Dissociated neurons of the pupal honeybee brain in cell culture

Primary cell cultures were prepared from specific regions of the pupal honeybee brain which are involved in proboscis extension learning. Defined areas could be dissociated purely by mechanical treatment. We show that cultured neurons regenerate new neurites and remain viable for up to three weeks in a serum-free, chemically-defined medium. Several labelling techniques were employed to identify subpopulations of cultured neurons. For example, acetylcholinesterase staining; fluorescent beads to distinguish identified cell populations of co-cultured brain areas; various markers for surface antigens such as a monoclonal antibody to olfactory projection neurons of the antennoglomerular tracts and monopolar cells of the optic lobes, as well as anti-HRP immunoreactivity and alpha-bungarotoxin binding; and various antisera for detecting transmitter phenotype. The appearance of transmitter-immunoreactive cells agreed closely with that expected from their known distribution in situ. Our results suggest that cultured cells retain surface properties and transmitter phenotype of their in vivo counterparts, despite differences in basic morphology. Thus our culture system provides the important initial step for future in vitro investigations of the cellular and electrophysiological properties of neurons mediating proboscis extension learning.