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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">02395</article-id><article-id pub-id-type="doi">10.7554/eLife.02395</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Neuroscience</subject></subj-group></article-categories><title-group><article-title>Shared mushroom body circuits underlie visual and olfactory memories in <italic>Drosophila</italic></article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-11025"><name><surname>Vogt</surname><given-names>Katrin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-11026"><name><surname>Schnaitmann</surname><given-names>Christopher</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="pa1">‡</xref><xref ref-type="other" rid="par-6"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-11027"><name><surname>Dylla</surname><given-names>Kristina V</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa2">§</xref><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-11028"><name><surname>Knapek</surname><given-names>Stephan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-2209"><name><surname>Aso</surname><given-names>Yoshinori</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-10190"><name><surname>Rubin</surname><given-names>Gerald M</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-9299"><name><surname>Tanimoto</surname><given-names>Hiromu</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff3"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-7"/><xref ref-type="other" rid="par-8"/><xref ref-type="other" rid="par-9"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution>Max-Planck-Institute of Neurobiology</institution>, <addr-line><named-content content-type="city">Martinsried</named-content></addr-line>, <country>Germany</country></aff><aff id="aff2"><institution>Janelia Farm Research Campus, Howard Hughes Medical Institute</institution>, <addr-line><named-content content-type="city">Ashburn</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Graduate School of Life Sciences</institution>, <institution>Tohoku University</institution>, <addr-line><named-content content-type="city">Sendai</named-content></addr-line>, <country>Japan</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Ramaswami</surname><given-names>Mani</given-names></name><role>Reviewing editor</role><aff><institution>Trinity College Dublin</institution>, <country>Ireland</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>hiromut@m.tohoku.ac.jp</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn><fn fn-type="present-address" id="pa1"><label>‡</label><p>Neurobiologie/Tierphysiologie, Institut für Biologie 1, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany</p></fn><fn fn-type="present-address" id="pa2"><label>§</label><p>Department of Biology–Neurobiology, University of Konstanz, Konstanz, Germany</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>19</day><month>08</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e02395</elocation-id><history><date date-type="received"><day>26</day><month>01</month><year>2014</year></date><date date-type="accepted"><day>07</day><month>07</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Vogt et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Vogt et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife02395.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.02395.001</object-id><p>In nature, animals form memories associating reward or punishment with stimuli from different sensory modalities, such as smells and colors. It is unclear, however, how distinct sensory memories are processed in the brain. We established appetitive and aversive visual learning assays for <italic>Drosophila</italic> that are comparable to the widely used olfactory learning assays. These assays share critical features, such as reinforcing stimuli (sugar reward and electric shock punishment), and allow direct comparison of the cellular requirements for visual and olfactory memories. We found that the same subsets of dopamine neurons drive formation of both sensory memories. Furthermore, distinct yet partially overlapping subsets of mushroom body intrinsic neurons are required for visual and olfactory memories. Thus, our results suggest that distinct sensory memories are processed in a common brain center. Such centralization of related brain functions is an economical design that avoids the repetition of similar circuit motifs.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.001">http://dx.doi.org/10.7554/eLife.02395.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.02395.002</object-id><title>eLife digest</title><p>Animals tend to associate good and bad things with certain visual scenes, smells and other kinds of sensory information. If we get food poisoning after eating a new food, for example, we tend to associate the taste and smell of the new food with feelings of illness. This is an example of a negative ‘associative memory’, and it can persist for months, even when we know that our sickness was not caused by the new food itself but by some foreign body that should not have been in the food. The same is true for positive associative memories.</p><p>It is known that many associative memories contain information from more than one of the senses. Our memory of a favorite food, for instance, includes its scent, color and texture, as well as its taste. However, little is known about the ways in which information from the different senses is processed in the brain. Does each sense have its own dedicated memory circuit, or do multiple senses converge to the same memory circuit?</p><p>A number of studies have used olfactory (smell) and visual stimuli to study the basic neuroscience that underpins associative memories in fruit flies. The olfactory experiments traditionally use sugar and electric shocks to induce positive and negative associations with various scents. However, the visual experiments use other methods to induce associations with colors. This means that it is difficult to combine and compare the results of olfactory and visual experiments.</p><p>Now, Vogt, Schnaitmann et al. have developed a transparent grid that can be used to administer electric shocks in visual experiments. This allows direct comparisons to be made between the neuronal processing of visual associative memories and the neural processing of olfactory associative memories.</p><p>Vogt, Schnaitmann et al. showed that both visual and olfactory stimuli are modulated in the same subset of dopamine neurons for positive associative memories. Similarly, another subset of dopamine neurons was found to drive negative memories of both the visual and olfactory stimuli. The work of Vogt, Schnaitmann et al. shows that associative memories are processed by a centralized circuit that receives both visual and olfactory inputs, thus reducing the number of memory circuits needed for such memories.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.002">http://dx.doi.org/10.7554/eLife.02395.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>associative memory</kwd><kwd>dopamine neurons</kwd><kwd>visual learning</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>D. melanogaster</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100002347</institution-id><institution>Bundesministerium für Bildung und Forschung</institution></institution-wrap></funding-source><award-id>Bernstein Focus Neurobiology of Learning 01GQ0932</award-id><principal-award-recipient><name><surname>Tanimoto</surname><given-names>Hiromu</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft</institution></institution-wrap></funding-source><award-id>TA552/5-1</award-id><principal-award-recipient><name><surname>Tanimoto</surname><given-names>Hiromu</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001700</institution-id><institution>Ministry of Education, Culture, Sports, Science, and Technology/Japan Society for the Promotion of Science</institution></institution-wrap></funding-source><award-id>KAKENHI 25890003</award-id><principal-award-recipient><name><surname>Tanimoto</surname><given-names>Hiromu</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100004189</institution-id><institution>Max-Planck-Gesellschaft</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Tanimoto</surname><given-names>Hiromu</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000011</institution-id><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Aso</surname><given-names>Yoshinori</given-names></name><name><surname>Rubin</surname><given-names>Gerald M</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001645</institution-id><institution>Boehringer Ingelheim Fonds</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Schnaitmann</surname><given-names>Christopher</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001691</institution-id><institution>Ministry of Education, Culture, Sports, Science, and Technology/Japan Society for the Promotion of Science</institution></institution-wrap></funding-source><award-id>KAKENHI 26120705</award-id><principal-award-recipient><name><surname>Tanimoto</surname><given-names>Hiromu</given-names></name></principal-award-recipient></award-group><award-group id="par-8"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001700</institution-id><institution>Ministry of Education, Culture, Sports, Science, and Technology/Japan Society for the Promotion of Science</institution></institution-wrap></funding-source><award-id>KAKENHI 26250001</award-id><principal-award-recipient><name><surname>Tanimoto</surname><given-names>Hiromu</given-names></name></principal-award-recipient></award-group><award-group id="par-9"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001691</institution-id><institution>Ministry of Education, Culture, Sports, Science, and Technology/Japan Society for the Promotion of Science</institution></institution-wrap></funding-source><award-id>KAKENHI 26119503</award-id><principal-award-recipient><name><surname>Tanimoto</surname><given-names>Hiromu</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>One memory center in the fly brain processes distinct appetitive and aversive associative memories of olfactory and visual cues using shared local circuits.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>When animals encounter reward or harm, they form associations with concomitant environmental cues. Such associative memories allow an animal to predict upcoming events and to choose an appropriate behavior. Memory induced by appetitive and aversive events is usually not restricted to a single sensory cue. For example, a traumatic event drives aversive associative memories of concurrent auditory and visual stimuli in rats (<xref ref-type="bibr" rid="bib12">Campeau and Davis, 1995</xref>). The same appetitive and aversive reinforcers drive both olfactory and visual memories in insects, while associative memories with different modalities are formed using the same neurotransmitter system (<xref ref-type="bibr" rid="bib80">Unoki et al., 2005</xref>, <xref ref-type="bibr" rid="bib81">2006</xref>). However, the circuit mechanisms underlying memories of different sensory modalities driven by the same reinforcing stimulus are unknown. Two alternative circuit organizations are possible: each sensory modality may feed into a dedicated memory circuit, or representations of different sensory stimuli (e.g., olfactory and visual) may undergo associative modulation in a shared set of neurons in the brain (<xref ref-type="bibr" rid="bib91">Zars, 2010</xref>).</p><p>Cellular mechanisms underlying associative learning have been intensely studied in various animals, including <italic>Drosophila</italic> (<xref ref-type="bibr" rid="bib32">Keene and Waddell, 2007</xref>). However, comparisons between memories of different sensory modalities have led to contradictory results. For example, the mushroom bodies (MBs) are required for olfactory and gustatory memories (<xref ref-type="bibr" rid="bib27">Heisenberg et al., 1985</xref>; <xref ref-type="bibr" rid="bib16">Davis, 1993</xref>; <xref ref-type="bibr" rid="bib26">Heisenberg, 2003</xref>; <xref ref-type="bibr" rid="bib48">Masek and Scott, 2010</xref>), but according to previous studies, not for a visual memory task (<xref ref-type="bibr" rid="bib27">Heisenberg et al., 1985</xref>; <xref ref-type="bibr" rid="bib87">Wolf et al., 1998</xref>; <xref ref-type="bibr" rid="bib75">Tang and Guo, 2001</xref>; <xref ref-type="bibr" rid="bib93">Zhang et al., 2007</xref>). Nevertheless, other studies suggest that visual information is indeed processed in the MBs (<xref ref-type="bibr" rid="bib4">Barth and Heisenberg, 1997</xref>; <xref ref-type="bibr" rid="bib45">Liu et al., 1999</xref>; <xref ref-type="bibr" rid="bib10">Brembs and Wiener, 2006</xref>; <xref ref-type="bibr" rid="bib83">van Swinderen et al., 2009</xref>). These discrepancies are difficult to resolve because many of these studies, especially those comparing stimuli with a different physical nature (e.g., olfactory vs visual), employed different behavioral tasks (e.g., flight orientation or binary choice by walking flies) and/or conditioning designs (<xref ref-type="bibr" rid="bib10">Brembs and Wiener, 2006</xref>; <xref ref-type="bibr" rid="bib9">Brembs and Plendl, 2008</xref>; <xref ref-type="bibr" rid="bib62">Pitman et al., 2009</xref>; <xref ref-type="bibr" rid="bib56">Ofstad et al., 2011</xref>). We reasoned that a more informative comparison might be obtained using comparable learning paradigms (<xref ref-type="bibr" rid="bib65">Scherer et al., 2003</xref>; <xref ref-type="bibr" rid="bib20">Gerber et al., 2004a</xref>; <xref ref-type="bibr" rid="bib23">Guo and Guo, 2005</xref>; <xref ref-type="bibr" rid="bib29">Hori et al., 2006</xref>; <xref ref-type="bibr" rid="bib54">Mota et al., 2011</xref>).</p><p>We previously developed appetitive and aversive visual conditioning assays (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). During appetitive training, flies receive one of the two color stimuli together with a sugar reward, whereas the other color is presented without a reward. When they are given the choice between the two colors in a subsequent test, flies show significant conditioned approach to the previously rewarded color. The same paradigm was also used with application of acid punishment during training instead of sugar reward, leading to conditioned avoidance. This visual conditioning assay, appetitive learning in particular, shares several critical features with the well-studied olfactory conditioning assay (<xref ref-type="bibr" rid="bib76">Tempel et al., 1983</xref>; <xref ref-type="bibr" rid="bib71">Schwaerzel et al., 2003</xref>), including the conditioning design, sugar-soaked filter paper as the reward, and the use of a binary choice between two conditioned stimuli scored as an alteration in the distribution of freely moving flies. Thus, our experimental design allows direct comparison of the mechanisms underlying appetitive visual memories with those of olfactory memories.</p><p>Studies that have found distinct neuromodulator circuits underlying appetitive and aversive memories for one modality have succeeded by restricting the critical difference to reward vs punishment (<xref ref-type="bibr" rid="bib71">Schwaerzel et al., 2003</xref>; <xref ref-type="bibr" rid="bib20">Gerber et al., 2004a</xref>; <xref ref-type="bibr" rid="bib84">Vergoz et al., 2007</xref>; <xref ref-type="bibr" rid="bib28">Honjo and Furukubo-Tokunaga, 2009</xref>; <xref ref-type="bibr" rid="bib85">von Essen et al., 2011</xref>). However, there is no established aversive visual learning assay employing the widely used potent aversive reinforcer, electric shock, in the same way as in aversive olfactory conditioning (<xref ref-type="bibr" rid="bib64">Quinn et al., 1974</xref>; <xref ref-type="bibr" rid="bib78">Tully and Quinn, 1985</xref>). To meet this need, we implemented electric shock punishment into our learning assay by devising a transparent shock grid that is placed beneath the flies. This allows us to pair electric shock with the same visual stimuli as used in appetitive learning (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). Using these appetitive and aversive visual learning assays, we examined the roles of distinct aminergic neurons and found a common requirement of dopamine neurons in visual and olfactory learning. Furthermore, we demonstrate a role for the MB for appetitive and aversive visual memories, suggesting significant commonality in the neuronal mechanisms underlying memories of different sensory modalities.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Electric shock punishment induces aversive visual memories</title><p>We previously developed an appetitive visual learning assay that shares critical features with olfactory conditioning (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>; <xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). In our assay, the visual stimuli (LEDs; <xref ref-type="fig" rid="fig1">Figure 1D</xref>) are projected from below through translucent sugar-soaked filter paper, the appetitive reinforcer used in olfactory conditioning. However, the commonly used aversive reinforcer, electric shock, is more difficult to integrate, as a metal grid beneath the fly would disrupt visual stimulation from below that is used in appetitive conditioning.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02395.003</object-id><label>Figure 1.</label><caption><title>Modular appetitive and aversive visual learning.</title><p>(<bold>A</bold>–<bold>C</bold>) Experimental setups for appetitive and aversive visual learning. Scheme shows single components (<bold>B</bold>) of exchangeable conditioning arenas for sugar reward (<bold>A</bold>) and electric shock punishment (<bold>C</bold>) that share the same light source and video camera (<bold>B</bold>). (<bold>B</bold>) Appetitive setup: cylindrical Fluon-coated arena closed from top with opaque lid during training or transparent lid during test. Exchangeable Petri dish on the bottom to present sugar or water soaked filter paper during training and neutral filter paper during test. Filter paper is clamped in the dish by a plastic ring. Aversive setup: the circular arena consists of a transparent electric shock grid, removable Fluon-coated plastic ring and transparent lid. The cylinder on top isolates each setup from the others and creates a similar closed visual scene as in the appetitive setup. (<bold>D</bold>) Visual stimulus source with one blue and one green high power LED per quadrant. (<bold>E</bold>) The conditioning arena with the transparent electric shock grid and a magnification with visual stimulation and a fly. Alternating stripes marked by + and − symbols indicate electric shock application. (<bold>F</bold>) Aversive visual memory depends on shock intensity (One-way ANOVA, p < 0.001). Flies show significant memory from 15 V (One sample <italic>t</italic> test, p < 0.001) <italic>n</italic> = 15. No difference in performance is found among 30–120 V (<italic>post-hoc</italic> pairwise comparisons p > 0.05) <italic>n</italic> = 16–30. Further parametric behavioral analyses for aversive conditioning are shown in <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.003">http://dx.doi.org/10.7554/eLife.02395.003</ext-link></p></caption><graphic xlink:href="elife02395f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02395.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Conditioning with electric shock induces significant aversive visual memory.</title><p>(<bold>A</bold>) For aversive visual memory a single training trial leads to significant aversive memory (One sample <italic>t</italic> test, p < 0.001). Further repetition of training does not lead to improvement in learning (one way ANOVA, p > 0.1). <italic>n</italic> = 16. (<bold>B</bold>) A single shock (1 s) before test facilitates the memory performance at 25°C (0 V; One sample <italic>t</italic> test, p > 0.05, 30–120 V; One sample <italic>t</italic> test, p < 0.001), <italic>n</italic> = 16. Bars and error bars represent mean and SEM, respectively. (<bold>C</bold>) Green preference of flies after aversive conditioning with 60 V at 25°C. During every second of the test green preference is calculated independently for both reciprocals (Blue+/Green+ represent punished color during training).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.004">http://dx.doi.org/10.7554/eLife.02395.004</ext-link></p></caption><graphic xlink:href="elife02395fs001"/></fig></fig-group></p><p>We solved this problem by fabricating a shock grid from a transparent low-resistance material, indium tin-oxide (ITO; <xref ref-type="fig" rid="fig1">Figure 1B,C</xref>). An alternating electrode pattern was laser-etched into a thin layer of ITO on a glass plate (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). Other parameters of the assay were replicated from appetitive conditioning, except that the arena height was reduced so that flies could not escape the electric shock.</p><p>To characterize shock punishment using the transparent grid, we subjected flies to visual conditioning with four training trials as for appetitive training (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>). During one training trial, each visual stimulus (green/blue color) was alternately presented for 1 min to the flies, one of them paired with punishment (‘Materials and methods’). The electric shock served as potent aversive reinforcement and induced aversive visual memory at a signal to noise ratio comparable to visual memories in other paradigms (<xref ref-type="fig" rid="fig1">Figure 1F</xref>, <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>; <xref ref-type="bibr" rid="bib51">Menne and Spatz, 1977</xref>; <xref ref-type="bibr" rid="bib86">Wolf and Heisenberg, 1991</xref>). We found that conditioned avoidance increased with ascending voltages. A plateau was reached at approximately 30 V, and the performance did not change with more intense shock (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). Thus, we applied 60 V for all subsequent experiments. For aversive conditioning, a single shock pulse was applied 5 s before the beginning of the test to arouse the flies (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>; <xref ref-type="bibr" rid="bib51">Menne and Spatz, 1977</xref>; <xref ref-type="bibr" rid="bib19">Gerber and Hendel, 2006</xref>). Video analysis of the whole test period showed that the choice behavior stabilized within roughly 20 s (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1C</xref>). Over 90 s of the test, flies' preference for a previously shock-paired visual stimulus is decreased in comparison to an unpaired visual stimulus (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1C</xref>). Together with the previously developed appetitive memory assay, these behavioral tools allow us to compare the neural requirements of appetitive and aversive visual memory, as well as visual and olfactory memories.</p></sec><sec id="s2-2"><title>Different sets of dopamine neurons drive appetitive and aversive visual memories</title><p>Monoamine neurons were previously shown to signal reinforcement during olfactory memory formation in <italic>Drosophila</italic> (<xref ref-type="bibr" rid="bib71">Schwaerzel et al., 2003</xref>; <xref ref-type="bibr" rid="bib14">Claridge-Chang et al., 2009</xref>; <xref ref-type="bibr" rid="bib2">Aso et al., 2012</xref>; <xref ref-type="bibr" rid="bib11">Burke et al., 2012</xref>; <xref ref-type="bibr" rid="bib43">Liu et al., 2012</xref>; <xref ref-type="bibr" rid="bib72">Sitaraman et al., 2012</xref>) and other insects (<xref ref-type="bibr" rid="bib25">Hammer, 1993</xref>; <xref ref-type="bibr" rid="bib80">Unoki et al., 2005</xref>; <xref ref-type="bibr" rid="bib84">Vergoz et al., 2007</xref>). In order to identify reinforcement signaling neurons for visual memories, we therefore blocked distinct sets of aminergic neurons by expressing <italic>shibire</italic><sup><italic>ts1</italic></sup> (<italic>shi</italic><sup><italic>ts1</italic></sup>; <xref ref-type="bibr" rid="bib35">Kitamoto, 2001</xref>) and assessed these neurons' role in appetitive and aversive conditioning. To target these aminergic neurons, we chose <italic>TDC2-GAL4</italic>, <italic>TH-GAL4</italic> and <italic>DDC-GAL4</italic> driver lines that label different subsets of tyramine/octopamine and dopamine neurons (<xref ref-type="bibr" rid="bib40">Li et al., 2000</xref>; <xref ref-type="bibr" rid="bib18">Friggi-Grelin et al., 2003</xref>; <xref ref-type="bibr" rid="bib15">Cole et al., 2005</xref>). We found that the requirements of these neurons for appetitive and aversive visual memories are strikingly similar to those in olfactory memories. Blocking octopamine/tyramine neurons with <italic>TDC2-GAL4</italic> did not cause a significant defect in sucrose reward or shock punishment memory (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). In contrast, the blockade of a large fraction of dopamine neurons with <italic>TH-GAL4</italic> selectively reduced aversive memory (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). As in olfactory learning (<xref ref-type="bibr" rid="bib43">Liu et al., 2012</xref>), the blockade with <italic>DDC-GAL4</italic> that labels different sets of dopamine and serotonin neurons substantially impaired appetitive memory, but not aversive memory (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). The blockade with <italic>DDC-GAL4</italic> did not significantly affect the reflexive choice of sugar, while blocking the dopamine system with <italic>TH-GAL4</italic> caused prolonged hyperactivity that indirectly influenced shock avoidance (<xref ref-type="bibr" rid="bib38">Lebestky et al., 2009</xref>; <xref ref-type="table" rid="tbl1">Table 1</xref>).<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.02395.005</object-id><label>Figure 2.</label><caption><title>Different dopamine neurons are required for appetitive and aversive memory acquisition.</title><p>(<bold>A</bold>) Different aminergic neurons are continuously blocked with corresponding GAL4 drivers. The blockade with <italic>TH-GAL4</italic> and <italic>DDC-GAL4</italic> selectively impaired aversive (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons, p < 0.001) and appetitive memories (Kruskal–Wallis test, <italic>post-hoc</italic> pairwise comparisons, p < 0.001), respectively. Blocking octopamine and tyramine neurons does not significantly impair memory (<italic>post hoc</italic> pairwise comparisons p > 0.05). <italic>n</italic> = 8–45. (<bold>B</bold> and <bold>C</bold>) Scheme of the temperature shift to block the output of corresponding neurons during training (<bold>B</bold>) or test (<bold>C</bold>). (<bold>D</bold> and <bold>E</bold>) Output of <italic>DDC-GAL4</italic> labeled neurons is only necessary in appetitive training (one-way ANOVA, <italic>post hoc</italic> pairwise comparisons, p < 0.05) but dispensable during test (one-way ANOVA, p > 0.05). <italic>n</italic> = 13–38. (<bold>F</bold> and <bold>G</bold>) Similarly, output of <italic>TH-GAL4</italic> labeled neurons is only necessary during aversive training (Kruskal–Wallis test, <italic>post hoc</italic> pairwise comparisons, p < 0.01) but dispensable during test (one-way ANOVA, p > 0.05). <italic>n</italic> = 12–16. All flies were starved prior to the experiments. Memory of the experimental group is compared to performances of the corresponding control groups. Only the most conservative statistical result of multiple pairwise comparisons is stated. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.005">http://dx.doi.org/10.7554/eLife.02395.005</ext-link></p></caption><graphic xlink:href="elife02395f002"/></fig><table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.02395.006</object-id><label>Table 1.</label><caption><p>Sugar and shock response of the lines with impaired visual memories</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.006">http://dx.doi.org/10.7554/eLife.02395.006</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Genotype</th><th>Sugar response Mean ± SEM</th><th>Shock response Mean ± SEM</th><th><xref ref-type="table-fn" rid="tblfn2">*</xref> p < 0.05</th><th>Control data for</th></tr></thead><tbody><tr><td><italic>shi/+</italic></td><td>–</td><td>−0.429 ± 0.056</td><td/><td><xref ref-type="fig" rid="fig2 fig3">Figures 2 and 3</xref></td></tr><tr><td><italic>shi/TH</italic></td><td>–</td><td>−0.189 ± 0.019</td><td>*</td><td><xref ref-type="fig" rid="fig2">Figure 2</xref></td></tr><tr><td><italic>+/TH</italic></td><td>–</td><td>−0.386 ± 0.060</td><td/><td><xref ref-type="fig" rid="fig2">Figure 2</xref></td></tr><tr><td><italic>shi/MB504B</italic></td><td>–</td><td>−0.382 ± 0.041</td><td/><td><xref ref-type="fig" rid="fig3">Figure 3</xref></td></tr><tr><td><italic>+/MB504B</italic></td><td>–</td><td>−0.385 ± 0.051</td><td/><td><xref ref-type="fig" rid="fig3">Figure 3</xref></td></tr><tr><td><italic>shi/+</italic></td><td>0.718 ± 0.020</td><td>–</td><td/><td><xref ref-type="fig" rid="fig2">Figure 2</xref></td></tr><tr><td><italic>shi/DDC</italic></td><td>0.729 ± 0.027</td><td>–</td><td/><td><xref ref-type="fig" rid="fig2">Figure 2</xref></td></tr><tr><td><italic>+/DDC</italic></td><td>0.725 ± 0.017</td><td>–</td><td/><td><xref ref-type="fig" rid="fig2">Figure 2</xref></td></tr><tr><td><italic>shi/+</italic></td><td>0.436 ± 0.053</td><td>–</td><td/><td><xref ref-type="fig" rid="fig3">Figure 3</xref></td></tr><tr><td><italic>shi/R58E02</italic></td><td>0.544 ± 0.033</td><td>–</td><td/><td><xref ref-type="fig" rid="fig3">Figure 3</xref></td></tr><tr><td><italic>+/R58E02</italic></td><td>0.563 ± 0.036</td><td>–</td><td/><td><xref ref-type="fig" rid="fig3">Figure 3</xref></td></tr><tr><td><italic>CS</italic></td><td>0.547 ± 0.041</td><td>−0.471 ± 0.044</td><td/><td><xref ref-type="fig" rid="fig4">Figure 4</xref></td></tr><tr><td><italic>dumb</italic><sup><italic>2</italic></sup></td><td>0.634 ± 0.042</td><td>−0.289 ± 0.031</td><td>*</td><td><xref ref-type="fig" rid="fig4">Figure 4</xref></td></tr><tr><td><italic>dumb</italic><sup><italic>2</italic></sup><italic>/MB247, dumb</italic><sup><italic>2</italic></sup></td><td>–</td><td>−0.255 ± 0.028</td><td>*</td><td><xref ref-type="fig" rid="fig4">Figure 4</xref></td></tr><tr><td><italic>+/MB247</italic></td><td>–</td><td>−0.452 ± 0.067</td><td/><td><xref ref-type="fig" rid="fig4">Figure 4</xref></td></tr><tr><td><italic>shi/+</italic></td><td>0.569 ± 0.039 <xref ref-type="table-fn" rid="tblfn1">†</xref></td><td>−0.359 ± 0.034</td><td/><td><xref ref-type="fig" rid="fig5">Figure 5</xref></td></tr><tr><td><italic>shi/201y</italic></td><td>0.444 ± 0.039</td><td>−0.401 ± 0.033</td><td/><td><xref ref-type="fig" rid="fig5">Figure 5</xref></td></tr><tr><td><italic>+/201y</italic></td><td>0.649 ± 0.064</td><td>−0.353 ± 0.033</td><td/><td><xref ref-type="fig" rid="fig5">Figure 5</xref></td></tr><tr><td><italic>shi/+</italic></td><td>0.569 ± 0.039 <xref ref-type="table-fn" rid="tblfn1">†</xref></td><td>−0.387 ± 0.029</td><td/><td><xref ref-type="fig" rid="fig5">Figure 5</xref></td></tr><tr><td><italic>shi/MB247</italic></td><td>0.535 ± 0.018</td><td>−0.297 ± 0.038</td><td/><td><xref ref-type="fig" rid="fig5">Figure 5</xref></td></tr><tr><td><italic>+/MB247</italic></td><td>0.575 ± 0.048</td><td>−0.384 ± 0.028</td><td/><td><xref ref-type="fig" rid="fig5">Figure 5</xref></td></tr><tr><td><italic>shi/+</italic></td><td>0.548 ± 0.024</td><td>−0.366 ± 0.031</td><td/><td><xref ref-type="fig" rid="fig7">Figure 7</xref></td></tr><tr><td><italic>shi/MB010B</italic></td><td>0.526 ± 0.061</td><td>−0.393 ± 0.052</td><td/><td><xref ref-type="fig" rid="fig7">Figure 7</xref></td></tr><tr><td><italic>+/MB010B</italic></td><td>0.591 ± 0.048</td><td>−0.353 ± 0.040</td><td/><td><xref ref-type="fig" rid="fig7">Figure 7</xref></td></tr><tr><td><italic>shi/MB009B</italic></td><td>0.689 ± 0.067</td><td>−0.368 ± 0.028</td><td/><td><xref ref-type="fig" rid="fig7">Figure 7</xref></td></tr><tr><td><italic>+/MB009B</italic></td><td>0.739 ± 0.017</td><td>−0.373 ± 0.035</td><td/><td><xref ref-type="fig" rid="fig7">Figure 7</xref></td></tr></tbody></table><table-wrap-foot><fn><p>No significant defect in naïve sugar preference is detected among the experimental groups and the corresponding control groups (one-way ANOVA, p > 0.05), <italic>n</italic> = 4–8. No significant defect in naïve shock avoidance is detected among the experimental groups and the corresponding control groups (one-way ANOVA, p > 0.05), except for <italic>shi/TH</italic>, <italic>dumb2</italic> and <italic>dumb2/MB247, dumb2</italic> (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons, p < 0.05). <italic>n</italic> = 6–10. Consistent with a study by <xref ref-type="bibr" rid="bib38">Lebestky et al. (2009)</xref>, we observe prolonged arousal after shocking these flies, and this hyperactivity rather than shock sensitivity is a likely cause of the reduced avoidance. Indeed, the <italic>DopR</italic><sup><italic>+</italic></sup> expression in the MB rescues visual memories, even though shock avoidance is still not intact.</p></fn><fn id="tblfn1"><label>†</label><p>The identical data is represented, as these two sets of measurements were performed in parallel.</p></fn><fn id="tblfn2"><label>*</label><p>indicates p-value lower than 0.05. Corrected p-values for shock avoidance: shi/TH vs. shi/+: p = 0.006, shi/TH vs. +/TH: p = 0.037; CS vs. dumb<sup>2</sup>: p = 0.01, dumb<sup>2</sup>/MB247,dumb<sup>2</sup> vs. CS/MB247: p = 0.0492</p></fn></table-wrap-foot></table-wrap></p><p>We next analyzed the temporal requirements for neurons labeled in <italic>TH-GAL4</italic> and <italic>DDC-GAL4</italic> in our learning paradigm. We measured visual memories for 30 min retention and transiently blocked the neurons either during training (<xref ref-type="fig" rid="fig2">Figure 2B</xref>) when reinforcers were presented or during retrieval of the memory (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). The blockade with <italic>DDC-GAL4</italic> during training severely impaired appetitive memory, whereas the same blockade after the training did not significantly affect memory (<xref ref-type="fig" rid="fig2">Figure 2D,E</xref>). Similarly, the neurons labeled in <italic>TH-GAL4</italic> were required specifically during acquisition of aversive memory (<xref ref-type="fig" rid="fig2">Figure 2F,G</xref>). These results suggest that the neurons differentially labeled with <italic>DDC-GAL4</italic> and <italic>TH-GAL4</italic> mediate the formation of appetitive and aversive visual memories, likely acting as reinforcement signals. As specific subsets of dopamine neurons in <italic>TH-GAL4</italic> and <italic>DDC-GAL4</italic> have been shown to signal sugar reward and shock punishment for olfactory memories (<xref ref-type="bibr" rid="bib14">Claridge-Chang et al., 2009</xref>; <xref ref-type="bibr" rid="bib2">Aso et al., 2012</xref>; <xref ref-type="bibr" rid="bib11">Burke et al., 2012</xref>; <xref ref-type="bibr" rid="bib43">Liu et al., 2012</xref>), we genetically dissected these populations further to identify the essential neurons for visual memories.</p><p>To functionally restrict the neurons in <italic>DDC-GAL4</italic> and <italic>TH-GAL4</italic> into smaller subsets, we selected two specific GAL4 driver lines for dopamine neurons: <italic>MB504B</italic> and <italic>R58E02. R58E02-GAL4</italic> drives GAL4 expression in the PAM cluster neurons that signal reward for olfactory memory (<xref ref-type="fig" rid="fig3">Figure 3A,C</xref>; <xref ref-type="bibr" rid="bib43">Liu, et al., 2012</xref>). This driver co-expresses with <italic>DDC-GAL4</italic>, but rarely with <italic>TH-GAL4,</italic> in the PAM cluster (<xref ref-type="bibr" rid="bib43">Liu et al., 2012</xref>). <italic>MB504B</italic> is a Split GAL4 line we generated to specifically label four individual dopamine neurons in the PPL1 cluster: MB-MP1, MB-MV1, MB-V1, and the neuron that projects to the tip of the α lobe (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). These neurons are a subset of <italic>TH-GAL4</italic> and have been shown, using a less specific line, to induce aversive olfactory memory (<xref ref-type="bibr" rid="bib2">Aso et al., 2012</xref>). We found that the blockade of these neurons with <italic>shi</italic><sup><italic>ts1</italic></sup> indeed impaired aversive or appetitive visual memory, respectively (<xref ref-type="fig" rid="fig3">Figure 3C–F</xref>), but did not significantly affect the reflexive choice of sugar and shock (<xref ref-type="table" rid="tbl1">Table 1</xref>). Thus, we conclude that visual and olfactory memories share neuronal substrates for appetitive and aversive reinforcements.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.02395.007</object-id><label>Figure 3.</label><caption><title>Different sets of dopamine neurons projecting to the MB are necessary for appetitive and aversive memories.</title><p>(<bold>A</bold> and <bold>B</bold>) Expression patterns of <italic>R58E02-GAL4</italic> (PAM-Cluster) and <italic>MB504B-GAL4</italic> (PPL1-Cluster) in the MB region (outlined) are visualized by mCD8::GFP (green). Neuropil counterstaining with antibody against Synapsin (magenta). Scalebar = 50 µm. (<bold>C</bold> and <bold>D</bold>) Blocking <italic>R58E02-GAL4</italic> (<bold>C</bold>), but not <italic>MB504B-GAL4</italic> (<bold>D</bold>) subsets of dopamine neurons impairs appetitive memories, respectively (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons, p < 0.001). <italic>n</italic> = 10–21. (<bold>E</bold> and <bold>F</bold>) Blocking <italic>MB504B-GAL4</italic> (<bold>E</bold>), but not <italic>R58E02-GAL4</italic> (<bold>F</bold>) subsets of dopamine neurons impairs aversive memories, respectively (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons, p < 0.001). <italic>n</italic> = 11–21. All flies were starved prior to the experiments. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.007">http://dx.doi.org/10.7554/eLife.02395.007</ext-link></p></caption><graphic xlink:href="elife02395f003"/></fig></p><p>To examine whether the activity of these neurons directly drives memories, or carries a regulatory role, we exerted direct control over neuronal activity with <italic>R58E02-GAL4</italic> and <italic>MB504B-GAL4</italic> using a temperature-sensitive cation channel dTRPA1 (<xref ref-type="bibr" rid="bib24">Hamada et al., 2008</xref>). We paired one of the visual stimuli with thermo-activation of GAL4-expressing neurons by raising ambient temperature to 31°C and subsequently measured the flies' color preference (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). Thermo-activation of the PAM and PPL1 cluster neurons with <italic>R58E02-GAL4</italic> and <italic>MB504B-GAL4</italic> was sufficient to induce appetitive and aversive memories, respectively (<xref ref-type="fig" rid="fig4">Figure 4B,C</xref>). Based on these results we conclude that different subsets of dopamine neurons supply appetitive and aversive reinforcement information for visual as well as olfactory memories.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.02395.008</object-id><label>Figure 4.</label><caption><title>Different sets of dopamine neurons projecting to the MB are sufficient for appetitive and aversive memories.</title><p>(<bold>A</bold>) Scheme of reinforcement replacement. One visual stimulus is paired with temperature elevation (31°C) during training, leading to activation of <italic>dTrpA1</italic>-expressing neurons. (<bold>B</bold> and <bold>C</bold>) Thermo-activation with <italic>R58E02-GAL4</italic> (PAM) and <italic>MB504B-GAL4</italic> (PPL1) induces appetitive (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons test, p < 0.01) and aversive visual memories (Kruskal–Wallis test, <italic>post-hoc</italic> pairwise comparisons test, p < 0.05), respectively. <italic>n</italic> = 6–18. (<bold>D</bold>) DopR null mutant <italic>dumb</italic><sup><italic>2</italic></sup> (which also allows <italic>dumb</italic> expression via GAL4) shows a strong defect in appetitive and aversive memory (Kruskal–Wallis test, <italic>post-hoc</italic> pairwise comparisons test, p < 0.001). Expression of DopR<sup><italic>+</italic></sup> in the MB restores both forms of visual memory of the <italic>dumb</italic><sup><italic>2</italic></sup> mutant (Kruskal–Wallis test, <italic>post-hoc</italic> pairwise comparisons test, p < 0.05). <italic>n</italic> = 8–16. Visual cue discrimination for <italic>dumb</italic><sup><italic>2</italic></sup> mutant flies is shown in <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>. All flies were starved prior to the experiments. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.008">http://dx.doi.org/10.7554/eLife.02395.008</ext-link></p></caption><graphic xlink:href="elife02395f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02395.009</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Visual stimulus preference in the memory test after aversive conditioning.</title><p>The blue and green bars represent the punished color during training in both reciprocals (Blue+ and Green+). Deviation from zero shows bias in visual choice, thus animals are able to discriminate visual stimuli in test. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.009">http://dx.doi.org/10.7554/eLife.02395.009</ext-link></p></caption><graphic xlink:href="elife02395fs002"/></fig></fig-group></p><p>Since DopR, a D1-like dopamine receptor, is required for olfactory memories (<xref ref-type="bibr" rid="bib34">Kim et al., 2007</xref>; <xref ref-type="bibr" rid="bib43">Liu et al., 2012</xref>; <xref ref-type="bibr" rid="bib63">Qin et al., 2012</xref>), we hypothesized that it is also required for visual memories. Consistent with our results from the block of dopamine neurons, we found severe appetitive and aversive visual memory defects in the mutant for DopR (<italic>dumb</italic><sup><italic>2</italic></sup>; <xref ref-type="bibr" rid="bib34">Kim et al., 2007</xref>, <xref ref-type="fig" rid="fig4">Figure 4D</xref>, see <xref ref-type="table" rid="tbl1">Table 1</xref>; <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref> for controls). As both the PAM neurons in <italic>R58E02-GAL4</italic> and the PPL1 neurons in <italic>MB504B-GAL4</italic> terminate in the MBs (<xref ref-type="fig" rid="fig3">Figure 3A,B</xref>), we hypothesized that their output is transmitted to MB intrinsic neurons, Kenyon cells (KCs), through DopR. To express DopR<sup>+</sup> in the mutant background, we made use of the PiggyBac insertion mutant (<italic>dumb</italic><sup><italic>2</italic></sup>) that contains UAS in the first intron of the <italic>DopR</italic> gene allowing GAL4-dependent expression of the gene (<xref ref-type="bibr" rid="bib34">Kim et al., 2007</xref>). Selective expression of <italic>DopR</italic><sup><italic>+</italic></sup> in the KCs using <italic>MB247-GAL4</italic> significantly rescued the memory defect of the mutant (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). Altogether, these results suggest that the same sets of dopamine neurons convey reward and punishment signals to the MBs to induce appetitive and aversive memories of the different sensory modalities.</p></sec><sec id="s2-3"><title>Kenyon cells are required for visual memories</title><p>If visual information is modulated by converging dopamine signals in the MBs, the output of KCs should be essential for visual memories. To test this hypothesis, we used two distinct GAL4 drivers labeling α/β and γ KCs, <italic>201y</italic> (<xref ref-type="bibr" rid="bib90">Yang et al., 1995</xref>) and <italic>MB247</italic> (<xref ref-type="bibr" rid="bib92">Zars et al., 2000</xref>), to express <italic>shi</italic><sup><italic>ts1</italic></sup> and continuously block the output of KCs during training and test. Both appetitive and aversive memories in the experimental groups were significantly impaired (<xref ref-type="fig" rid="fig5">Figure 5A</xref>, see <xref ref-type="table" rid="tbl1">Table 1</xref> and <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref> for controls). To control for expression of <italic>shi</italic><sup><italic>ts1</italic></sup> outside the MBs, we blocked GAL4 transactivation of <italic>201y</italic> in the MBs using <italic>MB247-GAL80</italic> (<xref ref-type="bibr" rid="bib37">Krashes et al., 2007</xref>). Addition of <italic>MB247-GAL80</italic> fully restored the impaired memories (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). Thus, we conclude that visual memories require the output of KCs.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.02395.010</object-id><label>Figure 5.</label><caption><title>MBs are necessary for visual memories.</title><p>(<bold>A</bold>) Blocking output of KCs labeled with <italic>201y-GAL4</italic> and <italic>MB247-GAL4</italic> leads to significant impairment in both appetitive and aversive memories (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons, p < 0.05). <italic>n</italic> = 10–14. <italic>MB247-GAL80</italic> restores impaired memory with <italic>201y-GAL4</italic> (<italic>post-hoc</italic> pairwise comparisons, p > 0.05). <italic>n</italic> = 10–14. Visual cue discrimination of these genotypes is shown in <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>. (<bold>B</bold>) Conditioning protocol with operant component and with visual context maintained between training and test (compare to protocol in <bold>A</bold>). Visual memories with the modified protocol require intact MBs (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons, p < 0.05). <italic>n</italic> = 14–15. <xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref> shows another example for the requirement of MBs with a modified aversive conditioning protocol. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.010">http://dx.doi.org/10.7554/eLife.02395.010</ext-link></p></caption><graphic xlink:href="elife02395f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02395.011</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Visual stimulus preference in the memory test after aversive conditioning.</title><p>The blue and green bars represent the punished color during training in both reciprocals (Blue+ and Green+). Deviation from zero shows bias in visual choice, thus animals are able to discriminate visual stimuli in test. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.011">http://dx.doi.org/10.7554/eLife.02395.011</ext-link></p></caption><graphic xlink:href="elife02395fs003"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02395.012</object-id><label>Figure 5—figure supplement 2.</label><caption><title>Shock before test is dispensable at high temperature, however, requirement of MBs remains.</title><p>Test at 33°C does not require shock (Control groups: One sample <italic>t</italic> test, p < 0.01). <italic>n</italic> = 10–11. Visual memories without shock before test require intact MBs (one-way ANOVA, <italic>post-hoc</italic> pairwise comparison, p < 0.05). <italic>n</italic> = 10–11. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.012">http://dx.doi.org/10.7554/eLife.02395.012</ext-link></p></caption><graphic xlink:href="elife02395fs004"/></fig></fig-group></p><p>The MBs have been reported to be dispensable for some forms of visual memory, especially in the ‘flight simulator’ (<xref ref-type="bibr" rid="bib87">Wolf et al., 1998</xref>; <xref ref-type="bibr" rid="bib56">Ofstad et al., 2011</xref>) and to be required only when the learning context is changed between training and test (<xref ref-type="bibr" rid="bib45">Liu et al., 1999</xref>; <xref ref-type="bibr" rid="bib60">Peng et al., 2007</xref>). Our conditioning design also involves a change in the context of visual stimulation: the entire conditioning arena is homogeneously illuminated during training, whereas green and blue lights are presented in the four quadrants of the arena in the test (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). To eliminate this context change, we modified the conditioning design by simultaneously presenting both visual cues throughout training and test (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). This necessarily introduced an ‘operant’ component to the training similar to that of the standard flight simulator learning; flies can avoid electric shock by staying away from the paired color. <italic>201y-GAL4</italic> flies with blocked MBs displayed no visual memory, whereas the control flies significantly avoided the punished color (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). Thus we conclude that visual memory in our assay requires the MBs independent of the conditioning design (classical vs operant) and of context changes between training and test. Also the additional arousal of flies prior to the test by a single pulse of electric shock did not change the requirement for MB output (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>).</p></sec><sec id="s2-4"><title>Distinct but overlapping subsets of Kenyon cells are required for visual and olfactory memories</title><p>Visual and olfactory memories use the same MB-projecting dopamine neurons. We therefore asked whether the post-synaptic MB neurons are also shared, using <italic>c305-GAL4</italic> (<xref ref-type="bibr" rid="bib37">Krashes et al., 2007</xref>), <italic>17D-GAL4</italic> (<xref ref-type="bibr" rid="bib47">Martin et al., 1998</xref>) and <italic>201y-GAL4</italic> to inactivate selective KC subsets during aversive visual and olfactory conditioning. Blocking the α′/β′ neurons with <italic>c305a-GAL4</italic> selectively impaired olfactory memory (<xref ref-type="fig" rid="fig6">Figure 6A,B</xref>). In contrast, the consequences of the blockades with <italic>201y-GAL4</italic> (α/β and γ neurons) and <italic>17D-GAL4</italic> (α/β neurons) were the same in visual and olfactory memories. <italic>17D-GAL4/shi</italic><sup><italic>ts1</italic></sup> flies had no significant perturbation of either memory, while the blockade of the α/β and γ neurons with <italic>201y-GAL4</italic> strongly impaired both visual and olfactory memories (<xref ref-type="fig" rid="fig6">Figure 6A,B</xref>). Hence, visual and olfactory memories recruit partially overlapping KC subsets. The specific contribution of αʹ/βʹ neurons to olfactory learning is consistent with the preferential representation of olfactory inputs to these neurons (<xref ref-type="bibr" rid="bib79">Turner et al., 2008</xref>).<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.02395.013</object-id><label>Figure 6.</label><caption><title>Overlapping, yet distinct sets of MB-lobes are needed for aversive visual and olfactory learning.</title><p>(<bold>A</bold>) Only blocking output of neurons labeled with <italic>201y-GAL4</italic> (α/β and γ lobes) during visual conditioning leads to memory impairment (one-way ANOVA, <italic>post-hoc</italic> pairwise comparison, p < 0.01), <italic>n</italic> = 12–19. (<bold>B</bold>) In contrast, in aversive olfactory memory, blocking output of neurons labeled with <italic>201y-GAL4</italic> (α/β and γ lobes) and <italic>c305-GAL4</italic> (αʹ/βʹ lobes) leads to significant memory impairment (one-way ANOVA, <italic>post-hoc</italic> pairwise comparison, p < 0.001). <italic>n</italic> = 10–22. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.013">http://dx.doi.org/10.7554/eLife.02395.013</ext-link></p></caption><graphic xlink:href="elife02395f006"/></fig></p></sec><sec id="s2-5"><title>The γ lobe neurons are important for visual memory</title><p>In olfactory memories, the different lobes of the MBs have specific functions. To map the contributions of MB lobes to visual learning, we took a suite of Split GAL4 drivers that specifically label different lobes (<xref ref-type="fig" rid="fig7">Figure 7A</xref>, ‘Materials and methods’ section). Blocking the output of the γ lobe neurons (<xref ref-type="fig" rid="fig7">Figures 7A</xref>, <italic>MB009B-GAL4</italic>, see <xref ref-type="table" rid="tbl1">Table 1</xref> and <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref> for controls) as well as the entire KC population (<xref ref-type="fig" rid="fig7">Figures 7A</xref>, <italic>MB010B-GAL4</italic>, see <xref ref-type="table" rid="tbl1">Table 1</xref> and <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref> for controls) impaired both appetitive and aversive visual memories (<xref ref-type="fig" rid="fig7">Figure 7B</xref>).<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.02395.014</object-id><label>Figure 7.</label><caption><title>MB γ lobes are required for visual memory.</title><p>(<bold>A</bold>) Partial projections of expression patterns of Split GAL4 lines are visualized by mCD8::GFP (green). Neuropil counterstaining with antibody against Synapsin (magenta). Scalebar = 100 µm. <italic>MB010B-GAL4</italic> labels all lobes of the MB, <italic>MB009B-GAL4</italic> labels γ lobes, <italic>MB186B-GAL4</italic> labels α′/β′ lobes, <italic>MB008B-GAL4</italic> labels α/β lobes. (<bold>B</bold>) Blocking output of specific MB-lobe subsets during appetitive conditioning showed that γ lobes are specifically required (Kruskal–Wallis test, <italic>post-hoc</italic> pairwise comparisons, p < 0.05). <italic>n</italic> = 10–23. In aversive conditioning we found similar requirement of the γ lobes (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons, p < 0.05). <italic>n</italic> = 8–23. Visual cue discrimination of these genotypes is shown in <xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.014">http://dx.doi.org/10.7554/eLife.02395.014</ext-link></p></caption><graphic xlink:href="elife02395f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.02395.015</object-id><label>Figure 7—figure supplement 1.</label><caption><title>Visual stimulus preference in the memory test after aversive conditioning.</title><p>The blue and green bars represent the punished color during training in both reciprocals (Blue+ and Green+). Deviation from zero shows bias in visual choice, thus animals are able to discriminate visual stimuli in test. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.015">http://dx.doi.org/10.7554/eLife.02395.015</ext-link></p></caption><graphic xlink:href="elife02395fs005"/></fig></fig-group></p><p>Blocking the output of α′/β′ neurons (<xref ref-type="fig" rid="fig7">Figures 7A</xref>, <italic>MB186B-GAL4</italic>) or α/β neurons (<xref ref-type="fig" rid="fig7">Figures 7A</xref>, <italic>MB008B-GAL4</italic>) did not significantly reduce the performance compared to the controls (<xref ref-type="fig" rid="fig7">Figure 7B</xref>), although <italic>MB008B-GAL4/UAS-shi</italic><sup><italic>ts1</italic></sup> flies showed a tendency toward impairment. The required KC subsets for appetitive and aversive memories were strikingly similar. This suggests that the output of specific subsets of KCs representing visual information is differentially modulated by reward or punishment.</p></sec><sec id="s2-6"><title>MB output is required during training and test</title><p>Next, we explored the temporal requirements of MB output for the formation and retrieval of visual memory. We transiently blocked the output of a broad population of KCs using <italic>MB010B-GAL4</italic> (<xref ref-type="fig" rid="fig8">Figure 8A–D</xref>, all lobes) (<xref ref-type="bibr" rid="bib7">Bräcker et al., 2013</xref>) and <italic>MB247-GAL4</italic> (<xref ref-type="fig" rid="fig8">Figure 8A,B,E,F</xref>; α/β and γ lobes) and found requirement for KCs in memory acquisition and retrieval. Interestingly, the transiently blocked output of a smaller set of KCs using <italic>201y-GAL4</italic> (α/β and γ lobes) <italic>and MB009B-GAL4</italic> (γ lobes) revealed a selective requirement for the retrieval but not the formation of aversive visual memory (<xref ref-type="fig" rid="fig8">Figure 8A,B,G,J</xref>). Output of KCs labeled by <italic>MB247-GAL4</italic> (α/β and γ lobes) was also required for acquisition and retrieval of appetitive memory (<xref ref-type="fig" rid="fig8">Figure 8A,B,K,L</xref>). We thus conclude that different KCs mediate acquisition and retrieval of visual memories.<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.02395.016</object-id><label>Figure 8.</label><caption><title>MB output is needed during visual memory acquisition and retrieval.</title><p>(<bold>A</bold> and <bold>B</bold>) Scheme of the temperature shift to block the output of corresponding neurons during training (<bold>A</bold>) or test (<bold>B</bold>). (<bold>C</bold> and <bold>D</bold>) Output of neurons labeled with <italic>MB010B-GAL4</italic> is necessary during aversive training and test (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons, p < 0.05), <italic>n</italic> = 7–13. (<bold>E</bold> and <bold>F</bold>) Output of neurons labeled with <italic>MB247-GAL4</italic> is necessary during aversive training and test (one-way ANOVA, <italic>post-hoc</italic> pairwise comparison, p < 0.05). <italic>n</italic> = 10–16. (<bold>G</bold> and <bold>H</bold>) Output of neurons labeled with <italic>201y-GAL4</italic> is dispensable during training (one-way ANOVA, p > 0.05), <italic>n</italic> = 20–22, but necessary during aversive test (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons, p < 0.01) <italic>n</italic> = 12–17. (<bold>I</bold> and <bold>J</bold>) Output of neurons labeled with <italic>MB009B-GAL4</italic> is dispensable during training (one-way ANOVA, p > 0.1), <italic>n</italic> = 8, but necessary during aversive test (one-way ANOVA, <italic>post-hoc</italic> pairwise comparisons, p < 0.05) <italic>n</italic> = 8. (<bold>K</bold> and <bold>L</bold>) Output of <italic>MB247-</italic>labeled neurons is needed during appetitive training and test (one-way ANOVA, <italic>post-hoc</italic> pairwise comparison, p < 0.05). <italic>n</italic> = 10–28. Bars and error bars represent mean and SEM, respectively.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.016">http://dx.doi.org/10.7554/eLife.02395.016</ext-link></p></caption><graphic xlink:href="elife02395f008"/></fig></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><sec id="s3-1"><title>High-throughput aversive visual conditioning</title><p>Devising a transparent electric shock grid module made it possible to apply the same visual stimulation in aversive and appetitive conditioning assays. We also developed an integrated platform for fully automated high-throughput data acquisition using customized software to control the presentation of electric shock and visual stimuli while making video recordings of behavior (<xref ref-type="fig" rid="fig1">Figure 1</xref>; <xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>, <xref ref-type="bibr" rid="bib68">2013</xref>). In our assays, memory performance is based on altered visual preference in walking flies, a task likely to be less demanding than the constant flight required for flight simulator learning. These advantages facilitate behavioral examination of many genotypes.</p></sec><sec id="s3-2"><title>Associative memories of different modalities share mushroom body circuits</title><p>Circuits underlying olfactory and visual memory can be optimally compared when the sugar reward and electric shock punishment are matched between the two modalities. We found that visual and olfactory memories share the same subsets of dopamine neurons that convey reinforcing signals (<xref ref-type="fig" rid="fig2 fig3 fig4">Figures 2,3 and 4</xref>). This shared requirement of the transmitter system between visual and olfactory learning has been described in crickets (<xref ref-type="bibr" rid="bib80">Unoki et al., 2005</xref>, <xref ref-type="bibr" rid="bib81">2006</xref>; <xref ref-type="bibr" rid="bib52">Mizunami et al., 2009</xref>). However, the pharmacological manipulation used in these studies does not allow further circuit dissection.</p><p>For electric shock reinforcement, identified neurons in the PPL1 cluster, such as MB-MP1, MB-MV1 and MB-V1, drive aversive memories in both visual and olfactory learning (<xref ref-type="fig" rid="fig3 fig4">Figures 3F and 4C</xref>; <xref ref-type="bibr" rid="bib14">Claridge-Chang et al., 2009</xref>; <xref ref-type="bibr" rid="bib3">Aso et al., 2010</xref>, <xref ref-type="bibr" rid="bib2">2012</xref>), while the MB-M3 neurons in the PAM cluster seem to be involved specifically in aversive olfactory memory (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, data not shown) (<xref ref-type="bibr" rid="bib3">Aso et al., 2010</xref>, <xref ref-type="bibr" rid="bib2">2012</xref>). Thus, overlapping sets of dopamine neurons appear to represent electric shock punishment in both visual and olfactory learning with olfactory aversive memory probably recruiting a larger set. We previously showed that the MB-M3 neurons induce aversive olfactory memory that increases stability of other memory components (<xref ref-type="bibr" rid="bib2">Aso et al., 2012</xref>). Olfactory memories last longer than visual memories (data not shown; <xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>) potentially due to the recruitment of additional dopamine neurons.</p><p>In appetitive conditioning, PAM cluster neurons play crucial roles in both olfactory and visual memories (<xref ref-type="fig" rid="fig2">Figure 2A,D,E</xref>, <xref ref-type="fig" rid="fig3">Figure 3C,E</xref>, <xref ref-type="fig" rid="fig4">Figure 4B</xref>; <xref ref-type="bibr" rid="bib11">Burke et al., 2012</xref>; <xref ref-type="bibr" rid="bib43">Liu et al., 2012</xref>). Which cell types in these clusters are involved and whether there is a cellular distinction between olfactory and visual memory requires further analysis at the single cell level. Most importantly, all these neurons convey dopamine signals to restricted subdomains of the MB. The blockade of octopamine neurons did not impair appetitive visual memories with sucrose (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). The involvement of octopamine neurons may be more substantial when non-nutritious sweet taste rewards are used, as has been shown in olfactory learning (<xref ref-type="bibr" rid="bib11">Burke et al., 2012</xref>).</p><p>In addition to these shared reinforcement circuits in the MB, the necessity of MB output for visual memory acquisition and retrieval is also consistent with olfactory conditioning (<xref ref-type="fig" rid="fig8">Figure 8</xref>; <xref ref-type="bibr" rid="bib17">Dubnau et al., 2001</xref>; <xref ref-type="bibr" rid="bib49">McGuire et al., 2001</xref>; <xref ref-type="bibr" rid="bib70">Schwaerzel et al., 2002</xref>; <xref ref-type="bibr" rid="bib37">Krashes et al., 2007</xref>), although the recruited KC subsets are not identical for different modalities (<xref ref-type="fig" rid="fig6">Figure 6</xref>). Taken together, these results suggest that the MBs harbor associative plasticity for visual memories and support the conclusion that similar coincidence detection mechanisms are used to form memories within the MBs (<xref ref-type="fig" rid="fig9">Figure 9</xref>) (<xref ref-type="bibr" rid="bib26">Heisenberg, 2003</xref>; <xref ref-type="bibr" rid="bib21">Gerber et al., 2004b</xref>; <xref ref-type="bibr" rid="bib63">Qin et al., 2012</xref>). Centralization of similar brain functions spares the cost of maintaining similar circuit motifs in different brain areas and may be an evolutionary conserved design of information processing. Such converging inputs of different stimuli into a multisensory area have even been described in humans (<xref ref-type="bibr" rid="bib5">Beauchamp et al., 2008</xref>).<fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.02395.017</object-id><label>Figure 9.</label><caption><title>Circuit model of olfactory and visual short-term memories.</title><p>Visual and olfactory information is conveyed to partially overlapping sets (γ lobe neurons) of KCs. Olfactory input to the calyx (CA) via projection neurons (PN) is well characterized, whereas the visual input to the MB has not been identified yet. Output of KCs, representing olfactory and visual information is locally modulated by the different subsets of dopamine neurons (PAM, PPL1) to form appetitive and aversive memories.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.02395.017">http://dx.doi.org/10.7554/eLife.02395.017</ext-link></p></caption><graphic xlink:href="elife02395f009"/></fig></p><p>‘Flight simulator’ visual learning was shown to require the central complex but not the MBs (<xref ref-type="bibr" rid="bib87">Wolf et al., 1998</xref>; <xref ref-type="bibr" rid="bib44">Liu et al., 2006</xref>; <xref ref-type="bibr" rid="bib58">Pan et al., 2009</xref>). Although this appears to contradict our study, we note that there are important differences between the behavioral paradigms employed. In the flight simulator, a single tethered flying <italic>Drosophila</italic> is trained to associate a specific visual cue with a laser beam punishment, to later on avoid flying towards this cue in the test. Although we controlled for visual context consistency and the ‘operant component’ of the flight simulator training, any other difference could account for the differential requirement of brain structures. Given that flies during flight show octopamine-mediated modulation of neurons in the optic lobe (<xref ref-type="bibr" rid="bib73">Suver et al., 2012</xref>), similar state-dependent mechanisms might underlie different requirement of higher brain centers. Thus, it is critical to design comparable memory paradigms.</p></sec><sec id="s3-3"><title>Differentiated sensory representations in the mushroom body?</title><p>This study together with the results in associative taste learning (<xref ref-type="bibr" rid="bib48">Masek and Scott, 2010</xref>; <xref ref-type="bibr" rid="bib31">Keene and Masek, 2012</xref>) highlights the fact that the role of the MB in associative learning is not restricted to one sensory modality or reinforcer (<xref ref-type="fig" rid="fig9">Figure 9</xref>). We found that olfactory and visual memories recruit overlapping, yet partly distinct, sets of Kenyon cells (<xref ref-type="fig" rid="fig6 fig9">Figures 6,9</xref>). In contrast to the well-described olfactory projection neurons, visual inputs to the MB remain unidentified. No anatomical evidence has been reported in <italic>Drosophila</italic> for direct connections between optic lobes and MBs (<xref ref-type="bibr" rid="bib57">Otsuna and Ito, 2006</xref>; <xref ref-type="bibr" rid="bib55">Mu et al., 2012</xref>) although such connections are found in other insects (<xref ref-type="bibr" rid="bib53">Mobbs, 1982</xref>; <xref ref-type="bibr" rid="bib66">Schildberger, 1984</xref>; <xref ref-type="bibr" rid="bib41">Li and Strausfeld, 1997</xref>; <xref ref-type="bibr" rid="bib22">Gronenberg and Hölldobler, 1999</xref>; <xref ref-type="bibr" rid="bib59">Paulk and Gronenberg, 2008</xref>; <xref ref-type="bibr" rid="bib42">Lin and Strausfeld, 2012</xref>). Also afferents originating in the protocerebrum were found to provide multi-modal input to the MB lobes of cockroaches (<xref ref-type="bibr" rid="bib41">Li and Strausfeld, 1997</xref>). Thus, <italic>Drosophila</italic> MBs may receive indirect visual input from optic lobes, and the identification of such a visual pathway would significantly contribute to our understanding of the MB circuits.</p><p>Given the general requirement of the γ lobe neurons (<xref ref-type="fig" rid="fig7">Figure 7</xref>), visual and olfactory cues may be both represented in the γ neurons. Consistently, the dopamine neurons that convey appetitive and aversive memories heavily project to the γ lobe (<xref ref-type="fig" rid="fig3">Figure 3A,B</xref>; <xref ref-type="bibr" rid="bib14">Claridge-Chang et al., 2009</xref>; <xref ref-type="bibr" rid="bib3">Aso et al., 2010</xref>, <xref ref-type="bibr" rid="bib2">2012</xref>; <xref ref-type="bibr" rid="bib11">Burke et al., 2012</xref>; <xref ref-type="bibr" rid="bib43">Liu et al., 2012</xref>). In olfactory conditioning, the γ lobe was shown to contribute mainly to short-term memory (<xref ref-type="bibr" rid="bib92">Zars et al., 2000</xref>; <xref ref-type="bibr" rid="bib30">Isabel et al., 2004</xref>; <xref ref-type="bibr" rid="bib6">Blum et al., 2009</xref>; <xref ref-type="bibr" rid="bib77">Trannoy et al., 2011</xref>; <xref ref-type="bibr" rid="bib63">Qin et al., 2012</xref>). This converging evidence from olfactory and visual memories suggests a general role for the γ lobe in short-lasting memories across different sensory modalities (<xref ref-type="fig" rid="fig6 fig7 fig9">Figures 6,7 and 9</xref>). Previous studies found that the MB is also involved in sensorimotor gating of visual stimuli or visual selective attention (<xref ref-type="bibr" rid="bib82">van Swinderen and Greenspan, 2003</xref>; <xref ref-type="bibr" rid="bib88">Xi et al., 2008</xref>; <xref ref-type="bibr" rid="bib83">van Swinderen et al., 2009</xref>). Therefore, the MB circuits for visual associative memories might be required for sensorimotor gating and attention.</p><p>Interestingly, the contribution of the α′/β′ lobes is selective for olfactory memories (<xref ref-type="fig" rid="fig6">Figure 6</xref>; <xref ref-type="bibr" rid="bib37">Krashes et al., 2007</xref>; <xref ref-type="bibr" rid="bib13">Cervantes-Sandoval et al., 2013</xref>). This Kenyon cell class is more specialized to odor representation, as the cells have the broadest odor tuning and the lowest response threshold among the three Kenyon cell types (<xref ref-type="bibr" rid="bib79">Turner et al., 2008</xref>).</p><p>The role of α/β neurons in visual memories is also limited (<xref ref-type="fig" rid="fig6 fig7">Figures 6A and 7B</xref>). The α/β neurons might play more modulatory roles in specific visual memory tasks, such as context generalization, facilitation of operant learning and occasion setting (<xref ref-type="bibr" rid="bib45">Liu et al., 1999</xref>; <xref ref-type="bibr" rid="bib75">Tang and Guo, 2001</xref>; <xref ref-type="bibr" rid="bib10">Brembs and Wiener, 2006</xref>; <xref ref-type="bibr" rid="bib8">Brembs, 2009</xref>; <xref ref-type="bibr" rid="bib94">Zhang et al., 2013</xref>). This modulatory role of the α/β neurons is corroborated in olfactory learning, where they are preferentially required for long-lasting memories (<xref ref-type="bibr" rid="bib50">McGuire et al., 2003</xref>; <xref ref-type="bibr" rid="bib30">Isabel et al., 2004</xref>; <xref ref-type="bibr" rid="bib6">Blum et al., 2009</xref>; <xref ref-type="bibr" rid="bib77">Trannoy et al., 2011</xref>; <xref ref-type="bibr" rid="bib33">Keleman et al., 2012</xref>; <xref ref-type="bibr" rid="bib89">Xie et al., 2013</xref>).</p><p>Differentiated but overlapping sensory representations by KCs may be conserved among insect species. In honeybees, different sensory modalities are represented in spatially segregated areas of the calyx, whereas the basal ring region receives visual and olfactory inputs (<xref ref-type="bibr" rid="bib53">Mobbs, 1982</xref>; <xref ref-type="bibr" rid="bib74">Strausfeld, 2002</xref>). The MB might thus have evolved to represent the sensory space of those modalities that are subject to associative modulation.</p></sec></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Flies and genetic crosses</title><p>Flies were reared at 25°C, at 60% relative humidity under a 12–12 hr light–dark cycle on a standard cornmeal-based food. As all transgenes were inserted into the <italic>w</italic><sup><italic>−</italic></sup> mutant genome, the X chromosomes of strains were replaced with that of wild-type Canton-S (<italic>w</italic><sup><italic>+</italic></sup>). We used F<sub>1</sub> progenies of crosses between females of genotypes <italic>UAS-dTrpA1</italic> (<xref ref-type="bibr" rid="bib24">Hamada et al., 2008</xref>), <italic>UAS-shi</italic><sup><italic>ts</italic></sup> (<xref ref-type="bibr" rid="bib35">Kitamoto, 2001</xref>), <italic>MB247-GAL80;UAS-shi</italic><sup><italic>ts</italic></sup> (<xref ref-type="bibr" rid="bib37">Krashes et al., 2007</xref>), <italic>UAS-mCD8::GFP</italic> (<xref ref-type="bibr" rid="bib39">Lee and Luo, 1999</xref>) or WT-females and males of genotypes <italic>TH-GAL4</italic> (<xref ref-type="bibr" rid="bib18">Friggi-Grelin et al., 2003</xref>), <italic>DDC-GAL4</italic> (<xref ref-type="bibr" rid="bib40">Li et al., 2000</xref>), <italic>R58E02-GAL4</italic> (<xref ref-type="bibr" rid="bib43">Liu et al., 2012</xref>), <italic>TDC2-GAL4</italic> (<xref ref-type="bibr" rid="bib15">Cole et al., 2005</xref>), <italic>MB247-GAL4</italic> (<xref ref-type="bibr" rid="bib92">Zars et al., 2000</xref>), <italic>c305a-GAL4</italic> (<xref ref-type="bibr" rid="bib37">Krashes et al., 2007</xref>)<italic>, 17D-GAL4</italic> (<xref ref-type="bibr" rid="bib47">Martin et al., 1998</xref>), <italic>201y-GAL4</italic> (<xref ref-type="bibr" rid="bib90">Yang et al., 1995</xref>), or Canton-S males. The expression patterns of drivers for KCs were compared previously (<xref ref-type="bibr" rid="bib1">Aso et al., 2009</xref>). The <italic>dumb</italic><sup><italic>2</italic></sup> null mutant was used to localize the cells that receive dopamine signals (<xref ref-type="bibr" rid="bib34">Kim et al., 2007</xref>).</p><p>To identify a role for dopamine neurons and specific lobes of the MB in visual learning we utilized specific Split GAL4 lines. Split GAL4 lines have high specificity in expression pattern, since here the DNA-binding domain (DBD) and the activation domain (AD) of the GAL4-protein were independently targeted by different promoters. In this way, the UAS transgene is only expressed where the expression patterns of the two enhancers intersect and therefore the functional GAL4-protein can be reconstituted (<xref ref-type="bibr" rid="bib46">Luan et al., 2006</xref>; <xref ref-type="bibr" rid="bib61">Pfeiffer et al., 2010</xref>). We used F<sub>1</sub> progenies of crosses between females of genotypes <italic>UAS-shi</italic><sup><italic>ts</italic></sup> (<xref ref-type="bibr" rid="bib35">Kitamoto, 2001</xref>), <italic>UAS-mCD8::GFP</italic> (<xref ref-type="bibr" rid="bib39">Lee and Luo, 1999</xref>), <italic>UAS-dTrpA1</italic> (<xref ref-type="bibr" rid="bib24">Hamada et al., 2008</xref>) or WT-females and males of genotypes <italic>MB504B-GAL4</italic>, <italic>MB010B-GAL4</italic> (<xref ref-type="bibr" rid="bib7">Bräcker et al., 2013</xref>), <italic>MB009B-GAL4</italic>, <italic>MB008B-GAL4</italic>, <italic>MB186B-GAL4</italic> (<xref ref-type="bibr" rid="bib7">Bräcker et al., 2013</xref>) or Canton-S males. Split GAL4 lines were generated using the vectors described in <xref ref-type="bibr" rid="bib61">Pfeiffer et al. (2010)</xref> by inserting <italic>R52H03-p65ADZp</italic> into attp40 and <italic>TH-ZpGAL4DBD</italic> into attP2 (<italic>MB504B-GAL4</italic>), <italic>R13F02-p65ADZp</italic> into attP40 and <italic>R52H09-ZpGAL4DBD</italic> into attP2 (<italic>MB010B-GAL4</italic>), <italic>R13F02-p65ADZp</italic> into attP40 and <italic>R45H04-ZpGAL4DBD</italic> into attP2 (<italic>MB009B-GAL4</italic>), <italic>R13F02-p65ADZp</italic> into attP40 and <italic>R44E04-ZpGAL4DBD</italic> into attP2 (<italic>MB008B-GAL4</italic>) and <italic>R52H09-p65ADZp</italic> into attP40 and <italic>R34A03-ZpGAL4DBD</italic> into attP2 (<italic>MB186B-GAL4</italic>). Detailed methods for generating and evaluating MB Split GAL4 driver lines will be described elsewhere (Aso et al., in preparation).</p><p>As we were unable to distinguish genotype or sex in our behavioral videos, we sorted flies by genotype under CO<sub>2</sub> anesthesia at least 2 days prior to experiments. Hence, for appetitive conditioning experiments 2–4 day old flies were starved in moistened empty vials to approximately 20% mortality (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). For aversive conditioning, starvation was not applied unless otherwise explicitly stated. Behavioral experiments each used 30–40 mixed males and females under dim red light in a custom-made plastic box, containing a heating element on the bottom and a fan for air circulation.</p></sec><sec id="s4-2"><title>Apparatus for appetitive conditioning and visual stimulation</title><p>Our appetitive conditioning paradigm was as previously described (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>, <xref ref-type="bibr" rid="bib68">2013</xref>), except we used LED (instead of an LCD screen) to present visual stimuli (green and blue light) from beneath the fly (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>) (<xref ref-type="bibr" rid="bib68">Schnaitmann et al., 2013</xref>). We constructed a stimulation module using computer-controlled high-power LEDs with peak wavelengths 452 nm and 520 nm (Seoul Z-Power RGB LED, Korea) or 456 nm and 520 nm (H-HP803NB, and H-HP803PG, 3W Hexagon Power LEDs, Roithner Lasertechnik, Austria) for blue and green stimulation, respectively. LEDs were housed in a base (144 mm below the arena), which allowed homogeneous illumination of a filter paper as a screen (<xref ref-type="fig" rid="fig1">Figure 1D</xref>) (<xref ref-type="bibr" rid="bib68">Schnaitmann et al., 2013</xref>). Using a custom-made software and controlling device we were able to illuminate four quadrants of the arena independently when required (<xref ref-type="bibr" rid="bib68">Schnaitmann et al., 2013</xref>). For separate illumination of each quadrant, the light paths of LEDs were separated by light-tight walls in a cylinder with air ducts (<xref ref-type="fig" rid="fig1">Figure 1A–C</xref>). The intensities were controlled by current and calibrated using a luminance meter BM-9 (Topcon Technohouse Corporation, Japan) or a PR-655 SpectraScan Spectroradiometer, Chatsworth, CA, USA,: 14.1 Cd/m<sup>2</sup> s (blue) and 70.7 Cd/m<sup>2</sup> s (green) (<xref ref-type="bibr" rid="bib68">Schnaitmann et al., 2013</xref>). Each quadrant was equipped with an IR-LED (850 nm), that was used for background illumination, for example, during the preference/avoidance test.</p></sec><sec id="s4-3"><title>Apparatus for aversive conditioning</title><p>For aversive electric shock conditioning, we developed a new apparatus module containing an arena with a transparent shock grid (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). The arena itself consisted of the transparent shock grid on the bottom, a plastic ring as a wall and a glass lid. The shock grid was a custom-made ITO-coated glass plate (9 × 9 cm; Diamond Coatings Ltd., UK). ITO is a conductive transparent substance. A grid was laser-etched onto the ITO glass in order to insulate the positive and negative electrodes (lanes in the grid were 1.6 mm spaced 0.1 mm apart, Lasermicronics GmbH, Germany). We applied alternating current. The two halves of the grid can be independently controlled. The plastic ring (wall) and the glass lid were coated with diluted Fluon (10%; Fluon GP1, Whitford Plastics Ltd., UK) to prevent flies from walking on the lid and wall. Consequently, flies were forced to stay on the shock grid on the bottom of the arena. A filter paper was clamped underneath the shock grid and served as a screen.</p><p>A trigger (Universal-Impulsgenerator UPG 100, ELV Elektronik AG, Leer, Germany) for activating the custom-made electric shock generator was controlled by the same custom-made software used to control the LEDs (<xref ref-type="bibr" rid="bib68">Schnaitmann et al., 2013</xref>). During the test phase, the shock arena was video recorded from above with a CMOS camera (Firefly MV, Point Grey, Richmond, Canada) controlled by custom-made software (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). Four setups were run in parallel.</p></sec><sec id="s4-4"><title>Behavioral protocols for appetitive and aversive learning</title><p>We used equivalent experimental designs for appetitive and aversive conditioning; in each, differential conditioning was followed by binary choice without reinforcement (<xref ref-type="fig" rid="fig1">Figure 1</xref>, <xref ref-type="fig" rid="fig5">Figure 5A</xref>; <xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). Briefly, in a single conditioning experiment, approximately 40 flies were introduced into the arena using an aspirator. During a training trial, the whole arena was illuminated with alternating green and blue light (60 s each; conditioned stimuli = CS), one of the colors was paired with reinforcement (unconditioned stimulus = US).</p><p>For appetitive conditioning, filter paper soaked in high concentration (2 M) of sucrose and dried was presented as a reward (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). For aversive conditioning, one second of electric shock (AC 60 V) was applied 12 times in 60 s during CS+ (CS paired with reinforcement) presentation. The consecutive CS+ and CS− presentations were interspersed with 12 s intervals without illumination (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). Training trials were repeated four times per experiment if not otherwise stated (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>).</p><p>In the test, administered 60 s after the end of the last training session, flies were allowed to choose between blue and green, which were each presented in two diagonally opposite quadrants of the arena (unless otherwise stated). The distribution of the flies was video recorded for 90 s at 1 frame per second (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). No US was presented in the test period. For aversive conditioning, a 1 s shock pulse (90 V) was applied 5 s before the beginning of the test to arouse the flies (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>). However when testing flies at high temperature (33°C) this additional shock was dispensable for aversive memory retrieval (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>). Two groups trained with reciprocal CS–US pairing (Green+/Blue− and Blue+/Green−) were trained in the same setup consecutively. The difference in visual stimulus preference between the two groups was then used to calculate a learning index for each video frame (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). Reinforcement was paired with the first visual stimulus in half of the experiments, and with the second in the remaining experiments, to cancel any effect of order (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>).</p><p>Control responses to sugar and shock were measured as described previously (<xref ref-type="bibr" rid="bib69">Schnaitmann et al., 2010</xref>). The arenas used for appetitive and aversive conditioning were backlit with IR-LEDs, and flies were given a choice between two halves of the arena, one with the US presented as in the training and one without US. Their behavior was recorded for 60 s using the same video setup. A preference index was calculated by subtracting the numbers of flies on the US half from the numbers on the control half, divided by the total number of flies.</p><p>By use of a heating element and fan we were able to raise the temperature around the apparatus to a constant 33°C. In temperature shift experiments flies were transferred into moistened empty vials and kept in darkness, while the temperature was adjusted from permissive (25/26°C) to restrictive (31/33°C) or vice versa. The test was performed 30–40 min after training and started 60 s after reintroduction of the flies.</p></sec><sec id="s4-5"><title>Reinforcement substitution with thermo-activation by TrpA1</title><p>We established a new behavioral protocol for reinforcement substitution for visual memories using <italic>dTrpA1</italic> expression as in olfactory conditioning (<xref ref-type="bibr" rid="bib3">Aso et al., 2010</xref>). Flies were ‘trained’ as for conditioning, but the conditioned visual stimulus was paired not with sugar or shock but with high temperature that leads to thermo-activation of dTRPA1-expressing neurons (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). Flies were transferred between two plastic vials with different visual stimuli (blue and green as for conditioning) and different temperatures (24°C and 31°C). 5 s before the onset of the visual stimulation, flies were gently tapped into the vial on the corresponding apparatus. The two CS presentations in each training trial were intermitted by a 60 s interval at 24°C. After four training trials, flies were kept at 24°C for 60 s in the transfer vial and 60 s in the dark test apparatus before testing at 24°C. Control flies not expressing <italic>dTrpA1</italic> and wild-type flies did not show conditioned visual preference (<xref ref-type="fig" rid="fig4">Figure 4B,C</xref>). Significant memory in this assay is thus driven by appetitive or aversive reinforcement signals from thermo-activation.</p></sec><sec id="s4-6"><title>Aversive conditioning without context change</title><p>In contrast to the standard classical conditioning protocol, CS+ and CS− were simultaneously presented in the two halves of the arena during training and test (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). The half of the arena displaying CS+ was electrified. Flies were allowed to choose between the differently cued two halves for 20 s, and this training trial was repeated eight times with an inter-trial interval of 20 s without stimulus. The sides of CS/US presentation were pseudo-randomized to avoid potential prediction of the next side to be shocked. During the test, the two halves were illuminated as in training, but without shock, creating a shared context between training and test.</p></sec><sec id="s4-7"><title>Olfactory conditioning</title><p>Standard olfactory conditioning was applied (<xref ref-type="bibr" rid="bib78">Tully and Quinn, 1985</xref>; <xref ref-type="bibr" rid="bib70">Schwaerzel et al., 2002</xref>). Differential conditioning with two odors (3-octanol and benzaldehyde) followed by binary choice without reinforcement. Each odor presentation lasted 60 s, and one of the odors was paired with 12 pulses of electric shock (100 V DC). Immediately after training, flies were tested for memory performance by measuring conditioned odor avoidance in the T-maze.</p></sec><sec id="s4-8"><title>Statistics</title><p>Statistical analyses were performed with Prism5 software (GraphPad). Groups that did not violate the assumption of normal distribution (Shapiro–Wilk test) and homogeneity of variance (Bartlett's test) were analyzed with parametric statistics: one-sample <italic>t</italic>-test or one-way analysis of variance followed by the planned pairwise multiple comparisons (Bonferroni). Experiments with data that were significantly different from the assumptions above were analyzed with non-parametric tests, such as Mann–Whitney test or Kruskal–Wallis test followed by Dunn's multiple pair-wise comparison. The significance level of statistical tests was set to 0.05. Only the most conservative statistical result of multiple pairwise comparisons is indicated.</p></sec><sec id="s4-9"><title>Immunohistochemistry</title><p>Adult fly brains were dissected, fixed and stained using standard protocols (<xref ref-type="bibr" rid="bib1">Aso et al., 2009</xref>). Synapsin antibody (<xref ref-type="bibr" rid="bib36">Klagges et al., 1996</xref>) combined with Cy3-conjugated goat anti-mouse antibody were used to visualize the neuropil. Anti-GFP antibody was used to increase the intensity of the GFP signal (rabbit polyclonal to GFP [Invitrogen] with Alexa Fluor488-conjugated goat anti-rabbit as the secondary antibody). Frontal optical sections of whole-mount brains were sampled with a confocal microscope (Olympus FV1000). Images of the confocal stacks were analyzed with the open-source software Fiji (<xref ref-type="bibr" rid="bib67">Schindelin et al., 2012</xref>).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We are thankful to Anja B Friedrich for providing confocal data for <italic>R58E02-GAL4</italic> and Igor Siwanowicz for the setup sketch.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>KV, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>CS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>KVD, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>SK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>YA, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con6"><p>GMR, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con7"><p>HT, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aso</surname><given-names>Y</given-names></name><name><surname>Grübel</surname><given-names>K</given-names></name><name><surname>Busch</surname><given-names>S</given-names></name><name><surname>Friedrich</surname><given-names>AB</given-names></name><name><surname>Siwanowicz</surname><given-names>I</given-names></name><name><surname>Tanimoto</surname><given-names>H</given-names></name></person-group><year>2009</year><article-title>The mushroom body of adult <italic>Drosophila</italic> characterized by GAL4 drivers</article-title><source>Journal of Neurogenetics</source><volume>23</volume><fpage>156</fpage><lpage>172</lpage><pub-id pub-id-type="doi">10.1080/01677060802471718</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aso</surname><given-names>Y</given-names></name><name><surname>Herb</surname><given-names>A</given-names></name><name><surname>Ogueta</surname><given-names>M</given-names></name><name><surname>Siwanowicz</surname><given-names>I</given-names></name><name><surname>Templier</surname><given-names>T</given-names></name><name><surname>Friedrich</surname><given-names>AB</given-names></name><name><surname>Ito</surname><given-names>K</given-names></name><name><surname>Scholz</surname><given-names>H</given-names></name><name><surname>Tanimoto</surname><given-names>H</given-names></name></person-group><year>2012</year><article-title>Three dopamine pathways induce aversive odor memories with different stability</article-title><source>PLOS Genetics</source><volume>8</volume><fpage>e1002768</fpage><pub-id pub-id-type="doi">10.1371/journal.pgen.1002768</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aso</surname><given-names>Y</given-names></name><name><surname>Siwanowicz</surname><given-names>I</given-names></name><name><surname>Bräcker</surname><given-names>L</given-names></name><name><surname>Ito</surname><given-names>K</given-names></name><name><surname>Kitamoto</surname><given-names>T</given-names></name><name><surname>Tanimoto</surname><given-names>H</given-names></name></person-group><year>2010</year><article-title>Specific dopaminergic neurons for the formation of labile aversive memory</article-title><source>Current Biology</source><volume>20</volume><fpage>1445</fpage><lpage>1451</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2010.06.048</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Barth</surname><given-names>M</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name></person-group><year>1997</year><article-title>Vision affects mushroom bodies and central complex in <italic>Drosophila melanogaster</italic></article-title><source>Learning & Memory</source><volume>4</volume><fpage>219</fpage><lpage>229</lpage><pub-id pub-id-type="doi">10.1101/lm.4.2.219</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Beauchamp</surname><given-names>MS</given-names></name><name><surname>Yasar</surname><given-names>NE</given-names></name><name><surname>Frye</surname><given-names>RE</given-names></name><name><surname>Ro</surname><given-names>T</given-names></name></person-group><year>2008</year><article-title>Touch, sound and vision in human superior temporal sulcus</article-title><source>NeuroImage</source><volume>41</volume><fpage>1011</fpage><lpage>1020</lpage><pub-id pub-id-type="doi">10.1016/j.neuroimage.2008.03.015</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Blum</surname><given-names>AL</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Cressy</surname><given-names>M</given-names></name><name><surname>Dubnau</surname><given-names>J</given-names></name></person-group><year>2009</year><article-title>Short- and long-term memory in <italic>Drosophila</italic> require cAMP signaling in distinct neuron types</article-title><source>Current Biology</source><volume>19</volume><fpage>1341</fpage><lpage>1350</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2009.07.016</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bräcker</surname><given-names>LB</given-names></name><name><surname>Siju</surname><given-names>KP</given-names></name><name><surname>Varela</surname><given-names>N</given-names></name><name><surname>Aso</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>M</given-names></name><name><surname>Hein</surname><given-names>I</given-names></name><name><surname>Vasconcelos</surname><given-names>ML</given-names></name><name><surname>Grunwald Kadow</surname><given-names>IC</given-names></name></person-group><year>2013</year><article-title>Essential role of the mushroom body in context-dependent CO<sub>2</sub> avoidance in <italic>Drosophila</italic></article-title><source>Current Biology</source><volume>23</volume><fpage>1228</fpage><lpage>1234</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2013.05.029</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brembs</surname><given-names>B</given-names></name></person-group><year>2009</year><article-title>Mushroom bodies regulate habit formation in <italic>Drosophila</italic></article-title><source>Current Biology</source><volume>19</volume><fpage>1351</fpage><lpage>1355</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2009.06.014</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brembs</surname><given-names>B</given-names></name><name><surname>Plendl</surname><given-names>W</given-names></name></person-group><year>2008</year><article-title>Double dissociation of PKC and AC manipulations on operant and classical learning in <italic>Drosophila</italic></article-title><source>Current Biology</source><volume>18</volume><fpage>1168</fpage><lpage>1171</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2008.07.041</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brembs</surname><given-names>B</given-names></name><name><surname>Wiener</surname><given-names>J</given-names></name></person-group><year>2006</year><article-title>Context and occasion setting in <italic>Drosophila</italic> visual learning</article-title><source>Learning & Memory</source><volume>13</volume><fpage>618</fpage><lpage>628</lpage><pub-id pub-id-type="doi">10.1101/lm.318606</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Burke</surname><given-names>CJ</given-names></name><name><surname>Huetteroth</surname><given-names>W</given-names></name><name><surname>Owald</surname><given-names>D</given-names></name><name><surname>Perisse</surname><given-names>E</given-names></name><name><surname>Krashes</surname><given-names>MJ</given-names></name><name><surname>Das</surname><given-names>G</given-names></name><name><surname>Gohl</surname><given-names>D</given-names></name><name><surname>Silies</surname><given-names>M</given-names></name><name><surname>Certel</surname><given-names>S</given-names></name><name><surname>Waddell</surname><given-names>S</given-names></name></person-group><year>2012</year><article-title>Layered reward signalling through octopamine and dopamine in <italic>Drosophila</italic></article-title><source>Nature</source><volume>492</volume><fpage>433</fpage><lpage>437</lpage><pub-id pub-id-type="doi">10.1038/nature11614</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Campeau</surname><given-names>S</given-names></name><name><surname>Davis</surname><given-names>M</given-names></name></person-group><year>1995</year><article-title>Involvement of the central nucleus and basolateral complex of the amygdala in fear conditioning measured with fear-potentiated startle in rats trained concurrently with auditory and visual conditioned stimuli</article-title><source>The Journal of Neuroscience</source><volume>15</volume><fpage>2301</fpage><lpage>2311</lpage></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cervantes-Sandoval</surname><given-names>I</given-names></name><name><surname>Martin-Pena</surname><given-names>A</given-names></name><name><surname>Berry</surname><given-names>JA</given-names></name><name><surname>Davis</surname><given-names>RL</given-names></name></person-group><year>2013</year><article-title>System-like consolidation of olfactory memories in <italic>Drosophila</italic></article-title><source>The Journal of Neuroscience</source><volume>33</volume><fpage>9846</fpage><lpage>9854</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.0451-13.2013</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Claridge-Chang</surname><given-names>A</given-names></name><name><surname>Roorda</surname><given-names>RD</given-names></name><name><surname>Vrontou</surname><given-names>E</given-names></name><name><surname>Sjulson</surname><given-names>L</given-names></name><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Hirsh</surname><given-names>J</given-names></name><name><surname>Miesenbock</surname><given-names>G</given-names></name></person-group><year>2009</year><article-title>Writing memories with light-addressable reinforcement circuitry</article-title><source>Cell</source><volume>139</volume><fpage>405</fpage><lpage>415</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2009.08.034</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cole</surname><given-names>SH</given-names></name><name><surname>Carney</surname><given-names>GE</given-names></name><name><surname>McClung</surname><given-names>CA</given-names></name><name><surname>Willard</surname><given-names>SS</given-names></name><name><surname>Taylor</surname><given-names>BJ</given-names></name><name><surname>Hirsh</surname><given-names>J</given-names></name></person-group><year>2005</year><article-title>Two functional but non-complementing <italic>Drosophila</italic> tyrosine decarboxylase genes: distinct roles for neural tyramine and octopamine in female fertility</article-title><source>The Journal of Biological Chemistry</source><volume>280</volume><fpage>14948</fpage><lpage>14955</lpage><pub-id pub-id-type="doi">10.1074/jbc.M414197200</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Davis</surname><given-names>RL</given-names></name></person-group><year>1993</year><article-title>Mushroom bodies and <italic>Drosophila</italic> learning</article-title><source>Neuron</source><volume>11</volume><fpage>1</fpage><lpage>14</lpage><pub-id pub-id-type="doi">10.1016/0896-6273(93)90266-T</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dubnau</surname><given-names>J</given-names></name><name><surname>Grady</surname><given-names>L</given-names></name><name><surname>Kitamoto</surname><given-names>T</given-names></name><name><surname>Tully</surname><given-names>T</given-names></name></person-group><year>2001</year><article-title>Disruption of neurotransmission in <italic>Drosophila</italic> mushroom body blocks retrieval but not acquisition of memory</article-title><source>Nature</source><volume>411</volume><fpage>476</fpage><lpage>480</lpage><pub-id pub-id-type="doi">10.1038/35078077</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Friggi-Grelin</surname><given-names>F</given-names></name><name><surname>Coulom</surname><given-names>H</given-names></name><name><surname>Meller</surname><given-names>M</given-names></name><name><surname>Gomez</surname><given-names>D</given-names></name><name><surname>Hirsh</surname><given-names>J</given-names></name><name><surname>Birman</surname><given-names>S</given-names></name></person-group><year>2003</year><article-title>Targeted gene expression in <italic>Drosophila</italic> dopaminergic cells using regulatory sequences from tyrosine hydroxylase</article-title><source>Journal of Neurobiology</source><volume>54</volume><fpage>618</fpage><lpage>627</lpage><pub-id pub-id-type="doi">10.1002/neu.10185</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gerber</surname><given-names>B</given-names></name><name><surname>Hendel</surname><given-names>T</given-names></name></person-group><year>2006</year><article-title>Outcome expectations drive learned behaviour in larval <italic>Drosophila</italic></article-title><source>Proceedings Biological Sciences</source><volume>273</volume><fpage>2965</fpage><lpage>2968</lpage><pub-id pub-id-type="doi">10.1098/rspb.2006.3673</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gerber</surname><given-names>B</given-names></name><name><surname>Scherer</surname><given-names>S</given-names></name><name><surname>Neuser</surname><given-names>K</given-names></name><name><surname>Michels</surname><given-names>B</given-names></name><name><surname>Hendel</surname><given-names>T</given-names></name><name><surname>Stocker</surname><given-names>RF</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name></person-group><year>2004a</year><article-title>Visual learning in individually assayed <italic>Drosophila</italic> larvae</article-title><source>The Journal of Experimental Biology</source><volume>207</volume><fpage>179</fpage><lpage>188</lpage><pub-id pub-id-type="doi">10.1242/jeb.00718</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gerber</surname><given-names>B</given-names></name><name><surname>Tanimoto</surname><given-names>H</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name></person-group><year>2004b</year><article-title>An engram found? evaluating the evidence from fruit flies</article-title><source>Current Opinion in Neurobiology</source><volume>14</volume><fpage>737</fpage><lpage>744</lpage><pub-id pub-id-type="doi">10.1016/j.conb.2004.10.014</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gronenberg</surname><given-names>W</given-names></name><name><surname>Hölldobler</surname><given-names>B</given-names></name></person-group><year>1999</year><article-title>Morphologic representation of visual and antennal information in the ant brain</article-title><source>The Journal of Comparative Neurology</source><volume>412</volume><fpage>229</fpage><lpage>240</lpage><pub-id pub-id-type="doi">10.1002/(SICI)1096-9861(19990920)412:23.0.CO;2-E</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Guo</surname><given-names>J</given-names></name><name><surname>Guo</surname><given-names>A</given-names></name></person-group><year>2005</year><article-title>Crossmodal interactions between olfactory and visual learning in <italic>Drosophila</italic></article-title><source>Science</source><volume>309</volume><fpage>307</fpage><lpage>310</lpage><pub-id pub-id-type="doi">10.1126/science.1111280</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hamada</surname><given-names>FN</given-names></name><name><surname>Rosenzweig</surname><given-names>M</given-names></name><name><surname>Kang</surname><given-names>K</given-names></name><name><surname>Pulver</surname><given-names>SR</given-names></name><name><surname>Ghezzi</surname><given-names>A</given-names></name><name><surname>Jegla</surname><given-names>TJ</given-names></name><name><surname>Garrity</surname><given-names>PA</given-names></name></person-group><year>2008</year><article-title>An internal thermal sensor controlling temperature preference in <italic>Drosophila</italic></article-title><source>Nature</source><volume>454</volume><fpage>217</fpage><lpage>220</lpage><pub-id pub-id-type="doi">10.1038/nature07001</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hammer</surname><given-names>M</given-names></name></person-group><year>1993</year><article-title>An identified neuron mediates the unconditioned stimulus in associative olfactory learning in honeybees</article-title><source>Nature</source><volume>366</volume><fpage>59</fpage><lpage>63</lpage><pub-id pub-id-type="doi">10.1038/366059a0</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Heisenberg</surname><given-names>M</given-names></name></person-group><year>2003</year><article-title>Mushroom body memoir: from maps to models</article-title><source>Nature Reviews Neuroscience</source><volume>4</volume><fpage>266</fpage><lpage>275</lpage><pub-id pub-id-type="doi">10.1038/nrn1074</pub-id></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Heisenberg</surname><given-names>M</given-names></name><name><surname>Borst</surname><given-names>A</given-names></name><name><surname>Wagner</surname><given-names>S</given-names></name><name><surname>Byers</surname><given-names>D</given-names></name></person-group><year>1985</year><article-title><italic>Drosophila</italic> mushroom body mutants are deficient in olfactory learning</article-title><source>Journal of Neurogenetics</source><volume>2</volume><fpage>1</fpage><lpage>30</lpage><pub-id pub-id-type="doi">10.3109/01677068509100140</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Honjo</surname><given-names>K</given-names></name><name><surname>Furukubo-Tokunaga</surname><given-names>K</given-names></name></person-group><year>2009</year><article-title>Distinctive neuronal networks and biochemical pathways for appetitive and aversive memory in <italic>Drosophila</italic> larvae</article-title><source>The Journal of Neuroscience</source><volume>29</volume><fpage>852</fpage><lpage>862</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.1315-08.2009</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hori</surname><given-names>S</given-names></name><name><surname>Takeuchi</surname><given-names>H</given-names></name><name><surname>Arikawa</surname><given-names>K</given-names></name><name><surname>Kinoshita</surname><given-names>M</given-names></name><name><surname>Ichikawa</surname><given-names>N</given-names></name><name><surname>Sasaki</surname><given-names>M</given-names></name><name><surname>Kubo</surname><given-names>T</given-names></name></person-group><year>2006</year><article-title>Associative visual learning, color discrimination, and chromatic adaptation in the harnessed honeybee <italic>Apis mellifera L</italic></article-title><source>Journal of Comparative Physiology A, Neuroethology, Sensory, Neural, and Behavioral Physiology</source><volume>192</volume><fpage>691</fpage><lpage>700</lpage><pub-id pub-id-type="doi">10.1007/s00359-005-0091-4</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Isabel</surname><given-names>G</given-names></name><name><surname>Pascual</surname><given-names>A</given-names></name><name><surname>Preat</surname><given-names>T</given-names></name></person-group><year>2004</year><article-title>Exclusive consolidated memory phases in <italic>Drosophila</italic></article-title><source>Science</source><volume>304</volume><fpage>1024</fpage><lpage>1027</lpage><pub-id pub-id-type="doi">10.1126/science.1094932</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Keene</surname><given-names>AC</given-names></name><name><surname>Masek</surname><given-names>P</given-names></name></person-group><year>2012</year><article-title>Optogenetic induction of aversive taste memory</article-title><source>Neuroscience</source><volume>222</volume><fpage>173</fpage><lpage>180</lpage><pub-id pub-id-type="doi">10.1016/j.neuroscience.2012.07.028</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Keene</surname><given-names>AC</given-names></name><name><surname>Waddell</surname><given-names>S</given-names></name></person-group><year>2007</year><article-title><italic>Drosophila</italic> olfactory memory: single genes to complex neural circuits</article-title><source>Nature Reviews Neuroscience</source><volume>8</volume><fpage>341</fpage><lpage>354</lpage><pub-id pub-id-type="doi">10.1038/nrn2098</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Keleman</surname><given-names>K</given-names></name><name><surname>Vrontou</surname><given-names>E</given-names></name><name><surname>Kruttner</surname><given-names>S</given-names></name><name><surname>Yu</surname><given-names>JY</given-names></name><name><surname>Kurtovic-Kozaric</surname><given-names>A</given-names></name><name><surname>Dickson</surname><given-names>BJ</given-names></name></person-group><year>2012</year><article-title>Dopamine neurons modulate pheromone responses in <italic>Drosophila</italic> courtship learning</article-title><source>Nature</source><volume>489</volume><fpage>145</fpage><lpage>149</lpage><pub-id pub-id-type="doi">10.1038/nature11345</pub-id></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kim</surname><given-names>YC</given-names></name><name><surname>Lee</surname><given-names>HG</given-names></name><name><surname>Han</surname><given-names>KA</given-names></name></person-group><year>2007</year><article-title>D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in <italic>Drosophila</italic></article-title><source>The Journal of Neuroscience</source><volume>27</volume><fpage>7640</fpage><lpage>7647</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.1167-07.2007</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kitamoto</surname><given-names>T</given-names></name></person-group><year>2001</year><article-title>Conditional modification of behavior in <italic>Drosophila</italic> by targeted expression of a temperature-sensitive shibire allele in defined neurons</article-title><source>Journal of Neurobiology</source><volume>47</volume><fpage>81</fpage><lpage>92</lpage><pub-id pub-id-type="doi">10.1002/neu.1018</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Klagges</surname><given-names>BR</given-names></name><name><surname>Heimbeck</surname><given-names>G</given-names></name><name><surname>Godenschwege</surname><given-names>TA</given-names></name><name><surname>Hofbauer</surname><given-names>A</given-names></name><name><surname>Pflugfelder</surname><given-names>GO</given-names></name><name><surname>Reifegerste</surname><given-names>R</given-names></name><name><surname>Reisch</surname><given-names>D</given-names></name><name><surname>Schaupp</surname><given-names>M</given-names></name><name><surname>Buchner</surname><given-names>S</given-names></name><name><surname>Buchner</surname><given-names>E</given-names></name></person-group><year>1996</year><article-title>Invertebrate synapsins: a single gene codes for several isoforms in <italic>Drosophila</italic></article-title><source>The Journal of Neuroscience</source><volume>16</volume><fpage>3154</fpage><lpage>3165</lpage></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Krashes</surname><given-names>MJ</given-names></name><name><surname>Keene</surname><given-names>AC</given-names></name><name><surname>Leung</surname><given-names>B</given-names></name><name><surname>Armstrong</surname><given-names>JD</given-names></name><name><surname>Waddell</surname><given-names>S</given-names></name></person-group><year>2007</year><article-title>Sequential use of mushroom body neuron subsets during <italic>Drosophila</italic> odor memory processing</article-title><source>Neuron</source><volume>53</volume><fpage>103</fpage><lpage>115</lpage><pub-id pub-id-type="doi">10.1016/j.neuron.2006.11.021</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lebestky</surname><given-names>T</given-names></name><name><surname>Chang</surname><given-names>JS</given-names></name><name><surname>Dankert</surname><given-names>H</given-names></name><name><surname>Zelnik</surname><given-names>L</given-names></name><name><surname>Kim</surname><given-names>YC</given-names></name><name><surname>Han</surname><given-names>KA</given-names></name><name><surname>Wolf</surname><given-names>FW</given-names></name><name><surname>Perona</surname><given-names>P</given-names></name><name><surname>Anderson</surname><given-names>DJ</given-names></name></person-group><year>2009</year><article-title>Two different forms of arousal in <italic>Drosophila</italic> are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits</article-title><source>Neuron</source><volume>64</volume><fpage>522</fpage><lpage>536</lpage><pub-id pub-id-type="doi">10.1016/j.neuron.2009.09.031</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>T</given-names></name><name><surname>Luo</surname><given-names>L</given-names></name></person-group><year>1999</year><article-title>Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis</article-title><source>Neuron</source><volume>22</volume><fpage>451</fpage><lpage>461</lpage><pub-id pub-id-type="doi">10.1016/S0896-6273(00)80701-1</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Chaney</surname><given-names>S</given-names></name><name><surname>Roberts</surname><given-names>IJ</given-names></name><name><surname>Forte</surname><given-names>M</given-names></name><name><surname>Hirsh</surname><given-names>J</given-names></name></person-group><year>2000</year><article-title>Ectopic G-protein expression in dopamine and serotonin neurons blocks cocaine sensitization in <italic>Drosophila melanogaster</italic></article-title><source>Current Biology</source><volume>10</volume><fpage>211</fpage><lpage>214</lpage><pub-id pub-id-type="doi">10.1016/S0960-9822(00)00340-7</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Strausfeld</surname><given-names>NJ</given-names></name></person-group><year>1997</year><article-title>Morphology and sensory modality of mushroom body extrinsic neurons in the brain of the cockroach, <italic>Periplaneta americana</italic></article-title><source>The Journal of Comparative Neurology</source><volume>387</volume><fpage>631</fpage><lpage>650</lpage><pub-id pub-id-type="doi">10.1002/(SICI)1096-9861(19971103)387:43.0.CO;2-3</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lin</surname><given-names>C</given-names></name><name><surname>Strausfeld</surname><given-names>NJ</given-names></name></person-group><year>2012</year><article-title>Visual inputs to the mushroom body calyces of the whirligig beetle <italic>Dineutus sublineatus</italic>: modality switching in an insect</article-title><source>The Journal of Comparative Neurology</source><volume>520</volume><fpage>2562</fpage><lpage>2574</lpage><pub-id pub-id-type="doi">10.1002/cne.23158</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>C</given-names></name><name><surname>Placais</surname><given-names>PY</given-names></name><name><surname>Yamagata</surname><given-names>N</given-names></name><name><surname>Pfeiffer</surname><given-names>BD</given-names></name><name><surname>Aso</surname><given-names>Y</given-names></name><name><surname>Friedrich</surname><given-names>AB</given-names></name><name><surname>Siwanowicz</surname><given-names>I</given-names></name><name><surname>Rubin</surname><given-names>GM</given-names></name><name><surname>Preat</surname><given-names>T</given-names></name><name><surname>Tanimoto</surname><given-names>H</given-names></name></person-group><year>2012</year><article-title>A subset of dopamine neurons signals reward for odour memory in <italic>Drosophila</italic></article-title><source>Nature</source><volume>488</volume><fpage>512</fpage><lpage>516</lpage><pub-id pub-id-type="doi">10.1038/nature11304</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>G</given-names></name><name><surname>Seiler</surname><given-names>H</given-names></name><name><surname>Wen</surname><given-names>A</given-names></name><name><surname>Zars</surname><given-names>T</given-names></name><name><surname>Ito</surname><given-names>K</given-names></name><name><surname>Wolf</surname><given-names>R</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name><name><surname>Liu</surname><given-names>L</given-names></name></person-group><year>2006</year><article-title>Distinct memory traces for two visual features in the <italic>Drosophila</italic> brain</article-title><source>Nature</source><volume>439</volume><fpage>551</fpage><lpage>556</lpage><pub-id pub-id-type="doi">10.1038/nature04381</pub-id></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>L</given-names></name><name><surname>Wolf</surname><given-names>R</given-names></name><name><surname>Ernst</surname><given-names>R</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name></person-group><year>1999</year><article-title>Context generalization in <italic>Drosophila</italic> visual learning requires the mushroom bodies</article-title><source>Nature</source><volume>400</volume><fpage>753</fpage><lpage>756</lpage><pub-id pub-id-type="doi">10.1038/23456</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Luan</surname><given-names>H</given-names></name><name><surname>Peabody</surname><given-names>NC</given-names></name><name><surname>Vinson</surname><given-names>CR</given-names></name><name><surname>White</surname><given-names>BH</given-names></name></person-group><year>2006</year><article-title>Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression</article-title><source>Neuron</source><volume>52</volume><fpage>425</fpage><lpage>436</lpage><pub-id pub-id-type="doi">10.1016/j.neuron.2006.08.028</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Martin</surname><given-names>JR</given-names></name><name><surname>Ernst</surname><given-names>R</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name></person-group><year>1998</year><article-title>Mushroom bodies suppress locomotor activity in <italic>Drosophila melanogaster</italic></article-title><source>Learning & Memory</source><volume>5</volume><fpage>179</fpage><lpage>191</lpage></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Masek</surname><given-names>P</given-names></name><name><surname>Scott</surname><given-names>K</given-names></name></person-group><year>2010</year><article-title>Limited taste discrimination in <italic>Drosophila</italic></article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>107</volume><fpage>14833</fpage><lpage>14838</lpage><pub-id pub-id-type="doi">10.1073/pnas.1009318107</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McGuire</surname><given-names>SE</given-names></name><name><surname>Le</surname><given-names>PT</given-names></name><name><surname>Davis</surname><given-names>RL</given-names></name></person-group><year>2001</year><article-title>The role of <italic>Drosophila</italic> mushroom body signaling in olfactory memory</article-title><source>Science</source><volume>293</volume><fpage>1330</fpage><lpage>1333</lpage><pub-id pub-id-type="doi">10.1126/science.1062622</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McGuire</surname><given-names>SE</given-names></name><name><surname>Le</surname><given-names>PT</given-names></name><name><surname>Osborn</surname><given-names>AJ</given-names></name><name><surname>Matsumoto</surname><given-names>K</given-names></name><name><surname>Davis</surname><given-names>RL</given-names></name></person-group><year>2003</year><article-title>Spatiotemporal rescue of memory dysfunction in <italic>Drosophila</italic></article-title><source>Science</source><volume>302</volume><fpage>1765</fpage><lpage>1768</lpage><pub-id pub-id-type="doi">10.1126/science.1089035</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Menne</surname><given-names>D</given-names></name><name><surname>Spatz</surname><given-names>HC</given-names></name></person-group><year>1977</year><article-title>Colour vision in <italic>Drosophila melanogaster</italic></article-title><source>Journal of Comparative Physiology A, Neuroethology, Sensory, Neural, and Behavioral Physiology</source><volume>114</volume><fpage>301</fpage><lpage>312</lpage><pub-id pub-id-type="doi">10.1007/BF00657325</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mizunami</surname><given-names>M</given-names></name><name><surname>Unoki</surname><given-names>S</given-names></name><name><surname>Mori</surname><given-names>Y</given-names></name><name><surname>Hirashima</surname><given-names>D</given-names></name><name><surname>Hatano</surname><given-names>A</given-names></name><name><surname>Matsumoto</surname><given-names>Y</given-names></name></person-group><year>2009</year><article-title>Roles of octopaminergic and dopaminergic neurons in appetitive and aversive memory recall in an insect</article-title><source>BMC Biology</source><volume>7</volume><fpage>46</fpage><pub-id pub-id-type="doi">10.1186/1741-7007-7-46</pub-id></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mobbs</surname><given-names>PG</given-names></name></person-group><year>1982</year><article-title>The brain of the honeybee <italic>Apis mellifera. I</italic>. The connections and spatial organization of the mushroom bodies</article-title><source>Philosophical Transactions of the Royal Society of London</source><fpage>309</fpage><lpage>354</lpage></element-citation></ref><ref id="bib54"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mota</surname><given-names>T</given-names></name><name><surname>Roussel</surname><given-names>E</given-names></name><name><surname>Sandoz</surname><given-names>JC</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><year>2011</year><article-title>Visual conditioning of the sting extension reflex in harnessed honeybees</article-title><source>The Journal of Experimental Biology</source><volume>214</volume><fpage>3577</fpage><lpage>3587</lpage><pub-id pub-id-type="doi">10.1242/jeb.062026</pub-id></element-citation></ref><ref id="bib55"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mu</surname><given-names>L</given-names></name><name><surname>Ito</surname><given-names>K</given-names></name><name><surname>Bacon</surname><given-names>JP</given-names></name><name><surname>Strausfeld</surname><given-names>NJ</given-names></name></person-group><year>2012</year><article-title>Optic glomeruli and their inputs in <italic>Drosophila</italic> share an organizational ground pattern with the antennal lobes</article-title><source>The Journal of Neuroscience</source><volume>32</volume><fpage>6061</fpage><lpage>6071</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.0221-12.2012</pub-id></element-citation></ref><ref id="bib56"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ofstad</surname><given-names>TA</given-names></name><name><surname>Zuker</surname><given-names>CS</given-names></name><name><surname>Reiser</surname><given-names>MB</given-names></name></person-group><year>2011</year><article-title>Visual place learning in <italic>Drosophila melanogaster</italic></article-title><source>Nature</source><volume>474</volume><fpage>204</fpage><lpage>207</lpage><pub-id pub-id-type="doi">10.1038/nature10131</pub-id></element-citation></ref><ref id="bib57"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Otsuna</surname><given-names>H</given-names></name><name><surname>Ito</surname><given-names>K</given-names></name></person-group><year>2006</year><article-title>Systematic analysis of the visual projection neurons of <italic>Drosophila melanogaster</italic>. <italic>I</italic>. Lobula-specific pathways</article-title><source>The Journal of Comparative Neurology</source><volume>497</volume><fpage>928</fpage><lpage>958</lpage><pub-id pub-id-type="doi">10.1002/cne.21015</pub-id></element-citation></ref><ref id="bib58"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pan</surname><given-names>Y</given-names></name><name><surname>Zhou</surname><given-names>Y</given-names></name><name><surname>Guo</surname><given-names>C</given-names></name><name><surname>Gong</surname><given-names>H</given-names></name><name><surname>Gong</surname><given-names>Z</given-names></name><name><surname>Liu</surname><given-names>L</given-names></name></person-group><year>2009</year><article-title>Differential roles of the fan-shaped body and the ellipsoid body in <italic>Drosophila</italic> visual pattern memory</article-title><source>Learning & Memory</source><volume>16</volume><fpage>289</fpage><lpage>295</lpage><pub-id pub-id-type="doi">10.1101/lm.1331809</pub-id></element-citation></ref><ref id="bib59"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Paulk</surname><given-names>AC</given-names></name><name><surname>Gronenberg</surname><given-names>W</given-names></name></person-group><year>2008</year><article-title>Higher order visual input to the mushroom bodies in the bee, <italic>Bombus impatiens</italic></article-title><source>Arthropod Structure & Development</source><volume>37</volume><fpage>443</fpage><lpage>458</lpage><pub-id pub-id-type="doi">10.1016/j.asd.2008.03.002</pub-id></element-citation></ref><ref id="bib60"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Peng</surname><given-names>Y</given-names></name><name><surname>Xi</surname><given-names>W</given-names></name><name><surname>Zhang</surname><given-names>W</given-names></name><name><surname>Zhang</surname><given-names>K</given-names></name><name><surname>Guo</surname><given-names>A</given-names></name></person-group><year>2007</year><article-title>Experience improves feature extraction in <italic>Drosophila</italic></article-title><source>The Journal of Neuroscience</source><volume>27</volume><fpage>5139</fpage><lpage>5145</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.0472-07.2007</pub-id></element-citation></ref><ref id="bib61"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pfeiffer</surname><given-names>BD</given-names></name><name><surname>Ngo</surname><given-names>TT</given-names></name><name><surname>Hibbard</surname><given-names>KL</given-names></name><name><surname>Murphy</surname><given-names>C</given-names></name><name><surname>Jenett</surname><given-names>A</given-names></name><name><surname>Truman</surname><given-names>JW</given-names></name><name><surname>Rubin</surname><given-names>GM</given-names></name></person-group><year>2010</year><article-title>Refinement of tools for targeted gene expression in <italic>Drosophila</italic></article-title><source>Genetics</source><volume>186</volume><fpage>735</fpage><lpage>755</lpage><pub-id pub-id-type="doi">10.1534/genetics.110.119917</pub-id></element-citation></ref><ref id="bib62"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pitman</surname><given-names>JL</given-names></name><name><surname>DasGupta</surname><given-names>S</given-names></name><name><surname>Krashes</surname><given-names>MJ</given-names></name><name><surname>Leung</surname><given-names>B</given-names></name><name><surname>Perrat</surname><given-names>PN</given-names></name><name><surname>Waddell</surname><given-names>S</given-names></name></person-group><year>2009</year><article-title>There are many ways to train a fly</article-title><source>Fly</source><volume>3</volume><fpage>3</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.4161/fly.3.1.7726</pub-id></element-citation></ref><ref id="bib63"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Qin</surname><given-names>H</given-names></name><name><surname>Cressy</surname><given-names>M</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Coravos</surname><given-names>JS</given-names></name><name><surname>Izzi</surname><given-names>SA</given-names></name><name><surname>Dubnau</surname><given-names>J</given-names></name></person-group><year>2012</year><article-title>Gamma neurons mediate dopaminergic input during aversive olfactory memory formation in <italic>Drosophila</italic></article-title><source>Current Biology</source><volume>22</volume><fpage>608</fpage><lpage>614</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2012.02.014</pub-id></element-citation></ref><ref id="bib64"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Quinn</surname><given-names>WG</given-names></name><name><surname>Harris</surname><given-names>WA</given-names></name><name><surname>Benzer</surname><given-names>S</given-names></name></person-group><year>1974</year><article-title>Conditioned behavior in <italic>Drosophila melanogaster</italic></article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>71</volume><fpage>708</fpage><lpage>712</lpage><pub-id pub-id-type="doi">10.1073/pnas.71.3.708</pub-id></element-citation></ref><ref id="bib65"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Scherer</surname><given-names>S</given-names></name><name><surname>Stocker</surname><given-names>RF</given-names></name><name><surname>Gerber</surname><given-names>B</given-names></name></person-group><year>2003</year><article-title>Olfactory learning in individually assayed <italic>Drosophila</italic> larvae</article-title><source>Learning & Memory</source><volume>10</volume><fpage>217</fpage><lpage>225</lpage><pub-id pub-id-type="doi">10.1101/lm.57903</pub-id></element-citation></ref><ref id="bib66"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schildberger</surname><given-names>K</given-names></name></person-group><year>1984</year><article-title>Multimodal interneurons in the cricket brain: properties of identified extrinsic mushroom body cells</article-title><source>Journal of Comparative Physiology A, Neuroethology, Sensory, Neural, and Behavioral Physiology</source><volume>154</volume><fpage>71</fpage><lpage>79</lpage><pub-id pub-id-type="doi">10.1007/BF00605392</pub-id></element-citation></ref><ref id="bib67"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schindelin</surname><given-names>J</given-names></name><name><surname>Arganda-Carreras</surname><given-names>I</given-names></name><name><surname>Frise</surname><given-names>E</given-names></name><name><surname>Kaynig</surname><given-names>V</given-names></name><name><surname>Longair</surname><given-names>M</given-names></name><name><surname>Pietzsch</surname><given-names>T</given-names></name><name><surname>Preibisch</surname><given-names>S</given-names></name><name><surname>Rueden</surname><given-names>C</given-names></name><name><surname>Saalfeld</surname><given-names>S</given-names></name><name><surname>Schmid</surname><given-names>B</given-names></name><name><surname>Tinevez</surname><given-names>JY</given-names></name><name><surname>White</surname><given-names>DJ</given-names></name><name><surname>Hartenstein</surname><given-names>V</given-names></name><name><surname>Eliceiri</surname><given-names>K</given-names></name><name><surname>Tomancak</surname><given-names>P</given-names></name><name><surname>Cardona</surname><given-names>A</given-names></name></person-group><year>2012</year><article-title>Fiji: an open-source platform for biological-image analysis</article-title><source>Nature Methods</source><volume>9</volume><fpage>676</fpage><lpage>682</lpage><pub-id pub-id-type="doi">10.1038/nmeth.2019</pub-id></element-citation></ref><ref id="bib68"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schnaitmann</surname><given-names>C</given-names></name><name><surname>Garbers</surname><given-names>C</given-names></name><name><surname>Wachtler</surname><given-names>T</given-names></name><name><surname>Tanimoto</surname><given-names>H</given-names></name></person-group><year>2013</year><article-title>Color discrimination with broadband photoreceptors</article-title><source>Current Biology</source><volume>23</volume><fpage>2375</fpage><lpage>2382</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2013.10.037</pub-id></element-citation></ref><ref id="bib69"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schnaitmann</surname><given-names>C</given-names></name><name><surname>Vogt</surname><given-names>K</given-names></name><name><surname>Triphan</surname><given-names>T</given-names></name><name><surname>Tanimoto</surname><given-names>H</given-names></name></person-group><year>2010</year><article-title>Appetitive and aversive visual learning in freely moving <italic>Drosophila</italic></article-title><source>Frontiers in Behavioral Neuroscience</source><volume>4</volume><fpage>10</fpage><pub-id pub-id-type="doi">10.3389/fnbeh.2010.00010</pub-id></element-citation></ref><ref id="bib70"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schwaerzel</surname><given-names>M</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name><name><surname>Zars</surname><given-names>T</given-names></name></person-group><year>2002</year><article-title>Extinction antagonizes olfactory memory at the subcellular level</article-title><source>Neuron</source><volume>35</volume><fpage>951</fpage><lpage>960</lpage><pub-id pub-id-type="doi">10.1016/S0896-6273(02)00832-2</pub-id></element-citation></ref><ref id="bib71"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schwaerzel</surname><given-names>M</given-names></name><name><surname>Monastirioti</surname><given-names>M</given-names></name><name><surname>Scholz</surname><given-names>H</given-names></name><name><surname>Friggi-Grelin</surname><given-names>F</given-names></name><name><surname>Birman</surname><given-names>S</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name></person-group><year>2003</year><article-title>Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in <italic>Drosophila</italic></article-title><source>The Journal of Neuroscience</source><volume>23</volume><fpage>10495</fpage><lpage>10502</lpage></element-citation></ref><ref id="bib72"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sitaraman</surname><given-names>D</given-names></name><name><surname>LaFerriere</surname><given-names>H</given-names></name><name><surname>Birman</surname><given-names>S</given-names></name><name><surname>Zars</surname><given-names>T</given-names></name></person-group><year>2012</year><article-title>Serotonin is critical for rewarded olfactory short-term memory in <italic>Drosophila</italic></article-title><source>Journal of Neurogenetics</source><volume>26</volume><fpage>238</fpage><lpage>244</lpage><pub-id pub-id-type="doi">10.3109/01677063.2012.666298</pub-id></element-citation></ref><ref id="bib73"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Suver</surname><given-names>MP</given-names></name><name><surname>Mamiya</surname><given-names>A</given-names></name><name><surname>Dickinson</surname><given-names>H</given-names></name></person-group><year>2012</year><article-title>Octopamine neurons mediate flight-induced modulation of visual processing in <italic>Drosophila</italic></article-title><source>Current Biology</source><volume>22</volume><fpage>2294</fpage><lpage>2302</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2012.10.034</pub-id></element-citation></ref><ref id="bib74"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Strausfeld</surname><given-names>NJ</given-names></name></person-group><year>2002</year><article-title>Organization of the honey bee mushroom body: representation of the calyx within the vertical and gamma lobes</article-title><source>The Journal of Comparative Neurology</source><volume>450</volume><fpage>4</fpage><lpage>33</lpage><pub-id pub-id-type="doi">10.1002/cne.10285</pub-id></element-citation></ref><ref id="bib75"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tang</surname><given-names>S</given-names></name><name><surname>Guo</surname><given-names>A</given-names></name></person-group><year>2001</year><article-title>Choice behavior of <italic>Drosophila</italic> facing contradictory visual cues</article-title><source>Science</source><volume>294</volume><fpage>1543</fpage><lpage>1547</lpage><pub-id pub-id-type="doi">10.1126/science.1058237</pub-id></element-citation></ref><ref id="bib76"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tempel</surname><given-names>BL</given-names></name><name><surname>Bonini</surname><given-names>N</given-names></name><name><surname>Dawson</surname><given-names>DR</given-names></name><name><surname>Quinn</surname><given-names>WG</given-names></name></person-group><year>1983</year><article-title>Reward learning in normal and mutant <italic>Drosophila</italic></article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>80</volume><fpage>1482</fpage><lpage>1486</lpage><pub-id pub-id-type="doi">10.1073/pnas.80.5.1482</pub-id></element-citation></ref><ref id="bib77"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Trannoy</surname><given-names>S</given-names></name><name><surname>Redt-Clouet</surname><given-names>C</given-names></name><name><surname>Dura</surname><given-names>JM</given-names></name><name><surname>Preat</surname><given-names>T</given-names></name></person-group><year>2011</year><article-title>Parallel processing of appetitive short- and long-term memories in <italic>Drosophila</italic></article-title><source>Current Biology</source><volume>21</volume><fpage>1647</fpage><lpage>1653</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2011.08.032</pub-id></element-citation></ref><ref id="bib78"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tully</surname><given-names>T</given-names></name><name><surname>Quinn</surname><given-names>WG</given-names></name></person-group><year>1985</year><article-title>Classical conditioning and retention in normal and mutant <italic>Drosophila melanogaster</italic></article-title><source>Journal of Comparative Physiology A, Sensory, Neural, and Behavioral Physiology</source><volume>157</volume><fpage>263</fpage><lpage>277</lpage><pub-id pub-id-type="doi">10.1007/BF01350033</pub-id></element-citation></ref><ref id="bib79"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Turner</surname><given-names>GC</given-names></name><name><surname>Bazhenov</surname><given-names>M</given-names></name><name><surname>Laurent</surname><given-names>G</given-names></name></person-group><year>2008</year><article-title>Olfactory representations by <italic>Drosophila</italic> mushroom body neurons</article-title><source>Journal of Neurophysiology</source><volume>99</volume><fpage>734</fpage><lpage>746</lpage><pub-id pub-id-type="doi">10.1152/jn.01283.2007</pub-id></element-citation></ref><ref id="bib80"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Unoki</surname><given-names>S</given-names></name><name><surname>Matsumoto</surname><given-names>Y</given-names></name><name><surname>Mizunami</surname><given-names>M</given-names></name></person-group><year>2005</year><article-title>Participation of octopaminergic reward system and dopaminergic punishment system in insect olfactory learning revealed by pharmacological study</article-title><source>The European Journal of Neuroscience</source><volume>22</volume><fpage>1409</fpage><lpage>1416</lpage><pub-id pub-id-type="doi">10.1111/j.1460-9568.2005.04318.x</pub-id></element-citation></ref><ref id="bib81"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Unoki</surname><given-names>S</given-names></name><name><surname>Matsumoto</surname><given-names>Y</given-names></name><name><surname>Mizunami</surname><given-names>M</given-names></name></person-group><year>2006</year><article-title>Roles of octopaminergic and dopaminergic neurons in mediating reward and punishment signals in insect visual learning</article-title><source>The European Journal of Neuroscience</source><volume>24</volume><fpage>2031</fpage><lpage>2038</lpage><pub-id pub-id-type="doi">10.1111/j.1460-9568.2006.05099.x</pub-id></element-citation></ref><ref id="bib82"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Swinderen</surname><given-names>B</given-names></name><name><surname>Greenspan</surname><given-names>RJ</given-names></name></person-group><year>2003</year><article-title>Salience modulates 20-30 Hz brain activity in <italic>Drosophila</italic></article-title><source>Nature Neuroscience</source><volume>6</volume><fpage>579</fpage><lpage>586</lpage><pub-id pub-id-type="doi">10.1038/nn1054</pub-id></element-citation></ref><ref id="bib83"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Swinderen</surname><given-names>B</given-names></name><name><surname>McCartney</surname><given-names>A</given-names></name><name><surname>Kauffman</surname><given-names>S</given-names></name><name><surname>Flores</surname><given-names>K</given-names></name><name><surname>Agrawal</surname><given-names>K</given-names></name><name><surname>Wagner</surname><given-names>J</given-names></name><name><surname>Paulk</surname><given-names>A</given-names></name></person-group><year>2009</year><article-title>Shared visual attention and memory systems in the <italic>Drosophila</italic> brain</article-title><source>PLOS ONE</source><volume>4</volume><fpage>e5989</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0005989</pub-id></element-citation></ref><ref id="bib84"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vergoz</surname><given-names>V</given-names></name><name><surname>Roussel</surname><given-names>E</given-names></name><name><surname>Sandoz</surname><given-names>JC</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><year>2007</year><article-title>Aversive learning in honeybees revealed by the olfactory conditioning of the sting extension reflex</article-title><source>PLOS ONE</source><volume>2</volume><fpage>e288</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0000288</pub-id></element-citation></ref><ref id="bib85"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>von Essen</surname><given-names>AM</given-names></name><name><surname>Pauls</surname><given-names>D</given-names></name><name><surname>Thum</surname><given-names>AS</given-names></name><name><surname>Sprecher</surname><given-names>SG</given-names></name></person-group><year>2011</year><article-title>Capacity of visual classical conditioning in <italic>Drosophila</italic> larvae</article-title><source>Behavioral Neuroscience</source><volume>125</volume><fpage>921</fpage><lpage>929</lpage><pub-id pub-id-type="doi">10.1037/a0025758</pub-id></element-citation></ref><ref id="bib86"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wolf</surname><given-names>R</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name></person-group><year>1991</year><article-title>Basic organization of operant behavior as revealed in <italic>Drosophila</italic> flight orientation</article-title><source>Journal of Comparative Physiology A, Sensory, Neural, and Behavioral Physiology</source><volume>169</volume><fpage>699</fpage><lpage>705</lpage><pub-id pub-id-type="doi">10.1007/BF00194898</pub-id></element-citation></ref><ref id="bib87"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wolf</surname><given-names>R</given-names></name><name><surname>Wittig</surname><given-names>T</given-names></name><name><surname>Liu</surname><given-names>L</given-names></name><name><surname>Wustmann</surname><given-names>G</given-names></name><name><surname>Eyding</surname><given-names>D</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name></person-group><year>1998</year><article-title><italic>Drosophila</italic> mushroom bodies are dispensable for visual, tactile, and motor learning</article-title><source>Learning & Memory</source><volume>5</volume><fpage>166</fpage><lpage>178</lpage></element-citation></ref><ref id="bib88"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Xi</surname><given-names>W</given-names></name><name><surname>Peng</surname><given-names>Y</given-names></name><name><surname>Guo</surname><given-names>J</given-names></name><name><surname>Ye</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>K</given-names></name><name><surname>Yu</surname><given-names>F</given-names></name><name><surname>Guo</surname><given-names>A</given-names></name></person-group><year>2008</year><article-title>Mushroom bodies modulate salience-based selective fixation behavior in <italic>Drosophila</italic></article-title><source>The European Journal of Neuroscience</source><volume>27</volume><fpage>1441</fpage><lpage>1451</lpage><pub-id pub-id-type="doi">10.1111/j.1460-9568.2008.06114.x</pub-id></element-citation></ref><ref id="bib89"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Xie</surname><given-names>Z</given-names></name><name><surname>Huang</surname><given-names>C</given-names></name><name><surname>Ci</surname><given-names>B</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Zhong</surname><given-names>Y</given-names></name></person-group><year>2013</year><article-title>Requirement of the combination of mushroom body gamma lobe and alpha/beta lobes for the retrieval of both aversive and appetitive early memories in <italic>Drosophila</italic></article-title><source>Learning & Memory</source><volume>20</volume><fpage>474</fpage><lpage>481</lpage><pub-id pub-id-type="doi">10.1101/lm.031823.113</pub-id></element-citation></ref><ref id="bib90"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname><given-names>MY</given-names></name><name><surname>Armstrong</surname><given-names>JD</given-names></name><name><surname>Vilinsky</surname><given-names>I</given-names></name><name><surname>Strausfeld</surname><given-names>NJ</given-names></name><name><surname>Kaiser</surname><given-names>K</given-names></name></person-group><year>1995</year><article-title>Subdivision of the <italic>Drosophila</italic> mushroom bodies by enhancer-trap expression patterns</article-title><source>Neuron</source><volume>15</volume><fpage>45</fpage><lpage>54</lpage><pub-id pub-id-type="doi">10.1016/0896-6273(95)90063-2</pub-id></element-citation></ref><ref id="bib91"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zars</surname><given-names>T</given-names></name></person-group><year>2010</year><article-title>Short-term memories in <italic>Drosophila</italic> are governed by general and specific genetic systems</article-title><source>Learning & Memory</source><volume>17</volume><fpage>246</fpage><lpage>251</lpage><pub-id pub-id-type="doi">10.1101/lm.1706110</pub-id></element-citation></ref><ref id="bib92"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zars</surname><given-names>T</given-names></name><name><surname>Fischer</surname><given-names>M</given-names></name><name><surname>Schulz</surname><given-names>R</given-names></name><name><surname>Heisenberg</surname><given-names>M</given-names></name></person-group><year>2000</year><article-title>Localization of a short-term memory in <italic>Drosophila</italic></article-title><source>Science</source><volume>288</volume><fpage>672</fpage><lpage>675</lpage><pub-id pub-id-type="doi">10.1126/science.288.5466.672</pub-id></element-citation></ref><ref id="bib93"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhang</surname><given-names>K</given-names></name><name><surname>Guo</surname><given-names>JZ</given-names></name><name><surname>Peng</surname><given-names>Y</given-names></name><name><surname>Xi</surname><given-names>W</given-names></name><name><surname>Guo</surname><given-names>A</given-names></name></person-group><year>2007</year><article-title>Dopamine-mushroom body circuit regulates saliency-based decision-making in <italic>Drosophila</italic></article-title><source>Science</source><volume>316</volume><fpage>1901</fpage><lpage>1904</lpage><pub-id pub-id-type="doi">10.1126/science.1137357</pub-id></element-citation></ref><ref id="bib94"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Ren</surname><given-names>Q</given-names></name><name><surname>Guo</surname><given-names>A</given-names></name></person-group><year>2013</year><article-title>Parallel pathways for cross-modal memory retrieval in <italic>Drosophila</italic></article-title><source>The Journal of Neuroscience</source><volume>33</volume><fpage>8784</fpage><lpage>8793</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.4631-12.2013</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02395.018</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Ramaswami</surname><given-names>Mani</given-names></name><role>Reviewing editor</role><aff><institution>Trinity College Dublin</institution>, <country>Ireland</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Shared mushroom body circuits underlie visual and olfactory memories in <italic>Drosophila</italic>” for consideration at <italic>eLife.</italic> Your article has been favorably evaluated by a Senior editor, a Reviewing editor (Mani Ramaswami) and 2 reviewers.</p><p>The Reviewing editor and the two reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>By developing behavioral assays for aversive and appetitive visual learning, the authors compare the circuitry required for visual learning with that previously established for olfactory learning. The question is interesting and assessed with the extremely good reagents and technologies expected from the authors. The conclusions mentioned in the Abstract describing dopaminergic neurons and mushroom body neurons required for visual learning, as well as how they compare with those required for olfactory learning, are interesting, significant, and deserve to be published in <italic>eLife</italic>.</p><p>1) One consensus experimental requirement is to provide clear information on the specificity of PAM DA neurons are for sugar learning and the PPL1 DA neurons for shock learning. This will require (reciprocal type) experiments to show that PAM DA neurons are not required for visual forms of shock learning and that PPL1 DA neurons are not required for visual forms of sugar learning. The fact that specificity has been demonstrated for olfactory learning is appreciated, but this needs to be demonstrated for visual learning as well.</p><p>2) The authors should consider a deeper discussion of the comparative roles of gamma lobes in short-term olfactory and visual forms of learning. If the authors have data on whether the gamma lobes are required for acquisition and/or retrieval of visual memory, then it will be a useful addition to the paper.</p><p>3) The manuscript is well written for a select group of <italic>Drosophila</italic> neuroscientists, intimately familiar with learning. The manuscript text will benefit from being edited to make it appropriate for a broader readership of <italic>eLife.</italic></p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.02395.019</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) One consensus experimental requirement is to provide clear information on the specificity of PAM DA neurons are for sugar learning and the PPL1 DA neurons for shock learning. This will require (reciprocal type) experiments to show that PAM DA neurons are not required for visual forms of shock learning and that PPL1 DA neurons are not required for visual forms of sugar learning. The fact that specificity has been demonstrated for olfactory learning is appreciated, but this needs to be demonstrated for visual learning as well</italic>.</p><p>We performed additional experiments and blocked different subsets of dopamine neurons with <italic>MB504B-GAL4</italic> and <italic>R58E02-GAL4</italic> and tested for appetitive and aversive memories, respectively. The dopamine neurons labeled in <italic>R58E02-GAL4</italic> are not required for aversive visual memory, and the dopamine neurons labeled in <italic>MB504B-GAL4</italic> are not required for appetitive visual memory either. Thus, we confirmed the differential requirement of the dopamine subsets in the PAM and PPL1 clusters for appetitive and aversive visual learning. We included these data in <xref ref-type="fig" rid="fig3">Figure 3</xref>. For practical reasons we split the original figure into two. Thus, selective requirement of dopamine neurons is now shown in <xref ref-type="fig" rid="fig3">Figure 3</xref>, whereas sufficiency of dopamine neurons is shown in new <xref ref-type="fig" rid="fig4">Figure 4</xref>.</p><p><italic>2) The authors should consider a deeper discussion of the comparative roles of gamma lobes in short-term olfactory and visual forms of learning. If the authors have data on whether the gamma lobes are required for acquisition and/or retrieval of visual memory, then it will be a useful addition to the paper</italic>.</p><p>As the reviewers pointed out, the contribution of the gamma lobe for olfactory conditioning is mainly to short-term memory (<xref ref-type="bibr" rid="bib30">Isabel et al., 2004</xref>; <xref ref-type="bibr" rid="bib6">Blum et al., 2009</xref>; <xref ref-type="bibr" rid="bib77">Trannoy et al., 2011</xref>). Particularly, dopamine input to the gamma lobe through DopR is sufficient to restore the memory defect (<xref ref-type="bibr" rid="bib63">Qin et al., 2012</xref>), and Rutabaga function (supposedly downstream of DopR) in the gamma lobes is sufficient to restore short-term memory (<xref ref-type="bibr" rid="bib92">Zars et al., 2000</xref>). Our study showed for the first time that gamma lobes are also essential for visual memory. This converging evidence suggests the general role for the formation of short-term memory. We added this discussion in the revision.</p><p>Based on the reviewers’ suggestion we examined the temporal requirement of the gamma neurons by performing an additional set of experiments using <italic>MB009B-GAL4</italic>, which specifically labels the gamma neurons. We found that output of gamma lobe KCs is only required during test. This is consistent with the results obtained with <italic>201y-GAL4</italic>. Given that the outputs of the dopamine neurons heavily project to the gamma lobe and are only required during training, short-term visual memory is likely formed inside the MB gamma lobes. We now compiled all temperature shift experiments with MB drivers in <xref ref-type="fig" rid="fig8">Figure 8</xref> for a better comparison between lines.</p><p><italic>3) The manuscript is well written for a select group of</italic> Drosophila <italic>neuroscientists, intimately familiar with learning. The manuscript text will benefit from being edited to make it appropriate for a broader readership of</italic> eLife<italic>.</italic></p><p>Thanks to this comment, we now include elaborated explanation and discussion about general information on associative memory, previous findings, different learning assays, and the contrast of memories in different sensory modalities.</p></body></sub-article></article> |