New Research

Cognition and Behavior

RNA from Trained Aplysia Can Induce an

Epigenetic Engram for Long-Term Sensitization in
Untrained Aplysia
ⴱ ⴱ ⴱ
Alexis Bédécarrats,1, Shanping Chen,1, Kaycey Pearce,1, Diancai Cai,1 and David L. Glanzman1,2,3

DOI:http://dx.doi.org/10.1523/ENEURO.0038-18.2018
1
Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA 90095,
2
Department of Neurobiology, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles,
CA 90095, and 3Integrative Center for Learning and Memory, Brain Research Institute, David Geffen School of
Medicine at University of California, Los Angeles, Los Angeles, CA 90095

Visual Abstract
Trained donor

Tail shocks

Trained RNA

V
i

Naive recipient Sensory neurons

24 h 24 h

+30 s

Siphon touch V
i

Sensitized withdrawal reflex Increased neuronal excitability

Significance Statement
It is generally accepted that long-term memory (LTM) is encoded as alterations in synaptic strength. An
alternative model, however, proposes that LTM is encoded by epigenetic changes. Noncoding RNAs (ncRNAs)
can mediate epigenetic modifications. Therefore, RNA from a trained animal might be capable of producing
learning-like behavioral change in an untrained animal. Here, it is demonstrated that the memory for long-term
sensitization (LTS) in the marine mollusk Aplysia can be successfully transferred by injecting RNA from sensitized
into naïve animals. Moreover, a specific cellular alteration that underlies sensitization in Aplysia, sensory neuron
hyperexcitability, can be reproduced by exposing sensory neurons in vitro to RNA from trained animals. The
results provide support for a nonsynaptic, epigenetic model of memory storage in Aplysia.

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The precise nature of the engram, the physical substrate of memory, remains uncertain. Here, it is reported that
RNA extracted from the central nervous system of Aplysia given long-term sensitization (LTS) training induced
sensitization when injected into untrained animals; furthermore, the RNA-induced sensitization, like training-
induced sensitization, required DNA methylation. In cellular experiments, treatment with RNA extracted from
trained animals was found to increase excitability in sensory neurons, but not in motor neurons, dissociated from
naïve animals. Thus, the behavioral, and a subset of the cellular, modifications characteristic of a form of
nonassociative long-term memory (LTM) in Aplysia can be transferred by RNA. These results indicate that RNA
is sufficient to generate an engram for LTS in Aplysia and are consistent with the hypothesis that RNA-induced
epigenetic changes underlie memory storage in Aplysia.
Key words: Aplysia; epigenetics; learning and memory; memory transfer; RNA; sensitization

Introduction epigenetic alterations (Peschansky and Wahlestedt, 2014;

A major goal of modern neuroscience is to determine Savell et al., 2016). This raises the intriguing possibility that
the identity of the engram, the physical memory trace constituents of LTM may be transferred from a trained to an
(Semon, 1921). At present, it is widely accepted that long- untrained animal by RNA. Here, we tested this possibility in
term memory (LTM) is stored by learning-induced modifica- the case of LTS in Aplysia.
tions of synaptic connections (Mayford et al., 2012;
Takeuchi et al., 2014). But theoretical considerations (Hol- Materials and Methods
liday, 1999; Gallistel and Balsam, 2014) and recent exper- Behavioral training and testing
imental evidence (Chen et al., 2014; Johansson et al., Adult Aplysia californica (80 –120 g) were obtained from
2014; Ryan et al., 2015) support the idea that LTM is Alacrity Marine Biological Services and initially housed in
stored within the cell bodies of neurons. Previously, it was a 50-gallon aquarium filled with cooled (12–14°C), aerated
reported that the memory for long-term sensitization (LTS) seawater. For the experiments, the animals were placed
in Aplysia (Pinsker et al., 1973) involves an early, protein individually into custom-built Plexiglas chambers that
synthesis-dependent priming component that can persist were continuously perfused with cooled (14°C) seawater.
independently of memory-related behavioral and synaptic One day before training, each animal was implanted bi-
alterations; the priming component permits LTM to be laterally with Teflon-coated platinum wires (0.008-inch
reinstated following its disruption by reconsolidation coated diameter, A-M Systems). For this procedure, the
blockade, or to be induced by partial training after impair- animal was anesthetized by cooling in cold seawater (4°C)
ment of memory consolidation by retrograde amnesia for 13 min. Wires, prepared by removing the Teflon from
(Chen et al., 2014; Pearce et al., 2017). The molecular the ends with forceps, were threaded through a 20-gauge
identity of the memory priming component is unknown, needle, which was used to insert the wire into the animal’s
but appears to involve epigenetic modifications (Zovkic tail. Following this procedure, the animal was placed into
et al., 2013). Noncoding RNAs (ncRNAs), which play im- the experimental chamber, where it was given 24 h to re-
portant roles in memory formation (Rajasethupathy et al., cover and acclimate to the chamber. The siphon-withdrawal
2009, 2012; Fiumara et al., 2015; Guven-Ozkan et al., reflex (SWR) was tested as follows: The siphon was lightly
2016; Tan et al., 2017), represent a major mechanism for stimulated with a soft, flexible probe and the duration of
the resulting SWR was timed. Timing of the SWR began
Received January 21, 2018; accepted April 27, 2018; First published May 14, once the siphon had retracted completely beneath the
2018.
parapodia and ended as soon as the siphon reappeared.
The authors declare no competing financial interests.
Author contributions: D.L.G., S.C., D.C., K.P., and A.B. designed research; Responses were given a score of 1.0 s if the siphon did
K.P., S.C., and A.B. performed research; D.C. and A.B. contributed unpub- not withdraw completely into the parapodia. Three pre-
lished reagents/analytic tools; D.C. and A.B. analyzed data; D.L.G. wrote the tests were delivered once every 10 min, beginning 25 min
paper. before the start of training (Figs. 1A, 2A). Sensitization
This work was supported by research grants to D.L.G. from the National
Institute of Neurological Disorders and Stroke [National Institutes of Health
training comprised two rounds of training separated by 24
(NIH) R01 NS029563], the National Institute of Mental Health (NIH R01 h. Each round of training consisted of five bouts of tail
MH096120), and the National Science Foundation (IOS 1121690) as well as by shocks delivered at 20-min intervals. During each bout of
a postdoctoral fellowship from the Fyssen Foundation (A.B.). training, the animal received three trains; the intertrain
*A.B., S.C., and K.P. contributed equally to this work.
interval was 2 s. Each train was 1 s in duration and
Acknowledgements: We thank A. C. Roberts for statistical advice; S. Api-
chon, B. Cheema, M. Kimbrough, D. Miresmaili, A. Rangchi, R. Sumner, X. consisted of shocks (10-ms pulse duration, 40 Hz, 120 V)
Zhao, and A. Zobi for assistance with the behavioral training; and W. C. delivered to the animal’s tail via a Grass stimulator (S88,
Abraham, A. D. Grinnell, J. Koester, F. B. Krasne, and A. C. Roberts for helpful Astro-Med) connected to the platinum wires. A single
comments on this manuscript. posttest of the SWR, performed exactly as the pretests,
Correspondence should be addressed to David L. Glanzman at the above
address, E-mail: [email protected].
was made at 48 h after the start of training. The testing
DOI:http://dx.doi.org/10.1523/ENEURO.0038-18.2018 and training were conducted by different experimenters,
Copyright © 2018 Bédécarrats et al. and the tester was blind to the experimental treatment of
This is an open-access article distributed under the terms of the Creative the animal.
Commons Attribution 4.0 International license, which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is In the experiments involving RNA injections (see Re-
properly attributed. sults), naïve animals were given three pretests, identical

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Posttest
A Pretest 5XTraining 5XTraining Posttest
C Pretest

RNA injection

-25 -15 -5 0 20 40 60 80 24 48 -30 -20 -10 0 24

RNA extraction Minutes Hours

Minutes Hours

B *** D
60 50 **
50 40
40

SWR (s)
SWR (s)

30
30
20
20

10 10

0 0
Control Trained Control RNA Trained RNA

Figure 1. RNA extracted from sensitization-trained donor animals induces long-term enhancement of the SWR in recipient Aplysia.
A, Experimental protocol for inducing LTS in the donor animals. B, Mean posttest duration of the SWR in the untrained control (1.2 ⫾
0.1 s, n ⫽ 31) and trained (56.4 ⫾ 2.0 s, n ⫽ 34) groups. The trained group exhibited significant sensitization, as indicated by the
comparison with control group (Mann–Whitney test, U ⫽ 496, p ⬍ 0.001). C, Experimental protocol for the RNA injection experiments.
The first pretest occurred 2–3 h after the posttest for the behavioral training (A). D, Mean duration of the SWR measured at ⬃24 h after
the injection of RNA for the control RNA (5.4 ⫾ 3.9 s, n ⫽ 7) and trained RNA (38.0 ⫾ 4.6 s, n ⫽ 7) groups. The two groups differed
significantly (U ⫽ 30, p ⬍ 0.003). Furthermore, Wilcoxon tests indicated that the difference between the pretest and posttest for the
trained RNA group was significant (W ⫽ 28, p ⬍ 0.02), whereas it was not significant for the control RNA group (p ⬎ 0.2). The bar
graphs in this and the following figures display means ⫾ SEM; ⴱp ⬍ 0.05, ⴱⴱp ⬍ 0.01, ⴱⴱⴱp ⬍ 0.001, n.s., nonsignificant.

to those that preceded the sensitization training, at 30, RNA and drug preparation and injection
20, and 10 min before the injection (Figs. 1C, 2C). A To prepare a single RNA injection, the pleural-pedal and
single posttest of the SWR was performed at 24 h after abdominal ganglia were removed from four to five
the injection. sensitization-trained animals, or from four to five untrained

Pretest Posttest
A Pretest 5XTraining 5XTraining Posttest
C
RNA

-25 -15 -5 0 20 40 60 80 24 48 -30 -20 -10 0 24

RNA extraction RG / Veh

Minutes Hours Minutes Hours

B 60
*** D 50
*
50 40
40
SWR (s)
SWR (s)

30
30
20
20

10 10

0 0
Pre Post RNA-Veh RNA-RG
Figure 2. DNA methylation is required for RNA-induced enhancement of the SWR. A, Experimental protocol for inducing sensitization in
the second donor group. B, Mean posttest duration of the SWR (n ⫽ 38). The training produced sensitization (mean posttest SWR ⫽ 56.4 ⫾
1.4 s, and mean pretest SWR ⫽ 1.1 ⫾ 0.1 s; W ⫽ 741, p ⬍ 0.001). C, Experimental protocol for testing the effect of DNMT inhibition on
RNA-induced enhancement of the SWR. RG-108/vehicle was injected into animals 5–10 min after the RNA injection. D, Mean postinjection
duration of the SWR in the RNA-Veh (n ⫽ 3) and RNA-RG (n ⫽ 7) groups. The mean duration of the SWR in the RNA-Veh group (35.7 ⫾
7.7 s) was significantly longer than that in the RNA-RG group (1.4 ⫾ 0.3 s; U ⫽ 27, p ⬍ 0.02). Moreover, the posttest SWR was sensitized
compared to the pretest reflex in the RNA-Veh group (paired t test, p ⬍ 0.05), but not in the RNA-RG group (p ⬎ 0.4).

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controls, immediately after the 48-h posttest. The total potential was current clamped at –50 mV. The action
RNA was then extracted from the dissected ganglia. The potential (AP) firing threshold was determined by injecting
ganglia were initially homogenized in TRIzol reagent for 30 2-s current pulses of incremental intensity (0.1 nA for the
s; typically, 1 ml TRIzol was used to homogenize the sensory neurons and 0.01 nA for the motor neurons). Cells
central ganglia from two animals. For every 1 ml TRIzol were injected with a 2-s steady pulse of suprathreshold
reagent, 200 ␮l chloroform was added and mixed by positive current for the measurements of neuronal excit-
vortexing for 15 s. After incubation at room temperature ability (Liu et al., 2011). In the case of the sensory neurons,
for 5–10 min, the sample was centrifuged at 12,000 ⫻ g current pulses of 0.5, 1.0, or 2.0 nA were used depending
for 15 min. The upper aqueous phase was transferred into on whether the initial firing threshold was ⬍0.5, ⱖ0.5, or
a new tube. The sample was then centrifuged for 10 min ⱖ1.0 nA, respectively. Sensory neurons were excluded
at 4°C after addition of 500 ␮l isopropanol to precipitate from the analysis if their resting membrane potential was
the RNA. The resulting RNA pellets were washed with more depolarized than –35 mV. To test the excitability of
70% ethanol and centrifuged for 2 min at 4°C. After being motor neurons, positive current pulses of 0.1, 0.2, or 0.3
air-dried for 10 min, the RNA pellet from each tube was nA were used when the initial spike threshold was ⬍0.1,
dissolved in 30 ␮l DIH2O; then the RNA from ganglia ⱖ0.1, or ⱖ0.2 nA, respectively. Motor neurons whose
dissected from trained animals (typically, from four ani- membrane potentials were more depolarized than –30 mV
mals) was combined, or the RNA from ganglia dissected were excluded. After the electrophysiological measurements
from untrained animals was combined, into a single tube, were completed, the microelectrodes were removed from
and the RNA concentration was measured using Nano the neurons, and the cell cultures were treated with RNA-
Drop (Thermo Fisher ND-1000). After the RNA concentra- containing medium or vehicle solution (see Results). Twenty-
tion had been determined, 70 ␮g of the combined RNA four hours later, the neurons were reimpaled and their
was aliquoted and ASW was added to this aliquot to attain electrophysiological properties remeasured.
a volume of 100 ␮l; this solution was then injected into the In the experiments involving sensorimotor cocultures,
hemocoel of an animal via its neck. Each recipient animal the amplitude of the monosynaptic EPSP evoked by a
therefore received 70 ␮g of either RNA from trained ani- single presynaptic AP was assessed on day 1 of the exper-
mals or RNA from control animals. iment. For this purpose, the presynaptic sensory neuron and
The DNA methyltransferase (DNMT) inhibitor RG108 postsynaptic motor neuron in the coculture were impaled
(Sigma) was dissolved in DMSO to a concentration of 25 with sharp microelectrodes. To prevent the motor neuron
mM. To inhibit DNMT, a volume of 100 ␮l/100 g of body from spontaneously firing during testing, the neuron’s
weight of RG108 was injected intrahemocoelically into membrane potential was held at – 80 to – 85 mV by pass-
each animal (Fig. 2C). ing negative current (0.3– 0.8 nA) into the cell via the
recording microelectrode using the bridge circuit of the
Cell culturing and electrophysiological amplifier. An initial EPSP was elicited through brief intra-
measurements cellular stimulation of the sensory neuron using a positive
Pleural sensory neurons and small siphon (LFS) motor current pulse (20 ms, 0.2– 0.8 nA). After the pretest, the
neurons were individually dissociated from adult animals microelectrodes were removed from the sensory and mo-
and placed into cell culture (Rayport and Schacher, 1986; tor neurons, and the recording medium was replaced with
Lin and Glanzman, 1994). Some of the cell cultures com- cell culture medium. Then the coculture was treated either
prised isolated neurons, either exclusively sensory or ex- with RNA-containing medium or control medium (see Re-
clusively motor neurons; others comprised synaptically sults). The sensory and motor neurons were reimpaled
coupled pairs of neurons, each consisting of a single with microelectrodes and the amplitude of the monosyn-
sensory neuron and a single motor neuron. The cell cul- aptic EPSP reassessed 24 h later.
ture medium was composed of 50% Aplysia sterile he- RNA/vehicle treatment of cell cultures
molymph and 50% Leibowitz-15 (L-15, Sigma). During Following the initial electrophysiological measurements
electrophysiological recording the cell cultures were per- on day 1, the recording medium was washed out with
fused with 50% ASW and 50% L-15 (recording medium). normal cell culture medium. The cultures were then ran-
The recordings from isolated neurons were made using domly assigned to treatment with RNA from trained ani-
dissociated neurons that had been in culture for 5 d at the mals (trained RNA group), RNA from untrained animals
start of the experiments. For the experiments on synap- (control RNA group), or vehicle. For the RNA treatments, 1
tically coupled pairs of neurons (sensorimotor cocultures), ␮g of RNA was added to each cell culture dish, yielding a
the neurons were in culture for 3 d before the initial concentration of 0.5 ␮g of RNA per 1 ml of cell culture
recordings. The neurons were impaled with sharp mi- medium. The RNA from the trained animals, the RNA from
cropipettes (20 –30 M⍀) filled with 1.5 M potassium ace- the control animals, or the vehicle was added to the cell
tate, 0.5 M potassium chloride, and 0.01 M HEPES (pH culture dish and left in the dish for 24 h, after which it was
7.2). The recorded voltage signals were amplified with an washed out with the recording medium for 30 min, and the
Axoclamp 2B amplifier (Molecular Devices), digitalized posttest electrophysiological measurements made.
with an ITC-18 (Instrutech), and acquired and stored using
Axograph software. Statistical analyses
During the measurements of the biophysical properties The statistical analyses of the data were performed
of isolated sensory and motor neurons, the cell membrane using SigmaStat (Systat Software). Nonparametric tests

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Table 1. Statistical table

Data structure Type of test Power (␣ ⫽ 0.05)
a (Fig. 1B) Non-normally distributed Mann–Whitney test Not applicable
b (Fig. 1D) Non-normally distributed Mann–Whitney test Not applicable
c (Fig. 1D) Non-normally distributed Wilcoxon test Not applicable
d (Fig. 1D) Non-normally distributed Wilcoxon test Not applicable
e (Fig. 2B) Non-normally distributed Wilcoxon test Not applicable
f (Fig. 2D) Non-normally distributed Mann–Whitney test Not applicable
g (Fig. 2D) Normally distributed Paired t test 0.647
h (Fig. 2D) Non-normally distributed Wilcoxon test Not applicable
i (Fig. 3B) Non-normally distributed Kruskal–Wallis test followed by Dunn’s test Not applicable
j (Fig. 3D) Non-normally distributed Kruskal–Wallis test Not applicable
k (Fig. 4B) Non-normally distributed Levene’s test Not applicable
l (Fig. 4B) Non-normally distributed Kruskal–Wallis test Not applicable

were used to assess the statistical significance of differ- posttest SWR was not significantly prolonged compared
ences whenever necessitated due to non-normality of the to the pretest SWR in animals that received the injection
data or to the violation of the assumption of homogeneity of RNA from the untrained donors. Thus, only the RNA
of variance among experimental groups. Mann–Whitney U from sensitized animals appeared to induce reflex en-
tests were used for comparisons of two independent hancement in the recipient snails.
groups. A paired t test or a Wilcoxon rank-sum test was
used to compare two dependent groups. When three Inhibition of DNA methylation blocks the behavioral
independent groups were involved, the significance of the effect of RNA from sensitized donor animals in the
overall group differences was initially assessed with a recipients
one-way ANOVA or a Kruskal–Wallis test. Given that the Both the consolidation and maintenance of the LTM
group differences were significant, Dunn’s post hoc tests for sensitization in Aplysia depend on DNA methylation
were used for pairwise comparisons. Normality of the (Rajasethupathy et al., 2012; Pearce et al., 2017). To
distribution were tested with a Shapiro–Wilk test. Lev- determine whether the RNA-mediated behavioral en-
ene’s test centered to the mean (car package) was used hancement similarly required DNA methylation, we exam-
with R software to test for homogeneity of variance in the ined whether inhibiting DNA methylation disrupted the
synaptic experiments. All reported levels of significance
sensitizing effect of the RNA from trained animals. Aplysia
represent two-tailed values. The statistical analyses are
were again given 2 d of sensitization training, which pro-
summarized in Table 1.
duced LTS, and afterward RNA was extracted from their
Results central ganglia (Fig. 2A,B). The RNA was then injected into
two groups of naïve snails; 5–10 min later, one of these
Injection of RNA from sensitization-trained donor groups (RNA-RG group) was also given an intrahemocoe-
animals causes enhancement of the withdrawal lic injection of the DNMT inhibitor RG-108 (Brueckner
reflex in untrained recipients et al., 2005; Pearce et al., 2017), whereas the other (RNA-
To generate the RNA used for memory transfer, individ- Veh group) was given an injection of the vehicle solution
ual Aplysia were given sensitization training consisting of
(Fig. 2C). The RNA-Veh group exhibited significant en-
spaced bouts of tail shocks for two consecutive days (Fig.
hancement of the SWR 24 h later; by contrast, the
1A). The training produced clear LTS, as indicated by the
RNA-RG group did not show behavioral enhancement
significant enhancement of the SWR 24 h after the second
(Fig. 2D). Therefore, DNA methylation is required for RNA-
day of training (48-h posttest) in the trained group of animals
induced enhancement of the SWR, as it is for tail shock-
(Fig. 1B). Immediately after the 48-h posttest, RNA was
extracted from the central nervous system (pleural, pedal induced LTS of the reflex (Pearce et al., 2017).
and abdominal ganglia) of the control and trained animals.
The extracted RNA was then injected intrahemocoelically RNA from sensitized animals induces increased
into other naïve Aplysia (recipient animals; Fig. 1C). (Note excitability in sensory neurons dissociated from
that occasional batches of wild-caught Aplysia did not naïve animals
sensitize. The behavioral data from these animals were A significant advantage of Aplysia as a model system
excluded from the analysis, and RNA was not extracted for mechanistic analyses of learning and memory is the
from them.) The duration of the SWR in the recipients was wealth of extant knowledge regarding the biological bases of
measured 24 h after the RNA injection. The SWR was sensitization in this organism (Kandel, 2001; Byrne and
significantly enhanced in the trained RNA group of ani- Hawkins, 2015). Accordingly, we tested whether RNA ex-
mals compared to the control RNA group (Fig. 1D). Fur- tracted from sensitization-trained animals caused cellular
thermore, a within-group comparison indicated that the alternations that mimic those known to result from re-
posttest duration of the reflex was significantly longer peated tail shocks. To ascertain whether the cellular
than the pretest duration in the animals that received the changes induced by RNA from sensitized animals mimic
injection of the RNA from trained donors; by contrast, the shock-induced cellular changes, we made use of sensory

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and motor neurons of the withdrawal circuit in dissociated RNA from sensitized animals has a variable effect on
cell culture (Lin and Glanzman, 1994). synaptic strength in sensorimotor cocultures
In response to a prolonged pulse of depolarizing intra- LTS in Aplysia involves long-term facilitation (LTF) of the
cellular current, Aplysia sensory neurons exhibit spike monosynaptic connection between the sensory and mo-
“accommodation”: they fire at the beginning of, but not tor neurons of the withdrawal circuit (Frost et al., 1985).
throughout, the current pulse (Klein et al., 1986). Long- Accordingly, we examined the effects of RNA from trained
lasting sensitization of the defensive withdrawal reflex is and untrained donors on the strength of sensorimotor
accompanied by a long-term increase in the excitability of synapses in dissociated cell culture (Montarolo et al.,
the somata of central sensory neurons in the withdrawal 1986; Cai et al., 2008). There was no long-term effect of
circuit (Walters, 1987); this enhanced excitability is reflected 24-h incubation with RNA from trained animals, RNA from
as anti-accommodation, an increase in the number of APs control animals, or the vehicle on the mean EPSP evoked
evoked by a prolonged pulse of positive current (Cleary in the postsynaptic motor neurons by a presynaptic AP
et al., 1998). To test whether RNA extracted from trained (Fig. 4). Nonetheless, although the mean EPSPs in the
Aplysia alters sensory neuron accommodation, we used three experimental groups did not differ significantly, the
isolated sensory neurons in dissociated cell culture. The variances among the EPSPs in the three groups were sig-
neurons were initially impaled with sharp microelectrodes nificantly unequal due to the greater variance in the EPSPs
and the number of APs evoked by a 2-s intracellular pulse for the synapses treated with RNA from sensitization-
of suprathreshold positive current quantified (Fig. 3A). trained animals. Inspection of the synaptic data revealed
Following this pretest, the sensory neurons were treated that the RNA from trained donors produced large en-
for 24 h with RNA from trained donors or RNA from hancement of a subset of the sensorimotor synapses.
untrained donors. Other sensory neurons were treated Such enhancement was never observed for synapses
with an equivalent amount of the vehicle alone. The next treated with RNA from untrained animals or for synapses
day, the RNA/vehicle was washed out of the culture treated with the vehicle.
dishes with cell recording medium, and the neurons were
reimpaled and reinjected with the same suprathreshold Discussion
current to measure potential changes in excitability. The We have shown that RNA from sensitization-trained
current injections produced significantly more APs in sen- Aplysia contains critical components of the engram for
sory neurons treated with RNA from sensitized animals LTS, as indicated by its ability to induce sensitization-like
than in sensory neurons treated with either vehicle or RNA behavioral enhancement when injected into naïve recipi-
from control animals (Fig. 3B). There was no significant ent animals. Importantly, the RNA-induced sensitization,
difference in excitability between the sensory neurons like the LTS induced by noxious stimulation, requires DNA
treated with control RNA and those treated with the ve- methylation for its consolidation (Pearce et al., 2017; Fig.
hicle. Anti-accommodation is known to result from a de- 2). Several of our cellular and behavioral results further
crease in cyclic AMP-dependent potassium currents in argue that this putative transference of memory from
Aplysia sensory neurons, and, in particular, to reduction of donor animals to the recipients cannot be easily ascribed
the slowly-inactivating S-type current (Klein et al., 1986; to nonspecific effects of the donor RNA. First, the control
Goldsmith and Abrams, 1992); thus, the RNA from RNA (RNA extracted from untrained donors) did not pro-
sensitization-trained animals may enhance the excitability of duce sensitization of the SWR (Fig. 1). Second, the RNA
sensory neurons through modulation of the same current from trained donors had an opposing effect on the excit-
that is modulated by electrical shocks to the body wall of ability of cultured sensory neurons from that of untrained
Aplysia. donors (Fig. 3A,B). Third, the changes produced by the
RNA from sensitized Aplysia were selective for sensory
RNA from sensitized animals does not increase the neurons; the biophysical properties of motor neurons
excitability of dissociated motor neurons were unaltered by the RNA from sensitized donors (Fig.
To ascertain the specificity of the cellular effects of the 3C,D). Admittedly, the alterations we observed in the
RNA treatment, we examined the effects of applying RNA biophysical properties of cultured sensory neurons after
from trained or control animals to isolated small siphon treatment with RNA from sensitized animals are unlikely to
(LFS) motor neurons in dissociated cell culture. A previous fully account for the behavioral changes produced in the
study of LTS in Aplysia showed that, in contrast to the intact recipient animals by injections of RNA from trained
effects observed in sensory neurons, in motor neurons donors; nonetheless, because these biophysical altera-
LTS was not accompanied by a significant increase in the tions mimic those found in intact animals after LTS train-
number of APs evoked to intracellular injection of a pro- ing (Walters, 1987; Cleary et al., 1998), they would be
longed pulse of suprathreshold current (Cleary et al., 1998). expected to contribute substantially to the RNA-induced
Thus, the induction of LTS does not produce an overall sensitization.
increase in the excitability of motor neurons. Similarly, we It is interesting that the RNA from sensitization-trained
observed no effect of the RNA from sensitization-trained animals appeared to produce strong facilitation only in a
animals on excitability-related properties of isolated mo- subset of sensorimotor synapses (Fig. 4). We do not
tor neurons in cell culture (Fig. 3C,D). This result indicates understand the reason for the variability of the synaptic
that the modulation of neuronal excitability by RNA from effect of the RNA from trained animals. One possibility is
sensitized animals was specific to the sensory neurons. that there is an as-yet unappreciated inhomogeneity

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Sensory neurons Sensory neurons

A B
Pretest Posttest
Vehicle
150
*
n.s.
**

% Change in AP number
100
Stim.

Control RNA 50

-50

Trained RNA

Vehicle

Trained RNA
Control RNA
-100

Motor neurons Motor neurons

C D
Pretest Posttest
Vehicle
150

100
% Change in AP number

n.s.
Stim.

Control RNA 50

-50
Trained RNA
Vehicle

Trained RNA
Control RNA

-100

Figure 3. Treatment with RNA from trained animals increases excitability in dissociated sensory neurons but not in dissociated motor
neurons. A, Sample electrophysiological traces from excitability tests on sensory neurons. Scale bars: 20 mV, 0.25 s. B, Changes in
the excitability of the sensory neurons induced by RNA/vehicle treatment. The mean change in evoked APs in each group was: vehicle ⫽
–17.29 ⫾ 12.86% (n ⫽ 19); control RNA ⫽ –35.76 ⫾ 19.88% (n ⫽ 16); and trained RNA ⫽ 56.66 ⫾ 22.07% (n ⫽ 19). The group
differences were significant (Kruskal–Wallis; H ⫽ 11.81, p ⬍ 0.04). Dunn’s post hoc tests indicated that the increased firing in the
trained RNA group was greater than that in the vehicle group (q ⫽ 2.44, p ⬍ 0.05) and control RNA group (q ⫽ 3.25, p ⬍ 0.004),
respectively. The difference between vehicle and control RNA groups was not significant (p ⬎ 0.9). C, Sample traces from tests of
motor neuron excitability. Scale bars: 25 mV, 0.25 s. D, Summary of posttreatment changes in the excitability of motor neurons. The
mean changes were: vehicle group ⫽ –29.28 ⫾ 19.16% (n ⫽ 15); control RNA group ⫽ 5.278 ⫾ 34.36% (n ⫽ 12); and trained RNA
group ⫽ –1.136 ⫾ 34.01% (n ⫽ 14). The group differences in excitability were insignificant (p ⬎ 0.7).

among the population of pleural sensory neurons and/or (Meadows et al., 2016; see also Meadows et al., 2015),
small siphon motor neurons that were used for the sen- than synapse-specific LTF. Of course, these possibilities
sorimotor cocultures; according to this idea, only some of are not mutually exclusive.
the dissociated neurons had the capacity to express the Overall, the cellular changes caused by the RNA from
long-term changes that contribute to LTF. Another possi- trained animals were admittedly modest compared to the
bility is that the epigenetic alterations, particularly DNA behavioral changes. But this is not unexpected; the de-
methylation, that result from treatment with the RNA from fensive withdrawal reflexes in Aplysia are regulated by
sensitized animals more reliably induce cell-wide altera- interneuronal neural circuits, in addition to the monosyn-
tions, such as changes in intrinsic neuronal excitability aptic sensorimotor connections (Cleary et al., 1995). In-

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A B
Pretest Posttest

% Change in EPSP amplitude

600
Vehicle 500
400
Control RNA 300
200
100
Trained RNA 0
-100
-200 Vehicle Control RNA Trained RNA

Figure 4. Exposure of in vitro sensorimotor synaptic connections to RNA from trained animals enhanced the strength of a
subpopulation of synapses. A, Representative records of EPSPs evoked in motor neurons by a single presynaptic AP before and 24
h after the RNA/vehicle treatments. Scale bars: 5 mV, 0.1s. B, Box and whiskers plots showing the distribution of posttreatment
changes in EPSP amplitude in the three experimental groups. The boxes delineate the second and third quartiles, the horizontal lines
in the boxes represent the medians, and the vertical bars (whiskers) show the extent of the data spread. The crosses indicate the
means, whereas individual data points are represented by circles. Mean posttreatment changes in EPSP amplitudes were: vehicle
group ⫽ –23.38 ⫾ 10.59% (n ⫽ 23); control RNA group ⫽ –21.32 ⫾ 10.23% (n ⫽ 34); and trained RNA group ⫽ 22.71 ⫾ 26.70% (n ⫽
32). A Kruskal–Wallis test revealed no significant differences among the groups with respect to the mean changes in EPSP amplitude
(p ⬎ 0.8). Note, however, that five of the 32 synapses treated with RNA from trained animals showed an increase of ⬎150%, whereas
none of the synapses treated with vehicle or RNA from control animals showed an increase of this magnitude. A Levene’s test
confirmed that the three groups displayed significantly unequal variances (F(2,86) ⫽ 5.883, p ⬍ 0.005).

jections of the RNA from sensitized donors may well have cles (Smalheiser, 2007; Valadi et al., 2007; Tkach and
produced modifications of interneuronal pathways within Théry, 2016; Ashley et al., 2018; Pastuzyn et al., 2018). If,
the animals that contributed to behavioral sensitization. In as we believe, ncRNAs in the RNA extracted from sensi-
addition, it is important to note that the RNA was removed tized animals were transferred to Aplysia neurons, per-
from the donors 48 h after training; indeed, the RNA from haps via extracellular vesicles, they likely caused one or
trained animals produced a greater increase in the excit- more epigenetic effects that contributed to the induction
ability of cultured sensory neurons at 48 h posttraining and maintenance of LTM (Fig. 2).
than long-term training with serotonin (Liu et al., 2011, There have been prior reports of the successful transfer
their Fig. 6). of LTM from trained donor animals to naïve recipients via
Our data indicate that essential components of the cannibalism (McConnell, 1962) or RNA injection (Babich
engram for LTM in Aplysia can be transferred to untrained et al., 1965; Jacobson et al., 1965; Albert, 1966; Braud,
animals, or to neurons in culture, via RNA. This finding 1970). However, these early claims have long been viewed
raises two questions: (1) Which specific RNA(s) mediate(s) with skepticism due to numerous failures to replicate the
the memory transfer? and (2) How does the naked RNA memory transfer effect (Hartry et al., 1964; Gross and
get from the hemolymph/cell culture medium into Aplysia Carey, 1965; Byrne et al., 1966; Luttges et al., 1966;
neurons? Regarding the first question, although we do not Walker, 1966; Walker and Milton, 1966; McGaugh, 1967).
know the identity of the memory-bearing molecules at The negative results convinced many that the positive
present, we believe it is likely that they are ncRNAs. Note reports of memory transfer were attributable to lack of
that previous results have implicated ncRNAs, notably proper controls for training-induced factors such as stress
microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs; or arousal, and/or the influence of poorly defined aspects
Rajasethupathy et al., 2009, 2012; Fiumara et al., 2015), in of the experimental methods used (time between the RNA
LTM in Aplysia. Long ncRNAs (lncRNAs) represent other injection and behavioral testing of the recipients, specific
potential candidate memory transfer molecules (Mercer method of RNA extraction, etc.; McGaugh, 1967; Setlow,
et al., 2008). Regarding the second question, recent evi- 1997).
dence has revealed potential pathways for the passage of A major advantage of our study over earlier studies of
cell-free, extracellular RNA from body fluids into neurons. memory transfer is that we used a type of learning, sen-
Thus, miRNAs, for example, have been detected in many sitization of the defensive withdrawal reflex in Aplysia, the
different types of body fluids, including blood plasma; and cellular and molecular basis of which is exceptionally well
cell-free extracellular miRNAs can become encapsulated characterized (Kandel, 2001; Kandel, 2012; Byrne and
within exosomes or attached to proteins of the Argonaut Hawkins, 2015). The extensive knowledge base regarding
(AGO) family, thereby rendering the miRNAs resistant to sensitization in Aplysia enabled us to show that the RNA
degradation by extracellular nucleases (Turchinovich et al., from sensitized donors not only produced sensitization-
2012, 2013). Moreover, miRNA-containing exosomes have like behavioral change in the naïve recipients, but also
been reported to pass freely through the blood-brain barrier caused specific electrophysiological alterations of cul-
(Ridder et al., 2014; Xu et al., 2017). And it is now appreci- tured neurons that mimic those observed in sensitized
ated that RNAs can be exchanged between cells of the animals. The cellular changes observed after exposure of
body, including between neurons, via extracellular vesi- cultured neurons to RNA from trained animals significantly

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 New Research 9 of 11

strengthens the case for positive memory transfer in our Frost WN, Castellucci VF, Hawkins RD, Kandel ER (1985) Monosyn-
study. aptic connections made by the sensory neurons of the gill- and
siphon-withdrawal reflex in Aplysia participate in the storage of
Another difference between our study and earlier at-
long-term memory for sensitization. Proc Natl Acad Sci USA 82:
tempts at memory transfer via RNA is that there is now at 8266 –8269. Medline
hand a mechanism, unknown 40 years ago, whereby RNA Gallistel CR, Balsam PD (2014) Time to rethink the neural mecha-
can powerfully influence the function of neurons: epige- nisms of learning and memory. Neurobiol Learn Mem 108:136 –
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the subject of vigorous investigation (Landry et al., 2013; sory neurons. Proc Natl Acad Sci USA 89:11481–11485. Medline
Gross CG, Carey FM (1965) Transfer of learned response by RNA
Sweatt, 2013; Fischer, 2014; Nestler, 2014; Smalheiser, injection: failure of attempts to replicate. Science 150:1749. Med-
2014; Marshall and Bredy, 2016). Our demonstration that line
inhibition of DNA methylation blocks the memory transfer Guven-Ozkan T, Busto GU, Schutte SS, Cervantes-Sandoval I,
effect (Fig. 2) supports the hypothesis that the behavioral O’Dowd DK, Davis RL (2016) MiR-980 is a memory suppressor
and cellular effects of RNA from sensitized Aplysia in our microRNA that regulates the autism-susceptibility gene A2bp1.
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