Int. J. Mol. Sci. 2022, 23, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/ijms
Cold shock disrupts massed training-elicited memory in Dro-
Anna Bourouliti 1, 2 and Efthimios M.C Skoulakis1
1Institute for Fundamental Biomedical Research, Biomedical Sciences Research Center “Alexander Fleming”,
Vari, 16672 Greece
2 Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, 68100
* Correspondence: firstname.lastname@example.org
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 Dro-
sophila 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.
Keywords: Memory; Anesthesia Resistant Memory; Olfactory conditioning; Massed conditioning;
Unconsolidated memories are labile and disrupted by amnestic agents in all animals
tested [1, 2]. In Drosophila, a brief cold shock immediately following negatively reinforced
olfactory conditioning results in complete memory loss of the association. However, if
delivered a couple of hours post-training it is incompletely disruptive, with the residual
memory termed Anesthesia Resistant Memory (ARM), as it persists the apparently
anesthetic, immobilizing cold shock  and is independent of protein synthesis [2, 4].
ARM appears to consolidate relatively rapidly as it is partially labile minutes after
conditioning  and stable by 2 hours post-training [3, 6]. A protein synthesis
independent memory also emerges after multiple consecutive rounds (massed
conditioning-MC) of negatively reinforced olfactory conditioning . Although cold
shock treatment after a single round of conditioning is typically used to probe 3-hour
memory and the MC protocol is utilized for 24-hour memory assessment, the presumed
protein synthesis independence has led to these two memory types to be called ARM.
Despite evidence suggesting that both of these operationally and temporally distinct
memory types engage common, or similar mechanisms [4, 7, 8], it is unclear whether they
reflect the same cognitive outcome assayed at different time points, or are in fact distinct
memory types. While both cold-shock and 5 or 10 -round MC protocols are widely used
to address questions regarding ARM [3, 4, 7], data acquired by one training method are
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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
properties is formed with both methods, we hypothesized that if ARM is equivalent or
the same as MC yielded 24-hour memory, a cold shock delivered 2 hours post-training
should have a similar effect on both. However, this notion would not be supported, if this
amnestic treatment impairs 24-hour MC memory. To this end, we conditioned wild type
Drosophila to associate an aversive odor with electric foot-shock by training with 5 MC
rounds and subjected them to a single cold shock prior to testing, or 2 hours after training.
We have been using a 5 round MC , because in our hands it affords higher resolution
than more intensive protocols that may yield “ceiling” effects. We report that 2 hours
post training, 5-round MC yields a cold-shock sensitive memory along with ARM.
Conditioning was performed using the classical aversive conditioning paradigm
which pairs an aversive odor (conditioned stimulus-CS+) with electric foot-shocks (the
unconditioned stimulus-US), while a second equally aversive odor explicitly unpaired
with the foot-shock (CS-) serves as control . Memory immediately after one condition-
ing round contains an ARM component [5, 10]. However, the defining brief cold shock
typically used to reveal the non-labile ARM memory component 3 hours after a single
round of conditioning [3, 11], has not been applied after 5 rounds of MC to our knowledge.
Cold shock immediately after one round of training disrupts memory completely .
However, this immediate effect cannot be addressed in our experiments as the time it
takes to deliver five training rounds unavoidably leads to testing twenty minutes after the
first foot-shock /odor association. Therefore, in all our experiments cold shock is delivered
at the indicated times after the last round of training.
As shown in Fig. 1A, 8-minute memory after five rounds of MC is also largely labile
and disrupted by a brief cold shock. Unexpectedly, a 2-min cold shock 2 hours after 5
rounds of MC resulted in a significant reduction of 3-hour memory (Fig 1B). This effect is
not specific to the w1118 strain, as an identical memory decrease was uncovered in Canton
S flies (Fig 1B). Therefore, memory elicited by 5 rounds of MC does not yield solely ARM,
but rather both cold shock-sensitive and cold shock-insensitive memory components. It
follows then, that consolidated memory after MC is not the same as that after a single
round of training. Unsurprisingly, given the kinetics of the protein synthesis sensitive
Long-Term Memory (LTM) , 5 rounds of spaced conditioning, a paradigm where the
training rounds are spaced apart by fifteen-minutes, which leads to LTM formation [2, 3,
6], also yielded a consolidated and a labile memory component 3 hours post-training (Fig
1C). This suggests that both massed and spaced conditioning-elicited memories are com-
posed of labile components even at 3 hours post-training. The residual memory persisting
amnestic treatment after spaced training is likely ARM as suggested previously .
Are consolidated memories 2 hours post conditioning with a single, or 5 MC rounds
equivalent, or proportional to the training intensity? To directly compare the memory lev-
els , we trained flies with either 1 round or 5 MC rounds and administered cold shock 2
hours later. 3-hour memory of untreated 5 MC trained animals was significantly different
from that of 1-round-trained flies (Fig 1D), indicating that the intensive MC training leads
to more robust three-hour memory. However, cold shock resilient memories were not sig-
nificantly different, suggesting that the consolidated ARM component is not affected by
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-
solidated post-cold shock memory was calculated, which demonstrated that although
there is no statistically significant difference between the two paradigms, the mean abso-
lute value of memory decline is higher for that yielded by 5-round MC (Fig1E). This likely
reflects the elevated labile memory yielded by MC.
Figure 1. Memory acquired by massed training is cold-shock sensitive. The graphs show mean performance ±SEM
consequent to the treatments detailed above. Star and pound symbols indicate significant differences as detailed below.
(A) 8-minute (immediate) memory of w1118 animals is inhibited by a 2-minute by cold shock (ANOVA F(1 ,20)=144.82,
p<4.8x10-10). (B) Three-hour memory produced by massed training is significantly reduced by cold shock treatment in
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
w1118 animals after spaced training is significantly affected by cold shock (ANOVA F(1,19)=10.87, p=0.004). (D) Three-hour
memory in w1118 flies after one training round is different from memory formed after five consecutive rounds [(ANOVA
F(3,40)=23.05, p=1.7x10-8). Subsequent analysis using LSM-planned comparisons revealed that the difference between the
two groups is indeed significant (p=0.003)]. The performace of cold shocked flies was significantly different from that
of untreated animals after one or 5 MC rounds (pound and star signs respectively, p<0.0001) . However, memories
resilient to cold shock were not significantly different (p=0.1856). (E) Differnce of indexes shown in (D) between
treated and non-treated groups that were simultaneously trained with five or one training rounds are not significantly
different (ANOVA F(1,20)= 1.79, p=0.1965).
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
memory consolidating with slow kinetics. Hence, a cold shock was administered 1 hour
before 24-hour memory assessment after MC to investigate memory stability at this point.
This pre-testing cold shock did not affect memory, as performance was not different from
similarly trained flies not subjected to the amnestic treatment (Fig 2A). This result demon-
strates that MC-elicited memory was consolidated at 23 hours post-training and verifies
that cold shock treatment does not generally affect recall. In contrast, a cold shock deliv-
ered 2 hours after 5-round MC resulted in significantly reduced 24-hour memory com-
pared to that of non-cold shocked flies or animals subjected to the amnestic treatment 1
hour prior to testing (Fig 2B). Therefore, memory elicited by 5-round MC includes a sig-
nificant labile component at 2 hours post-training, which when blocked is reflected in
compromised 24-hour olfactory associative memory. Collectively, these results strongly
suggest that 5-round MC yields a memory type that consolidates slowly, being labile at 3
hours, whereas ARM is not .
Figure 2. Memory acquired by massed training is resistant to cold shock at 23 hours. Graphs show mean performance
±SEM consequent to the treatments detailed above. Star and pound symbols indicate significant differences as detailed
below. (A) 24-hour memory elicited by MC is not affected by cold shock delivered 23 hours post-training. Because
variances were different, the unpaired parametric Welch’s t test was used to compare means. (t=0.0039, df=18.23, n=13.
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
if animals are cold shocked 2 hours post-training, but not if cold shock is delivered 1 hour before testing. [ANOVA
F(2,44)=7.992, p=0.001. Subsequent comparisons using LSM-planned comparisons to non-cold shocked animals revealed
significant differences in the performance of animals cold-shocked 2 hours post-training (p=0.0006, star) and from those
cold shocked at 23 hrs (p=0044, pound), but not from animals cold shocked 23 hours post-training (p=0.5759)].
Massed conditioning (MC) in a negatively reinforced olfactory conditioning task
yields a translation independent 24-hour memory. Since 1995 when the protocol was first
reported, it has been assumed that massed conditioning-yielded memory is equivalent to
3-hour memory elicited by a single round of conditioning and revealed as resilient to a
cold shock 2 hours post-training. 3-hour ARM and MC-elicited 24 hour memory are both
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
components [3, 4, 12]. Clearly however, unlike ARM, 5 round MC-elicited memory is not
resistant to amnestic treatment 2 hours post-training (Fig 1E). Moreover, the amnestic
treatment at 2 hours post-training nearly eliminates 24-hour memory of the event, further
supporting the notion that unlike ARM, this MC elicited memory is not consolidated at
Therefore, MC elicits a distinct type of slow-consolidating memory that remains sen-
sitive to the amnestic cold shock two hours after conditioning, unlike the amnestic re-
sistant memory present two hours after a single round of conditioning. We argue there-
fore that the two memories are distinct and that 5-round MC ostensibly does not yield
ARM alone, but a labile memory as well. We suggest the term Protein Synthesis Inde-
pendent Memory (PSIM) for the labile memory elicited by MC protocols, and ARM for
the cold shock resistant 3-hour memory after a single round of training to distinguish
them. The collective evidence presented herein strongly suggests that 1 training round
elicited ARM is not equivalent to memory yielded by 5 round and most likely 10 round
MC, which yields ARM and PSIM and therefore, the terms should not be used inter-
Even though a number of genes and molecular pathways have been reported to func-
tion in ARM, evidence supporting their involvement comes largely from one of the two
assays, either 3-hour memory after cold shock (ARM) or after MC, with few tested in both
assays [4, 7, 13] uncovering similar defects. However, for these and others that have been
characterized solely via MC protocols the effect of cold shock 2 hours post-training on 24-
hour memory has not been assessed, so it remains an open question as to whether defects
in these mutants result from the ARM or the PSIM component. Archetypical mutants such
as radish with clear deficits in ARM [12, 14], have not been subjected to our knowledge to
amnestic treatments after MC, so their reported 24-hour memory deficit after 10-round
MC , may also harbor a PSIM deficit. Furthermore, it would be interesting to investigate
whether PSIM is affected or parallels the labile memory compromised in mutants like am-
nesiac [15, 16]. Common molecular components of PSIM and other labile memory types
would suggest at least partially overlapping molecular mechanisms, yet perhaps distinct
enough to differentiate the two processes, a hypothesis currently under investigation.
4. Materials and Methods
Drosophila culture and strains. Cantonized w1118 and Canton S wild type strains were
cultured in wheat-flour-sugar food as previously described  and raised in a 12h
night/dark cycle, at 25oC and 50% humidity.
Behavioral experiments. 2–4-day old flies were used in all experiments, which were
performed at 25oC and 55%-65% humidity under dim red light. Aversive olfactory condi-
tioning utilized 90 Volt electric foot-shocks as unconditioned stimuli (US), paired to one
of the aversive odorants 5% Benzaldehyde (BNZ), or 50% Octanol (OCT) diluted in Iso-
propyl Myristate as conditioned stimuli (CS). One training cycle consisted of 12 CS/US
pairings of 1.25 seconds with a 4-second interstimulus interval, followed by 30 seconds of
rest before presenting another odor in the absence of shock. Either odor was paired with
shock, while the other served as control. Massed conditioning (MC) involved five
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
except the interval between cycles was 15 minutes. Cold-shock treatment was adminis-
tered as described previously  at 1min, 2, or 23hrs after the final training round, as in-
dicated in each experiment. Memory testing involved simultaneous presentation of both
odors for 90sec as described . To calculate Δ, the difference between labile and consoli-
dated memories, two groups of animals were simultaneously trained with 1 round of
training and half were subjected to cold shock while the others were not. Δ was calculated
as the performance difference between simultaneously trained untreated and cold-
shocked animals. A similar method was used to calculate Δ for animals trained with 5
Data analysis. Raw data analysis was performed with the JMP7 software (SAS Insti-
tute Inc.). Statistical comparisons were performed as detailed in the figure legend. Com-
parison between two groups was carried out with ANOVA and subsequent LSM-planned
comparisons or, in cases of different variances between groups, unpaired parametric
Welch’s t test when variances of the measurements were unequal. Statistical details are
presented in Table 1. Graphs were created with the GraphPad Prism 8.0.1 software and
show means ±SEM.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 9
Mean ± SEM
Fig. 1A ANOVA F(1,20) = 144.8201 p=4.8x10-10
76.04 ± 3.16
Fig. 1B ANOVA F(1,24) = 30.7419 p=1.4x10-5
39.52 ± 2.08
18.46 ± 3.18
ANOVA F(1,22) = 12.4188 p= 0.0021
Canton S (-cs)
38.39 ± 3.89
Canton S (+cs)
19.42 ± 3.72
Fig. 1C ANOVA F(1,19) = 10.8714 p=0.0043
51.58 ± 4.49
34.93 ± 2.59
Fig. 1D ANOVA F(3,40) = 23.0502 p=1.7x10-8
42.46 ± 2.22
18.94 ± 3.37
30.78 ± 1.72
13.86 ± 3.01
30.78 ± 1.72
13.86 ± 3.01
18.94 ± 3.37
13.86 ± 3.01
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
Fig. 2A ANOVA F(1,22) = 1.57x10-5 p=0.9969
Welch-corrected t=0.00395711, df=18.23
20.22 ± 4.26
20.20 ± 2.26
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
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
Canton S (+cs@23h)
18.13 ± 2.95
Canton S (+cs@23h)
18.13 ± 2.95
Canton S (+cs@2)
8.15 ± 2.11
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
2B are not with the relevant control, but rather between treatments.
Author Contributions:. “Conceptualization, AB and EMCS.; methodology: AB and EMCS.; formal
analysis: AB ; writing—original draft preparation, AB. writing—review and editing: EMCS; super-
vision: EMCS. All authors have read and agreed to the published version of the manuscript.”.
Funding: This Research was supported by a grant from Fondation Santé to EMCS.
Data Availability Statement: All relevant data are presented within this manuscript
Acknowledgments: The authors would like to thank M. Loizou for technical help, Prof I. Marou-
lakou for advice and Fondation Santé for support.
Conflicts of Interest: The authors declare no conflict of interest.
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