1. Neuroscience

Differential conditioning produces merged long-term memory in Drosophila

  1. Bohan Zhao
  2. Jiameng Sun
  3. Qian Li
  4. Yi Zhong  Is a corresponding author
  1. School of Life Sciences, IDG/McGovern Institute for Brain Research, and MOE Key Laboratory for Protein Science, Tsinghua University, China
  2. Tsinghua-Peking Center for Life Sciences, China
https://doi.org/10.7554/eLife.66499
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  1. Bohan Zhao
  2. Jiameng Sun
  3. Qian Li
  4. Yi Zhong
(2021)
Differential conditioning produces merged long-term memory in Drosophila
eLife 10:e66499.
https://doi.org/10.7554/eLife.66499
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Abstract

Multiple spaced trials of aversive differential conditioning can produce two independent long-term memories (LTMs) of opposite valence. One is an aversive memory for avoiding the conditioned stimulus (CS+), and the other is a safety memory for approaching the non-conditioned stimulus (CS–). Here, we show that a single trial of aversive differential conditioning yields one merged LTM (mLTM) for avoiding both CS+ and CS–. Such mLTM can be detected after sequential exposures to the shock-paired CS+ and -unpaired CS–, and be retrieved by either CS+ or CS–. The formation of mLTM relies on triggering aversive-reinforcing dopaminergic neurons and subsequent new protein synthesis. Expressing mLTM involves αβ Kenyon cells and corresponding approach-directing mushroom body output neurons, in which similar-amplitude long-term depression of responses to CS+ and CS– seems to signal the mLTM. Our results suggest that animals can develop distinct strategies for occasional and repeated threatening experiences.

Introduction

To survive in a complex environment, animals need to learn from threatening experiences to avoid potential dangers. From invertebrates to humans, aversive differential conditioning is widely used to study memories produced by threatening experiences (Carew et al., 1983; Corches et al., 2019; Gentile et al., 1986; Schneider et al., 1999; Tully and Quinn, 1985). After repetitive spaced trials of conditioning, animals form two complementary long-term memories (LTMs) of opposite valence (Jacob and Waddell, 2020; Pollak et al., 2008; Pollak et al., 2010), including the aversive memory to the conditioned stimulus (CS+) and the rewarding memory to the non-conditioned stimulus (CS–) (Figure 1—figure supplement 1). Such complementary LTMs result in enhanced long-lasting discrimination between CS+ and CS– through guiding avoidance to CS+ and approach to CS–. However, it remains unclear whether and how occasional threatening experiences, such as single-trial conditionings, would induce long-lasting changes in future escape behavior.

From invertebrates to humans, experience-dependent long-lasting behavioral modifications mainly rely on the formation of LTMs (Kandel et al., 2014). In Drosophila, there are at least two categories of aversive olfactory LTMs that last for more than 7 days. One is the spaced training-induced LTM that can be observed after repetitive spaced training with inter-trial rests (multiple trials with a 15 min rest interval between each), but not after either single-trial training or repetitive massed training without interval (Tully et al., 1994). Forming such aversive LTM requires new protein synthesis and the paired posterior lateral 1 (PPL1) cluster of dopaminergic neurons (DANs) to depress the connection between odor-activated Kenyon cells (KCs) in the mushroom body (MB) αβ lobe and downstream α2sc (MB-V2) MB output neurons (MBONs) (Aso et al., 2014a; Aso et al., 2014b; Cognigni et al., 2018; Modi et al., 2020; Pascual and Préat, 2001; Plaçais et al., 2012). The other is a recently reported context-dependent LTM that forms after single-trial training, which does not require protein synthesis-dependent consolidation (Zhao et al., 2019). The expression of context-dependent LTM relies on multisensory integration in the lateral horn and is not affected by blocking KCs. However, all these observations derived from the same design principle that evaluates memory performance through testing the discrimination between CS+ and CS– (Tully and Quinn, 1985). Thus, direct responses to CS+ and CS– have been largely overlooked.

In the current study, we introduced a third-odor test, in which flies were given a choice between either CS+ and a novel odor, or CS– and a novel odor. We therefore identified that the single-trial differential conditioning produces a merged LTM (mLTM) guiding avoidances of both CS+ and CS– for several days after training. The encoding and expression of such mLTM involve new protein synthesis, PPL1 DANs, αβ KCs, and α2sc MBONs. These findings suggest that animals utilize distinct escape strategies for facing occasional and repeated dangers.

Results

Single-trial training produces aversive LTMs to both CS+ and CS–

We first plotted the memory retention curve at various time points after training (Figure 1A). Consistent with previous reports (Davis and Zhong, 2017; Shuai et al., 2010; Tully et al., 1994), flies exposed to single-trial conditioning rapidly lost their ability to discriminate CS+ and CS– within 1 day. However, when trained flies were instead tested between either CS+ and a novel odor, or CS– and a novel odor, they gradually exhibited significant avoidances of both CS+ and CS– from 12 hr to more than 7 days after training (Figure 1B). Consistently, testing CS+ or CS– versus air also revealed training-induced long-lasting avoidances of CS+ and CS– (Figure 1—figure supplement 2).

Figure 1 with 2 supplements see all
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Long-term avoidances of both conditioned stimulus (CS+) and non-conditioned stimulus (CS–) induced by aversive differential conditioning.

(A) Single-trial differential conditioning induced discriminative memory that was forgotten within 1 day (n = 6–8). (B) Conditioned flies exhibited long-lasting avoidances of both CS+ and CS– (n = 8–10). (C) Cycloheximide (CXM) treatment abolished 1 day avoidances of CS+ and CS– (n = 6). (D) The 1 day avoidances were tested after cold-shock anesthesia at different time points following training. Aversive memory to CS+ and CS– requires a multi-hour consolidation process (n = 6–8). All data shown are presented as mean ± SEM. *p < 0.05.

Figure 1—source data 1

Behavioral data of each group in Figure 1.

https://cdn.elifesciences.org/articles/66499/elife-66499-fig1-data1-v2.xlsx

Based on these observations, we wondered whether such aversive LTMs require new protein synthesis to consolidate. Results show that such avoidances resulted from the formation of protein synthesis-dependent LTMCS+ and LTMCS– because the administration of cycloheximide (CXM), a protein synthesis inhibitor, prevented the avoidances of both CS+ and CS– after 1 day (Figure 1C). The time course of the consolidation of such LTMs could be revealed by cold-shock treatment, which is known to abolish labile memories but leave consolidated LTM intact (Krashes and Waddell, 2008; Li et al., 2016; Tully et al., 1994). As shown in Figure 1D, it takes more than 3 hr for consolidation to be completed.

The long-term avoidances of CS+ and CS– seem to be derived from the same mLTM

The above results prompted us to investigate whether LTMCS+ and LTMCS– are based on two parallel LTMs of the same valence, or the same LTM that can be retrieved by either odor. Two lines of evidence suggested that LTMCS+ and LTMCS– are derived from the same memory component.

First, LTMCS+ and LTMCS– were both extinguished by the re-exposure to either CS+ or CS– alone (Figure 2A). Trained flies were re-exposed to three cycles of either CS+ or CS– for 1 min with 1 min inter-trial intervals immediately before testing. Such treatment significantly reduced both LTMCS+ and LTMCS–, as compared to those re-exposed to air, suggesting the LTMCS+ and LTMCS– are both retrievable for either one of CS+ and CS–.

Figure 2 with 1 supplement see all
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A merged long-term memory (mLTM) underlies the long-term avoidances of conditioned stimulus (CS+) and non-conditioned stimulus (CS–).

(A) Three trials of re-exposure to either CS+ or CS– alone can impair both CS+ avoidance and CS– avoidance 1 day after training (n = 6). (B) Prolonging the inter-trial interval (ITI) between CS+ and CS– to more than 5 min significantly impaired 1 day avoidances (n = 6–8). (C) Changing the sequence of CS+ and CS– during training did not affect 1 day avoidances (n = 6). All data shown are presented as mean ± SEM. *p < 0.05. NS, non-significant.

Figure 2—source data 1

Behavioral data of each group in Figure 2.

https://cdn.elifesciences.org/articles/66499/elife-66499-fig2-data1-v2.xlsx

Second, LTMCS+ and LTMCS– were both abolished when the temporal interval between CS+ and CS– during training was prolonged to more than 5 min (Figure 2B), suggesting that the formation of LTMCS+ and LTMCS– depends on the contiguity of two shock-paired CS+ and CS– exposures, instead of temporally separated exposures. In addition, changing the sequence of exposure to CS+ and CS– did not affect either LTMCS+ or LTMCS– (Figure 2C), but training with CS+ or CS– alone failed to induce the LTMCS+ or LTMCS– (Figure 2—figure supplement 1).

Together, these results strongly suggest that, instead of two parallel LTMs, observed LTMCS+ and LTMCS– are more likely to be derived from the same memory component. Since it was observable after two separated memory curves (CS+ and CS–) merged over time, we termed it as mLTM.

The encoding of mLTM requires aversive-reinforcing DANs

Then we further investigated the neural network mechanisms underlying such mLTM. In Drosophila, associative olfactory memories are mainly encoded by the DANs (Aso and Rubin, 2016; Claridge-Chang et al., 2009; Cognigni et al., 2018) through alterations of the connections between KCs and MBONs (Aso et al., 2014a; Aso et al., 2014b; Cognigni et al., 2018; Dubnau and Chiang, 2013; Modi et al., 2020; Pascual and Préat, 2001; Plaçais et al., 2012). We therefore tested whether DANs involve in mLTM encoding by comparing 24 hr memory in control flies with that in flies whose synaptic outputs from different clusters of DANs were blocked during training. For this purpose, we expressed the dominant-negative temperature-sensitive UAS-Shibirets1 (Shits)-encoded dynamin (Kitamoto, 2001) driven by R58E02-Gal4, which labels the protocerebral anterior medial (PAM) cluster of DANs, and R52H03-Gal4 and TH-Gal4, which both label PPL1 DANs (Figure 3—figure supplement 1A,B and C). We specifically blocked output from DANs during training by raising the temperature of flies from 23°C to 32°C. Flies were then returned to 23°C and later tested for 1 day memory. The results showed that blocking the release of neurotransmitter from PPL1 DANs, but not PAM DANs, during training impaired mLTM (Figure 3A), suggesting that the neuromodulation of PPL1 DANs is necessary for mLTM formation.

Figure 3 with 2 supplements see all
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Expression of the merged long-term memory (mLTM) requires the αβ Kenyon cells (KCs).

(A) Blocking paired posterior lateral 1 (PPL1) dopaminergic neurons (DANs) (R52H03-Gal4 or TH-Gal4) but not protocerebral anterior medial (PAM) DANs (R58E02-Gal4) during training significantly impaired mLTM (n = 6–8). (B) Blocking mushroom body (MB) during testing using R13F02-Gal4>UAS-Shits impaired the mLTM (n = 6–8). (C) The mLTM was impaired by blocking αβ KCs (C739-Gal4), but not that of α’β’ KCs (VT30604-Gal4) or γ KCs (5-HT1B-Gal4) (n = 6–8). All data shown are presented as mean ± SEM. *p < 0.05.

Figure 3—source data 1

Behavioral data of each group in Figure 3.

https://cdn.elifesciences.org/articles/66499/elife-66499-fig3-data1-v2.xlsx

Retrieval of mLTM requires output of αβ KCs

We then examined the role of KCs in mLTM retrieval. We imposed the block by raising temperature to 32°C from 23°C, 15 min before the test. Inhibition of the synaptic output of all KCs (R13F02-Gal4>UAS-Shits) through exposure to a restrictive temperature during the test abolished mLTM (Figure 3B and Figure 3—figure supplement 1D). We further tested the effects of blocking three distinct subgroups of KCs (Figure 3C), namely, those located within the MB αβ lobe (αβ KCs), which were labeled with C739-Gal4; those located within the α’β’ lobe (α’β’ KCs), which were labeled with VT30604-Gal4; and those located within the γ lobe (γ KCs), which were labeled with 5-HT1B-Gal4 (Figure 3—figure supplement 1E-G). The results showed that mLTM did not express when the synaptic output of αβ KCs was blocked, whereas inhibition of α’β’ KCs and γ KCs had no significant effect (Figure 3C).

The α2sc cluster of MBONs is required for mLTM expression

Then we further tried to identify which cluster of MBONs from αβ lobe of MB is required for mLTM. Prior studies on the structure of MB network have uncovered that the PPL1 DANs mainly integrate to the α2 and α3 compartments of the vertical α lobe of MB, and corresponding MBONs (α2sc and α3 clusters) have been reported to be important to aversive spaced training-induced LTM expression (Aso et al., 2014a; Aso et al., 2014b; Bouzaiane et al., 2015; Dubnau and Chiang, 2013; Jacob and Waddell, 2020; Modi et al., 2020; Pascual and Préat, 2001; Plaçais et al., 2012; Schwaerzel et al., 2003). We therefore attempted to block α2sc MBONs (R71D08-Gal4) and α3 MBONs (G0239-Gal4) during testing, respectively (Figure 4—figure supplement 1A,B). Results showed that mLTM expression was significantly impaired by blocking α2sc MBONs, but not α3 MBONs (Figure 4A,B). This conclusion was further strengthened by the result that inhibiting the neural activity of α2sc MBONs using heat-inducible expression of the potassium channel Kir2.1 decreased the mLTM retrieval (Figure 4—figure supplement 2). Thus, these results suggest that retrieving mLTM specifically relies on the output of α2sc MBONs.

Figure 4 with 2 supplements see all
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Expression of the merged long-term memory (mLTM) requires α2sc mushroom body output neurons (MBONs).

(A) Blocking α2sc MBONs using R71D08-Gal4>UAS-Shits impaired mLTM expression (n = 6–10). (B) Blocking α3 MBONs using G0239-Gal4>UAS-Shits did not affect mLTM expression (n = 6). All data shown are presented as mean ± SEM. *p < 0.05. NS, non-significant.

Figure 4—source data 1

Behavioral data of each group in Figure 4.

https://cdn.elifesciences.org/articles/66499/elife-66499-fig4-data1-v2.xlsx

Differential conditioning induces long-lasting depression of CS+ and CS– odor-evoked responses in α2sc

We then imaged odor-evoked calcium responses in the dendritic field of α2sc MBONs to CS+ and CS– through expressing jGCamP7f, a calcium-sensitive fluorescent protein (Dana et al., 2019), driven by R71D08-Gal4. To account for variance between different odors, the responses of trained flies were calibrated to the average responses of naïve flies to corresponding odors. Consistent with results of the behavioral assay, 1 day after training, odor-evoked calcium responses of α2sc MBONs to CS+ and CS– were both significantly reduced when compared to responses to the novel odor (Figure 5A,B). Moreover, such depression of CS+ and CS– odor-evoked responses can be abolished by prolonging the temporal interval between CS+ and CS– to 10 min during training (Figure 5C), which is consistent with the result of behavioral assay (Figure 2B). Interestingly, when we recorded the calcium responses immediately after training, the depressed responses to CS+ and CS– were observed, suggesting the mLTM should be encoded during training (Figure 5—figure supplement 1). Together, our data showed that the aversion to CS+ and CS– are both linked to the neural plasticity conferred by the same MBON.

Figure 5 with 1 supplement see all
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The merged long-term memory (mLTM) can be recorded as depressed odor-evoked responses in α2sc mushroom body output neurons (MBONs).

(A) Left: training and imaging protocols. The imaging plane for α2sc MBONs is shown. The novel odor-evoked response, conditioned stimulus (CS+) odor-evoked response, and non-conditioned stimulus (CS–) odor-evoked response were calibrated to the average responses of corresponding odors in naïve flies. Right: the mLTM can be recorded as depressed odor-specific responses in α2sc MBONs (n = 9). (B) Prolonging the inter-trial interval (ITI) between CS+ and CS– during training abolished the depressed odor-specific responses (n = 8). All data shown are presented as mean ± SEM. *p < 0.05. NS, non-significant.

Figure 5—source data 1

Calcium imaging data of each group in Figure 5.

https://cdn.elifesciences.org/articles/66499/elife-66499-fig5-data1-v2.xlsx

Discussion

In the current study, the use of third-odor test leads to a conclusion that single-trial training produces an mLTM for guiding flies to avoid both CS+ and CS– for more than 7 days. Three categories of evidence in support of this conclusion are outlined below.

First, throughout our study, the amplitudes of long-term avoidances of CS+ and CS– are always at a similar level under various conditions, including pharmacological treatment (Figure 1C), cold-shock treatment (Figure 1D), odor re-exposure (Figure 2A), paradigm alteration (Figure 2B and C), and neural circuitry manipulations (Figures 3 and 4). Second, re-exposure to either one of CS+ and CS– alone can extinguish both CS+ avoidance and CS– avoidance (Figure 2A). Third, the long-term avoidances of CS+ and CS– can be recorded as the depression of odor-evoked responses in the same α2sc MBONs (Figure 4), meanwhile, CS+ avoidance and CS– avoidance both involve the same PPL1 DANs, αβ KCs, and α2sc MBONs (Figures 3 and 4A). Thus, CS+ avoidance and CS– avoidance derive from the same aversive mLTM, instead of two parallel LTMs of the same valence. The significance of these findings is further discussed below.

Combining with a recent report that uses a similar third-odor test to dissect LTMs induced by multi-trial spaced training (Jacob and Waddell, 2020), we are led to conclude that spaced multi-trial aversive differential conditioning produces two independent LTMs of opposite valence for avoiding CS+ and approaching CS–, whereas single-trial aversive differential conditioning yields one mLTM that guides avoidances of both CS+ and CS–. Thus, animals can develop distinct escape strategies for different categories of dangers. When the same dangerous situation has been experienced repeatedly, animals would remember the detailed information to guide behavior in the next similar situation. However, when the dangerous event has only been experienced occasionally, animals would choose to avoid all potentially dangerous cues as a more reserved survival strategy.

Moreover, the differences between single-trial training-induced mLTM and multi-trial training-induced complementary LTMs lead us to ask: how these differences are induced by different training sessions? Jacob and Waddell, 2020 reported that multi-trial spaced training induces depressed responses to CS+ in α2sc MBONs and α3 MBONs are required for aversive LTM to CS+, whereas the modulated responses to CS– in β’2mp MBONs and γ3, γ3β’1 MBONs appears to be responsible for the safety memory to CS–. In contrast, here we found that single-trial training is sufficient to induce the depressed responses to both CS+ and CS– in α2sc MBONs. Therefore, our results suggest a lower threshold and specificity of the plasticity between KCs-α2sc MBONs, compared to KCs-α3 MBONs, KCs-β’2mp MBONs, and KCs-γ3, γ3β’1 MBONs connections. Consequently, changing these synaptic connections requires involving more training sessions.

Materials and methods

Fly strains

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All flies (Drosophila melanogaster) were raised on standard cornmeal medium at 23°C and 60% relative humidity under a 12 hr light-dark cycle as described (Shuai et al., 2010). The control strain was w1118(isoCJ1). UAS-Shibirets1 and UAS-Kir2.1;tub-gal80ts were extant stocks in the laboratory. Gal4 lines used have been described previously: TH-Gal4 (Friggi-Grelin et al., 2003), C739-Gal4 (McGuire et al., 2001), R58E02-Gal4, R52H03-Gal4, R13F02-Gal4, and R71D08-Gal4 (Jenett et al., 2012), 5HT1b-Gal4 (Yuan et al., 2005), G0239-Gal4 (Pai et al., 2013), and VT30604-Gal4 (#200228) (Wu et al., 2013). UAS-GCaMP7f was a gift from Yi Sun, Westlake University.

Behavioral assays

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All flies were raised at 23°C and mixed-sex populations of 2- to 5-day-old flies were used in all experiments. The Pavlovian olfactory aversive conditioning procedure was performed as described previously in a behavioral room at 23°C and 60% relative humidity described (Shuai et al., 2010).

During training, roughly 80 flies successfully received the following stimuli in a training tube, which contained a copper grid: air for 90 s, an odor paired with 12 pulses of 60 V electric shock (CS+) for 1 min, air for 45 s, a second odor without pairing the electric shock (CS–) for 60 s, and finally air for 45 s. 3-Octanol (OCT, 10–15 µl in 10 ml mineral oil), 4-methylcyclohexanol (MCH, 10 µl in 10 ml mineral oil), and ethyl acetate (EA, 10 µl in 10 ml mineral oil) were used as standard odorants. This process describes a single training trial. Six sequential trials with 15 min intervals constitute spaced training. For memory retrieval, trained flies were transferred into a T-maze, where they were allowed 2 min to choose between two odors (CS+ and novel odor, or CS– and novel odor).

Odor performance was quantified by a PI (performance index) calculated based on the fraction of flies in the two T-maze arms. A PI of 100 indicated that all flies avoid the odor exposed during training (either CS+ or CS–), while a PI of 0 indicated no odor preference, as reflected by 50:50 distributions between the arms. To balance naïve odor bias, two reciprocal groups were trained and tested simultaneously and the testing odors were always OCT and MCH, one serves as the CS+ or CS– and the other serves as the novel odor alternatively. One complete PI was generated from the average PI of a couple of rotate training and testing. Specifically, for the testing of CS+ performance, either OCT or MCH was alternately used as CS+, and EA was CS–; whereas for the testing of CS– performance, either OCT or MCH was alternately used as CS–, and EA was CS+. For 3 min memory, flies were tested immediately after training. For longer memory retention, flies were placed in a fresh vial with the same contents as the vial they had been kept in before the training until the test.

For odor avoidance testing, a similar PI was calculated based on the fraction of flies in the two T-maze arms. A PI of 100 indicated that all flies choose the air arm, while a PI of 0 indicated no preference.

For cold-shock anesthesia, flies were transferred into pre-chilled plastic vials and kept on ice for 2 min as described previously (Shuai et al., 2010).

For cycloheximide (CXM) feeding, flies were provided food with (CXM+) or without (CXM–) 35 mM CXM (Sigma-Aldrich) dissolved in control solution, and 5% (wt/vol) glucose, for 16 hr before and after training until memory retention was tested.

For neural inactivation experiments using UAS-Shits, crosses were reared at 23°C to avoid unintended inactivation. Flies were shifted to 32°C 15 min in advance for neural inhibition during training or test.

For odor re-exposure, flies were exposed to three cycles of conditioned odor (either CS+ or CS–) for 1 min with 1 min interval as described previously (Wang et al., 2019). All mentioned re-exposure treatments were performed immediately before testing.

Immunohistochemistry

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Flies were quickly anesthetized on ice and whole brains were dissected in ice-cold PBS within 5 min, then stained as described (Shuai et al., 2010). Brains were fixed in 4% paraformaldehyde in PBS for 30 min on ice. Brains were incubated for at least 72 hr with the primary antibodies anti-GFP (chicken, 1:2000; Abcam, Cambridge, UK), anti-nc82 (mouse, 1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), and anti-DsRed (rabbit, 1:500; Takara Bio, Kyoto, Japan). Brains were washed three times again in PBS with 0.2% Triton X-100 and transferred into secondary antibody solution (anti-chicken Alexa Fluor 488, 1:200; anti-mouse Alexa Fluor 647, 1:200; anti-rabbit Alexa Fluor 647, 1:200; Molecular Probes, Eugene, OR) and incubated for 48 hr at 4°C. Images were obtained using a Zeiss LSM710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).

In vivo two-photon calcium imaging

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Two- to five-day-old female flies were used. After anesthetized in a plastic vial on ice for 15–20 s, flies were then gently inserted into a hole of a thin plastic rectangular plate and stabilized in the hole by glue. In a saline bath, the area surrounding the region of interest was surgically removed to expose the dorsal side of the brain. Fat and air sacs were gently removed to give a clear view of the brain. For calcium response imaging, the 40× water immersion objective lens (NA=1.0; Zeiss) were lowered near the exposed brain.

Imaging was performed on a Zeiss LSM 7MP two-photon laser scanning microscope with an imaging wavelength at 910 nm (Carl Zeiss AG). The 512 × 512 pixel images were acquired at 2.6 Hz. In each trial, 10 s of baseline was recorded, followed by an odor-evoked response recording. Two to three minutes of rest were given in between trials when multiple trials were applied. GCaMP responses and the average value within 10 s before and during stimulus were quantified using custom software written in MatLab (MathWorks, Natick, MA). For the brain region of interest during the experimental period, the average fluorescence value, Fave, was then converted to ΔF/F0 using the formula ΔF/F0 = (Fave-F0)/F0, where F0 is the baseline fluorescence value, measured as the average of 10 s before the stimulus. Flies were trained using CS+ (OCT, 10 µl in 10 ml mineral oil) and CS– (MCH 10 µl in 10 ml mineral oil), and IA (10 µl in 10 ml mineral oil) was used as novel odor. To gain the calibrated calcium responses of trained flies, the ratios of ΔF/F0 to different odors of trained and naïve flies were calculated. Specifically, all odor-evoked responses after training were divided to the mean responses to the same odors of same number naïve flies. Thus, a calibrated ΔF/F0 of 1.0 indicated no difference in the odor-evoked responses to the correspondence odor between trained and naïve group.

Statistics

Statistics were performed with GraphPad Prism software (version 7; GraphPad Software, San Diego, CA). All data satisfied the assumption of normal distribution (one-sample Kolmogorov–Smirnov test). Comparisons between two groups were performed using two-tailed t-tests. Comparisons of multiple groups were performed using one-way or two-way analysis of variance (ANOVA) tests followed by Bonferroni correction for multiple comparisons. p-Values less than 0.05 were considered statistically significant and are marked with an asterisk in figures; NS indicates non-significant differences (p > 0.05).

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

References

Decision letter

  1. K VijayRaghavan
    Senior and Reviewing Editor; National Centre for Biological Sciences, Tata Institute of Fundamental Research, India
  2. Ann-Shyn Chiang
    Reviewer; National Tsing Hua University, Taiwan

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

This work reveals a novel form of Drosophila long-term memory that is of potential interest to most neuroscientists working on various animals. Classical protein-synthesis-dependent long-term memory forms only after repetitive spaced trials of olfactory conditioning. The authors discovered that flies also form a "blurred" or "vague" protein-synthesis-dependent long-term memory which distinguishes experienced two odors from a third naive odor after single-trial training. This merged long-term memory lacking the event details likely occurs in most animals since long-lasting memory of occasional threatening experiences for future escape behavior is crucial for survival.

Decision letter after peer review:

Thank you for submitting your article "Differential Conditioning Produces Merged Long-term Memory in Drosophila" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by K VijayRaghavan as the Senior and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ann-Shyn Chiang (Reviewer #1).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The authors should keep these essential revisions in mind while attending to the reviewers' comments which are appended below.

The final model presented in the Zhong manuscript presents an integrated view of the results obtained by them and by Jacob and Wadell, Neuron 2020. To do this it is important to replicate some of the essential results from Jacob et al. In addition the reviewers put a high value on manuscript data that replicates data, in the same or complementary ways from other laboratories. Please keep these points in mind while preparing your revisions.

1. By using cell-type-specific Gal4 lines that are readily available, target neurons for "the avoiding CS+ memory", "the early approaching CS- memory" and "the late avoiding CS- memory" should be identified. For example, PPL1-DANs and PAM-DANs expressed in TH-Gal4 can be respectively tested with specific split-Gal4 lines.

2. In Figure 2B, when CS- was presented 20min after CS+ even the mLTM of CS+ vs novel odor was impaired, the result means that the formation of CS+ mLTM requires the presence of CS- within a certain time window. This raises an alternative interpretation of the data that CS- presented 20min after CS+ actually serves as interference for the formation of CS+ mLTM, rather than the merged CS+/CS- memory. As to distinguish these two hypotheses, training the flies with only CS+ without delivering CS-, and then test between CS+ and novel odor should be able to address the question. If presenting CS+ alone fails to form LTM of CS+ vs the third odor, then CS- presented within a certain time window is necessary. Otherwise, CS- presented 20min after CS- may serve as an interference.

3. In Figure 4B, the author measured the odor responses of CS+, CS-, and novel odor in MBON-a2sc one day after association, and found that both CS+ and CS- showed depression response. To strengthen the link between the functional response of MBON-a2sc and behavioral phenotype, conditions abolished mLTM by reactivation or prolong odor delivery interval (Figure 2A and 2B) should also be tested to see whether the memory trace are also abolished. Also, what is the responses immediately after 1X training? As CS- mLTM changes from appetitive to avoidance within the first 24 hours after training, the calcium responses of MBON-a2 during this period are necessary for understanding the circuit mechanism of mLTM dynamics.

4. (Point 1 of Reviewer 2) The observation that presentation of a novel third odor leads to mLTM after only a single session of aversive conditioning is intriguing. Authors describe in their methods using three odors for their experiments (as CS+, CS- or novel), but did not alternate/rotate the different combination pairings used as the "novel" one. A panel of odors as "novel", not listed in the manuscript, should be tested which will strengthen the larger conceptual framework and impact. In addition, the authors should perform at least a subset of the experiment using air during testing rather than a 3rd odor.

5. (Point6 of Reviewer 2) More experimentation and discussion regarding the differences between single-trial conditioning to form mLTM and spaced conditioning to form complementary LTM, is required. The authors contrast/merge their behavioral results with those published by Jacob et al., (2020). The authors should reproduce the essence of those found by Jacob et al., and publish them in this paper. Replication of experimental results across labs is very important, especially for behavioral outcomes and when models are constructed using results obtained by other investigators. The authors allude to the two pairs of DAN that project to α2sc MBN for this plasticity, but did not specifically mention those DAN (lines 220-221) nor elaborate on this speculation.

Reviewer #1 (Recommendations for the authors):

The finding of mLTM opens many questions. Knowing it is impractical to address all these questions in one study, I thus suggest only essential experiments to strengthen the report.

1. By using cell-type specific Gal4 lines that are readily available, target neurons for "the avoiding CS+ memory", "the early approaching CS- memory" and "the late avoiding CS- memory" should be identified. For example, PPL1-DANs and PAM-DANs expressed in TH-Gal4 can be respectively tested with specific split-Gal4 lines.

2. In Figure 2B, when CS- was presented 20min after CS+ even the mLTM of CS+ vs novel odor was impaired, the result means that the formation of CS+ mLTM requires the present of CS- within certain time window. This raises an alternative interpretation of the data that CS- presented 20min after CS+ actually serves as an interference for the formation of CS+ mLTM, rather than the merged CS+/CS- memory. As to distinguish these two hypothesis, training the flies with only CS+ without delivering CS-, and then test between CS+ and novel odor should be able to address the question. If presenting CS+ alone fails to form LTM of CS+ vs the third odor, then CS- presented within certain time window is necessary. Otherwise, CS- presented 20min after CS- may serve as an interference.

3. In Figure 4B, the author measured the odor responses of CS+, CS-, and novel odor in MBON-a2sc one day after association, and found that both CS+ and CS- showed depression response. To strengthen the link between functional response of MBON-a2sc and behavior phenotype, conditions abolished mLTM by reactivation or prolong odor delivery interval (Figure 2A and 2B) should also be tested to see whether the memory trace are also abolished. Also, what is the responses immediately after 1X training? As CS- mLTM changes from appetitive to avoidance within the first 24 hours after training, the calcium responses of MBON-a2 during this period are necessary for understanding the circuit mechanism of mLTM dynamics.

In summary, the discovery of mLTM is interesting. However, to sustain the authors' claims that the same neurons are involved in both CS+/CS-avoidance, the authors need to use specific split-Gal4 lines expressed in only the target neurons. Also, to claim this is a merged LTM, authors need to address how the trained flies turn from the initial CS- approaching memory to late CS- avoidance memory. Without additional data to address these critical issues, the authors should largely tune down their claims.

Reviewer #2 (Recommendations for the authors):

1) Citations or references are not included when the phrase "described previously" is used (lines 238, 248, 275, 287).

2) Provide more detail on odor re-exposure methods (lines 283-284). When was this administered?

3) Explain with more detail by "calibrated calcium responses" in methods (lines 316-318).

4) Include sample size used for experiments. It is difficult to count individual data points when variability of data is tight.

5) Figure 3A cartoon – α'3 lobe ( α='3 tip) should be in magenta – innervated by PPL1 DAN.

6) suggest "unconditioned stimulus" rather than "non-conditioned stimulus"

7) organization of references when listed in the text – suggest organizing them chronologically or alphabetically. Currently there is random organization.

8) Figure 2A – why use the term "reactivation" rather than "extinction trials?"

9) The Abstract and the Introduction use the extremely vague phrase, "…that animals utilize escape strategies for facing occasional and repeated dangers…" Either bring this point home for the reader with specific and concrete examples in the Discussion or modify/eliminate the sentence.

10) A good overhaul of the referencing throughout the manuscript is warranted. One glaring omission of perhaps several references that should be offered is in support of the sentence, "In Drosophila, aversive olfactory memories are mainly encoded by the PPL1 cluster of DANs," (line 124). Schwaerzel et al., 2003, and others.

https://doi.org/10.7554/eLife.66499.sa1

Author response

Essential revisions:

The authors should keep these essential revisions in mind while attending to the reviewers' comments which are appended below.

The final model presented in the Zhong manuscript presents an integrated view of the results obtained by them and by Jacob and Wadell, Neuron 2020. To do this it is important to replicate some of the essential results from Jacob et al. In addition the reviewers put a high value on manuscript data that replicates data, in the same or complementary ways from other laboratories. Please keep these points in mind while preparing your revisions.

1. By using cell-type-specific Gal4 lines that are readily available, target neurons for "the avoiding CS+ memory", "the early approaching CS- memory" and "the late avoiding CS- memory" should be identified. For example, PPL1-DANs and PAM-DANs expressed in TH-Gal4 can be respectively tested with specific split-Gal4 lines.

We thank the reviewer for this suggestion. In the revised manuscript, we added the suggested experiments with two more specific Gal4 lines, R58E02 for PAM-DANs and R52H03 for PPL1-DANs (Figure 3—figure supplement 1A, B). The 24-h avoidances to CS+ and CS– were both impaired by blocking PPL1-, but not PAM-DANs (Figure 3A). Interestingly, although the early avoidance to CS+ was impaired specifically by blocking PPL1-DANs, the immediate response to CS– was affected oppositely when PPL1-DANs or PAM-DANs were blocked (Figure 3—figure supplement 2). These data support our theoretical model in the revised edition that the response to CS– is the sum of two opposite memory components: the hours-lasting safety-memory to CS– and the aversive mLTM (Figure 6B).

2. In Figure 2B, when CS- was presented 20min after CS+ even the mLTM of CS+ vs novel odor was impaired, the result means that the formation of CS+ mLTM requires the presence of CS- within a certain time window. This raises an alternative interpretation of the data that CS- presented 20min after CS+ actually serves as interference for the formation of CS+ mLTM, rather than the merged CS+/CS- memory. As to distinguish these two hypotheses, training the flies with only CS+ without delivering CS-, and then test between CS+ and novel odor should be able to address the question. If presenting CS+ alone fails to form LTM of CS+ vs the third odor, then CS- presented within a certain time window is necessary. Otherwise, CS- presented 20min after CS- may serve as an interference.

As suggested by the reviewer, we have performed new experiments with absolute training (training without CS– or CS+) in the revision, and such training failed to induce 24-h avoidances (Figure 2—figure supplement 1). The obtained data support presenting CS+ and CS– are both necessary for forming mLTM. Moreover, this conclusion can be further strengthened by the result that mLTM was gradually decreased with the prolonged intervals between CS+ and CS– (Figure 2B).

3. In Figure 4B, the author measured the odor responses of CS+, CS-, and novel odor in MBON-a2sc one day after association, and found that both CS+ and CS- showed depression response. To strengthen the link between the functional response of MBON-a2sc and behavioral phenotype, conditions abolished mLTM by reactivation or prolong odor delivery interval (Figure 2A and 2B) should also be tested to see whether the memory trace are also abolished. Also, what is the responses immediately after 1X training? As CS- mLTM changes from appetitive to avoidance within the first 24 hours after training, the calcium responses of MBON-a2 during this period are necessary for understanding the circuit mechanism of mLTM dynamics.

We provide new data in revision that training with 10-min temporal intervals fails to form the long-term depression in α2sc MBONs (Figure 5). In addition, we found that the depression of odor-evoked responses to CS+ and CS– can be observed immediately after training (Figure 5—figure supplement 1A). Our data suggest that the merged memory should be encoded during training (Figure 5—figure supplement 1B) and then consolidated within 8 hours (Figure 1D).

4. (Point 1 of Reviewer 2) The observation that presentation of a novel third odor leads to mLTM after only a single session of aversive conditioning is intriguing. Authors describe in their methods using three odors for their experiments (as CS+, CS- or novel), but did not alternate/rotate the different combination pairings used as the "novel" one. A panel of odors as "novel", not listed in the manuscript, should be tested which will strengthen the larger conceptual framework and impact. In addition, the authors should perform at least a subset of the experiment using air during testing rather than a 3rd odor.

We appreciate that the reviewer pointed out our mistake. We added a detailed description of the odor combination protocol in the Methods (line 244-257). A panel of odors, OCT and MCH, were used as the novel odor alternatively throughout our study. In addition, we performed the suggested experiment and observed increased 24-h avoidance to CS+ and CS– when they are compared with air (Figure 1—figure supplement 2), which is consistent with our findings.

5. (Point6 of Reviewer 2) More experimentation and discussion regarding the differences between single-trial conditioning to form mLTM and spaced conditioning to form complementary LTM, is required. The authors contrast/merge their behavioral results with those published by Jacob et al., (2020). The authors should reproduce the essence of those found by Jacob et al., and publish them in this paper. Replication of experimental results across labs is very important, especially for behavioral outcomes and when models are constructed using results obtained by other investigators. The authors allude to the two pairs of DAN that project to α2sc MBN for this plasticity, but did not specifically mention those DAN (lines 220-221) nor elaborate on this speculation.

We thank the reviewer for this suggestion and replicated the essential result of Jacob et al., (2020). Consistently, we observed complementary 24-h memories were formed after repetitive spaced training (Figure 1—figure supplement 1). Moreover, as suggested by the reviewer, we added more discussion of the differences between mLTM and complementary LTMs, and potential neural mechanisms (line 239-249).

Reviewer #1 (Recommendations for the authors):

The finding of mLTM opens many questions. Knowing it is impractical to address all these questions in one study, I thus suggest only essential experiments to strengthen the report.

1. By using cell-type specific Gal4 lines that are readily available, target neurons for "the avoiding CS+ memory", "the early approaching CS- memory" and "the late avoiding CS- memory" should be identified. For example, PPL1-DANs and PAM-DANs expressed in TH-Gal4 can be respectively tested with specific split-Gal4 lines.

We thank the reviewer for this suggestion. In the revised manuscript, we added the suggested experiments with two more specific Gal4 lines, R58E02 for PAM-DANs and R52H03 for PPL1-DANs (Figure 3—figure supplement 1A, B). The 24-h avoidances to CS+ and CS– were both impaired by blocking PPL1-, but not PAM-DANs (Figure 3A). Interestingly, although the early avoidance to CS+ was impaired specifically by blocking PPL1-DANs, the immediate response to CS– was affected oppositely when PPL1-DANs or PAM-DANs were blocked (Figure 3—figure supplement 2). These data support our theoretical model in the revised edition that the response to CS– is the sum of two opposite memory components: the hours-lasting safety-memory to CS– and the aversive mLTM (Figure 6B).

2. In Figure 2B, when CS- was presented 20min after CS+ even the mLTM of CS+ vs novel odor was impaired, the result means that the formation of CS+ mLTM requires the present of CS- within certain time window. This raises an alternative interpretation of the data that CS- presented 20min after CS+ actually serves as an interference for the formation of CS+ mLTM, rather than the merged CS+/CS- memory. As to distinguish these two hypothesis, training the flies with only CS+ without delivering CS-, and then test between CS+ and novel odor should be able to address the question. If presenting CS+ alone fails to form LTM of CS+ vs the third odor, then CS- presented within certain time window is necessary. Otherwise, CS- presented 20min after CS- may serve as an interference.

As suggested by the reviewer, we have performed new experiments with absolute training (training without CS– or CS+) in the revision, and such training failed to induce 24-h avoidances (Figure 2—figure supplement 1). The obtained data support presenting CS+ and CS– are both necessary for forming mLTM. Moreover, this conclusion can be further strengthened by the result that mLTM was gradually decreased with the prolonged intervals between CS+ and CS– (Figure 2B).

3. In Figure 4B, the author measured the odor responses of CS+, CS-, and novel odor in MBON-a2sc one day after association, and found that both CS+ and CS- showed depression response. To strengthen the link between functional response of MBON-a2sc and behavior phenotype, conditions abolished mLTM by reactivation or prolong odor delivery interval (Figure 2A and 2B) should also be tested to see whether the memory trace are also abolished. Also, what is the responses immediately after 1X training? As CS- mLTM changes from appetitive to avoidance within the first 24 hours after training, the calcium responses of MBON-a2 during this period are necessary for understanding the circuit mechanism of mLTM dynamics.

We provide new data in revision that training with 10-min temporal intervals fails to form the long-term depression in α2sc MBONs (Figure 5). In addition, we found that the depression of odor-evoked responses to CS+ and CS– can be observed immediately after training (Figure 5—figure supplement 1A). Our data suggest that the merged memory should be encoded during training (Figure 5—figure supplement 1B) and then consolidated within 8 hours (Figure 1D).

Reviewer #2 (Recommendations for the authors):

1) Citations or references are not included when the phrase "described previously" is used (lines 238, 248, 275, 287).

2) Provide more detail on odor re-exposure methods (lines 283-284). When was this administered?

3) Explain with more detail by "calibrated calcium responses" in methods (lines 316-318).

4) Include sample size used for experiments. It is difficult to count individual data points when variability of data is tight.

5) Figure 3A cartoon – α'3 lobe ( α='3 tip) should be in magenta – innervated by PPL1 DAN.

We thank the reviewer for these helpful suggestions and modified the text and figures accordingly.

6) suggest "unconditioned stimulus" rather than "non-conditioned stimulus"

Considering the “unconditioned stimulus” has been widely defined as the reward or the punishment (in this case, the electric shock) in memory studies, here we used the “non-conditioned stimulus” to indicate the CS–.

7) organization of references when listed in the text – suggest organizing them chronologically or alphabetically. Currently there is random organization.

The list of references was organized alphabetically based on the first author’s last name in the manuscript as the requirement of eLife.

8) Figure 2A – why use the term "reactivation" rather than "extinction trials?"

We modified the figure accordingly in revision (Figure 2A).

9) The Abstract and the Introduction use the extremely vague phrase, "…that animals utilize escape strategies for facing occasional and repeated dangers…" Either bring this point home for the reader with specific and concrete examples in the Discussion or modify/eliminate the sentence.

As the reviewer suggested, we added examples in the revised Discussion (line 233-237).

10) A good overhaul of the referencing throughout the manuscript is warranted. One glaring omission of perhaps several references that should be offered is in support of the sentence, "In Drosophila, aversive olfactory memories are mainly encoded by the PPL1 cluster of DANs," (line 124). Schwaerzel et al., 2003, and others.

We appreciate that the reviewer pointed out our mistake. We updated references in revision.

https://doi.org/10.7554/eLife.66499.sa2

Article and author information

Author details

  1. Bohan Zhao

    1. School of Life Sciences, IDG/McGovern Institute for Brain Research, and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China
    2. Tsinghua-Peking Center for Life Sciences, Beijing, China
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing, Behavioral Experiments
    Contributed equally with
    Jiameng Sun
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9177-1278

  2. Jiameng Sun

    1. School of Life Sciences, IDG/McGovern Institute for Brain Research, and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China
    2. Tsinghua-Peking Center for Life Sciences, Beijing, China
    Contribution
    Formal analysis, Investigation, Calcium Imaging and Immunostaining
    Contributed equally with
    Bohan Zhao
    Competing interests
    No competing interests declared

  3. Qian Li

    1. School of Life Sciences, IDG/McGovern Institute for Brain Research, and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China
    2. Tsinghua-Peking Center for Life Sciences, Beijing, China
    Contribution
    Formal analysis, Funding acquisition, Writing - original draft
    Competing interests
    No competing interests declared

  4. Yi Zhong

    1. School of Life Sciences, IDG/McGovern Institute for Brain Research, and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China
    2. Tsinghua-Peking Center for Life Sciences, Beijing, China
    Contribution
    Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Writing - review and editing
    For correspondence
    zhongyithu@tsinghua.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7927-5976

Funding

National Science Foundation of China (31970955)

  • Qian Li

Tsinghua-Peking Center for Life Sciences

  • Yi Zhong

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank BDSC and VDRC, and Dr Yi Sun for fly stocks. We thank Bo Lei, Yijun Niu, Dr Xuchen Zhang, Dr Wantong Hu, Dr Dongqin Cai, Dr Yunchuan Zhang, Ning Huang, Shiqiang Hu, Yikai Tang, Shunan Wang, and Dr Stella Christie for helpful discussions; Bowen Chen for software supply; Lianzhang Wang for facility supply. This work was supported by grants from the National Science Foundation of China (31970955, to QL), and the Tsinghua-Peking Center for Life Sciences.

Senior and Reviewing Editor

  1. K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

Reviewer

  1. Ann-Shyn Chiang, National Tsing Hua University, Taiwan

Publication history

  1. Received: January 13, 2021
  2. Preprint posted: January 14, 2021 (view preprint)
  3. Accepted: July 18, 2021
  4. Accepted Manuscript published: July 19, 2021 (version 1)
  5. Version of Record published: August 6, 2021 (version 2)

Copyright

© 2021, Zhao et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Bohan Zhao
  2. Jiameng Sun
  3. Qian Li
  4. Yi Zhong
(2021)
Differential conditioning produces merged long-term memory in Drosophila
eLife 10:e66499.
https://doi.org/10.7554/eLife.66499

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