Cold Shock Disrupts Massed Training-Elicited Memory in Drosophila

Abstract

Memory consolidation is a time-dependent process occurring over hours, days, or longer in different species and requires protein synthesis. An apparent exception is a memory type in Drosophila elicited by a single olfactory conditioning episode, which ostensibly consolidates quickly, rendering it resistant to disruption by cold anesthesia a few hours post-training. This anesthesia-resistant memory (ARM), is independent of protein synthesis. Protein synthesis independent memory can also be elicited in Drosophila by multiple massed cycles of olfactory conditioning, and this led to the prevailing notion that both of these operationally distinct training regimes yield ARM. Significantly, we show that, unlike bona fide ARM, massed conditioning-elicited memory remains sensitive to the amnestic treatment two hours post-training and hence it is not ARM. Therefore, there are two protein synthesis-independent memory types in Drosophila.

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Int. J. Mol. Sci. 2022, 23, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/ijms
Communication
1
Cold shock disrupts massed training-elicited memory in Dro-
2
sophila.
3
Anna Bourouliti 1, 2 and Efthimios M.C Skoulakis1
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1Institute for Fundamental Biomedical Research, Biomedical Sciences Research Center “Alexander Fleming”,
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Vari, 16672 Greece
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2 Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, 68100
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Greece
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* Correspondence: skoulakis@fleming.gr
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Abstract: Memory consolidation is a time dependent process occurring over hours, days, or longer
10
in different species and requires protein synthesis. An apparent exception is a memory type in Dro-
11
sophila elicited by a single olfactory conditioning episode, which ostensibly consolidates quickly,
12
rendering it resistant to disruption by cold anesthesia a few hours post training. This Anesthesia
13
Resistant Memory (ARM), is independent of protein synthesis. Protein synthesis independent
14
memory can also be elicited in Drosophila by multiple massed cycles of olfactory conditioning and
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this led to the prevailing notion that both of these operationally distinct training regimes yield ARM.
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Significantly, we show that unlike bona fide ARM, massed conditioning-elicited memory remains
17
sensitive to the amnestic treatment two hours post training and hence it is not ARM. Therefore, there
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are two protein synthesis-independent memory types in Drosophila.
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Keywords: Memory; Anesthesia Resistant Memory; Olfactory conditioning; Massed conditioning;
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Drosophila
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1. Introduction
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Unconsolidated memories are labile and disrupted by amnestic agents in all animals
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tested [1, 2]. In Drosophila, a brief cold shock immediately following negatively reinforced
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olfactory conditioning results in complete memory loss of the association. However, if
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delivered a couple of hours post-training it is incompletely disruptive, with the residual
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memory termed Anesthesia Resistant Memory (ARM), as it persists the apparently
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anesthetic, immobilizing cold shock [3] and is independent of protein synthesis [2, 4].
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ARM appears to consolidate relatively rapidly as it is partially labile minutes after
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conditioning [5] and stable by 2 hours post-training [3, 6]. A protein synthesis
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independent memory also emerges after multiple consecutive rounds (massed
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conditioning-MC) of negatively reinforced olfactory conditioning [3]. Although cold
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shock treatment after a single round of conditioning is typically used to probe 3-hour
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memory and the MC protocol is utilized for 24-hour memory assessment, the presumed
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protein synthesis independence has led to these two memory types to be called ARM.
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Despite evidence suggesting that both of these operationally and temporally distinct
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memory types engage common, or similar mechanisms [4, 7, 8], it is unclear whether they
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reflect the same cognitive outcome assayed at different time points, or are in fact distinct
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memory types. While both cold-shock and 5 or 10 -round MC protocols are widely used
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to address questions regarding ARM [3, 4, 7], data acquired by one training method are
41
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Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 2 of 9
not typically cross-checked with the other. To elucidate whether memory with the same
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properties is formed with both methods, we hypothesized that if ARM is equivalent or
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the same as MC yielded 24-hour memory, a cold shock delivered 2 hours post-training
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should have a similar effect on both. However, this notion would not be supported, if this
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amnestic treatment impairs 24-hour MC memory. To this end, we conditioned wild type
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Drosophila to associate an aversive odor with electric foot-shock by training with 5 MC
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rounds and subjected them to a single cold shock prior to testing, or 2 hours after training.
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We have been using a 5 round MC [4], because in our hands it affords higher resolution
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than more intensive protocols that may yield “ceiling” effects. We report that 2 hours
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post training, 5-round MC yields a cold-shock sensitive memory along with ARM.
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2. Results
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Conditioning was performed using the classical aversive conditioning paradigm
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which pairs an aversive odor (conditioned stimulus-CS+) with electric foot-shocks (the
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unconditioned stimulus-US), while a second equally aversive odor explicitly unpaired
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with the foot-shock (CS-) serves as control [9]. Memory immediately after one condition-
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ing round contains an ARM component [5, 10]. However, the defining brief cold shock
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typically used to reveal the non-labile ARM memory component 3 hours after a single
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round of conditioning [3, 11], has not been applied after 5 rounds of MC to our knowledge.
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Cold shock immediately after one round of training disrupts memory completely [11].
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However, this immediate effect cannot be addressed in our experiments as the time it
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takes to deliver five training rounds unavoidably leads to testing twenty minutes after the
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first foot-shock /odor association. Therefore, in all our experiments cold shock is delivered
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at the indicated times after the last round of training.
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As shown in Fig. 1A, 8-minute memory after five rounds of MC is also largely labile
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and disrupted by a brief cold shock. Unexpectedly, a 2-min cold shock 2 hours after 5
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rounds of MC resulted in a significant reduction of 3-hour memory (Fig 1B). This effect is
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not specific to the w1118 strain, as an identical memory decrease was uncovered in Canton
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S flies (Fig 1B). Therefore, memory elicited by 5 rounds of MC does not yield solely ARM,
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but rather both cold shock-sensitive and cold shock-insensitive memory components. It
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follows then, that consolidated memory after MC is not the same as that after a single
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round of training. Unsurprisingly, given the kinetics of the protein synthesis sensitive
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Long-Term Memory (LTM) [3], 5 rounds of spaced conditioning, a paradigm where the
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training rounds are spaced apart by fifteen-minutes, which leads to LTM formation [2, 3,
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6], also yielded a consolidated and a labile memory component 3 hours post-training (Fig
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1C). This suggests that both massed and spaced conditioning-elicited memories are com-
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posed of labile components even at 3 hours post-training. The residual memory persisting
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amnestic treatment after spaced training is likely ARM as suggested previously [3].
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Are consolidated memories 2 hours post conditioning with a single, or 5 MC rounds
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equivalent, or proportional to the training intensity? To directly compare the memory lev-
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els , we trained flies with either 1 round or 5 MC rounds and administered cold shock 2
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hours later. 3-hour memory of untreated 5 MC trained animals was significantly different
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from that of 1-round-trained flies (Fig 1D), indicating that the intensive MC training leads
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to more robust three-hour memory. However, cold shock resilient memories were not sig-
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nificantly different, suggesting that the consolidated ARM component is not affected by
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Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 9
training intensity. The above observations were also apparent when the relative non-con-
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solidated post-cold shock memory was calculated, which demonstrated that although
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there is no statistically significant difference between the two paradigms, the mean abso-
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lute value of memory decline is higher for that yielded by 5-round MC (Fig1E). This likely
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reflects the elevated labile memory yielded by MC.
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Figure 1. Memory acquired by massed training is cold-shock sensitive. The graphs show mean performance ±SEM
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consequent to the treatments detailed above. Star and pound symbols indicate significant differences as detailed below.
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(A) 8-minute (immediate) memory of w1118 animals is inhibited by a 2-minute by cold shock (ANOVA F(1 ,20)=144.82,
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p<4.8x10-10). (B) Three-hour memory produced by massed training is significantly reduced by cold shock treatment in
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both w1118 (ANOVA F(1,24)=30.74, p<1.4x10-5) and Canton S (ANOVA F(1,22 )=12.41, p=0.002) animals. (C) 3-hour memory of
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w1118 animals after spaced training is significantly affected by cold shock (ANOVA F(1,19)=10.87, p=0.004). (D) Three-hour
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memory in w1118 flies after one training round is different from memory formed after five consecutive rounds [(ANOVA
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F(3,40)=23.05, p=1.7x10-8). Subsequent analysis using LSM-planned comparisons revealed that the difference between the
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two groups is indeed significant (p=0.003)]. The performace of cold shocked flies was significantly different from that
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of untreated animals after one or 5 MC rounds (pound and star signs respectively, p<0.0001) . However, memories
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resilient to cold shock were not significantly different (p=0.1856). (E) Differnce of indexes shown in (D) between
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treated and non-treated groups that were simultaneously trained with five or one training rounds are not significantly
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different (ANOVA F(1,20)= 1.79, p=0.1965).
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Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 9
We hypothesized that the cold shock sensitive component after MC may reflect
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memory consolidating with slow kinetics. Hence, a cold shock was administered 1 hour
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before 24-hour memory assessment after MC to investigate memory stability at this point.
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This pre-testing cold shock did not affect memory, as performance was not different from
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similarly trained flies not subjected to the amnestic treatment (Fig 2A). This result demon-
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strates that MC-elicited memory was consolidated at 23 hours post-training and verifies
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that cold shock treatment does not generally affect recall. In contrast, a cold shock deliv-
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ered 2 hours after 5-round MC resulted in significantly reduced 24-hour memory com-
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pared to that of non-cold shocked flies or animals subjected to the amnestic treatment 1
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hour prior to testing (Fig 2B). Therefore, memory elicited by 5-round MC includes a sig-
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nificant labile component at 2 hours post-training, which when blocked is reflected in
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compromised 24-hour olfactory associative memory. Collectively, these results strongly
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suggest that 5-round MC yields a memory type that consolidates slowly, being labile at 3
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hours, whereas ARM is not [3].
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120
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Figure 2. Memory acquired by massed training is resistant to cold shock at 23 hours. Graphs show mean performance
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±SEM consequent to the treatments detailed above. Star and pound symbols indicate significant differences as detailed
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below. (A) 24-hour memory elicited by MC is not affected by cold shock delivered 23 hours post-training. Because
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variances were different, the unpaired parametric Welch’s t test was used to compare means. (t=0.0039, df=18.23, n=13.
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p=0.9969 for w1118 and t=0.3129, df=12.50, n=11. p=0.7595 for Canton S). (B) 24-hr memory elicited by MC is compromised
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if animals are cold shocked 2 hours post-training, but not if cold shock is delivered 1 hour before testing. [ANOVA
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F(2,44)=7.992, p=0.001. Subsequent comparisons using LSM-planned comparisons to non-cold shocked animals revealed
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significant differences in the performance of animals cold-shocked 2 hours post-training (p=0.0006, star) and from those
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cold shocked at 23 hrs (p=0044, pound), but not from animals cold shocked 23 hours post-training (p=0.5759)].
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3. Discussion
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Massed conditioning (MC) in a negatively reinforced olfactory conditioning task
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yields a translation independent 24-hour memory. Since 1995 when the protocol was first
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reported, it has been assumed that massed conditioning-yielded memory is equivalent to
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3-hour memory elicited by a single round of conditioning and revealed as resilient to a
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cold shock 2 hours post-training. 3-hour ARM and MC-elicited 24 hour memory are both
136

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 9
independent of protein synthesis [2, 3] and are reported to engage common molecular
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components [3, 4, 12]. Clearly however, unlike ARM, 5 round MC-elicited memory is not
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resistant to amnestic treatment 2 hours post-training (Fig 1E). Moreover, the amnestic
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treatment at 2 hours post-training nearly eliminates 24-hour memory of the event, further
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supporting the notion that unlike ARM, this MC elicited memory is not consolidated at
141
that time.
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Therefore, MC elicits a distinct type of slow-consolidating memory that remains sen-
143
sitive to the amnestic cold shock two hours after conditioning, unlike the amnestic re-
144
sistant memory present two hours after a single round of conditioning. We argue there-
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fore that the two memories are distinct and that 5-round MC ostensibly does not yield
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ARM alone, but a labile memory as well. We suggest the term Protein Synthesis Inde-
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pendent Memory (PSIM) for the labile memory elicited by MC protocols, and ARM for
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the cold shock resistant 3-hour memory after a single round of training to distinguish
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them. The collective evidence presented herein strongly suggests that 1 training round
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elicited ARM is not equivalent to memory yielded by 5 round and most likely 10 round
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MC, which yields ARM and PSIM and therefore, the terms should not be used inter-
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changeably.
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Even though a number of genes and molecular pathways have been reported to func-
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tion in ARM, evidence supporting their involvement comes largely from one of the two
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assays, either 3-hour memory after cold shock (ARM) or after MC, with few tested in both
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assays [4, 7, 13] uncovering similar defects. However, for these and others that have been
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characterized solely via MC protocols the effect of cold shock 2 hours post-training on 24-
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hour memory has not been assessed, so it remains an open question as to whether defects
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in these mutants result from the ARM or the PSIM component. Archetypical mutants such
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as radish with clear deficits in ARM [12, 14], have not been subjected to our knowledge to
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amnestic treatments after MC, so their reported 24-hour memory deficit after 10-round
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MC [3], may also harbor a PSIM deficit. Furthermore, it would be interesting to investigate
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whether PSIM is affected or parallels the labile memory compromised in mutants like am-
164
nesiac [15, 16]. Common molecular components of PSIM and other labile memory types
165
would suggest at least partially overlapping molecular mechanisms, yet perhaps distinct
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enough to differentiate the two processes, a hypothesis currently under investigation.
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4. Materials and Methods
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Drosophila culture and strains. Cantonized w1118 and Canton S wild type strains were
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cultured in wheat-flour-sugar food as previously described [4] and raised in a 12h
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night/dark cycle, at 25oC and 50% humidity.
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Behavioral experiments. 2–4-day old flies were used in all experiments, which were
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performed at 25oC and 55%-65% humidity under dim red light. Aversive olfactory condi-
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tioning utilized 90 Volt electric foot-shocks as unconditioned stimuli (US), paired to one
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of the aversive odorants 5% Benzaldehyde (BNZ), or 50% Octanol (OCT) diluted in Iso-
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propyl Myristate as conditioned stimuli (CS). One training cycle consisted of 12 CS/US
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pairings of 1.25 seconds with a 4-second interstimulus interval, followed by 30 seconds of
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rest before presenting another odor in the absence of shock. Either odor was paired with
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shock, while the other served as control. Massed conditioning (MC) involved five
180

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 9
consecutive training cycles with 30 seconds between cycles. Spaced training was identical
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except the interval between cycles was 15 minutes. Cold-shock treatment was adminis-
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tered as described previously [4] at 1min, 2, or 23hrs after the final training round, as in-
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dicated in each experiment. Memory testing involved simultaneous presentation of both
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odors for 90sec as described [4]. To calculate Δ, the difference between labile and consoli-
185
dated memories, two groups of animals were simultaneously trained with 1 round of
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training and half were subjected to cold shock while the others were not. Δ was calculated
187
as the performance difference between simultaneously trained untreated and cold-
188
shocked animals. A similar method was used to calculate Δ for animals trained with 5
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rounds MC.
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Data analysis. Raw data analysis was performed with the JMP7 software (SAS Insti-
191
tute Inc.). Statistical comparisons were performed as detailed in the figure legend. Com-
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parison between two groups was carried out with ANOVA and subsequent LSM-planned
193
comparisons or, in cases of different variances between groups, unpaired parametric
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Welch’s t test when variances of the measurements were unequal. Statistical details are
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presented in Table 1. Graphs were created with the GraphPad Prism 8.0.1 software and
196
show means ±SEM.
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Group
Mean ± SEM
P Value
Fig. 1A ANOVA F(1,20) = 144.8201 p=4.8x10-10
w1118 (-cs)
76.04 ± 3.16
w1118 (+cs)
17.22± 3.73
<0.0001
Fig. 1B ANOVA F(1,24) = 30.7419 p=1.4x10-5
w1118 (-cs)
39.52 ± 2.08
w1118 (+cs)
18.46 ± 3.18
<0.0001
ANOVA F(1,22) = 12.4188 p= 0.0021
Canton S (-cs)
38.39 ± 3.89
Canton S (+cs)
19.42 ± 3.72
0.0021
Fig. 1C ANOVA F(1,19) = 10.8714 p=0.0043
w1118 (-cs)
51.58 ± 4.49
w1118 (+cs)
34.93 ± 2.59
0.0043
Fig. 1D ANOVA F(3,40) = 23.0502 p=1.7x10-8
w1118 (5x-cs)
42.46 ± 2.22
w1118 (5x+cs)
18.94 ± 3.37
<0.0001
w1118 (1x-cs)
30.78 ± 1.72
0.0037
w1118 (1x+cs)
13.86 ± 3.01
<0.0001
w1118 (1x-cs)
30.78 ± 1.72
w1118 (1x+cs)
13.86 ± 3.01
<0.0001
w1118 (5x+cs)
18.94 ± 3.37
w1118 (1x+cs)
13.86 ± 3.01
0.1856
Fig. 1E ANOVA F(1,20) = 1.7990 p=0.1965
w1118 (Δ5x) = (5x-cs)-(5x+cs)
23.52 ± 3.38
w1118 (Δ1x) = (1x-cs)-(1x+cs)
16.92 ± 3.58
0.1965
Fig. 2A ANOVA F(1,22) = 1.57x10-5 p=0.9969
Welch-corrected t=0.00395711, df=18.23
w1118 (-cs)
20.22 ± 4.26
w1118 (+cs)
20.20 ± 2.26
0.9969
ANOVA F(1,26) = 0.0979 p=0.7576
Welch-corrected t=0.3129, df=12.50
Canton S (-cs)
15.71 ± 1.33
Canton S (+cs)
16.94 ± 3.72
0.7595
Fig. 2B ANOVA F(2,44) = 7.9924 p=0.0012
Canton S (-cs)
20.02 ± 1.94
Canton S (+cs@2h)
8.15 ± 2.11
0.0006
Canton S (+cs@23h)
18.13 ± 2.95
0.5759
Canton S (+cs@23h)
18.13 ± 2.95
Canton S (+cs@2)
8.15 ± 2.11
0.0044

Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 9
Table 1. Statistical comparisons. Note the final two comparisons in analysis regarding Fig.1D and
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2B are not with the relevant control, but rather between treatments.
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Author Contributions:. “Conceptualization, AB and EMCS.; methodology: AB and EMCS.; formal
231
analysis: AB ; writing—original draft preparation, AB. writing—review and editing: EMCS; super-
232
vision: EMCS. All authors have read and agreed to the published version of the manuscript.”.
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Funding: This Research was supported by a grant from Fondation Santé to EMCS.
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Data Availability Statement: All relevant data are presented within this manuscript
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Acknowledgments: The authors would like to thank M. Loizou for technical help, Prof I. Marou-
236
lakou for advice and Fondation Santé for support.
237
Conflicts of Interest: The authors declare no conflict of interest.
238
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