Abstract Adaptive decision-making depends on the formation of novel memories. In Drosophila, the mushroom body (MB) is the site of associative olfactory long-term memory (LTM) storage. However, due to the sparse and stochastic representation of olfactory information in Kenyon cells (KCs), genetic access to individual LTMs remains elusive. Here, we develop a cAMP response element (CRE)-activity–dependent memory engram label (CAMEL) tool that genetically tags KCs responding to the conditioned stimulus (CS). CAMEL activity depends on protein-synthesis–dependent aversive LTM conditioning and reflects the time course of CRE binding protein 2 (CREB2) activity during natural memory formation. We demonstrate that inhibition of LTM-induced CAMEL neurons reduces memory expression and that artificial optogenetic reactivation is sufficient to evoke aversive behavior phenocopying memory recall. Together, our data are consistent with CAMEL neurons marking a subset of engram KCs encoding individual memories. This study provides new insights into memory circuitry organization and an entry point towards cellular and molecular understanding of LTM storage.

Citation: Siegenthaler D, Escribano B, Bräuler V, Pielage J (2019) Selective suppression and recall of long-term memories in Drosophila. PLoS Biol 17(8): e3000400. https://doi.org/10.1371/journal.pbio.3000400 Academic Editor: Josh Dubnau, Stony Brook University, UNITED STATES Received: January 13, 2019; Accepted: July 22, 2019; Published: August 27, 2019 Copyright: © 2019 Siegenthaler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by the Deutsche Forschungsgemeinschaft (www.dfg.de) (INST 248/222-1 FUGG, to JP) and the Forschungsinitiative Rheinland Pfalz (www.uni-kl.de/biocomp) (BioComp 2.0, to JP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: AD, activation domain; ARM, anesthesia-resistant memory; AS, avoidance score; CAMEL, CRE-activity–dependent memory engram label; CRE, cAMP response element; CREB2, cAMP response element binding protein 2; CS, conditioned stimulus; DAN, dopaminergic neuron; DBD, DNA binding domain; EGFP, enhanced GFP; Flp, Flippase; GFP, green fluorescent protein; IBA, Isobutyl-acetate; KC, Kenyon cell; LTM, long-term memory; MB, mushroom body; MBON, MB output neuron; MCH, 4-Methyl-cylohexanol; MTM, mid-term memory; OCT, 3-Octanol; PerI, Performance Index; PI, Preference Index; PN, projection neuron; SEM, standard error of the mean; STM, short-term memory; TNT, tetanus toxin; UAS, upstream activation sequence

Introduction Successful adaptation to aversive stimuli through the formation of associative memories is essential for the survival of most animals. The physical correlate of these memories, the memory engram, represents long-lasting alterations of neurons and circuits that enable precise recall of stored information to modulate behavior [1–3]. Drosophila aversive olfactory conditioning has been successfully used in the past to unravel the molecular mechanisms underlying long-term associative memory [4–7]. The mushroom body (MB), the site of associative olfactory learning in insects, consists of 7 types (αβc, αβs, αβp, α’β’m, α’β’ap, γm, and γd) of Kenyon cells (KCs) that project into specific layers in the α/β, α’/β’, and γ MB lobes that represent functional domains dedicated to different aspects of memory acquisition, storage, and retrieval [8–19]. Olfactory information from approximately 50 antennal lobe glomeruli is provided in a stochastic manner via projection neurons (PNs) to approximately 2,000 KCs [20–24]. KCs then transmit the information to only 34 MB output neurons (MBONs) in 15 anatomically defined lobular compartments [18,19]. Consistent with this anatomical architecture, MBONs are broadly tuned to odors, whereas olfactory sensory representation in KCs is sparse and thus well suited for memory association [25–28]. The KC > MBON synaptic compartments are selectively innervated by dopaminergic neurons (DANs) that provide reinforcement signals of positive or negative valence during associative memory formation [18,29–32]. Temporal pairing of an odor with an aversive stimulus results in KC activity that coincides with dopamine release and induces synaptic depression of the KC > MBON synapse in a compartment-specific manner [33–35]. Because MBONs are known to direct approach or avoidance behavior [36,37], KC > MBON plasticity is thought to be sufficient to modulate behavior and store olfactory memories [38,39]. The recent complete synaptic reconstruction of the adult MB α lobe [40] and of the entire adult brain [41] in combination with genetic access to MBONs and DANs [18] provide a unique framework to unravel the cellular and circuit mechanisms underlying long-term memory (LTM). Prior studies demonstrated that one subtype of KCs, the αβ surface neurons (500 cells), is essential for LTM recall [9], indicating that olfactory LTMs are stored within these neurons. The stochastic PN > KC synaptic connectivity and the sparse representation of olfactory information within a genetically identical subpopulation of KCs currently precludes the identification and characterization of the KCs encoding individual LTMs. In mice, activity-dependent techniques based on the transcriptional activity of c-Fos enabled successful tagging and manipulation of engram cells [42–44], and a similar approach has recently been described in Drosophila [45]. In addition to c-Fos, cellular consolidation of LTM requires protein synthesis and activity of the cAMP response element (CRE)-binding protein CREB in mice, Aplysia and Drosophila MBs [5,45–56]. In this study, we generate a novel engram-tagging tool based on CRE-dependent transcriptional activity to specifically manipulate KCs encoding individual LTMs.

Discussion In this study, we established a novel tool enabling genetic access to KCs encoding individual aversive olfactory LTMs in Drosophila. In mice and flies, prior studies utilized expression of the immediate-early gene c-Fos to successfully tag populations of neurons containing memory engram cells [42–45]. Here, we aimed to generate a tool enabling specific tagging of LTMs. Therefore, we designed the CAMEL tool to directly report CREB2 activity that is essential not only for induction but also for the consolidation and maintenance of LTMs [5,52,55,56]. Thus, in contrast to the activity-based methods, this tool should be suitable to endogenously tag, monitor, and manipulate engram cells over prolonged periods of engram persistence. In accordance with these features, we observed long-lasting LTM-specific CAMEL induction in CS+-activated cells. Silencing and activation experiments showed that these CAMEL-tagged cells are necessary and sufficient for aversive LTM expression. Importantly, in these experiments, CAMEL activity reflected the time frames of physiological CREB2-dependent LTM formation and maintenance [55,59]. This time course, together with the observation that innate CAMEL activity decreased upon food-associated odor deprivation, indicates that innate CAMEL activity may reflect natural memory formation. Variable CAMEL activity between flies may thus underlie the plasticity-driven interanimal differences in odor tuning previously observed at the level of MBONs [25]. In aged flies and upon LTM induction, we observed a significant right-to-left brain hemisphere bias of CAMEL tagging. While the relevance of this right-side bias for LTM remains to be explored, it is consistent with the observation that asymmetry of a central brain structure named asymmetric body is required for effective LTM performance [65]. It will be interesting to test how asymmetries of memory coding at the level of KCs may contribute to directional decision-making during olfactory 2-choice assays. Our GFP- and GCaMP-based observational experiments revealed conditioning-dependent CAMEL activity in a relatively low number of cells in comparison to prior studies estimating putative engram cell number in Drosophila [45,66]. These studies utilized either artificial memory induction in randomly labeled KCs [66] or c-Fos-activity–based labeling [45] to estimate engram cell size shortly after training. Thus, differences in the technical approaches and in the time point of memory engram size evaluation likely contribute to these discrepancies. Furthermore, due to constraints of the CAMEL design, we likely underestimate the actual number of KCs encoding individual LTMs. Because the CAMEL tool reports LTM-dependent CREB2 activity without amplification, our GFP- and GCamP6f-based observational assays likely fail to capture all engram cells. The enzymatic and channel properties of TNT and Chrimson in our functional assays potentially modulated the activity of engram cells not identified in the observational assays. Second, in mice, a preferential recruitment of CREB-overexpressing cells into the memory engram has been described and used to tag engram cells (memory allocation) [67,68]. Accordingly, in our system, endogenously tagged CAMEL cells might have been allocated to the engram and thereby potentially contributed to memory performance in the activation and silencing assays. Finally, it is important to note that our evaluation of aversive LTM is based on population analyses that currently prevent conclusions regarding successful LTM formation in individual flies. At a circuit level, the impairment of 2- and 5-day memory after CAMEL-cell silencing demonstrated that only a small subset of KCs is required for Drosophila LTM expression. The intact memory at day 7 despite multiple days of engram cell silencing argues that KC engram output is dispensable for LTM storage. Recent studies demonstrated that aversive STMs are encoded by depression of KC > MBONapproach synapses [33,34,36]. As a consequence, CS+ exposure during memory recall leads to a differential activation of approach and avoidance output channels resulting in net avoidance (“balance” model [38,39]). Our data are in general agreement with such a balance model for LTM; silencing of CAMEL neurons selectively eliminates KCs with biased output from the circuitry and thus disrupts memory recall. Optogenetic activation of these biased KCs induces aversive behavior via a preferential activation of aversive MBONs indicating that compartment-specific LTM-dependent synaptic changes [34,35] uphold artificial reactivation. Because silencing of individual aversive MBONs does not impair memory performance [36], it is likely that engram KCs simultaneously activate multiple aversive MBONs to induce aversive behavior. Support for such a model is provided by the EM reconstruction that demonstrated extensive synaptic connectivity between individual KCs and all MBONs in analyzed MB compartments [40]. We find that the effects on memory are mediated by a surprisingly small number of KCs in accordance with the observation that insect KC > MBON synapses are strong [69]. Moreover, the high number of KC > KC synapses [40,41] and converging KC > MBON synapses (or rosettes [40]) may provide amplification mechanisms to enhance the impact of individual KCs on circuit output and behavior. Together, our study advances our understanding of memory circuitry and function and provides a novel entry point to unravel the cellular and synaptic changes underlying LTM. Because CREB represents a general, evolutionary conserved biochemical LTM switch, CAMEL might be applicable to a wide range of memory studies in different systems.

Materials and methods Fly stocks Flies were kept on standard fly food at 22°C and 70% humidity unless otherwise noted. Flies subjected to aversive conditioning and GFP-labeling experiments were reared and collected at 18°C and 70% humidity in standard fly bottles. One day before experiments, flies were split into standard fly vials and shifted to 22°C. Fly stocks used in this study are as follows: elav-split-Gal4DBD [70] (BDSC23867), elav-splitGal4AD [70] (BDSC23868), 2xUAS-EGFP [71] (BDSC 23867), R21B06-splitGal4DBD [18] (MB364B), 10xUAS-mCD8GFP [72] (BDSC 32186), UAS-TNTG [73] (BDSC 28838), UAS-TNTQ4 [73] (BDSC 28839), UAS-Flp [74] (BDSC 4539), 13xLexAop-mCD8GFP [72] (BDSC 32205), Brp-FRTstopFRT-V5-2A-LexA [75], hs-CREB2b [5] (17–2), 20xUAS-ChrimsonVENUS [64] (BDSC 55135), Gr66a-Gal4 [76], MB077B [18], 20xUAS-GCaMP6f [77] (BDSC 42747), and 10xUAS-tdTomatomyr [72] (BDSC 32223). BDSC stocks were obtained from the Bloomington Drosophila Stock Center, Indiana University, Bloomington. Molecular biology For the generation of the CAMEL tool, we annealed complementary DNA sequences (Microsynth AG, Balgach, Switzerland) containing restriction sites and varying numbers of CRE sites (S3 Table). The double-stranded DNAs were inserted into pENTR vectors (Thermo Fisher, Waltham, MA) using restriction ligation. CRE constructs were then cloned into the pBPp65ADZpUw vector [72] via gateway cloning (Thermo Fisher, Waltham, MA). All cloning steps were verified by sequencing. Transgenic flies were generated using the attP2 landing site using the phi-C31 system [78]. Aversive olfactory conditioning Aversive conditioning was performed as described by Tully and colleagues [49] under weak red light at 20°C to 22°C and 75% humidity using 2- to 7-day-old flies. We used a custom-made conditioning device allowing parallel training of 8 groups of flies (details available on request). Flies were loaded into custom-made copper grid tubes and exposed to a constant air stream (750 mL/min). To expose flies to odors, the air stream was odorized via air suction (750 mL/min) through odor containing mineral oil (1:250, Thermo Fisher, Waltham, MA, CAS #8042-47-5). After 2-minute resting periods (air only), flies were simultaneously exposed to CS+ and electric shocks (twelve 1.5-second 90 V shocks with 3.5-second intervals) for 60 seconds. Subsequently, flies were exposed to air for 45 seconds and then to the CS− for 60 seconds. This training cycle was repeated 5 times in 15-minute intervals for spaced training and 10 times in 45-second intervals for massed training. For unpaired conditioning, flies received 60-second electric shocks (twelve 1.5-second 90 V shocks with 3.5-second intervals) in the absence of odor presentation. After 5-minute recovery in constant airflow, flies were exposed to 60 seconds of the first odor, 45 seconds of air, and 60 seconds of the second odor. Five training cycles were performed in 9-minute intervals to keep the overall duration of the experiment identical to the spaced training protocol. Flies were kept at 18°C for the indicated amount of time before memory test in the T-maze [49]. For testing, flies were brought to the decision point of a T-maze at which they could choose for 2 minutes between CS+ and CS− (1:1,000) containing arms. Odors used for conditioning were 4-MCH (Merck, Darmstadt, Germany, CAS #589-91-3) and OCT (Merck, Darmstadt, Germany, CAS #589-98-0). For each experiment, 2 groups of flies were conditioned with reversed odor pairings such that each odor served one time as CS+ and one time as CS−. For each group, Performance Indices (PerIs) were calculated as follows: Averaging the 2 PerIs of reciprocal groups results in the final PerI. For Chrimson experiments, flies were conditioned in parallel using either the paired (spaced) or unpaired protocol and kept at 18°C for the indicated amount of time until testing. The unpaired group in Fig 5G was shocked only once 5 minutes before odor presentations. Flies were kept in the dark on all-trans-retinal containing food (0.5 mM, Sigma-Aldrich) for 2 days before testing (see “Light preference test”). For GCaMP6f experiments, flies were paired (spaced) and unpaired conditioned in parallel as described above and subsequently stored at 18°C for 3 to 4 days before performing live imaging (see “In vivo calcium imaging”). For direct comparison of memory performance across days (S4B Fig, S4C Fig and S4D Fig), we calculated a forgetting factor (F) as follows: Odor acuity and shock responsiveness Odor acuity and shock responsiveness was performed in a T-maze as described by Keene and colleagues [79]. For odor acuity, flies were given 2 minutes to choose between an odor (OCT or MCH) diluted in mineral oil (1:1,000) and pure mineral oil. To test shock responsiveness, flies could freely choose for 60 seconds between a voltage-carrying shock tube (twelve 1.5-second 90 V shocks with 3.5-second intervals) and a nonconnected shock tube under constant airflow. Avoidance scores (ASs) were calculated as follows: Experimental tubes (odor, shock) and control tubes (no odor, no shock) were alternately placed into the right and left slot of the T-maze. GFP-labeling experiments For GFP-labeling experiments after conditioning, 2- to 5-day-old flies were conditioned as described and stored at 22°C for 2 days before brain dissection. For odor-reduced environment experiment, late pupae were washed and transferred to vials containing odor-reduced food (1M D-Sorbitol, 2% Agarose) or standard food at 22°C. Three- to 4-day-old flies were dissected and analyzed for GFP expression. In forskolin feeding experiments, 1- to 3-day-old flies were transferred onto food containing 10 μM forskolin (Enzo Life Sciences GmbH, Lörrach, Germany) or onto standard food containing identical volumes of vehicle (PBS) and stored for 3 to 4 days at 22°C before dissection. For heat shock-CREB2b experiments, 1- to 3-day-old flies were subjected to 3 heat shocks (37°C water bath, 30 minutes) in 24-hour intervals prior to dissection. To quantify GFP-labeling experiments, cell bodies of MB neurons were counted using a PL APO 20× oil immersion objective (Leica Microsystems GmbH, Wetzlar, Germany). Immunohistochemistry and microscopy Dissection and antibody labeling was performed as described previously [80]. Briefly, flies were incubated in 4% PFA + 0.2% Triton-X 100 (Merck, Darmstadt, Germany) for 3.5 hours at 4°C and then washed for 3 × 30 minutes in PBS + 0.2% Triton-X 100 at room temperature prior to brain dissection. Dissected brains were incubated in primary antibodies for 2 to 5 days and in secondary antibodies for 2 days at 4°C. Afterwards, brains were washed for at least 3 × 30 minutes. The following antibodies were used in this study: rabbit anti-GFP (A6455, Thermo Fisher, Waltham, MA) (1:1,000); rabbit anti-Dlg [81] (1:30,000); rat anti-CD8a (MCD0800, Thermo Fisher, Waltham, MA) (1:500); mouse anti-Brp (nc82, Developmental Studies Hybridoma Bank, Iowa City, Iowa) (1:200); rabbit anti-dsRed (632496, Takara, Kyoto, Japan) (1:500); mouse anti-V5 (960–25, Thermo Fisher, Waltham, MA) (1:500); and Alexa488, 568, and 647 coupled secondary antibodies (Thermo Fisher, Waltham, MA) (1:1,000). Brains were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were captured using a Zeiss LSM 700 laser scanning confocal microscope with a 25× (NA 0.8) or 40× (NA 1.3) oil immersion objective (Carl Zeiss Microscopy GmbH, Jena, Germany). Images were processed using Imaris (Bitplane AG, www.imaris.oxinst.com) and Adobe Photoshop software (Adobe; www.adobe.com). Light preference test A light preference test was performed in a custom-built choice arena as described by Aso and colleagues and by Klapoetke and colleagues [36,64]. Briefly, approximately 20 flies were placed into the arena under constant airflow (150 mL/min) at 20°C and 60% humidity. The arena was controlled using Arduino UNO and Matlab to expose the flies to the following light exposure protocol: 30 seconds of no light, 30 seconds of red light in 2 quadrants, 30 seconds of no light, and 30 seconds of red light in the other 2 quadrants. The amount of 20 μW/mm2 red light (627 nm) intensity was used for all experiments. Flies were monitored and counted using infrared light and an infrared camera (Rasperry Pi Camera V2.1). Light PI of 1 time point was calculated as follows: For one experiment, the total PI is the average of 10 PIs from the last 5 seconds of the 2 light-on episodes. ΔPI represents the difference between the PI of the paired group minus the PI of the unpaired group. In vivo calcium imaging Flies were anaesthetized on ice for 10 to 15 seconds and mounted on a custom-built chamber using paraffin wax (Merck, Darmstadt, Germany) and a dental waxing device. The head, proboscis, and legs were immobilized with wax, and the chamber was filled with ice-cold sugar-free HL3-like saline [82]. To access the brain, a small coronal window through the fly’s head cuticle was opened. Images were acquired using an Ultima Investigator Multiphoton Imaging System (Bruker, Billerica, MA) powered by Prairie View Imaging Software (Bruker, Billerica, MA) and a Chameleon Vision II excitation laser (Coherent, Palo Alto, CA) at 940 nm. Imaging was performed using a 20× water immersion objective (NA 1.0 XLUMPLFLN, Olympus, Tokyo, Japan). All images were uniformly acquired with a galvo scanner at a 16× optical zoom and a dwell time of 1.4–1.6 μs and were formatted to 256 × 256 pixels of 16 bit resulting in a 7 Hz frame rate. For odor presentation, we used a custom-built olfactory presentation device similar to previously described setups [83]. We used manual airflow meters (Cole Parmer, Vernon Hills, IL) to adjust a carrier stream (0.4 LPM), an odor stream (0.1 LPM), and a replacement stream (0.1 LPM). For stimulus presentation, a dual synchronous 3-way valve (SV360T041, NResearch, West Caldwell, NJ) was used to switch between the replacement stream and the odor stream that was channeled through odor-containing vials (1:1,000 in mineral oil). The final tubing segment (approximately 25 cm) was fixed on the custom-made imaging chamber and oriented towards the anterior part of the fly’s head. For Ca2+ imaging of all R21B06 neurons, flies (elavAD∩R21B06DBD > GCaMP6f, tdTomatomyr) were prepared as described. Data were acquired as described in previous studies [28] in 20-second sweeps. We used Fiji custom macros to extract responsive cells by subtracting the first frame (baseline) from the peak activity frame. We presented each stimulus 3 times (OCT, MCH) and counted a cell as stimulus specific when 2 or more of the presentations of the same stimulus resulted in neuronal activity. For CAMEL-cell imaging, paired and unpaired conditioned flies (6xCREAD∩R21B06DBD > GCaMP6f, tdTomatomyr) were alternately prepared and imaged on the same day. Due to technical issues with the olfactory presentation device, 2 complete days were excluded from analysis. We tested each KC manually with 1 odor pulse per odor and then started an automated odor presentation routine consisting of OCT, MCH, benzaldehyde (Merck, Darmstadt, Germany, CAS #100-52-7), and IBA (Merck, Darmstadt, Germany, CAS #110-19-0). We used Fiji [84] and custom recursive batch processing algorithms to enable batch image registration with TurboReg [85] of the whole data set. Images were segmented using a combined approach of Fiji recursive algorithms and manual ROI selection (whole cell body). After segmentation, in each time frame the mean fluorescence intensity of the background was subtracted from the complete image. Custom Matlab (MathWorks, www.mathworks.com) routines were programmed to calculate a normalized trace (f-f 0 /f 0 ) such that the baseline is the average mean intensity of the first 28 frames. From the normalized trace, a 5-point running average smoothed trace was generated to extract the peak in each response window. If the peak (within 0–4.5 seconds post stimulus) was 2.33 times greater than the standard deviation of the baseline, it was scored as a response to the stimulus [27]. The same analysis was performed manually. To correct for false positive and false negative responses, in a few cases, response and baseline windows were manually adjusted. Statistics Statistical analysis was performed using GraphPad Prism 7.0 software. Normality of data was tested using D’Agostino Pearson omnibus and Shapiro-Wilk normality tests. Data sets that were significantly different from normal distribution were analyzed using nonparametric tests (Mann-Whitney test and Kruskal-Wallis test) instead of parametric tests (unpaired t test and ordinary one-way ANOVA). To correct for multiple comparisons between relevant groups, the Bonferroni (after ANOVA) and Dunn’s (after Kruskal-Wallis) tests were used. More detailed information on statistics and sample sizes are listed in S1 Table for main figures and S2 Table for Supporting Information figures.

Acknowledgments We thank FlyLight, the Bloomington Stock Center (Indiana), S. Lawrence Zipursky, and Jerry C. P. Yin for flies and the Developmental Hybridoma Bank (Iowa) for antibodies. We are grateful to Chunsu Xu for critical help with behavioral experiments, Yoshinori Aso for blueprints of the light preference arena and advice, and Anne Thyssen for realization of the arena.