AN ORGANOTYPIC COCULTURE OF EMBRYONIC RAT BRAIN-STEM AND TONGUE OR SKELETAL-MUSCLE

Transverse sections of rat embryo brainstem (embryonic age 18 - 19 days) containing the brainstem motor nuclei were explanted, together with a small piece of tongue or skeletal muscle, in a plasma clot and maintained in culture for 2 - 3 weeks using the roller tube technique. The results show that brainstem motoneurons survived, differentiated and innervated newly formed multinucleated myotubes which displayed large synchronized contractions after 1 week in culture. Muscle fibre contractions could be induced by excitatory amino acid applications and suppressed by curarization. Muscle fibres differentiated normally. During the first week they showed diffuse acetylcholinesterase positivity and multi-innervation. During the second and third weeks the number of motor end-plates was greatly reduced and transverse striation was observed. In the presence of muscle fibres, the brainstem thinned out and spread, becoming one or two cell layers thick, and the motoneurons tended to migrate towards the muscle fibres, becoming clearly observable in the living culture. When the muscle explant was not present, the brainstem explant did not spread and remained several cell layers thick, while acetylcholinesterase-positive cells, presumed to be motoneurons, tended to disappear. The preparation described is well suited for electrophysiological studies of differentiating motoneurons and offers direct access to their dendritic tree, a most desirable feature for patch-clamp or multisite optical recording.