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_18.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_19.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.
<figure><img src="image_17.png" /><figcaption>**Figure 11.** Activation of APL only in the presence or only in the absence of the odor has differential effects on odor preference. A, Examination of the preference scores (PREF) underlying the associative memory scores from Figure 10 reveals that odor preference scores are higher after paired than after unpaired training with odor and fructose reward (black-filled box plots to the left), a difference that is abolished when testing is conducted in the presence of the training reward (right-most colored plots). This is adaptive because learned search for the reward is obsolete in its presence. These preference scores can thus be pooled to serve as the baseline odor preference cleared of associative memory (stippled line). This reveals that odor preference scores are higher than the baseline after paired training and lower than the baseline after unpaired training. B, C, The same on activation of APL during training (blue square), whether only during odor presentation (B) or unpaired from odor presentation (C). Remarkably, baseline levels of odor preference differ between these three training conditions (D); compared with the control condition without APL activation, baseline odor preference scores are increased when APL is activated together with odor presentation, and decreased when APL is activated unpaired from odor presentation. The sample sizes and the genotype are given within the figure. A-C, MWW comparisons between groups with a BH correction: \*p < 0.05; NS, p > 0.05. D, Different letters indicate significant differences between groups in MWW comparisons, also with a BH correction: p < 0.05. A-C, MWW comparisons to baseline levels of odor preference, 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 Figures 9 and 10.</figcaption></figure>

In third-instar larvae of the genotype APLi/Syt1:SNAP>mCD8::GFP/TLN:CLIP, presynaptic staining was mostly found in the calyx (Fig. 8A'), whereas postsynaptic staining was distributed in the calyx and the lobes (Fig. 8A''), consistent with previous observations (Fig. 4) (Masuda-Nakagawa et al., 2014). As early as 6 h after puparium formation, we detected presynaptic structures that were more punctate (Fig. 8B',C') and observed fewer postsynaptic structures overall (Fig. 8B'',C''), consistent with the previously reported pruning of APL secondary neurites during pupal stages (Mayseless et al., 2018). Interestingly, at 12 h after puparium formation, a stage where APL pruning is almost at its peak (Mayseless et al., 2018), we could still detect both presynaptic and postsynaptic structures (Fig. 8D-D'',) although some postsynaptic structures were observed detached from the neurite (Fig. 8D-D'',D'', yellow arrowhead). Nonetheless, the polarized organization of APL in third-instar larvae was no longer observed in the adult stage, as we detected both presynaptic and postsynaptic markers across both the calyx and the lobes of the adult mush-room bodies (Fig. 8E-F'') (C. L. Wu et al., 2013; Lin et al., 2014).

Together, our results indicate that, whereas APL is regionally polarized throughout larval stages, it undergoes rearrangement during metamorphosis to give rise to a regionally more diffuse organization. In addition, the DPM neuron, one of the main synaptic partners of APL involved in memory consolidation in adults, does not exist in larvae (Pitman et al., 2011; C. L. Wu et al., 2011; Eichler et al., 2017; Saumweber et al., 2018). In light of these differences, and despite the rich insights recently gained into the function of APL in adults (Inada et al., 2017; Zhou et al., 2019; Amin et al., 2020; Apostolopoulou and Lin, 2020; Kanellopoulos et al., 2020; Yamagata et al., 2021), a detailed look into the function of the larval APL neuron seemed warranted.

## Memory scores are abolished on activating APL throughout odor-fructose training

We first asked whether the optogenetic activation of APL affects associative memory formation. Third-instar larvae were trained

in a standard Pavlovian conditioning paradigm, using an odor (AM) as the conditioned stimulus and a fructose reward as the unconditioned stimulus (Scherer et al., 2003; Neuser et al., 2005; Saumweber et al., 2011; Michels et al., 2017). 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 and the fructose reward (unpaired training). After training, both groups were tested for their odor preference. A difference in odor preference between paired and unpaired training thus reflects associative memory, and is quantified by the memory score. According to convention, positive memory scores reflect appetitive associative memory, whereas negative scores reveal aversive memory (Eq. 2; see Materials and Methods). Notably, paired and unpaired training both establish associative memory, yet of opposite “sign.” After paired training, the odor predicts the occurrence of the reward, leading to an associative increase in odor preference. In contrast, unpaired training establishes the odor as a predictor of the nonoccurrence of the reward and supports an associative decrease in odor preference (for a detailed discussion, see Schleyer et al., 2018).

We repeated an experiment from Saumweber et al. (2018), in which APL was optogenetically activated throughout odor-fructose training (Fig. 9). Here, we confirmed their report that odor-fructose memory scores in the experimental genotype (APL>ChR2XXL) were reduced to chance levels and were reduced relative to both genetic controls, heterozygous for either only the effector (+>ChR2XXL) or the driver (APL>) construct alone (Fig. 9A). The same abolishment of memory scores was observed in a shortened, one-trial version of this experiment (Fig. 9B). For practical reasons, this shortened experimental design was used throughout the rest of our study. In addition, the expression of ChR2XXL in APL was directly confirmed by immunohistochemistry (Fig. 9C; Movie 7). Critically, the behavior of experimentally naive larvae toward the odor (i.e., innate odor preference) was unaffected by APL activation (Fig. 9D) (Saumweber et al., 2018). Further, as shown here from offline analyses of video tracking data, APL activation did not
<figure><img src="image_20.png" /><figcaption>**Figure 11.** Activation of APL only in the presence or only in the absence of the odor has differential effects on odor preference. A, Examination of the preference scores (PREF) underlying the associative memory scores from Figure 10 reveals that odor preference scores are higher after paired than after unpaired training with odor and fructose reward (black-filled box plots to the left), a difference that is abolished when testing is conducted in the presence of the training reward (right-most colored plots). This is adaptive because learned search for the reward is obsolete in its presence. These preference scores can thus be pooled to serve as the baseline odor preference cleared of associative memory (stippled line). This reveals that odor preference scores are higher than the baseline after paired training and lower than the baseline after unpaired training. B, C, The same on activation of APL during training (blue square), whether only during odor presentation (B) or unpaired from odor presentation (C). Remarkably, baseline levels of odor preference differ between these three training conditions (D); compared with the control condition without APL activation, baseline odor preference scores are increased when APL is activated together with odor presentation, and decreased when APL is activated unpaired from odor presentation. The sample sizes and the genotype are given within the figure. A-C, MWW comparisons between groups with a BH correction: \*p < 0.05; NS, p > 0.05. D, Different letters indicate significant differences between groups in MWW comparisons, also with a BH correction: p < 0.05. A-C, MWW comparisons to baseline levels of odor preference, 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 Figures 9 and 10.</figcaption></figure>

In third-instar larvae of the genotype APLi/Syt1:SNAP>mCD8::GFP/TLN:CLIP, presynaptic staining was mostly found in the calyx (Fig. 8A'), whereas postsynaptic staining was distributed in the calyx and the lobes (Fig. 8A''), consistent with previous observations (Fig. 4) (Masuda-Nakagawa et al., 2014). As early as 6 h after puparium formation, we detected presynaptic structures that were more punctate (Fig. 8B',C') and observed fewer postsynaptic structures overall (Fig. 8B'',C''), consistent with the previously reported pruning of APL secondary neurites during pupal stages (Mayseless et al., 2018). Interestingly, at 12 h after puparium formation, a stage where APL pruning is almost at its peak (Mayseless et al., 2018), we could still detect both presynaptic and postsynaptic structures (Fig. 8D-D'',) although some postsynaptic structures were observed detached from the neurite (Fig. 8D-D'',D'', yellow arrowhead). Nonetheless, the polarized organization of APL in third-instar larvae was no longer observed in the adult stage, as we detected both presynaptic and postsynaptic markers across both the calyx and the lobes of the adult mushroom bodies (Fig. 8E-F'') (C. L. Wu et al., 2013; Lin et al., 2014).

Together, our results indicate that, whereas APL is regionally polarized throughout larval stages, it undergoes rearrangement during metamorphosis to give rise to a regionally more diffuse organization. In addition, the DPM neuron, one of the main synaptic partners of APL involved in memory consolidation in adults, does not exist in larvae (Pitman et al., 2011; C. L. Wu et al., 2011; Eichler et al., 2017; Saumweber et al., 2018). In light of these differences, and despite the rich insights recently gained into the function of APL in adults (Inada et al., 2017; Zhou et al., 2019; Amin et al., 2020; Apostolopoulou and Lin, 2020; Kanellopoulos et al., 2020; Yamagata et al., 2021), a detailed look into the function of the larval APL neuron seemed warranted.

## Memory scores are abolished on activating APL throughout odor-fructose training

We first asked whether the optogenetic activation of APL affects associative memory formation. Third-instar larvae were trained

in a standard Pavlovian conditioning paradigm, using an odor (AM) as the conditioned stimulus and a fructose reward as the unconditioned stimulus (Scherer et al., 2003; Neuser et al., 2005; Saumweber et al., 2011; Michels et al., 2017). 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 and the fructose reward (unpaired training). After training, both groups were tested for their odor preference. A difference in odor preference between paired and unpaired training thus reflects associative memory, and is quantified by the memory score. According to convention, positive memory scores reflect appetitive associative memory, whereas negative scores reveal aversive memory (Eq. 2; see Materials and Methods). Notably, paired and unpaired training both establish associative memory, yet of opposite “sign.” After paired training, the odor predicts the occurrence of the reward, leading to an associative increase in odor preference. In contrast, unpaired training establishes the odor as a predictor of the nonoccurrence of the reward and supports an associative decrease in odor preference (for a detailed discussion, see Schleyer et al., 2018).

We repeated an experiment from Saumweber et al. (2018), in which APL was optogenetically activated throughout odor-fructose training (Fig. 9). Here, we confirmed their report that odor-fructose memory scores in the experimental genotype (APL>ChR2XXL) were reduced to chance levels and were reduced relative to both genetic controls, heterozygous for either only the effector (+>ChR2XXL) or the driver (APL>) construct alone (Fig. 9A). The same abolishment of memory scores was observed in a shortened, one-trial version of this experiment (Fig. 9B). For practical reasons, this shortened experimental design was used throughout the rest of our study. In addition, the expression of ChR2XXL in APL was directly confirmed by immunohistochemistry (Fig. 9C; Movie 7). Critically, the behavior of experimentally naive larvae toward the odor (i.e., innate odor preference) was unaffected by APL activation (Fig. 9D) (Saumweber et al., 2018). Further, as shown here from offline analyses of video tracking data, APL activation did not
<figure><img src="image_18.png" /><figcaption>**Figure 12.** Activating APL has a rewarding effect. A, Animals were trained by presenting odor either paired with, or unpaired from, activation of APL using ChR2XXL as the effector through blue light illumination (blue square). The effect of APL activation as a reward is quantified by positive memory scores, differing significantly from the genetic controls. B, Preference scores (PREF) underlying the associative memory scores in A. Preference scores from the genetic controls were pooled to serve as the baseline preference (stippled line), revealing that preference scores are higher than the baseline after paired training and lower than the baseline after unpaired training. C, Procedure as in A, except that APL was optogenetically silenced using GtACR1 as the effector through green light illumination (green square). The effect of APL silencing as a punishment is quantified by negative memory scores, differing significantly from the genetic controls. D, Preference scores (PREF) underlying the associative memory scores in C. Stippled line indicates the pooled preference scores of the genetic controls as a baseline. E, Larvae of the experimental genotype (APL>ChR2XXL) were trained as described in A and tested either immediately after training (retention interval 0 min) or 5, 10, or 20 min after training. Expression of odor-APL memory was observed immediately after training and was still detectable at a 5 min retention interval; it was significantly reduced compared with immediate testing when assessed at 5, 10, or 20 min retention intervals. F, Larvae of the experimental genotype (APL>ChR2XXL) were trained as in A (i.e., paired or unpaired) but with modifications of the paradigm in accordance with Weiglein et al. (2021). Specifically, odor presentation and APL activation lasted for 30 s each with different timings relative to their onset (interstimulus interval [ISI]): the odor was presented before the APL activation (negative ISI values), during the APL activation (ISI 0), or after the APL activation (positive ISI values); in all cases, reciprocal training involved odor presentation unpaired from APL activation. Three training trials were performed, followed by the test of odor preference. Memory scores differed according to the ISI. G, Repetition of the experiment from F, but for simultaneous presentation of odor and APL activation (ISI 0), including genetic controls. Positive memory scores for the experimental genotype (APL>ChR2XXL) indicate that a brief</figcaption></figure>
<figure><img src="image_21.png" /><figcaption>**Figure 12.** Activating APL has a rewarding effect. A, Animals were trained by presenting odor either paired with, or unpaired from, activation of APL using ChR2XXL as the effector through blue light illumination (blue square). The effect of APL activation as a reward is quantified by positive memory scores, differing significantly from the genetic controls. B, Preference scores (PREF) underlying the associative memory scores in A. Preference scores from the genetic controls were pooled to serve as the baseline preference (stippled line), revealing that preference scores are higher than the baseline after paired training and lower than the baseline after unpaired training. C, Procedure as in A, except that APL was optogenetically silenced using GtACR1 as the effector through green light illumination (green square). The effect of APL silencing as a punishment is quantified by negative memory scores, differing significantly from the genetic controls. D, Preference scores (PREF) underlying the associative memory scores in C. Stippled line indicates the pooled preference scores of the genetic controls as a baseline. E, Larvae of the experimental genotype (APL>ChR2XXL) were trained as described in A and tested either immediately after training (retention interval 0 min) or 5, 10, or 20 min after training. Expression of odor-APL memory was observed immediately after training and was still detectable at a 5 min retention interval; it was significantly reduced compared with immediate testing when assessed at 5, 10, or 20 min retention intervals. F, Larvae of the experimental genotype (APL>ChR2XXL) were trained as in A (i.e., paired or unpaired) but with modifications of the paradigm in accordance with Weiglein et al. (2021). Specifically, odor presentation and APL activation lasted for 30 s each with different timings relative to their onset (interstimulus interval [ISI]): the odor was presented before the APL activation (negative ISI values), during the APL activation (ISI 0), or after the APL activation (positive ISI values); in all cases, reciprocal training involved odor presentation unpaired from APL activation. Three training trials were performed, followed by the test of odor preference. Memory scores differed according to the ISI. G, Repetition of the experiment from F, but for simultaneous presentation of odor and APL activation (ISI 0), including genetic controls. Positive memory scores for the experimental genotype (APL>ChR2XXL) indicate that a brief</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>**Figure 13.** Locomotor “footprint” of odor-APL memory. A, The behavior of larvae of the genotype APL>ChR2XXL was video-recorded after paired or unpaired training with odor and APL activation. B, Larvae showed a higher preference for the odor after paired training than after unpaired training; dataset split into 100 bins (1.8 s, each), showing the median of odor preferences across Petri dishes over time. C, Larvae from the paired group spent more time on the odor side than on the nonodor side during testing, whereas the contrary was observed for the unpaired group. D, Paired-trained larvae exhibited more HCs when crawling away from the odor than when moving toward it; the opposite was observed for the unpaired group. E, Larvae from the paired group oriented their HCs more in the direction of the odor compared with larvae from the unpaired group. F, The run speed when heading toward versus when heading away from the odor did not differ between paired- and unpaired-trained animals. B–F, Analyses are based on data available from the experiments shown in Figures 12A, E and 14A. Similar results were observed using Chrimson as the optogenetic effector (not shown). The sample sizes (number of biological replications) and the genotype are given within the figure. C–E, Significant differences between groups in MWW comparisons: \*p<0.05. F, NS indicates the absence of significance between groups in MWW comparisons: NS, 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 Figures 9-12.</figcaption></figure>

affect the modulation of locomotor patterns by which these odor preferences were realized (i.e., modulations of HC rate and direction, but not of run speed: Fig. 9E–I).

## Activating APL either in the presence or in the absence of the odor reduces memory scores

As argued in Saumweber et al. (2018), the abolishment of memory scores on activation of APL during the complete training phase (Fig. 9) may arise because APL provides an inhibitory GABAergic signal onto the KCs (Fig. 5) (Masuda-Nakagawa et al., 2014). Taking the argument to the extreme, the activation of

←

stimulation of APL is sufficient to be rewarding, an effect that was not observed in the genetic controls. The sample sizes and the genotypes are indicated within the figure. A, C, G, Different letters indicate significant differences between groups in MWW comparisons with a BH correction: \*p<0.05. E, Significant differences between groups in MWW comparisons with a BH correction: \*p<0.05. F, KW multiple-group comparison: \*p<0.05. A, C, E, G, OSS comparisons to chance levels (i.e., to zero), also with a BH correction: \#p<0.05. B, MWW comparisons to the control baseline (i.e., to the pooled preference scores from the genetic controls), 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 Figures 9-11.

APL would silence the KCs, preventing a proper odor representation in the mushroom body and thereby also preventing odor-fructose memory formation thereafter. If so, memory formation should be disrupted when APL is activated while the odor is presented, but should not be disrupted when APL is activated while the odor is not presented. To our surprise, however, in both cases, odor-fructose memory scores were partially reduced compared with a control condition in which APL was not activated at all (Fig. 10). In regard to these residual memory scores, we considered the interpretation of odor-fructose memory as a learned search for the fructose reward (Saumweber et al., 2011; Schleyer et al., 2011). This interpretation implies that memory is behaviorally expressed if the sought-for fructose reward is indeed absent during the test, but that memory is not expressed if the testing is conducted in the presence of the sought-for fructose reward. This was indeed the case in all three conditions, namely (1) when APL was not activated during training at all, and (2) when APL was activated during odor presentation or (3) in the absence of odor (Fig. 10, right); innate olfactory behavior is not changed in the presence of fructose or other tastants (Schleyer et al., 2011). In other words, the residual memory scores after APL activation during either period of the training also reflect a search for the fructose reward.
<figure><img src="image_22.png" /><figcaption>**Figure 13.** Locomotor “footprint” of odor-APL memory. A, The behavior of larvae of the genotype APL>ChR2XXL was video-recorded after paired or unpaired training with odor and APL activation. B, Larvae showed a higher preference for the odor after paired training than after unpaired training; dataset split into 100 bins (1.8 s, each), showing the median of odor preferences across Petri dishes over time. C, Larvae from the paired group spent more time on the odor side than on the nonodor side during testing, whereas the contrary was observed for the unpaired group. D, Paired-trained larvae exhibited more HCs when crawling away from the odor than when moving toward it; the opposite was observed for the unpaired group. E, Larvae from the paired group oriented their HCs more in the direction of the odor compared with larvae from the unpaired group. F, The run speed when heading toward versus when heading away from the odor did not differ between paired- and unpaired-trained animals. B–F, Analyses are based on data available from the experiments shown in Figures 12A, E and 14A. Similar results were observed using Chrimson as the optogenetic effector (not shown). The sample sizes (number of biological replications) and the genotype are given within the figure. C–E, Significant differences between groups in MWW comparisons: \*p<0.05. F, NS indicates the absence of significance between groups in MWW comparisons: NS, 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 Figures 9-12.</figcaption></figure>

affect the modulation of locomotor patterns by which these odor preferences were realized (i.e., modulations of HC rate and direction, but not of run speed: Fig. 9E–I).

## Activating APL either in the presence or in the absence of the odor reduces memory scores

As argued in Saumweber et al. (2018), the abolishment of memory scores on activation of APL during the complete training phase (Fig. 9) may arise because APL provides an inhibitory GABAergic signal onto the KCs (Fig. 5) (Masuda-Nakagawa et al., 2014). Taking the argument to the extreme, the activation of

←

stimulation of APL is sufficient to be rewarding, an effect that was not observed in the genetic controls. The sample sizes and the genotypes are indicated within the figure. A, C, G, Different letters indicate significant differences between groups in MWW comparisons with a BH correction: \*p<0.05. E, Significant differences between groups in MWW comparisons with a BH correction: \*p<0.05. F, KW multiple-group comparison: \*p<0.05. A, C, E, G, OSS comparisons to chance levels (i.e., to zero), also with a BH correction: \#p<0.05. B, MWW comparisons to the control baseline (i.e., to the pooled preference scores from the genetic controls), 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 Figures 9-11.

APL would silence the KCs, preventing a proper odor representation in the mushroom body and thereby also preventing odor-fructose memory formation thereafter. If so, memory formation should be disrupted when APL is activated while the odor is presented, but should not be disrupted when APL is activated while the odor is not presented. To our surprise, however, in both cases, odor-fructose memory scores were partially reduced compared with a control condition in which APL was not activated at all (Fig. 10). In regard to these residual memory scores, we considered the interpretation of odor-fructose memory as a learned search for the fructose reward (Saumweber et al., 2011; Schleyer et al., 2011). This interpretation implies that memory is behaviorally expressed if the sought-for fructose reward is indeed absent during the test, but that memory is not expressed if the testing is conducted in the presence of the sought-for fructose reward. This was indeed the case in all three conditions, namely (1) when APL was not activated during training at all, and (2) when APL was activated during odor presentation or (3) in the absence of odor (Fig. 10, right); innate olfactory behavior is not changed in the presence of fructose or other tastants (Schleyer et al., 2011). In other words, the residual memory scores after APL activation during either period of the training also reflect a search for the fructose reward.
<figure><img src="image_20.png" /><figcaption>**Figure 14.** Activation of APL during testing prevents the behavioral expression of appetitive odor-APL memory. A, Repetition of the experiment from Figure 12A, confirming that APL activation has a rewarding effect (black-filled box plots). Activating APL during testing as well prevented the behavioral expression of appetitive odor-APL memory (blue-filled box plots). B, Larvae were trained and tested as in A, except that in a differential conditioning protocol, OCT was used as a second odor (yellow cloud) in all training trials in which AM (black cloud) was not presented. Presenting one of the two odors paired with APL activation induced odor-specific appetitive memory; as in A, testing the animals while activating APL prevented the behavioral expression of memory. C, Larvae were trained and tested as in Figure 12A either with AM (black cloud) or OCT (yellow cloud) as a single odor. Compared with AM, larvae showed lower appetitive memory scores when OCT was used as a conditioned stimulus. D, Larvae were trained as in C and tested either with the same trained odor (AM or OCT), or with the respective other novel odor (AM or OCT). Testing larvae with the novel odor led to generalization decrement. E, The behavior of experimentally naive larvae toward AM (black cloud) was tested either without APL activation or with APL activation during the test. Naïve odor preference in the experimental group was unaffected by APL activation (APL>ChR2XXL) (see also Fig. 9D), with the caveat that it did differ from the effector (+>ChR2XXL), but not from the driver control (APL>). F, Same as in E, except that OCT was used as a single odor (yellow cloud). Naïve odor preference in the experimental group was unaffected by APL activation (APL>ChR2XXL), and did not differ from the genetic controls. G, Same as in E, F, except that OCT was used as a second odor (yellow cloud). Again, naïve odor preference in the experimental group was unaffected by APL activation (APL>ChR2XXL) and did not differ from the genetic controls. The sample sizes and the genotypes are given within the figure. A–D, OSS comparisons to chance levels (i.e., to zero) with a BH correction: $^{#}p<0.05$. A, B, D, E, Different letters indicate significant differences between groups in MWW comparisons, also with a BH correction: $p<0.05$. C, Significant differences between groups in MWW comparisons. NS indicates the absence of significance between groups in MWW comparisons with a BH correction in F, and in KW comparisons in G (NS, $p>0.05$). The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2. Other details as in Figures 9-13.</figcaption></figure>

## Differential effects of activating APL only in the presence or only in the absence of the odor

As mentioned, it was unexpected that odor-fructose memory was impaired by activation of APL during the odor-absent periods of training. In order to understand this result, we separately analyzed odor preferences underlying the memory scores from Figure 10. In all 3 cases — (1) when APL was not activated at all during training, (2) when it was activated while the odor was presented, and (3) when it was activated while the odor was not presented — odor preference scores after paired versus unpaired training were indistinguishable from each other when the fructose reward was present during testing (Fig. 11A–C, open boxes). In other words, in all cases, learned search ceased once

the sought-for reward was found. As discussed in detail by Schleyer et al. (2018), this allows the odor preferences after paired and unpaired training to be pooled to determine baseline levels of odor preference, cleared of associative memory (Fig. 11A–C, stippled lines). In all 3 cases, these baseline preference scores were intermediate between the paired-trained and the unpaired-trained animals that were tested in the absence of fructose, consistent with earlier reports (Schleyer et al., 2018). This is adaptive because after paired training the larvae search for fructose where the odor is, whereas after unpaired training they search for fructose where the odor is not, and accordingly in either case their search is suppressed in the presence of the sought-for fructose. Important for the
<figure><img src="image_23.png" /><figcaption>**Figure 14.** Activation of APL during testing prevents the behavioral expression of appetitive odor-APL memory. A, Repetition of the experiment from Figure 12A, confirming that APL activation has a rewarding effect (black-filled box plots). Activating APL during testing as well prevented the behavioral expression of appetitive odor-APL memory (blue-filled box plots). B, Larvae were trained and tested as in A, except that in a differential conditioning protocol, OCT was used as a second odor (yellow cloud) in all training trials in which AM (black cloud) was not presented. Presenting one of the two odors paired with APL activation induced odor-specific appetitive memory; as in A, testing the animals while activating APL prevented the behavioral expression of memory. C, Larvae were trained and tested as in Figure 12A either with AM (black cloud) or OCT (yellow cloud) as a single odor. Compared with AM, larvae showed lower appetitive memory scores when OCT was used as a conditioned stimulus. D, Larvae were trained as in C and tested either with the same trained odor (AM or OCT), or with the respective other novel odor (AM or OCT). Testing larvae with the novel odor led to generalization decrement. E, The behavior of experimentally naive larvae toward AM (black cloud) was tested either without APL activation or with APL activation during the test. Naïve odor preference in the experimental group was unaffected by APL activation (APL>ChR2XXL) (see also Fig. 9D), with the caveat that it did differ from the effector (+>ChR2XXL), but not from the driver control (APL>). F, Same as in E, except that OCT was used as a single odor (yellow cloud). Naïve odor preference in the experimental group was unaffected by APL activation (APL>ChR2XXL), and did not differ from the genetic controls. G, Same as in E, F, except that OCT was used as a second odor (yellow cloud). Again, naïve odor preference in the experimental group was unaffected by APL activation (APL>ChR2XXL) and did not differ from the genetic controls. The sample sizes and the genotypes are given within the figure. A–D, OSS comparisons to chance levels (i.e., to zero) with a BH correction: $^{#}p<0.05$. A, B, D, E, Different letters indicate significant differences between groups in MWW comparisons, also with a BH correction: $p<0.05$. C, Significant differences between groups in MWW comparisons. NS indicates the absence of significance between groups in MWW comparisons with a BH correction in F, and in KW comparisons in G (NS, $p>0.05$). The source data and results of all statistical tests are documented in Extended Data Figure 3-1 and Table 2. Other details as in Figures 9-13.</figcaption></figure>

## Differential effects of activating APL only in the presence or only in the absence of the odor

As mentioned, it was unexpected that odor-fructose memory was impaired by activation of APL during the odor-absent periods of training. In order to understand this result, we separately analyzed odor preferences underlying the memory scores from Figure 10. In all 3 cases — (1) when APL was not activated at all during training, (2) when it was activated while the odor was presented, and (3) when it was activated while the odor was not presented — odor preference scores after paired versus unpaired training were indistinguishable from each other when the fructose reward was present during testing (Fig. 11A–C, open boxes). In other words, in all cases, learned search ceased once

the sought-for reward was found. As discussed in detail by Schleyer et al. (2018), this allows the odor preferences after paired and unpaired training to be pooled to determine baseline levels of odor preference, cleared of associative memory (Fig. 11A–C, stippled lines). In all 3 cases, these baseline preference scores were intermediate between the paired-trained and the unpaired-trained animals that were tested in the absence of fructose, consistent with earlier reports (Schleyer et al., 2018). This is adaptive because after paired training the larvae search for fructose where the odor is, whereas after unpaired training they search for fructose where the odor is not, and accordingly in either case their search is suppressed in the presence of the sought-for fructose. Important for the
<figure><img src="image_21.png" /><figcaption>**Figure 15.** Activating APL with Crimson has a rewarding effect. A, The rewarding effect of APL activation was confirmed using Crimson as the effector and red light illumination (red square), and quantified through positive memory scores in the experimental group (APL>Chrimson), differing significantly from the genetic controls. Transgenic flies were raised on standard food supplemented with retinal (100 mM final concentration). B, Preference scores (PREF) underlying the associative memory scores in A. Preference scores from the genetic controls were pooled to serve as the baseline odor preference (stippled line), revealing that preference scores are higher than the baseline after paired training and lower than the baseline after unpaired training. C, Larvae of the experimental genotype (APL>Chrimson) were trained and tested after being raised on food either supplemented with retinal (final concentration in EtOH [99.9%] 100 mM), or without retinal (food medium supplemented with EtOH only). The rewarding effect of APL activation was observed in retinal-fed animals but was not observed without retinal feeding. D, The behavioral expression of odor-APL memory was reduced but not abolished by testing the animals while APL was activated (red-filled box plot). E, The behavior of experimentally naive larvae of the genotype APL>Chrimson toward AM (black cloud) was tested, either without APL activation or with APL activated during testing (red square). Naive odor preference remained unaffected by APL activation. A, C, D, E, The sample sizes and the genotypes are given within the figure. A, Different letters indicate significant differences between groups in MWW comparisons with a BH correction: *p*< 0.05. C, D, Significant differences between groups in MWW comparisons, also with a BH correction: \*\*p*< 0.05. E, NS indicates the absence of significance between groups in MWW comparisons: NS, *p* > 0.05. A, C, D, OSS comparisons to chance levels (i.e., to zero) with a BH correction: \*\*p*< 0.05. B, MWW comparisons to the control baseline (i.e., to the pooled preference scores from the genetic controls), 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 Figures 9-14.</figcaption></figure>

current context, however, is that these baseline levels varied strikingly with the contingency between APL activation and odor presentation (Fig. 11D): compared with the control baseline scores when APL was not activated at all (stippled line in Fig. 11A and plotted in Fig. 11D, left), the baseline scores were increased when APL was activated in the presence of the odor (stippled line in Fig. 11B; plotted in Fig. 11D, middle), and were decreased when APL was activated in the absence of the odor (stippled line in Fig. 11C; plotted in Fig. 11D, right). In other words, activation of APL paired with odor increased odor preferences, whereas activation of APL unpaired from odor presentation decreased odor preferences (Fig. 11D) — as if, above and beyond the fructose that we intended to be the only reward in these experiments, activation of APL also had a rewarding effect. The next experiment tested this hypothesis.

## Activating APL has a rewarding effect

To test our hypothesis that optogenetically activating APL has a rewarding effect, animals were trained by presenting the odor either paired or unpaired with APL activation instead of with the fructose reward. This established positive memory scores in the experimental genotype, differing from the genetic controls (Fig. 12A). An analysis of the underlying odor preferences showed that these positive memory scores resulted from both an increase in odor preferences after paired training and a decrease in odor preferences after unpaired training, relative to the baseline odor preferences in the genetic controls (Fig. 12B). Thus, APL activation during training has a rewarding effect and can establish appetitive, associative memory. In turn, optogenetically silencing APL leads to an aversive memory (Fig. 12C); in this case, the apparent trend for opposite effects of paired versus unpaired training did not reach statistical significance (Fig.
<figure><img src="image_24.png" /><figcaption>**Figure 15.** Activating APL with Crimson has a rewarding effect. A, The rewarding effect of APL activation was confirmed using Crimson as the effector and red light illumination (red square), and quantified through positive memory scores in the experimental group (APL>Chrimson), differing significantly from the genetic controls. Transgenic flies were raised on standard food supplemented with retinal (100 mM final concentration). B, Preference scores (PREF) underlying the associative memory scores in A. Preference scores from the genetic controls were pooled to serve as the baseline odor preference (stippled line), revealing that preference scores are higher than the baseline after paired training and lower than the baseline after unpaired training. C, Larvae of the experimental genotype (APL>Chrimson) were trained and tested after being raised on food either supplemented with retinal (final concentration in EtOH [99.9%] 100 mM), or without retinal (food medium supplemented with EtOH only). The rewarding effect of APL activation was observed in retinal-fed animals but was not observed without retinal feeding. D, The behavioral expression of odor-APL memory was reduced but not abolished by testing the animals while APL was activated (red-filled box plot). E, The behavior of experimentally naive larvae of the genotype APL>Chrimson toward AM (black cloud) was tested, either without APL activation or with APL activated during testing (red square). Naive odor preference remained unaffected by APL activation. A, C, D, E, The sample sizes and the genotypes are given within the figure. A, Different letters indicate significant differences between groups in MWW comparisons with a BH correction: *p*< 0.05. C, D, Significant differences between groups in MWW comparisons, also with a BH correction: \*\*p*< 0.05. E, NS indicates the absence of significance between groups in MWW comparisons: NS, *p* > 0.05. A, C, D, OSS comparisons to chance levels (i.e., to zero) with a BH correction: \*\*p*< 0.05. B, MWW comparisons to the control baseline (i.e., to the pooled preference scores from the genetic controls), 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 Figures 9-14.</figcaption></figure>

current context, however, is that these baseline levels varied strikingly with the contingency between APL activation and odor presentation (Fig. 11D): compared with the control baseline scores when APL was not activated at all (stippled line in Fig. 11A and plotted in Fig. 11D, left), the baseline scores were increased when APL was activated in the presence of the odor (stippled line in Fig. 11B; plotted in Fig. 11D, middle), and were decreased when APL was activated in the absence of the odor (stippled line in Fig. 11C; plotted in Fig. 11D, right). In other words, activation of APL paired with odor increased odor preferences, whereas activation of APL unpaired from odor presentation decreased odor preferences (Fig. 11D) — as if, above and beyond the fructose that we intended to be the only reward in these experiments, activation of APL also had a rewarding effect. The next experiment tested this hypothesis.

## Activating APL has a rewarding effect

To test our hypothesis that optogenetically activating APL has a rewarding effect, animals were trained by presenting the odor either paired or unpaired with APL activation instead of with the fructose reward. This established positive memory scores in the experimental genotype, differing from the genetic controls (Fig. 12A). An analysis of the underlying odor preferences showed that these positive memory scores resulted from both an increase in odor preferences after paired training and a decrease in odor preferences after unpaired training, relative to the baseline odor preferences in the genetic controls (Fig. 12B). Thus, APL activation during training has a rewarding effect and can establish appetitive, associative memory. In turn, optogenetically silencing APL leads to an aversive memory (Fig. 12C); in this case, the apparent trend for opposite effects of paired versus unpaired training did not reach statistical significance (Fig.
A

<figure><img src="image_22.png" /></figure>

B

<figure><img src="image_23.png" /></figure>

C

<figure><img src="image_24.png" /></figure>

D

<figure><img src="image_25.png" /></figure>

**Figure 16.** Manipulating activity in the calyx MBONs has no reinforcing effect. A, Larvae were trained such that odor was presented either paired or unpaired with the silencing of the two calyx MBONs, using GtACR1 as the effector and green light illumination (green square). Silencing the two calyx MBONs together was seen to have no rewarding or punishing effect, as larvae of the experimental genotype (MBONa1,a2>GtACR1) did not behave differently from the genetic controls. B, Activating the two calyx MBONs together likewise had no rewarding or punishing effect. C, D, Silencing (C) or activating (D) the two calyx MBONs separately had no reinforcing effect, either. The sample sizes and the genotypes are given within the figure. NS indicates the absence of significance between groups in MWW comparisons: NS, *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 Figures 9-15. Expression patterns of the calyx MBON drivers used in C, D are shown in Extended Data Figure 16-1.

12D). Throughout the rest of the study, we decided to focus on the rewarding effect of APL on its activation. We found that odor-APL memory was transient and lasted for<10 min (Fig. 12E), as is the case for odor-fructose memory after one training trial (Weiglein et al., 2019) and for appetitive olfactory memories formed by optogenetic activation of large sets of KCs

(Lyutova et al., 2019). A rewarding effect of APL activation was likewise observed when a brief-stimulation protocol was used (Fig. 12F,G). In addition, offline analysis of video-recorded larval locomotion revealed that the same aspects of larval locomotion were modulated by odor-APL memory (Fig. 13) as previously shown for similarly strong odor-taste reward associative memories, namely, the rate of HCs and their orientation but not run speed (Schleyer et al., 2015b, 2020; Paisios et al., 2017; Saumweber et al., 2018; Thane et al., 2019). Inspired by what has been reported on fructose as a taste reward (Schleyer et al., 2015a) (see also Figs. 10, 11), we reasoned that, if odor-APL memory scores reflect a learned search for the training reward (which is APL activation in the present case), these memory scores should be abolished if the sought-for reward is present during the test. We therefore repeated the experiment from Figure 12A and added an experimental condition whereby APL was also activated during testing. This prevented the behavioral expression of appetitive odor-APL memory (Fig. 14A) (for a similar effect of DAN activation, see Schleyer et al., 2020). The same was observed for a two-odor, differential conditioning version of the paradigm, using OCT as the second odor (Fig. 14B). Compared with AM, OCT turned out less salient as a conditioned stimulus (Fig. 14C). Interestingly, when these odors were used for training but testing was conducted either with the same trained odor or with the respectively other novel odor, we observed a generalization decrement in both cases (Fig. 14D), suggesting that the established memories are at least partially specific for the trained odor. We find it striking that APL activation is effective as a reward, even in differential conditioning, because it implies that an associable, odor-specific representation can be established in the mushroom body under the condition of an optogenetically activated APL neuron. In line with our earlier results from Figure 9D, naive odor preferences were unaffected by APL activation (Fig. 14E-G). Thus, APL activation has two kinds of effect previously reported for taste rewards: it both induces associative memory when paired with odor during training (Figs. 12, 14; with the same locomotor “footprint” as for taste rewards: Fig. 13), and it terminates the search behavior that is based on this memory during the test (Fig. 14). These two effects of reward are adaptive because they help the animals to search for the reward, and prevent them from drifting away from a reward once it is found, respectively. Both of these reward-like effects of APL activation, plus the opposite effects of paired versus unpaired training and the lack of any effect of APL activation on naive odor preference, were confirmed using Chrimson as the effector (Fig. 15A-E), although the effect of APL activation to terminate search behavior during testing was only partial for Chrimson (Fig. 15D).

## Manipulating activity in the calyx MBONs has no reinforcing effect

Considering the circuit mechanisms by which APL activation exerts a rewarding effect, we first focused on the calyx MBONs to which APL is presynaptic (MBONa1 and MBONa2; also known as “Odd” neurons: Figs. 6F, 7B; Movie 6) (Slater et al., 2015; Eichler et al., 2017; Saumweber et al., 2018). We reasoned that, if activation of the GABAergic APL neuron exerts its rewarding effect by inhibiting the calyx MBONs, then optogenetically silencing these MBONs should also have a rewarding effect. Using the chloride channel GtACR1 as the effector, however, this was found not to be the case (Fig. 16A). We then
<figure><img src="image_25.png" /><figcaption>**Figure 16.** Manipulating activity in the calyx MBONs has no reinforcing effect. A, Larvae were trained such that odor was presented either paired or unpaired with the silencing of the two calyx MBONs, using GtACR1 as the effector and green light illumination (green square). Silencing the two calyx MBONs together was seen to have no rewarding or punishing effect, as larvae of the experimental genotype (MBONa1,a2>GtACR1) did not behave differently from the genetic controls. B, Activating the two calyx MBONs together likewise had no rewarding or punishing effect. C, D, Silencing (C) or activating (D) the two calyx MBONs separately had no reinforcing effect, either. The sample sizes and the genotypes are given within the figure. NS indicates the absence of significance between groups in MWW comparisons: NS, *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 Figures 9-15. Expression patterns of the calyx MBON drivers used in C, D are shown in Extended Data Figure 16-1.</figcaption></figure>

12D). Throughout the rest of the study, we decided to focus on the rewarding effect of APL on its activation. We found that odor-APL memory was transient and lasted for<10 min (Fig. 12E), as is the case for odor-fructose memory after one training trial (Weiglein et al., 2019) and for appetitive olfactory memories formed by optogenetic activation of large sets of KCs

(Lyutova et al., 2019). A rewarding effect of APL activation was likewise observed when a brief-stimulation protocol was used (Fig. 12F,G). In addition, offline analysis of video-recorded larval locomotion revealed that the same aspects of larval locomotion were modulated by odor-APL memory (Fig. 13) as previously shown for similarly strong odor-taste reward associative memories, namely, the rate of HCs and their orientation but not run speed (Schleyer et al., 2015b, 2020; Paisios et al., 2017; Saumweber et al., 2018; Thane et al., 2019). Inspired by what has been reported on fructose as a taste reward (Schleyer et al., 2015a) (see also Figs. 10, 11), we reasoned that, if odor-APL memory scores reflect a learned search for the training reward (which is APL activation in the present case), these memory scores should be abolished if the sought-for reward is present during the test. We therefore repeated the experiment from Figure 12A and added an experimental condition whereby APL was also activated during testing. This prevented the behavioral expression of appetitive odor-APL memory (Fig. 14A) (for a similar effect of DAN activation, see Schleyer et al., 2020). The same was observed for a two-odor, differential conditioning version of the paradigm, using OCT as the second odor (Fig. 14B). Compared with AM, OCT turned out less salient as a conditioned stimulus (Fig. 14C). Interestingly, when these odors were used for training but testing was conducted either with the same trained odor or with the respectively other novel odor, we observed a generalization decrement in both cases (Fig. 14D), suggesting that the established memories are at least partially specific for the trained odor. We find it striking that APL activation is effective as a reward, even in differential conditioning, because it implies that an associable, odor-specific representation can be established in the mushroom body under the condition of an optogenetically activated APL neuron. In line with our earlier results from Figure 9D, naive odor preferences were unaffected by APL activation (Fig. 14E-G). Thus, APL activation has two kinds of effect previously reported for taste rewards: it both induces associative memory when paired with odor during training (Figs. 12, 14; with the same locomotor “footprint” as for taste rewards: Fig. 13), and it terminates the search behavior that is based on this memory during the test (Fig. 14). These two effects of reward are adaptive because they help the animals to search for the reward, and prevent them from drifting away from a reward once it is found, respectively. Both of these reward-like effects of APL activation, plus the opposite effects of paired versus unpaired training and the lack of any effect of APL activation on naive odor preference, were confirmed using Chrimson as the effector (Fig. 15A-E), although the effect of APL activation to terminate search behavior during testing was only partial for Chrimson (Fig. 15D).

## Manipulating activity in the calyx MBONs has no reinforcing effect

Considering the circuit mechanisms by which APL activation exerts a rewarding effect, we first focused on the calyx MBONs to which APL is presynaptic (MBONa1 and MBONa2; also known as “Odd” neurons: Figs. 6F, 7B; Movie 6) (Slater et al., 2015; Eichler et al., 2017; Saumweber et al., 2018). We reasoned that, if activation of the GABAergic APL neuron exerts its rewarding effect by inhibiting the calyx MBONs, then optogenetically silencing these MBONs should also have a rewarding effect. Using the chloride channel GtACR1 as the effector, however, this was found not to be the case (Fig. 16A). We then
<figure><img src="image_26.png" /><figcaption>**Figure 17.** Inhibition of dopamine synthesis impairs odor-APL memory. A systemic pharmacological approach was used to disrupt dopamine synthesis (Thoener et al., 2021). A, Sketch of dopamine biosynthesis. The enzyme tyrosine hydroxylase (TH) converts the amino acid L-tyrosine to L-DOPA. In the next step, the enzyme dopa-decarboxylase (DDC) converts L-DOPA to dopamine. Application of 3IY inhibits the TH enzyme. B, Third-instar APL>ChR2XXL larvae were transferred from their food vials to a yeast solution either without 3IY or supplemented with 3IY. After 4 h of such feeding, the animals were trained and tested as in Figure 12A. C, Relative to control larvae, the 3IY-fed larvae exhibited impaired odor-APL memory scores. D, Same as in C, except that the yeast solution was prepared (1) without additional substances, (2) with 3IY added, (3) with 3IY plus L-DOPA (a dopamine precursor), or (4) with L-DOPA only, at the concentrations indicated. Again, relative to control larvae, reduced memory scores were observed in 3IY-fed larvae; feeding them additionally with L-DOPA rescued that memory impairment, leading to scores similar to those of the control animals. L-DOPA alone had no impact on odor-APL memory. The sample sizes and the genotypes are given within the figure. C, D, OSS comparisons to chance levels (i.e., to zero) with a BH correction: \*p<0.05. C, Significant differences between groups in MWW comparisons with a BH correction: \*p<0.05. D, 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 Figures 9-16.</figcaption></figure>

considered the possibility that, unlike the KCs (Fig. 5), the calyx MBONs might actually be activated rather than inhibited by activation of APL, for example, through GABA-induced chloride spikes as reported for insect motoneurons (Ryglewski et al., 2017) or by yet-to-be-identified excitatory transmitters released by APL. We thus repeated the same experiment, but this time activated the MBONs: again, no rewarding effect was observed on such manipulation (Fig. 16B). Before ruling out the involvement of the two calyx MBONs in the rewarding effect of APL, however, it was important to test for the effects of manipulating each of them separately. This is because activation of MBONa1 and MBONa2 induces approach and avoidance, respectively (Eschbach et al., 2021). We therefore reasoned that they might exert a rewarding and punishing effect, respectively, which would sum to zero when both these MBONs were manipulated together. However, neither silencing nor activating either one of the calyx MBONs yielded evidence of such oppositely reinforcing effects (Fig. 16C,D; Extended Data Fig. 16-1); we included groups tested in the presence of light because we reasoned that, similar to what has been observed for gustatory punishment (Gerber and Hendel, 2006; Selcho et al., 2009; Schleyer et al., 2011; Widmann et al., 2016; Weber et al., 2023), this might promote aversive memory expression. These results suggest that the rewarding effect of APL activation does not involve APL-to-MBONa1/a2 connections. Given the role of dopamine in conveying reward signals in larval *Drosophila* (Selcho et al., 2009; Rohwedder et al., 2016; Thoener et al., 2021), we next inquired into the dopamine dependency of APL's rewarding effect.

### Inhibition of dopamine synthesis impairs odor-APL memory

We used a systemic pharmacological approach to acutely disrupt dopamine synthesis. This was done by inhibiting the enzyme tyrosine hydroxylase (TH), which is rate-limiting for dopamine synthesis (Neckameyer and White, 1993; Bainton et al., 2000; Fernandez et al., 2017; Thoener et al., 2021). The TH-inhibitor 3IY was added to the larval food at a dose that

<figure><img src="image_27.png" /><figcaption>**Figure 18.** Ablation of dopaminergic pPAM neurons impairs odor-APL memory. Larvae were trained and tested as in Figure 12A. Optogenetic activation of APL and simultaneous ablation of the pPAM neurons (APL<sub>55008</sub>>ChR2XXL; pPAM>>reaper) reduced odor-APL memory scores relative to genetic controls (APL<sub>55008</sub>>ChR2XXL; pPAM>>+ and APL<sub>55008</sub>>ChR2XXL; >>reaper). The sample sizes and the genotypes are given within the figure. Different letters indicate significant differences between groups in MWW comparisons with a BH correction (p<0.05). \*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. Other details as in Figures 9-17.</figcaption></figure>

leaves intact task-relevant behavioral faculties (i.e., innate odor preference and locomotion) (Thoener et al., 2021). When 4 h later the larvae were trained and tested for odor-APL memory, they exhibited reduced memory scores (Fig. 17A-
<figure><img src="image_26.png" /><figcaption>**Figure 17.** Inhibition of dopamine synthesis impairs odor-APL memory. A systemic pharmacological approach was used to disrupt dopamine synthesis (Thoener et al., 2021). A, Sketch of dopamine biosynthesis. The enzyme tyrosine hydroxylase (TH) converts the amino acid L-tyrosine to L-DOPA. In the next step, the enzyme dopa-decarboxylase (DDC) converts L-DOPA to dopamine. Application of 3IY inhibits the TH enzyme. B, Third-instar APL>ChR2XXL larvae were transferred from their food vials to a yeast solution either without 3IY or supplemented with 3IY. After 4 h of such feeding, the animals were trained and tested as in Figure 12A. C, Relative to control larvae, the 3IY-fed larvae exhibited impaired odor-APL memory scores. D, Same as in C, except that the yeast solution was prepared (1) without additional substances, (2) with 3IY added, (3) with 3IY plus L-DOPA (a dopamine precursor), or (4) with L-DOPA only, at the concentrations indicated. Again, relative to control larvae, reduced memory scores were observed in 3IY-fed larvae; feeding them additionally with L-DOPA rescued that memory impairment, leading to scores similar to those of the control animals. L-DOPA alone had no impact on odor-APL memory. The sample sizes and the genotypes are given within the figure. C, D, OSS comparisons to chance levels (i.e., to zero) with a BH correction: \*p<0.05. C, Significant differences between groups in MWW comparisons with a BH correction: \*p<0.05. D, 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 Figures 9-16.</figcaption></figure>

considered the possibility that, unlike the KCs (Fig. 5), the calyx MBONs might actually be activated rather than inhibited by activation of APL, for example, through GABA-induced chloride spikes as reported for insect motoneurons (Ryglewski et al., 2017) or by yet-to-be-identified excitatory transmitters released by APL. We thus repeated the same experiment, but this time activated the MBONs: again, no rewarding effect was observed on such manipulation (Fig. 16B). Before ruling out the involvement of the two calyx MBONs in the rewarding effect of APL, however, it was important to test for the effects of manipulating each of them separately. This is because activation of MBONa1 and MBONa2 induces approach and avoidance, respectively (Eschbach et al., 2021). We therefore reasoned that they might exert a rewarding and punishing effect, respectively, which would sum to zero when both these MBONs were manipulated together. However, neither silencing nor activating either one of the calyx MBONs yielded evidence of such oppositely reinforcing effects (Fig. 16C,D; Extended Data Fig. 16-1); we included groups tested in the presence of light because we reasoned that, similar to what has been observed for gustatory punishment (Gerber and Hendel, 2006; Selcho et al., 2009; Schleyer et al., 2011; Widmann et al., 2016; Weber et al., 2023), this might promote aversive memory expression. These results suggest that the rewarding effect of APL activation does not involve APL-to-MBONa1/a2 connections. Given the role of dopamine in conveying reward signals in larval *Drosophila* (Selcho et al., 2009; Rohwedder et al., 2016; Thoener et al., 2021), we next inquired into the dopamine dependency of APL's rewarding effect.

### Inhibition of dopamine synthesis impairs odor-APL memory

We used a systemic pharmacological approach to acutely disrupt dopamine synthesis. This was done by inhibiting the enzyme tyrosine hydroxylase (TH), which is rate-limiting for dopamine synthesis (Neckameyer and White, 1993; Bainton et al., 2000; Fernandez et al., 2017; Thoener et al., 2021). The TH-inhibitor 3IY was added to the larval food at a dose that

<figure><img src="image_27.png" /><figcaption>**Figure 18.** Ablation of dopaminergic pPAM neurons impairs odor-APL memory. Larvae were trained and tested as in Figure 12A. Optogenetic activation of APL and simultaneous ablation of the pPAM neurons (APL<sub>55008</sub>>ChR2XXL; pPAM>>reaper) reduced odor-APL memory scores relative to genetic controls (APL<sub>55008</sub>>ChR2XXL; pPAM>>+ and APL<sub>55008</sub>>ChR2XXL; >>reaper). The sample sizes and the genotypes are given within the figure. Different letters indicate significant differences between groups in MWW comparisons with a BH correction (p<0.05). \*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. Other details as in Figures 9-17.</figcaption></figure>

leaves intact task-relevant behavioral faculties (i.e., innate odor preference and locomotion) (Thoener et al., 2021). When 4 h later the larvae were trained and tested for odor-APL memory, they exhibited reduced memory scores (Fig. 17A-
A

<figure><img src="image_28.png" /></figure>

B

<figure><img src="image_29.png" /></figure>

C

<figure><img src="image_30.png" /></figure>

D

<figure><img src="image_31.png" /><figcaption>**Figure 19.** Analysis of connectivity from APL, via KCs, to the DANs/OANs/MBINs. A, Fraction of input synapses from APL left to each left-hemisphere KC (APL left >KC left), and from each of these KCs to DAN-i1 left (KC left >DAN-i1 left) and DAN-i1 right (KC left >DAN-i1 right). All APL-KC connections are ipsilateral, excepting the “freak” KC indicated with *. Bottom, The median fraction of input synapses is shown for the synapses from APL left on KCs (APL left >KCs left), and for the KCs synapsing to either DAN-i1 left (KC left >DAN-i1 left) or DAN-i1 right (KC left >DAN-i1 right), or to both DAN-i1 left and right (KC left >DAN-i1 right + left). B, The impact of APL left on the DAN-i1s via the left-hemisphere KCs is estimated by the product of the FI synapses shown in A. Schematic represents the connectivity between APL, DAN-i1, and the KCs. Bottom, The median of these products is shown. The raw data underlying corresponding analyses of the right-hemisphere APL are shown in Extended Data Figure 3-1. C, Products, summed across KCs and hemispheres, as a proxy of the total possible impact of both APLs, via KCs, on the indicated DANs, OANs, and MBINs. D, Bar graphs and color-coded map of the mushroom body based on the data in C, suggesting a stronger negative impact of APL (1) on activity in the input neurons to the vertical lobe (compartments e, f, g) compared with the medial lobe input neurons (compartments i, j, k) (indicated with bold magenta or green edges and filled bars, respectively); (2) on activity in the DL1-DANs (DAN-c1, -d1, -f1, -g1, and MBIN-e2: Selcho et al., 2009; Rohwedder et al., 2016; Saumweber et al., 2018; but see Eichler et al., 2017) compared</figcaption></figure>
A

<figure><img src="image_28.png" /></figure>

B

<figure><img src="image_29.png" /></figure>

C

<figure><img src="image_30.png" /></figure>

D

<figure><img src="image_31.png" /><figcaption>**Figure 19.** Analysis of connectivity from APL, via KCs, to the DANs/OANs/MBINs. A, Fraction of input synapses from APL left to each left-hemisphere KC (APL left >KC left), and from each of these KCs to DAN-i1 left (KC left >DAN-i1 left) and DAN-i1 right (KC left >DAN-i1 right). All APL-KC connections are ipsilateral, excepting the “freak” KC indicated with *. Bottom, The median fraction of input synapses is shown for the synapses from APL left on KCs (APL left >KCs left), and for the KCs synapsing to either DAN-i1 left (KC left >DAN-i1 left) or DAN-i1 right (KC left >DAN-i1 right), or to both DAN-i1 left and right (KC left >DAN-i1 right + left). B, The impact of APL left on the DAN-i1s via the left-hemisphere KCs is estimated by the product of the FI synapses shown in A. Schematic represents the connectivity between APL, DAN-i1, and the KCs. Bottom, The median of these products is shown. The raw data underlying corresponding analyses of the right-hemisphere APL are shown in Extended Data Figure 3-1. C, Products, summed across KCs and hemispheres, as a proxy of the total possible impact of both APLs, via KCs, on the indicated DANs, OANs, and MBINs. D, Bar graphs and color-coded map of the mushroom body based on the data in C, suggesting a stronger negative impact of APL (1) on activity in the input neurons to the vertical lobe (compartments e, f, g) compared with the medial lobe input neurons (compartments i, j, k) (indicated with bold magenta or green edges and filled bars, respectively); (2) on activity in the DL1-DANs (DAN-c1, -d1, -f1, -g1, and MBIN-e2: Selcho et al., 2009; Rohwedder et al., 2016; Saumweber et al., 2018; but see Eichler et al., 2017) compared</figcaption></figure>
C). In a repetition of this experiment, we showed that this reduction in memory was rescued in larvae that were additionally fed with the dopamine precursor L-DOPA (Fig. 17D; notably, L-DOPA alone did not increase memory scores: Fig. 17D). These results suggest that the rewarding effect of APL activation involves a dopaminergic process.

## Ablation of the dopaminergic pPAM neurons impairs odor-APL memory

We next sought to identify the dopaminergic neurons that mediate the rewarding effect of APL activation. Given the role of the dopaminergic pPAM neurons in larval reward learning (Rohwedder et al., 2016; Saumweber et al., 2018; Schleyer et al., 2020; Thoener et al., 2022), we combined the GAL4/UAS with the lexA/lexAop systems to optogenetically activate APL (APL-GAL4>UAS-ChR2XXL) in animals expressing the pro-apoptotic *reaper* gene in the pPAM neurons, leading to their ablation (pPAM-lexA>lexAop-reaper). Larvae of the experimental group (APL<sub>55D08</sub>>ChR2XXL; pPAM>>reaper) showed reduced odor-APL memory scores relative to genetic controls that lacked either the reaper effector or the pPAM driver for ablation (APL<sub>55D08</sub>> ChR2XXL; pPAM>>+ and APL<sub>55D08</sub>>ChR2XXL; >>reaper, respectively) (Fig. 18). Together, these results suggest that the rewarding effect of the activation of APL comes about, in part, by engaging a dopaminergic and pPAM-dependent process.

## Discussion

The present study consolidates and broadens our knowledge of the morphology of the larval APL, of its GABAergic nature and its capacity to inhibit mushroom body KCs, of the polarity and topology of its chemical synapses, its development through metamorphosis, and of the specificity of a transgenic driver strain for studying it (Figs. 2-8) (Masuda-Nakagawa et al., 2014; Eichler et al., 2017; Saumweber et al., 2018). All these findings are consistent with APL playing a role in the sparsening of neuronal activity across the mushroom body (Masuda-Nakagawa et al., 2014; adults: Lin et al., 2014; Amin et al., 2020) and establish APL as one of the most comprehensively described neurons in *Drosophila*. The present study further uncovers unexpected functional complexity by revealing a rewarding effect of optogenetically activating APL (Fig. 12A). Our experiments were then designed to study key features of this effect and how it comes about (Figs. 12C-18).

## Features of the rewarding effect of APL activation

Optogenetic activation of APL induces associative reward learning about odors after only one training trial (Figs. 12-15). This is similar to odor-sugar associative learning and to the rewarding effect of activating DAN-i1 (Weiglein et al., 2019; Thoener et al., 2022). Likewise, similar to fructose and DAN-i1 as rewards (Saumweber et al., 2011; Schleyer et al., 2011, 2018, 2020), activation of APL leads to an increase in odor preference when it is paired with odor presentation and to a decrease in odor preference when it is unpaired from odor presentation (Figs. 12B,

←

with the pPAM-DANs (DAN-i1, -j1, -k1) (magenta or green hatched bars, respectively); and (3) on activity in DAN-f1 compared with DAN-i1 (magenta or green hatched bars, respectively). The absence of DAN-h1 in first-instar larvae is indicated by the gray fill of the h compartment. MBIN-I1 is not considered in these analyses because it innervates multiple compartments in first-instar larvae. Magenta and green represent elements of the system preferentially involved in aversive and appetitive processing, respectively.

15B). In other words, the respective memories are appetitive as they are about the reward: After paired training, the larvae search for the reward where the odor *is*, whereas after unpaired training, adaptively, they search for the reward precisely where the odor is *not*. Associative learning by APL activation has a symmetrical “temporal fingerprint,” with a fairly narrow temporal window for presenting the odor (Fig. 12F,G). This mirrors what was found for DAN-d1 in the aversive domain (Weiglein et al., 2021), adding to the heterogeneity of internal reinforcement signals (Saumweber et al., 2018; Weiglein et al., 2021; Thoener et al., 2022; adults: Aso and Rubin, 2016; König et al., 2018; Handler et al., 2019).

The appetitive memories established by activating APL decay within minutes (Fig. 12E), as do memories for odor-fructose, odor-DAN-i1, and odor-KC association (Neuser et al., 2005; Kleber et al., 2016; Lyutova et al., 2019; Weiglein et al., 2019; Thoener et al., 2022). Similar to odor-fructose memories and odor-DAN-i1 memories (Paisios et al., 2017; Schleyer et al., 2020), APL-induced memories are expressed as modulations of turning, but not of run speed (Fig. 13).

In addition to establishing appetitive associative memory during training, the activation of APL can also terminate learned search behavior during testing (Figs. 14A,B, 15D), without affecting task-relevant innate olfactory behavior (Figs. 14E–G, 15E). This is in line with reports on taste rewards (Schleyer et al., 2015a) and on DAN-i1 activation as a reward (Schleyer et al., 2020).

The rewarding effect of APL activation offers a new perspective on the result from Saumweber et al. (2018), who showed that activating APL throughout the complete training phase abolishes odor-fructose memory scores, an effect that we replicated (Fig. 9A,B). In these experiments, a fructose reward is presented paired with or unpaired from the odor. The protocol of APL activation engages an additional, much stronger reward signal throughout training. Thus, the animals are rewarded regardless of the presence or absence of the odor, which overrides the learning of the relationship between odor and fructose. Thus, the rewarding effect of activating APL is compatible with the previous results from Saumweber et al. (2018), and the resulting appetitive, associative memories resemble those established by DANs or taste reinforcement.

## Possible mechanisms of odor-APL learning: notes upfront

Our results confirm that the larval APL neuron is GABAergic (Fig. 4A-B") (Masuda-Nakagawa et al., 2014). Tests for the presence of octopamine, acetylcholine, and glutamate in the larval APL turned out negative (Masuda-Nakagawa et al., 2014; Eichler et al., 2017). This matches a recent transcriptome analysis in adults (Aso et al., 2019), whereas two earlier reports had suggested the presence of GABA, octopamine, and glutamate in the adult APL (C. L. Wu et al., 2013; H. Li et al., 2017). In the absence of evidence suggesting otherwise for the larval APL, the following discussion considers only GABAergic signaling.

GABA binding to ionotropic GABA-A receptors and the ensuing chloride influx confer the typical inhibitory effect of GABA on postsynaptic neurons. Accordingly, APL activation reduces activity in KCs (Fig. 5G–I). However, effects of GABA via metabotropic receptors may also have a role in postsynaptic activity. In motoneurons of pupal *Drosophila*, moreover, GABA-induced spikelets have been observed, arguably because of a relatively positive reversal potential for chloride during this life stage (Ryglewski et al., 2017; mammals: Ben-Ari, 2002). Thus, it cannot be excluded that GABA release from APL leads to
C). In a repetition of this experiment, we showed that this reduction in memory was rescued in larvae that were additionally fed with the dopamine precursor L-DOPA (Fig. 17D; notably, L-DOPA alone did not increase memory scores: Fig. 17D). These results suggest that the rewarding effect of APL activation involves a dopaminergic process.

## Ablation of the dopaminergic pPAM neurons impairs odor-APL memory

We next sought to identify the dopaminergic neurons that mediate the rewarding effect of APL activation. Given the role of the dopaminergic pPAM neurons in larval reward learning (Rohwedder et al., 2016; Saumweber et al., 2018; Schleyer et al., 2020; Thoener et al., 2022), we combined the GAL4/UAS with the lexA/lexAop systems to optogenetically activate APL (APL-GAL4>UAS-ChR2XXL) in animals expressing the pro-apoptotic *reaper* gene in the pPAM neurons, leading to their ablation (pPAM-lexA>lexAop-reaper). Larvae of the experimental group (APL<sub>55D08</sub>>ChR2XXL; pPAM>>reaper) showed reduced odor-APL memory scores relative to genetic controls that lacked either the reaper effector or the pPAM driver for ablation (APL<sub>55D08</sub>> ChR2XXL; pPAM>>+ and APL<sub>55D08</sub>>ChR2XXL; >>reaper, respectively) (Fig. 18). Together, these results suggest that the rewarding effect of the activation of APL comes about, in part, by engaging a dopaminergic and pPAM-dependent process.

## Discussion

The present study consolidates and broadens our knowledge of the morphology of the larval APL, of its GABAergic nature and its capacity to inhibit mushroom body KCs, of the polarity and topology of its chemical synapses, its development through metamorphosis, and of the specificity of a transgenic driver strain for studying it (Figs. 2-8) (Masuda-Nakagawa et al., 2014; Eichler et al., 2017; Saumweber et al., 2018). All these findings are consistent with APL playing a role in the sparsening of neuronal activity across the mushroom body (Masuda-Nakagawa et al., 2014; adults: Lin et al., 2014; Amin et al., 2020) and establish APL as one of the most comprehensively described neurons in *Drosophila*. The present study further uncovers unexpected functional complexity by revealing a rewarding effect of optogenetically activating APL (Fig. 12A). Our experiments were then designed to study key features of this effect and how it comes about (Figs. 12C-18).

## Features of the rewarding effect of APL activation

Optogenetic activation of APL induces associative reward learning about odors after only one training trial (Figs. 12-15). This is similar to odor-sugar associative learning and to the rewarding effect of activating DAN-i1 (Weiglein et al., 2019; Thoener et al., 2022). Likewise, similar to fructose and DAN-i1 as rewards (Saumweber et al., 2011; Schleyer et al., 2011, 2018, 2020), activation of APL leads to an increase in odor preference when it is paired with odor presentation and to a decrease in odor preference when it is unpaired from odor presentation (Figs. 12B,

←

with the pPAM-DANs (DAN-i1, -j1, -k1) (magenta or green hatched bars, respectively); and (3) on activity in DAN-f1 compared with DAN-i1 (magenta or green hatched bars, respectively). The absence of DAN-h1 in first-instar larvae is indicated by the gray fill of the h compartment. MBIN-h1 is not considered in these analyses because it innervates multiple compartments in first-instar larvae. Magenta and green represent elements of the system preferentially involved in aversive and appetitive processing, respectively.

15B). In other words, the respective memories are appetitive as they are about the reward: After paired training, the larvae search for the reward where the odor *is*, whereas after unpaired training, adaptively, they search for the reward precisely where the odor is *not*. Associative learning by APL activation has a symmetrical “temporal fingerprint,” with a fairly narrow temporal window for presenting the odor (Fig. 12F,G). This mirrors what was found for DAN-d1 in the aversive domain (Weiglein et al., 2021), adding to the heterogeneity of internal reinforcement signals (Saumweber et al., 2018; Weiglein et al., 2021; Thoener et al., 2022; adults: Aso and Rubin, 2016; König et al., 2018; Handler et al., 2019).

The appetitive memories established by activating APL decay within minutes (Fig. 12E), as do memories for odor-fructose, odor-DAN-i1, and odor-KC association (Neuser et al., 2005; Kleber et al., 2016; Lyutova et al., 2019; Weiglein et al., 2019; Thoener et al., 2022). Similar to odor-fructose memories and odor-DAN-i1 memories (Paisios et al., 2017; Schleyer et al., 2020), APL-induced memories are expressed as modulations of turning, but not of run speed (Fig. 13).

In addition to establishing appetitive associative memory during training, the activation of APL can also terminate learned search behavior during testing (Figs. 14A,B, 15D), without affecting task-relevant innate olfactory behavior (Figs. 14E–G, 15E). This is in line with reports on taste rewards (Schleyer et al., 2015a) and on DAN-i1 activation as a reward (Schleyer et al., 2020).

The rewarding effect of APL activation offers a new perspective on the result from Saumweber et al. (2018), who showed that activating APL throughout the complete training phase abolishes odor-fructose memory scores, an effect that we replicated (Fig. 9A,B). In these experiments, a fructose reward is presented paired with or unpaired from the odor. The protocol of APL activation engages an additional, much stronger reward signal throughout training. Thus, the animals are rewarded regardless of the presence or absence of the odor, which overrides the learning of the relationship between odor and fructose. Thus, the rewarding effect of activating APL is compatible with the previous results from Saumweber et al. (2018), and the resulting appetitive, associative memories resemble those established by DANs or taste reinforcement.

## Possible mechanisms of odor-APL learning: notes upfront

Our results confirm that the larval APL neuron is GABAergic (Fig. 4A-B") (Masuda-Nakagawa et al., 2014). Tests for the presence of octopamine, acetylcholine, and glutamate in the larval APL turned out negative (Masuda-Nakagawa et al., 2014; Eichler et al., 2017). This matches a recent transcriptome analysis in adults (Aso et al., 2019), whereas two earlier reports had suggested the presence of GABA, octopamine, and glutamate in the adult APL (C. L. Wu et al., 2013; H. Li et al., 2017). In the absence of evidence suggesting otherwise for the larval APL, the following discussion considers only GABAergic signaling.

GABA binding to ionotropic GABA-A receptors and the ensuing chloride influx confer the typical inhibitory effect of GABA on postsynaptic neurons. Accordingly, APL activation reduces activity in KCs (Fig. 5G–I). However, effects of GABA via metabotropic receptors may also have a role in postsynaptic activity. In motoneurons of pupal *Drosophila*, moreover, GABA-induced spikelets have been observed, arguably because of a relatively positive reversal potential for chloride during this life stage (Ryglewski et al., 2017; mammals: Ben-Ari, 2002). Thus, it cannot be excluded that GABA release from APL leads to
<table><caption>Table 2. All statistical results grouped by figure number, figure panel, and statistical tests</caption><thead><tr><th>Figure</th><th>Panel</th><th>Data</th><th>Statistical test</th><th>Result</th></tr></thead><tbody><tr><td>3</td><td>E</td><td>Mean pixel intensity right (x) vs left (y) hemisphere</td><td>Pearson correlation</td><td>r = 0.974, p &lt; 0.0001</td></tr><tr><td rowspan="5">5</td><td>B</td><td>+&gt;ChR2XXL; KC&gt;GCaMP6m vs APL<sub>26602</sub>&gt;ChR2XXL; KC&gt;GCaMP6m</td><td rowspan="5">MWW</td><td>U = 284.5, p = 0.238<br>U = 257, p = 0.090<br>U = 108, p &lt; 0.001<br>U = 119, p &lt; 0.001<br>U = 429, p &lt; 0.01<br>U = 415, p = 0.04</td></tr><tr><td>C</td><td></td></tr><tr><td>E</td><td></td></tr><tr><td>F</td><td></td></tr><tr><td>H</td><td></td></tr><tr><td>I</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="10">6</td><td rowspan="10">D</td><td>APL left: axon; dendrite; neurite</td><td>KW</td><td>H (2, 5924) = 1008.2, p &lt; 0.001</td></tr><tr><td>APL left: axon vs dendrite</td><td rowspan="3">MWW with BH correction</td><td>U = 1558487, p &lt; 0.001<br>U = 74037, p = 0.9821<br>U = 27809, p &lt; 0.001</td></tr><tr><td>APL left: axon vs neurite</td><td></td></tr><tr><td>APL left: dendrite vs neurite</td><td></td></tr><tr><td>APL right: axon; dendrite; neurite</td><td>KW</td><td>H (2, 8477) = 1940.9, p &lt; 0.001</td></tr><tr><td>APL right: axon vs dendrite</td><td rowspan="3">MWW with BH correction</td><td>U = 3097808, p &lt; 0.001<br>U = 131161, p &lt; 0.01<br>U = 45183, p &lt; 0.001</td></tr><tr><td>APL right: axon vs neurite</td><td></td></tr><tr><td>APL right: dendrite vs neurite</td><td></td></tr><tr><td>APL left -&gt;KC vs KC -&gt;APL left</td><td>MWW</td><td>U = 27236.5, p &lt; 0.001</td></tr><tr><td>APL right -&gt;KC vs KC -&gt;APL right</td><td></td><td>U = 50181.5, p &lt; 0.001</td></tr><tr><td rowspan="10">7; Extended Data Figure 7-4<br>Extended Data Figure 7-4</td><td rowspan="10">A</td><td>APL-&gt;ChR2XXL; +&gt;ChR2XXL; APL-&gt; +</td><td>KW</td><td>H (2, 36) = 17.353, p &lt; 0.001</td></tr><tr><td>APL-&gt;ChR2XXL vs +&gt;ChR2XXL</td><td rowspan="3">MWW with BH correction</td><td>U = 5, p &lt; 0.001<br>U = 15, p = 0.001<br>U = 62, p = 0.583</td></tr><tr><td>APL-&gt;ChR2XXL vs APL-&gt; +<br>+&gt;ChR2XXL vs APL-&gt; +</td><td></td></tr><tr><td>APL-&gt;ChR2XXL vs 0<br>+&gt;ChR2XXL vs 0<br>APL-&gt; + vs 0</td><td>OSS test with BH correction</td><td>p = 0.146<br>p &lt; 0.001<br>p = 0.006</td></tr><tr><td rowspan="5">B</td><td>APL-&gt;ChR2XXL; +&gt;ChR2XXL; APL-&gt; +</td><td>KW</td><td>H (2, 90) = 28.063, p &lt; 0.001</td></tr><tr><td>APL-&gt;ChR2XXL vs +&gt;ChR2XXL</td><td rowspan="3">MWW with BH correction</td><td>U = 119, p &lt; 0.001<br>U = 165, p &lt; 0.001<br>U = 421, p = 0.673</td></tr><tr><td>APL-&gt;ChR2XXL vs APL-&gt; +<br>+&gt;ChR2XXL vs APL-&gt; +</td><td></td></tr><tr><td>APL-&gt;ChR2XXL vs 0<br>+&gt;ChR2XXL vs 0<br>APL-&gt; + vs 0</td><td>OSS test with BH correction</td><td>p = 1<br>p &lt; 0.001<br>p &lt; 0.001</td></tr><tr><td>D</td><td>No light vs light</td><td>MWW</td><td>U = 389.5, p = 0.974<br>U = 313, p = 0.381<br>U = 363, p = 0.993<br>U = 363, p = 0.993<br>U = 337, p = 0.646</td></tr><tr><td>F</td><td></td><td></td><td></td><td></td></tr><tr><td>G</td><td></td><td></td><td></td><td></td></tr><tr><td>H</td><td></td><td></td><td></td><td></td></tr><tr><td>I</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="10">10</td><td rowspan="10">A</td><td>No light; light odor trial; light nonodor trial; no light test@fru; light odor trial test@fru; light nonodor trial test@fru</td><td>KW</td><td>H (5, 150) = 56.412, p &lt; 0.001</td></tr><tr><td>No light vs light odor trial</td><td rowspan="3">MWW with BH correction</td><td>U = 88, p &lt; 0.001<br>U = 119, p &lt; 0.001<br>U = 292.5, p = 0.705</td></tr><tr><td>No light vs light nonodor trial</td><td></td></tr><tr><td>Light odor trial vs light nonodor trial</td><td></td></tr><tr><td>No light test@fru vs light odor trial test@fru</td><td></td><td>U = 291, p = 0.683<br>U = 292, p = 0.700<br>U = 259, p = 0.304</td></tr><tr><td>No light vs no light test@fru</td><td></td><td>U = 54, p &lt; 0.001<br>U = 162, p = 0.004<br>U = 146, p = 0.001</td></tr><tr><td>Light odor trial vs light odor trial test@fru</td><td></td><td>OSS test with BH correction</td><td>p &lt; 0.001<br>p &lt; 0.001<br>p = 0.004<br>p = 1</td></tr><tr><td>Light nonodor trial vs light nonodor trial test@fru</td><td></td><td></td><td>p = 0.690<br>p = 1</td></tr><tr><td>No light vs 0</td><td></td><td></td><td></td></tr><tr><td>Light odor trial vs 0</td><td></td><td></td><td></td></tr><tr><td>Light nonodor trial vs 0</td><td></td><td></td><td></td></tr><tr><td>No light test@fru vs 0</td><td></td><td></td><td></td></tr><tr><td>Light odor trial test@fru vs 0</td><td></td><td></td><td></td></tr><tr><td>Light nonodor trial test@fru vs 0</td><td></td><td></td><td></td></tr><tr><td rowspan="10">11</td><td rowspan="10">B</td><td>PREF no light; no light test@fru</td><td>KW</td><td>H (3, 100) = 51.684, p &lt; 0.001</td></tr><tr><td>PREF paired vs unpaired no light</td><td rowspan="3">MWW with BH correction</td><td>U = 10.5, p &lt; 0.001<br>U = 308.5, p = 0.946<br>U = 228, p &lt; 0.001</td></tr><tr><td>PREF paired vs unpaired light test@fru</td><td></td></tr><tr><td>PREF paired no light vs baseline</td><td></td></tr><tr><td>PREF unpaired no light vs baseline</td><td></td><td>U = 155, p &lt; 0.001</td></tr><tr><td>PREF light odor trial; light odor trial test@fru</td><td>KW</td><td>H (3, 100) = 22.014, p &lt; 0.001</td></tr><tr><td>PREF paired vs unpaired light odor trial</td><td rowspan="3">MWW with BH correction</td><td>U = 73, p &lt; 0.001<br>U = 272, p = 0.438<br>U = 431, p = 0.029</td></tr><tr><td>PREF paired vs unpaired light odor trial test@fru</td><td></td></tr><tr><td>PREF paired light odor trial vs baseline</td><td></td></tr><tr><td>PREF unpaired light odor trial vs baseline</td><td></td><td>U = 358, p = 0.002</td></tr><tr><td>C</td><td>PREF light nonodor trial; light nonodor trial test@fru</td><td>KW</td><td>H (3, 100) = 14.67, p = 0.002</td></tr></tbody></table>

(Table continues)
<table><caption>Table 2. All statistical results grouped by figure number, figure panel, and statistical tests</caption><thead><tr><th>Figure</th><th>Panel</th><th>Data</th><th>Statistical test</th><th>Result</th></tr></thead><tbody><tr><td>3</td><td>E</td><td>Mean pixel intensity right (x) vs left (y) hemisphere</td><td>Pearson correlation</td><td>r = 0.974, p &lt; 0.0001</td></tr><tr><td rowspan="6">5</td><td>B</td><td>+&gt;ChR2XXL; KC&gt;GCaMP6m vs APL<sub>26602</sub>&gt;ChR2XXL; KC&gt;GCaMP6m</td><td rowspan="6">MWW</td><td>U = 284.5, p = 0.238<br>U = 257, p = 0.090<br>U = 108, p &lt; 0.001<br>U = 119, p &lt; 0.001<br>U = 429, p &lt; 0.01<br>U = 415, p = 0.04</td></tr><tr><td>C</td><td></td></tr><tr><td>E</td><td></td></tr><tr><td>F</td><td></td></tr><tr><td>H</td><td></td></tr><tr><td>I</td><td></td></tr><tr><td rowspan="12">6</td><td rowspan="12">D</td><td>APL left: axon; dendrite; neurite</td><td>KW</td><td>H (2, 5924) = 1008.2, p &lt; 0.001</td></tr><tr><td>APL left: axon vs dendrite</td><td rowspan="4">MWW with BH correction</td><td>U = 1558487, p &lt; 0.001<br>U = 74037, p = 0.9821<br>U = 27809, p &lt; 0.001</td></tr><tr><td>APL left: axon vs neurite</td><td></td></tr><tr><td>APL left: dendrite vs neurite</td><td></td></tr><tr><td>APL right: axon; dendrite; neurite</td><td>KW</td><td>H (2, 8477) = 1940.9, p &lt; 0.001</td></tr><tr><td>APL right: axon vs dendrite</td><td rowspan="3">MWW with BH correction</td><td>U = 3097808, p &lt; 0.001<br>U = 131161, p &lt; 0.01<br>U = 45183, p &lt; 0.001</td></tr><tr><td>APL right: axon vs neurite</td><td></td></tr><tr><td>APL right: dendrite vs neurite</td><td></td></tr><tr><td>APL left -&gt;KC vs KC -&gt;APL left</td><td>MWW</td><td>U = 27236.5, p &lt; 0.001</td></tr><tr><td>APL right -&gt;KC vs KC -&gt;APL right</td><td></td><td>U = 50181.5, p &lt; 0.001</td></tr><tr><td rowspan="12">7; Extended Data Figure 7-4<br>Extended Data Figure 7-4</td><td rowspan="12">A</td><td>APL-&gt;ChR2XXL; +&gt;ChR2XXL; APL-&gt; +</td><td>KW</td><td>H (2, 36) = 17.353, p &lt; 0.001</td></tr><tr><td>APL-&gt;ChR2XXL vs +&gt;ChR2XXL</td><td rowspan="4">MWW with BH correction</td><td>U = 5, p &lt; 0.001<br>U = 15, p = 0.001<br>U = 62, p = 0.583</td></tr><tr><td>APL-&gt;ChR2XXL vs APL-&gt; +<br>+&gt;ChR2XXL vs APL-&gt; +</td><td></td></tr><tr><td>APL-&gt;ChR2XXL vs 0<br>+&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p = 0.146<br>p &lt; 0.001<br>p = 0.006</td></tr><tr><td>APL-&gt; + vs 0</td><td></td></tr><tr><td rowspan="6">B</td><td>APL-&gt;ChR2XXL; +&gt;ChR2XXL; APL-&gt; +</td><td>KW</td><td>H (2, 90) = 28.063, p &lt; 0.001</td></tr><tr><td>APL-&gt;ChR2XXL vs +&gt;ChR2XXL</td><td rowspan="4">MWW with BH correction</td><td>U = 119, p &lt; 0.001<br>U = 165, p &lt; 0.001<br>U = 421, p = 0.673</td></tr><tr><td>APL-&gt;ChR2XXL vs APL-&gt; +<br>+&gt;ChR2XXL vs APL-&gt; +</td><td></td></tr><tr><td>APL-&gt;ChR2XXL vs 0<br>+&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p = 1<br>p &lt; 0.001<br>p &lt; 0.001</td></tr><tr><td>APL-&gt; + vs 0</td><td></td></tr><tr><td rowspan="6">D</td><td rowspan="6">F</td><td>No light vs light</td><td rowspan="6">MWW</td><td>U = 389.5, p = 0.974<br>U = 313, p = 0.381<br>U = 363, p = 0.993<br>U = 363, p = 0.993<br>U = 337, p = 0.646</td></tr><tr><td>G</td><td></td></tr><tr><td>H</td><td></td></tr><tr><td>I</td><td></td></tr><tr><td rowspan="12">10</td><td rowspan="12">A</td><td>No light; light odor trial; light nonodor trial; no light test@fru; light odor trial test@fru; light nonodor trial test@fru</td><td>KW</td><td>H (5, 150) = 56.412, p &lt; 0.001</td></tr><tr><td>No light vs light odor trial</td><td rowspan="4">MWW with BH correction</td><td>U = 88, p &lt; 0.001<br>U = 119, p &lt; 0.001<br>U = 292.5, p = 0.705<br>U = 291, p = 0.683</td></tr><tr><td>No light vs light nonodor trial</td><td></td></tr><tr><td>Light odor trial vs light nonodor trial</td><td></td></tr><tr><td>No light test@fru vs light odor trial test@fru</td><td></td></tr><tr><td>No light test@fru vs light nonodor trial test@fru</td><td></td><td>U = 292, p = 0.700<br>U = 259, p = 0.304<br>U = 54, p &lt; 0.001<br>U = 162, p = 0.004<br>U = 146, p = 0.001</td></tr><tr><td>Light odor trial test@fru vs light nonodor trial test@fru</td><td></td><td></td></tr><tr><td>No light vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.001<br>p &lt; 0.001<br>p = 0.004<br>p = 1<br>p = 0.690<br>p = 1</td></tr><tr><td>Light odor trial vs 0</td><td></td></tr><tr><td>Light nonodor trial vs 0</td><td></td></tr><tr><td>No light test@fru vs 0</td><td></td></tr><tr><td>Light odor trial test@fru vs 0</td><td></td></tr><tr><td>Light nonodor trial test@fru vs 0</td><td></td></tr><tr><td rowspan="12">11</td><td rowspan="12">B</td><td>PREF no light; no light test@fru</td><td>KW</td><td>H (3, 100) = 51.684, p &lt; 0.001</td></tr><tr><td>PREF paired vs unpaired no light</td><td rowspan="4">MWW with BH correction</td><td>U = 10.5, p &lt; 0.001<br>U = 308.5, p = 0.946<br>U = 228, p &lt; 0.001<br>U = 155, p &lt; 0.001</td></tr><tr><td>PREF paired vs unpaired light test@fru</td><td></td></tr><tr><td>PREF paired no light vs baseline</td><td></td></tr><tr><td>PREF unpaired no light vs baseline</td><td></td></tr><tr><td>PREF light odor trial; light odor trial test@fru</td><td>KW</td><td>H (3, 100) = 22.014, p &lt; 0.001</td></tr><tr><td>PREF paired vs unpaired light odor trial</td><td rowspan="4">MWW with BH correction</td><td>U = 73, p &lt; 0.001<br>U = 272, p = 0.438<br>U = 431, p = 0.029<br>U = 358, p = 0.002</td></tr><tr><td>PREF paired vs unpaired light odor trial test@fru</td><td></td></tr><tr><td>PREF paired light odor trial vs baseline</td><td></td></tr><tr><td>PREF unpaired light odor trial vs baseline</td><td></td></tr><tr><td rowspan="2">C</td><td rowspan="2">C</td><td>PREF light nonodor trial; light nonodor trial test@fru</td><td>KW</td><td>H (3, 100) = 14.67, p = 0.002</td></tr><tr><td></td><td></td><td></td></tr></tbody></table>

(Table continues)
<table><caption>Table 2. Continued</caption><thead><tr><th>Figure</th><th>Panel</th><th>Data</th><th>Statistical test</th><th>Result</th></tr></thead><tbody><tr><td rowspan="12">12</td><td rowspan="12">A</td><td>PREF paired vs unpaired light nonodor trial</td><td>MWW with BH correction</td><td>U = 137, p &lt; 0.001</td></tr><tr><td>PREF paired vs unpaired light nonodor trial test@fru</td><td>MWW with BH correction</td><td>U = 264.5, p = 0.356</td></tr><tr><td>PREF paired light nonodor trial vs baseline</td><td>MWW with BH correction</td><td>U = 419.5, p = 0.021</td></tr><tr><td>PREF unpaired light nonodor trial vs baseline</td><td>MWW with BH correction</td><td>U = 408, p = 0.015</td></tr><tr><td>PREF no light test@fru; light odor trial test@fru; light nonodor trial test@fru</td><td>KW</td><td>H (2, 150) = 88.073, p &lt; 0.001</td></tr><tr><td>No light test@fru vs light odor trial test@fru</td><td>MWW with BH correction</td><td>U = 513, p &lt; 0.001</td></tr><tr><td>No light test@fru vs light nonodor trial test@fru</td><td>MWW with BH correction</td><td>U = 376, p &lt; 0.001</td></tr><tr><td>Light odor trial test@fru vs light nonodor trial test@fru</td><td>MWW with BH correction</td><td>U = 20.5, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt; +</td><td>KW</td><td>H (2, 36) = 22.524, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs +&gt;ChR2XXL</td><td>MWW with BH correction</td><td>U = 0, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 3, p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 64, p = 0.665</td></tr><tr><td>APL&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p = 0.387</td></tr><tr><td>APL&gt; + vs 0</td><td>OSS test with BH correction</td><td>p = 1</td></tr><tr><td rowspan="4">B</td><td>APL&gt;ChR2XXL paired; APL&gt;ChR2XXL unpaired; +&gt;ChR2XXL paired; +&gt;ChR2XXL unpaired; APL&gt; + paired; APL&gt; + unpaired</td><td>KW</td><td>H (5, 72) = 30.41, p &lt; 0.0001</td></tr><tr><td>APL&gt;ChR2XXL paired vs baseline</td><td>MWW with BH correction</td><td>U = 49, p &lt; 0.0001</td></tr><tr><td>APL&gt;ChR2XXL unpaired vs baseline</td><td>MWW with BH correction</td><td>U = 117.5, p &lt; 0.002</td></tr><tr><td>+&gt;ChR2XXL paired vs +&gt;ChR2XXL unpaired</td><td>MWW with BH correction</td><td>U = 64.00, p = 0.665</td></tr><tr><td>APL&gt; + paired vs APL&gt; + unpaired</td><td>MWW with BH correction</td><td>U = 69.50, p = 0.908</td></tr><tr><td rowspan="5">C</td><td>APL&gt;GtACR1; +&gt;GtACR1; APL&gt; +</td><td>KW</td><td>H (2, 72) = 7.756, p = 0.02</td></tr><tr><td>APL&gt;GtACR1 vs +&gt;GtACR1</td><td>MWW with BH correction</td><td>U = 165, p = 0.011</td></tr><tr><td>APL&gt;GtACR1 vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 178, p = 0.023</td></tr><tr><td>+&gt;GtACR1 vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 287.5, p = 0.990</td></tr><tr><td>APL&gt;GtACR1 vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.01</td></tr><tr><td rowspan="3">D</td><td>+&gt;GtACR1 vs 0</td><td>OSS test with BH correction</td><td>p = 0.840</td></tr><tr><td>APL&gt; + vs 0</td><td>OSS test with BH correction</td><td>p = 0.670</td></tr><tr><td>APL&gt;GtACR1 paired; APL&gt;GtACR1 unpaired; +&gt;GtACR1 paired; +&gt;GtACR1 unpaired; APL&gt; + paired; APL&gt; + unpaired</td><td>KW</td><td>H (5, 144) = 10.650, p = 0.059</td></tr><tr><td rowspan="10">E</td><td>Retention interval 0; 5; 10; 20 min</td><td>KW</td><td>H (3, 120) = 51.554, p &lt; 0.001</td></tr><tr><td>0 vs 5 min</td><td>MWW with BH correction</td><td>U = 207, p &lt; 0.001</td></tr><tr><td>0 vs 10 min</td><td>MWW with BH correction</td><td>U = 93, p &lt; 0.001</td></tr><tr><td>0 vs 20 min</td><td>MWW with BH correction</td><td>U = 24, p &lt; 0.001</td></tr><tr><td>0 min vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>5 min vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>10 min vs 0</td><td>OSS test with BH correction</td><td>p = 0.585</td></tr><tr><td>20 min vs 0</td><td>OSS test with BH correction</td><td>p = 0.585</td></tr><tr><td>ISI &lt; 120; &lt; 60; &lt; 30; &lt; 10; 0; 10; 30; 60; 120</td><td>KW</td><td>H (8, 216) = 23.4, p = 0.003</td></tr><tr><td rowspan="5">F</td><td>APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt; +</td><td>KW</td><td>H (2, 36) = 13.839, p = 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs +&gt;ChR2XXL</td><td>MWW with BH correction</td><td>U = 21, p = 0.003</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 14, p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 58, p = 0.436</td></tr><tr><td>APL&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p = 0.006</td></tr><tr><td rowspan="2">G</td><td>+&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p = 1</td></tr><tr><td>APL&gt; + vs 0</td><td>OSS test with BH correction</td><td>p = 0.388</td></tr><tr><td>13</td><td>C</td><td>Paired vs unpaired</td><td>MWW</td><td>U = 399, p &lt; 0.001</td></tr><tr><td>D</td><td></td><td></td><td>MWW</td><td>U = 270, p &lt; 0.001</td></tr><tr><td>E</td><td></td><td></td><td>MWW</td><td>U = 419, p &lt; 0.001</td></tr><tr><td>F</td><td></td><td></td><td>MWW</td><td>U = 791, p = 0.104</td></tr><tr><td rowspan="10">14</td><td rowspan="10">A</td><td>APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt; +; APL&gt;ChR2XXL test@light; +&gt;ChR2XXL test@light; APL&gt; + test@light</td><td>KW</td><td>H (5, 96) = 37.772, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs +&gt;ChR2XXL</td><td>MWW with BH correction</td><td>U = 7, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 17, p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 103, p = 0.356</td></tr><tr><td>APL&gt;ChR2XXL test@light vs +&gt;ChR2XXL test@light</td><td>MWW with BH correction</td><td>U = 94, p = 0.207</td></tr><tr><td>APL&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>MWW with BH correction</td><td>U = 68, p = 0.025</td></tr><tr><td>+&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>MWW with BH correction</td><td>U = 103, p = 0.356</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt;ChR2XXL test@light</td><td>MWW with BH correction</td><td>U = 10, p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs +&gt;ChR2XXL test@light</td><td>MWW with BH correction</td><td>U = 122, p = 0.836</td></tr><tr><td>APL&gt; + vs APL&gt; + test@light</td><td>MWW with BH correction</td><td>U = 127, p = 0.985</td></tr><tr><td>APL&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p = 0.804</td></tr><tr><td>APL&gt; + vs 0</td><td>OSS test with BH correction</td><td>p = 0.210</td></tr></tbody></table>

(Table continues)
<table><caption>Table 2. Continued</caption><thead><tr><th>Figure</th><th>Panel</th><th>Data</th><th>Statistical test</th><th>Result</th></tr></thead><tbody><tr><td rowspan="12">12</td><td rowspan="12">A</td><td>PREF paired vs unpaired light nonodor trial</td><td>MWW with BH correction</td><td>U = 137, p &lt; 0.001</td></tr><tr><td>PREF paired vs unpaired light nonodor trial test@fru</td><td>MWW with BH correction</td><td>U = 264.5, p = 0.356</td></tr><tr><td>PREF paired light nonodor trial vs baseline</td><td>MWW with BH correction</td><td>U = 419.5, p = 0.021</td></tr><tr><td>PREF unpaired light nonodor trial vs baseline</td><td>MWW with BH correction</td><td>U = 408, p = 0.015</td></tr><tr><td>PREF no light test@fru; light odor trial test@fru; light nonodor trial test@fru</td><td>KW</td><td>H (2, 150) = 88.073, p &lt; 0.001</td></tr><tr><td>No light test@fru vs light odor trial test@fru</td><td>MWW with BH correction</td><td>U = 513, p &lt; 0.001</td></tr><tr><td>No light test@fru vs light nonodor trial test@fru</td><td>MWW with BH correction</td><td>U = 376, p &lt; 0.001</td></tr><tr><td>Light odor trial test@fru vs light nonodor trial test@fru</td><td>MWW with BH correction</td><td>U = 20.5, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL; + &gt;ChR2XXL; APL&gt; +</td><td>KW</td><td>H (2, 36) = 22.524, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs + &gt;ChR2XXL</td><td>MWW with BH correction</td><td>U = 0, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 3, p &lt; 0.001</td></tr><tr><td>+ &gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 64, p = 0.665</td></tr><tr><td>APL&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>+ &gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p = 0.387</td></tr><tr><td>APL&gt; + vs 0</td><td>OSS test with BH correction</td><td>p = 1</td></tr><tr><td rowspan="4">B</td><td>APL&gt;ChR2XXL paired; APL&gt;ChR2XXL unpaired; + &gt;ChR2XXL paired; + &gt;ChR2XXL unpaired; APL&gt; + paired; APL&gt; + unpaired</td><td>KW</td><td>H (5, 72) = 30.41, p &lt; 0.0001</td></tr><tr><td>APL&gt;ChR2XXL paired vs baseline</td><td>MWW with BH correction</td><td>U = 49, p &lt; 0.0001</td></tr><tr><td>APL&gt;ChR2XXL unpaired vs baseline</td><td>MWW with BH correction</td><td>U = 117.5, p &lt; 0.002</td></tr><tr><td>+ &gt;ChR2XXL paired vs + &gt;ChR2XXL unpaired</td><td>MWW with BH correction</td><td>U = 64.00, p = 0.665</td></tr><tr><td>APL&gt; + paired vs APL&gt; + unpaired</td><td>MWW with BH correction</td><td>U = 69.50, p = 0.908</td></tr><tr><td rowspan="5">C</td><td>APL&gt;GtACR1; + &gt;GtACR1; APL&gt; +</td><td>KW</td><td>H (2, 72) = 7.756, p = 0.02</td></tr><tr><td>APL&gt;GtACR1 vs + &gt;GtACR1</td><td>MWW with BH correction</td><td>U = 165, p = 0.011</td></tr><tr><td>APL&gt;GtACR1 vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 178, p = 0.023</td></tr><tr><td>+ &gt;GtACR1 vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 287.5, p = 0.990</td></tr><tr><td>APL&gt;GtACR1 vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.01</td></tr><tr><td rowspan="3">D</td><td>+ &gt;GtACR1 vs 0</td><td>OSS test with BH correction</td><td>p = 0.840</td></tr><tr><td>APL&gt; + vs 0</td><td>OSS test with BH correction</td><td>p = 0.670</td></tr><tr><td>APL&gt;GtACR1 paired; APL&gt;GtACR1 unpaired; + &gt;GtACR1 paired; + &gt;GtACR1 unpaired; APL&gt; + paired; APL&gt; + unpaired</td><td>KW</td><td>H (5, 144) = 10.650, p = 0.059</td></tr><tr><td rowspan="10">E</td><td>Retention interval 0; 5; 10; 20 min</td><td>KW</td><td>H (3, 120) = 51.554, p &lt; 0.001</td></tr><tr><td>0 vs 5 min</td><td>MWW with BH correction</td><td>U = 207, p &lt; 0.001</td></tr><tr><td>0 vs 10 min</td><td>MWW with BH correction</td><td>U = 93, p &lt; 0.001</td></tr><tr><td>0 vs 20 min</td><td>MWW with BH correction</td><td>U = 24, p &lt; 0.001</td></tr><tr><td>0 min vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>5 min vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>10 min vs 0</td><td>OSS test with BH correction</td><td>p = 0.585</td></tr><tr><td>20 min vs 0</td><td>OSS test with BH correction</td><td>p = 0.585</td></tr><tr><td>ISI &lt;120; &lt;60; &lt;30; &lt;10; 0; 10; 30; 60; 120</td><td>KW</td><td>H (8, 216) = 23.4, p = 0.003</td></tr><tr><td rowspan="5">F</td><td>APL&gt;ChR2XXL; + &gt;ChR2XXL; APL&gt; +</td><td>KW</td><td>H (2, 36) = 13.839, p = 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs + &gt;ChR2XXL</td><td>MWW with BH correction</td><td>U = 21, p = 0.003</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 14, p &lt; 0.001</td></tr><tr><td>+ &gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 58, p = 0.436</td></tr><tr><td>APL&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p = 0.006</td></tr><tr><td rowspan="2">G</td><td>+ &gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p = 1</td></tr><tr><td>APL&gt; + vs 0</td><td>OSS test with BH correction</td><td>p = 0.388</td></tr><tr><td>13</td><td>C</td><td>Paired vs unpaired</td><td>MWW</td><td>U = 399, p &lt; 0.001</td></tr><tr><td>D</td><td></td><td></td><td>MWW</td><td>U = 270, p &lt; 0.001</td></tr><tr><td>E</td><td></td><td></td><td>MWW</td><td>U = 419, p &lt; 0.001</td></tr><tr><td>F</td><td></td><td></td><td>MWW</td><td>U = 791, p = 0.104</td></tr><tr><td rowspan="10">14</td><td rowspan="10">A</td><td>APL&gt;ChR2XXL; + &gt;ChR2XXL; APL&gt; +; APL&gt;ChR2XXL test@light; + &gt;ChR2XXL test@light; APL&gt; + test@light</td><td>KW</td><td>H (5, 96) = 37.772, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs + &gt;ChR2XXL</td><td>MWW with BH correction</td><td>U = 7, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 17, p &lt; 0.001</td></tr><tr><td>+ &gt;ChR2XXL vs APL&gt; +</td><td>MWW with BH correction</td><td>U = 103, p = 0.356</td></tr><tr><td>APL&gt;ChR2XXL test@light vs + &gt;ChR2XXL test@light</td><td>MWW with BH correction</td><td>U = 94, p = 0.207</td></tr><tr><td>APL&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>MWW with BH correction</td><td>U = 68, p = 0.025</td></tr><tr><td>+ &gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>MWW with BH correction</td><td>U = 103, p = 0.356</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt;ChR2XXL test@light</td><td>MWW with BH correction</td><td>U = 10, p &lt; 0.001</td></tr><tr><td>+ &gt;ChR2XXL vs + &gt;ChR2XXL test@light</td><td>MWW with BH correction</td><td>U = 122, p = 0.836</td></tr><tr><td>APL&gt; + vs APL&gt; + test@light</td><td>MWW with BH correction</td><td>U = 127, p = 0.985</td></tr><tr><td>APL&gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>+ &gt;ChR2XXL vs 0</td><td>OSS test with BH correction</td><td>p = 0.804</td></tr><tr><td>APL&gt; + vs 0</td><td>OSS test with BH correction</td><td>p = 0.210</td></tr></tbody></table>

(Table continues)
<table><caption>Table 2. Continued</caption><thead><tr><th>Figure</th><th>Panel</th><th>Data</th><th>Statistical test</th><th>Result</th></tr></thead><tbody><tr><td></td><td></td><td>APL&gt;ChR2XXL test@light vs 0</td><td></td><td>p = 0.210</td></tr><tr><td></td><td></td><td>+&gt;ChR2XXL test@light vs 0</td><td></td><td>p = 0.210</td></tr><tr><td></td><td></td><td>APL&gt;+ test@light vs 0</td><td></td><td>p = 0.210</td></tr><tr><td rowspan="15">B</td><td rowspan="15">APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt;+; APL&gt;ChR2XXL test@light; +&gt;ChR2XXL test@light; APL&gt;+ test@light</td><td>APL&gt;ChR2XXL vs +&gt;ChR2XXL</td><td rowspan="5">MWW with BH correction</td><td>U = 15, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>U = 12, p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs APL&gt; +</td><td>U = 109, p = 0.486</td></tr><tr><td>APL&gt;ChR2XXL test@light vs +&gt;ChR2XXL test@light</td><td>U = 121, p = 0.806</td></tr><tr><td>APL&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 88, p = 0.136</td></tr><tr><td>+&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 90, p = 0.157</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt;ChR2XXL test@light</td><td>U = 38, p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs +&gt;ChR2XXL test@light</td><td>U = 119, p = 0.749</td></tr><tr><td>APL&gt;+ vs APL&gt; + test@light</td><td>U = 126, p = 0.955</td></tr><tr><td>APL&gt;ChR2XXL vs 0</td><td rowspan="5">OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs 0</td><td>p = 0.804</td></tr><tr><td>APL&gt;+ vs 0</td><td>p = 0.454</td></tr><tr><td>APL&gt;ChR2XXL test@light vs 0</td><td>p = 0.804</td></tr><tr><td>+&gt;ChR2XXL test@light vs 0</td><td>p = 1</td></tr><tr><td>APL&gt;+ test@light vs 0</td><td></td><td>p = 0.077</td></tr><tr><td rowspan="4">C</td><td rowspan="4">AM/AM vs OCT/OCT</td><td>AM/AM vs OCT/OCT</td><td rowspan="2">MWW</td><td>U = 85, p = 0.0019</td></tr><tr><td>AM/AM vs 0</td><td>p &lt; 0.0001</td></tr><tr><td>OCT/OCT vs 0</td><td>p = 0.0118</td></tr><tr><td>OCT/OCT vs 0</td><td></td></tr><tr><td rowspan="10">D</td><td rowspan="10">AM/AM; OCT/OCT; AM/OCT; OCT/AM</td><td>AM/AM vs OCT/OCT</td><td rowspan="2">MWW with BH correction</td><td>H (3, 118) = 19.34, p &lt; 0.001</td></tr><tr><td>AM/AM vs OCT/OCT</td><td>U = 319.00, p = 0.054</td></tr><tr><td>AM/AM vs AM/OCT</td><td>U = 241.00, p = 0.003</td></tr><tr><td>OCT/OCT vs OCT/AM</td><td>U = 280.00, p = 0.019</td></tr><tr><td>AM/AM vs 0</td><td rowspan="4">OSS test with BH correction</td><td>p &lt; 0.0001</td></tr><tr><td>OCT/OCT vs 0</td><td>p &lt; 0.0001</td></tr><tr><td>AM/OCT vs 0</td><td>p = 0.0009</td></tr><tr><td>OCT/AM vs 0</td><td>p = 0.0002</td></tr><tr><td rowspan="10">E</td><td rowspan="10">APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt;+; APL&gt;ChR2XXL test@light; +&gt;ChR2XXL test@light; APL&gt;+ test@light</td><td>APL&gt;ChR2XXL vs +&gt;ChR2XXL</td><td rowspan="5">MWW with BH correction</td><td>U = 391.5, p = 0.391</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>U = 361, p = 0.191</td></tr><tr><td>+&gt;ChR2XXL vs APL&gt; +</td><td>U = 289, p = 0.017</td></tr><tr><td>APL&gt;ChR2XXL test@light vs +&gt;ChR2XXL test@light</td><td>U = 239, p = 0.002</td></tr><tr><td>APL&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 388, p = 0.363</td></tr><tr><td>+&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 285.5, p = 0.015</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt;ChR2XXL test@light</td><td>U = 398, p = 0.761</td></tr><tr><td>+&gt;ChR2XXL vs +&gt;ChR2XXL test@light</td><td>U = 360, p = 0.188</td></tr><tr><td>APL&gt;+ vs APL&gt; + test@light</td><td>U = 331.5, p = 0.081</td></tr><tr><td rowspan="10">F</td><td rowspan="10">APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt;+; APL&gt;ChR2XXL test@light; +&gt;ChR2XXL test@light; APL&gt;+ test@light</td><td>APL&gt;ChR2XXL vs +&gt;ChR2XXL</td><td rowspan="5">MWW with BH correction</td><td>U = 264, p = 0.628</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>U = 269.5, p = 0.710</td></tr><tr><td>+&gt;ChR2XXL vs APL&gt; +</td><td>U = 244.5, p = 0.375</td></tr><tr><td>APL&gt;ChR2XXL test@light vs +&gt;ChR2XXL test@light</td><td>U = 277.5, p = 0.836</td></tr><tr><td>APL&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 162, p &lt; 0.01</td></tr><tr><td>+&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 167, p = 0.013</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt;ChR2XXL test@light</td><td>U = 275.5, p = 0.804</td></tr><tr><td>+&gt;ChR2XXL vs +&gt;ChR2XXL test@light</td><td>U = 208.5, p = 0.885</td></tr><tr><td>APL&gt;+ vs APL&gt; + test@light</td><td>U = 184.5, p = 0.033</td></tr><tr><td>APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt;+; APL&gt;ChR2XXL test@light; +&gt;ChR2XXL test@light; APL&gt;+ test@light</td><td></td><td>H (5, 184) = 4.43, p = 0.489</td></tr><tr><td rowspan="5">G</td><td rowspan="5">APL&gt;Chrimson; +&gt;Chrimson; APL&gt;+</td><td>APL&gt;Chrimson vs +&gt;Chrimson</td><td rowspan="2">MWW with BH correction</td><td>U = 24, p = 0.002</td></tr><tr><td>APL&gt;Chrimson vs +&gt;Chrimson</td><td>U = 25, p = 0.004</td></tr><tr><td>APL&gt;Chrimson vs APL&gt; +</td><td rowspan="3">OSS test with BH correction</td><td>U = 74, p = 0.849</td></tr><tr><td>+&gt;Chrimson vs APL&gt; +</td><td>p = 0.003</td></tr><tr><td>APL&gt;Chrimson vs 0</td><td>p = 0.581</td></tr><tr><td>APL&gt;+ vs 0</td><td></td><td>p = 0.774</td></tr><tr><td rowspan="2">B</td><td rowspan="2">APL&gt;Chrimson paired; APL&gt;Chrimson unpaired; +&gt;Chrimson paired; +&gt;Chrimson unpaired; APL&gt;+ paired; APL&gt;+ unpaired</td><td>APL&gt;Chrimson vs 0</td><td></td><td>H (5, 76) = 26.67, p &lt; 0.001</td></tr><tr><td>APL&gt;+ vs 0</td><td></td><td>p = 0.003</td></tr></tbody></table>

(Table continues)
<table><caption>Table 2. Continued</caption><thead><tr><th>Figure</th><th>Panel</th><th>Data</th><th>Statistical test</th><th>Result</th></tr></thead><tbody><tr><td></td><td></td><td>APL&gt;ChR2XXL test@light vs 0</td><td></td><td>p = 0.210</td></tr><tr><td></td><td></td><td>+&gt;ChR2XXL test@light vs 0</td><td></td><td>p = 0.210</td></tr><tr><td></td><td></td><td>APL&gt;+ test@light vs 0</td><td></td><td>p = 0.210</td></tr><tr><td rowspan="14">B</td><td rowspan="14">APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt;+; APL&gt;ChR2XXL test@light; +&gt;ChR2XXL test@light; APL&gt;+ test@light</td><td>APL&gt;ChR2XXL vs +&gt;ChR2XXL</td><td rowspan="4">MWW with BH correction</td><td>U = 15, p &lt; 0.001</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>U = 12, p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs APL&gt; +</td><td>U = 109, p = 0.486</td></tr><tr><td>APL&gt;ChR2XXL test@light vs +&gt;ChR2XXL test@light</td><td>U = 121, p = 0.806</td></tr><tr><td>APL&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 88, p = 0.136</td></tr><tr><td>+&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 90, p = 0.157</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt;ChR2XXL test@light</td><td>U = 38, p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs +&gt;ChR2XXL test@light</td><td>U = 119, p = 0.749</td></tr><tr><td>APL&gt;+ vs APL&gt; + test@light</td><td>U = 126, p = 0.955</td></tr><tr><td>APL&gt;ChR2XXL vs 0</td><td rowspan="5">OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>+&gt;ChR2XXL vs 0</td><td>p = 0.804</td></tr><tr><td>APL&gt;+ vs 0</td><td>p = 0.454</td></tr><tr><td>APL&gt;ChR2XXL test@light vs 0</td><td>p = 0.804</td></tr><tr><td>+&gt;ChR2XXL test@light vs 0</td><td>p = 1</td></tr><tr><td>APL&gt;+ test@light vs 0</td><td></td><td>p = 0.077</td></tr><tr><td rowspan="4">C</td><td rowspan="4">AM/AM vs OCT/OCT</td><td>AM/AM vs OCT/OCT</td><td rowspan="2">MWW</td><td>U = 85, p = 0.0019</td></tr><tr><td>AM/AM vs 0</td><td>p &lt; 0.0001</td></tr><tr><td>OCT/OCT vs 0</td><td>p = 0.0118</td></tr><tr><td>OCT/OCT vs 0</td><td></td></tr><tr><td rowspan="10">D</td><td rowspan="10">AM/AM; OCT/OCT; AM/OCT; OCT/AM</td><td>AM/AM vs OCT/OCT</td><td rowspan="2">MWW with BH correction</td><td>H (3, 118) = 19.34, p &lt; 0.001</td></tr><tr><td>AM/AM vs OCT/OCT</td><td>U = 319.00, p = 0.054</td></tr><tr><td>AM/AM vs AM/OCT</td><td>U = 241.00, p = 0.003</td></tr><tr><td>OCT/OCT vs OCT/AM</td><td>U = 280.00, p = 0.019</td></tr><tr><td>AM/AM vs 0</td><td rowspan="4">OSS test with BH correction</td><td>p &lt; 0.0001</td></tr><tr><td>OCT/OCT vs 0</td><td>p &lt; 0.0001</td></tr><tr><td>AM/OCT vs 0</td><td>p = 0.0009</td></tr><tr><td>OCT/AM vs 0</td><td>p = 0.0002</td></tr><tr><td rowspan="14">E</td><td rowspan="14">APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt;+; APL&gt;ChR2XXL test@light; +&gt;ChR2XXL test@light; APL&gt;+ test@light</td><td>APL&gt;ChR2XXL vs +&gt;ChR2XXL</td><td rowspan="4">MWW with BH correction</td><td>U = 391.5, p = 0.391</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>U = 361, p = 0.191</td></tr><tr><td>+&gt;ChR2XXL vs APL&gt; +</td><td>U = 289, p = 0.017</td></tr><tr><td>APL&gt;ChR2XXL test@light vs +&gt;ChR2XXL test@light</td><td>U = 239, p = 0.002</td></tr><tr><td>APL&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 388, p = 0.363</td></tr><tr><td>+&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 285.5, p = 0.015</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt;ChR2XXL test@light</td><td>U = 398, p = 0.761</td></tr><tr><td>+&gt;ChR2XXL vs +&gt;ChR2XXL test@light</td><td>U = 360, p = 0.188</td></tr><tr><td>APL&gt;+ vs APL&gt; + test@light</td><td>U = 331.5, p = 0.081</td></tr><tr><td rowspan="14">F</td><td rowspan="14">APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt;+; APL&gt;ChR2XXL test@light; +&gt;ChR2XXL test@light; APL&gt;+ test@light</td><td>APL&gt;ChR2XXL vs +&gt;ChR2XXL</td><td rowspan="4">MWW with BH correction</td><td>U = 264, p = 0.628</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt; +</td><td>U = 269.5, p = 0.710</td></tr><tr><td>+&gt;ChR2XXL vs APL&gt; +</td><td>U = 244.5, p = 0.375</td></tr><tr><td>APL&gt;ChR2XXL test@light vs +&gt;ChR2XXL test@light</td><td>U = 277.5, p = 0.836</td></tr><tr><td>APL&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 162, p &lt; 0.01</td></tr><tr><td>+&gt;ChR2XXL test@light vs APL&gt; + test@light</td><td>U = 167, p = 0.013</td></tr><tr><td>APL&gt;ChR2XXL vs APL&gt;ChR2XXL test@light</td><td>U = 275.5, p = 0.804</td></tr><tr><td>+&gt;ChR2XXL vs +&gt;ChR2XXL test@light</td><td>U = 208.5, p = 0.885</td></tr><tr><td>APL&gt;+ vs APL&gt; + test@light</td><td>U = 184.5, p = 0.033</td></tr><tr><td>APL&gt;ChR2XXL; +&gt;ChR2XXL; APL&gt;+; APL&gt;ChR2XXL test@light; +&gt;ChR2XXL test@light; APL&gt;+ test@light</td><td></td><td>H (5, 184) = 4.43, p = 0.489</td></tr><tr><td rowspan="5">G</td><td rowspan="5">APL&gt;Chrimson; +&gt;Chrimson; APL&gt;+</td><td>APL&gt;Chrimson vs +&gt;Chrimson</td><td rowspan="2">MWW with BH correction</td><td>U = 24, p = 0.002</td></tr><tr><td>APL&gt;Chrimson vs +&gt;Chrimson</td><td>U = 25, p = 0.004</td></tr><tr><td>APL&gt;Chrimson vs APL&gt; +</td><td rowspan="3">OSS test with BH correction</td><td>U = 74, p = 0.849</td></tr><tr><td>+&gt;Chrimson vs APL&gt; +</td><td>p = 0.003</td></tr><tr><td>APL&gt;Chrimson vs 0</td><td>p = 0.581</td></tr><tr><td>APL&gt;+ vs 0</td><td></td><td>p = 0.774</td></tr><tr><td rowspan="2">B</td><td rowspan="2">APL&gt;Chrimson paired; APL&gt;Chrimson unpaired; +&gt;Chrimson paired; +&gt;Chrimson unpaired; APL&gt;+ paired; APL&gt;+ unpaired</td><td>APL&gt;Chrimson vs 0</td><td></td><td>H (5, 76) = 26.67, p &lt; 0.001</td></tr><tr><td>APL&gt;+ vs 0</td><td></td><td>p = 0.003</td></tr></tbody></table>

(Table continues)
<table><caption>Table 2. Continued</caption><thead><tr><th>Figure</th><th>Panel</th><th>Data</th><th>Statistical test</th><th>Result</th></tr></thead><tbody><tr><td rowspan="10">16</td><td rowspan="4">C</td><td>APL&gt;Chrimson paired vs baseline</td><td rowspan="4">MWW with BH correction</td><td>U = 17.50, p = 0.011</td></tr><tr><td>APL&gt;Chrimson unpaired vs baseline</td><td>U = 81.50, p &lt; 0.0001</td></tr><tr><td>+ &gt;Chrimson paired vs + &gt;Chrimson unpaired</td><td>U = 67.00, p = 0.383</td></tr><tr><td>APL + paired vs APL + unpaired</td><td>U = 65.00, p = 0.707</td></tr><tr><td rowspan="3">D</td><td>Retinal vs EtOH</td><td rowspan="3">MWW</td><td>U = 5, p &lt; 0.001</td></tr><tr><td>Retinal vs 0</td><td>p &lt; 0.001</td></tr><tr><td>EtOH vs 0</td><td>p = 1</td></tr><tr><td rowspan="3">E</td><td>APL&gt;Chrimson vs APL&gt;Chrimson test@light</td><td rowspan="3">MWW</td><td>U = 290, p = 0.001</td></tr><tr><td>APL&gt;Chrimson vs 0</td><td>p &lt; 0.001</td></tr><tr><td>APL&gt;Chrimson test@light vs 0</td><td>p = 0.013</td></tr><tr><td>E</td><td>APL&gt;Chrimson test@dark vs APL&gt;Chrimson test@light</td><td>MWW</td><td>U = 370, p = 0.239</td></tr><tr><td>A</td><td>MBONa1,a2&gt;GtACR1; + &gt;GtACR1; MBONa1,a2&gt; +</td><td>KW</td><td>H (2, 36) = 0.758, p = 0.684</td></tr><tr><td>B</td><td>MBONa1,a2&gt;ChR2XXL; + &gt;ChR2XXL; MBONa1,a2&gt; +</td><td></td><td>H (2, 48) = 4.844, p = 0.088</td></tr><tr><td>C</td><td>MBONa1&gt;GtACR1; MBONa2&gt;GtACR1; MBONa1&gt;GtACR1 test@light; MBONa2&gt;GtACR1 test@light</td><td></td><td>H (3, 48) = 0.404, p = 0.939</td></tr><tr><td>D</td><td>MBONa1&gt;ChR2XXL; MBONa2&gt;ChR2XXL; MBONa1&gt;ChR2XXL test@light; MBONa2&gt;ChR2XXL test@light</td><td></td><td>H (1, 50) = 0.0377, p = 0.846</td></tr><tr><td rowspan="6">17</td><td rowspan="3">C</td><td>3IY [0 mg.ml<sup>-1</sup>] vs [5 mg.ml<sup>-1</sup>]</td><td rowspan="3">MWW</td><td>U = 180, p = 0.026</td></tr><tr><td>3IY [0 mg.ml<sup>-1</sup>] vs 0</td><td>p &lt; 0.001</td></tr><tr><td>3IY [5 mg.ml<sup>-1</sup>] vs 0</td><td>p &lt; 0.001</td></tr><tr><td rowspan="10">D</td><td>Control; 3IY [5 mg.ml<sup>-1</sup>]; 3IY [5 mg.ml<sup>-1</sup>] + L-DOPA [10 mg.ml<sup>-1</sup>]; L-DOPA [10 mg.ml<sup>-1</sup>]</td><td>KW</td><td>H (3, 104) = 20.974, p &lt; 0.001</td></tr><tr><td>Control vs 3IY [5 mg.ml<sup>-1</sup>]</td><td rowspan="3">MWW</td><td>U = 122, p &lt; 0.001</td></tr><tr><td>Control vs 3IY [5 mg.ml<sup>-1</sup>] + L-DOPA [10 mg.ml<sup>-1</sup>]</td><td>U = 267, p = 0.196</td></tr><tr><td>Control vs L-DOPA [10 mg.ml<sup>-1</sup>]</td><td>U = 309, p = 0.60</td></tr><tr><td>3IY [5 mg.ml<sup>-1</sup>] vs 3IY [5 mg.ml<sup>-1</sup>] + L-DOPA [10 mg.ml<sup>-1</sup>]</td><td>U = 171, p = 0.002</td></tr><tr><td>Control vs 0</td><td rowspan="3">OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>3IY [5 mg.ml<sup>-1</sup>] vs 0</td><td>p &lt; 0.001</td></tr><tr><td>3IY [5 mg.ml<sup>-1</sup>] + L-DOPA [10 mg.ml<sup>-1</sup>] vs 0</td><td>p &lt; 0.001</td></tr><tr><td rowspan="7">18</td><td rowspan="7">A</td><td>L-DOPA [10 mg.ml<sup>-1</sup>] vs 0</td><td></td><td>p &lt; 0.001</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt;reaper | APL55D08&gt;ChR2XXL; pPAM&gt; + | APL55D08&gt;ChR2XXL; + &gt;reaper</td><td>KW</td><td>H (2, 79) = 30.85, p &lt; 0.001</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt;reaper vs APL55D08&gt;ChR2XXL; pPAM&gt; +</td><td rowspan="3">MWW with BH correction</td><td>U = 60, p &lt; 0.0001</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt;reaper vs APL55D08&gt;ChR2XXL; + &gt;reaper</td><td>U = 180.5, p = 0.002</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt; + vs APL55D08&gt;ChR2XXL; + &gt;reaper</td><td>U = 159, p &lt; 0.001</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt;reaper vs 0</td><td rowspan="3">OSS test with BH correction</td><td>p = 0.009</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt; + vs 0</td><td>p &lt; 0.001</td></tr><tr><td>APL55D08&gt;ChR2XXL; + &gt;reaper vs 0</td><td>p &lt; 0.001</td></tr></tbody></table>

excitatory effects in a minority of KCs or in non-KC targets of APL. Furthermore, a postinhibitory rebound activation may occur in the target neurons of APL, a widely observed physiological phenomenon (Huguenard and McCormick, 2007; for evidence in adult *Drosophila* after days of APL activation: Apostolopoulou and Lin, 2020). With these caveats in mind, the discussion below maintains the conventional notion of GABA as mediating inhibition.

The fact that activation of APL can establish appetitive memory for an associated odor means that, even under conditions of GABAergic, inhibitory input, an associable odor representation can be established across the KCs. Indeed, these representations can be odor-specific (Fig. 14B,D). The scenario could be that, under baseline conditions, odors strongly activate “their” subset of KCs while the other KCs are inactive or mildly inhibited. Upon optogenetic APL activation, however, odors only mildly activate an even sparser set of KCs while most other KCs would be strongly inhibited. Interestingly, associable odor representations can be established also under conditions of optogenetically increased levels of activity across the KCs (Lytova et al., 2019).

We are not aware of data suggesting that GABA can have a direct associative memory-trace-inducing effect. Rather, our results suggest that the rewarding effect of APL activation comes about, at least in part, by engaging a dopaminergic reward signal from the pPAM neurons (Figs. 17, 18). We will therefore focus on plausible pathways from APL toward these pPAM neurons.

## From APL to dopaminergic pPAM neurons?

The larval APL has presynapses only in the calyx (Figs. 4, 6-8; Movie 3). The postsynaptic partners of APL include the KCs (see next paragraph) and the two calyx MBONs (Eichler et al., 2017; Saumweber et al., 2018). One of these calyx MBONs promotes approach when optogenetically activated (MBON-a1), whereas the other promotes avoidance (MBON-a2) (Eschbach et al., 2021). Both calyx MBONs give rise to indirect feedback to DANs, including to the punishing DAN-d1 and the rewarding DAN-il neuron of the pPAM cluster (Eschbach et al., 2020). However, presenting an odor together with activating or silencing the two calyx MBONs, alone or in combination, did not establish either appetitive or aversive associative odor memory (Fig. 16). Thus, although it is a connectomic possibility, there is no evidence for a reinforcing APL-MBONa1/a2-pPAM loop. What about a loop from APL via the KCs to the pPAM neurons?

A first intuition is that an APL-KC-pPAM loop is an unlikely candidate. This is because, bearing the discussed caveats in mind, APL inhibits KCs, while KCs in turn excite DANs (Lytova et al., 2019; adults: Cervantes-Sandoval et al., 2017). Thus, APL activation should reduce KC activity, which in turn reduces drive to the pPAMs. However, it might not be the absolute level of activity in the pPAM neurons that matters, but rather whether these are relatively less affected than the punishing DANs. In other words, is the negative impact that APL exerts on DAN activity
<table><caption>Table 2. Continued</caption><thead><tr><th>Figure</th><th>Panel</th><th>Data</th><th>Statistical test</th><th>Result</th></tr></thead><tbody><tr><td rowspan="14">16</td><td rowspan="4">C</td><td>APL&gt;Chrimson paired vs baseline</td><td rowspan="4">MWW with BH correction</td><td>U = 17.50, p = 0.011</td></tr><tr><td>APL&gt;Chrimson unpaired vs baseline</td><td>U = 81.50, p &lt; 0.0001</td></tr><tr><td>+&gt;Chrimson paired vs +&gt;Chrimson unpaired</td><td>U = 67.00, p = 0.383</td></tr><tr><td>APL&gt; paired vs APL&gt; unpaired</td><td>U = 65.00, p = 0.707</td></tr><tr><td rowspan="3">D</td><td>Retinal vs EtOH</td><td rowspan="3">MWW</td><td>U = 5, p &lt; 0.001</td></tr><tr><td>Retinal vs 0</td><td>p &lt; 0.001</td></tr><tr><td>EtOH vs 0</td><td>p = 1</td></tr><tr><td rowspan="3">E</td><td>APL&gt;Chrimson vs APL&gt;Chrimson test@light</td><td rowspan="3">MWW</td><td>U = 290, p = 0.001</td></tr><tr><td>APL&gt;Chrimson vs 0</td><td>p &lt; 0.001</td></tr><tr><td>APL&gt;Chrimson test@light vs 0</td><td>p = 0.013</td></tr><tr><td>F</td><td>APL&gt;Chrimson test@dark vs APL&gt;Chrimson test@light</td><td>MWW</td><td>U = 370, p = 0.239</td></tr><tr><td>A</td><td>MBONa1,a2&gt;GtACR1; +&gt;GtACR1; MBONa1,a2&gt; +</td><td>KW</td><td>H (2, 36) = 0.758, p = 0.684</td></tr><tr><td>B</td><td>MBONa1,a2&gt;ChR2XXL; +&gt;ChR2XXL; MBONa1,a2&gt; +</td><td></td><td>H (2, 48) = 4.844, p = 0.088</td></tr><tr><td>C</td><td>MBONa1&gt;GtACR1; MBONa2&gt;GtACR1; MBONa1&gt;GtACR1 test@light; MBONa2&gt;GtACR1 test@light</td><td></td><td>H (3, 48) = 0.404, p = 0.939</td></tr><tr><td>D</td><td>MBONa1&gt;ChR2XXL; MBONa2&gt;ChR2XXL; MBONa1&gt;ChR2XXL test@light; MBONa2&gt;ChR2XXL test@light</td><td></td><td>H (1, 50) = 0.0377, p = 0.846</td></tr><tr><td rowspan="10">17</td><td rowspan="3">C</td><td>3IY [0 mg·ml<sup>-1</sup>] vs [5 mg·ml<sup>-1</sup>]</td><td rowspan="3">MWW</td><td>U = 180, p = 0.026</td></tr><tr><td>3IY [0 mg·ml<sup>-1</sup>] vs 0</td><td>p &lt; 0.001</td></tr><tr><td>3IY [5 mg·ml<sup>-1</sup>] vs 0</td><td>p &lt; 0.001</td></tr><tr><td rowspan="8">D</td><td>Control; 3IY [5 mg·ml<sup>-1</sup>]; 3IY [5 mg·ml<sup>-1</sup>] + L-DOPA [10 mg·ml<sup>-1</sup>]; L-DOPA [10 mg·ml<sup>-1</sup>]</td><td>KW</td><td>H (3, 104) = 20.974, p &lt; 0.001</td></tr><tr><td>Control vs 3IY [5 mg·ml<sup>-1</sup>]</td><td rowspan="3">MWW</td><td>U = 122, p &lt; 0.001</td></tr><tr><td>Control vs 3IY [5 mg·ml<sup>-1</sup>] + L-DOPA [10 mg·ml<sup>-1</sup>]</td><td>U = 267, p = 0.196</td></tr><tr><td>Control vs L-DOPA [10 mg·ml<sup>-1</sup>]</td><td>U = 309, p = 0.60</td></tr><tr><td>3IY [5 mg·ml<sup>-1</sup>] vs 3IY [5 mg·ml<sup>-1</sup>] + L-DOPA [10 mg·ml<sup>-1</sup>]</td><td>U = 171, p = 0.002</td></tr><tr><td>Control vs 0</td><td rowspan="3">OSS test with BH correction</td><td>p &lt; 0.001</td></tr><tr><td>3IY [5 mg·ml<sup>-1</sup>] vs 0</td><td>p &lt; 0.001</td></tr><tr><td>3IY [5 mg·ml<sup>-1</sup>] + L-DOPA [10 mg·ml<sup>-1</sup>] vs 0</td><td>p &lt; 0.001</td></tr><tr><td rowspan="10">18</td><td rowspan="10">A</td><td>L-DOPA [10 mg·ml<sup>-1</sup>] vs 0</td><td></td><td>p &lt; 0.001</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt;reaper | APL55D08&gt;ChR2XXL; pPAM&gt; + | APL55D08&gt;ChR2XXL; +&gt;reaper</td><td>KW</td><td>H (2, 79) = 30.85, p &lt; 0.001</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt;reaper vs APL55D08&gt;ChR2XXL; pPAM&gt; +</td><td rowspan="3">MWW with BH correction</td><td>U = 60, p &lt; 0.0001</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt;reaper vs APL55D08&gt;ChR2XXL; +&gt;reaper</td><td>U = 180.5, p = 0.002</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt; + vs APL55D08&gt;ChR2XXL; +&gt;reaper</td><td>U = 159, p &lt; 0.001</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt;reaper vs 0</td><td rowspan="3">OSS test with BH correction</td><td>p = 0.009</td></tr><tr><td>APL55D08&gt;ChR2XXL; pPAM&gt; + vs 0</td><td>p &lt; 0.001</td></tr><tr><td>APL55D08&gt;ChR2XXL; +&gt;reaper vs 0</td><td>p &lt; 0.001</td></tr></tbody></table>

excitatory effects in a minority of KCs or in non-KC targets of APL. Furthermore, a postinhibitory rebound activation may occur in the target neurons of APL, a widely observed physiological phenomenon (Huguenard and McCormick, 2007; for evidence in adult *Drosophila* after days of APL activation: Apostolopoulou and Lin, 2020). With these caveats in mind, the discussion below maintains the conventional notion of GABA as mediating inhibition.

The fact that activation of APL can establish appetitive memory for an associated odor means that, even under conditions of GABAergic, inhibitory input, an associable odor representation can be established across the KCs. Indeed, these representations can be odor-specific (Fig. 14B,D). The scenario could be that, under baseline conditions, odors strongly activate “their” subset of KCs while the other KCs are inactive or mildly inhibited. Upon optogenetic APL activation, however, odors only mildly activate an even sparser set of KCs while most other KCs would be strongly inhibited. Interestingly, associable odor representations can be established also under conditions of optogenetically increased levels of activity across the KCs (Lytova et al., 2019).

We are not aware of data suggesting that GABA can have a direct associative memory-trace-inducing effect. Rather, our results suggest that the rewarding effect of APL activation comes about, at least in part, by engaging a dopaminergic reward signal from the pPAM neurons (Figs. 17, 18). We will therefore focus on plausible pathways from APL toward these pPAM neurons.

## From APL to dopaminergic pPAM neurons?

The larval APL has presynapses only in the calyx (Figs. 4, 6-8; Movie 3). The postsynaptic partners of APL include the KCs (see next paragraph) and the two calyx MBONs (Eichler et al., 2017; Saumweber et al., 2018). One of these calyx MBONs promotes approach when optogenetically activated (MBON-a1), whereas the other promotes avoidance (MBON-a2) (Eschbach et al., 2021). Both calyx MBONs give rise to indirect feedback to DANs, including to the punishing DAN-d1 and the rewarding DAN-il neuron of the pPAM cluster (Eschbach et al., 2020). However, presenting an odor together with activating or silencing the two calyx MBONs, alone or in combination, did not establish either appetitive or aversive associative odor memory (Fig. 16). Thus, although it is a connectomic possibility, there is no evidence for a reinforcing APL-MBONa1/a2-pPAM loop. What about a loop from APL via the KCs to the pPAM neurons?

A first intuition is that an APL-KC-pPAM loop is an unlikely candidate. This is because, bearing the discussed caveats in mind, APL inhibits KCs, while KCs in turn excite DANs (Lytova et al., 2019; adults: Cervantes-Sandoval et al., 2017). Thus, APL activation should reduce KC activity, which in turn reduces drive to the pPAMs. However, it might not be the absolute level of activity in the pPAM neurons that matters, but rather whether these are relatively less affected than the punishing DANs. In other words, is the negative impact that APL exerts on DAN activity
stronger for the punishing DANs than for the rewarding DANs? We therefore queried the larval connectome for the possible impact of APL, via the KCs, on the input neurons of the mushroom body (Fig. 19). It turns out that APL can indeed impact the inputs to the vertical lobe more strongly than those to the medial lobe, suggesting a stronger negative impact of APL on activity in the aversive than the appetitive lobe system (Eschbach et al., 2020). Considering the impact on DL1-DANs versus pPAM-DANs (Rohwedder et al., 2016), as well as comparing the impact on DAN-fl versus DAN-il (Thoener et al., 2022), reveals a similar picture of a stronger impact on aversive pathways.

An alternative could be that, similar to the situation in adults for APL and the DPM neuron (C. L. Wu et al., 2011), there is direct signaling from APL to rewarding pPAM-DANs via electrical synapses in the lobes (Figs. 3, 4, 6, 7) and that fewer or no such electrical synapses exist for the punishing DANs.

In conclusion, we report a case of complex circuit function in a numerically simple brain, and demonstrate the capacity of a central-brain GABAergic neuron to engage dopaminergic reward signaling when optogenetically activated.

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* Heisenberg M (2003) Mushroom body memoir: from maps to models. Nat Rev Neurosci 4:266–275.

* Herranz H, Weng R, Cohen SM (2014) Crosstalk between epithelial and mesenchymal tissues in tumorigenesis and imaginal disc development. Curr Biol 24:1476–1484.

* Homberg U, Kingan TG, Hildebrand JG (1987) Immunocytochemistry of GABA in the brain and suboesophageal ganglion of *Manduca sexta*. Cell Tissue Res 248:1–24.

* Honegger KS, Campbell RA, Turner GC (2011) Cellular-resolution population imaging reveals robust sparse coding in the *Drosophila* mushroom body. J Neurosci 31:11772–11785.

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* Jenett A, Rubin GM, Ngo TT, Shepherd D, Murphy C, Dionne H, Pfeiffer BD, Cavallaro A, Hall D, Jeter J, Iyer N (2012) A GAL4-driver line resource for Drosophila neurobiology. Cell Rep 2:991–1001.

* Kanellopoulos AK, Mariano V, Spinazzi M, Woo YJ, McLean C, Pech U, Li KW, Armstrong JD, Giangrande A, Callaerts P, Smit AB, Abrahams BS, Fiala A, Achsel T, Bagni C (2020) Aralar sequesters GABA into hyperactive mitochondria, causing social behavior deficits. Cell 180:1178–1197. e20.

* Kaun KR, Azanchi R, Maung Z, Hirsh J, Heberlein U (2011) A *Drosophila* model for alcohol reward. Nat Neurosci 14:612–619.

* Klapoetke NC, et al. (2014) Independent optical excitation of distinct neural populations. Nat Methods 11:338–346.

* Kleber J, Chen Y, Michels B, Saumweber T, Schleyer M, Kähne T, Buchner E, Gerber B (2016) Synapsin is required to 'boost' memory strength for highly salient events. Learn Mem 23:9–20.

* Kobler O, Weiglein A, Hartung K, Chen YC, Gerber B, Thomas U (2021) A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval *Drosophila*. J Neurogenet 35:306–319.

* Kohl J, Ng J, Cachero S, Ciabatti E, Dolan MJ, Sutcliffe B, Tozer A, Ruehle S, Krueger D, Frechter S, Branco T, Tripodi M, Jefferis GS (2014) Ultrafast tissue staining with chemical tags. Proc Natl Acad Sci USA 111:E3805–E3814.

* König C, Khalili A, Ganesan M, Nishu AP, Gomez AP, Niewalda T, Gerber B, Aso Y, Yarali A (2018) Reinforcement signalling of punishment vs. relief in fruit flies. Learn Mem 25:247–257.

* König C, Khalili A, Niewalda T, Gao S, Gerber B (2019) An optogenetic analogue of second-order reinforcement in *Drosophila*. Biol Lett 15:20190084.

* Lee PT, Lin HW, Chang YH, Fu TF, Dubnau J, Hirsh J, Lee T, Chiang AS (2011) Serotonin-mushroom body circuit modulating the formation of anesthesia-resistant memory in *Drosophila*. Proc Natl Acad Sci USA 108:13794–13799.
stronger for the punishing DANs than for the rewarding DANs? We therefore queried the larval connectome for the possible impact of APL, via the KCs, on the input neurons of the mushroom body (Fig. 19). It turns out that APL can indeed impact the inputs to the vertical lobe more strongly than those to the medial lobe, suggesting a stronger negative impact of APL on activity in the aversive than the appetitive lobe system (Eschbach et al., 2020). Considering the impact on DL1-DANs versus pPAM-DANs (Rohwedder et al., 2016), as well as comparing the impact on DAN-fl versus DAN-il (Thoener et al., 2022), reveals a similar picture of a stronger impact on aversive pathways.

An alternative could be that, similar to the situation in adults for APL and the DPM neuron (C. L. Wu et al., 2011), there is direct signaling from APL to rewarding pPAM-DANs via electrical synapses in the lobes (Figs. 3, 4, 6, 7) and that fewer or no such electrical synapses exist for the punishing DANs.

In conclusion, we report a case of complex circuit function in a numerically simple brain, and demonstrate the capacity of a central-brain GABAergic neuron to engage dopaminergic reward signaling when optogenetically activated.

## References

* Amin H, Apostolopoulou AA, Suárez-Grimalt R, Vrontou E, Lin AC (2020) Localized inhibition in the *Drosophila* mushroom body. Elife 9:e56954.

* Apostolopoulou AA, Lin AC (2020) Mechanisms underlying homeostatic plasticity in the *Drosophila* mushroom body in vivo. Proc Natl Acad Sci USA 117:16606–16615.

* Aso Y, Rubin GM (2016) Dopaminergic neurons write and update memories with cell-type-specific rules. Elife 5:e16135.

* Aso Y, Hattori D, Yu Y, Johnston RM, Iyer NA, Ngo TT, Dionne H, Abbott LF, Axel R, Tanimoto H, Rubin GM (2014) The neuronal architecture of the mushroom body provides a logic for associative learning. Elife 3: e04577.

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* Ben-Ari Y (2002) Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neurosci 3:728–739.

* Cervantes-Sandoval I, Phan A, Chakraborty M, Davis RL (2017) Reciprocal synapses between mushroom body and dopamine neurons form a positive feedback loop required for learning. Elife 6:e23789.

* Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS (2013) Ultra-sensitive fluorescent proteins for imaging neuronal activity. Nature 499:295–300.

* Dawydow A, Gueta R, Ljaschenko D, Ullrich S, Hermann M, Ehmann N, Gao S, Fiala A, Langenhan T, Nagel G, Kittel RJ (2014) Channelrhodopsin-2-XXL, a powerful optogenetic tool for low-light applications. Proc Natl Acad Sci USA 111:13972–13977.

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