Characterization of a D2-like dopamine receptor (AmDOP3) in honey bee, Apis mellifera.

Dopamine is an important neurotransmitter in vertebrate and invertebrate nervous systems and is widely distributed in the brain of the honey bee, Apis mellifera. We report here the functional characterization and cellular localization of the putative dopamine receptor gene, Amdop3, a cDNA clone isolated and identified in previous studies as AmBAR3 (Apis mellifera Biogenic Amine Receptor 3). The Amdop3 cDNA encodes a 694 amino acid protein, AmDOP3. Comparison of AmDOP3 to Drosophila melanogaster sequences indicates that it is orthologous to the D2-like dopamine receptor, DD2R. Using AmDOP3 receptors expressed in HEK293 cells we show that of the endogenous biogenic amines, dopamine is the most potent AmDOP3 agonist, and that activation of AmDOP3 receptors results in down regulation of intracellular levels of cAMP, a property characteristic of D2-like dopamine receptors. In situ hybridization reveals that Amdop3 is widely expressed in the brain but shows a pattern of expression that differs from that of either Amdop1 or Amdop2, both of which encode D1-like dopamine receptors. Nonetheless, overlaps in the distribution of cells expressing Amdop1, Amdop2 and Amdop3 mRNAs suggest the likelihood of D1:D2 receptor interactions in some cells, including subpopulations of mushroom body neurons.

Developmental expression of a tyramine receptor gene in the brain of the honey bee, Apis mellifera.

This study reveals that the tyramine receptor gene, Amtyr1, is expressed in the developing brain, as well as in the brain of the adult worker honey bee. Changes in levels of Amtyr1 expression were examined using Northern analysis. Age-related increases in Amtyr1 transcript levels were observed not only during metamorphic adult development, but also in the brain of the adult worker bee. RNA in situ hybridization revealed the pattern of Amtyr1 expression. Cell bodies staining intensely for tyramine receptor-gene transcript were observed throughout the somata rind, with well-defined clusters of cells associated with developing mushroom bodies, optic lobes, and antennal lobes of the brain. Staining for Amtyr1 transcript was particularly intense within the three major divisions of mushroom body intrinsic neurons (outer compact, noncompact, and inner compact cells), suggesting that Amtyr1 is highly expressed in these structures. Activation of AmTYR1 receptors heterologously expressed in insect (Spodoptera frugiperda) cells led to a reduction in intracellular levels of cAMP similar to that reported for AmTYR1 receptors expressed in mammalian (HEK 293) cells (Blenau et al. [2000] J Neurochem 74:900-908). Taken together, these results suggest that AmTYR1 receptors may play a role in the developing brain as well as in the brain of the adult worker bee. The actions of tyramine are likely to be mediated, at least in part, via the cAMP-signaling pathway.

Developmental changes in expression patterns of two dopamine receptor genes in mushroom bodies of the honeybee, Apis mellifera.

The expression patterns of two dopamine receptor genes, Amdop1 and Amdop2, in the developing mushroom bodies of the honeybee brain were determined by using in situ hybridisation. Both genes were expressed throughout pupal development, but their patterns of expression in the three major divisions of mushroom body intrinsic neurons (outer compact cells, noncompact cells, and inner compact cells) were quite distinct. Amdop1 expression could be detected in all three mushroom body cell groups throughout development. Staining for Amdop1 mRNA was particularly intense in newly born Kenyon cells, suggesting that levels of Amdop1 expression are higher in newborn cells than in more mature mushroom body neurons. This was not the case for Amdop2. Amdop2 expression in the mushroom bodies was restricted to inner and outer compact cells during most of pupal development, appearing in noncompact cells only late in metamorphosis or at adult eclosion. In contrast to the case with Amdop1, staining for Amdop2 mRNA was observed in glial cells. Expression of Amdop2 in glial cells was detected only at early stages of glial cell development, when the cells are reported to be actively dividing. This study not only implicates dopamine in the development of honeybee mushroom bodies but also suggests different roles for the two dopamine receptors investigated.

Analysis of two D1-like dopamine receptors from the honey bee Apis mellifera reveals agonist-independent activity.

Dopamine is found in many invertebrate organisms, including insects, however, the mechanisms through which this amine operates remain unclear. We have expressed two dopamine receptors cloned from honey bee (AmDOP1 and AmDOP2) in insect cells (Spodoptera frugiperda), and compared their pharmacology directly using production of cAMP as a functional assay. In each assay, AmDOP1 receptors required lower concentrations of dopamine and 6,7-ADTN for maximal activation than AmDOP2 receptors. Conversely, butaclamol and cis(Z)-flupentixol were more potent at blocking the cAMP response mediated through AmDOP2 than AmDOP1 receptors. Expression of AmDOP1, but not AmDOP2, receptors significantly increased levels of cAMP even in the absence of ligand. This constitutive activity was blocked by cis(Z)-flupentixol. This work provides the first evidence of a constitutively activated dopamine receptor in invertebrates and suggests that although AmDOP1 and AmDOP2 share much less homology than their vertebrate counterparts, they display a number of functional parallels with the mammalian D1-like dopamine receptors.