# Rewarding Capacity of Optogenetically Activating a Giant GABAergic Central-Brain Interneuron in Larval Drosophila

Nino Mancini, Juliane Thoener, Esmeralda Tafani, Dennis Pauls, Oded Mayseless, Martin Strauch, Katharina Eichler, Andrew Champion, Oliver Kobler, Denise Weber, et al.

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## To cite this version:

Nino Mancini, Juliane Thoener, Esmeralda Tafani, Dennis Pauls, Oded Mayseless, et al.. Rewarding Capacity of Optogenetically Activating a Giant GABAergic Central-Brain Interneuron in Larval Drosophila. Journal of Neuroscience, 2023, 43 (44), pp.7393-7428. 10.1523/JNEUROSCI.2310-22.2023. hal-04287180

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# Rewarding Capacity of Optogenetically Activating a Giant GABAergic Central-Brain Interneuron in Larval *Drosophila*

Nino Mancini,¹ Juliane Thoener,¹ Esmeralda Tafani,¹ Dennis Pauls,² Oded Mayseless,⁴ Martin Strauch,⁵
¹Katharina Eichler,⁶ Andrew Champion,⁷,⁸ Oliver Kobler,⁹ Denise Weber,³ Edanur Sen,¹ Aliće Weiglein,¹
Volker Hartenstein,¹⁰ Charalampos-Chrysovalantis Chytoudis-Peroudis,¹¹ Tihana Jovanic,¹¹ Andreas S. Thum,³
Astrid Rohwedder,³ Michael Schleyer,¹ and Bertram Gerber¹,¹²,¹³

¹Leibniz Institute for Neurobiology, Department Genetics of Learning and Memory, Magdeburg, 39118, Germany, ²Department of Animal Physiology, Institute of Biology, Leipzig University, Leipzig, 04103, Germany, ³Department of Genetics, Institute of Biology, Leipzig University, Leipzig, 04103, Germany, ⁴Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 7610001, Israel, ⁵Institute of Imaging and Computer Vision, RWTH Aachen University, Aachen, 52074, Germany, ⁶Institute of Neurobiology, University of Puerto Rico Medical Science Campus, Old San Juan, Puerto Rico, 00901, ⁷Department of Physiology, Development and Neuroscience, Cambridge University, Cambridge, CB2 3EL, United Kingdom, ⁸Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, 20147, Virginia, ⁹Leibniz Institute for Neurobiology, Combinatorial Neuroimaging Core Facility, Magdeburg, 39118, Germany, ¹⁰University of California, Department of Molecular, Cell and Developmental Biology, Los Angeles, California 90095-1606, ¹¹Université Paris-Saclay, Centre National de la Recherche Scientifique, Institut des neurosciences Paris-Saclay, Saclay, 91400, France, ¹²Center for Behavioral Brain Sciences, Magdeburg, 39106, Germany, and ¹³Institute for Biology, Otto von Guericke University, Magdeburg, 39120, Germany

Larvae of the fruit fly *Drosophila melanogaster* are a powerful study case for understanding the neural circuits underlying behavior. Indeed, the numerical simplicity of the larval brain has permitted the reconstruction of its synaptic connectome, and genetic tools for manipulating single, identified neurons allow neural circuit function to be investigated with relative ease and precision. We focus on one of the most complex neurons in the brain of the larva (of either sex), the GABAergic anterior paired lateral neuron (APL). Using behavioral and connectomic analyses, optogenetics, Ca²⁺ imaging, and pharmacology, we study how APL affects associative olfactory memory. We first provide a detailed account of the structure, regional polarity, connectivity, and metamorphic development of APL, and further confirm that optogenetic activation of APL has an inhibiting effect on its main targets, the mushroom body Kenyon cells. All these findings are consistent with the previously identified function of APL in the sparsening of sensory representations. To our surprise, however, we found that optogenetically activating APL can also have a strong rewarding effect. Specifically, APL activation together with odor presentation establishes an odor-specific, appetitive, associative short-term memory, whereas naive olfactory behavior remains unaffected. An acute, systemic inhibition of dopamine synthesis as well as an ablation of the dopaminergic pPAM neurons impair reward learning through APL activation. Our findings provide a study case of complex circuit function in a numerically simple brain, and suggest a previously unrecognized capacity of central-brain GABAergic neurons to engage in dopaminergic reinforcement.

**Key words:** dopamine; mushroom body; olfaction; optogenetics; reward

Received Dec. 17, 2022; revised July 19, 2023; accepted Aug. 26, 2023.

Author contributions: N.M., D.P., O.M., M.S., K.E., O.K., V.H., A.S.T., M.S., and B.G. designed research; N.M., J.T., E.T., D.P., O.M., M.S., K.E., A.C., O.K., D.W., E.S., A.W., V.H., C.C.C.P., T.J., and A.R. performed research; N.M., J.T., E.T., D.P., O.M., M.S., K.E., A.C., O.K., D.W., E.S., A.W., V.H., C.C.C.P., T.J., A.S.T., A.R., M.S., and B.G. analyzed data; N.M. and B.G. wrote the first draft of the paper; N.M., J.T., E.T., D.P., O.M., M.S., K.E., A.C., O.K., D.W., E.S., A.W., V.H., C.C.C.P., T.J., A.S.T., A.R., M.S., and B.G. edited the paper; N.M. and B.G. wrote the paper; A.S.T. and A.R. contributed unpublished reagents/analytic tools.

This work was supported by Otto von Guericke University Magdeburg, Wissenschaftsgemeinschaft Gottfried Wilhelm Leibniz Institute for Neurobiology (LIN), and Deutsche Forschungsgemeinschaft (DFG) (DFG-GE 1091/4-1 and FOR 2705 Mushroom body to B.G.; PA1979/3-1 to D.P.; DFG-TH 1584/8-1 and DFG-TH 1584/7-1 to A.S.T.); German Federal Ministry of Education and Research (BMBF) (BMBF-NSF CRCNS *Encoding reward expectation in Drosophila* to B.G.); National Institutes of Health Grant R01 NS054814 to V.H.; and Agence Nationale de la Recherche ANR-21-NEUC-0002 (CRCNS *DrosoExpect*) and ANR-17-CE37-0019 to T.J. We thank A. Bates (Cambridge) for providing the script allowing compartment boundaries to be generated on the dendrograms; H. Tanimoto (U Tohoku), M. Stopfer (National Institutes of Health), Y. Aso (HHMI Janelia), A. Lin (U Sheffield), O. Schuldiner (Weizmann), C. Duch (U Mainz), G. Tavosanis (U Bonn), A. Khalili (U Göttingen), C. König (LIN), B. Michels (LIN), and N. Toshima (LIN) for discussions; and A. Ciuraszkiewicz (LIN), K. Hartung (LIN), M.L. Rodriguez Lopez (LIN), B. Kracht (LIN), T. Niewalda (LIN),

M. Paisios (LIN), H. Reim (LIN), and H. Wickborn (LIN) and the workshop team at the LIN for technical assistance.

N. Mancini's present address: Max Planck Florida Institute for Neuroscience, Jupiter, Florida 33458.

A. Weiglein's present address: Institute of Anatomy, Otto von Guericke University, 39120 Magdeburg, Germany.

O. Mayseless' present address: Biozentrum, University of Basel, 4056 Basel, Switzerland.

M. Schleyer's present address: Hokkaido University, Institute for the Advancement of Higher Education, Hokkaido 060-0808, Japan.

A. Champion's present address: Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, United Kingdom and MRC Laboratory of Molecular Biology, Cambridge, CB2 00H, United Kingdom.

K. Eichler's present address: Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, United Kingdom.

The authors declare no competing financial interests.

Correspondence should be addressed to Nino Mancini at nino.mancini@mpfi.org or Bertram Gerber at bertram.gerber@lin-magdeburg.de.

https://doi.org/10.1523/JNEUROSCI.2310-22.2023

Copyright © 2023 the authors
Behavioral/Cognitive

# Rewarding Capacity of Optogenetically Activating a Giant GABAergic Central-Brain Interneuron in Larval *Drosophila*

Nino Mancini,¹ Juliane Thoener,¹ Esmeralda Tafani,¹ Dennis Pauls,² Oded Mayseless,⁴ Martin Strauch,⁵
¹Katharina Eichler,⁶ Andrew Champion,⁷,⁸ Oliver Kobler,⁹ Denise Weber,³ Edanur Sen,¹ Aliće Weiglein,¹
Volker Hartenstein,¹⁰ Charalampos-Chrysovalantis Chytoudis-Peroudis,¹¹ Tihana Jovanic,¹¹ Andreas S. Thum,³
Astrid Rohwedder,³ Michael Schleyer,¹ and Bertram Gerber¹,¹²,¹³

¹Leibniz Institute for Neurobiology, Department Genetics of Learning and Memory, Magdeburg, 39118, Germany, ²Department of Animal Physiology, Institute of Biology, Leipzig University, Leipzig, 04103, Germany, ³Department of Genetics, Institute of Biology, Leipzig University, Leipzig, 04103, Germany, ⁴Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 7610001, Israel, ⁵Institute of Imaging and Computer Vision, RWTH Aachen University, Aachen, 52074, Germany, ⁶Institute of Neurobiology, University of Puerto Rico Medical Science Campus, Old San Juan, Puerto Rico, 00901, ⁷Department of Physiology, Development and Neuroscience, Cambridge University, Cambridge, CB2 3EL, United Kingdom, ⁸Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, 20147, Virginia, ⁹Leibniz Institute for Neurobiology, Combinatorial Neuroimaging Core Facility, Magdeburg, 39118, Germany, ¹⁰University of California, Department of Molecular, Cell and Developmental Biology, Los Angeles, California 90095-1606, ¹¹Université Paris-Saclay, Centre National de la Recherche Scientifique, Institut des neurosciences Paris-Saclay, Saclay, 91400, France, ¹²Center for Behavioral Brain Sciences, Magdeburg, 39106, Germany, and ¹³Institute for Biology, Otto von Guericke University, Magdeburg, 39120, Germany

Larvae of the fruit fly *Drosophila melanogaster* are a powerful study case for understanding the neural circuits underlying behavior. Indeed, the numerical simplicity of the larval brain has permitted the reconstruction of its synaptic connectome, and genetic tools for manipulating single, identified neurons allow neural circuit function to be investigated with relative ease and precision. We focus on one of the most complex neurons in the brain of the larva (of either sex), the GABAergic anterior paired lateral neuron (APL). Using behavioral and connectomic analyses, optogenetics, Ca²⁺ imaging, and pharmacology, we study how APL affects associative olfactory memory. We first provide a detailed account of the structure, regional polarity, connectivity, and metamorphic development of APL, and further confirm that optogenetic activation of APL has an inhibiting effect on its main targets, the mushroom body Kenyon cells. All these findings are consistent with the previously identified function of APL in the sparsening of sensory representations. To our surprise, however, we found that optogenetically activating APL can also have a strong rewarding effect. Specifically, APL activation together with odor presentation establishes an odor-specific, appetitive, associative short-term memory, whereas naive olfactory behavior remains unaffected. An acute, systemic inhibition of dopamine synthesis as well as an ablation of the dopaminergic pPAM neurons impair reward learning through APL activation. Our findings provide a study case of complex circuit function in a numerically simple brain, and suggest a previously unrecognized capacity of central-brain GABAergic neurons to engage in dopaminergic reinforcement.

**Key words:** dopamine; mushroom body; olfaction; optogenetics; reward

Received Dec. 17, 2022; revised July 19, 2023; accepted Aug. 26, 2023.

Author contributions: N.M., D.P., O.M., M.S., K.E., O.K., V.H., A.S.T., M.S., and B.G. designed research; N.M., J.T., E.T., D.P., O.M., M.S., K.E., A.C., O.K., D.W., E.S., A.W., V.H., C.C.C.P., T.J., and A.R. performed research; N.M., J.T., E.T., D.P., O.M., M.S., K.E., A.C., O.K., D.W., E.S., A.W., V.H., C.C.C.P., T.J., A.S.T., A.R., M.S., and B.G. analyzed data; N.M. and B.G. wrote the first draft of the paper; N.M., J.T., E.T., D.P., O.M., M.S., K.E., A.C., O.K., D.W., E.S., A.W., V.H., C.C.C.P., T.J., A.S.T., A.R., M.S., and B.G. edited the paper; N.M. and B.G. wrote the paper; A.S.T. and A.R. contributed unpublished reagents/analytic tools.

This work was supported by Otto von Guericke University Magdeburg, Wissenschaftsgemeinschaft Gottfried Wilhelm Leibniz Institute for Neurobiology (LIN), and Deutsche Forschungsgemeinschaft (DFG) (DFG-GE 1091/4-1 and FOR 2705 Mushroom body to B.G.; PA1979/3-1 to D.P.; DFG-TH 1584/8-1 and DFG-TH 1584/7-1 to A.S.T.); German Federal Ministry of Education and Research (BMBF) (BMBF-NSF CRCNS *Encoding reward expectation in Drosophila* to B.G.); National Institutes of Health Grant R01 NS054814 to V.H.; and Agence Nationale de la Recherche ANR-21-NEUC-0002 (CRCNS *DrosoExpect*) and ANR-17-CE37-0019 to T.J. We thank A. Bates (Cambridge) for providing the script allowing compartment boundaries to be generated on the dendrograms; H. Tanimoto (U Tohoku), M. Stopfer (National Institutes of Health), Y. Aso (HHMI Janelia), A. Lin (U Sheffield), O. Schuldiner (Weizmann), C. Duch (U Mainz), G. Tavosanis (U Bonn), A. Khalili (U Göttingen), C. König (LIN), B. Michels (LIN), and N. Toshima (LIN) for discussions; and A. Ciuraszkiewicz (LIN), K. Hartung (LIN), M.L. Rodriguez Lopez (LIN), B. Kracht (LIN), T. Niewalda (LIN),

M. Paisios (LIN), H. Reim (LIN), and H. Wickborn (LIN) and the workshop team at the LIN for technical assistance.

N. Mancini's present address: Max Planck Florida Institute for Neuroscience, Jupiter, Florida 33458.

A. Weiglein's present address: Institute of Anatomy, Otto von Guericke University, 39120 Magdeburg, Germany.

O. Mayseless' present address: Biozentrum, University of Basel, 4056 Basel, Switzerland.

M. Schleyer's present address: Hokkaido University, Institute for the Advancement of Higher Education, Hokkaido 060-0808, Japan.

A. Champion's present address: Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, United Kingdom and MRC Laboratory of Molecular Biology, Cambridge, CB2 00H, United Kingdom.

K. Eichler's present address: Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, United Kingdom.

The authors declare no competing financial interests.

Correspondence should be addressed to Nino Mancini at nino.mancini@mpfi.org or Bertram Gerber at bertram.gerber@lin-magdeburg.de.

https://doi.org/10.1523/JNEUROSCI.2310-22.2023

Copyright © 2023 the authors
## Significance Statement

The single, identified giant anterior paired lateral (APL) neuron is one of the most complex neurons in the insect brain. It is GABAergic and contributes to the sparsening of neuronal activity in the mushroom body, the memory center of insects. We provide the most detailed account yet of the structure of APL in larval *Drosophila* as a neurogenetically accessible study case. We further reveal that, contrary to expectations, the experimental activation of APL can exert a rewarding effect, likely via dopaminergic reward pathways. The present study both provides an example of unexpected circuit complexity in a numerically simple brain, and reports an unexpected effect of activity in central-brain GABAergic circuits.

## Introduction

Larvae of the fruit fly *Drosophila melanogaster*, which naturally live on overripe fruit, provide a powerful model for investigating the neurogenetic bases of learning and memory (Gerber and Stocker, 2007; Widmann et al., 2018; Thum and Gerber, 2019; Eschbach and Zlatic, 2020). Their small size and low number of neurons have allowed their chemical synapse connectome to be reconstructed, revealing unexpected complexity. In the mushroom body, which is a higher brain structure for sensory integration and memory in insects (Heisenberg, 2003), more than half of the classes of synaptic connections had previously escaped attention (Fig. 1D) (Eichler et al., 2017; Eschbach et al., 2020, 2021; adults: Takemura et al., 2017; F. Li et al., 2020). For instance, dopaminergic mushroom body input neurons (DANs) not only relay ascending information to local compartments along the elongated axonal fibers of the mushroom body intrinsic Kenyon cells (KCs), but also integrate local information from the KCs and recurrent signals originating from mushroom body output neurons (MBONs; these likewise respect compartmental boundaries). Similar complexity is observed for octopaminergic input neurons (OANs) and for input neurons using as yet unidentified signaling (Eichler et al., 2017; Saumweber et al., 2018; Eschbach et al., 2020, 2021; Schleyer et al., 2020). Collectively, this should prepare us for more surprises regarding mushroom body function. Here we study one of the most complex mushroom body neurons of larvae, the anterior paired lateral (APL) neuron.

APL is an embryonic-born and hemispherically unique interneuron that can be identified from the earliest larval stage on, throughout metamorphosis and in adults (Eichler et al., 2017; Mayseless et al., 2018; Saumweber et al., 2018). It receives most of its input from, and provides GABAergic output to, the cholinergic KCs, suggesting a role in sparsening sensory representation within the mushroom body (Masuda-Nakagawa et al., 2014; adults: Honegger et al., 2011; Lin et al., 2014; Amin et al., 2020; Prisco et al., 2021; further insects: Homberg et al., 1987; Grünewald, 1999; Papadopoulou et al., 2011). In contrast to most other aspects of mushroom body connectivity, however, there are major differences in APL connectivity between larvae and adults.

In adults, APL innervates all 15 mushroom body compartments and the calyx, where the KCs receive input from sensory projection neurons (Tanaka et al., 2008; Aso et al., 2014). In larvae, APL also innervates the calyx, but only 6 of the 10 compartments (Fig. 1B) (Eichler et al., 2017; Saumweber et al., 2018). In adults, APL connects reciprocally with the KCs in the calyx and in all the compartments (C. L. Wu et al., 2013; Takemura et al., 2017; Zheng et al., 2018; Scheffer et al., 2020), whereas in larvae such reciprocal connections exist only in the calyx, and only KC-to-APL synapses are found otherwise (Masuda-Nakagawa et al.,

2014; Eichler et al., 2017; Saumweber et al., 2018). In adults, APL is electrically coupled to the dorsal paired median neuron (DPM), a local interneuron that innervates all the compartments but not the calyx (Pitman et al., 2011; C. L. Wu et al., 2011). DPM is serotonergic, coreleases GABA, and can express the amnesiac peptide (Waddell et al., 2000; Lee et al., 2011; Haynes et al., 2015; Turrel et al., 2018). Strikingly, DPM is absent in larvae (Eichler et al., 2017; Saumweber et al., 2018).

These differences caution against extrapolating between findings on APL in larvae and adults, since functions of APL other than a sparsening of KC activity have been described in adults (Liu et al., 2007; Liu and Davis, 2009; Ren et al., 2012; Y. Wu et al., 2012; Lin et al., 2014). In this context, we provide a comprehensive account of the structure of the larval APL neuron, the spatial arrangement of its synapses, its physiological effect on KC activity, and its metamorphic development. Investigating its role in Pavlovian conditioning, we discover that, surprisingly, optogenetic activation of APL exerts a rewarding effect. This effect is studied in detail and is shown to involve a dopamine-dependent process.

## Materials and Methods

### *Drosophila strains*

*Drosophila melanogaster* were kept and maintained on standard medium, in mass culture at 25°C, 60%-70% humidity, in a 12/12 h light/dark cycle. We used randomly chosen third-instar, feeding-stage larvae of both sexes, aged 5 d (120 h) after egg laying, unless mentioned otherwise. The strains used in this study and their genotypes are listed in Table 1.

### *Full-body fluorescence microscopy*

To allow a full-body assessment of transgene expression from the APL-specific split-GAL4 SS01671 driver (henceforth APL-GAL4) (Saumweber et al., 2018), it was crossed to w*; UAS-mCherry-CAAX (abbreviated as UAS-mCherry-CAAX; Bloomington Stock Center #59021) (Sens et al., 2010) to express the mCherry-CAAX reporter. Double-heterozygous third-instar progeny (abbreviated as APL>mCherry-CAAX) were analyzed for fluorescence signals under a light-sheet microscope (see next paragraph). Genetic controls were heterozygous for either the GAL4 element (APL>+) or the UAS element (+>mCherry-CAAX). To obtain the driver control, APL-GAL4 was crossed to y¹w¹ (Bloomington Stock Center #1495). As regards the effector control, a strain lacking the GAL4 domains but containing the two split-GAL4 landing sites (attP40/attP2) (Pfeiffer et al., 2010) was crossed to UAS-mCherry-CAAX.

Experimental procedures follow Kobler et al. (2021). In brief, third-instar larvae were first bleached (4% sodium hypochlorite, Roth, order #9062.1) for 10 min at room temperature. After washing (3 × 5 min in distilled water, dH₂O), they were fixed in 4% PFA in 0.1 M PB at pH 9, with gentle shaking overnight at 4°C. Fixed samples were briefly rinsed 3 × with 0.1 M PB containing 0.2% Triton X-100 (PBT), then washed 2 × 60 min and left overnight at 4°C in PBT at pH 9. Dehydration the following day used a graded ethanol (EtOH) series (60 min in 10% and 25% EtOH, followed by 30 min in 50%, 60%, 80% (all at pH 9), and 2 × 100%
## Significance Statement

The single, identified giant anterior paired lateral (APL) neuron is one of the most complex neurons in the insect brain. It is GABAergic and contributes to the sparsening of neuronal activity in the mushroom body, the memory center of insects. We provide the most detailed account yet of the structure of APL in larval *Drosophila* as a neurogenetically accessible study case. We further reveal that, contrary to expectations, the experimental activation of APL can exert a rewarding effect, likely via dopaminergic reward pathways. The present study both provides an example of unexpected circuit complexity in a numerically simple brain, and reports an unexpected effect of activity in central-brain GABAergic circuits.

## Introduction

Larvae of the fruit fly *Drosophila melanogaster*, which naturally live on overripe fruit, provide a powerful model for investigating the neurogenetic bases of learning and memory (Gerber and Stocker, 2007; Widmann et al., 2018; Thum and Gerber, 2019; Eschbach and Zlatic, 2020). Their small size and low number of neurons have allowed their chemical synapse connectome to be reconstructed, revealing unexpected complexity. In the mushroom body, which is a higher brain structure for sensory integration and memory in insects (Heisenberg, 2003), more than half of the classes of synaptic connections had previously escaped attention (Fig. 1D) (Eichler et al., 2017; Eschbach et al., 2020, 2021; adults: Takemura et al., 2017; F. Li et al., 2020). For instance, dopaminergic mushroom body input neurons (DANs) not only relay ascending information to local compartments along the elongated axonal fibers of the mushroom body intrinsic Kenyon cells (KCs), but also integrate local information from the KCs and recurrent signals originating from mushroom body output neurons (MBONs; these likewise respect compartmental boundaries). Similar complexity is observed for octopaminergic input neurons (OANs) and for input neurons using as yet unidentified signaling (Eichler et al., 2017; Saumweber et al., 2018; Eschbach et al., 2020, 2021; Schleyer et al., 2020). Collectively, this should prepare us for more surprises regarding mushroom body function. Here we study one of the most complex mushroom body neurons of larvae, the anterior paired lateral (APL) neuron.

APL is an embryonic-born and hemispherically unique interneuron that can be identified from the earliest larval stage on, throughout metamorphosis and in adults (Eichler et al., 2017; Mayseless et al., 2018; Saumweber et al., 2018). It receives most of its input from, and provides GABAergic output to, the cholinergic KCs, suggesting a role in sparsening sensory representation within the mushroom body (Masuda-Nakagawa et al., 2014; adults: Honegger et al., 2011; Lin et al., 2014; Amin et al., 2020; Prisco et al., 2021; further insects: Homberg et al., 1987; Grünewald, 1999; Papadopoulou et al., 2011). In contrast to most other aspects of mushroom body connectivity, however, there are major differences in APL connectivity between larvae and adults.

In adults, APL innervates all 15 mushroom body compartments and the calyx, where the KCs receive input from sensory projection neurons (Tanaka et al., 2008; Aso et al., 2014). In larvae, APL also innervates the calyx, but only 6 of the 10 compartments (Fig. 1B) (Eichler et al., 2017; Saumweber et al., 2018). In adults, APL connects reciprocally with the KCs in the calyx and in all the compartments (C. L. Wu et al., 2013; Takemura et al., 2017; Zheng et al., 2018; Scheffer et al., 2020), whereas in larvae such reciprocal connections exist only in the calyx, and only KC-to-APL synapses are found otherwise (Masuda-Nakagawa et al.,

2014; Eichler et al., 2017; Saumweber et al., 2018). In adults, APL is electrically coupled to the dorsal paired median neuron (DPM), a local interneuron that innervates all the compartments but not the calyx (Pitman et al., 2011; C. L. Wu et al., 2011). DPM is serotonergic, coreleases GABA, and can express the amnesiac peptide (Waddell et al., 2000; Lee et al., 2011; Haynes et al., 2015; Turrel et al., 2018). Strikingly, DPM is absent in larvae (Eichler et al., 2017; Saumweber et al., 2018).

These differences caution against extrapolating between findings on APL in larvae and adults, since functions of APL other than a sparsening of KC activity have been described in adults (Liu et al., 2007; Liu and Davis, 2009; Ren et al., 2012; Y. Wu et al., 2012; Lin et al., 2014). In this context, we provide a comprehensive account of the structure of the larval APL neuron, the spatial arrangement of its synapses, its physiological effect on KC activity, and its metamorphic development. Investigating its role in Pavlovian conditioning, we discover that, surprisingly, optogenetic activation of APL exerts a rewarding effect. This effect is studied in detail and is shown to involve a dopamine-dependent process.

## Materials and Methods

### *Drosophila strains*

*Drosophila melanogaster* were kept and maintained on standard medium, in mass culture at 25°C, 60%-70% humidity, in a 12/12 h light/dark cycle. We used randomly chosen third-instar, feeding-stage larvae of both sexes, aged 5 d (120 h) after egg laying, unless mentioned otherwise. The strains used in this study and their genotypes are listed in Table 1.

### *Full-body fluorescence microscopy*

To allow a full-body assessment of transgene expression from the APL-specific split-GAL4 SS01671 driver (henceforth APL-GAL4) (Saumweber et al., 2018), it was crossed to w*; UAS-mCherry-CAAX (abbreviated as UAS-mCherry-CAAX; Bloomington Stock Center #59021) (Sens et al., 2010) to express the mCherry-CAAX reporter. Double-heterozygous third-instar progeny (abbreviated as APL>mCherry-CAAX) were analyzed for fluorescence signals under a light-sheet microscope (see next paragraph). Genetic controls were heterozygous for either the GAL4 element (APL>+) or the UAS element (+>mCherry-CAAX). To obtain the driver control, APL-GAL4 was crossed to y¹w¹ (Bloomington Stock Center #1495). As regards the effector control, a strain lacking the GAL4 domains but containing the two split-GAL4 landing sites (attP40/attP2) (Pfeiffer et al., 2010) was crossed to UAS-mCherry-CAAX.

Experimental procedures follow Kobler et al. (2021). In brief, third-instar larvae were first bleached (4% sodium hypochlorite, Roth, order #9062.1) for 10 min at room temperature. After washing (3 × 5 min in distilled water, dH₂O), they were fixed in 4% PFA in 0.1 M PB at pH 9, with gentle shaking overnight at 4°C. Fixed samples were briefly rinsed 3 × with 0.1 M PB containing 0.2% Triton X-100 (PBT), then washed 2 × 60 min and left overnight at 4°C in PBT at pH 9. Dehydration the following day used a graded ethanol (EtOH) series (60 min in 10% and 25% EtOH, followed by 30 min in 50%, 60%, 80% (all at pH 9), and 2 × 100%
<figure><img src="image_1.png" /></figure>

*pPAM cluster

**DL1 cluster

**Figure 1.** Overview of the larval body and brain and connectivity of the APL neuron in the mushroom body. A, Schematic overview of the larval body, adapted from Demerec and Kaufmann (1940). B, Sketch of one larval brain hemisphere with the mushroom body, highlighting its intrinsic KCs and organization in 10 compartments plus the calyx (letters a-k indicate the suffixes used to indicate the regions innervated by mushroom body extrinsic neurons): CX, Calyx; IP and LP, intermediate and lower peduncle; LA, lateral appendix; UVL, IVL, and LVL: upper, intermediate, and lower vertical lobe; SHA, shaft; UT, IT, and LT, upper, intermediate, and lower toe of the medial lobe. Adapted from Saumweber et al. (2018). C, The larval APL neuron collects input (<> mostly from the KCs both in the calyx and in a subset of the compartments in the lobes, and delivers output (arrowhead) mostly to KCs and almost exclusively in the calyx. Adapted from Saumweber et al. (2018). D, Simplified diagram of the connectivity of APL and of circuits underlying associative odor-reward learning. Within the calyx, a given odor (cloud) leads to the activation of a sparse, odor-specific pattern of KCs established through input from the projection neurons (PN). Within the lobes, a subset of modulatory dopaminergic neurons (Reward DAN) carry taste reward signals to the KCs, which send their axonal projection to avoidance-promoting MBONs (Avoidance MBON). Conversely, a different set of dopaminergic neurons (Punishment DAN) carry aversive signals to the KCs, which send their axonal projection to approach-promoting MBONs (Approach MBON). Reward and punishment DANs belong to the pPAM and DL1 cluster of
<figure><img src="image_3.png" /></figure>

*pPAM cluster

**DL1 cluster

<caption>**Figure 1.** Overview of the larval body and brain and connectivity of the APL neuron in the mushroom body. A, Schematic overview of the larval body, adapted from Demerec and Kaufmann (1940). B, Sketch of one larval brain hemisphere with the mushroom body, highlighting its intrinsic KCs and organization in 10 compartments plus the calyx (letters a-k indicate the suffixes used to indicate the regions innervated by mushroom body extrinsic neurons): CX, Calyx; IP and LP, intermediate and lower peduncle; LA, lateral appendix; UVL, IVL, and LVL: upper, intermediate, and lower vertical lobe; SHA, shaft; UT, IT, and LT, upper, intermediate, and lower toe of the medial lobe. Adapted from Saumweber et al. (2018). C, The larval APL neuron collects input (<> mostly from the KCs both in the calyx and in a subset of the compartments in the lobes, and delivers output (arrowhead) mostly to KCs and almost exclusively in the calyx. Adapted from Saumweber et al. (2018). D, Simplified diagram of the connectivity of APL and of circuits underlying associative odor-reward learning. Within the calyx, a given odor (cloud) leads to the activation of a sparse, odor-specific pattern of KCs established through input from the projection neurons (PN). Within the lobes, a subset of modulatory dopaminergic neurons (Reward DAN) carry taste reward signals to the KCs, which send their axonal projection to avoidance-promoting MBONs (Avoidance MBON). Conversely, a different set of dopaminergic neurons (Punishment DAN) carry aversive signals to the KCs, which send their axonal projection to approach-promoting MBONs (Approach MBON). Reward and punishment DANs belong to the pPAM and DL1 cluster of</caption>
EtOH). Samples were then cleared by replacing EtOH with ethyl cinnamate (ECi; ethyl 3-phenyl-2-propenoate; Sigma-Aldrich, order #112372-100G). After 30-60 min, the ECi was refreshed once, and the samples stored at room temperature in black boxes in a desiccator.

The samples were placed ventral side up in ECi-cleared phytagel blocks (1 × 1 × 1 cm, Sigma-Aldrich, P8169). These were placed in a sample holder, which in turn was fixed on a mounting suspension to fit into a high-precision quartz glass cuvette filled with ECi and optically accessible with an UltraMicroscope II light-sheet microscope (Miltenyi Biotec). The microscope was equipped with a Zyla 4.2 PLUS sCMOS camera (Oxford Instruments) and a tube for infinity-corrected objective lenses. An EXW-12 laser (NKT Photonics) was used for excitation through triple-sheet optics to illuminate samples from one side. Excitation and emission filters (AHF Analysentechnik AG) were used as indicated in Figure 2 and Movie 1.

Tiled image stacks were acquired with a LVMI-Fluor 12× objective (Miltenyi Biotec) with a format of 2048 × 2048. Using ImSpector software (version 7.1.4, Miltenyi Biotec), a 10% overlap of tiles was set for stitching.

Image files were processed with Imaris software (version 9.8, Bitplane), including file conversion, stitching, further processing, and rendering. The ortho slicer tool was used to restrict volumes in the *z* direction to improve the representation of structures that were otherwise covered in the context of the whole body. All 3D images were generated using the snapshot function. 2D maximum-intensity projections were generated in Fiji (Schindelin et al., 2012).

Movie 1 was produced in Imaris with the key frame animation tool; Adobe Premiere Pro 2020 (version 14.9.0, Adobe) was used for cutting and labeling.

## Immunohistochemistry

All the antibodies used in this study are listed in Table 1.

**Transgene expression pattern of the SS01671 driver strain.** To validate specific expression in the larval APL neuron of the APL-GAL4 driver strain (Saumweber et al., 2018), it was crossed to UAS-ChR2XXL::tdtomato to express a tomato-tagged version of ChR2XXL (FlyBase ID: FBtp0131815) (Saumweber et al., 2018). Double-heterozygous third-instar progeny (abbreviated as APL>ChR2XXL::tdtomato) were dissected in ice-cold Ringer's solution, and the brains were fixed for 30 min in 10% formaldehyde dissolved in PBS (pH 7.2, P4417, Sigma-Aldrich) at room temperature. After consecutive washing steps (3 × 10 min each) in PBT (0.3% Triton X-100 [CAS: 9036-19-5, Roth] in PBS), the brains were blocked in 5% NGS solution (005-000-121, Jackson ImmunoResearch Laboratories; in PBS) for 2 h at room temperature. To provide a reference staining of fiber tracts (including the mushroom bodies), tissues were incubated overnight at 4°C with a primary monoclonal mouse anti-FASII antibody (AB_528235, DSHB) diluted 1:50 in blocking solution containing 4% NGS in PBS. After six washes (10 min each) in PBS, the tissues were treated overnight at 4°C with a secondary polyclonal goat anti-mouse AlexaFluor-488 antibody (A11001, Invitrogen) diluted 1:200 in PBS. The brains were then

←

dopaminergic neurons, respectively, as indicated. In addition to its connections with the KCs, the APL neuron establishes synaptic contacts with the calyx MBONs (MBON-a1 and -a2) as well as with a subset of PNs (for additional connections between APL and mushroom body extrinsic neurons that are omitted here, see Figs. 6, 7). During odor-taste reward associative learning, the coincidence between the odor and the reward signal at the KCs is thought to lead to a presynaptic depression of the synapses between the odor-activated KCs and avoidance-promoting MBONs, whereas the synapses of these KCs with approach-promoting MBONs in other compartments remain unchanged (the contribution of MBON-a1/a2 to learned behavior is unclear, as indicated by the stippled lines). Future processing of the learned odor is thus biased in favor of approach. The same rationale is thought to apply for odor-punishment learning, occurring at the synapses between the KCs and approach MBONs. The electron microscopy reconstruction of a first-instar larval nervous system additionally revealed unexpected connections from KCs toward mushroom body input neurons (MBINs), including DANs, as well as MBIN/DAN-to-MBON synapses; KC-to-KC and MBON-to-MBIN connections are not displayed. Arrows indicate synaptic contacts between neurons.

washed in PBS (6 × 10 min each) and mounted in Vectashield (Vector Laboratories) on a cover slip. Signal detection from the tomato-tag of ChR2XXL (labeling the APL neuron) did not require antibodies; rather, the tomato fluorescence signal was detected directly under the microscope. Image *z* stacks were acquired with a Leica TCS SP8 confocal microscope (Leica Mikrosysteme Vertrieb) at a format of 1024 × 1024. Image processing was performed using Imaris software (version 9.72, Bitplane).

To visualize the larval APL neuron together with the mushroom bodies, we crossed the APL-GAL4 driver with a recombined effector/enhancer effector strain that includes a UAS-mIFP-T2A-HO1 effector (abbreviated as mIFP; Bloomington Stock Center #64181) (Yu et al., 2015) recombined with the enhancer effector construct MB247>mCherry-CAAX (Kobler et al., 2021). Third-instar larval progeny (abbreviated as APL>mIFP/MB247>mCherry-CAAX) were dissected in ice-cold Ca²⁺-free saline solution and fixed for 24 h in 4% PFA (J19943, Alfa Aesar; in PBS) at 4°C. After six washes (3 × brief; 3 × 10 min) in 0.3% PBT, the brains were mounted in Vectashield (Vector Laboratories) on a cover slip. Signal detection from the mCherry-CAAX reporter (labeling the mushroom bodies) and the mIFP reporter (labeling APL) did not require antibodies for signal amplification. The image *z* stack was acquired with a Leica TCS SP8 confocal microscope (Leica Mikrosysteme Vertrieb) at a format of 1024 × 1024. The corresponding Movie 2 was produced in Imaris (version 9.72, Bitplane).

To examine the interhemispheric symmetry in the morphology of APL, the APL-GAL4 driver was crossed to UAS-mCD8::GFP (Bloomington Stock Center #5137) (Lee and Luo, 1999) as the effector. Third-instar larvae were put on ice and dissected in PBS. The brains were fixed in 4% PFA for 20 min at room temperature. After a succession of washing steps (3 × brief; 1 × 5 min; 3 × 15 min; 1 × 90 min) in 3% PBT (3% Triton X-100 [CAS: 9002-93-1, Sigma-Aldrich] in PBS) on ice, the brains were blocked with 5% NGS (G9023, Sigma-Aldrich) in PBT for 1 h at room temperature and incubated for 48 h with primary antibodies at 4°C. The brains were then washed (2 × brief; 3 × 15 min; 1 × 60 min; on ice; 1 × 30 min at room temperature) in 3% PBT before application of the secondary antibodies for at least 24 h at 4°C. After a final set of washing steps (3 × brief; 3 × 5 min; 2 × 15 min) in 3% PBT, the brains were mounted on poly-L-lysin-coated cover slips (following the Janelia FlyLight recipe), dehydrated by a series of increasing concentrations of EtOH (1× brief in distilled water; 1 × 10 min 30% EtOH; 1 × 10 min 50% EtOH; 1 × 10 min 75% EtOH; 1 × 10 min 95% EtOH; 3 × 10 min 100% EtOH) and cleared (3 × 5 min) in xylene (247642, CAS: 1330-20-7, Sigma-Aldrich). Finally, the brains were mounted in DPX mounting medium (dibutyl phthalate in xylene; 06522, Sigma-Aldrich) and left in darkness for at least 24 h before imaging.

The primary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) a polyclonal rabbit anti-GFP antibody (A6455, Invitrogen) diluted 1:1000 in 3% PBT (for APL staining), (3) a monoclonal mouse 4F3 anti-DLG antibody (AB_528203, Developmental Studies Hybridoma Bank) diluted 1:200 in 3% PBT (for mushroom body staining), and (4) a monoclonal rat anti-N-Cadherin antibody (DN-Ex #8-s, Developmental Studies Hybridoma Bank) diluted 1:50 in 3% PBT (for neuropil staining).

The secondary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) polyclonal goat anti-rabbit AlexaFluor-488 (A11008, Invitrogen), (3) polyclonal goat anti-mouse AlexaFluor-568 (A10037, Invitrogen), and (4) polyclonal goat anti-rat AlexaFluor-647 (712-605-153, Jackson ImmunoResearch Laboratories), all diluted 1:500 in 3% PBT. Confocal microscopy was conducted on a Zeiss LSM800 confocal laser scanning microscope with ZEN 2.3 software. Image *z* stacks were acquired with a LSM800 confocal microscope (Zeiss) at a format of 1024 × 1024. Image processing was performed using Imaris software (version 9.72, Bitplane).

To compare the coverage of the mushroom body calyx and compartments between the APL neuron of each hemisphere, mean pixel intensities were measured using ImageJ (version 1.53c, Fiji ImageJ). Grayscale maximum intensity projections of the GFP-channel (labeling APL membranes) were created, whereas the DLG channel (labeling the mushroom bodies) served as a template for orientation. The mushroom body calyx and compartments were
EtOH). Samples were then cleared by replacing EtOH with ethyl cinnamate (ECi; ethyl 3-phenyl-2-propenoate; Sigma-Aldrich, order #112372-100G). After 30-60 min, the ECi was refreshed once, and the samples stored at room temperature in black boxes in a desiccator.

The samples were placed ventral side up in ECi-cleared phytagel blocks (1 × 1 × 1 cm, Sigma-Aldrich, P8169). These were placed in a sample holder, which in turn was fixed on a mounting suspension to fit into a high-precision quartz glass cuvette filled with ECi and optically accessible with an UltraMicroscope II light-sheet microscope (Miltenyi Biotec). The microscope was equipped with a Zyla 4.2 PLUS sCMOS camera (Oxford Instruments) and a tube for infinity-corrected objective lenses. An EXW-12 laser (NKT Photonics) was used for excitation through triple-sheet optics to illuminate samples from one side. Excitation and emission filters (AHF Analysentechnik AG) were used as indicated in Figure 2 and Movie 1.

Tiled image stacks were acquired with a LVMI-Fluor 12× objective (Miltenyi Biotec) with a format of 2048 × 2048. Using ImSpector software (version 7.1.4, Miltenyi Biotec), a 10% overlap of tiles was set for stitching.

Image files were processed with Imaris software (version 9.8, Bitplane), including file conversion, stitching, further processing, and rendering. The ortho slicer tool was used to restrict volumes in the *z* direction to improve the representation of structures that were otherwise covered in the context of the whole body. All 3D images were generated using the snapshot function. 2D maximum-intensity projections were generated in Fiji (Schindelin et al., 2012).

Movie 1 was produced in Imaris with the key frame animation tool; Adobe Premiere Pro 2020 (version 14.9.0, Adobe) was used for cutting and labeling.

## Immunohistochemistry

All the antibodies used in this study are listed in Table 1.

**Transgene expression pattern of the SS01671 driver strain.** To validate specific expression in the larval APL neuron of the APL-GAL4 driver strain (Saumweber et al., 2018), it was crossed to UAS-ChR2XXL::tdtomato to express a tomato-tagged version of ChR2XXL (FlyBase ID: FBtp0131815) (Saumweber et al., 2018). Double-heterozygous third-instar progeny (abbreviated as APL>ChR2XXL::tdtomato) were dissected in ice-cold Ringer's solution, and the brains were fixed for 30 min in 10% formaldehyde dissolved in PBS (pH 7.2, P4417, Sigma-Aldrich) at room temperature. After consecutive washing steps (3 × 10 min each) in PBT (0.3% Triton X-100 [CAS: 9036-19-5, Roth] in PBS), the brains were blocked in 5% NGS solution (005-000-121, Jackson ImmunoResearch Laboratories; in PBS) for 2 h at room temperature. To provide a reference staining of fiber tracts (including the mushroom bodies), tissues were incubated overnight at 4°C with a primary monoclonal mouse anti-FASII antibody (AB_528235, DSHB) diluted 1:50 in blocking solution containing 4% NGS in PBS. After six washes (10 min each) in PBS, the tissues were treated overnight at 4°C with a secondary polyclonal goat anti-mouse AlexaFluor-488 antibody (A11001, Invitrogen) diluted 1:200 in PBS. The brains were then

←

dopaminergic neurons, respectively, as indicated. In addition to its connections with the KCs, the APL neuron establishes synaptic contacts with the calyx MBONs (MBON-a1 and -a2) as well as with a subset of PNs (for additional connections between APL and mushroom body extrinsic neurons that are omitted here, see Figs. 6, 7). During odor-taste reward associative learning, the coincidence between the odor and the reward signal at the KCs is thought to lead to a presynaptic depression of the synapses between the odor-activated KCs and avoidance-promoting MBONs, whereas the synapses of these KCs with approach-promoting MBONs in other compartments remain unchanged (the contribution of MBON-a1/a2 to learned behavior is unclear, as indicated by the stippled lines). Future processing of the learned odor is thus biased in favor of approach. The same rationale is thought to apply for odor-punishment learning, occurring at the synapses between the KCs and approach MBONs. The electron microscopy reconstruction of a first-instar larval nervous system additionally revealed unexpected connections from KCs toward mushroom body input neurons (MBINs), including DANs, as well as MBIN/DAN-to-MBON synapses; KC-to-KC and MBON-to-MBIN connections are not displayed. Arrows indicate synaptic contacts between neurons.

washed in PBS (6 × 10 min each) and mounted in Vectashield (Vector Laboratories) on a cover slip. Signal detection from the tomato-tag of ChR2XXL (labeling the APL neuron) did not require antibodies; rather, the tomato fluorescence signal was detected directly under the microscope. Image *z* stacks were acquired with a Leica TCS SP8 confocal microscope (Leica Mikrosysteme Vertrieb) at a format of 1024 × 1024. Image processing was performed using Imaris software (version 9.72, Bitplane).

To visualize the larval APL neuron together with the mushroom bodies, we crossed the APL-GAL4 driver with a recombined effector/enhancer effector strain that includes a UAS-mIFP-T2A-HO1 effector (abbreviated as mIFP; Bloomington Stock Center #64181) (Yu et al., 2015) recombined with the enhancer effector construct MB247>mCherry-CAAX (Kobler et al., 2021). Third-instar larval progeny (abbreviated as APL>mIFP/MB247>mCherry-CAAX) were dissected in ice-cold Ca²⁺-free saline solution and fixed for 24 h in 4% PFA (J19943, Alfa Aesar; in PBS) at 4°C. After six washes (3 × brief; 3 × 10 min) in 0.3% PBT, the brains were mounted in Vectashield (Vector Laboratories) on a cover slip. Signal detection from the mCherry-CAAX reporter (labeling the mushroom bodies) and the mIFP reporter (labeling APL) did not require antibodies for signal amplification. The image *z* stack was acquired with a Leica TCS SP8 confocal microscope (Leica Mikrosysteme Vertrieb) at a format of 1024 × 1024. The corresponding Movie 2 was produced in Imaris (version 9.72, Bitplane).

To examine the interhemispheric symmetry in the morphology of APL, the APL-GAL4 driver was crossed to UAS-mCD8::GFP (Bloomington Stock Center #5137) (Lee and Luo, 1999) as the effector. Third-instar larvae were put on ice and dissected in PBS. The brains were fixed in 4% PFA for 20 min at room temperature. After a succession of washing steps (3 × brief; 1 × 5 min; 3 × 15 min; 1 × 90 min) in 3% PBT (3% Triton X-100 [CAS: 9002-93-1, Sigma-Aldrich] in PBS) on ice, the brains were blocked with 5% NGS (G9023, Sigma-Aldrich) in PBT for 1 h at room temperature and incubated for 48 h with primary antibodies at 4°C. The brains were then washed (2 × brief; 3 × 15 min; 1 × 60 min; on ice; 1 × 30 min at room temperature) in 3% PBT before application of the secondary antibodies for at least 24 h at 4°C. After a final set of washing steps (3 × brief; 3 × 5 min; 2 × 15 min) in 3% PBT, the brains were mounted on poly-L-lysin-coated cover slips (following the Janelia FlyLight recipe), dehydrated by a series of increasing concentrations of EtOH (1× brief in distilled water; 1 × 10 min 30% EtOH; 1 × 10 min 50% EtOH; 1 × 10 min 75% EtOH; 1 × 10 min 95% EtOH; 3 × 10 min 100% EtOH) and cleared (3 × 5 min) in xylene (247642, CAS: 1330-20-7, Sigma-Aldrich). Finally, the brains were mounted in DPX mounting medium (dibutyl phthalate in xylene; 06522, Sigma-Aldrich) and left in darkness for at least 24 h before imaging.

The primary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) a polyclonal rabbit anti-GFP antibody (A6455, Invitrogen) diluted 1:1000 in 3% PBT (for APL staining), (3) a monoclonal mouse 4F3 anti-DLG antibody (AB_528203, Developmental Studies Hybridoma Bank) diluted 1:200 in 3% PBT (for mushroom body staining), and (4) a monoclonal rat anti-N-Cadherin antibody (DN-Ex #8-s, Developmental Studies Hybridoma Bank) diluted 1:50 in 3% PBT (for neuropil staining).

The secondary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) polyclonal goat anti-rabbit AlexaFluor-488 (A11008, Invitrogen), (3) polyclonal goat anti-mouse AlexaFluor-568 (A10037, Invitrogen), and (4) polyclonal goat anti-rat AlexaFluor-647 (712-605-153, Jackson ImmunoResearch Laboratories), all diluted 1:500 in 3% PBT. Confocal microscopy was conducted on a Zeiss LSM800 confocal laser scanning microscope with ZEN 2.3 software. Image *z* stacks were acquired with a LSM800 confocal microscope (Zeiss) at a format of 1024 × 1024. Image processing was performed using Imaris software (version 9.72, Bitplane).

To compare the coverage of the mushroom body calyx and compartments between the APL neuron of each hemisphere, mean pixel intensities were measured using ImageJ (version 1.53c, Fiji ImageJ). Grayscale maximum intensity projections of the GFP-channel (labeling APL membranes) were created, whereas the DLG channel (labeling the mushroom bodies) served as a template for orientation. The mushroom body calyx and compartments were
<figure><img src="image_2.png" /><figcaption>**Figure 2.** The APL-GAL4 driver does not express outside the brain. **A–B''**, Maximum intensity projection of fluorescence signals from an entire larva of the genotype APL>**mCherry-CAAX** acquired on a light-sheet microscope using a 12× objective (top view, rostral to the left). Dashed boxes represent the CNS, shown enlarged for a volume-restricted view in **(B–B'')**. λ exc and λ em indicate filter band passes used for excitation and emission, respectively (merged in **A'''**, **B''**). CX, Mushroom body calyx; CB, cell body of the APL neuron; vL and mL, innervation of APL in the vertical and medial lobe of the mushroom body, respectively. Scale bars: **A–A''**, 200 µm; **B–B''**, 50 µm. See also Movie 2. **C–F''**, Same as in **A–B''**, but for the effector control (+>**mCherry-CAAX**) (**C–D''**) and driver control (APL>+) (**E–F''**), respectively. Arrowhead points to autofluorescence signals from the pharynx (visible also in **B–B''** but omitted for clarity). Fluorescence signals that can be observed across wavelengths and genotypes reflect autofluorescence (including from food particles in the gut) and allow bodily detail to be discerned. Fluorescence reflecting the expression of mCherry-CAAX was observed only in the brain and only the APL neuron (**A'''**, **B''**, magenta signals; Movie 1) (compare **B'**, **D'**, **F'**).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** The APL-GAL4 driver does not express outside the brain. **A–B''**, Maximum intensity projection of fluorescence signals from an entire larva of the genotype APL>**mCherry-CAAX** acquired on a light-sheet microscope using a 12× objective (top view, rostral to the left). Dashed boxes represent the CNS, shown enlarged for a volume-restricted view in **(B–B'')**. λ exc and λ em indicate filter band passes used for excitation and emission, respectively (merged in **A'''**, **B''**). CX, Mushroom body calyx; CB, cell body of the APL neuron; vL and mL, innervation of APL in the vertical and medial lobe of the mushroom body, respectively. Scale bars: **A–A''**, 200 µm; **B–B''**, 50 µm. See also Movie 2. **C–F''**, Same as in **A–B''**, but for the effector control (+>**mCherry-CAAX**) (**C–D''**) and driver control (APL>+) (**E–F''**), respectively. Arrowhead points to autofluorescence signals from the pharynx (visible also in **B–B''** but omitted for clarity). Fluorescence signals that can be observed across wavelengths and genotypes reflect autofluorescence (including from food particles in the gut) and allow bodily detail to be discerned. Fluorescence reflecting the expression of mCherry-CAAX was observed only in the brain and only the APL neuron (**A'''**, **B''**, magenta signals; Movie 1) (compare **B'**, **D'**, **F'**).</figcaption></figure>
<figure><img src="image_3.png" /></figure>

**Movie 1.** Volume rendering of fluorescence signals in a third-instar larva of the genotype APL>*mCherry*-*CAAX* at the indicated combinations of excitation and emission wavelengths (same specimen as in Fig. 2A–B'''). The movie starts in the brain region from a dorsal view (rostral to the left), and after rotation zooms out to show the full body. The data were acquired with a 12× objective; grid spacing: 200 µm. [View online]

<figure><img src="image_4.png" /></figure>

**Movie 2.** 3D rendering of the APL neuron (green) and the mushroom bodies (magenta) in a third-instar larval brain. Genotype: APL>*mIFP/MB247>*-*mCherry*-*CAAX*. The data were acquired with a 40× oil objective; grid spacing: 20 µm. [View online]

selected using the ROI Manager function; mean gray values are documented in Extended Data Figure 3-1.

To validate the expression of the ChR2XXL effector protein used for activating APL, the APL-GAL4 driver was crossed to the UAS-ChR2XXL effector (Bloomington Stock Center #58374) (Dawydow et al., 2014). Following the procedure of Schleyer et al. (2020), brains of third-instar larval progeny (abbreviated as APL>ChR2XXL) were dissected in ice-cold Ca²⁺-free saline solution and fixed in Bouin's solution (HT10132, Sigma-Aldrich) for 7 min at room temperature. After six successive washing steps (3 × brief; 3 × 15 min) in 0.2% PBT, the brains were incubated overnight at 4°C with a primary monoclonal mouse anti-ChR2 antibody (610180, ProGen Biotechnik) diluted 1:100 in 0.2% PBT. The brains were then washed (3 × 10 min each) in 0.2% PBT and incubated for 1 h at room temperature with a secondary polyclonal donkey anti-mouse Cy3 antibody (715-165-150, Jackson ImmunoResearch Laboratories) diluted 1:300 in 0.2% PBT. Finally, the samples were washed (3 × 10 min each) in 0.2% PBT and mounted in Vectashield (Vector Laboratories) on a cover slip. Image z stacks were acquired with a Leica TCS SP8 confocal microscope (Leica Mikrosysteme Vertrieb) at a format of 1024 × 1024. Image processing was performed using Imaris software (version 9.72, Bitplane).

<figure><img src="image_5.png" /></figure>

**Movie 3.** 3D rendering and segmentation of presynaptic (green) and postsynaptic (magenta) regions of the APL neuron in a third-instar larval brain, based on data shown in Figure 4C–C". Genotype: APL>*Dsyd-1::GFP/DenMark*. The data were acquired with a 63× glycerol objective; grid spacing: 20 µm. [View online]

*GABA staining.* To confirm the presence of GABA in APL, the APL-GAL4 driver was crossed to a UAS-CsChrimson::mVenus effector (Bloomington Stock Center #55135) (Klapoetke et al., 2014), and third-instar progeny (abbreviated as APL>Chrimson) were dissected in PBS. Signal detection from the mVenus tag of the Chrimson transgene allows visualization of APL membranes without antibodies under the fluorescence microscope. The brains were fixed for 20 min with 4% PFA in 3% PBT on ice. After successive washing steps (2 × brief; 1 × 5 min; 3 × 15 min; 1 × 2 h) in 3% PBT, the brains were blocked for 1-2 h in 2% NGS solution (S-1000, Vector Laboratories; in PBS) on ice. After two overnight incubations at 4°C with the primary antibodies, the brains were rinsed (2 × brief; 1 × 5 min; 3 × 15 min; 1 × 2 h) in 3% PBT and incubated overnight with the secondary antibodies at 4°C. The preparations were finally washed (2 × brief; 1 × 5 min; 5 × 15 min) in 3% PBT, mounted in Vectashield (Vector Laboratories) on a cover slip, and scanned under a LSM510 confocal microscope (Zeiss) at a format of 1024 × 1024. Image processing was performed using Imaris software (version 9.72, Bitplane).

The primary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) a monoclonal rat anti-N-Cadherin antibody (DN-Ex #8-s, Developmental Studies Hybridoma Bank) diluted 1:50 in 3% PBT (for neuropil staining), and (3) a polyclonal rabbit anti-GABA antibody (A2052, Sigma-Aldrich) diluted 1:500 in 3% PBT.

The secondary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) a polyclonal Cy3-conjugated goat anti-rat antibody (A10522, Invitrogen) diluted 1:200 in 3% PBT, and (3) a polyclonal Cy5-conjugated goat anti-rabbit antibody (A10523, Invitrogen) diluted 1:200 in 3% PBT.

*APL regional synaptic polarity.* To analyze the regional synaptic polarity of the larval APL neuron, the APL-GAL4 driver was crossed to a double effector with both UAS-Dsyd-1::GFP (Owald et al., 2015) and UAS-DenMark (Bloomington Stock Center #33062) (Nicolaï et al., 2010). Third-instar progeny (abbreviated as APL>Dsyd-1::GFP/DenMark) were dissected, fixed, dehydrated, and mounted as described in the preceding section. Image processing was performed, and the corresponding Movie 3 was generated using Imaris software (version 9.72, Bitplane).

The primary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) a monoclonal rat anti-N-Cadherin antibody (DN-Ex #8-s, Developmental Studies Hybridoma Bank) diluted 1:50 in 3% PBT (for neuropil staining), (3) a polyclonal FITC-conjugated goat anti-GFP antibody (ab 6662, Abcam) diluted 1:1000 in 3% PBT (for visualization of the GFP-tag from Dsyd-1::GFP to label presynaptic regions), and (4) a polyclonal rabbit anti-DsRed antibody (632496,
<figure><img src="image_5.png" /></figure>

**Movie 1.** Volume rendering of fluorescence signals in a third-instar larva of the genotype APL>mACherry-CAAX at the indicated combinations of excitation and emission wavelengths (same specimen as in Fig. 2A–B′′′). The movie starts in the brain region from a dorsal view (rostral to the left), and after rotation zooms out to show the full body. The data were acquired with a 12× objective; grid spacing: 200 µm. [View online]

<figure><img src="image_6.png" /></figure>

**Movie 2.** 3D rendering of the APL neuron (green) and the mushroom bodies (magenta) in a third-instar larval brain. Genotype: APL>mlFP/MB247>mACherry-CAAX. The data were acquired with a 40× oil objective; grid spacing: 20 µm. [View online]

selected using the ROI Manager function; mean gray values are documented in Extended Data Figure 3-1.

To validate the expression of the ChR2XXL effector protein used for activating APL, the APL-GAL4 driver was crossed to the UAS-ChR2XXL effector (Bloomington Stock Center #58374) (Dawydow et al., 2014). Following the procedure of Schleyer et al. (2020), brains of third-instar larval progeny (abbreviated as APL>ChR2XXL) were dissected in ice-cold Ca²⁺-free saline solution and fixed in Bouin's solution (HT10132, Sigma-Aldrich) for 7 min at room temperature. After six successive washing steps (3 × brief; 3 × 15 min) in 0.2% PBT, the brains were incubated overnight at 4°C with a primary monoclonal mouse anti-ChR2 antibody (610180, ProGen Biotechnik) diluted 1:100 in 0.2% PBT. The brains were then washed (3 × 10 min each) in 0.2% PBT and incubated for 1 h at room temperature with a secondary polyclonal donkey anti-mouse Cy3 antibody (715-165-150, Jackson ImmunoResearch Laboratories) diluted 1:300 in 0.2% PBT. Finally, the samples were washed (3 × 10 min each) in 0.2% PBT and mounted in Vectashield (Vector Laboratories) on a cover slip. Image z stacks were acquired with a Leica TCS SP8 confocal microscope (Leica Mikrosysteme Vertrieb) at a format of 1024 × 1024. Image processing was performed using Imaris software (version 9.72, Bitplane).

<figure><img src="image_7.png" /></figure>

**Movie 3.** 3D rendering and segmentation of presynaptic (green) and postsynaptic (magenta) regions of the APL neuron in a third-instar larval brain, based on data shown in Figure 4C–C′. Genotype: APL>Dsyd-1::GFP/DenMark. The data were acquired with a 63× glycerol objective; grid spacing: 20 µm. [View online]

**GABA staining.** To confirm the presence of GABA in APL, the APL-GAL4 driver was crossed to a UAS-CsChrimson:mVenus effector (Bloomington Stock Center #55135) (Klapoetke et al., 2014), and third-instar progeny (abbreviated as APL>Chrimson) were dissected in PBS. Signal detection from the mVenus tag of the Chrimson transgene allows visualization of APL membranes without antibodies under the fluorescence microscope. The brains were fixed for 20 min with 4% PFA in 3% PBT on ice. After successive washing steps (2 × brief; 1 × 5 min; 3 × 15 min; 1 × 2 h) in 3% PBT, the brains were blocked for 1-2 h in 2% NGS solution (S-1000, Vector Laboratories; in PBS) on ice. After two overnight incubations at 4°C with the primary antibodies, the brains were rinsed (2 × brief; 1 × 5 min; 3 × 15 min; 1 × 2 h) in 3% PBT and incubated overnight with the secondary antibodies at 4°C. The preparations were finally washed (2 × brief; 1 × 5 min; 5 × 15 min) in 3% PBT, mounted in Vectashield (Vector Laboratories) on a cover slip, and scanned under a LSM510 confocal microscope (Zeiss) at a format of 1024 × 1024. Image processing was performed using Imaris software (version 9.72, Bitplane).

The primary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) a monoclonal rat anti-N-Cadherin antibody (DN-Ex #8-s, Developmental Studies Hybridoma Bank) diluted 1:50 in 3% PBT (for neuropil staining), and (3) a polyclonal rabbit anti-GABA antibody (A2052, Sigma-Aldrich) diluted 1:500 in 3% PBT.

The secondary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) a polyclonal Cy3-conjugated goat anti-rat antibody (A10522, Invitrogen) diluted 1:200 in 3% PBT, and (3) a polyclonal Cy5-conjugated goat anti-rabbit antibody (A10523, Invitrogen) diluted 1:200 in 3% PBT.

**APL regional synaptic polarity.** To analyze the regional synaptic polarity of the larval APL neuron, the APL-GAL4 driver was crossed to a double effector with both UAS-Dsyd-1::GFP (Owald et al., 2015) and UAS-DenMark (Bloomington Stock Center #33062) (Nicolaï et al., 2010). Third-instar progeny (abbreviated as APL>Dsyd-1::GFP/DenMark) were dissected, fixed, dehydrated, and mounted as described in the preceding section. Image processing was performed, and the corresponding Movie 3 was generated using Imaris software (version 9.72, Bitplane).

The primary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) a monoclonal rat anti-N-Cadherin antibody (DN-Ex #8-s, Developmental Studies Hybridoma Bank) diluted 1:50 in 3% PBT (for neuropil staining), (3) a polyclonal FITC-conjugated goat anti-GFP antibody (ab 6662, Abcam) diluted 1:1000 in 3% PBT (for visualization of the GFP-tag from Dsyd-1::GFP to label presynaptic regions), and (4) a polyclonal rabbit anti-DsRed antibody (632496,
Clontech) diluted 1:200 in 3% PBT (for detecting the DenMark signal to label postsynaptic regions).

The secondary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) a polyclonal Cy3-conjugated goat anti-rat antibody (A10522, Invitrogen) diluted 1:200 in 3% PBT, and (3) a polyclonal Cy5-conjugated goat anti-rabbit antibody (A10523, Invitrogen) diluted 1:200 in 3% PBT.

### Chemical tagging for tracking APL development

Chemical tagging provides an alternative method to immunohistochemistry for labeling specific cells and structures in tissues. The tag-based approach uses genetically driven, enzyme-based protein “tags” that are expressed in specific cells and that covalently bind small fluorescent substrates, resulting in fast and specific tissue staining with low background signals (Kohl et al., 2014; Sutcliffe et al., 2017; Meissner et al., 2018).

We used such tagging to track the regional synaptic polarity of APL during development. Specifically, we used the synaptic reporters synaptotagmin fused to the chemical tag SNAPm (Syt1-SNAPm) to label presynaptic regions, and telencephalin fused to CLIPm (TLN-CLIPm) to label postsynaptic regions (Kohl et al., 2014). The effectors UAS-Syt1: SNAP (Bloomington Stock Center #58379) (Kohl et al., 2014), UAS-TLN:CLIP (Bloomington Stock Center #58382) (Kohl et al., 2014), and UAS-mCD8::GFP (for labeling APL) (Lin et al., 2014) were used together with the intersectional driver APLi-GAL4 (NP2631-GAL4, GH146-FLP, tubP-FRT-GAL80-FRT) for specific expression in both larval and adult APL neurons (Lin et al., 2014; Mayseless et al., 2018), since APL-GAL4 does not cover the adult APL neurons (not shown).

Our procedures followed Kohl et al. (2014). In brief, brains of third-instar larvae, pupae (6 or 12 h after puparium formation), and adults of the genotype APLi/Syt1:SNAP>mCD8::GFP/TLN:CLIP were dissected in ice-cold PB (0.1 M) and fixed in 4% PFA at room temperature for 20 min. The brains were permeabilized and washed (3 × 10 min) in PBT (0.3% Triton X-100 in PBS). Chemical tag ligands were then applied in a 300 µl volume on a nutator for 15 min, at room temperature. The chemical substrates were SNAP-tag ligands (SNAP surface 549 - BG 549 [NEB, S9112S]) and CLIP-tag ligands (CLIP surface 647 - BC 647 [NEB, S9234S]) at final concentrations of 1 µM in 0.3% PBT. To minimize cross-reactivity, the SNAP-tag ligands were applied 10 min before the CLIP-tag ligands. To label APL, the brains were immunostained: three consecutive washing steps (10 min each) in 0.3% PBT were followed by 30 min incubation in blocking solution with 5% NGS (005-000-121, Jackson ImmunoResearch Laboratories) in PBT. The brains were then incubated overnight with a primary antibody mixture consisting of the following: (1) 5% NGS diluted in 0.3% PBT and (2) a polyclonal chicken anti-GFP antibody (AB_10000240, Aves Labs) diluted 1:500. After five consecutive washing steps (3 × brief; 2 × 20 min) in 0.3% PBT, the brains were incubated for 2 h at room temperature with a polyclonal secondary FITC-conjugated goat anti-chicken antibody (A16055, Invitrogen) diluted 1:300 in 0.3% PBT. The preparations were mounted on slides in Slow Fade (Invitrogen), and examined under a confocal microscope (Zeiss LSM 800). Image processing was performed using Imaris software (version 9.2 Bitplane).

### Volume reconstruction of APL from an EM dataset

We added radial volume annotations to an existing skeleton reconstruction of the APL neuron in both hemispheres (Eichler et al., 2017) from an electron microscopy dataset of a 6-h-old first-instar larva (Ohyama et al., 2015). More details of the neuron reconstructions can be found in Eichler et al. (2017). Volume annotations were made manually using the web-based software CATMAID (Saalfeld et al., 2009; Schneider-Mizell et al., 2016), which was extended with a tool to allow for rapid graphical annotations of the radii of contiguous cable segments with similar radius. Radial annotations were used to create a volumetric representation of the cells' morphology as conical frustum compartments. The radii were placed so as to preserve the approximate volume of the irregularly shaped processes while accounting for the anisotropic image resolution of 3.8 nm × 3.8 nm × 50 nm. We defined the axon and dendrite of both APL neurons as the two synapse-rich areas along the arbor separated from the neurite by the high Strahler branch point nearest to the cell

body. Reconstructed neurons and their synapses were analyzed using the natverse package (http://natverse.org/) (Bates et al., 2020) in R (version 3.6.2) and plotted using Blender (version 2.79) with the CATMAID-to-Blender plugin (https://github.com/schlegelp/CATMAID-to-Blender) (Schlegel et al., 2016).

### Dendrogram representations of APL synapses and branching

Neuron dendrograms are simplified, but topologically correct, 2D representations of neurons with complex morphologies (Strauch et al., 2018). As relative branch lengths and synapse location are preserved, dendrograms can be used to visualize the spatial distribution of synapses in an easily readable way. The APL dendrograms are derived from existing electron microscopy reconstructions (Eichler et al., 2017) and were created following established computational methods (Strauch et al., 2018). Additionally, mushroom body calyx and compartment boundaries were superimposed onto the dendrograms, based on the projection patterns of mushroom body extrinsic neurons (Saumweber et al., 2018). We used the natverse toolbox for R (Bates et al., 2020) and custom code (A. Bates, University of Cambridge) to extract the synaptic coordinates from the CATMAID L1 dataset and to generate an envelope that surrounds the synapses formed by the mushroom body extrinsic neurons belonging to the calyx and each given compartment. Boundaries were plotted onto the dendrograms as hulls around the synapses located within the respective region. When these extended across several dendrogram branches, they were connected by dashed lines.

To analyze the relative distribution of APL-to-KC and KC-to-APL synapses in the calyx, we followed a procedure described in detail in Schleyer et al. (2020). In brief, we computed geodesic distances between synapses (i.e., “cable length” distances along the neuron’s branches). Based on the geodesic distances, a clustering algorithm served to partition all synapses, regardless of their type, into local synapse clusters, that is, regions of high synapse density (domains). For each domain, the distances were then evaluated from the APL-to-KC (or KC-to-APL) synapses to the cluster’s centroid point, which served as a measure for the spatial distribution of APL-to-KC (or KC-to-APL) within the domain.

### Functional imaging

The functional imaging methods follow those described in greater detail previously (Selcho et al., 2017; Lyutova et al., 2019). In brief, to monitor intracellular $Ca^{2+}$ levels of KCs in response to optogenetic activation of the APL neuron, the lexA-lexAop system was used to express the fluorescent $Ca^{2+}$ reporter GCaMP6m in KCs (effector lexAOp-GCaMP6m; Bloomington Stock Center #44276) (Chen et al., 2013); driver R14H06-lexA (Bloomington Stock Center #52482) (Pfeiffer et al., 2010), and the GAL4-UAS system was used to express ChR2-XXL in APL (effector UAS-ChR2XXL; Bloomington Stock Center #58374) (Dawydow et al., 2014); driver R26G02-GAL4 (Bloomington Stock Center #48065) (Saumweber et al., 2018). The R26G02-GAL4 driver covers APL plus additional cells outside the mushroom body; it was chosen because the more specific APL-GAL4 driver, as a split-GAL4 strain, could not be combined with the lexA-lexAop system. Using classical genetics, we generated strains that carried a combination of the drivers (R14H06-lexA/R26G02-GAL4) or the effectors (lexAop-GCaMP6m/UAS-ChR2XXL) (Lyutova et al., 2019). These strains were crossed, and from the progeny (abbreviated as APL<sub>26G02</sub>>ChR2XXL; KC>>GCaMP6m) larval brains were dissected and placed in a Petri dish containing 405 µl hemolymph-like HL3.1 Ringer solution. Images with ROIs around the calyx and KCs were recorded with an Axio Examiner D1 microscope (Zeiss) using a W Plan-Apochromat 20 × 1.0 DIC (UV) VIS-IR objective and a Axiocam 506 camera (Zeiss). We monitored fluorescence intensity on pulsed 475 nm light (Colibri LED, Zeiss) at an intensity of 1.8 mW/cm² for a first observation period, followed by pulses at an intensity of 4.1 mW/cm² for a second observation period immediately thereafter. The light pulses were of 80 ms duration and were given at a 2 s onset-onset interval. We used larvae without the APL driver as the genetic control (abbreviated as +>ChR2XXL; KC>>GCaMP6m).

To see whether APL activation would be potent enough to reduce the very high intracellular $Ca^{2+}$ levels that result from cholinergic stimulation of the KCs, we used carbamylcholine (CAS: 51-83-2, Sigma-
Clontech) diluted 1:200 in 3% PBT (for detecting the DenMark signal to label postsynaptic regions).

The secondary antibody mixture consisted of the following: (1) 2% NGS diluted 1:25 in 3% PBT, (2) a polyclonal Cy3-conjugated goat anti-rat antibody (A10522, Invitrogen) diluted 1:200 in 3% PBT, and (3) a polyclonal Cy5-conjugated goat anti-rabbit antibody (A10523, Invitrogen) diluted 1:200 in 3% PBT.

### Chemical tagging for tracking APL development

Chemical tagging provides an alternative method to immunohistochemistry for labeling specific cells and structures in tissues. The tag-based approach uses genetically driven, enzyme-based protein “tags” that are expressed in specific cells and that covalently bind small fluorescent substrates, resulting in fast and specific tissue staining with low background signals (Kohl et al., 2014; Sutcliffe et al., 2017; Meissner et al., 2018).

We used such tagging to track the regional synaptic polarity of APL during development. Specifically, we used the synaptic reporters synaptotagmin fused to the chemical tag SNAPm (Syt1-SNAPm) to label presynaptic regions, and telencephalin fused to CLIPm (TLN-CLIPm) to label postsynaptic regions (Kohl et al., 2014). The effectors UAS-Syt1: SNAP (Bloomington Stock Center #58379) (Kohl et al., 2014), UAS-TLN:CLIP (Bloomington Stock Center #58382) (Kohl et al., 2014), and UAS-mCD8::GFP (for labeling APL) (Lin et al., 2014) were used together with the intersectional driver APLi-GAL4 (NP2631-GAL4, GH146-FLP, tubP-FRT-GAL80-FRT) for specific expression in both larval and adult APL neurons (Lin et al., 2014; Mayseless et al., 2018), since APL-GAL4 does not cover the adult APL neurons (not shown).

Our procedures followed Kohl et al. (2014). In brief, brains of third-instar larvae, pupae (6 or 12 h after puparium formation), and adults of the genotype APLi/Syt1:SNAP>mCD8::GFP/TLN:CLIP were dissected in ice-cold PB (0.1 M) and fixed in 4% PFA at room temperature for 20 min. The brains were permeabilized and washed (3 × 10 min) in PBT (0.3% Triton X-100 in PBS). Chemical tag ligands were then applied in a 300 µl volume on a nutator for 15 min, at room temperature. The chemical substrates were SNAP-tag ligands (SNAP surface 549 - BG 549 [NEB, S9112S]) and CLIP-tag ligands (CLIP surface 647 - BC 647 [NEB, S9234S]) at final concentrations of 1 µM in 0.3% PBT. To minimize cross-reactivity, the SNAP-tag ligands were applied 10 min before the CLIP-tag ligands. To label APL, the brains were immunostained: three consecutive washing steps (10 min each) in 0.3% PBT were followed by 30 min incubation in blocking solution with 5% NGS (005-000-121, Jackson ImmunoResearch Laboratories) in PBT. The brains were then incubated overnight with a primary antibody mixture consisting of the following: (1) 5% NGS diluted in 0.3% PBT and (2) a polyclonal chicken anti-GFP antibody (AB_10000240, Aves Labs) diluted 1:500. After five consecutive washing steps (3 × brief; 2 × 20 min) in 0.3% PBT, the brains were incubated for 2 h at room temperature with a polyclonal secondary FITC-conjugated goat anti-chicken antibody (A16055, Invitrogen) diluted 1:300 in 0.3% PBT. The preparations were mounted on slides in Slow Fade (Invitrogen), and examined under a confocal microscope (Zeiss LSM 800). Image processing was performed using Imaris software (version 9.2 Bitplane).

### Volume reconstruction of APL from an EM dataset

We added radial volume annotations to an existing skeleton reconstruction of the APL neuron in both hemispheres (Eichler et al., 2017) from an electron microscopy dataset of a 6-h-old first-instar larva (Ohyama et al., 2015). More details of the neuron reconstructions can be found in Eichler et al. (2017). Volume annotations were made manually using the web-based software CATMAID (Saalfeld et al., 2009; Schneider-Mizell et al., 2016), which was extended with a tool to allow for rapid graphical annotations of the radii of contiguous cable segments with similar radius. Radial annotations were used to create a volumetric representation of the cells' morphology as conical frustum compartments. The radii were placed so as to preserve the approximate volume of the irregularly shaped processes while accounting for the anisotropic image resolution of 3.8 nm × 3.8 nm × 50 nm. We defined the axon and dendrite of both APL neurons as the two synapse-rich areas along the arbor separated from the neurite by the high Strahler branch point nearest to the cell

body. Reconstructed neurons and their synapses were analyzed using the natverse package (http://natverse.org/) (Bates et al., 2020) in R (version 3.6.2) and plotted using Blender (version 2.79) with the CATMAID-to-Blender plugin (https://github.com/schlegelp/CATMAID-to-Blender) (Schlegel et al., 2016).

### Dendrogram representations of APL synapses and branching

Neuron dendrograms are simplified, but topologically correct, 2D representations of neurons with complex morphologies (Strauch et al., 2018). As relative branch lengths and synapse location are preserved, dendrograms can be used to visualize the spatial distribution of synapses in an easily readable way. The APL dendrograms are derived from existing electron microscopy reconstructions (Eichler et al., 2017) and were created following established computational methods (Strauch et al., 2018). Additionally, mushroom body calyx and compartment boundaries were superimposed onto the dendrograms, based on the projection patterns of mushroom body extrinsic neurons (Saumweber et al., 2018). We used the natverse toolbox for R (Bates et al., 2020) and custom code (A. Bates, University of Cambridge) to extract the synaptic coordinates from the CATMAID L1 dataset and to generate an envelope that surrounds the synapses formed by the mushroom body extrinsic neurons belonging to the calyx and each given compartment. Boundaries were plotted onto the dendrograms as hulls around the synapses located within the respective region. When these extended across several dendrogram branches, they were connected by dashed lines.

To analyze the relative distribution of APL-to-KC and KC-to-APL synapses in the calyx, we followed a procedure described in detail in Schleyer et al. (2020). In brief, we computed geodesic distances between synapses (i.e., “cable length” distances along the neuron’s branches). Based on the geodesic distances, a clustering algorithm served to partition all synapses, regardless of their type, into local synapse clusters, that is, regions of high synapse density (domains). For each domain, the distances were then evaluated from the APL-to-KC (or KC-to-APL) synapses to the cluster’s centroid point, which served as a measure for the spatial distribution of APL-to-KC (or KC-to-APL) within the domain.

### Functional imaging

The functional imaging methods follow those described in greater detail previously (Selcho et al., 2017; Lyutova et al., 2019). In brief, to monitor intracellular $Ca^{2+}$ levels of KCs in response to optogenetic activation of the APL neuron, the lexA-lexAop system was used to express the fluorescent $Ca^{2+}$ reporter GCaMP6m in KCs (effector lexAOp-GCaMP6m; Bloomington Stock Center #44276) (Chen et al., 2013); driver R14H06-lexA (Bloomington Stock Center #52482) (Pfeiffer et al., 2010), and the GAL4-UAS system was used to express ChR2-XXL in APL (effector UAS-ChR2XXL; Bloomington Stock Center #58374) (Dawydow et al., 2014); driver R26G02-GAL4 (Bloomington Stock Center #48065) (Saumweber et al., 2018). The R26G02-GAL4 driver covers APL plus additional cells outside the mushroom body; it was chosen because the more specific APL-GAL4 driver, as a split-GAL4 strain, could not be combined with the lexA-lexAop system. Using classical genetics, we generated strains that carried a combination of the drivers (R14H06-lexA/R26G02-GAL4) or the effectors (lexAop-GCaMP6m/UAS-ChR2XXL) (Lyutova et al., 2019). These strains were crossed, and from the progeny (abbreviated as APL<sub>26G02</sub>>ChR2XXL; KC>GCaMP6m) larval brains were dissected and placed in a Petri dish containing 405 µl hemolymph-like HL3.1 Ringer solution. Images with ROIs around the calyx and KCs were recorded with an Axio Examiner D1 microscope (Zeiss) using a W Plan-Apochromat 20 × 1.0 DIC (UV) VIS-IR objective and a Axiocam 506 camera (Zeiss). We monitored fluorescence intensity on pulsed 475 nm light (Colibri LED, Zeiss) at an intensity of 1.8 mW/cm² for a first observation period, followed by pulses at an intensity of 4.1 mW/cm² for a second observation period immediately thereafter. The light pulses were of 80 ms duration and were given at a 2 s onset-onset interval. We used larvae without the APL driver as the genetic control (abbreviated as +>ChR2XXL; KC>GCaMP6m).

To see whether APL activation would be potent enough to reduce the very high intracellular $Ca^{2+}$ levels that result from cholinergic stimulation of the KCs, we used carbamylcholine (CAS: 51-83-2, Sigma-
Aldrich). Specifically, we monitored fluorescence intensity under pulsed 475 nm light as described above for a total of 8 min and after the first 2 min manually bath-applied 45 µl carbamylcholine, dissolved in HL3.1 to a final concentration of 10⁻⁴ M.

We present the fluorescence intensity (ΔF/F₀) across the observation period normalized to the values at its beginning (Normalized ΔF/F₀) as the baseline. To analyze the differences between the experimental conditions, we determined, for each calycal ROI/KC, the maximum difference from the baseline, as well as the normalized area under the curve.

## Behavioral assays

*Experimental setup.* Larvae were trained and tested on Petri dishes (9 cm inner diameter; Sarstedt) filled either with 1% agarose only (CAS: 9012-36-6, Roth) or with 1% agarose containing 2 mol/L D-fructose (99% purity; CAS: 57-48-7, Roth) as the taste reward (+). Once the contents had solidified, the dishes were covered with their lids and left at 4°C until the experiment started, and for a maximum of 2 weeks.

As the odors, n-amyl acetate (AM; CAS: 628-63-7, Merck) diluted 1:20 in paraffin oil (CAS: 042-47-5, AppliChem) and 1-octanol (OCT, undiluted; CAS: 111-87-5, Sigma-Aldrich) were used. The paraffin oil is without behavioral effect as an odor (Saumweber et al., 2011). Before the experiments, 10 µl of the respective odor was added to odor containers (5 mm inner diameter) covered by perforated lids (5-10 holes of 0.5 mm diameter each). Larvae were collected from their food vial with a brush, briefly rinsed in tap water, and used immediately for behavioral experiments.

Behavioral assays were conducted in a light-shielded, custom-built box, as described by Schleyer et al. (2020). In brief, the box contained a 24 × 12 LED array light table (Solarox) with a 6-mm-thick Plexiglas diffusion panel placed above it, providing constant light conditions and intensity for the activation of light-gated ion channels expressed in neurons of interest (see Genotypes and methods for optophysiology). Larvae in Petri dishes were placed onto the diffusion panel and were surrounded by a translucent polyethylene ring. The ring featured 30 infrared LEDs mounted behind to deliver light (invisible to the animals) allowing behavioral recording for offline tracking analysis (see Video recording and tracking of locomotion).

*Odor-fructose reward association.* A two-group, reciprocal conditioning paradigm was used following standard procedures (Scherer et al., 2003; Neuser et al., 2005; Saumweber et al., 2011; for a detailed manual: Michels et al., 2017). In brief, one group of larvae received the odor presented together with the fructose reward (paired training), whereas a second group received separate presentations of the odor alone and the fructose reward alone (unpaired training).

For paired training, a cohort of ~30 larvae was placed at the center of a Petri dish filled with agarose that was mixed with fructose as the reward (+). Two containers were filled with the odor (AM+) and placed on opposite sides of the Petri dish. The lid was then closed, and the larvae were allowed to move freely for 2.5 min. The larvae were then removed and placed on a fresh, pure-agarose Petri dish in the presence of two empty containers (EM), the lid was closed, and the larvae could again move freely for 2.5 min. This training cycle was performed once only, unless mentioned otherwise. The sequence of training was alternated across replications: that is, for half of the cases, we started with AM as described above (AM+/EM), and for the other half with EM (EM/AM+). After training, the larvae were tested for their odor preference. Specifically, the animals were transferred to the center of a fresh, pure-agarose Petri dish (i.e., without fructose reward, unless mentioned otherwise) featuring one AM container on one side, and one EM container on the other side. After 3 min, the number of larvae on the AM side (#AM), the EM side (#EM), as well as on the middle “neutral” stripe (10 mm), was counted, and the olfactory preference score (PREF) was calculated as follows:

$$ \mathrm{PREF} = \frac{\#AM - \#EM}{\#Total} \qquad (1) $$

Thus, preference scores may range from 1 to -1, with positive values showing preference for AM, and negative values indicating avoidance of

AM. Larvae crawling up onto the lid or onto the odorant containers during the test (<5%) were discarded from the analysis.

For unpaired training, the procedure was the same, except that the odor and the reward were presented separately to the animals. That is, after collection the larvae were placed on a fresh, pure-agarose Petri dish in the presence of two containers both filled with AM. Then, the larvae were transferred onto a fresh agarose Petri dish with fructose added, in the presence of two empty containers (EM+). Again, the training sequence started with AM (AM/EM+) in half of the cases, and in the other half of the cases with EM (EM+/AM). The larvae were then tested for their AM preference, and the olfactory preference score was calculated as for the paired group (Eq. 1).

Associative memory is indicated by a difference in preference for AM after paired training compared with the reciprocal, unpaired training. These differences in AM preference were quantified by the associative memory score as follows:

$$ \text{Memory score} = \frac{\text{PREF (Paired)} - \text{PREF (Unpaired)}}{2} \qquad (2) $$

Thus, memory scores may range from 1 to -1, with positive values indicating appetitive associative memory, and negative values indicating aversive associative memory. These experiments were combined with optogenetic APL activation (see Genotypes and methods for optophysiology).

*Odor-APL association.* Based on early results in this study (see Results), we suspected that optogenetic activation of the APL neuron might have a rewarding effect. Therefore, the associative learning paradigm described above was modified by using optogenetic APL activation (+) instead of a fructose reward (i.e., no real reward was presented). In the paired group, AM was presented together with continuous 2.5 min light stimulation to activate APL, whereas empty containers were subsequently presented in darkness, also for 2.5 min (AM+/EM). In the unpaired group, the larvae were exposed to odor and light separately (AM/EM+). This training cycle was performed once only, with the training sequence alternated across repetitions as described in the preceding section. After training, the larvae were tested on a fresh, pure-agarose Petri dish, and their odor preference as well as the memory score were calculated as detailed above (Eqs. 1, 2).

In addition, a differential two-odor version of the paradigm using APL activation as the reinforcer was used. This was performed as described above, except that, instead of using empty containers (EM), the containers were filled with OCT (undiluted). Thus, differential conditioning followed the logical structure of training being either AM+/OCT or in the reciprocal case AM/OCT+ (again, the training sequence was alternated across repetitions of the experiments). The larvae were then tested for their choice between AM and OCT on a fresh, pure-agarose Petri dish, and the data were analyzed, with due adjustment, as detailed above (Eqs. 1, 2). In this case, positive memory score values thus indicate odor-specific appetitive associative memory, whereas negative memory score values indicate odor-specific aversive associative memory.

Whenever variations on the above paradigms were used, these are mentioned in Results.

*Innate olfactory behavior.* The odor preference of experimentally native larvae was assayed following standard procedures (Saumweber et al., 2011). Cohorts of ~30 animals were transferred onto a pure-agarose plate in the presence of one odor-filled container and another empty container placed on opposite sides of the plate. Odor preference was calculated after 3 min following Equation 1. To probe whether APL activation has an effect on innate olfactory behavior, the test was conducted either without light stimulation, or with light stimulation.

*Genotypes and methods for optophysiology.* For the experiments on APL activation, we used third-instar transgenic larvae expressing either ChR2XXL or Chrimson in APL. To this end, APL-GAL4 was crossed to UAS-ChR2XXL or to UAS-CsChrimson::mVenus as the effector. Double-heterozygous progeny (abbreviated as APL>ChR2XXL or APL>Chrimson) were used for activation of the APL neuron; larvae heterozygous for either the GAL4 element (APL>+) or the UAS element
Aldrich). Specifically, we monitored fluorescence intensity under pulsed 475 nm light as described above for a total of 8 min and after the first 2 min manually bath-applied 45 µl carbamylcholine, dissolved in HL3.1 to a final concentration of 10⁻⁴ M.

We present the fluorescence intensity (ΔF/F₀) across the observation period normalized to the values at its beginning (Normalized ΔF/F₀) as the baseline. To analyze the differences between the experimental conditions, we determined, for each calycal ROI/KC, the maximum difference from the baseline, as well as the normalized area under the curve.

## Behavioral assays

*Experimental setup.* Larvae were trained and tested on Petri dishes (9 cm inner diameter; Sarstedt) filled either with 1% agarose only (CAS: 9012-36-6, Roth) or with 1% agarose containing 2 mol/L D-fructose (99% purity; CAS: 57-48-7, Roth) as the taste reward (+). Once the contents had solidified, the dishes were covered with their lids and left at 4°C until the experiment started, and for a maximum of 2 weeks.

As the odors, n-amyl acetate (AM; CAS: 628-63-7, Merck) diluted 1:20 in paraffin oil (CAS: 042-47-5, AppliChem) and 1-octanol (OCT, undiluted; CAS: 111-87-5, Sigma-Aldrich) were used. The paraffin oil is without behavioral effect as an odor (Saumweber et al., 2011). Before the experiments, 10 µl of the respective odor was added to odor containers (5 mm inner diameter) covered by perforated lids (5-10 holes of 0.5 mm diameter each). Larvae were collected from their food vial with a brush, briefly rinsed in tap water, and used immediately for behavioral experiments.

Behavioral assays were conducted in a light-shielded, custom-built box, as described by Schleyer et al. (2020). In brief, the box contained a 24 × 12 LED array light table (Solarox) with a 6-mm-thick Plexiglas diffusion panel placed above it, providing constant light conditions and intensity for the activation of light-gated ion channels expressed in neurons of interest (see Genotypes and methods for optophysiology). Larvae in Petri dishes were placed onto the diffusion panel and were surrounded by a translucent polyethylene ring. The ring featured 30 infrared LEDs mounted behind to deliver light (invisible to the animals) allowing behavioral recording for offline tracking analysis (see Video recording and tracking of locomotion).

*Odor-fructose reward association.* A two-group, reciprocal conditioning paradigm was used following standard procedures (Scherer et al., 2003; Neuser et al., 2005; Saumweber et al., 2011; for a detailed manual: Michels et al., 2017). In brief, one group of larvae received the odor presented together with the fructose reward (paired training), whereas a second group received separate presentations of the odor alone and the fructose reward alone (unpaired training).

For paired training, a cohort of ~30 larvae was placed at the center of a Petri dish filled with agarose that was mixed with fructose as the reward (+). Two containers were filled with the odor (AM+) and placed on opposite sides of the Petri dish. The lid was then closed, and the larvae were allowed to move freely for 2.5 min. The larvae were then removed and placed on a fresh, pure-agarose Petri dish in the presence of two empty containers (EM), the lid was closed, and the larvae could again move freely for 2.5 min. This training cycle was performed once only, unless mentioned otherwise. The sequence of training was alternated across replications: that is, for half of the cases, we started with AM as described above (AM+/EM), and for the other half with EM (EM/AM+). After training, the larvae were tested for their odor preference. Specifically, the animals were transferred to the center of a fresh, pure-agarose Petri dish (i.e., without fructose reward, unless mentioned otherwise) featuring one AM container on one side, and one EM container on the other side. After 3 min, the number of larvae on the AM side (#AM), the EM side (#EM), as well as on the middle “neutral” stripe (10 mm), was counted, and the olfactory preference score (PREF) was calculated as follows:

$$ \text{PREF} = \frac{\#AM - \#EM}{\#Total} \qquad (1) $$

Thus, preference scores may range from 1 to -1, with positive values showing preference for AM, and negative values indicating avoidance of

AM. Larvae crawling up onto the lid or onto the odorant containers during the test (<5%) were discarded from the analysis.

For unpaired training, the procedure was the same, except that the odor and the reward were presented separately to the animals. That is, after collection the larvae were placed on a fresh, pure-agarose Petri dish in the presence of two containers both filled with AM. Then, the larvae were transferred onto a fresh agarose Petri dish with fructose added, in the presence of two empty containers (EM+). Again, the training sequence started with AM (AM/EM+) in half of the cases, and in the other half of the cases with EM (EM+/AM). The larvae were then tested for their AM preference, and the olfactory preference score was calculated as for the paired group (Eq. 1).

Associative memory is indicated by a difference in preference for AM after paired training compared with the reciprocal, unpaired training. These differences in AM preference were quantified by the associative memory score as follows:

$$ \text{Memory score} = \frac{\text{PREF (Paired)} - \text{PREF (Unpaired)}}{2} \qquad (2) $$

Thus, memory scores may range from 1 to -1, with positive values indicating appetitive associative memory, and negative values indicating aversive associative memory. These experiments were combined with optogenetic APL activation (see Genotypes and methods for optophysiology).

*Odor-APL association.* Based on early results in this study (see Results), we suspected that optogenetic activation of the APL neuron might have a rewarding effect. Therefore, the associative learning paradigm described above was modified by using optogenetic APL activation (+) instead of a fructose reward (i.e., no real reward was presented). In the paired group, AM was presented together with continuous 2.5 min light stimulation to activate APL, whereas empty containers were subsequently presented in darkness, also for 2.5 min (AM+/EM). In the unpaired group, the larvae were exposed to odor and light separately (AM/EM+). This training cycle was performed once only, with the training sequence alternated across repetitions as described in the preceding section. After training, the larvae were tested on a fresh, pure-agarose Petri dish, and their odor preference as well as the memory score were calculated as detailed above (Eqs. 1, 2).

In addition, a differential two-odor version of the paradigm using APL activation as the reinforcer was used. This was performed as described above, except that, instead of using empty containers (EM), the containers were filled with OCT (undiluted). Thus, differential conditioning followed the logical structure of training being either AM+/OCT or in the reciprocal case AM/OCT+ (again, the training sequence was alternated across repetitions of the experiments). The larvae were then tested for their choice between AM and OCT on a fresh, pure-agarose Petri dish, and the data were analyzed, with due adjustment, as detailed above (Eqs. 1, 2). In this case, positive memory score values thus indicate odor-specific appetitive associative memory, whereas negative memory score values indicate odor-specific aversive associative memory.

Whenever variations on the above paradigms were used, these are mentioned in Results.

*Innate olfactory behavior.* The odor preference of experimentally native larvae was assayed following standard procedures (Saumweber et al., 2011). Cohorts of ~30 animals were transferred onto a pure-agarose plate in the presence of one odor-filled container and another empty container placed on opposite sides of the plate. Odor preference was calculated after 3 min following Equation 1. To probe whether APL activation has an effect on innate olfactory behavior, the test was conducted either without light stimulation, or with light stimulation.

*Genotypes and methods for optophysiology.* For the experiments on APL activation, we used third-instar transgenic larvae expressing either ChR2XXL or Chrimson in APL. To this end, APL-GAL4 was crossed to UAS-ChR2XXL or to UAS-CsChrimson::mVenus as the effector. Double-heterozygous progeny (abbreviated as APL>ChR2XXL or APL>Chrimson) were used for activation of the APL neuron; larvae heterozygous for either the GAL4 element (APL>+) or the UAS element
(+>ChR2XXL or +>Chrimson) were used as the driver and the effector genetic control, respectively. To obtain the driver controls, APL-GAL4 was crossed to w¹¹¹⁸ (Bloomington Stock Center #3605, #5905, #6326). As regards the effector controls, a strain lacking the GAL4 domains but containing the two split-GAL4 landing sites (attP40/attP2) was crossed to UAS-ChR2XXL or UAS-CsChrimson: mVenus. For experiments using Crimson, the flies were raised on food supplemented with all-trans retinal (100 mM final concentration; cat: R2500; CAS: 116-31-4, Sigma-Aldrich), unless mentioned otherwise.

For the experiments on MBON activation, UAS-ChR2XXL was crossed to one of the three following drivers: (1) R36G04-GAL4, covering the two calyx MBONs in each hemisphere, plus additional cells in the ventral nerve cord (Bloomington Stock Center #49940; abbreviated as MBONa1,a2-GAL4) (Saumweber et al., 2018); (2) the split-GAL4 line SS02006, covering only one calyx MBON in each hemisphere (kindly provided by M. Zlatic, University of Cambridge; abbreviated as MBONa1-GAL4) (Eschbach et al., 2021); (3) SS01417, covering one, or in some cases both, of the calyx MBONs in each hemisphere (Extended Data Fig. 16-1; kindly provided by M. Zlatic, University of Cambridge; abbreviated as MBONa2-GAL4) (Eschbach et al., 2021). Again, double-heterozygous progeny (abbreviated as MBONa1,a2>ChR2XXL, MBONa1>ChR2XXL or MBONa2>ChR2XXL) were used for activating the calyx MBONs; driver and effector control larvae were obtained as detailed in the preceding paragraph.

For simultaneous activation of APL and ablation of the pPAM neurons, the lexA-lexAop system was used to express the pro-apoptotic reaper gene in the pPAM neurons (effector lexAop-reaper) (Herranz et al., 2014); driver R58E02-lexA (Bloomington Stock Center #52740) (Lyutova et al., 2019), and the GAL4-UAS system was used to express ChR2-XXL in APL (effector UAS-ChR2XXL; Bloomington Stock Center #58374) (Dawydow et al., 2014); driver R55D08-GAL4 (Bloomington Stock Center #39115) (Saumweber et al., 2018). The R55D08-GAL4 driver covers APL plus additional cells outside the mushroom body; it was chosen because the more specific APL-GAL4 driver, as a split-GAL4 strain, could not be combined with the lexA-lexAop system. Using classical genetics, we generated strains that carried a combination of the drivers (R58E02-lexA/R55D08-GAL4) or the effectors (lexAop-reaper/UAS-ChR2XXL) (Lyutova et al., 2019). These strains were crossed, and the progeny obtained (abbreviated as APL₅₅D₀₈>ChR2XXL; pPAM>reaper) were used for the experiment. As regards single effector controls, a strain lacking lexAop-reaper but containing UAS-ChR2XXL was crossed to R58E02-lexA/R55D08-GAL4 (abbreviated as APL₅₅D₀₈>ChR2XXL; pPAM>+). As regards single driver controls, a strain lacking R58E02-lexA but containing R55D08-GAL4 was crossed to lexAop-reaper/UAS-ChR2XXL (abbreviated as APL₅₅D₀₈>ChR2XXL; +>reaper).

The above-mentioned custom-built box (see Experimental setup) was equipped for illumination from a blue LED light table when ChR2XXL was used (wavelength: 470 nm; intensity: 120 µW/cm²; Solarox), or from a red LED light table when Crimson was used (wavelength: 630 nm; intensity: 350 µW/cm²; Solarox).

For silencing experiments, the light-gated chloride channel GtACR1 was used. Specifically, APL-GAL4, R36G04-GAL4, SS02006-GAL4, or SS01417-GAL4 was crossed to UAS-GtACR1::YFP (Bloomington Stock Center #9736; kindly provided by R. Kittel, University of Leipzig) (König et al., 2019). Double-heterozygous progeny (APL>GtACR1, MBONa1, a2>GtACR1, MBONa1>GtACR1 or MBONa2>GtACR1) were used for silencing the respective neurons; driver and effector control larvae were obtained as described in the preceding paragraphs. A green LED light table (wavelength: 520 nm; intensity: 2003 µW/cm²; Solarox) was used for illumination.

In all cases, the timing of illumination is mentioned for each experiment in Results. As all effectors are sensitive to daylight, the breeding of all transgenic animals was performed in darkness, effectuated by black covers wrapped around the food vials. All behavioral experiments were conducted in parallel for the respective experimental group and genetic controls; investigators were blind with respect to genotypes.

**Video recording and tracking of locomotion.** For a subset of experiments, larval behavior was video-recorded throughout the test and

analyzed as described by Paisios et al. (2017). In brief, four behavioral features were analyzed in relation to odor:

First, the olfactory preference (PREF time, in s) was calculated as follows:

$$PREF time = \frac{Time spent on AM side - Time spent on EM side}{Total duration} \quad (3)$$

Thus, preference scores may range from 1 to −1, with positive scores indicating that larvae spent more time on the odor side, and negative values indicating more time spent on the nonodor side, representing approach and avoidance, respectively.

Second, the head cast (HC) rate modulation was calculated as follows:

$$HC rate modulation = \frac{\#HC/s(away from AM) - \#HC/s(toward AM)}{\#HC/s(away from AM) + \#HC/s(toward AM)} \quad (4)$$

Thus, positive scores indicate odor approach; that is, the larvae make more HCs when crawling away from the odor than when crawling toward it. Conversely, negative scores indicate odor avoidance.

Third, the HC reorientation (°) was calculated as follows:

$$HC reorientation = abs(before HC) - abs(after HC) \quad (5)$$

The absolute heading angle (abs) indicates how the larva's head is oriented relative to the odor. For instance, at abs 180° or 0°, the odor is located behind or in front of the animal, respectively. Thus, positive values indicate odor approach; that is, the HC directs the larva toward the odor instead of away from it. Conversely, negative values indicate odor avoidance.

Fourth, the run speed modulation was calculated as follows:

### Run speed modulation

$$= \frac{\text{Run speed toward AM} - \text{Run speed away from AM}}{\text{Run speed toward AM} + \text{Run speed away from AM}} \quad (6)$$

Thus, positive values for run speed modulation indicate that animals slow down whenever they head away from the odor, and speed up whenever they move toward it, indicating approach. Conversely, negative values indicate avoidance.

### Pharmacological manipulation of dopamine synthesis

To test whether the dopaminergic system is implicated in odor-APL associative learning, a systemic pharmacological approach was used to disrupt dopamine synthesis (Neckameyer and White, 1993; Kaun et al., 2011; Thoener et al., 2021). This approach was combined with behavioral experiments using optogenetic APL activation as the reinforcer (see Odor-APL association) and followed the procedures described by Thoener et al. (2021). In brief, a 0.5 mg/ml yeast solution was produced and kept for up to 1 week at 4°C. The dopamine synthesis inhibitor 3-iodo-L-tyrosine (3IY; CAS: 70-78-0, Sigma-Aldrich; concentration: 5 mg/ml) was added to samples of 2 ml yeast solution. In the instances mentioned in Results, the dopamine precursor 3,4-dihydroxyphenylalanine (L-DOPA; CAS: 59-92-7, Sigma-Aldrich; concentration: 10 mg/ml) was added to a yeast solution with or without 3IY. After mixing on a shaker for 1 h, the solutions were transferred into vials containing two pieces of PET mesh. Third-instar progeny of the APL-GAL4 driver crossed to UAS-ChR2XXL (APL>ChR2XXL) were transferred from their food vials to the respective yeast solutions. After a feeding period of 4 h at 25°C and 60%-70% relative humidity, the larvae were briefly washed in water and immediately used in behavioral experiments.

### Connectivity analyses from an EM dataset

A connectivity analysis was performed using the open-source web-based platform CATMAID (Saalfeld et al., 2009; Schneider-Mizell et al., 2016)
(+>ChR2XXL or +>Chrimson) were used as the driver and the effector genetic control, respectively. To obtain the driver controls, APL-GAL4 was crossed to w¹¹¹⁸ (Bloomington Stock Center #3605, #5905, #6326). As regards the effector controls, a strain lacking the GAL4 domains but containing the two split-GAL4 landing sites (attP40/attP2) was crossed to UAS-ChR2XXL or UAS-CsChrimson: mVenus. For experiments using Crimson, the flies were raised on food supplemented with all-trans retinal (100 mM final concentration; cat: R2500; CAS: 116-31-4, Sigma-Aldrich), unless mentioned otherwise.

For the experiments on MBON activation, UAS-ChR2XXL was crossed to one of the three following drivers: (1) R36G04-GAL4, covering the two calyx MBONs in each hemisphere, plus additional cells in the ventral nerve cord (Bloomington Stock Center #49940; abbreviated as MBONa1,a2-GAL4) (Saumweber et al., 2018); (2) the split-GAL4 line SS02006, covering only one calyx MBON in each hemisphere (kindly provided by M. Zlatic, University of Cambridge; abbreviated as MBONa1-GAL4) (Eschbach et al., 2021); (3) SS01417, covering one, or in some cases both, of the calyx MBONs in each hemisphere (Extended Data Fig. 16-1; kindly provided by M. Zlatic, University of Cambridge; abbreviated as MBONa2-GAL4) (Eschbach et al., 2021). Again, double-heterozygous progeny (abbreviated as MBONa1,a2>ChR2XXL, MBONa1>ChR2XXL or MBONa2>ChR2XXL) were used for activating the calyx MBONs; driver and effector control larvae were obtained as detailed in the preceding paragraph.

For simultaneous activation of APL and ablation of the pPAM neurons, the lexA-lexAop system was used to express the pro-apoptotic reaper gene in the pPAM neurons (effector lexAop-reaper) (Herranz et al., 2014); driver R58E02-lexA (Bloomington Stock Center #52740) (Lyutova et al., 2019), and the GAL4-UAS system was used to express ChR2-XXL in APL (effector UAS-ChR2XXL; Bloomington Stock Center #58374) (Dawydow et al., 2014); driver R55D08-GAL4 (Bloomington Stock Center #39115) (Saumweber et al., 2018). The R55D08-GAL4 driver covers APL plus additional cells outside the mushroom body; it was chosen because the more specific APL-GAL4 driver, as a split-GAL4 strain, could not be combined with the lexA-lexAop system. Using classical genetics, we generated strains that carried a combination of the drivers (R58E02-lexA/R55D08-GAL4) or the effectors (lexAop-reaper/UAS-ChR2XXL) (Lyutova et al., 2019). These strains were crossed, and the progeny obtained (abbreviated as APL₅₅D₀₈>ChR2XXL; pPAM>reaper) were used for the experiment. As regards single effector controls, a strain lacking lexAop-reaper but containing UAS-ChR2XXL was crossed to R58E02-lexA/R55D08-GAL4 (abbreviated as APL₅₅D₀₈>ChR2XXL; pPAM>+). As regards single driver controls, a strain lacking R58E02-lexA but containing R55D08-GAL4 was crossed to lexAop-reaper/UAS-ChR2XXL (abbreviated as APL₅₅D₀₈>ChR2XXL; +>reaper).

The above-mentioned custom-built box (see Experimental setup) was equipped for illumination from a blue LED light table when ChR2XXL was used (wavelength: 470 nm; intensity: 120 µW/cm²; Solarox), or from a red LED light table when Crimson was used (wavelength: 630 nm; intensity: 350 µW/cm²; Solarox).

For silencing experiments, the light-gated chloride channel GtACR1 was used. Specifically, APL-GAL4, R36G04-GAL4, SS02006-GAL4, or SS01417-GAL4 was crossed to UAS-GtACR1::YFP (Bloomington Stock Center #9736; kindly provided by R. Kittel, University of Leipzig) (König et al., 2019). Double-heterozygous progeny (APL>GtACR1, MBONa1, a2>GtACR1, MBONa1>GtACR1 or MBONa2>GtACR1) were used for silencing the respective neurons; driver and effector control larvae were obtained as described in the preceding paragraphs. A green LED light table (wavelength: 520 nm; intensity: 2003 µW/cm²; Solarox) was used for illumination.

In all cases, the timing of illumination is mentioned for each experiment in Results. As all effectors are sensitive to daylight, the breeding of all transgenic animals was performed in darkness, effectuated by black covers wrapped around the food vials. All behavioral experiments were conducted in parallel for the respective experimental group and genetic controls; investigators were blind with respect to genotypes.

**Video recording and tracking of locomotion.** For a subset of experiments, larval behavior was video-recorded throughout the test and

analyzed as described by Paisios et al. (2017). In brief, four behavioral features were analyzed in relation to odor:

First, the olfactory preference (PREF time, in s) was calculated as follows:

$$PREF\ time = \frac{Time\ spent\ on\ AM\ side - Time\ spent\ on\ EM\ side}{Total\ duration} \quad (3)$$

Thus, preference scores may range from 1 to -1, with positive scores indicating that larvae spent more time on the odor side, and negative values indicating more time spent on the nonodor side, representing approach and avoidance, respectively.

Second, the head cast (HC) rate modulation was calculated as follows:

$$HC\ rate\ modulation = \frac{\#HC/s(away\ from\ AM) - \#HC/s(toward\ AM)}{\#HC/s(away\ from\ AM) + \#HC/s(toward\ AM)} \quad (4)$$

Thus, positive scores indicate odor approach; that is, the larvae make more HCs when crawling away from the odor than when crawling toward it. Conversely, negative scores indicate odor avoidance.

Third, the HC reorientation (°) was calculated as follows:

$$HC\ reorientation = abs(\text{before HC}) - abs(\text{after HC}) \quad (5)$$

The absolute heading angle (abs) indicates how the larva's head is oriented relative to the odor. For instance, at abs 180° or 0°, the odor is located behind or in front of the animal, respectively. Thus, positive values indicate odor approach; that is, the HC directs the larva toward the odor instead of away from it. Conversely, negative values indicate odor avoidance.

Fourth, the run speed modulation was calculated as follows:

### Run speed modulation

$$= \frac{\text{Run speed toward AM} - \text{Run speed away from AM}}{\text{Run speed toward AM} + \text{Run speed away from AM}} \quad (6)$$

Thus, positive values for run speed modulation indicate that animals slow down whenever they head away from the odor, and speed up whenever they move toward it, indicating approach. Conversely, negative values indicate avoidance.

### Pharmacological manipulation of dopamine synthesis

To test whether the dopaminergic system is implicated in odor-APL associative learning, a systemic pharmacological approach was used to disrupt dopamine synthesis (Neckameyer and White, 1993; Kaun et al., 2011; Thoener et al., 2021). This approach was combined with behavioral experiments using optogenetic APL activation as the reinforcer (see Odor-APL association) and followed the procedures described by Thoener et al. (2021). In brief, a 0.5 mg/ml yeast solution was produced and kept for up to 1 week at 4°C. The dopamine synthesis inhibitor 3-iodo-L-tyrosine (3IY; CAS: 70-78-0, Sigma-Aldrich; concentration: 5 mg/ml) was added to samples of 2 ml yeast solution. In the instances mentioned in Results, the dopamine precursor 3,4-dihydroxyphenylalanine (L-DOPA; CAS: 59-92-7, Sigma-Aldrich; concentration: 10 mg/ml) was added to a yeast solution with or without 3IY. After mixing on a shaker for 1 h, the solutions were transferred into vials containing two pieces of PET mesh. Third-instar progeny of the APL-GAL4 driver crossed to UAS-ChR2XXL (APL>ChR2XXL) were transferred from their food vials to the respective yeast solutions. After a feeding period of 4 h at 25°C and 60%-70% relative humidity, the larvae were briefly washed in water and immediately used in behavioral experiments.

### Connectivity analyses from an EM dataset

A connectivity analysis was performed using the open-source web-based platform CATMAID (Saalfeld et al., 2009; Schneider-Mizell et al., 2016)
<table><caption>Table 1. Table containing the key reagents (fly strains, antibodies, software) used in this study</caption><thead><tr><th>Reagent or resource</th><th>Source or reference</th><th>Identifiers</th><th>Additional information</th></tr></thead><tbody><tr><td>Fly strains</td><td></td><td></td><td></td></tr><tr><td>SS01671-GAL4 (APL-GAL4; split-GAL4 driver covering specifically APL in larvae)</td><td>Saumweber et al., 2018</td><td></td><td></td></tr><tr><td>APLi-GAL4 (intersectional driver covering specifically APL in larvae and adults)</td><td>Lin et al., 2014; Mayseless et al., 2018</td><td></td><td></td></tr><tr><td>R36G04-GAL4 (MBONa1,a2-GAL4; GAL4 driver covering the calyx MBONs, plus additional neurons in the VNC in larvae)</td><td>Saumweber et al., 2018</td><td>BDSC #49940</td><td>Chrs III</td></tr><tr><td>SS02006-GAL4 (MBONa1-GAL4; split-GAL4 driver covering specifically one of the two calyx MBONs in larvae)</td><td>Eschbach et al., 2020; kindly provided by M. Zlatic, University of Cambridge</td><td></td><td></td></tr><tr><td>SS01417-GAL4 (MBONa2-GAL4; split-GAL4 driver covering one, or in some cases both calyx MBONs in larvae)</td><td>Eschbach et al., 2020; kindly provided by M. Zlatic, University of Cambridge</td><td></td><td></td></tr><tr><td>R26G02-GAL4 (GAL4 driver covering APL, plus additional neurons in the VNC)</td><td>Jenett et al., 2012; Saumweber et al., 2018</td><td>BDSC #48065</td><td>Chrs III</td></tr><tr><td>R55D08-GAL4 (GAL4 driver covering APL, plus additional neurons in the VNC)</td><td>Jenett et al., 2012; Saumweber et al., 2018</td><td>BDSC #39115</td><td>Chrs III</td></tr><tr><td>R14H06-LexA (LexA driver covering the KCs in larvae)</td><td>Pfeiffer et al., 2010; lexA Driver Collection of Rubin Laboratory at Janelia Farm</td><td>BDSC #52482</td><td>Chrs II</td></tr><tr><td>R58E02-LexA (LexA driver covering the pPAM neurons in larvae)</td><td>Pfeiffer et al., 2010; lexA Driver Collection of Rubin Laboratory at Janelia Farm</td><td>BDSC #52740</td><td>Chrs II</td></tr><tr><td>UAS-ChR2-XXL (optogenetic effector)</td><td>Dawydow et al., 2014</td><td>BDSC #58374</td><td>Chrs II</td></tr><tr><td>UAS-ChR2-XXL-td::tomato (reporter/optogenetic effector)</td><td>Saumweber et al., 2018</td><td>FlyBase ID: FBtbp0131815</td><td>Chrs II</td></tr><tr><td>UAS-mCherry-CAAX (reporter effector)</td><td>Sens et al., 2010; Kobler et al., 2021</td><td>BDSC #59021</td><td>Chrs II</td></tr><tr><td>UAS-mMFP-T2A-H01 (reporter effector)</td><td>Yu et al., 2015; Kobler et al., 2021</td><td>BDSC #64181</td><td>Chrs III</td></tr><tr><td>UAS-mMFP/MB247-mCherry-CAAX (recombined reporter effector/enhancer>effector)</td><td>Kobler et al., 2021</td><td></td><td></td></tr><tr><td>UAS-mCD8::GFP (reporter effector)</td><td>Lee and Luo, 1999</td><td>BDSC #5137</td><td>Chrs II</td></tr><tr><td>20xUAS-IVS-CsChrimson::mVenus (reporter/optogenetic effector)</td><td>Klapoetke et al., 2014</td><td>BDSC #55136</td><td>Chrs III</td></tr><tr><td>UAS-GtACR1::YFP (reporter/optogenetic effector)</td><td>Kindly provided by R. Kittel, Würzburg; König et al., 2019</td><td>BDSC #9736</td><td>Chrs II</td></tr><tr><td>lexAop-GCaMP6m (reporter effector)</td><td>Chen et al., 2013; Lyutova et al., 2019</td><td>BDSC #44276</td><td>Chrs III</td></tr><tr><td>lexAop-reaper (pro-apoptotic effector)</td><td>Herranz et al., 2014; Lyutova et al., 2019</td><td></td><td>Chrs III</td></tr><tr><td>UAS-Dsyd-1::GFP (presynaptic reporter)</td><td>Owald et al., 2015</td><td></td><td>Chrs III</td></tr><tr><td>UAS-DenMark (postsynaptic reporter)</td><td>Nicolaï et al., 2010</td><td>BDSC #33062</td><td>Chrs II</td></tr><tr><td>UAS-Syt1:SNAP (presynaptic TAG reporter)</td><td>Kohl et al., 2014</td><td>BDSC #58379</td><td>Chrs III</td></tr><tr><td>UAS-TLN:CLIP (postsynaptic TAG reporter)</td><td>Kohl et al., 2014</td><td>BDSC #58382</td><td>Chrs III</td></tr><tr><td>w1118</td><td></td><td>BDSC #3605, #5905, #6326</td><td></td></tr><tr><td>y1w1</td><td></td><td>BDSC #1495</td><td></td></tr><tr><td>attP40/attP2</td><td>Pfeiffer et al., 2010</td><td></td><td></td></tr><tr><td>Antibodies</td><td></td><td></td><td></td></tr><tr><td>Primary monoclonal mouse anti-FASII</td><td>DSHB</td><td>1D4 anti-Fasciclin II; AB_528235</td><td>1:50</td></tr><tr><td>Primary monoclonal mouse anti-ChR2</td><td>ProGen Biotechnik</td><td>610180</td><td>1:100</td></tr><tr><td>Primary polyclonal rabbit anti-GFP</td><td>Invitrogen</td><td>A6455</td><td>1:1000</td></tr><tr><td>Primary polyclonal FITC-conjugated goat anti-GFP</td><td>Abcam</td><td>ab 6662</td><td>1:1000</td></tr><tr><td>Primary monoclonal mouse 4F3 anti-DLG</td><td>Hybridoma</td><td>AB_528203</td><td>1:200</td></tr><tr><td>Primary polyclonal rabbit anti-DsRed</td><td>Clontech</td><td>632496</td><td>1:200</td></tr><tr><td>Primary monoclonal rat anti-N-Cadherin</td><td>Hybridoma</td><td>DN-Ex #8-s</td><td>1:50</td></tr><tr><td>Primary polyclonal rabbit anti-GABA</td><td>Sigma-Aldrich</td><td>A2052</td><td>1:500</td></tr><tr><td>Primary polyclonal chicken anti-GFP</td><td>Aves Labs</td><td>AB_10000240</td><td>1:500</td></tr><tr><td>Secondary polyclonal goat anti-rabbit AlexaFluor-488</td><td>Invitrogen</td><td>A11008</td><td>1:500</td></tr><tr><td>Secondary polyclonal goat anti-mouse AlexaFluor-568</td><td>Invitrogen</td><td>A10037</td><td>1:500</td></tr><tr><td>Secondary polyclonal goat anti-rat AlexaFluor-647</td><td>Jackson ImmunoResearch Laboratories</td><td>712-605-153</td><td>1:500</td></tr><tr><td>Secondary polyclonal donkey anti-mouse Cy3</td><td>Jackson ImmunoResearch Laboratories</td><td>715-165-150</td><td>1:300</td></tr><tr><td>Secondary polyclonal goat anti-mouse AlexaFluor-488</td><td>Invitrogen</td><td>A11001</td><td>1:200</td></tr><tr><td>Secondary polyclonal goat anti-rabbit Cy5</td><td>Invitrogen</td><td>A10523</td><td>1:200</td></tr><tr><td>Secondary polyclonal goat anti-rat Cy3</td><td>Invitrogen</td><td>A10522</td><td>1:200</td></tr><tr><td>Secondary polyclonal FITC-conjugated goat anti-chicken</td><td>Invitrogen</td><td>A16055</td><td>1:300</td></tr><tr><td>Chemical TAG ligands (chemical substrates)</td><td></td><td></td><td></td></tr><tr><td>SNAP-tag ligands (SNAP surface 549 - BG 549)</td><td>NEB</td><td>S9112S</td><td></td></tr><tr><td>CLIP-tag ligands (CLIP surface 647 - BC 647)</td><td>NEB</td><td>S9234S</td><td></td></tr><tr><td>Odors</td><td></td><td></td><td></td></tr><tr><td>AM</td><td>Merck</td><td>628-63-7</td><td></td></tr><tr><td>OCT</td><td>Sigma-Aldrich</td><td>111-87-5</td><td></td></tr><tr><td>Drugs</td><td></td><td></td><td></td></tr><tr><td>Carbamylcholine</td><td>Sigma-Aldrich</td><td>51-83-2</td><td></td></tr></tbody></table>

(Table continues)
<table><caption>Table 1. Table containing the key reagents (fly strains, antibodies, software) used in this study</caption><thead><tr><th>Reagent or resource</th><th>Source or reference</th><th>Identifiers</th><th>Additional information</th></tr></thead><tbody><tr><td>Fly strains</td><td></td><td></td><td></td></tr><tr><td>SS01671-GAL4 (APL-GAL4; split-GAL4 driver covering specifically APL in larvae)</td><td>Saumweber et al., 2018</td><td></td><td></td></tr><tr><td>APLi-GAL4 (intersectional driver covering specifically APL in larvae and adults)</td><td>Lin et al., 2014; Mayseless et al., 2018</td><td></td><td></td></tr><tr><td>R36G04-GAL4 (MBONa1,a2-GAL4; GAL4 driver covering the calyx MBONs, plus additional neurons in the VNC in larvae)</td><td>Saumweber et al., 2018</td><td>BDSC #49940</td><td>Chrs III</td></tr><tr><td>SS02006-GAL4 (MBONa1-GAL4; split-GAL4 driver covering specifically one of the two calyx MBONs in larvae)</td><td>Eschbach et al., 2020; kindly provided by M. Zlatic, University of Cambridge</td><td></td><td></td></tr><tr><td>SS01417-GAL4 (MBONa2-GAL4; split-GAL4 driver covering one, or in some cases both calyx MBONs in larvae)</td><td>Eschbach et al., 2020; kindly provided by M. Zlatic, University of Cambridge</td><td></td><td></td></tr><tr><td>R26G02-GAL4 (GAL4 driver covering APL, plus additional neurons in the VNC)</td><td>Jenett et al., 2012; Saumweber et al., 2018</td><td>BDSC #48065</td><td>Chrs III</td></tr><tr><td>R55D08-GAL4 (GAL4 driver covering APL, plus additional neurons in the VNC)</td><td>Jenett et al., 2012; Saumweber et al., 2018</td><td>BDSC #39115</td><td>Chrs III</td></tr><tr><td>R14H06-LexA (LexA driver covering the KCs in larvae)</td><td>Pfeiffer et al., 2010; lexA Driver Collection of Rubin Laboratory at Janelia Farm</td><td>BDSC #52482</td><td>Chrs II</td></tr><tr><td>R58E02-LexA (LexA driver covering the pPAM neurons in larvae)</td><td>Pfeiffer et al., 2010; lexA Driver Collection of Rubin Laboratory at Janelia Farm</td><td>BDSC #52740</td><td>Chrs II</td></tr><tr><td>UAS-ChR2-XXL (optogenetic effector)</td><td>Dawydow et al., 2014</td><td>BDSC #58374</td><td>Chrs II</td></tr><tr><td>UAS-ChR2-XXL-td::tomato (reporter/optogenetic effector)</td><td>Saumweber et al., 2018</td><td>FlyBase ID: FBtbp0131815</td><td>Chrs II</td></tr><tr><td>UAS-mCherry-CAAX (reporter effector)</td><td>Sens et al., 2010; Kobler et al., 2021</td><td>BDSC #59021</td><td>Chrs II</td></tr><tr><td>UAS-mIFP-T2A-H01 (reporter effector)</td><td>Yu et al., 2015; Kobler et al., 2021</td><td>BDSC #64181</td><td>Chrs III</td></tr><tr><td>UAS-mIFP/MB247-mCherry-CAAX (recombined reporter effector/enhancer effector)</td><td>Kobler et al., 2021</td><td></td><td></td></tr><tr><td>UAS-mCD8::GFP (reporter effector)</td><td>Lee and Luo, 1999</td><td>BDSC #5137</td><td>Chrs II</td></tr><tr><td>20xUAS-IVS-CsChrimson::mVenus (reporter/optogenetic effector)</td><td>Klapoetke et al., 2014</td><td>BDSC #55136</td><td>Chrs III</td></tr><tr><td>UAS-GtACR1::YFP (reporter/optogenetic effector)</td><td>Kindly provided by R. Kittel, Würzburg; König et al., 2019</td><td>BDSC #9736</td><td>Chrs II</td></tr><tr><td>lexAop-GCaMP6m (reporter effector)</td><td>Chen et al., 2013; Lyutova et al., 2019</td><td>BDSC #44276</td><td>Chrs III</td></tr><tr><td>lexAop-reaper (pro-apoptotic effector)</td><td>Herranz et al., 2014; Lyutova et al., 2019</td><td></td><td>Chrs III</td></tr><tr><td>UAS-Dsyd-1::GFP (presynaptic reporter)</td><td>Owald et al., 2015</td><td></td><td>Chrs III</td></tr><tr><td>UAS-DenMark (postsynaptic reporter)</td><td>Nicolaï et al., 2010</td><td>BDSC #33062</td><td>Chrs II</td></tr><tr><td>UAS-Syt1:SNAP (presynaptic TAG reporter)</td><td>Kohl et al., 2014</td><td>BDSC #58379</td><td>Chrs III</td></tr><tr><td>UAS-TLN:CLIP (postsynaptic TAG reporter)</td><td>Kohl et al., 2014</td><td>BDSC #58382</td><td>Chrs III</td></tr><tr><td>w1118</td><td></td><td>BDSC #3605, #5905, #6326</td><td></td></tr><tr><td>y1w1</td><td></td><td>BDSC #1495</td><td></td></tr><tr><td>attP40/attP2</td><td>Pfeiffer et al., 2010</td><td></td><td></td></tr><tr><td>Antibodies</td><td></td><td></td><td></td></tr><tr><td>Primary monoclonal mouse anti-FASII</td><td>DSHB</td><td>1D4 anti-Fasciclin II; AB_528235</td><td>1:50</td></tr><tr><td>Primary monoclonal mouse anti-ChR2</td><td>ProGen Biotechnik</td><td>610180</td><td>1:100</td></tr><tr><td>Primary polyclonal rabbit anti-GFP</td><td>Invitrogen</td><td>A6455</td><td>1:1000</td></tr><tr><td>Primary polyclonal FITC-conjugated goat anti-GFP</td><td>Abcam</td><td>ab 6662</td><td>1:1000</td></tr><tr><td>Primary monoclonal mouse 4F3 anti-DLG</td><td>Hybridoma</td><td>AB_528203</td><td>1:200</td></tr><tr><td>Primary polyclonal rabbit anti-DsRed</td><td>Clontech</td><td>632496</td><td>1:200</td></tr><tr><td>Primary monoclonal rat anti-N-Cadherin</td><td>Hybridoma</td><td>DN-Ex #8-s</td><td>1:50</td></tr><tr><td>Primary polyclonal rabbit anti-GABA</td><td>Sigma-Aldrich</td><td>A2052</td><td>1:500</td></tr><tr><td>Primary polyclonal chicken anti-GFP</td><td>Aves Labs</td><td>AB_10000240</td><td>1:500</td></tr><tr><td>Secondary polyclonal goat anti-rabbit AlexaFluor-488</td><td>Invitrogen</td><td>A11008</td><td>1:500</td></tr><tr><td>Secondary polyclonal goat anti-mouse AlexaFluor-568</td><td>Invitrogen</td><td>A10037</td><td>1:500</td></tr><tr><td>Secondary polyclonal goat anti-rat AlexaFluor-647</td><td>Jackson ImmunoResearch Laboratories</td><td>712-605-153</td><td>1:500</td></tr><tr><td>Secondary polyclonal donkey anti-mouse Cy3</td><td>Jackson ImmunoResearch Laboratories</td><td>715-165-150</td><td>1:300</td></tr><tr><td>Secondary polyclonal goat anti-mouse AlexaFluor-488</td><td>Invitrogen</td><td>A11001</td><td>1:200</td></tr><tr><td>Secondary polyclonal goat anti-rabbit Cy5</td><td>Invitrogen</td><td>A10523</td><td>1:200</td></tr><tr><td>Secondary polyclonal goat anti-rat Cy3</td><td>Invitrogen</td><td>A10522</td><td>1:200</td></tr><tr><td>Secondary polyclonal FITC-conjugated goat anti-chicken</td><td>Invitrogen</td><td>A16055</td><td>1:300</td></tr><tr><td>Chemical TAG ligands (chemical substrates)</td><td></td><td></td><td></td></tr><tr><td>SNAP-tag ligands (SNAP surface 549 - BG 549)</td><td>NEB</td><td>S9112S</td><td></td></tr><tr><td>CLIP-tag ligands (CLIP surface 647 - BC 647)</td><td>NEB</td><td>S9234S</td><td></td></tr><tr><td>Odors</td><td></td><td></td><td></td></tr><tr><td>AM</td><td>Merck</td><td>628-63-7</td><td></td></tr><tr><td>OCT</td><td>Sigma-Aldrich</td><td>111-87-5</td><td></td></tr><tr><td>Drugs</td><td></td><td></td><td></td></tr><tr><td>Carbamylcholine</td><td>Sigma-Aldrich</td><td>51-83-2</td><td></td></tr></tbody></table>

(Table continues)
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Reagent or resource</th><th>Source or reference</th><th>Identifiers</th><th>Additional information</th></tr></thead><tbody><tr><td>3IY</td><td>Sigma-Aldrich</td><td>70-78-0</td><td></td></tr><tr><td>L-DOPA</td><td>Sigma-Aldrich</td><td>59-92-7</td><td></td></tr><tr><td>Software</td><td></td><td></td><td></td></tr><tr><td>Fiji ImageJ 1.53c</td><td>National Institutes of Health</td><td>SCR_002285</td><td></td></tr><tr><td>CATMAID</td><td>Saalfeld et al., 2009; Schneider-Mizell et al., 2016</td><td></td><td></td></tr><tr><td>Imaris 9.72, 9.8</td><td>Oxford Instruments</td><td>SCR_007370</td><td></td></tr><tr><td>ImSpector 7.1.4</td><td>Miltenyi Biotec</td><td></td><td></td></tr><tr><td>R 3.3.2</td><td>Development Core Team 2016</td><td></td><td></td></tr><tr><td>Statistika 13</td><td>StatSoft</td><td>SCR_014213</td><td></td></tr><tr><td>Corel Draw 2019</td><td>Corel</td><td>SCR_013674</td><td></td></tr><tr><td>GraphPad Prism 6</td><td>GraphPad Software</td><td>SCR_002798</td><td></td></tr><tr><td>Adobe Premiere Pro 2020, version 14.9.0</td><td>Adobe</td><td></td><td></td></tr></tbody></table>

on published EM reconstruction data (Eichler et al., 2017; Eschbach et al., 2021). We used the fractions of inputs (FI) as a measure of the connectomic impact of an upstream neuron on its downstream partner (Ohyama et al., 2015; Schneider-Mizell et al., 2016). To analyze the connections that APL makes, via the KCs, with DANs/OANs/MBINs, we determined these fractions for each APL>KC>DAN/OAN/MBIN connection. For an indirect connection between, for example, neurons A and C via neuron B, the activations from A to B and from B to C were considered as independent; the impact of A on C can therefore be defined as the product of two probabilities (FI). Thus, we determine the total impact of A on C via B as follows:

$$iA > C = FI(B, A) * FI(C, B) \quad (7)$$

We computed the impact of the APLs of each hemisphere for each KC that is connected both to one of the APLs and any one of the DANs/OANs/MBINs (KCn) as follows:

$$i(KC_n)_{APL>DAN/OAN/MBIN} = FI(APL > KC_n) \times FI(KC_n > DAN/OAN/MBIN) \quad (8)$$

We then summed the impact of the respective APL, via the KCs, on each DAN/OAN/MBIN as follows:

$$I(KC_{total})_{APL>DAN/OAN/MBIN} = \sum_n i(KC_n)_{APL>DAN/OAN/MBIN} \quad (9)$$

Finally, the impact of the APLs of both hemispheres on DANs/OANs/MBINs of the mushroom body calyx and compartments were summed to compare the impact of APL between compartments and between the medial lobe and vertical lobe systems.

### Experimental design and statistical analyses

The source data are included in Extended Data Figure 3-1; the results of all statistical tests, performed in Statistica 13 unless mentioned otherwise (SCR_014213, StatSoft), are included in Table 2. Graphs, figures, and sketches were generated with Statistica 13, Corel Draw 2019 (SCR_013674, Corel), and GraphPad Prism 6 (SCR_002798, GraphPad Software); references are documented in Table 1.

To compare the calyx and compartmental coverage between the APL neuron of each hemisphere (see Fig. 3E), a Pearson correlation was performed; the data are displayed as a scatter plot.

The experiments in Figure 5 followed a two-group design with two genotypes (APL<sub>26G02</sub>>ChR2XXL; KC>GCaMP6m as the experimental genotype and +>ChR2XXL; KC>GCaMP6m as the genetic control). We analyzed changes in fluorescence intensity and plotted mean maximal ΔF/F0 (see Fig. 5B,E,H) and the area under the curve for each experiment after normalization (see Fig. 5C,F,I).

To compare the radii of the neurite, dendrite, and axon of APL in both hemispheres (see Fig. 6D), Kruskal-Wallis (KW) tests and Mann-Whitney-Wilcoxon (MWW) tests (total of three MWW tests per APL) were used for multiple and two-group comparisons, respectively (R Core Team, 2016). The Bonferroni-Holm (BH) correction was applied to maintain an error rate<5% (Holm, 1979). The data are displayed as violin plots, with bars showing the mean.

For the experiments in Figures 7, 9-19 and Extended Data Figure 7-4, the data are displayed as box plots, the middle line indicates the median, the box boundaries represent the 25% and 75% quantiles, and the whiskers represent the 10% and 90% quantiles; outliers are not displayed.

To compare the geodesic distances of synapses to the center of their respective center-surround structure on the left-hemisphere APL neuron (see Fig. 7D), we performed an MWW test between KC-to-APL and APL-to-KC synapses. Corresponding analyses for the right-hemisphere APL neuron can be found in Extended Data Figure 7-4.

For the behavioral results displayed in Figures 9-18, KW and MWW tests were used for multiple and two-group comparisons, respectively. For comparisons to chance levels (i.e., to zero), one-sample sign tests (OSS; corresponding to binom.test in R version 3.3.2) (R Core Team, 2016) were used. The BH correction was applied to maintain an error rate<5%. Sample sizes (biological replications) were chosen based on previous studies that had revealed moderate to mild effect sizes (Paisios et al., 2017; Saumweber et al., 2018) and are indicated in the figures. A sample size of N=1 included ~30 animals of both sexes for each reciprocally trained group, and ~30 animals of both sexes for all innate preference experiments. All behavioral experiments were performed in parallel for the respective experimental group and genetic controls; experimenters were blind to genotypes.

The experiments in Figures 9A, B, 12A, C, G, 15A, 16A, B, and 18 followed a three-group design with three genotypes (experimental genotype expressing ChR2XXL or GtACR1 or ChR2XXL/reaper, plus effector and driver controls). In case of significance, a KW test across all groups was followed by pairwise MWW tests between genotypes (three MWW tests in total). Comparisons to chance levels (i.e., to zero) were tested for each group by OSS tests; in Figure 16A, B, no OSS tests were performed.

The experiments in Figures 9D, F-I and 15E followed a two-group design with two test conditions (the presence or absence of light) for the experimental genotype expressing ChR2XXL (see Fig. 9D,F-I) or Chrimson (see Fig. 15E). MWW tests were performed between the two test conditions; no OSS tests were performed.

The experiment in Figure 10 followed a six-group design with two test conditions (the presence or absence of the fructose reward) for the experimental genotype expressing ChR2XXL. The animals received paired or unpaired odor-fructose training, either not activating APL during training at all, or activating APL during odor presentation, or in the absence of odor. A KW test across all groups was followed by pairwise MWW tests between groups within the same test condition, as well as between test conditions
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Reagent or resource</th><th>Source or reference</th><th>Identifiers</th><th>Additional information</th></tr></thead><tbody><tr><td>3IY</td><td>Sigma-Aldrich</td><td>70-78-0</td><td></td></tr><tr><td>L-DOPA</td><td>Sigma-Aldrich</td><td>59-92-7</td><td></td></tr><tr><td>Software</td><td></td><td></td><td></td></tr><tr><td>Fiji ImageJ 1.53c</td><td>National Institutes of Health</td><td>SCR_002285</td><td></td></tr><tr><td>CATMAID</td><td>Saalfeld et al., 2009; Schneider-Mizell et al., 2016</td><td></td><td></td></tr><tr><td>Imaris 9.72, 9.8</td><td>Oxford Instruments</td><td>SCR_007370</td><td></td></tr><tr><td>ImSpector 7.1.4</td><td>Miltenyi Biotec</td><td></td><td></td></tr><tr><td>R 3.3.2</td><td>Development Core Team 2016</td><td></td><td></td></tr><tr><td>Statistika 13</td><td>StatSoft</td><td>SCR_014213</td><td></td></tr><tr><td>Corel Draw 2019</td><td>Corel</td><td>SCR_013674</td><td></td></tr><tr><td>GraphPad Prism 6</td><td>GraphPad Software</td><td>SCR_002798</td><td></td></tr><tr><td>Adobe Premiere Pro 2020, version 14.9.0</td><td>Adobe</td><td></td><td></td></tr></tbody></table>

on published EM reconstruction data (Eichler et al., 2017; Eschbach et al., 2021). We used the fractions of inputs (FI) as a measure of the connectomic impact of an upstream neuron on its downstream partner (Ohyama et al., 2015; Schneider-Mizell et al., 2016). To analyze the connections that APL makes, via the KCs, with DANs/OANs/MBINs, we determined these fractions for each APL>KC>DAN/OAN/MBIN connection. For an indirect connection between, for example, neurons A and C via neuron B, the activations from A to B and from B to C were considered as independent; the impact of A on C can therefore be defined as the product of two probabilities (FI). Thus, we determine the total impact of A on C via B as follows:

$$iA > C = FI(B, A) * FI(C, B) \quad (7)$$

We computed the impact of the APLs of each hemisphere for each KC that is connected both to one of the APLs and any one of the DANs/OANs/MBINs (KCn) as follows:

$$i(KC_n)_{APL>DAN/OAN/MBIN} = FI(APL > KC_n) * FI(KC_n > DAN/OAN/MBIN) \quad (8)$$

We then summed the impact of the respective APL, via the KCs, on each DAN/OAN/MBIN as follows:

$$I(KC_{total})_{APL>DAN/OAN/MBIN} = \sum_n i(KC_n)_{APL>DAN/OAN/MBIN} \quad (9)$$

Finally, the impact of the APLs of both hemispheres on DANs/OANs/MBINs of the mushroom body calyx and compartments were summed to compare the impact of APL between compartments and between the medial lobe and vertical lobe systems.

### Experimental design and statistical analyses

The source data are included in Extended Data Figure 3-1; the results of all statistical tests, performed in Statistica 13 unless mentioned otherwise (SCR_014213, StatSoft), are included in Table 2. Graphs, figures, and sketches were generated with Statistica 13, Corel Draw 2019 (SCR_013674, Corel), and GraphPad Prism 6 (SCR_002798, GraphPad Software); references are documented in Table 1.

To compare the calyx and compartmental coverage between the APL neuron of each hemisphere (see Fig. 3E), a Pearson correlation was performed; the data are displayed as a scatter plot.

The experiments in Figure 5 followed a two-group design with two genotypes (APL<sub>26G02</sub>>ChR2XXL; KC>GCaMP6m as the experimental genotype and +>ChR2XXL; KC>GCaMP6m as the genetic control). We analyzed changes in fluorescence intensity and plotted mean maximal ΔF/F0 (see Fig. 5B,E,H) and the area under the curve for each experiment after normalization (see Fig. 5C,F,I).

To compare the radii of the neurite, dendrite, and axon of APL in both hemispheres (see Fig. 6D), Kruskal-Wallis (KW) tests and Mann-Whitney-Wilcoxon (MWW) tests (total of three MWW tests per APL) were used for multiple and two-group comparisons, respectively (R Core Team, 2016). The Bonferroni-Holm (BH) correction was applied to maintain an error rate<5% (Holm, 1979). The data are displayed as violin plots, with bars showing the mean.

For the experiments in Figures 7, 9-19 and Extended Data Figure 7-4, the data are displayed as box plots, the middle line indicates the median, the box boundaries represent the 25% and 75% quantiles, and the whiskers represent the 10% and 90% quantiles; outliers are not displayed.

To compare the geodesic distances of synapses to the center of their respective center-surround structure on the left-hemisphere APL neuron (see Fig. 7D), we performed an MWW test between KC-to-APL and APL-to-KC synapses. Corresponding analyses for the right-hemisphere APL neuron can be found in Extended Data Figure 7-4.

For the behavioral results displayed in Figures 9-18, KW and MWW tests were used for multiple and two-group comparisons, respectively. For comparisons to chance levels (i.e., to zero), one-sample sign tests (OSS; corresponding to binom.test in R version 3.3.2) (R Core Team, 2016) were used. The BH correction was applied to maintain an error rate<5%. Sample sizes (biological replications) were chosen based on previous studies that had revealed moderate to mild effect sizes (Paisios et al., 2017; Saumweber et al., 2018) and are indicated in the figures. A sample size of N=1 included ~30 animals of both sexes for each reciprocally trained group, and ~30 animals of both sexes for all innate preference experiments. All behavioral experiments were performed in parallel for the respective experimental group and genetic controls; experimenters were blind to genotypes.

The experiments in Figures 9A, B, 12A, C, G, 15A, 16A, B, and 18 followed a three-group design with three genotypes (experimental genotype expressing ChR2XXL or GtACR1 or ChR2XXL/reaper, plus effector and driver controls). In case of significance, a KW test across all groups was followed by pairwise MWW tests between genotypes (three MWW tests in total). Comparisons to chance levels (i.e., to zero) were tested for each group by OSS tests; in Figure 16A, B, no OSS tests were performed.

The experiments in Figures 9D, F-I and 15E followed a two-group design with two test conditions (the presence or absence of light) for the experimental genotype expressing ChR2XXL (see Fig. 9D,F-I) or Chrimson (see Fig. 15E). MWW tests were performed between the two test conditions; no OSS tests were performed.

The experiment in Figure 10 followed a six-group design with two test conditions (the presence or absence of the fructose reward) for the experimental genotype expressing ChR2XXL. The animals received paired or unpaired odor-fructose training, either not activating APL during training at all, or activating APL during odor presentation, or in the absence of odor. A KW test across all groups was followed by pairwise MWW tests between groups within the same test condition, as well as between test conditions
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Brain expression of the APL-GAL4 driver is restricted to the APL neuron. **A–A''**, 3D view of the expression pattern from the APL-GAL4 driver in a third-instar larval brain visualized using the fluorescence signal from the UAS-ChR2XXL::tdtomato effector (APL>ChR2XXL::tdtomato; green). Axon-rich regions of the mushroom body peduncle and lobes can be discerned as references after labeling with a primary monoclonal mouse anti-FASII antibody and a secondary polyclonal goat anti-mouse AlexaFluor-488 antibody (anti-FASII; magenta). Transgene expression is specific to the hemispherically unique APL neuron. The data were acquired with a 20× glycerol objective. Scale bar and grid spacing, 50 µm. **B–B''**, Same as in **A–A''**, providing a close-up view of the mushroom bodies. APL sends projections into the calyx and a subset of the compartments of the medial and vertical lobes. **B''**, White arrowheads point to the calyx, which is innervated by APL but is largely devoid of the axonal FASII marker. The data were acquired with a 63× glycerol objective. Scale bar and grid spacing, 20 µm. **C–D''**, Same as in **B–B''**, except that the APL-GAL4 driver was crossed to UAS-mCD8::GFP as the effector. APL membranes can be visualized after labeling with a primary polyclonal rabbit anti-GFP antibody and a secondary polyclonal goat anti-rabbit AlexaFluor-488 antibody (anti-GFP; green). The mushroom bodies are labeled by a primary monoclonal mouse anti-DLG antibody and a secondary polyclonal goat anti-mouse AlexaFluor-568 antibody (anti-DLG; magenta); neuropils can be discerned as a reference by a primary monoclonal rat anti-N-Cadherin antibody and a secondary polyclonal goat anti-rat AlexaFluor-647 antibody (anti-N-Cadherin; blue). Close-up analysis of the APL morphology revealed two, or in one case three, branches (white arrowheads in **C** and **D**, respectively) splitting from the primary neurite; notably, these numbers of branches do not differ between the two hemispheres (N = 11 brains). The data were acquired with a 16× glycerol objective. Scale bar and grid spacing, 20 µm. **E**, For each mushroom body calyx and compartment, the mean pixel intensities of APL labeling in the right hemisphere versus the left hemisphere are plotted (color code in accordance with the mushroom body schematic). The observed correlation indicates no interhemispheric difference in APL morphology. The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 3.** Brain expression of the APL-GAL4 driver is restricted to the APL neuron. **A–A''**, 3D view of the expression pattern from the APL-GAL4 driver in a third-instar larval brain visualized using the fluorescence signal from the UAS-ChR2XXL::tdtomato effector (APL>ChR2XXL::tdtomato; green). Axon-rich regions of the mushroom body peduncle and lobes can be discerned as references after labeling with a primary monoclonal mouse anti-FASII antibody and a secondary polyclonal goat anti-mouse AlexaFluor-488 antibody (anti-FASII; magenta). Transgene expression is specific to the hemispherically unique APL neuron. The data were acquired with a 20× glycerol objective. Scale bar and grid spacing, 50 µm. **B–B''**, Same as in **A–A''**, providing a close-up view of the mushroom bodies. APL sends projections into the calyx and a subset of the compartments of the medial and vertical lobes. **B''**, White arrowheads point to the calyx, which is innervated by APL but is largely devoid of the axonal FASII marker. The data were acquired with a 63× glycerol objective. Scale bar and grid spacing, 20 µm. **C–D''**, Same as in **B–B''**, except that the APL-GAL4 driver was crossed to UAS-mCD8::GFP as the effector. APL membranes can be visualized after labeling with a primary polyclonal rabbit anti-GFP antibody and a secondary polyclonal goat anti-rabbit AlexaFluor-488 antibody (anti-GFP; green). The mushroom bodies are labeled by a primary monoclonal mouse anti-DLG antibody and a secondary polyclonal goat anti-mouse AlexaFluor-568 antibody (anti-DLG; magenta); neuropils can be discerned as a reference by a primary monoclonal rat anti-N-Cadherin antibody and a secondary polyclonal goat anti-rat AlexaFluor-647 antibody (anti-N-Cadherin; blue). Close-up analysis of the APL morphology revealed two, or in one case three, branches (white arrowheads in **C** and **D**, respectively) splitting from the primary neurite; notably, these numbers of branches do not differ between the two hemispheres (N = 11 brains). The data were acquired with a 16× glycerol objective. Scale bar and grid spacing, 20 µm. **E**, For each mushroom body calyx and compartment, the mean pixel intensities of APL labeling in the right hemisphere versus the left hemisphere are plotted (color code in accordance with the mushroom body schematic). The observed correlation indicates no interhemispheric difference in APL morphology. The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 4.** The larval APL neuron is GABAergic and is presynaptic in the calyx and postsynaptic in both the calyx and the lobes. A–A", 3D view of the expression pattern from the APL-GAL4 driver in the third-instar larval brain visualized using the fluorescence signal from the Chrimson effector (APL>CsChrimson::mVenus; green). GABAergic signals can be visualized after labeling with a polyclonal rabbit anti-GABA antibody and a polyclonal Cy5-conjugated goat anti-rabbit antibody (anti-GABA; magenta). A", White arrowheads point to an overlap of the GABA signal and the fluorescence signal in the APL soma. Neuropil regions are visualized as a reference by using a primary monoclonal rat anti-N-Cadherin antibody and a secondary polyclonal goat anti-rat Cy3 antibody (anti-N-Cadherin; blue). The data were acquired with a 63× glycerol objective. Scale bar and grid spacing, 20 µm. B–B", Same as in A–A", providing a close-up view of the APL soma. B', White arrowhead points to the APL soma surrounded by additional GABAergic cells. The data were acquired with a 63× glycerol objective. Scale bar and grid spacing, 5 µm. C–C", The APL-GAL4 driver was crossed to a double effector with both UAS-Dsyd-1::GFP and UAS-DenMark to label the presynaptic and postsynaptic sites of the APL neuron in third-instar larvae. Presynaptic regions of APL can be visualized after labeling with a polyclonal FITC-conjugated goat anti-GFP antibody (anti-Dsyd-1::GFP; green). Postsynaptic regions are revealed after labeling with a primary polyclonal rabbit anti-DsRed antibody and a secondary polyclonal goat anti-rabbit Cy5 antibody (anti-DenMark; magenta). Neuropil regions are visualized as a reference by using a primary monoclonal rat anti-N-Cadherin antibody and a secondary polyclonal goat anti-rat Cy3 antibody (anti-N-Cadherin; blue). The presynaptic marker Dsyd-1 is mainly restricted to the calyx, whereas the postsynaptic marker DenMark localizes to both the calyx and a subset of the compartments in the lobes, confirming the regional synaptic polarities of the larval APL neuron (Masuda-Nakagawa et al., 2014; Eichler et al., 2017). The data were acquired with a 16× glycerol objective. Scale bar and grid spacing, 50 µm. D–D", Same as in C–C", providing a close-up view of the presynaptic and postsynaptic regions of APL. Scale bar and grid spacing, 25 µm. For a corresponding movie, see Movie 3.</figcaption></figure>

for a given kind of training regimen (nine MWW tests in total). Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests.

The preference scores shown in Figure 11A–C underlie the associative memory scores in Figure 10. The experiment followed a four-group design with two test conditions (the presence or absence of the fructose reward) for the experimental genotype expressing ChR2XXL. A KW test across all groups was followed by pairwise MWW tests between groups

that had received paired or unpaired odor-fructose training within the same test condition; in addition, MWW tests were performed between the respectively trained groups that were tested in the absence of fructose and the baseline odor preference scores (four MWW tests in total). Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests.

Figure 11D shows pooled preferences from Figure 11A–C and follows a three-group design for the experimental genotype expressing
<figure><img src="image_9.png" /><figcaption>**Figure 4.** The larval APL neuron is GABAergic and is presynaptic in the calyx and postsynaptic in both the calyx and the lobes. A–A", 3D view of the expression pattern from the APL-GAL4 driver in the third-instar larval brain visualized using the fluorescence signal from the Chrimson effector (APL>CsChrimson::mVenus; green). GABAergic signals can be visualized after labeling with a polyclonal rabbit anti-GABA antibody and a polyclonal Cy5-conjugated goat anti-rabbit antibody (anti-GABA; magenta). A", White arrowheads point to an overlap of the GABA signal and the fluorescence signal in the APL soma. Neuropil regions are visualized as a reference by using a primary monoclonal rat anti-N-Cadherin antibody and a secondary polyclonal goat anti-rat Cy3 antibody (anti-N-Cadherin; blue). The data were acquired with a 63× glycerol objective. Scale bar and grid spacing, 20 µm. B–B", Same as in A–A", providing a close-up view of the APL soma. B', White arrowhead points to the APL soma surrounded by additional GABAergic cells. The data were acquired with a 63× glycerol objective. Scale bar and grid spacing, 5 µm. C–C", The APL-GAL4 driver was crossed to a double effector with both UAS-Dsyd-1::GFP and UAS-DenMark to label the presynaptic and postsynaptic sites of the APL neuron in third-instar larvae. Presynaptic regions of APL can be visualized after labeling with a polyclonal FITC-conjugated goat anti-GFP antibody (anti-Dsyd-1::GFP; green). Postsynaptic regions are revealed after labeling with a primary polyclonal rabbit anti-DsRed antibody and a secondary polyclonal goat anti-rabbit Cy5 antibody (anti-DenMark; magenta). Neuropil regions are visualized as a reference by using a primary monoclonal rat anti-N-Cadherin antibody and a secondary polyclonal goat anti-rat Cy3 antibody (anti-N-Cadherin; blue). The presynaptic marker Dsyd-1 is mainly restricted to the calyx, whereas the postsynaptic marker DenMark localizes to both the calyx and a subset of the compartments in the lobes, confirming the regional synaptic polarities of the larval APL neuron (Masuda-Nakagawa et al., 2014; Eichler et al., 2017). The data were acquired with a 16× glycerol objective. Scale bar and grid spacing, 50 µm. D–D", Same as in C–C", providing a close-up view of the presynaptic and postsynaptic regions of APL. Scale bar and grid spacing, 25 µm. For a corresponding movie, see Movie 3.</figcaption></figure>

for a given kind of training regimen (nine MWW tests in total). Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests.

The preference scores shown in Figure 11A–C underlie the associative memory scores in Figure 10. The experiment followed a four-group design with two test conditions (the presence or absence of the fructose reward) for the experimental genotype expressing ChR2XXL. A KW test across all groups was followed by pairwise MWW tests between groups

that had received paired or unpaired odor-fructose training within the same test condition; in addition, MWW tests were performed between the respectively trained groups that were tested in the absence of fructose and the baseline odor preference scores (four MWW tests in total). Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests.

Figure 11D shows pooled preferences from Figure 11A–C and follows a three-group design for the experimental genotype expressing
<figure><img src="image_8.png" /><figcaption>**Figure 5.** Optogenetic activation of APL can reduce levels of activity in mushroom body KCs. **A–C,** At relatively low light intensity (from 0 to 270 s), activation of APL in isolated brain preparations of APL<sub>26G02</sub> > ChR2XXL; KC > GCaMP6m larvae (blue) had no major effect on intracellular $Ca^{2+}$ signals in the calycal ROIs/KCs (Normalized $\Delta F/F_0$) relative to genetic controls (+ > ChR2XXL; KC > GCaMP6m) (gray). For each calycal ROI/KC, the data are normalized to the beginning of the indicated observation period as a baseline and are plotted over time in **A** (showing mean ± SEM). The maximum difference from the baseline for each calycal ROI/KC is plotted in **B** (showing mean and data-point scatter). The area under the curve is shown in **C** (showing mean and data-point scatter). **D–F,** In contrast, compared with genetic controls, activation of APL by a more intense light in the same specimen (from 300 to 570 s) reduced $Ca^{2+}$ signals in the KCs. **G–I,** Using the same light intensity as in **D–F,** bath-application of the acetylcholine receptor agonist carbamylcholine (at 120 s) massively increased $Ca^{2+}$ signals in genetic controls (gray), an effect that was reduced to about half under conditions of APL activation (blue). Preparation and imaging according to Selcho et al. (2017) and Lyutova et al. (2019). The number of brains for genetic controls and APL activation, respectively, was 7 and 9 in **A–F** and 10 and 8 in **G–I;** the number of calycal ROIs/KCs was 27 and 26 in **A–C**, 26 and 25 in **D–F**, and 36 and 37 in **G–I**. MWW comparisons: NS, *p* > 0.05; \* *p* < 0.05. The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2.</figcaption></figure>

ChR2XXL. A KW test across all groups was followed by pairwise MWW tests between groups (three MWW tests in total); no OSS tests were performed.

The preference scores shown in Figures 12B, D and 15B underlie the associative memory scores in Figure 12A, C and Figure 15A, respectively. The experiment followed a three-group design for the experimental genotype expressing ChR2XXL (see Fig. 12B), GtACR1 (see Fig. 12C), or Chrimson (see Fig. 15B) and their respective genetic controls. After a KW test across all groups, MWW tests were performed between groups that had received paired or unpaired odor-APL activation training and the baseline odor preference scores (four MWW tests in total).

The experiment in Figure 12E followed a four-group design for the experimental genotype expressing ChR2XXL. After a KW test across all groups, MWW tests were performed between the group tested immediately after training (retention interval 0 min) and the groups tested 5, 10, or 20 min after training (three MWW tests in total). Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests.

The experiment in Figure 12F followed a nine-group design for the experimental genotype expressing ChR2XXL. A KW test was performed across all groups; no OSS tests were performed.

Figure 13B describes the odor preferences of larvae of the experimental genotype expressing ChR2XXL tested after paired or unpaired training, over time. No statistical analyses were performed; rather, the data were collated over time and were statistically compared between the two training groups in Figure 13C (see next paragraph).

The experiments in Figures 13C–F, 14C, 15C, D, and 17C followed a two-group design for the experimental genotype expressing ChR2XXL (see Figs. 13C–F, 14C, 17C) or Chrimson (see Fig. 15C,D). MWW tests were performed between the two groups. Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests (in Fig. 13C–F, no OSS tests were performed).

The experiments in Figure 14A, B, E, G followed a six-group design with three genotypes (experimental genotype expressing ChR2XXL, effector, and driver controls) and two test conditions (the presence or absence of light). A KW test across all the groups was performed first. In case of significance, pairwise MWW tests were performed between genotypes within the same test condition, as well as between test conditions for a given genotype (nine MWW tests in total). In Figure 14A, B, differences from chance levels (i.e., from zero) were tested for in each group by OSS tests; in Figure 14E, G, no OSS tests were performed.
<figure><img src="image_10.png" /><figcaption>**Figure 5.** Optogenetic activation of APL can reduce levels of activity in mushroom body KCs. **A–C,** At relatively low light intensity (from 0 to 270 s), activation of APL in isolated brain preparations of APL<sub>26G02</sub> > ChR2XXL; KC > GCaMP6m larvae (blue) had no major effect on intracellular $Ca^{2+}$ signals in the calycal ROIs/KCs (Normalized $\Delta F/F_0$) relative to genetic controls (+ > ChR2XXL; KC > GCaMP6m) (gray). For each calycal ROI/KC, the data are normalized to the beginning of the indicated observation period as a baseline and are plotted over time in **A** (showing mean ± SEM). The maximum difference from the baseline for each calycal ROI/KC is plotted in **B** (showing mean and data-point scatter). The area under the curve is shown in **C** (showing mean and data-point scatter). **D–F,** In contrast, compared with genetic controls, activation of APL by a more intense light in the same specimen (from 300 to 570 s) reduced $Ca^{2+}$ signals in the KCs. **G–I,** Using the same light intensity as in **D–F,** bath-application of the acetylcholine receptor agonist carbamylcholine (at 120 s) massively increased $Ca^{2+}$ signals in genetic controls (gray), an effect that was reduced to about half under conditions of APL activation (blue). Preparation and imaging according to Selcho et al. (2017) and Lyutova et al. (2019). The number of brains for genetic controls and APL activation, respectively, was 7 and 9 in **A–F** and 10 and 8 in **G–I;** the number of calycal ROIs/KCs was 27 and 26 in **A–C**, 26 and 25 in **D–F**, and 36 and 37 in **G–I**. MWW comparisons: NS, *p* > 0.05; \* *p* < 0.05. The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2.</figcaption></figure>

ChR2XXL. A KW test across all groups was followed by pairwise MWW tests between groups (three MWW tests in total); no OSS tests were performed.

The preference scores shown in Figures 12B, D and 15B underlie the associative memory scores in Figure 12A, C and Figure 15A, respectively. The experiment followed a three-group design for the experimental genotype expressing ChR2XXL (see Fig. 12B), GtACR1 (see Fig. 12C), or Chrimson (see Fig. 15B) and their respective genetic controls. After a KW test across all groups, MWW tests were performed between groups that had received paired or unpaired odor-APL activation training and the baseline odor preference scores (four MWW tests in total).

The experiment in Figure 12E followed a four-group design for the experimental genotype expressing ChR2XXL. After a KW test across all groups, MWW tests were performed between the group tested immediately after training (retention interval 0 min) and the groups tested 5, 10, or 20 min after training (three MWW tests in total). Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests.

The experiment in Figure 12F followed a nine-group design for the experimental genotype expressing ChR2XXL. A KW test was performed across all groups; no OSS tests were performed.

Figure 13B describes the odor preferences of larvae of the experimental genotype expressing ChR2XXL tested after paired or unpaired training, over time. No statistical analyses were performed; rather, the data were collated over time and were statistically compared between the two training groups in Figure 13C (see next paragraph).

The experiments in Figures 13C–F, 14C, 15C, D, and 17C followed a two-group design for the experimental genotype expressing ChR2XXL (see Figs. 13C–F, 14C, 17C) or Chrimson (see Fig. 15C,D). MWW tests were performed between the two groups. Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests (in Fig. 13C–F, no OSS tests were performed).

The experiments in Figure 14A, B, E, G followed a six-group design with three genotypes (experimental genotype expressing ChR2XXL, effector, and driver controls) and two test conditions (the presence or absence of light). A KW test across all the groups was performed first. In case of significance, pairwise MWW tests were performed between genotypes within the same test condition, as well as between test conditions for a given genotype (nine MWW tests in total). In Figure 14A, B, differences from chance levels (i.e., from zero) were tested for in each group by OSS tests; in Figure 14E, G, no OSS tests were performed.
<figure><img src="image_9.png" /><figcaption>**Figure 6.** Volume reconstruction of the larval APL neuron. A, Electron microscopy cross-section of the APL neuron in a first-instar larva. Points connected by lines represent the skeletonized reconstruction of the neuron (for details, see Eichler et al., 2017). Circles represent radius annotations for volume reconstruction. Scale bar, 500 nm. B, Reconstructed volume of the left- and the right-hemisphere APL neuron (green) in the context of the complete CNS (left; gray mesh), and in a close-up of the mushroom body region (right; magenta). For a corresponding movie, see Movie 4. C, Reconstructed volume of both APL neurons separated into axonal (yellow) and dendritic (red) regions, and the neurite and its branches (green). D, Quantification of the radii of the APL neurons, showing that the neurite is thicker than the axonal regions (significant only for the right-hemisphere APL neuron), which in turn are thicker than the dendritic regions. The data are displayed as violin plots. Bars represent mean. MWW comparisons between the APL regions with a BH correction: NS, *p* > 0.05; \* *p* < 0.05. The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2. E, F, Dots and triangles represent presynaptic and postsynaptic sites, respectively, selectively for different types of connected neuron, namely: (E) single-claw, multidaw, and young KCs; and (F) neurons with connections in the calyx (top row: olfactory PNs; OAN-a1/a2; MBON-a1/a2), as well as neurons that have been studied elsewhere in functional experiments, such as DAN-i1 (Saumweber et al., 2018; Schleyer et al., 2020), DAN-f1 (Eschbach et al., 2020; Weiglein et al., 2021), and DAN-k1 (Saumweber et al., 2018). Neurons with less than two synapses with APL in both hemispheres are shown as “Other.” B, C, A, Anterior; D, dorsal; M, medial. Corresponding 3D visualizations can be found in Movies 4, 5, 6.</figcaption></figure>

The experiments in Figure 16C, D followed a four-group design with two test conditions (the presence or absence of light) for the experimental genotype expressing GtACR1 (see Fig. 16C) or ChR2XXL (see Fig. 16D). A KW test was performed across all groups; no OSS tests were performed.

The experiments in Figures 14D and 17D followed a four-group design for the experimental genotype expressing ChR2XXL. In Figure 14D, a KW test across all groups was followed by MWW tests performed between the group of larvae trained/tested with AM and (1) the group
<figure><img src="image_11.png" /><figcaption>**Figure 6.** Volume reconstruction of the larval APL neuron. A, Electron microscopy cross-section of the APL neuron in a first-instar larva. Points connected by lines represent the skeletonized reconstruction of the neuron (for details, see Eichler et al., 2017). Circles represent radius annotations for volume reconstruction. Scale bar, 500 nm. B, Reconstructed volume of the left- and the right-hemisphere APL neuron (green) in the context of the complete CNS (left; gray mesh), and in a close-up of the mushroom body region (right; magenta). For a corresponding movie, see Movie 4. C, Reconstructed volume of both APL neurons separated into axonal (yellow) and dendritic (red) regions, and the neurite and its branches (green). D, Quantification of the radii of the APL neurons, showing that the neurite is thicker than the axonal regions (significant only for the right-hemisphere APL neuron), which in turn are thicker than the dendritic regions. The data are displayed as violin plots. Bars represent mean. MWW comparisons between the APL regions with a BH correction: NS, *p* > 0.05; \* *p* < 0.05. The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2. E, F, Dots and triangles represent presynaptic and postsynaptic sites, respectively, selectively for different types of connected neuron, namely: (E) single-claw, multidaw, and young KCs; and (F) neurons with connections in the calyx (top row: olfactory PNs; OAN-a1/a2; MBON-a1/a2), as well as neurons that have been studied elsewhere in functional experiments, such as DAN-i1 (Saumweber et al., 2018; Schleyer et al., 2020), DAN-f1 (Eschbach et al., 2020; Weiglein et al., 2021), and DAN-k1 (Saumweber et al., 2018). Neurons with less than two synapses with APL in both hemispheres are shown as “Other.” B, C, A, Anterior; D, dorsal; M, medial. Corresponding 3D visualizations can be found in Movies 4, 5, 6.</figcaption></figure>

The experiments in Figure 16C, D followed a four-group design with two test conditions (the presence or absence of light) for the experimental genotype expressing GtACR1 (see Fig. 16C) or ChR2XXL (see Fig. 16D). A KW test was performed across all groups; no OSS tests were performed.

The experiments in Figures 14D and 17D followed a four-group design for the experimental genotype expressing ChR2XXL. In Figure 14D, a KW test across all groups was followed by MWW tests performed between the group of larvae trained/tested with AM and (1) the group
<figure><img src="image_10.png" /><figcaption>**Movie 4.** Volume reconstruction of the left- and the right-hemisphere APL neuron (green) in a first-instar larval brain (CNS; gray), with the focus on APL's connectivity within the mushroom bodies (MB; magenta). “X” indicates synapses with any type of partner. Yellow spheres and red pyramids represent the location of presynaptic and postsynaptic sites of both APLs, respectively. A, Anterior; D, dorsal; M, medial. Based on the dataset from Eichler et al. (2017). See also Figure 6B. [View online]</figcaption></figure>

trained with AM/tested with OCT and (2) the group trained/tested with OCT; as well as between the group trained/tested with OCT and the group trained with OCT/tested with AM (three MWW tests in total). Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests. In Figure 17D, a KW test across all groups was followed by MWW tests performed between the untreated group and (1) the group fed 3IY, (2) the group fed 3IY plus L-DOPA, and (3) the group fed L-DOPA; as well as between the group fed 3IY and the group fed 3IY plus L-DOPA (four MWW tests in total). Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests.

# Results

## Organization of the APL neuron

We first investigated the expression pattern of the APL-GAL4 driver in third-instar larvae in the full-body context. This took advantage of a combination of autofluorescence signals with fluorescence from UAS-mCherry-CAAX as the effector construct. These signals can be detected conveniently under a light-sheet microscope on clearing the sample (Kobler et al., 2021). Fluorescence signals that can be observed across wavelengths and in both the experimental genotype (APL>mCherry-CAAX) and the genetic controls (+>mCherry-CAAX and APL>+ as the effector and the driver control, respectively) constitute autofluorescence and allow rich anatomic detail to be discerned (Fig. 2). We note that signals from fluorescent food particles contribute to individually variable signals along the alimentary canal (Kobler et al., 2021). Fluorescence reflecting the expression of mCherry-CAAX, however, was reproducibly seen specifically in a giant pair of cells in the mushroom body region (Fig. 2B''', D''', F''') that can be identified as APL (Movie 1). Thus, to the extent tested here and in the absence of a standard neuroanatomy against which full-body preparations can be systematically registered at high resolution, the effects observed using the APL-GAL4 driver are interpreted without reference to transgene expression elsewhere in the body.

Combining the APL-GAL4 driver with the UAS-ChR2XXL::tdtomato effector and using the resulting fluorescence signal confirm that within the CNS and the mushroom body APL-GAL4

<figure><img src="image_11.png" /><figcaption>**Movie 5.** Volume reconstruction of the left- and the right-hemisphere APL neuron in a first-instar larva and sites of presynapses and postsynapses with different subclasses of mushroom body intrinsic neurons (KCs), namely, single-claw, multidaw, and young KCs. Spheres and pyramids represent presynaptic and postsynaptic sites of APL, respectively. A, Anterior; D, dorsal; M, medial. Based on the dataset from Eichler et al. (2017). See also Figure 6E. [View online]</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Movie 6.** Volume reconstruction of the left- and the right-hemisphere APL neuron in a first-instar larva and sites of presynapses and postsynapses with different types of partner, separated into all subclasses of mushroom body intrinsic neurons (KCs) and the mushroom body extrinsic neurons indicated. Remaining neurons bearing less than two synapses with APL in both hemispheres are shown as “Other”; “X” indicates synapses with non-KCs. Spheres and pyramids represent presynaptic and postsynaptic sites, respectively. A, Anterior; D, dorsal; M, medial. Based on the dataset from Eichler et al. (2017). See also Figure 6F. [View online]</figcaption></figure>

specifically expresses in the APL neuron (Fig. 3A-B'') (Saumweber et al., 2018). In addition, APL-GAL4 was crossed to UAS-mIFP/MB247>mCherry-CAAX (Kobler et al., 2021) to label APL together with the mushroom body (Movie 2). In both cases, our results confirm that the primary neurite of APL splits to send projections separately into the calyx and the lobes of the mushroom bodies (Fig. 3B''; Movie 2) (Masuda-Nakagawa et al., 2014; Mayseless et al., 2018; Saumweber et al., 2018). This morphology was seen in 10 of 11 preparations of third-instar larval brains with the APL-driver and UAS-mCD8::GFP as the effector (Fig. 3C-C''). Only in one preparation did the primary neurite split into three branches in both hemispheres (Fig. 3D-D''). This made us wonder whether there is any variability in APL projections to the calyx or different lobe compartments, in particular, in the third-instar larvae
<figure><img src="image_12.png" /><figcaption>**Movie 4.** Volume reconstruction of the left- and the right-hemisphere APL neuron (green) in a first-instar larval brain (CNS; gray), with the focus on APL's connectivity within the mushroom bodies (MB; magenta). “X” indicates synapses with any type of partner. Yellow spheres and red pyramids represent the location of presynaptic and postsynaptic sites of both APLs, respectively. A, Anterior; D, dorsal; M, medial. Based on the dataset from Eichler et al. (2017). See also Figure 6B. [View online]</figcaption></figure>

trained with AM/tested with OCT and (2) the group trained/tested with OCT; as well as between the group trained/tested with OCT and the group trained with OCT/tested with AM (three MWW tests in total). Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests. In Figure 17D, a KW test across all groups was followed by MWW tests performed between the untreated group and (1) the group fed 3IY, (2) the group fed 3IY plus L-DOPA, and (3) the group fed L-DOPA; as well as between the group fed 3IY and the group fed 3IY plus L-DOPA (four MWW tests in total). Differences from chance levels (i.e., from zero) were tested for in each group by OSS tests.

# Results

## Organization of the APL neuron

We first investigated the expression pattern of the APL-GAL4 driver in third-instar larvae in the full-body context. This took advantage of a combination of autofluorescence signals with fluorescence from UAS-mCherry-CAAX as the effector construct. These signals can be detected conveniently under a light-sheet microscope on clearing the sample (Kobler et al., 2021). Fluorescence signals that can be observed across wavelengths and in both the experimental genotype (APL>mCherry-CAAX) and the genetic controls (+>mCherry-CAAX and APL>+ as the effector and the driver control, respectively) constitute autofluorescence and allow rich anatomic detail to be discerned (Fig. 2). We note that signals from fluorescent food particles contribute to individually variable signals along the alimentary canal (Kobler et al., 2021). Fluorescence reflecting the expression of mCherry-CAAX, however, was reproducibly seen specifically in a giant pair of cells in the mushroom body region (Fig. 2B''', D''', F''') that can be identified as APL (Movie 1). Thus, to the extent tested here and in the absence of a standard neuroanatomy against which full-body preparations can be systematically registered at high resolution, the effects observed using the APL-GAL4 driver are interpreted without reference to transgene expression elsewhere in the body.

Combining the APL-GAL4 driver with the UAS-ChR2XXL::tdtomato effector and using the resulting fluorescence signal confirm that within the CNS and the mushroom body APL-GAL4

<figure><img src="image_13.png" /><figcaption>**Movie 5.** Volume reconstruction of the left- and the right-hemisphere APL neuron in a first-instar larva and sites of presynapses and postsynapses with different subclasses of mushroom body intrinsic neurons (KCs), namely, single-claw, multidaw, and young KCs. Spheres and pyramids represent presynaptic and postsynaptic sites of APL, respectively. A, Anterior; D, dorsal; M, medial. Based on the dataset from Eichler et al. (2017). See also Figure 6E. [View online]</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Movie 6.** Volume reconstruction of the left- and the right-hemisphere APL neuron in a first-instar larva and sites of presynapses and postsynapses with different types of partner, separated into all subclasses of mushroom body intrinsic neurons (KCs) and the mushroom body extrinsic neurons indicated. Remaining neurons bearing less than two synapses with APL in both hemispheres are shown as “Other”; “X” indicates synapses with non-KCs. Spheres and pyramids represent presynaptic and postsynaptic sites, respectively. A, Anterior; D, dorsal; M, medial. Based on the dataset from Eichler et al. (2017). See also Figure 6F. [View online]</figcaption></figure>

specifically expresses in the APL neuron (Fig. 3A-B'') (Saumweber et al., 2018). In addition, APL-GAL4 was crossed to UAS-mIFP/MB247>mCherry-CAAX (Kobler et al., 2021) to label APL together with the mushroom body (Movie 2). In both cases, our results confirm that the primary neurite of APL splits to send projections separately into the calyx and the lobes of the mushroom bodies (Fig. 3B''; Movie 2) (Masuda-Nakagawa et al., 2014; Mayseless et al., 2018; Saumweber et al., 2018). This morphology was seen in 10 of 11 preparations of third-instar larval brains with the APL-driver and UAS-mCD8::GFP as the effector (Fig. 3C-C''). Only in one preparation did the primary neurite split into three branches in both hemispheres (Fig. 3D-D''). This made us wonder whether there is any variability in APL projections to the calyx or different lobe compartments, in particular, in the third-instar larvae
<figure><img src="image_13.png" /><figcaption>**Figure 7.** Dendrogram analysis of the larval APL neuron. A, Two-dimensional dendrogram of the APL neuron from the left hemisphere, based on an electron microscope reconstruction in a first-instar larva (data from Eichler et al., 2017). Branch lengths are preserved in a topologically correct manner. Colored envelopes represent the mushroom body calyx and compartments innervated by APL (see also Fig. 1B,C). High-resolution versions of this figure, for the APL neurons of both hemispheres, can be found in Extended Data Figure 7-1. B, Synapses at their topologically correct site on the left-hemisphere APL neuron with the mushroom body extrinsic neurons indicated. Dots and triangles represent presynaptic and postsynaptic sites of APL, respectively. For better readability, some symbols were displaced and their true locations indicated by a dashed line. High-resolution versions of this figure, for the APL neurons of both hemispheres, can be found in Extended Data Figure 7-2. C, Same as in B, but showing synaptic sites with the mushroom body intrinsic neurons, the KCs. Dark purple dots and bright purple triangles represent APL-to-KC and KC-to-APL synapses, respectively. High-resolution versions of this figure, for the APL neurons of both hemispheres, can be found in Extended Data Figure 7-3. D, Cluster analysis revealed that calycal synaptic sites of the left-hemisphere APL with the KCs are organized in four clusters (1-4). The accompanying quantification shows geodesic distances of synapses to the center of their respective center-surround structure on the left-hemisphere APL neuron. Most of the APL-to-KC synapses (dark purple dots) are observed toward the center of these clusters (dark square), whereas KC-to-APL synapses (bright purple triangles) are observed mainly in the surround. The data are displayed as box plots. Middle line indicates the median. Box boundaries represent the 25% and 75% quantiles. Whiskers represent the 10% and 90% quantiles. The sample sizes (number of synapses) are given within the figure. MWW comparisons between APL-to-KC and KC-to-APL synapses: *p*< 0.05. The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2. Corresponding analyses, in the context of the full dendrograms and for the APL neurons of both hemispheres, can be found in Extended Data Figure 7-4.</figcaption></figure>

that we intended to use later in our behavioral analyses. Across five
specimens of third-instar larval brains with the APL-driver and
UAS-mCD8::GFP as the effector, coverage of the calyx and of the
compartments was similar between the APL neuron of each hemi-
sphere (Fig. 3E). GFP signals were consistently strong in the calyx,
close to absent in the two peduncle compartments, weak in the
upper vertical lobe and the shaft of the medial lobe, and moderate
to strong in the other compartments (Fig. 3E). Taking the present
data together with previously published data, we conclude that the
larval APL innervates the calyx and 6 of the 10 compartments,
namely, the lateral appendix, the upper, intermediate, and lower
vertical lobe, as well as the upper and lower toe (Figs. 1C, 3)

(Masuda-Nakagawa et al., 2014; Eichler et al., 2017; Saumweber
et al., 2018).

We next confirmed that APL is GABAergic (Fig. 4A–B")
(Masuda-Nakagawa et al., 2014) and studied the regional organi-
zation of presynaptic and postsynaptic sites of APL using the
APL-GAL4 driver together with the double effector UAS-Dsyd-
1::GFP/UAS-DenMark (Owald et al., 2015). Our results show
that APL is presynaptic in the calyx, whereas it is postsynaptic in
both the calyx and the lobes (Fig. 4C–D"; Movie 3), confirming
earlier reports (Masuda-Nakagawa et al., 2014; Eichler et al.,
2017). Indeed, we also confirm a functionally inhibitory connec-
tion from APL to the KCs in the calyx. Combining transgene
<figure><img src="image_15.png" /><figcaption>**Figure 7.** Dendrogram analysis of the larval APL neuron. A, Two-dimensional dendrogram of the APL neuron from the left hemisphere, based on an electron microscope reconstruction in a first-instar larva (data from Eichler et al., 2017). Branch lengths are preserved in a topologically correct manner. Colored envelopes represent the mushroom body calyx and compartments innervated by APL (see also Fig. 1B,C). High-resolution versions of this figure, for the APL neurons of both hemispheres, can be found in Extended Data Figure 7-1. B, Synapses at their topologically correct site on the left-hemisphere APL neuron with the mushroom body extrinsic neurons indicated. Dots and triangles represent presynaptic and postsynaptic sites of APL, respectively. For better readability, some symbols were displaced and their true locations indicated by a dashed line. High-resolution versions of this figure, for the APL neurons of both hemispheres, can be found in Extended Data Figure 7-2. C, Same as in B, but showing synaptic sites with the mushroom body intrinsic neurons, the KCs. Dark purple dots and bright purple triangles represent APL-to-KC and KC-to-APL synapses, respectively. High-resolution versions of this figure, for the APL neurons of both hemispheres, can be found in Extended Data Figure 7-3. D, Cluster analysis revealed that calycal synaptic sites of the left-hemisphere APL with the KCs are organized in four clusters (1-4). The accompanying quantification shows geodesic distances of synapses to the center of their respective center-surround structure on the left-hemisphere APL neuron. Most of the APL-to-KC synapses (dark purple dots) are observed toward the center of these clusters (dark square), whereas KC-to-APL synapses (bright purple triangles) are observed mainly in the surround. The data are displayed as box plots. Middle line indicates the median. Box boundaries represent the 25% and 75% quantiles. Whiskers represent the 10% and 90% quantiles. The sample sizes (number of synapses) are given within the figure. MWW comparisons between APL-to-KC and KC-to-APL synapses: \*p\*< 0.05. The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2. Corresponding analyses, in the context of the full dendrograms and for the APL neurons of both hemispheres, can be found in Extended Data Figure 7-4.</figcaption></figure>

that we intended to use later in our behavioral analyses. Across five
specimens of third-instar larval brains with the APL-driver and
UAS-mCD8::GFP as the effector, coverage of the calyx and of the
compartments was similar between the APL neuron of each hemi-
sphere (Fig. 3E). GFP signals were consistently strong in the calyx,
close to absent in the two peduncle compartments, weak in the
upper vertical lobe and the shaft of the medial lobe, and moderate
to strong in the other compartments (Fig. 3E). Taking the present
data together with previously published data, we conclude that the
larval APL innervates the calyx and 6 of the 10 compartments,
namely, the lateral appendix, the upper, intermediate, and lower
vertical lobe, as well as the upper and lower toe (Figs. 1C, 3)

(Masuda-Nakagawa et al., 2014; Eichler et al., 2017; Saumweber
et al., 2018).

We next confirmed that APL is GABAergic (Fig. 4A–B")
(Masuda-Nakagawa et al., 2014) and studied the regional organi-
zation of presynaptic and postsynaptic sites of APL using the
APL-GAL4 driver together with the double effector UAS-Dsyd-
1::GFP/UAS-DenMark (Owald et al., 2015). Our results show
that APL is presynaptic in the calyx, whereas it is postsynaptic in
both the calyx and the lobes (Fig. 4C–D"; Movie 3), confirming
earlier reports (Masuda-Nakagawa et al., 2014; Eichler et al.,
2017). Indeed, we also confirm a functionally inhibitory connec-
tion from APL to the KCs in the calyx. Combining transgene
<figure><img src="image_16.png" /><figcaption>**Figure 8.** Regional synaptic polarity of APL across metamorphosis. Confocal maximum projection images of stainings for mCD8::GFP, Syt1::SNAP, and TLN::CLIP (Kohl et al., 2014), driven by the APL-specific intersectional driver APLi (Lin et al., 2014; Mayseless et al., 2018) at the following developmental times: (A–A'') third-instar larva (L3); (B–C'') 6 h after puparium formation (6 h APF: calyx: B–B''; lobes: C–C'''); (D–D'') 12 h APF; (E–F'') adult (calyx: E–E''; lobes: F–F'''). Brains were stained with a polyclonal chicken anti-GFP antibody to label the APL neuron (A–F). To label presynapses (A'–F') and postsynapses (A''–F''), the presynaptic reporter synaptotagmin was fused to the chemical tag SNAPm (Syt1-SNAPm), and the postsynaptic reporter telencephalin was fused to CLIPm (TLN-CLIPm), respectively (Kohl et al., 2014). Merged images are shown in A''–F''. In the third-instar larva, presynaptic staining was largely restricted to the calyx (A'), whereas postsynaptic staining was distributed in both the calyx and the lobes (A''). At 6 h APF, both presynaptic and postsynaptic staining are similarly distributed to that in the larvae (B'–C''); notably, presynaptic structures seem to be more punctate (B',C'), and fewer postsynaptic structures are detectable (B'',C''). As late as 12 h APF, both presynaptic and postsynaptic structures are still detectable (D',D''); postsynaptic structures appear to be detached from the neurite (D'',D'', yellow arrowhead). In adults, both presynaptic and postsynaptic markers are detectable in both the calyx and the lobes (E–F''). The data were acquired with a 40× oil objective. Scale bars, 20 µm.</figcaption></figure>

expression by the GAL4-UAS and the lexA-lexAop systems, we monitored changes in intracellular Ca²⁺ through changes in fluorescence intensity in the KCs of isolated brain preparations of controls (+>ChR2XXL; KC>GCaMP6m) and on optogenetic activation of APL (APL26G02>ChR2XXL; KC>GCaMP6m). At the relatively lower blue-light intensity (80 ms pulses every 2 s, at 1.8 mW/cm²), the control brains showed no specific response to the light; rather, fluorescence intensity decreased slightly across

the recording period (Fig. 5A, gray). Activation of APL resulted in a trend for yet further decreased levels of fluorescence (Fig. 5A–C, blue). We then exploited the observation that for increased light intensity (4.1 mW/cm²) fluorescence intensity increased under control conditions (Fig. 5D, gray), reflecting the light responsiveness of the KCs themselves or of neurons upstream of the KCs. Relative to these fluorescence signals observed in the controls, signals were reduced by APL activation (Fig. 5E,F).
<figure><img src="image_14.png" /><figcaption>**Figure 9.** Memory scores are abolished on activation of APL throughout training. A, Larvae were trained such that, in one group of animals, the odor AM (black cloud) was paired with the fructose reward (green fill of circle indicating a Petri dish), alternating with blank trials (open circle), whereas in a reciprocal group, the odor was presented unpaired from the fructose reward; here and throughout this study, the sequence of training events was as depicted in half of the cases, and in the reverse order in the other half of the cases. The APL neuron was optogenetically activated with blue light illumination (blue rectangle) during the complete training phase. The larvae from both groups were then tested for their odor preference, and associative memory was quantified by the memory score as the difference in preference between these reciprocally trained groups of animals. Double-heterozygous animals of the genotype APL>ChR2XXL were used for APL activation; larvae heterozygous for either the GAL4 (APL>+) or the effector (+>ChR2XXL) were used as the genetic controls. Optogenetic activation of the APL neuron during the complete training phase abolished associative memory scores. B, The same effects were observed in a shortened, one-training-cycle version of this experiment. C, Full projection of the expression pattern from the APL-GAL4 driver crossed to UAS-ChR2XXL in the third-instar larval brain. ChR2XXL is visualized by a primary monoclonal mouse anti-ChR2 antibody and a secondary polyclonal donkey anti-mouse Cy3 antibody. Confirming our results from Figures 2-4, this reveals strong and reliable transgene expression in the APL neuron of both hemispheres (anti-ChR2XXL; green). The data were acquired with a 63× glycerol objective. Scale bar and grid spacing, 20 µm. For a corresponding movie, see Movie 7. D, The behavior of experimentally naive</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>**Figure 9.** Memory scores are abolished on activation of APL throughout training. A, Larvae were trained such that, in one group of animals, the odor AM (black cloud) was paired with the fructose reward (green fill of circle indicating a Petri dish), alternating with blank trials (open circle), whereas in a reciprocal group, the odor was presented unpaired from the fructose reward; here and throughout this study, the sequence of training events was as depicted in half of the cases, and in the reverse order in the other half of the cases. The APL neuron was optogenetically activated with blue light illumination (blue rectangle) during the complete training phase. The larvae from both groups were then tested for their odor preference, and associative memory was quantified by the memory score as the difference in preference between these reciprocally trained groups of animals. Double-heterozygous animals of the genotype APL>ChR2XXL were used for APL activation; larvae heterozygous for either the GAL4 (APL>+) or the effector (+>ChR2XXL) were used as the genetic controls. Optogenetic activation of the APL neuron during the complete training phase abolished associative memory scores. B, The same effects were observed in a shortened, one-training-cycle version of this experiment. C, Full projection of the expression pattern from the APL-GAL4 driver crossed to UAS-ChR2XXL in the third-instar larval brain. ChR2XXL is visualized by a primary monoclonal mouse anti-ChR2 antibody and a secondary polyclonal donkey anti-mouse Cy3 antibody. Confirming our results from Figures 2-4, this reveals strong and reliable transgene expression in the APL neuron of both hemispheres (anti-ChR2XXL; green). The data were acquired with a 63× glycerol objective. Scale bar and grid spacing, 20 µm. For a corresponding movie, see Movie 7. D, The behavior of experimentally naive</figcaption></figure>
Remarkably, even high levels of fluorescence induced by pharmacologically stimulating KCs via the acetylcholine receptor agonist carbamylcholine in control brains (Fig. 5G, gray) were reduced by simultaneous APL activation (Fig. 5G–I). Together, these data confirm a functionally inhibitory effect of APL activation on the KCs in the calyx.

In terms of the organization of APL, the above results also match the situation in first-instar larvae, as shown here for a volume reconstruction of APL generated from the electron microscopy reconstruction of the mushroom body in Eichler et al. (2017) (Fig. 6A,B; Movie 4). Specifically, this volume reconstruction shows that the relatively slender axonal and dendritic branches of APL arise separately from a thicker neurite (Fig. 6C,D), similar to the locust homolog of APL, called GGN (for giant GABAergic neuron) (Papadopoulou et al., 2011; Ray et al., 2020). The electron microscope dataset of Eichler et al. (2017) further allowed the site of the synapses for the different classes of synaptic partners of APL to be mapped onto its volume reconstruction (Fig. 6E,F; Movies 5, 6). Furthermore, the connectomics data allowed dendrograms of APL to be derived, that is, 2D representations of APL preserving branch lengths and synaptic locations in a topologically correct manner (Fig. 7). Within such a topology, it can be discerned that, wherever they coexist, the synapses that APL has with mushroom body extrinsic neurons are not segregated from, but are intermingled with, the connections to the mushroom body intrinsic neurons, the KCs (Fig. 7B,C). In the lobes, the almost exclusively postsynaptic sites of APL are relatively sparse (Fig. 7B, C) and with some variation in topology between the APL neuron of the left and the right brain hemisphere (for the right-hemisphere APL neuron, see Extended Data Fig. 7-1). In the calyx, an analysis of cable length (geodesic distance between synapses) reveals that reciprocal synapses between APL and the KCs are arranged in four synapse-rich center-surround structures such that APL-to-KC synapses are found toward their center, whereas KC-to-APL synapses are located mainly in the surround (Fig. 7D).

## Regional synaptic polarity of APL across metamorphosis

Given the conserved regional synaptic polarity of APL across larval stages (see preceding section) and given that APL persists into adulthood yet in adults it is not regionally polarized (C. L. Wu et al., 2013; Lin et al., 2014; Mayseless et al., 2018; Saumweber et al., 2018), we examined how APL develops across metamorphosis. To this end, we used genetically encoded protein

←

larvae of the experimental genotype (APL>ChR2XXL) toward AM (black cloud) was tested, without APL being activated during testing or with APL activated (blue square). Naive odor preference was unaffected by APL activation. *E–I*, The behavior of larvae in *D* was video-recorded and analyzed offline as described by Paisios et al. (2017). *E*, A short sample from a video recording of a larva with successive runs and HCs. Displayed is the track of the mid-point. Magenta and orange dots represent right and left HCs, respectively. Specifically, four features of locomotion were analyzed in addition to the olfactory preference: that is, the time spent by the larvae on the odor and the nonodor side (*F*), the HC rate modulation (*G*), the HC reorientation (*H*), and the run speed modulation (*I*). In all cases, APL activation had no effect. The data are displayed as box plots. Middle line indicates the median. Box boundaries represent the 25% and 75% quantiles. Whiskers represent the 10% and 90% quantiles. The sample sizes (number of biological replications) and the genotypes are given within the figure. *A, B*, Different letters indicate significant differences between groups in MWW comparisons with a BH correction (*p* < 0.05), as specified in Experimental design and statistical analyses. *D, F–I*, NS indicates the absence of significance between groups in MWW comparisons: NS, *p* > 0.05. *A, B*, OSS comparisons to chance levels (i.e., to zero), also with a BH correction: \* *p* < 0.05. The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2.

<figure><img src="image_15.png" /><figcaption>**Movie 7.** 3D rendering of the expression pattern of ChR2XXL in APL in a third-instar larva, based on data shown in Figure 9C. Genotype: APL>ChR2XXL. The data were acquired with a 63× glycerol objective; grid spacing: 20 µm. [View online]</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>**Figure 10.** Activation of APL only in the presence or only in the absence of the odor reduces memory scores. Optogenetic activation of APL (blue square) either only when the odor was presented during training, or only when the odor was not presented during training, reduced memory scores to about half the level of control animals that did not receive any APL activation (black-filled box plots). Testing the animals in the presence of the training reward (i.e., fructose) abolished the behavioral expression of memory in all cases (green-filled box plots). The sample sizes and the genotype are given within the figure. \#OSS comparisons to chance levels (i.e., to zero) with a BH correction (\* *p* < 0.05); different letters indicate significant differences between groups in MWW comparisons also with a BH correction (*p* < 0.05). The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2. Other details as in Figure 9.</figcaption></figure>

"tags" coupled with chemical fluorophore ligands (Kohl et al., 2014; Sutcliffe et al., 2017; Meissner et al., 2018). The synaptic reporter synaptotagmin fused to the tag SNAPm (Syt1-SNAPm) allowed us to label presynapses, and the reporter telencephalin fused to the tag CLIPm (TLN-CLIPm) allowed us to label postsynapses (Kohl et al., 2014). These constructs were expressed in APL throughout development using the intersectional driver APLi-GAL4, which specifically expresses in APL of both larvae and adults (Lin et al., 2014; Mayseless et al., 2018) (as stated earlier, APL-GAL4 does not express in adult APLs). In addition, UAS-mCD8::GFP was expressed to visualize APL membranes.