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Dopamine can play opposing physiological roles depending on the receptor subtype. In the fruit fly Drosophila melanogaster, Dop1R1 and Dop2R encode the D1- and D2-like receptors, respectively, and are reported to oppositely regulate intracellular cAMP levels. Here, we profiled the expression and subcellular localization of endogenous Dop1R1 and Dop2R in specific cell types in the mushroom body circuit. For cell-type-specific visualization of endogenous proteins, we employed reconstitution of split-GFP tagged to the receptor proteins. We detected dopamine receptors at both presynaptic and postsynaptic sites in multiple cell types. Quantitative analysis revealed enrichment of both receptors at the presynaptic sites, with Dop2R showing a greater degree of localization than Dop1R1. The presynaptic localization of Dop1R1 and Dop2R in dopamine neurons suggests dual feedback regulation as autoreceptors. Furthermore, we discovered a starvation-dependent, bidirectional modulation of the presynaptic receptor expression in the protocerebral anterior medial (PAM) and posterior lateral 1 (PPL1) clusters, two distinct subsets of dopamine neurons, suggesting their roles in regulating appetitive behaviors. Our results highlight the significance of the co-expression of the two opposing dopamine receptors in the spatial and conditional regulation of dopamine responses in neurons.

CCHamide-2 (CCHa2) is a protostome excitatory peptide ortholog known for various arthropod species. In fruit flies, CCHa2 plays a crucial role in the endocrine system, allowing peripheral tissue to communicate with the central nervous system to ensure proper development and the maintenance of energy homeostasis. Since the formation of odor-sugar associative long-term memory (LTM) depends on the nutrient status in an animal, CCHa2 may play an essential role in linking memory and metabolic systems. Here we show that CCHa2 signals are important for consolidating appetitive memory by acting on the rewarding dopamine neurons. Genetic disruption of CCHa2 using mutant strains abolished appetitive LTM but not short-term memory (STM). A post-learning thermal suppression of CCHa2 expressing cells impaired LTM. In contrast, a post-learning thermal activation of CCHa2 cells stabilized STM induced by non-nutritious sugar into LTM. The receptor of CCHa2, CCHa2-R, was expressed in a subset of dopamine neurons that mediate reward for LTM. In accordance, the receptor expression in these dopamine neurons was required for LTM specifically. We thus concluded that CCHa2 conveys a sugar nutrient signal to the dopamine neurons for memory consolidation. Our finding establishes a direct interplay between brain reward and the putative endocrine system for long-term energy homeostasis.

Regulation of reward signaling in the brain is critical for appropriate judgement of the environment and self. In Drosophila, the protocerebral anterior medial (PAM) cluster dopamine neurons mediate reward signals. Here, we show that localized inhibitory input to the presynaptic terminals of the PAM neurons titrates olfactory reward memory and controls memory specificity. The inhibitory regulation was mediated by metabotropic gamma-aminobutyric acid (GABA) receptors clustered in presynaptic microdomain of the PAM boutons. Cell type-specific silencing the GABA receptors enhanced memory by augmenting internal reward signals. Strikingly, the disruption of GABA signaling reduced memory specificity to the rewarded odor by changing local odor representations in the presynaptic terminals of the PAM neurons. The inhibitory microcircuit of the dopamine neurons is thus crucial for both reward values and memory specificity. Maladaptive presynaptic regulation causes optimistic cognitive bias.

Summary: Neurotransmitters often have multiple receptors that induce distinct responses in receiving cells. Expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits. Here we describe a comprehensive library of reporter strains in which a convertible T2A-GAL4 cassette is inserted into endogenous neurotransmitter receptor genes of Drosophila. Using this library, we profile the expression of 75 neurotransmitter receptors in the brain. Cluster analysis reveals neurochemical segmentation of the brain, distinguishing higher brain centers from the rest. By recombinase-mediated cassette exchange, we convert T2A-GAL4 into split-GFP and Tango to visualize subcellular localization and activation of dopamine receptors in specific cell types. This reveals striking differences in their subcellular localization, which may underlie the distinct cellular responses to dopamine in different behavioral contexts. Our resources thus provide a versatile toolkit for dissecting the cellular organization and function of neurotransmitter systems in the fly brain. : Kondo et al. have generated a comprehensive library of convertible GAL4 knockin Drosophila strains for neurotransmitter receptor genes. The GAL4 lines can be converted into other reporters through the use of RMCE, providing a versatile toolkit. Expression profiling of receptor genes reveals neurochemical segmentation of the brain.

Dopamine modulates a variety of animal behaviors that range from sleep and learning to courtship and aggression. Besides its well-known phasic firing to natural reward, a substantial number of dopamine neurons (DANs) are known to exhibit ongoing intrinsic activity in the absence of an external stimulus. While accumulating evidence points at functional implications for these intrinsic “spontaneous activities” of DANs in cognitive processes, a causal link to behavior and its underlying mechanisms has yet to be elucidated. Recent physiological studies in the model organism Drosophila melanogaster have uncovered that DANs in the fly brain are also spontaneously active, and that this activity reflects the behavioral/internal states of the animal. Strikingly, genetic manipulation of basal DAN activity resulted in behavioral alterations in the fly, providing critical evidence that links spontaneous DAN activity to behavioral states. Furthermore, circuit-level analyses have started to reveal cellular and molecular mechanisms that mediate or regulate spontaneous DAN activity. Through reviewing recent findings in different animals with the major focus on flies, we will discuss potential roles of this physiological phenomenon in directing animal behaviors.

Massive activation of dopamine neurons is critical for natural reward and drug abuse. In contrast, the significance of their spontaneous activity remains elusive. In Drosophila melanogaster, depolarization of the protocerebral anterior medial (PAM) cluster dopamine neurons en masse signals reward to the mushroom body (MB) and drives appetitive memory. Focusing on the functional heterogeneity of PAM cluster neurons, we identified that a single class of PAM neurons, PAM-γ3, mediates sugar reward by suppressing their own activity. PAM-γ3 is selectively required for appetitive olfactory learning, while activation of these neurons in turn induces aversive memory. Ongoing activity of PAM-γ3 gets suppressed upon sugar ingestion. Strikingly, transient inactivation of basal PAM-γ3 activity can substitute for reward and induces appetitive memory. Furthermore, we identified the satiety-signaling neuropeptide Allatostatin A (AstA) as a key mediator that conveys inhibitory input onto PAM-γ3. Our results suggest the significance of basal dopamine release in reward signaling and reveal a circuit mechanism for negative regulation.

Dopamine signals reward in animal brains. A single presentation of a sugar reward to Drosophila activates distinct subsets of dopamine neurons that independently induce short- and long-term olfactory memories (STM and LTM, respectively). In this study, we show that a recurrent reward circuit underlies the formation and consolidation of LTM. This feedback circuit is composed of a single class of reward-signaling dopamine neurons (PAM-α1) projecting to a restricted region of the mushroom body (MB), and a specific MB output cell type, MBON-α1, whose dendrites arborize that same MB compartment. Both MBON-α1 and PAM-α1 neurons are required during the acquisition and consolidation of appetitive LTM. MBON-α1 additionally mediates the retrieval of LTM, which is dependent on the dopamine receptor signaling in the MB α/β neurons. Our results suggest that a reward signal transforms a nascent memory trace into a stable LTM using a feedback circuit at the cost of memory specificity.

Animals discriminate stimuli, learn their predictive value and use this knowledge to modify their behavior. In Drosophila, the mushroom body (MB) plays a key role in these processes. Sensory stimuli are sparsely represented by ∼2000 Kenyon cells, which converge onto 34 output neurons (MBONs) of 21 types. We studied the role of MBONs in several associative learning tasks and in sleep regulation, revealing the extent to which information flow is segregated into distinct channels and suggesting possible roles for the multi-layered MBON network. We also show that optogenetic activation of MBONs can, depending on cell type, induce repulsion or attraction in flies. The behavioral effects of MBON perturbation are combinatorial, suggesting that the MBON ensemble collectively represents valence. We propose that local, stimulus-specific dopaminergic modulation selectively alters the balance within the MBON network for those stimuli. Our results suggest that valence encoded by the MBON ensemble biases memory-based action selection.

Social insects exhibit sophisticated communication by means of pheromones, one example of which is the use of alarm pheromones to alert nestmates for colony defense. We review recent advances in the understanding of the processing of alarm pheromone information in the ant brain. We found that information about formic acid and n-undecane, alarm pheromone components, is processed in a set of specific glomeruli in the antennal lobe of the ant Camponotus obscuripes. Alarm pheromone information is then transmitted, via projection neurons, to the lateral horn and the calyces of the mushroom body of the protocerebrum. In the lateral horn, we found a specific area where terminal boutons of alarm pheromone-sensitive projection neurons are more densely distributed than in the rest of the lateral horn. Some neurons in the protocerebrum responded specifically to formic acid or n-undecane and they may participate in the control of behavioral responses to each pheromone component. Other neurons, especially those originating from the mushroom body lobe, responded also to non-pheromonal odors and may play roles in integration of pheromonal and non-pheromonal signals. We found that a class of neurons receive inputs in the lateral horn and the mushroom body lobe and terminate in a variety of premotor areas. These neurons may participate in the control of aggressive behavior, which is sensitized by alarm pheromones and is triggered by non-pheromonal sensory stimuli associated with a potential enemy. We propose that the alarm pheromone processing system has evolved by differentiation of a part of general odor processing system.

An important component in understanding central olfactory processing and coding in the insect brain relates to the characterization of the functional divisions between morphologically distinct types of projection neurons (PN). Using calcium imaging, we investigated how the identity, concentration and mixtures of odors are represented in axon terminals (boutons) of two types of PNs - lPN and mPN. In lPN boutons we found less concentration dependence, narrow tuning profiles at a high concentration, which may be optimized for fine, concentration-invariant odor discrimination. In mPN boutons, however, we found clear rising concentration dependence, broader tuning profiles at a high concentration, which may be optimized for concentration coding. In addition, we found more mixture suppression in lPNs than in mPNs, indicating lPNs better adaptation for synthetic mixture processing. These results suggest a functional division of odor processing in both PN types.

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons. [ABSTRACT FROM AUTHOR]

Tremendous evolutional success and the ecological dominance of social insects, including ants, termites and social bees, are due to their efficient social organizations and their underlying communication systems. Functional division into reproductive and sterile castes, cooperation in defending the nest, rearing the young and gathering food are all regulated by communication by means of various kinds of pheromones. No brain structures specifically involved in the processing of non-sexual pheromone have been physiologically identified in any social insects. By use of intracellular recording and staining techniques, we studied responses of projection neurons of the antennal lobe (primary olfactory centre) of ants to alarm pheromone, which plays predominant roles in colony defence. Among 23 alarm pheromone-sensitive projection neurons recorded and stained in this study, eight were uniglomerular projection neurons with dendrites in one glomerulus, a structural unit of the antennal lobe, and the remaining 15 were multiglomerular projection neurons with dendrites in multiple glomeruli. Notably, all alarm pheromone-sensitive uniglomerular projection neurons had dendrites in one of five ‘alarm pheromone-sensitive (AS)’ glomeruli that form a cluster in the dorsalmost part of the antennal lobe. All alarm pheromone-sensitive multiglomerular projection neurons had dendrites in some of the AS glomeruli as well as in glomeruli in the anterodorsal area of the antennal lobe. The results suggest that components of alarm pheromone are processed in a specific cluster of glomeruli in the antennal lobe of ants. [ABSTRACT FROM AUTHOR]

Communication by means of pheromones plays predominant roles in colony integration by social insects. However, almost nothing is known about pheromone processing in the brains of social insects. In this study, we successfully applied intracellular recording and staining techniques to anatomically and physiologically characterize brain neurons of the ant Camponotus obscuripes. We identified 42 protocerebral neurons that responded to undecane and/or formic acid, components of alarm pheromones that evoke attraction or evasive behavior, respectively. Notably, 30 (71%) of these neurons were efferent (output) or feedback neurons of the mushroom body, and many of these exhibited different responses to formic acid and undecane. Eight of the remaining 12 neurons had arborizations in the lateral and/or medial protocerebrum, which receive terminations of efferent neurons of the mushroom body and from which premotor descending neurons originate. The remaining four neurons were bilateral neurons that connect lateral accessory lobes or dorsal protocerebrums of both hemispheres. We suggest that the mushroom body of the ant participates in the processing of alarm pheromones. Seventeen (40%) of 42 neurons exhibited responses to nonpheromonal odors, indicating that the pheromonal and nonpheromonal signals are not fully segregated when they are processed in the protocerebrum. This may be related to modulatory functions of alarm pheromones, i.e., they change alertness of the ant and change responses to a variety of sensory stimuli. [ABSTRACT FROM AUTHOR]