Higher-order unimodal olfactory sensory preconditioning in Drosophila

  1. Juan Martinez-Cervantes
  2. Prachi Shah
  3. Anna Phan
  4. Isaac Cervantes-Sandoval  Is a corresponding author
  1. Department of Biology, Georgetown University, United States
  2. Department of Biological Sciences and Neuroscience & Mental Health Institute, University of Alberta, Canada
  3. Interdisciplinary Program in Neuroscience, Georgetown University, United States

Abstract

Learning and memory storage is a complex process that has proven challenging to tackle. It is likely that, in nature, the instructive value of reinforcing experiences is acquired rather than innate. The association between seemingly neutral stimuli increases the gamut of possibilities to create meaningful associations and the predictive power of moment-by-moment experiences. Here, we report physiological and behavioral evidence of olfactory unimodal sensory preconditioning in fruit flies. We show that the presentation of a pair of odors (S1 and S2) before one of them (S1) is associated with electric shocks elicits a conditional response not only to the trained odor (S1) but to the odor previously paired with it (S2). This occurs even if the S2 odor was never presented in contiguity with the aversive stimulus. In addition, we show that inhibition of the small G protein Rac1, a known forgetting regulator, facilitates the association between S1/S2 odors. These results indicate that flies can infer value to olfactory stimuli based on the previous associative structure between odors, and that inhibition of Rac1 lengthens the time window of the olfactory ‘sensory buffer’, allowing the establishment of associations between odors presented in sequence.

Editor's evaluation

This paper shows that Drosophila can perform olfactory unimodal sensory preconditioning, an example of higher-order conditioning that may guide behaviour through inferred value. This is of conceptual significance for the brain, behavioural, and to some extent, the social sciences, because it shows that a conditioned response to a stimulus can occur even when the stimulus itself was never paired with punishment, for example.

https://doi.org/10.7554/eLife.79107.sa0

Introduction

Learning and memory are fundamental for animal survival in dynamic and noisy environments and for the cognitive abilities of humans. A significant amount of what we know on the biological basis of learning and memory has come from studying classical conditioning, also known as Pavlovian conditioning (Heisenberg et al., 1985; Romanski et al., 1993; Uwano et al., 1995; LeDoux, 2000; Davis and Whalen, 2001; Dubnau et al., 2001; McGuire et al., 2001; Maren and Quirk, 2004; Fanselow and Poulos, 2005; Ehrlich et al., 2009; Johansen et al., 2010; Duvarci and Pare, 2014; Herry and Johansen, 2014). During classical conditioning, learning depends on the contiguity of conditioned and unconditioned stimuli. Studying classical conditioning has provided essential insights into the molecular, cellular, and circuit basis of how the brain transforms sensory information into memories and how it uses these memories to drive behavior. Nevertheless, in humans and other animals, the instructive value of naturally occurring reinforcing experiences is acquired rather than innately instructive and does not necessarily dependent on mere contiguity. For example, the value of money and its capacity to function as a reinforcer is learned rather than innate. Learning theory has postulated the idea that learned behavioral control uses two types of information. The first results from habits, policies, or cached values (e.g., Pavlovian conditioning – model-free learning) (Jones et al., 2012). This kind of information produces a rapid, efficient behavioral response but does not consider changes in the value of the expected outcome. The second type of information relies on the knowledge of the associative structure of the environment to infer value (model-based learning). In other words, acquiring new knowledge depends on creating an associative structure of external events, which is then used to create additional new meaningful associations (Daw et al., 2005; Jones et al., 2012; McDannald et al., 2012; Lucantonio et al., 2014; Sadacca et al., 2016). Several different types of higher-order conditioning are examples of this type of information. In higher-order conditioning procedures, neutral stimuli acquire the property to elicit conditional responses even though they have never been in contiguity with a reinforcer. Sensory preconditioning is one example of higher-order conditioning (Giurfa, 2013; Giurfa, 2015; Todd et al., 2016). In sensory preconditioning, two initially neutral stimuli (S1 and S2) are repeatedly presented in contiguity or in sequence (preconditioning phase); later, one of the stimuli (S1) is paired with a reinforcer (conditioning phase). After this, S2 will elicit a conditioned response even though it was never paired with the reinforcer, indicating that the preconditioning phase created an association between S1 and S2. The response to the preconditioned stimulus (S2) is different from the response to the reinforcer-paired S1 in that it is not based on a mere association; instead, it must infer value by virtue of knowledge of the associative structure of the task (Jones et al., 2012). Sensory preconditioning has been shown previously in bees and flies (Müller et al., 2000; Brembs and Heisenberg, 2001; Guo and Guo, 2005). Using a flight simulator (Guo and Guo, 2005), trained flies, using visual or olfactory cues to avoid certain flight directions. They reported sensory preconditioning using S1–S2 cues of different modalities (olfactory and visual). Here, we provide evidence of olfactory unimodal sensory preconditioning in fruit flies. We show that the presentation of a pair of odors (S1 and S2) before one of them (S1) is associated with electric shocks elicits a conditioned response not only to the trained odor (S1) but also to the odor it was previously paired with (S2). This occurs even if the S2 odor was never presented in contiguity with the negative reinforcer. These data provide evidence for unimodal sensory preconditioning in Drosophila. These findings open the door to better understand how a simple brain achieves sensory preconditioning with limited neurons and synapses, providing meaningful insights into how a more complex mammalian brain may solve similar problems.

Results and discussion

The Drosophila mushroom bodies (MB) are brain structures made up by the axons of the Kenyon cells (KC), and are known to play a central role in the encoding of olfactory memories (Heisenberg et al., 1985; Dubnau and Tully, 2001; McGuire et al., 2001). During associative memory acquisition, positive or negative values are assigned to odors by associated reward and punishment, respectively. This reinforcement is achieved by the coincident activation of a sparse number of KC by an odorant and the activation of dopaminergic neurons (DAN), which innervate the MB, by the unconditioned stimulus. Different DAN synapse onto discrete zones of the MB, resulting in 15 tile-like compartments of the MB. Each of these 15 tiled areas in turn synapse onto the dendrites of distinct corresponding mushroom bodies output neurons (MBONs). Activation of different MBONs are then known to alter behavioral approach or avoidance (Aso et al., 2014; Owald and Waddell, 2015; Roselli et al., 2021). The memory of an odor associated with an electric shock (classical conditioning) is known to be partially encoded as a decreased response specifically to the trained odor (CS+) in a particular MBON known as MBON-γ1pedc>α/β (Hige et al., 2015; Perisse et al., 2016; Cervantes-Sandoval et al., 2020). Conversely, odors that have not been associated with an electric shock do not normally demonstrate decreased responses in the MBON-γ1pedc>α/β neuron (Hige et al., 2015; Perisse et al., 2016; Cervantes-Sandoval et al., 2020). Thus, we were extremely surprised to report in a previous publication that flies expressing the dominant-negative form of Rac1N17 (Rac1DN) in adult KC using the temperature-dependent tool gal80ts (TARGET) (McGuire et al., 2003), displayed a depression to a non-shock associated odor in MBON-γ1pedc>α/β neurons (Cervantes-Sandoval et al., 2020). We decided to investigate the nature of this intriguing non-shock associated neural depression.

It has been reported that animals can link together neutral sensory cues presented in succession or simultaneously; if then one of these cues is associated with reinforcement, animals can display a response to the non-associated pre-linked cue; this is known as sensory preconditioning (Brogden, 1939; Jones et al., 2012). As mentioned above, this type of behavior is categorized as model-based learning where a value is inferred from the associative structure of the environment (Jones et al., 2012). It was also reported that expressing Rac1N17 in KC significantly enhances trace conditioning, in which an odor is associated with an electric shock presented many seconds after odor offset – suggesting that inhibition of Rac1 lengthens an olfactory ‘sensory buffer’ that later converges with the punishment signal (Shuai et al., 2011). Therefore, we reasoned that sensory preconditioning and Rac1 inhibition could explain the observed depression to the non-trained odor we previously reported (Cervantes-Sandoval et al., 2020) and replicated this finding in pilot experiments (Figure 1—figure supplement 1).

To test for the possibility that we were observing sensory preconditioning in MBON-γ1pedc>α/β neurons, we exposed animals to a single pairing of 5 s of 4-methylcyclohexanol (MCH) (S1) and 5 s of 3-octanol (OCT) (S2) odors, separated by an interstimulus interval (ISI) of 1 s, 30 s, or 5 min, before classical aversive olfactory conditioning occurred only to S1 by pairing MCH (S1) with an electric shock (20 s of odor with four 90 V, 1.25 s electric shocks). This is also known as forward conditioning. Post-training calcium responses to both MCH and OCT were then recorded 5 min after conditioning in MBON-γ1pedc>α/β neurons using the R12G04-lexA driver to express GCaMP6f (Figure 1A, B). These calcium responses in MBON-γ1pedc>α/β were compared with the odor responses in backwards conditioned animals which serve as our procedural control. Backwards conditioned animals receive all the same stimuli as forward conditioned animals, except that the electric shock occurs before the odor during what would be the ‘conditioning phase’, thus classical conditioning does not occur. Two naive odors, penthyl acetate (PA) and ethyl lactate (EL), were also given to the animals post-conditioning to ensure that the experimental methods did not result in generalized MBON-γ1pedc>α/β changes to all odors (Figure 1A, B).

Figure 1 with 1 supplement see all
One-second interstimulus interval (ISI) during preconditioning induces depression to non-trained, pre-paired odor S2 in control flies.

(A) Diagram of the in vivo, under the microscope training imaging setup, MBON-γ1pedc>α/β diagram, and Rac1N17 expression induction. (B) Diagram of the experimental setup (odor schedule): preconditioning was induced by presentation of 4-methylcyclohexanol (MCH) and 3-octanol (OCT) (S1 and S2) with 1-s, 30-s, or 5-min ISI; then flies were aversively trained to MCH (S1) and 5 min later, post-conditioning responses were recorded. Responses were compared to flies trained using backwards conditioning. (C) S1 (MCH) responses were completely depressed 5 min after training in control flies preconditioned with 1-s, 30-s, or 5-min ISI. Non-parametric Mann–Whitney test p ≤ 0.0044; n = 8–10. A significant inhibition of post-trained response to S2 (OCT) was observed in control flies preconditioned with 1-s ISI. Non-parametric Mann–Whitney test p = 0.035; n = 10. In contrast, S2 (OCT) responses in control flies preconditioned with 30-s or 5-min ISI showed no significant change. Non-parametric Mann–Whitney test p ≥ 0.0630, n = 8–10. Neither naive odor (penthyl acetate [PA] and ethyl lactate [EL]) responses showed any significant reduction in flies preconditioned with 1-s, 30-s, or 5-min ISI. Non-parametric Mann–Whitney test p ≥ 0.0726, n = 8–10. (D) S1 (MCH) responses were completely depressed 5 min after training in flies expressing Rac1N17 preconditioned with 1-s, 30-s, or 5-min ISI. Non-parametric Mann–Whitney test p ≤ 0.0009; n = 8–12. A significant inhibition of post-trained response to S2 (OCT) was observed in flies preconditioned using a 1- or 30-s ISI. Non-parametric Mann–Whitney test p ≤ 0.0031; n = 8–12. In contrast, S2 (OCT) responses in flies preconditioned with 5-min ISI showed no significant change. Non-parametric Mann–Whitney test p = 0.4234, n = 8–9. Neither naive odor (PA and EL) responses showed any significant reduction in flies preconditioned with 1-s, 30-s, or 5-min ISI. Non-parametric Mann–Whitney test p ≥ 0.0726, n = 8–12. Boxplots represent distribution of %∆F/Fo responses across the 5 s of odor presentation. The thick black bar below each trace represents the time of odor presentation. The asterisk's number shows N digits after the decimal.

Figure 1—source data 1

One-second interstimulus interval (ISI) during preconditioning induces depression to non-trained, pre-paired odor S2 in control flies.

https://cdn.elifesciences.org/articles/79107/elife-79107-fig1-data1-v2.xlsx

Using the typical imaging protocol with a 25- to 30-s ISI between the preconditioned odors, we and others (Hige et al., 2015; Cervantes-Sandoval et al., 2020) have shown there is no evidence of sensory preconditioning in the MBON-γ1pedc>α/β of control animals. That is, the MBON-γ1pedc>α/β does not display a decreased response to the S2 odor (Figure 1C), but does demonstrate the expected near-complete depression in response to the conditioned S1 odor. Lengthening the ISI to 5 min has the same effect on control flies (Figure 1C). Interestingly however, when we shorten the sensory preconditioning interval to 1 s, the MBON-γ1pedc>α/β neurons in control flies exhibit a decreased calcium response to the S2 odor, and to the S1 odor that is expected (Figure 1C). Throughout these experiments, MBON-γ1pedc>α/β responses to naive odors remained unchanged, indicating the decrease in calcium response to the S2 odor is specific to the preconditioning phase, and not the result of a generalized depression to all odors (Figure 1C). This suggests that shortening the ISI window during sensory preconditioning allows control flies to form some association between the S1 and S2 odors, thus resulting in an inferred predictive value of the S2 odor after animals were aversively conditioned to the S1 odor. These results demonstrate that flies show, at least at the physiological level, sensory preconditioning.

In contrast to control animals, flies expressing dominant-negative Rac1N17 in the KC had previously been found to exhibit a decreased calcium response to the S2 odor when using an ISI of 30 s (Figure 1D, Figure 1—figure supplement 1B). This was also true when the sensory preconditioning ISI was decreased to 1 s (Figure 1D). The data from our control flies led us to speculate that the timing of the S1/S2 ISI dictates whether an association between odors S1 and S2 (MCH and OCT) occurs during the sensory preconditioning stage. Secondly, from prior data on Rac1’s role in trace conditioning and our sensory preconditioning data, we suspected that Rac1 inhibition in the KC extends the time period that allows for the S1/S2 association to occur. If this was true, we predicted that extending the ISI should eliminate sensory preconditioning in Rac1DN flies. When the ISI was increased to 5 min, no sensory preconditioning was observed in the Rac1DN animals (Figure 1D). Again, naive odor responses in these flies were unchanged. This widening of the ISI window to allow for an S1/S2 association is dependent on the inhibition of Rac1, since flies with the same genotype but raised at 18°C to prevent the expression of Rac1N17 (using the TARGET system) showed the normal conditioning-induced odor depression to the S1 but no change to S2 with a preconditioning ISI window of 30 s (Cervantes-Sandoval et al., 2020).

We tested whether this suspected sensory preconditioning-induced calcium depression might be the result of increased habituation of the non-associated S2 odor. We recorded olfactory responses in flies exposed to the same odor protocol except that the unconditioned stimulus was excluded. Results showed no depression to either S1 or S2 odor presented (Figure 2—figure supplement 1A). Going even further, we confirmed this observation by recording and evaluating changes in calcium responses after 10 repeated presentations of both MCH and OCT. Data were analyzed as previously described (Hattori et al., 2017). The ratio between the mean of three initial responses and mean of three last responses in MBON-γ1pedc>α/β was not significantly different between control and experimental genotypes (Figure 2—figure supplement 1B). Thus, the reduced calcium response we see in MBON-γ1pedc>α/β to the classically conditioned S1 odor and the sensory preconditioned S2 odor cannot be explained by a general habituation to the odors due to the experimental protocol.

Rac1 has been implicated in memory forgetting, and it has been theorized that forgetting is fundamental for memory generalization (Richards and Frankland, 2017). In addition, (Hige et al., 2015) showed that a similar depression to the non-trained odor could result from a partial overlap in the neural representation of odors such as MCH and OCT. For these reasons, we tested whether the expression of Rac1N17 in KC may somehow increase that generalization between MCH and OCT, and that this may lead to the sensory preconditioned neural response that we are observing. For this, we trained animals using orthogonal odors, PA and EL, that are known to have little overlap in their KC representations. Surprisingly, similar results to MCH/OCT were obtained; we observed a depression to the non-trained S2 odor (EL) when Rac1 function is inhibited in KC (Figure 2). Thus, this physiological response to sensory preconditioning can be observed for different odors and odor pairs.

Figure 2 with 1 supplement see all
Rac1 inhibition induces depression to sensory preconditioned odor S2 in MBON-γ1pedc>α/β using a different odor pair with orthogonal representation.

(A) Diagram of experimental setup (odor schedule): preconditioning responses were obtained for penthyl acetate (PA) and ethyl lactate (EL) (S1 and S2, respectively); later flies were aversively trained to PA (S1) and 5 min after, post-conditioning responses were recorded. (B, C) S1 (PA) responses were completely depressed 5 min after training in both control flies and flies expressing Rac1N17. Non-parametric Wilcoxon-paired test p ≤ 0.0078; n = 8–11. No significant changes were detected to S2 (EL) for control animals. Non-parametric Wilcoxon-paired test p = 0.5771; n = 11. Nevertheless, a significant inhibition of post-trained response to S2 (EL) was observed in flies expressing Rac1N17. Non-parametric Wilcoxon-paired test p = 0.0391; n = 8. Boxplots represent distribution of %∆F/Fo responses across the 5 s of odor presentation. The thick black bar below each trace represents the time of odor presentation. The asterisk's number shows N digits after the decimal.

Figure 2—source data 1

Rac1 inhibition induces depression to sensory preconditioned odor S2 in MBON-γ1pedc>α/β using a different odor pair with orthogonal representation.

https://cdn.elifesciences.org/articles/79107/elife-79107-fig2-data1-v2.xlsx

To further challenge the assertion that the reduced calcium response in MBON-γ1pedc>α/β neurons to the S2 odor is a result of sensory preconditioning, we trained animals with forward or backwards conditioning but excluded the pre-training odor presentation of S1 and S2. When we eliminated the preconditioning association between the S1 and S2 odors, the results showed significant depression to the classically conditioned S1 odor that was associated with the shock, but no depression to any other odors (Figure 3). These results demonstrate that the depression to the non-trained S2 odor was not due to a general depression to odors or generalization of learned aversion. Instead, depression of the non-trained S2 odor is dependent on the animal learning the association between S1 and S2 during the preconditioning stage. These results further support the hypothesis that we are observing physiological sensory preconditioning.

Excluding preconditioning eliminates depression to the S2 odor.

(A) Diagram of experimental procedure (odor schedule): Flies were aversively trained to 4-methylcyclohexanol (MCH) (S1) and 5 min later post-conditioning responses to four odors were recorded (preconditioning was excluded). Responses were compared to flies trained using backwards conditioning. (B, C) S1 (MCH) responses were completely depressed 5 min after training in both control and flies expressing Rac1N17. Non-parametric Mann–Whitney test p ≤ 0.0147; n = 10–11. Neither 3-octanol (OCT), penthyl acetate (PA), nor ethyl lactate (EL) responses showed any significant reduction for both control and experimental flies. Non-parametric Mann–Whitney test p ≥ 0.072, n = 10–11. Boxplots represent distribution of %∆F/Fo responses across the 5 s of odor presentation. The asterisk's number shows N digits after the decimal.

Figure 3—source data 1

Excluding preconditioning eliminates depression to the S2 odor.

https://cdn.elifesciences.org/articles/79107/elife-79107-fig3-data1-v2.xlsx

We then tested if sensory preconditioning could be observed at the behavioral level. For this, we trained the flies using an Arduino microcontroller for precise odor delivery by controlling solenoids automatically. Using this Arduino system, we trained animals as follows (Figure 4—figure supplement 1A). Control flies were presented with a single pairing of the odors (5 s pulse each) with a 1-s ISI. Using the standard aversive olfactory conditioning paradigm, flies were then conditioned by the presentation of 1 min of S1 odor along with 12, 90 V shocks. Memory was tested right after training in a T-maze by presenting animals with either the S1 (shock-paired odor) and a novel odor (NO), or the S2 (non-shocked, pre-paired odor) and an NO. As in canonical aversive olfactory conditioning experiments, each behavioral experiment was conducted using the reciprocal odor as S1. The final performance index (PI) was calculated by averaging the PI for each odor used as S1. Results were compared to flies trained with backwards conditioning. Despite observing evidence of sensory preconditioning by functional imaging using a similar protocol, we could not observe a behavioral memory to the non-shocked pre-paired odor (S2) (Figure 4—figure supplement 1B). At a behavioral level, sensory preconditioning has previously been observed in animals after repeated presentation of a pair of sensory cues. Thus, we preconditioned flies with 10 repeated presentations of odor pairings (S1/S2) before training (Figure 4A). Using an ISI of 1 s, this resulted in a significant behavioral aversive memory expression to the S2 odor in control flies (Figure 4B), consistent with our predictions based on the sensory preconditioning physiological data. Furthermore, control flies did not demonstrate an aversive memory to the S2 odor when an ISI of 30 s was used (Figure 4B), as would be predicted from our physiological imaging data. These results also suggest that while a single odor pairing is sufficient to induce sensory preconditioned-related plasticity in the MBON-γ1pedc>α/β compartment, it is not enough for the S2 odor to drive the learned behavior. We suggest that the presentation of additional odor pairings recruits and induces sensory preconditioning-related plasticity in additional MB compartments, and it is the additive effect of multiple compartments that is necessary to drive the behavioral response. Similar phenomena have been observed before for classical conditioning where a short pairing of odor and shock – 1-s odor paired with 4 DAN photostimulation pulses – is enough to induce plasticity in MBON-γ1pedc>α/β compartment but not in MBON-α2sc. Longer training protocols – 1-min odor presentation paired with 120 DAN photostimulation pulses – induce plasticity in MBON-α2sc (Hige et al., 2015). Physiologically, preconditioning flies with MCH/OCT pairs repeated 10 times before training resulted in a robust depression in MBON-γ1pedc>α/β to the non-shocked pre-paired odor (CS2) in control flies (Figure 4—figure supplement 2A). These results were similar when PA/EL were used as the preconditioned odors (Figure 4—figure supplement 2B).

Figure 4 with 4 supplements see all
Repeated presentations of paired odors (S1/S2) induce behavioral sensory preconditioning.

(A) Flies were trained using an Arduino microcontroller for precise odor delivery: Flies were preconditioned by 10 repeated presentations of S1/S2 odor pairs with 1-s interstimulus interval (ISI). Later flies were aversively trained to 4-methylcyclohexanol (MCH) (S1) by pairing 1-min odor presentation along 12 90 V, 1.25 s shocks. Right after training memory was tested in a T-maze by presenting either S1 vs. a novel odor (NO) or S2 vs. an NO. Performance index (PI) was compared to flies trained using backwards conditioning. (B) Memory to S1 was significantly different from flies trained with backwards conditioning, after 10× preconditioning presentations using 1-s, 30-s, or 5-min ISI. Non-parametric Mann–Whitney test p < 0.0001, n = 8–12. In contrast, memory to S2 was significantly different from flies trained with backwards conditioning, with 10× preconditioning presentations only using 1-s ISI in control flies. Non-parametric Mann–Whitney test p = 0.0019, n = 12. Memory to S2 was not significantly different from backwards conditioning, with 10× preconditioning presentations using 30-s or 5-min ISI in control flies. Non-parametric Mann–Whitney test p > 0.999, n = 8. (C) Memory to S1 in flies expressing Rac1N17 was significantly different from flies trained with backwards conditioning, after 10× preconditioning presentations using 30-s or 5-min ISI. Non-parametric Mann–Whitney test p = 0.0002, n = 8. Memory to S2 in flies expressing Rac1N17 was significantly different from flies trained with backwards conditioning, with 10× preconditioning presentations when using a 30-s ISI. Non-parametric Mann–Whitney test p = 0.0191, n = 8. Memory in flies expressing Rac1N17 to S2 was not significantly different from backwards conditioning, with 10× preconditioning presentations when using 5-min ISI. Non-parametric Mann–Whitney test p = 0.8981, n = 8. The asterisk's number shows N digits after the decimal. The following source data and figure supplements are available for Figure 4.

Figure 4—source data 1

Repeated presentations of paired odors (S1/S2) induce behavioral sensory preconditioning.

https://cdn.elifesciences.org/articles/79107/elife-79107-fig4-data1-v2.xlsx

We next tested flies expressing Rac1N17 in KC for evidence of behavioral sensory preconditioning when using 30-s and 5-min ISI between S1 and S2. As would be predicted from our physiological data, while a 30-s ISI resulted in no aversive memory to S2 in control flies, a significant aversive memory was observed in flies expressing Rac1N17 (Figure 4B). This is consistent with our calcium imaging results in Figure 1B, in which a 30-s ISI results in a depression to S2 in Rac1N17 flies but not in control flies. Further lengthening the ISI to 5 min eliminated behavioral sensory preconditioning both in controls and in flies expressing Rac1N17 (Figure 4B), again mirroring the physiological results in the MBON-γ1pedc>α/β neuron (Figure 1B). Flies kept at 18°C to prevent expression of Rac1N17 behaved as control flies and did not demonstrate behavioral evidence of sensory preconditioning when using an ISI of 30 min (Figure 4—figure supplement 3). Finally, we tested if Rac1N17 expression in KC altered olfactory responses in the MB lobes. Interestingly, we could not find any change in the calcium response dynamics in KC γ lobes, even when recordings were performed at a higher speed (28 Hz) (Figure 4—figure supplement 4).

Taken together, these results indicate that flies can undergo unimodal sensory preconditioning and that the small G protein Rac1, previously implicated in memory forgetting, gates the windows for this S1–S2 association.

Ideas and speculations

Our results demonstrate that flies can infer value to non-reinforced odors based on the previous associative structure between odors. Memories are not discrete bits of events and associations that are added and accumulated in our brains. Rather, memories are dynamic; they undergo modifications and updates and are integrated into a coherent story that continuously incorporates new information to predict upcoming events better and to inform decision making. Within this complex associative structure, associations between seemingly valueless stimuli are fundamental. Here we present evidence that even the simple brain of a fly can form these associations.

How do these associations occur? Are S1–S2 associations the result of forming new or strengthening connections between KC following a Hebbian-like delay of synaptic reinforcement? If this is true, one could suggest a role for dopamine in this reinforcement. We know that DAN are essential for many types of reinforcement and even for non-associative learning like novelty detection (Hattori et al., 2017). Furthermore, we know dopamine neurons receive substantial feedback from both KC and MBON. Alternatively, S1–S2 associations could result from a shift in the stimulus representation during sensory preconditioning, probably by mechanisms similar to the hypothesis of excitability-dependent allocation of engram cells (or, in this case, neuronal representation) (Yiu et al., 2014; Josselyn and Frankland, 2018; Josselyn and Tonegawa, 2020).

Another interesting finding is that while a single odor pairing is sufficient to induce sensory preconditioned-related plasticity in the MBON-γ1pedc>α/β compartment, it is not enough for the S2 odor to drive the learned behavior. As mentioned above, we suggest that the presentation of additional odor pairings recruits and induces sensory preconditioning-related plasticity in additional MB compartments, and it is the additive effect of multiple compartments that is necessary to drive the behavioral response. Follow-up studies should include looking for the rules of sensory preconditioning-related plasticity in additional MB compartments. This finding has important implications for how memory systems work. There is accumulating evidence that memorable experiences are encoded in parallel, relatively independent memory traces, and it is the integration of these different memory traces that drives the most adequate behavior. Examples include the additive effect of aversive memory traces (Aso et al., 2012); the additive effect of contextual memory to aversive olfactory memories (Zhao et al., 2019); or the integration of parallel opposite memories during memory extinction (Felsenberg et al., 2017; Felsenberg et al., 2018).

Our data also demonstrate that inhibition of Rac1 by expressing its dominant-negative form in the KC lengthens the time for an olfactory ‘sensory buffer’, allowing the linking of odors presented in sequence even when separated by at least 30 s. Interestingly, calcium responses in KC are not altered by the inhibition of Rac1 (Figure 4—figure supplement 4). While calcium is a good proxy of neuronal activity within the KC, it does not necessarily directly translate to a readout of the neurotransmission output of the KC and its communication with downstream neurons.

A challenging question that remains for future study is on the nature of the circuit connections that link S1 and S2 during the sensory preconditioning phase. Once this S1–S2 link is formed, the key process by which we experimentally detect that link is as an aversive response to S2 during the testing phase. Does this aversive response to S2 result during the associative learning phase, in which the S1 presentation summons an S2 representation during CS/US pairing, and therefore, S2 forms a direct association with the punishment? Conversely, is the aversive response to S2 dependent on the memory retrieval phase, during which exposing flies to the S2 odor somehow recalls the S1-punishment association to influence the aversive response? This second possibility would imply that S2 is not directly associated with punishment. Deciphering the neural circuit rules that support unimodal sensory preconditioning in the relatively simple fly brain will help us to unravel how other animals, including humans, use our nervous systems to build models of the world.

Materials and methods

Drosophila husbandry

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Flies were cultured on standard medium at room temperature. Crosses, unless otherwise stated, were kept at 25°C and 70% relative humidity with a 12-hr light–dark cycle. The drivers used in this study include MB112C-splitgal4 (Aso et al., 2014) (RRID: BDSC_68263), R12G04-lexA (Jenett et al., 2012) (RRID: BDSC_52448), and R13F02-gal4 (Jenett et al., 2012) (RRID: BDSC_48571). Driver expression was verified by immunohistochemistry (Videos 13). Additional transgene stocks included uas-GCaMP6f (Chen et al., 2013) (RRID: BDSC_42747), lexAop-GCaMP6f (Chen et al., 2013) (RRID: BDSC_44277), uas-rac1N17 (Luo et al., 1994) (RRID: BDSC_6292), and tub-gal80ts (McGuire et al., 2003) (RRID: BDSC_7019).

Video 1
Expression patterns of MB112C-splitGal4 driver expressing GFP.

Brains where dissected and stained using anti-GFP and anti-nc82 as a counterstain.

Video 2
Expression patterns of R12G04-LexA driver expressing GFP.

Brains where dissected and stained using anti-GFP and anti-nc82 as a counterstain.

Video 3
Expression patterns of R13F02-Gal4 driver expressing GFP.

Brains where dissected and stained using anti-GFP and anti-nc82 as a counterstain.

To inhibit Rac1 activity in KC, the dominant-negative Rac1 (Rac1N17) was expressed in adult flies using the Target system. Briefly, to restrict expression of transgenes to adult animals, fly crosses were kept at 18°C during development. After eclosion, 1- to 2-day-old flies were transferred to 30°C for 3–4 days for the induction of expression. Flies were then transferred to 25°C 1 hr before imaging or behavior. The Target system was used because inhibiting Rac1 in KC during development results in lethality. Control flies were subjected to exactly the same protocol but they did not contain the UAS transgene. Additional controls were performed by keeping the crosses at 18°C during development and after eclosion; these flies were then transferred to 25°C 1 hr before imaging.

In vivo calcium imaging

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For measuring calcium responses following conditioning, odor or shock delivery, we processed flies as previously described with some modifications (Cervantes-Sandoval et al., 2017; Cervantes-Sandoval et al., 2020). Briefly, a single fly was gently aspirated without anesthesia into a metal pipette to immobilize the head using proboscis aspiration. Once the head is immobilized, using a micromanipulator, the fly was inserted in a narrow slot the width of their body in a custom-designed recording chamber. The head was then fixed by gluing the eyes to the chamber using melted myristic acid. After this the fly was released from the metal pipette and the proboscis fixed with myristic acid to avoid brain movement during proboscis extension. Using a syringe needle, a small, square section of dorsal cuticle was removed from the head to allow optical access to the brain. Fresh saline (103 mM NaCl, 3 mM KCl, 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1.5 mM CaCl2, MgCl2, 26 mM NaHCO3, 1 mM NaH2PO4, 10 mM trehalose, 7 mM sucrose, and 10 mM glucose [pH 7.2]) was perfused immediately across the brain to prevent desiccation and ensure the health of the fly. Then the fat bodies and trachea above the brain was removed. Using a 20× water-immersion objective and a Leica TCS SP8 II confocal microscope with a 488 nm argon laser, we imaged the MBON-γ1pedc>α/β neuron for 2 min at 2 Hz, during which stimuli was delivered starting at 30 s after imaging initiation. We used one HyD channel (510–550 nm) to detect GCaMP6f fluorescence. In order to detect small changes in the dynamics of odor responses in KC the recording speed was increased to 28 Hz.

Odor and shock presentation

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To deliver odors to flies under the microscope, a stream of air (500 ml/min) was diverted (via solenoids) from flowing through a clean 20 ml glass vial to instead flow through a 20-ml glass vial containing a 0.5 µl drop of pure odorant. This air stream was then serially diluted into a larger air stream (1500 ml/min) before traveling through Teflon tubing (~2.5 mm diameter) to reach the fly. Odors presented to the fly were continuously clear by a funnel connected to high vacuum pressure positioned right behind the recording chamber. To deliver shocks to flies under the microscope, a custom shock platform was made from shock grids used in the standard olfactory memory assays that consist of alternating +/− charged copper strips attached to an epoxy sheet. To simulate shock exposure given during the standard olfactory memory assay, the surface of the shock platform was positioned so that all six legs are touching but the fly could temporarily break contact by moving its legs. Both solenoids that control odor delivery and the Grass stimulator that delivers shocks were controlled by an Arduino microcontroller (Arduino Uno) with custom-made programs.

Training under microscope programs

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The regular training protocol followed for most experiments in the paper consisted of flies that were presented to preconditioning odors with 5 s of the first odor followed by variable ISI lengths (indicated in figures) during which clean air was presented, followed by 5 s of a second odor (non-associated odor) pre-training (MCH and OCT or PA and EL). The order of odor presentation was kept the same for all experiments and is indicated in each figure. Five minutes after these preconditioning recordings, flies were trained under the microscope by simultaneous presentation of a single 20-s odor pulse and four 90-V, 1.25-s electric shocks (5-s intershock interval). Five minutes after training, post-conditioning odor responses were recorded similarly to pre-responses. For control purposes, flies were trained with backwards training in which electric pulses were presented right before the onset of odor delivery.

Behavior

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Two- to five-day-old flies were used for all behavior experiments. Standard aversive olfactory conditioning experiments were performed as described (Beck et al., 2000) with some modifications. Precise control of odor delivery was achieved automatically using an Arduino microcontroller with custom-made programs to control a pair of solenoids. Briefly, a group of ~60 flies were loaded into a training tube where they received a preconditioned stimuli as indicated in each experiment. After preconditioning, flies were trained by 1 min of an odor paired with 12 pulses of 90-V electric shock (S1). We used OCT, MCH, PA, and EL as standard odorants. To measure memory, we transferred the flies into a T-maze where they were allowed 2 min to choose between two odors, either S1 and an NO or S2 and an NO.

Immunostaining

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Whole brains were isolated and processed with minor modifications from those described (Jenett et al., 2012). Brains were first incubated with primary antibodies including: rabbit polyclonal anti-GFP (1:1,000, Life Technologies Cat. # A11122, RRID: AB_10073917) and mouse monoclonal anti-nc82 (1:50, University of Iowa, DSHB, RRID: AB_2314866). Secondary antibodies included: anti-rabbit IgG conjugated to Alexa Fluor 488 (1:800, Life Technologies Cat. # A11008, RRID: AB_143165) and anti-mouse IgG conjugated to Alexa Fluor 633 (1:1000, Life Technologies Cat. # A21052, RRID: AB_141459). Images were collected using a 20× objective with a Leica TCS SP8 confocal microscope with 488 and 633 nm laser excitation.

Quantification and statistical analysis

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Fluorescence was acquired from a region of interest (ROI) drawn around the axon tract of MBON-γ1pedc>α/β. Baseline was calculated using a Matlab program as the mean fluorescence across the 5 s before each odor presentation. This baseline was then used to calculate %∆F/Fo for the complete recording. Bar graphs represent distribution of %∆F/Fo responses across the 5 s of odor presentation. Solid lines in fluorescence traces represent mean %∆F/Fo ± standard error (SE) (shaded area) across the odor responses.

Sample size was estimated using G*Power3.1 using preliminary data. All replicates in the manuscript were biological replicates. Statistics were performed using Prism 5 (GraphPad). All tests were two tailed and significance levels were set at α = 0.05. The figure legends present the p values and comparisons made for each experiment. Unless otherwise stated, non-parametric tests were used for all imaging data.

Data availability

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

References

    1. Beck CD
    2. Schroeder B
    3. Davis RL
    (2000)
    Learning performance of normal and mutant Drosophila after repeated conditioning trials with discrete stimuli
    The Journal of Neuroscience 20:2944–2953.
    1. Brogden WJ
    (1939) Sensory pre-conditioning
    Journal of Experimental Psychology 25:323–332.
    https://doi.org/10.1037/h0058944
    1. Giurfa M
    (2015) Learning and cognition in insects
    Wiley Interdisciplinary Reviews. Cognitive Science 6:383–395.
    https://doi.org/10.1002/wcs.1348

Decision letter

  1. Sonia Sen
    Reviewing Editor; Tata Institute for Genetics and Society, India
  2. K VijayRaghavan
    Senior Editor; National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

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.

Decision letter after peer review:

Thank you for submitting your article "Olfactory unimodal sensory preconditioning in Drosophila" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor. The reviewers have opted to remain anonymous.

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:

These results are both interesting and important to the field. We have a few suggestions that we think will help this bring out and strengthen the claims made.

1. The writing: In the current form, the writing makes the story accessible largely to a specialist audience. That flies can learn through inferred value will be of interest to a wider audience and so, we're suggesting that the authors revisit their writing to make the data more accessible to the reader. Specifically, we're requesting that they revisit the introduction and abstract to better equip the reader with the specifics of the paper. This would include a more accessible description of sensory preconditioning (SPC) and the paradigms that model it; its prior demonstration in other insects; and the MBONs. We're also recommending that the authors reorganise their text and figures according to the convention (matched order in text and figures), and fix the numerous typos. Finally, we're requesting them to expand on their methodology.

2. Reorganisation of the story: We felt that a reorganisation of the data would greatly improve readability and make for a more convincing story. Our main suggestion is to focus on presenting all the data necessary to demonstrate that SPC is indeed occurring together. Specific suggestions on how to do this are in the detailed reviews below.

3. Discrepancy between behavioural and physiological responses: We were unclear as to why the authors chose the novel odours and why they did not use wild-type flies (CS) for this. So, we're requesting that the authors present their rationale for using novel odours and for using before-after instead of between-group comparisons. We also recommend that they use CS to address some of the concerns that have been raised regarding the controls.

Reviewer #1 (Recommendations for the authors):

Please check the whole manuscript for typos. There are also many wrong labels in the figures.

It was a bit confusing that the authors start the result section with figure2B. Either the authors provide a better description for this unusual beginning or change the figure arrangement.

Also, the Materials and methods is quite short. I was wondering if the odors in the test were always presented in the same order. S1, then S2, then other odors? Is this order necessary to induce the S2 effect? Especially in the 30s gap experiment, flies might not show the memory if the authors would be present in the "wrong order" compared to preconditioning.

To show that Rac1 inhibition might affect the duration of odor representation, the authors might have performed functional imaging in KCs if feasible.

Another question that came up is why the authors use a novel odor as a control after conditioning in behavioral experiments. Maybe that is confusing for the animals? Might they perform better if the S1 and S2 would be tested against the solvent or an empty tube? This also might explain discrepancies between imaging data and behavioral data.

Reviewer #2 (Recommendations for the authors):

In this manuscript, the authors provide physiological and behavioral evidence to support that fruit flies can perform olfactory unimodal sensory preconditioning, an example of higher-order conditioning. Flies were first exposed to a pair of odors (S1 and S2) with a 1-s inter-stimulus interval (ISI) before conditioning. Then odor S1 was given a negative value through aversive olfactory conditioning. After conditioning, the non-reinforced odor S2 was also found to be linked to the negative value. When the ISI was changed from 1 s to 30 s, flies failed to link odor S2 with the negative value, unless Rac1 activity was inhibited. Such sensory preconditioning can be observed in an aversive memory trace in a pair of MBON-γ1pedc>α/β neurons as well as behavioral performance. These results indicate that a simple brain may have the ability to guide behavior through inferred value and Rac1 may have a pivotal role in such behavior. The findings of sensory preconditioning are interesting and important, however, the behavioral data should be further strengthened by adding more controls. And calcium imaging results only partially explain the behavioral data, more discussion should be provided. More detailed information about methods is required. The writing needs to be improved.

1) The writing of this manuscript needs to be greatly improved. The text does not match the figures in some places. There are many other errors throughout the text. It makes reading the manuscript very difficult and time-consuming.

a) In line 95, "and vivo calcium imaging" should be "and in vivo calcium imaging", and "pairing and odor" should be "pairing an odor".

b) In line 105, the result description is inconsistent with Figure 2B.

c) In line 124, Figure 2A should be Figure 1B.

d) In line 129, Figure 1B should be Figure 1C.

e) In line 148, there is no Figure 2C.

f) In Figure 3A, "Barwards" should be "Backwards".

g) The first time the abbreviation KC is mentioned, the full form should be provided.

2) Is sensory preconditioning stable and significant at the behavioral level? The behavioral performance of the sensory preconditioning is weak and demonstrated by comparing forward and backward conditioning groups. As a new behavioral paradigm, more control groups such as naïve, "odor only" and "shock only" groups should be tested using wild-type flies like Canton-S.

3) Is one-second ISI sufficient to clear odor S1 and switch to odor S2? Is it possible that flies indeed sense mixed odors of S1 and S2? Is preconditioning effect can also be observed when S1-S2 mixed odors are exposed before conditioning? More evidence should be provided. It may affect the interpretation of the results.

4) As the authors mentioned in lines 183-186, the preconditioning effect observed in physiological trace in MBON-γ1pedc>α/β neurons (Figures 1 and 2) is not sufficient to explain the behavioral preconditioning in Figures 3 and 4. This point should be discussed more in the "ideas and speculations" section.

Reviewer #3 (Recommendations for the authors):

A weakness is seen in the way the data and arguments are presented, because key data belonging together are not shown and analysed together (and are not shown in the same way), and because the final "Ideas and Speculations" section of the Results and Discussion section reads somewhat disjointed.

Another weakness is that a rationale should be presented under which circumstances before-after comparisons are used as arguments, and when between-group comparisons are used.

Last but not least, the reviewer believes a better job could be done to make the manuscript more accessible for a non-expert audience, for example regarding the definition of wild-type animals, a plain-language rationale for the use of the transgenes, the logic of the Gal80ts/ target system including whether expressing the Rac transgene entails a gain or loss of Rac function, and what, thus, the colour code for presenting the genotypes means in terms of Rac function.

I) My main recommendation is to present together what belongs together, focusing on the most important issue, namely whether the evidence for sensory pre-conditioning (SPC) is indeed convincing. I discuss this for the physiological data, but the same applies to the behavioural data as well.

I think you could present together with the data from Figure 1, Figure 2-S1 (in between what are now Figure 1B and C), and Figure 2-S2 (after what is now Figure 1C). This would allow you to make your points for all the three mentioned requirements for a demonstration of SPC in one panel – plus: the broader time window for SPC upon Rac inhibition (?!) would be very nicely visible.

To simplify, you could present all the traces of recordings as supplements throughout.

You could then present a separate figure with all (sic) pre-post comparisons for that panel; "all" meaning for MCH and for OCT, and for both the forward and the backward cases.

There are a number of additional and rather unfortunate arrangements of the figures that make it hard to follow, almost creating 'stroop effects'. In addition to what was mentioned above, examples include:

If you deal with Figure 2 before Figure 1 in the Results section, you should also present these figures in that order. By the way, it might be a good idea to label the experiment in Figure 2 as a 'Pilot experiment' and present it as a supplement. This is because it is inconclusive with respect to all the three requirements for demonstrating SPC mentioned above.

Be consistent in presenting eg the control genotype towards the top and the experimental genotype towards the bottom, as in Figure 1 (this is apparently swapped in Figure 2-S1 and Figure 2-S2, and rotated by 90 degrees in Figure 2).

In general, I find your arguments regarding the discrepancy in terms of how many trials of pre-conditioning are needed for SPC at the physiological and behavioural levels reasonable.

As mentioned before, the "Ideas and Speculations" section of the Results and Discussion section lacks clarity. I like the idea of the "novelty" activations of DANs by untrained odours as a signal to establish S1-S2 associations.

At the conceptual level, one of the main future questions, I think, would be whether in addition to the S1-S2 link being formed during the first training phase, the key process to experimentally detect that link as SPC takes place during the second training phase (i.e. S1 calls up an S2-representation during this second training phase, such that this S2-representation is associated with punishment; meaning conditioned responding is based on an S2-punishment association), or whether this happens at the moment of the final test (i.e. at the moment of testing S2 calls up S1, which in turn calls up the S1-punishment association; meaning there is no direct S2-punishment association). One could rephrase this as: does S1-S2 pattern completion take place during the second training phase, and/or at the moment of the final test?

II) Further recommendations

More clarity is needed about the time intervals between the two training phases, and between the second training phase and the final test.

In a number of instances, articles or verbs appear to be missing. Some language polishing is advised.

The Introduction does not inform the reader that there have been earlier demonstrations of SPC in insects/ invertebrates. This may be read as a claim of primacy, although the authors, quite appropriately, do not explicitly make such a claim. In other words, not-citing the relevant literature unwittingly leads to an inappropriate, implicit claim of primacy.

Better do not introduce new terminology on the run, such as "S-S associations" line 205.

Better remove the money example from the Introduction as it refers to second-order conditioning, distracting from SPC as the subject of the study.

Line 148 refers to Figure 2C, which does not appear to exist.

https://doi.org/10.7554/eLife.79107.sa1

Author response

Essential revisions:

Reviewer #1 (Recommendations for the authors):

Please check the whole manuscript for typos. There are also many wrong labels in the figures.

It was a bit confusing that the authors start the result section with figure2B. Either the authors provide a better description for this unusual beginning or change the figure arrangement.

We have rearranged all figures as suggested by Reviewer 1 and other Reviewers.

Also, the Materials and methods is quite short. I was wondering if the odors in the test were always presented in the same order. S1, then S2, then other odors? Is this order necessary to induce the S2 effect? Especially in the 30s gap experiment, flies might not show the memory if the authors would be present in the "wrong order" compared to preconditioning.

We have expanded the methods details. Yes, odors were presented in the same order every time as indicated in each figure.

To show that Rac1 inhibition might affect the duration of odor representation, the authors might have performed functional imaging in KCs if feasible.

We have performed this experiment, as suggested by the reviewer. We expressed Gcamp6f in KC using R13F02-lexA>lexAop-GCaMP6f in flies expressing or not Rac1N17 using R13F02-gal4 and tub-gal80ts. Results did not show significant difference between these groups.

Another question that came up is why the authors use a novel odor as a control after conditioning in behavioral experiments. Maybe that is confusing for the animals? Might they perform better if the S1 and S2 would be tested against the solvent or an empty tube? This also might explain discrepancies between imaging data and behavioral data.

When we started the behavioral experiments, we used the solvent as the Reviewers suggested. We did not observe sensory preconditioning when a single odor pair was presented during the preconditioning phase. We later thought that using a third, well-balanced, novel odor during the test would give us a better chance to observe mild changes in the PI. The reasoning was that when using solvent, the baseline of S2 odor avoidance starts high. Therefore, small changes in this odor avoidance would be harder to see, in other words there is a smaller dynamic range. We reasoned that using an initially well-balanced novel odor would move the assay's baseline to zero, increasing the dynamic range. As shown in the results, a single odor pair pre-presentation was also unsuccessful when using a novel odor. After this, we decided to change two parameters: (1) the number of repeated pair presentations was increased to ten, and (2) we started constructing an odor delivery system using an Arduino to precisely control the timing of odor delivery. This new approach resulted in evidence of sensory preconditioning only when the preconditioning was repeated ten times. After this, we did not go back and test the solvent.

Reviewer #2 (Recommendations for the authors):

In this manuscript, the authors provide physiological and behavioral evidence to support that fruit flies can perform olfactory unimodal sensory preconditioning, an example of higher-order conditioning. Flies were first exposed to a pair of odors (S1 and S2) with a 1-s inter-stimulus interval (ISI) before conditioning. Then odor S1 was given a negative value through aversive olfactory conditioning. After conditioning, the non-reinforced odor S2 was also found to be linked to the negative value. When the ISI was changed from 1 s to 30 s, flies failed to link odor S2 with the negative value, unless Rac1 activity was inhibited. Such sensory preconditioning can be observed in an aversive memory trace in a pair of MBON-γ1pedc>α/β neurons as well as behavioral performance. These results indicate that a simple brain may have the ability to guide behavior through inferred value and Rac1 may have a pivotal role in such behavior. The findings of sensory preconditioning are interesting and important, however, the behavioral data should be further strengthened by adding more controls. And calcium imaging results only partially explain the behavioral data, more discussion should be provided. More detailed information about methods is required. The writing needs to be improved.

As suggested by the Reviewer, all figures have been rearranged. Nevertheless, we have paid more attention to details to avoid any similar errors.

1) The writing of this manuscript needs to be greatly improved. The text does not match the figures in some places. There are many other errors throughout the text. It makes reading the manuscript very difficult and time-consuming.

We have corrected these mistakes.

a) In line 95, "and vivo calcium imaging" should be "and in vivo calcium imaging", and "pairing and odor" should be "pairing an odor".

This has been superseded.

b) In line 105, the result description is inconsistent with Figure 2B.

This has been superseded.

c) In line 124, Figure 2A should be Figure 1B.

This has been superseded.

d) In line 129, Figure 1B should be Figure 1C.

This has been superseded.

e) In line 148, there is no Figure 2C.

This has been superseded.

f) In Figure 3A, "Barwards" should be "Backwards".

This has been corrected.

g) The first time the abbreviation KC is mentioned, the full form should be provided.

This has been corrected.

2) Is sensory preconditioning stable and significant at the behavioral level? The behavioral performance of the sensory preconditioning is weak and demonstrated by comparing forward and backward conditioning groups. As a new behavioral paradigm, more control groups such as naïve, "odor only" and "shock only" groups should be tested using wild-type flies like Canton-S.

We used backwards conditioning because these control flies are exposed to all the same stimuli but in a different order. So it is more adequate than testing odors only and shock only. Furthermore, we continuously performed behavior with naïve animals to ensure we had a stable odor balance before experiments. Therefore naïve only control gives a behavioral π score of zero. Finally, during the imaging experiments, we did show that the presentation of odors only, backwards conditioning, and excluding the preconditioning phase all resulted in non-observable physiological sensory preconditioning.

3) Is one-second ISI sufficient to clear odor S1 and switch to odor S2? Is it possible that flies indeed sense mixed odors of S1 and S2? Is preconditioning effect can also be observed when S1-S2 mixed odors are exposed before conditioning? More evidence should be provided. It may affect the interpretation of the results.

The Reviewer makes a good point here. In the classical sensory preconditioning experiments, stimuli of two different modalities are usually presented together or with some overlap. Therefore, sensory preconditioning still occurs if stimuli are presented together. Because we are working with unimodal sensory preconditioning, we decided to avoid odor mixing to simplify possible interpretations.

During imaging, odors delivered to the flies are on a continuous flow of air. Therefore, the odors are pushed by clean air as soon as solenoids valves switch from odor back to clean air. In addition, odors are immediately cleared by a funnel connected to a high vacuum pressure right behind the recording chamber.

During behavior, because the system works with a closed continuous vacuum, odors are cleared immediately after being presented.

Finally, and more importantly, the Rac1 inhibition experiments show that sensory preconditioning can still occur even when odors are presented 30 s apart.

4) As the authors mentioned in lines 183-186, the preconditioning effect observed in physiological trace in MBON-γ1pedc>α/β neurons (Figures 1 and 2) is not sufficient to explain the behavioral preconditioning in Figures 3 and 4. This point should be discussed more in the "ideas and speculations" section.

We have included a discussion about this in “ideas and speculation” section.

Reviewer #3 (Recommendations for the authors):

A weakness is seen in the way the data and arguments are presented, because key data belonging together are not shown and analysed together (and are not shown in the same way), and because the final "Ideas and Speculations" section of the Results and Discussion section reads somewhat disjointed.

We have rearranged the data as suggested by the Reviewer. We have added specific points to the ideas and speculations section. In particular the disconnect between the physiological and behavioral data

Another weakness is that a rationale should be presented under which circumstances before-after comparisons are used as arguments, and when between-group comparisons are used.

In the Material and Methods section, we have added the rationale for selecting the different ways of analyzing the groups—especially the before-after vs. forward-backward comparisons.

Last but not least, the reviewer believes a better job could be done to make the manuscript more accessible for a non-expert audience, for example regarding the definition of wild-type animals, a plain-language rationale for the use of the transgenes, the logic of the Gal80ts/ target system including whether expressing the Rac transgene entails a gain or loss of Rac function, and what, thus, the colour code for presenting the genotypes means in terms of Rac function.

We have edited the manuscript in an attempt to make it more accessible for non-expert audiences as suggested by Reviewer 3.

I) My main recommendation is to present together what belongs together, focusing on the most important issue, namely whether the evidence for sensory pre-conditioning (SPC) is indeed convincing. I discuss this for the physiological data, but the same applies to the behavioural data as well.

I think you could present together with the data from Figure 1, Figure 2-S1 (in between what are now Figure 1B and C), and Figure 2-S2 (after what is now Figure 1C). This would allow you to make your points for all the three mentioned requirements for a demonstration of SPC in one panel – plus: the broader time window for SPC upon Rac inhibition (?!) would be very nicely visible.

To simplify, you could present all the traces of recordings as supplements throughout.

You could then present a separate figure with all (sic) pre-post comparisons for that panel; "all" meaning for MCH and for OCT, and for both the forward and the backward cases.

We have rearranged the data to be as close as possible to the Reviewer 3 suggestions.

There are a number of additional and rather unfortunate arrangements of the figures that make it hard to follow, almost creating 'stroop effects'. In addition to what was mentioned above, examples include:

If you deal with Figure 2 before Figure 1 in the Results section, you should also present these figures in that order. By the way, it might be a good idea to label the experiment in Figure 2 as a 'Pilot experiment' and present it as a supplement. This is because it is inconclusive with respect to all the three requirements for demonstrating SPC mentioned above.

Be consistent in presenting eg the control genotype towards the top and the experimental genotype towards the bottom, as in Figure 1 (this is apparently swapped in Figure 2-S1 and Figure 2-S2, and rotated by 90 degrees in Figure 2).

In general, I find your arguments regarding the discrepancy in terms of how many trials of pre-conditioning are needed for SPC at the physiological and behavioural levels reasonable.

As mentioned before, the "Ideas and Speculations" section of the Results and Discussion section lacks clarity. I like the idea of the "novelty" activations of DANs by untrained odours as a signal to establish S1-S2 associations.

At the conceptual level, one of the main future questions, I think, would be whether in addition to the S1-S2 link being formed during the first training phase, the key process to experimentally detect that link as SPC takes place during the second training phase (i.e. S1 calls up an S2-representation during this second training phase, such that this S2-representation is associated with punishment; meaning conditioned responding is based on an S2-punishment association), or whether this happens at the moment of the final test (i.e. at the moment of testing S2 calls up S1, which in turn calls up the S1-punishment association; meaning there is no direct S2-punishment association). One could rephrase this as: does S1-S2 pattern completion take place during the second training phase, and/or at the moment of the final test?

We acknowledge these comments from the Reviewer 3. He makes very interesting points. We have added these to the discussion and speculations section.

II) Further recommendations

More clarity is needed about the time intervals between the two training phases, and between the second training phase and the final test.

The time between the first and the second training phases is 5 minutes. The time between the second training phase and the test phase was 5 min. We made sure this is explicit in the material and methods.

In a number of instances, articles or verbs appear to be missing. Some language polishing is advised.

We have polished the English writing of the manuscript.

The Introduction does not inform the reader that there have been earlier demonstrations of SPC in insects/ invertebrates. This may be read as a claim of primacy, although the authors, quite appropriately, do not explicitly make such a claim. In other words, not-citing the relevant literature unwittingly leads to an inappropriate, implicit claim of primacy.

We have including previous reports of SPC in the new version of the manuscript.

Better do not introduce new terminology on the run, such as "S-S associations" line 205.

Better remove the money example from the Introduction as it refers to second-order conditioning, distracting from SPC as the subject of the study.

Line 148 refers to Figure 2C, which does not appear to exist.

We addressed these comments as suggested.

https://doi.org/10.7554/eLife.79107.sa2

Article and author information

Author details

  1. Juan Martinez-Cervantes

    Department of Biology, Georgetown University, Washington, DC, United States
    Contribution
    Data curation, Investigation
    Contributed equally with
    Prachi Shah
    Competing interests
    No competing interests declared
  2. Prachi Shah

    Department of Biology, Georgetown University, Washington, DC, United States
    Contribution
    Data curation, Formal analysis, Investigation
    Contributed equally with
    Juan Martinez-Cervantes
    Competing interests
    No competing interests declared
  3. Anna Phan

    Department of Biological Sciences and Neuroscience & Mental Health Institute, University of Alberta, Edmonton, Canada
    Contribution
    Data curation, Investigation, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
  4. Isaac Cervantes-Sandoval

    1. Department of Biology, Georgetown University, Washington, DC, United States
    2. Interdisciplinary Program in Neuroscience, Georgetown University, Washington, DC, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing
    For correspondence
    ic400@georgetown.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6372-7288

Funding

National Institute of Mental Health (R21MH117485-01A1)

  • Isaac Cervantes-Sandoval

Brain and Behavior Research Foundation (30442)

  • Isaac Cervantes-Sandoval

National Institute on Aging (T32AG071745)

  • Prachi Shah

Georgetown University (ID162838)

  • Isaac Cervantes-Sandoval

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

Acknowledgements

This work was supported by grants R21 MH117485-01A1 from the NIMH to IC-S, GU pilot research grant award ID162838 to IC-S, and BBRF Young Investigator Grant 30442. In addition, PS is supported NIA T32 AG071745 award.

Senior Editor

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

Reviewing Editor

  1. Sonia Sen, Tata Institute for Genetics and Society, India

Publication history

  1. Preprint posted: November 30, 2021 (view preprint)
  2. Received: March 31, 2022
  3. Accepted: September 18, 2022
  4. Accepted Manuscript published: September 21, 2022 (version 1)
  5. Version of Record published: October 13, 2022 (version 2)

Copyright

© 2022, Martinez-Cervantes, Shah 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. Juan Martinez-Cervantes
  2. Prachi Shah
  3. Anna Phan
  4. Isaac Cervantes-Sandoval
(2022)
Higher-order unimodal olfactory sensory preconditioning in Drosophila
eLife 11:e79107.
https://doi.org/10.7554/eLife.79107