Modulation of motor behavior by dopamine and the D1-like dopamine receptor AmDOP2 in the honey bee
Introduction
In invertebrates, numerous studies have shown that dopamine signaling affects locomotion (Chase et al., 2004, Draper et al., 2007, Lima and Miesenbock, 2005, Pendleton et al., 2002, Sawin et al., 2000, Yellman et al., 1997). Furthermore, dopamine affects locomotor behavior on several levels including modulation of the neuromuscular junction (Cooper and Neckameyer, 1999, Dasari and Cooper, 2004), regulation of central pattern generators (Puhl and Mesce, 2008, Svensson et al., 2001) and setting general arousal levels (Andretic et al., 2005, Kume et al., 2005). Thus, dopamine may act at multiple sites to influence locomotor behavior by affecting sensory information in the periphery, the regulation of central pattern generators and/or higher order processing of information in the brain.
Although the role of dopamine in modulating behavior has been studied extensively, the distinct molecular pathways through which dopamine acts to affect different behaviors are still not well understood. Both, vertebrates and invertebrates, have two distinct classes of dopamine receptors: D1-like receptors that increase intracellular cAMP levels when activated and D2-like receptors which cause a decrease in cAMP levels in the presence of dopamine (Mustard et al., 2005, Neve et al., 2004). In the past, establishing the role of specific dopamine receptors in behavior was difficult due to the lack of pharmacological agents that are selective for specific invertebrate dopamine receptor subtypes. In addition, dopamine signaling plays an important role during development (Neckameyer, 1996), suggesting that animals with mutations in their dopamine receptors may show changes in behavior due to developmental defects.
In this study, we use both pharmacological and molecular approaches to examine the role of dopamine signaling in regulating the behavior of freely moving adult honey bees. The role of dopamine in modulating behavior in honey bees was characterized via injections of dopamine or the general dopamine receptor antagonist flupenthixol into the hemolymph leading to global perturbations in dopamine signaling. In the honey bee, three distinct dopamine receptors, two D1-like receptors, AmDOP1 and AmDOP2 (Blenau et al., 1998, Humphries et al., 2003), and one D2-like receptor, AmDOP3 (Beggs et al., 2005) have been cloned and characterized. Each receptor has a unique expression pattern; however, each receptor is expressed in the mushroom bodies of the brain of adult workers (Beggs et al., 2005, Blenau et al., 1998, Humphries et al., 2003, Kurshan et al., 2003). In insects, the mushroom bodies of the brain have been implicated as regions that control and coordinate locomotor activity (Besson and Martin, 2005, Martin et al., 1998, Mizunami et al., 1998) making the role of dopamine signaling in the mushroom bodies of particular interest. The role of a D1-like dopamine receptor, AmDOP2, in the brain was investigated using RNAi mediated knockdown of expression. AmDOP2 is the ortholog of the Drosophila DAMB dopamine receptor, also known as DopR99B (Feng et al., 1996, Han et al., 1996). Injection of dsRNA corresponding to the sequence of the Amdop2 receptor gene into the mushroom bodies reduced Amdop2 mRNA levels and affected a subset of the behaviors influenced by treatment with the dopamine receptor antagonist flupenthixol. These results provide insight into the specific contributions of this receptor to motor behavior and show that the combination of pharmacological treatments with RNAi is a useful strategy for revealing the roles of biogenic amines in behavior.
Section snippets
Subjects
Honey bees used in this study were from the New World Carniolan population maintained at the Rothenbuhler Honey Bee Research Laboratory at Ohio State University. Pollen foragers were used exclusively in this study as biogenic amine levels vary in the brains of worker honey bees depending on their age and the behavioral task in which they specialize (Schulz and Robinson, 1999, Taylor et al., 1992, Wagener-Hulme et al., 1999). Therefore, using bees in one specific task group should minimize the
Results
Analysis of the behavioral profile of the control (buffer injected) bees revealed differences in the amount of time spent in each behavior during the course of the observation (time spent in each behavior versus interval after injection, F25,25 = 4.17, p < 0.0001). For example, compare the behavior of the buffer bees at the 20–25 min interval, when they spent the majority of their time walking, with their behavior at 35–40 min, when they spent less time walking and more time grooming. Post hoc
Discussion
Injection of dopamine or the dopamine receptor antagonist flupenthixol into the hemolymph had significant effects on motor behavior. Bees treated with dopamine or flupenthixol show a dose dependent U-shaped effect, where the behavioral effects are not linear with concentration. This non-linear effect of dose and response has been observed in other studies investigating the effects of biogenic amines on honey bee behavior (Barron et al., 2007, Fussnecker et al., 2006, Scheiner et al., 2002).
Acknowledgements
The authors thank Sue Cobey for maintaining the honey bee colonies. This work was supported by NIH (NIDA) grant DA017694 to JAM; NIH (NCRR) grant RR014166 to BHS; and by Ohio State University College of Biological Sciences Dean's Undergraduate Research Awards to PMP.
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