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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.

Reinforcement signals such as food reward and noxious punishment can change diverse behaviors. This holds true in fruit flies, Drosophila melanogaster, which can be conditioned by an odor a...

Summary The mushroom body (MB) of Drosophila melanogaster has multiple functions in controlling memory and behavior. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 However, circuit mechanisms that generate this functional diversity are largely unclear. Here, we systematically probed the behavioral contribution of each type of MB output neuron (MBON) by blocking during acquisition, retention, or retrieval of reward or punishment memories. We evaluated the contribution using two conditioned responses: memory-guided odor choice and odor source attraction. Quantitative analysis revealed that these conditioned odor responses are controlled by different sets of MBONs. We found that the valence of memory, rather than the transition of memory steps, has a larger impact on the patterns of required MBONs. Moreover, we found that the glutamatergic MBONs forming recurrent circuits commonly contribute to appetitive memory acquisition, suggesting a pivotal role of this circuit motif for reward processing. Our results provide principles how the MB output circuit processes associative memories of different valence and controls distinct memory-guided behaviors.

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.

The mushroom body (MB) in insects is known as a major center for associative learning and memory, although exact locations for the correlating memory traces remain to be elucidated. Here, we asked whether presynaptic boutons of olfactory projection neurons (PNs) in the main input site of the MB undergo neuronal plasticity during classical odor-reward conditioning and correlate with the conditioned behavior. We simultaneously measured Ca2+responses in the boutons and conditioned behavioral responses to learned odors in honeybees. We found that the absolute amount of the neural change for the rewarded but not for the unrewarded odor was correlated with the behavioral learning rate across individuals. The temporal profile of the induced changes matched with odor response dynamics of the MB-associated inhibitory neurons, suggestive of activity modulation of boutons by this neural class. We hypothesize the circuit-specific neural plasticity relates to the learned value of the stimulus and underlies the conditioned behavior of the bees.

Summary Background Drosophila learn to avoid odors that are paired with aversive stimuli. Electric shock is a potent aversive stimulus that acts via dopamine neurons to elicit avoidance of the associated odor. While dopamine signaling has been demonstrated to mediate olfactory electric shock conditioning, it remains unclear how this pathway is involved in other types of behavioral reinforcement, such as in learned avoidance of odors paired with increased temperature. Results To better understand the neural mechanisms of distinct aversive reinforcement signals, we here established an olfactory temperature conditioning assay comparable to olfactory electric shock conditioning. We show that the AC neurons, which are internal thermal receptors expressing dTrpA1, are selectively required for odor-temperature but not for odor-shock memory. Furthermore, these separate sensory pathways for increased temperature and shock converge onto overlapping populations of dopamine neurons that signal aversive reinforcement. Temperature conditioning appears to require a subset of the dopamine neurons required for electric shock conditioning. Conclusions We conclude that dopamine neurons integrate different noxious signals into a general aversive reinforcement pathway.

Animals approach stimuli that predict a pleasant outcome. After the paired presentation of an odour and a reward, Drosophila melanogaster can develop a conditioned approach towards that odour. Despite recent advances in understanding the neural circuits for associative memory and appetitive motivation, the cellular mechanisms for reward processing in the fly brain are unknown. Here we show that a group of dopamine neurons in the protocerebral anterior medial (PAM) cluster signals sugar reward by transient activation and inactivation of target neurons in intact behaving flies. These dopamine neurons are selectively required for the reinforcing property of, but not a reflexive response to, the sugar stimulus. In vivo calcium imaging revealed that these neurons are activated by sugar ingestion and the activation is increased on starvation. The output sites of the PAM neurons are mainly localized to the medial lobes of the mushroom bodies (MBs), where appetitive olfactory associative memory is formed. We therefore propose that the PAM cluster neurons endow a positive predictive value to the odour in the MBs. Dopamine in insects is known to mediate aversive reinforcement signals. Our results highlight the cellular specificity underlying the various roles of dopamine and the importance of spatially segregated local circuits within the MBs.

Social insects like ants exhibit sophisticated communication by means of pheromones, one example of which is the use of alarm pheromones to alert nestmates for colony defense. In the ant Camponotus obscuripes, we have reported that information about formic acid and n-undecane, alarm pheromone components, is processed in a set of specific glomeruli in the antennal lobe (primary olfactory center). Alarm pheromone signals are then transmitted, mainly via uniglomerular projection neurons (uni-PNs), to the protocerebrum (PR), where sensory signals are integrated to form motor commands for behavioral responses. In this study, we physiologically and morphologically characterized 63 alarm pheromone-sensitive PR neurons in ants by using intracellular recording and staining techniques. Most of the pheromone-sensitive PR neurons had dendrites in the mushroom body (MB), the lateral horn, or the medial PR. Some neurons with dendrites in these areas responded specifically to formic acid or n-undecane and may participate in the control of specific behavioral responses to each pheromone component. Other neurons responded also to non-pheromonal odors, in contrast to uni-PNs, most of which responded specifically to alarm pheromones. Responses to non-pheromonal odors were most prominent in efferent neurons of the MB lobe, suggesting that they may participate in integration of pheromonal and non-pheromonal information. We found a class of PR neurons that receives input in all of these pheromone-processing areas and terminates in a variety of premotor areas. These neurons may participate in the control of pheromone-sensitized aggressive behavior, which is triggered by non-pheromonal sensory stimuli associated with a potential enemy.

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.

The alarm pheromone of the ant Camponotus obscuripes (Formicinae) was identified and quantified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Comparisons between alarm pheromone components and extracts from the major exocrine gland of this ant species revealed that the sources of its alarm pheromone are Dufour's gland and the poison gland. Most components of Dufour's gland were saturated hydrocarbons. n-Undecane comprised more than 90% of all components and in a single Dufour's gland amounted to 19 microg. n-Decane and n-pentadecane were also included in the Dufour's gland secretion. Only formic acid was detected in the poison gland, in amounts ranging from 0.049 to 0.91 microl. This ant species releases a mixture of these substances, each of which has a different volatility and function. When the ants sensed formic acid, they eluded the source of the odor; however, they aggressively approached odors of n-undecane and n-decane, which are highly volatile. In contrast, n-pentadecane, which has the lowest volatility among the identified compounds, was shown to calm the ants. The volatilities of the alarm pheromone components were closely related to their roles in alarm communication. Highly volatile components vaporized rapidly and spread widely, and induced drastic reactions among the ants. As these components became diluted, the less volatile components calmed the excited ants. How the worker ants utilize this alarm communication system for efficient deployment of their nestmates in colony defense is also discussed herein.

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.

The honeybee Apis mellifera has a remarkable ability to detect and locate food sources during foraging, and to associate odor cues with food rewards. In the honeybee's olfactory system, sensory input is first processed in the antennal lobe (AL) network. Uniglomerular projection neurons (PNs) convey the sensory code from the AL to higher brain regions via two parallel but anatomically distinct pathways, the lateral and the medial antenno-cerebral tract (l- and m-ACT). Neurons innervating either tract show characteristic differences in odor selectivity, concentration dependence, and representation of mixtures. It is still unknown how this differential stimulus representation is achieved within the AL network. In this contribution, we use a computational network model to demonstrate that the experimentally observed features of odor coding in PNs can be reproduced by varying lateral inhibition and gain control in an otherwise unchanged AL network. We show that odor coding in the l-ACT supports detection and accurate identification of weak odor traces at the expense of concentration sensitivity, while odor coding in the m-ACT provides the basis for the computation and following of concentration gradients but provides weaker discrimination power. Both coding strategies are mutually exclusive, which creates a tradeoff between detection accuracy and sensitivity. The development of two parallel systems may thus reflect an evolutionary solution to this problem that enables honeybees to achieve both tasks during bee foraging in their natural environment, and which could inspire the development of artificial chemosensory devices for odor-guided navigation in robots.

Pheromones play major roles in intraspecific communication in many animals. Elaborated communication systems in eusocial insects provide excellent materials to study neural mechanisms for social pheromone processing. We previously reported that alarm pheromone information is processed in a specific cluster of glomeruli in the antennal lobe of the antCamponotus obscuripes. However, representation of alarm pheromone information in a secondary olfactory centre is unknown in any animal. Olfactory information in the antennal lobe is transmitted to secondary olfactory centres, including the lateral horn, by projection neurons (PNs). In this study, we compared distributions of terminal boutons of alarm pheromone-sensitive and -insensitive PNs in the lateral horn of ants. Distributions of their dendrites largely overlapped, but there was a region where boutons of pheromone-sensitive PNs, but not those of pheromone-insensitive PNs, were significantly denser than in the rest of the lateral horn. Moreover, most of a major type of pheromone-sensitive efferent neurons from the lateral horn extended dendritic branches in this region, suggesting specialization of this region for alarm pheromone processing. This study is the first study to demonstrate the presence of specialized areas for the processing of a non-sexual, social pheromone in the secondary olfactory centre in any animal.

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.