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Summary Animals continuously evaluate sensory information to decide on their next action. Different sensory cues, however, often demand opposing behavioral responses. How does the brain process conflicting sensory information during decision making? Here, we show that flies use neural substrates attributed to odor learning and memory, including the mushroom body (MB), for immediate sensory integration and modulation of innate behavior. Drosophila melanogaster must integrate contradictory sensory information during feeding on fermenting fruit that releases both food odor and the innately aversive odor CO 2 . Here, using this framework, we examine the neural basis for this integration. We have identified a local circuit consisting of specific glutamatergic output and PAM dopaminergic input neurons with overlapping innervation in the MB-β′2 lobe region, which integrates food odor and suppresses innate avoidance. Activation of food odor-responsive dopaminergic neurons reduces innate avoidance mediated by CO 2 -responsive MB output neurons. We hypothesize that the MB, in addition to its long recognized role in learning and memory, serves as the insect's brain center for immediate sensory integration during instantaneous decision making.

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.

To investigate how sensory information is processed, transformed, and stored within an olfactory system, we examined the anatomy of the input region, the calyx, of the mushroom bodies of Drosophila melanogaster. These paired structures are important for various behaviors, including olfactory learning and memory. Cells in the input neuropil, the calyx, are organized into an array of microglomeruli each comprising the large synaptic bouton of a projection neuron (PN) from the antennal lobe surrounded by tiny postsynaptic neurites from intrinsic Kenyon cells. Extrinsic neurons of the mushroom body also contribute to the organization of microglomeruli. We employed a combination of genetic reporters to identify single cells in the Drosophila calyx by light microscopy and compared these with cell shapes, synapses, and circuits derived from serial-section electron microscopy. We identified three morphological types of PN boutons, unilobed, clustered, and elongated; defined three ultrastructural types, with clear- or dense-core vesicles and those with a dark cytoplasm having both; reconstructed diverse dendritic specializations of Kenyon cells; and identified Kenyon cell presynaptic sites upon extrinsic neurons. We also report new features of calyx synaptic organization, in particular extensive serial synapses that link calycal extrinsic neurons into a local network, and the numerical proportions of synaptic contacts between calycal neurons. All PN bouton types had more ribbon than nonribbon synapses, dark boutons particularly so, and ribbon synapses were larger and with more postsynaptic elements (2–14) than nonribbon (1–10). The numbers of elements were in direct proportion to presynaptic membrane area. Extrinsic neurons exclusively had ribbon synapses. J. Comp. Neurol. 520:2185–2201, 2012. © 2012 Wiley Periodicals, Inc.

Previously, we demonstrated that visual and olfactory associative memories of Drosophila share mushroom body (MB) circuits (Vogt et al., 2014). Unlike for odor representation, the MB circuit for visual information has not been characterized. Here, we show that a small subset of MB Kenyon cells (KCs) selectively responds to visual but not olfactory stimulation. The dendrites of these atypical KCs form a ventral accessory calyx (vAC), distinct from the main calyx that receives olfactory input. We identified two types of visual projection neurons (VPNs) directly connecting the optic lobes and the vAC. Strikingly, these VPNs are differentially required for visual memories of color and brightness. The segregation of visual and olfactory domains in the MB allows independent processing of distinct sensory memories and may be a conserved form of sensory representations among insects.

There is mounting molecular evidence that bacteria belonging to the phylum Plancto- mycetes are abundant in marine sponges including members of the genus Aplysina. In an attempt to culture planctomycete bacteria from Aplysina sponges, 116 bacterial strains were isolated on selec- tive oligotrophic media. Screening of the strain collection by fluorescence in situ hybridization with the planctomycete-specific probe Pla46 yielded 3 positive candidates. Nearly complete sequencing of the respective 16S rRNA genes revealed that the isolates were affiliated with 2 distinct clusters of the genus Pirellula: 1 isolate was obtained from a Mediterranean sponge, 1 from a Caribbean sponge and a third from Caribbean seawater. To our knowledge this is the first report of cultured Planctomycetes from marine sponges. The isolates grew slowly on oligotrophic media and failed to grow on nutrient- rich media. Pirellula sp. Strain 797 was pink-pigmented while the other 2 isolates, 16 and 81, were non-pigmented. Transmission electron microscopy revealed a pear- or droplet-shaped cell morphol- ogy that is characteristic of the genus Pirellula. The application of strain-specific oligonucleotide probes to sponge tissue cryosections showed that the isolates contribute only a minor fraction to the total microbial community that is associated with Aplysina spp. sponges.

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.

Sponges of the Aplysinidae family contain large amounts of bacteria that are embedded within the sponge tissue matrix. In order to determine the stability and specificity of the Aplysina–microbe association, sponges were maintained in recirculating seawater aquariums for 11 days. One aquarium was left untreated, a second one contained 0.45 μm filtered seawater (starvation conditions) and the third one contained 0.45 μm filtered seawater plus antibiotics (antibiotics exposure). Changes in the microbial community were monitored using group-specific, 16S rRNA targeted oligonucleotide probes, by denaturing gradient gel electrophoresis and by electron microscopic observations. Furthermore, the changes in the natural product profile were monitored using high-performance liquid chromatography. The measured parameters showed that a large fraction of the sponge-associated microbial community could not be cleared under the given experimental conditions. Based on these cumulative results we postulate that a large fraction of sponge-associated bacteria resides permanently in the Aplysina aerophoba mesohyl pointing to a highly integrated interaction between the host sponge and associated microorganisms.

We have employed electronmicroscopical methods (SEM, TEM) to document the microbial community associated with the marine sponge Aplysina cavernicola (formerly Verongia cavernicola, class Demospongiae). Five dominant bacterial types were identified, three of which resemble the morphotypes originally described by Vacelet (1975). One bacterial type possesses morphological properties that are characteristic of the genus Planctomyces. In addition, morphologically uniform bacteria which reside inside the nuclei of host cells were observed. Using in situ hybridization with fluorescently labelled rRNA probes directed against known bacterial groups, the phylogenetic affiliation of the mesohyl bacteria was assessed. It could be shown that the vast majority of mesohyl bacteria belongs to the domain Bacteria with a low GC content. Among the Bacteria, the delta-Proteobacteria were most abundant, followed by the gamma-Proteobacteria and representatives of the Bacteroides cluster. Clusters of Gram-positive bacteria with a high GC content were also found consistently in low amounts. No hybridization signal was obtained with probes specific to the domain Archaea, to the alpha- and beta-Proteobacteria and to the Cytophaga/Flavobacterium cluster. This study describes for the first time the application of the “top-to-bottom approach” using 16S rRNA probes and in situ hybridization to assess the microbial diversity in Aplysina sponges.

SummaryOne of the challenges facing memory research is to combine network- and cellular-level descriptions of memory encoding. In this context, Drosophila offers the opportunity to decipher, down to single-cell resolution, memory-relevant circuits in connection with the mushroom bodies (MBs), prominent structures for olfactory learning and memory. Although the MB-afferent circuits involved in appetitive learning were recently described, the circuits underlying appetitive memory retrieval remain unknown. We identified two pairs of cholinergic neurons efferent from the MB α vertical lobes, named MB-V3, that are necessary for the retrieval of appetitive long-term memory (LTM). Furthermore, LTM retrieval was correlated to an enhanced response to the rewarded odor in these neurons. Strikingly, though, silencing the MB-V3 neurons did not affect short-term memory (STM) retrieval. This finding supports a scheme of parallel appetitive STM and LTM processing.

The mushroom body is required for a variety of behaviors of Drosophila melanogaster. Different types of intrinsic and extrinsic mushroom body neurons might underlie its functional diversity. There have been many GAL4 driver lines identified that prominently label the mushroom body intrinsic neurons, which are known as "Kenyon cells." Under one constant experimental condition, we analyzed and compared the the expression patterns of 25 GAL4 drivers labeling the mushroom body. As an internet resource, we established a digital catalog indexing representative confocal data of them. Further more, we counted the number of GAL4-positive Kenyon cells in each line. We found that approximately 2,000 Kenyon cells can be genetically labeled in total. Three major Kenyon cell subtypes, the gamma, alpha'/beta', and alpha/beta neurons, respectively, contribute to 33, 18, and 49% of 2,000 Kenyon cells. Taken together, this study lays groundwork for functional dissection of the mushroom body.

Sponges (class Porifera) are evolutionarily ancient metazoans that populate the tropical oceans in great abundances but also occur in temperate regions and even in freshwater. Sponges contain large numbers of bacteria that are embedded within the animal matrix. The phylogeny of these bacteria and the evolutionary age of the interaction are virtually unknown. In order to provide insights into the species richness of the microbial community of sponges, we performed a comprehensive diversity survey based on 190 sponge-derived 16S ribosomal DNA (rDNA) sequences. The sponges Aplysina aerophoba and Theonella swinhoei were chosen for construction of the bacterial 16S rDNA library because they are taxonomically distantly related and they populate nonoverlapping geographic regions. In both sponges, a uniform microbial community was discovered whose phylogenetic signature is distinctly different from that of marine plankton or marine sediments. Altogether 14 monophyletic, sponge-specific sequence clusters were identified that belong to at least seven different bacterial divisions. By definition, the sequences of each cluster are more closely related to each other than to a sequence from nonsponge sources. These monophyletic clusters comprise 70% of all publicly available sponge-derived 16S rDNA sequences, reflecting the generality of the observed phenomenon. This shared microbial fraction represents the smallest common denominator of the sponges investigated in this study. Bacteria that are exclusively found in certain host species or that occur only transiently would have been missed. A picture emerges where sponges can be viewed as highly concentrated reservoirs of so far uncultured and elusive marine microorganisms.