual neurons with a known identity in vitro. To label specific afferent cell types, we injected retrograde tracers (dextrans coupled to fluorescent dyes) into either the mid-line or the superior olivary nuclei (SON) of the isolated chicken brainstem in vitro. Mid-line injections resulted in stable labelling of neurons of the nucleus magnocellularis (NM), whereas injections into the SON retrogradely labelled neurons of the nucleus laminaris (NL). The fluorescent label survives the dissociation procedure and is detectable for at least 1 week in vitro. Only about 0.1% of all cells in vitro are pre-labelled. The auditory identity of the pre-labelled neurons was confirmed with calretinin immunocytochemistry and electrophysiological recordings, where the cells had typical firing patterns of auditory brainstem neurons. In the future, this method can be combined with single cell PCR to match nuclear origin, firing patterns and the expression of functional molecules in vitro.