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.
Wirth
Marcus J
MJ
Institute of Biology II, RWTH Aachen, Kopernikusstrasse 16, D-52074 Aachen, Germany. wirth@bio2.rwth-aachen.de
Kuenzel
Thomas
T
Luksch
Harald
H
Wagner
Hermann
H
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D001306:Auditory Pathways / Q000166:cytology* / Q000502:physiology; D001370:Axonal Transport / Q000502:physiology; D001931:Brain Mapping / Q000379:methods; D001933:Brain Stem / Q000166:cytology* / Q000502:physiology; D064032:Calbindin 2; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002478:Cells, Cultured; D002642:Chick Embryo; D018920:Coculture Techniques; D003911:Dextrans; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000378:metabolism; D007150:Immunohistochemistry; D008845:Microinjections / Q000379:methods; D009474:Neurons / Q000145:classification / Q000166:cytology* / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000295:instrumentation / Q000379:methods; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D064032:Calbindin 2; D003911:Dextrans; D005456:Fluorescent Dyes; D064030:S100 Calcium Binding Protein G; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid