Open Access
Kim, Youngcho
Graduate Program:
Integrative Biosciences
Doctor of Philosophy
Document Type:
Date of Defense:
June 26, 2007
Committee Members:
  • Kyung An Han, Committee Chair
  • Bernhard Luscher, Committee Member
  • Byron C Jones, Committee Member
  • Pamela J Mitchell, Committee Member
  • Richard W Ordway, Committee Member
  • Octopamine receptor
  • Dopamine receptor
  • Aversive conditioning
  • Reward conditioning
  • Associative learning
  • Drosophila
  • Classical Olfactory conditioning
Drosophila has robust behavioral plasticity to avoid or prefer the odor that predicts punishment or food reward, respectively. Both types of plasticity are mediated by the mushroom body (MB) neurons in the brain, in which various signaling molecules play crucial roles. However, important yet unresolved molecules are the receptors that initiate aversive or reward learning cascades in the MB. The dDA1 represents one of the Drosophila dopamine receptors that activate the cAMP cascade. To gain insights into the role of dDA1, we generated a polyclonal antibody against the unique sequence in dDA1 and investigated dDA1 distribution in the central nervous system (CNS) of Drosophila melanogaster. In both larval and adult CNS pronounced dDA1 immunoreactivity was present in the neuropil of the mushroom bodies, a brain structure crucial for learning and memory in insects, and four unpaired neurons in each thoracic segment. The adult CNS also exhibited intense dDA1 immunoreactivity in the central complex, a structure controlling higher-order motor function, moderate expression in several neurosecretory cells, and weak staining in two unpaired neurons in the mesothoracic neuromere. The dDA1 expression in these areas was only detected in the adult, but not in the third instars larval CNS. Given the findings that dDA1 and the octopamine receptor OAMB are highly expressed in the MB and use cAMP as a cellular signaling prompt us to test the roles of these receptors in associative learning using two classical conditioning paradigms: negatively and positively reinforced olfactory conditioning. Negatively reinforced olfactory conditioning has been well-characterized and flies show robust and reliable performance; however, olfactory conditioning utilizing positive reinforcement has shown to be less effective in that flies display low learning performance with highly variable scores. Therefore, we developed a novel assay for positively reinforced (appetitive) olfactory conditioning. In this assay, flies were involuntarily exposed to appetitive unconditioned stimulus sucrose along with conditioned stimulus odor during training and their preference to the odor previously associated with sucrose was measured to assess learning and memory capacities. After one training session, wild-type Canton S flies displayed reliable performance, which was enhanced after two training cycles with 1 or 15 min inter-training intervals. Higher performance scores were also obtained by increasing sucrose concentration. Memory in Canton-S flies decayed slowly when measured at 30 min, 1 h and 3 h after training, whereas memory had declined significantly at 6 h and 12 h post-training. When the octopamine-deficient t¥âh flies was challenged, they exhibited poor performance, validating the utility of this assay. As the Drosophila model offers vast genetic and transgenic resources, the new appetitive conditioning described here provides a useful tool for elucidating the molecular and cellular underpinnings of reward learning and memory. Similar to negatively reinforced conditioning, this reward conditioning represents classical olfactory conditioning. Thus, comparative analyses of learning and memory mutants in two assays may help dissect molecular and cellular components common or discrete to US information used in conditioning. With the new appetitive conditioning along with negatively reinforced (aversive) conditioning paradigms, we tested the roles of dDA1 and OAMB in both aversive and appetitive conditioning. We first identified two mutants dumb1 and dumb2 with abnormal dDA1 expression. When trained with the same conditioned stimuli, both dumb alleles showed negligible learning in electric shock-mediated conditioning while they exhibited moderately impaired learning in sugar-mediated conditioning. These phenotypes were not due to anomalous sensory modalities of dumb mutants because their olfactory acuity, shock reactivity, and sugar preference were comparable to those of control lines. Remarkably, the dumb mutant¡¯s impaired performance in both paradigms was fully rescued by reinstating dDA1 expression in the same subset of MB neurons, indicating the critical roles of the MB dDA1 in aversive as well as reward learning. Previous studies employing dopamine receptor antagonists implicate the involvement of D1/D5 receptors in various Pavlovian conditioning tasks in mammals; however, these have not been supported by the studies of D1- or D5-deficient animals. Our observations unambiguously clarify the critical roles of D1 dopamine receptor in aversive and appetitive learning. In contrast to dDA1, OAMB is the receptor that mediates appetitive, but not aversive, learning in Pavlovian olfactory conditioning. When trained with the same conditioned stimuli, oamb mutants showed severely impaired learning in sugar-mediated conditioning while they exhibited normal learning in electric shock-mediated conditioning. This learning defect was not caused by abnormal sensory processing of oamb mutants because their olfactory acuity and sugar preference were not affected. The oamb mutant¡¯s impaired performance in reward learning was fully rescued by restoring OAMB in the subset of the MB neurons that require dDA1 for normal appetitive learning, indicating the critical role of OAMB in the MB for appetitive learning. Thus, aversive and appetitive olfactory learning are likely mediated by the same subset of the MB neurons and dDA1 and OAMB together mediate reward memory formation.