Factors modulating dopamine receptor gene expression and calcium signalling in primary cell culture of honeybee Kenyon cells

Abstract

Signaling via the neuroamine dopamine is known to be important in insects for controlling behaviours such as sleep, arousal, motivation, locomotion, learning and memory. In the honeybee Apis mellifera pheromones and hormones can further modulate behaviour, and how these factors mediate their effects on the honeybee is of particular interest. However, faced with such a complex environment as the honeybee brain, it can be difficult to assess the individual effects of single factors. The major goals of this thesis were to determine whether mushroom body Kenyon cells in primary cell culture could be used to investigate the individual effects of hormones on dopamine receptor gene transcript levels as well as to detect the presence of a dopamine-induced calcium response in Kenyon cells of the honeybee.

Using cell culture it was found that the hormones 20-hydroxyecdysone (20HE) and juvenile hormone (JH) had a significant effect on the transcript levels of Amdop1, Amdop2, Amdop3, and the putative ecdysone/dopamine receptor gene Amgpcr19. The effects of 20HE were also found to be age dependent, with Kenyon cells from newly-emerged adult workers and pollen foragers displaying distinct differences in the transcript levels of the dopamine receptor genes. These results indicate that both 20HE and JH could contribute to age-related changes in dopamine signaling in the honeybee brain. Using the insight gained from the work on Kenyon cells in vitro, the potential effects of 20HE to modulate dopamine responses in the mushroom body calyces of the honeybee were investigated. This preliminary work showed that dopamine-induced changes in cAMP levels in mushroom body calyces of newly-emerged adult workers and in pollen foragers were increased in the presence of 10 nM of 20HE. In the absence of 20HE the response of newly-emerged adult worker and pollen forager mushroom body calyces to dopamine were different, with the application of dopamine reducing levels of cAMP in mushroom body calyces of pollen foragers. Finally, Kenyon cells in primary cell culture were used to investigate the hypothesis that dopamine receptor activation (in particular AmDOP2) increases intracellular calcium. A dopamine-induced calcium influx was identified. Consistent with the hypothesis that these responses are mediated by AmDOP2 receptors, the responses could be blocked by the receptor antagonists cis(Z)flupentixol and epinastine. The work described in this thesis demonstrates the potential for using Kenyon cells in vitro to gain a clearer picture of how dopamine modulates the functioning of neurons in the mushroom bodies of the honeybee brain. Development of this technique provides an important bridging tool between the whole brain of the honeybee which due to the complex nature of its neural networks make the role of individual agents difficult to define, and transfected cell cultures, in which receptors are not in their native environment.