Neurobiology of Learning and Memory 136 (2016) 47–53

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Neurobiology of Learning and Memory

journal homepage: www.elsevier.com/locate/ynlme

Mechanisms underlying long-term fear memory formation

from a metaplastic neuronal state
Ryan G. Parsons a,b,⇑, David L. Walker a, Michael Davis a
a
Emory University, Department of Psychiatry and Behavioral Sciences, United States
b
Stony Brook University, Department of Psychology and Neurosciences Institute, United States

a r t i c l e i n f o a b s t r a c t

Article history: We previously showed that a single weak fear conditioning trial, that does not produce a long-term fear
Received 2 February 2016 memory (LTM), appeared to prime memory formation such that when a second trial followed within a
Revised 15 August 2016 circumscribed time window a robust and long-lasting fear memory was formed. We also showed that this
Accepted 17 September 2016
priming effect could be blocked if we interfered with protein kinase A (PKA) signaling in the amygdala
Available online 19 September 2016
during the first conditioning trial. The goals of the current study were to determine if LTM formation after
the second trial depends on PKA signaling in the amygdala and to characterize the underlying memory
Keywords:
processes engaged during the second trial that allows for LTM formation. Our interpretation of the orig-
Fear
Memory
inal findings is that the second conditioning trial triggers LTM from a metaplastic state that is engaged by
Metaplasticity the first conditioning trial. However, it is also possible that the second conditioning trial acts as a remin-
Retrieval der of the first and engages a reconsolidation-like process. Several experiments were conducted to distin-
Consolidation guish between these two possibilities. We show that interfering with PKA signaling during the second
Amygdala conditioning trial disrupts memory formation. However, if a third trial follows the second or if the second
trial was presented without shock, the PKA inhibitor was no longer effective. Our findings demonstrate
that the induction of fear memory from a metaplastic state involves new learning that is distinct from
retrieval-dependent updating of memories.
Ó 2016 Published by Elsevier Inc.

1. Introduction ceptually similar to metaplasticity, a term used to characterize a

There is a growing recognition that memories are not always
formed upon a clean slate, but instead are often influenced by prior
and ongoing experience (Dudai, 2009; McKenzie & Eichenbaum,
2011). This is a fundamental aspect of memory formation, but
one that is poorly captured by most neurobiological studies of
learning and memory. This is especially true in studies that use
Pavlovian fear conditioning in which the vast majority of experi-
ments train animals in a single session with a few trials spaced sec-
onds to minutes apart. We recently developed a fear conditioning
paradigm which allows us to study how prior events can influence
subsequent learning. We found in rats that a single pairing of a
light cue with a mild shock, insufficient to support either short-
or long-term fear memory, primed future learning such that a sec-
ond trial delivered within a time window lasting from 60 min to
3 days resulted in the formation of a long-lasting and robust fear
memory (Parsons & Davis, 2012). Our behavioral results are con-
⇑ Corresponding author at: Department of Psychology and Neurosciences Insti-
tute, Stony Brook University, 100 Nichols Rd, Stony Brook, NY 11794, United States.
E-mail address: ryan.parsons@stonybrook.edu (R.G. Parsons).
set of in vitro findings in which an activity-dependent change in
neuronal state can influence future bouts of synaptic plasticity
(Hulme, Jones, & Abraham, 2013; Sehgal, Song, Ehlers, & Moyer,
2013).
Although a single trial did not produce a LTM, it did result in the
phosphorylation of several targets of protein kinase A (PKA) in the
basolateral amygdala (BLA). Thus, disrupting the activity of PKA in
the BLA during the first conditioning trial prevented the ability of
the second trial to produce LTM. In this same study, protein expres-
sion studies revealed that the second conditioning trial resulted in
an increase in the phosphorylation of proteins downstream of PKA.
However, we do not know if the LTM initiated by the second con-
ditioning trial also depends on PKA signaling.
The goals of the current study were (1) to determine if PKA activ-
ity during or soon after the second conditioning trial is also necessary
for the formation of LTM, and (2) to test if a memory impairment fol-
lowing the second trial can be distinguished from retrieval-based
disruptions in memory such as reconsolidation blockade. To this
end, Experiment 1 tested whether inhibiting PKA signaling in the
amygdala during the second conditioning trial would disrupt LTM
formation. The results from this experiment showed that blocking

http://dx.doi.org/10.1016/j.nlm.2016.09.009
1074-7427/Ó 2016 Published by Elsevier Inc.
48 R.G. Parsons et al. / Neurobiology of Learning and Memory 136 (2016) 47–53
PKA in the amygdala during the second conditioning trial did disrupt
LTM formation. Additionally, two experiments tested the possibility
that the memory impairments observed by blocking PKA signaling
during the second conditioning trial are the result of disrupting a
retrieval-based process such as memory reconsolidation (Nader,
2015; Nader, Schafe, & LeDoux, 2000).
In Experiment 2 we asked if blocking PKA activity during or
after a second conditioning trial would still disrupt memory forma-
tion if a third conditioning trial followed 24 h later. Experiment 3
was identical except that we used a more conventional reconsoli-
dation design where on the second day rats were presented with
the cue in the absence of shock (i.e. retrieval trial). We reasoned
that if the disruption in memory in the first experiment was a
reconsolidation deficit, then blocking PKA during the second trial
would disrupt the memory trace established by the first trial,
and a third trial would not support LTM. However, if the LTM
formed as a result of the second conditioning trial represents pro-
duction of a LTM from a metaplastic state, which lasts for several
days, then a third conditioning trial should still be able to support
LTM. Our results showed, in both of these experiments using a
third conditioning trial, rats given the PKA inhibitor around the
time of the second trial showed intact memory during testing.
We believe our results support the conclusion that LTM produced
as a result of the second trial depends on PKA signaling in the
amygdala, and it does so in a manner that is mechanistically dis-
tinct from retrieval-dependent memory updating.
2. Materials and methods
2.1. Subjects
Eighty-two male Sprague Dawley rats (300–350 g) obtained
from Charles River (Raleigh, NC) served as subjects. All rats were
approximately 2 months old upon arrival and were group housed
in a vivarium maintained on a 12 h light/dark cycle. Surgery and
behavioral testing began within two weeks after arrival and took
place during the light portion of the cycle. Food and water were
freely available. All procedures were carried out with approval of
the Emory University or Stony Brook University Institutional Ani-
mal Care and Use Committee.
2.2. Surgery
Before surgery, animals were anesthetized with IP injections of
dexdomitor (0.5 mg/kg) and ketamine (75 mg/kg) or ketamine
(87 mg/kg) and xylazine (13 mg/kg). Each rat received a subcuta-
neous injection of metacam (1 mg/kg) on the day of surgery and
another the day after surgery. Rats were implanted with 22-
gauge bilateral cannulae aimed at the amygdala (AP = 2.8/
L = ±5.2/V = 8.0). The cannulae were anchored to the skull using
stainless steel screws, superglue, and acrylic cement. Stainless
steel obturators, cut to be flush with the end of the guide cannulae,
were inserted to prevent blockage.
2.3. Drug preparation & infusion
The PKA inhibitor Rp-cAMPS (Tocris, Ellisville, MO USA) was
diluted with ACSF to a concentration of 36 lg/ll (Schafe &
LeDoux, 2000). Rats received bilateral infusions of Rp-cAMPS or
ACSF into the amygdala 30 min before or immediately after the
second conditioning trial. The total volume of the infusion
(0.5 ll/side) was given over 2 min and the injection cannula
remained in place for an additional 90 s to allow for diffusion away
from the tip of the injection cannulae, which were cut to extend
1 mm beyond the guide cannulae.
2.4. Apparatus
Rats were trained and tested in two identical 9  14  10 cm
(depth  width  height; internal dimensions) Plexiglas and
wire-mesh cages. Each cage was suspended between compression
springs within a steel frame and located within a custom-designed
60  79.5  59.5 cm sound-attenuating chamber lined with
6.3 mm thick Plexiglas. The floor of each cage consisted of four
6.0 mm diameter stainless steel bars spaced 18 mm apart.
Startle responses were evoked by 50-ms 95-dB white noise
bursts (5-ms rise-decay time, 0–22 kHz) generated by a Macintosh
G3 computer sound file, amplified by a Radio Shack amplifier
(Model MPA–200), and delivered through high frequency speakers
(Radio Shack Supertweeter) located in front of each cage. Back-
ground noise (60-dB wideband) was produced by an ACO Pacific
white noise generator (Model 3024) and was delivered through
the same speakers as those used to provide white noise bursts.
Sound level measurements were made with a Brüel & Kjaer model
2235 sound level meter (A scale; random input) with the micro-
phone (Type 4176) located 10 cm from the center of the speaker,
which approximates the distance of the rats ear from the speaker
during testing. Attached to the bottom of each cage was an Ende-
vco accelerometer (Model 2217E). Cage movement resulted in dis-
placement of the accelerometer that produced a voltage output
proportional to the velocity of cage movement. The accelerome-
ter’s output was amplified by an Endevco Model 104 amplifier
and digitized on a scale of 0–2500 units by an InstruNET device
(GW Instruments, Model 100B) interfaced to a Macintosh G3 com-
puter. Startle amplitude was defined as the maximal peak–to–peak
voltage that occurred during the first 200 ms after onset of the
startle-eliciting noise burst. Shock reactivity was similarly quanti-
fied using a 500 ms window, while activity levels were sampled by
measuring cage movement during a 500 ms window every 30 s
during the 5-min acclimation period.
The unconditioned stimulus for all experiments was a 0.5-s 0.4-
mA scrambled shock delivered through the floor bars. Shock inten-
sity was measured with a 1 kOhm resistor across a differential
channel of an oscilloscope in series with a 100 kOhm resistor con-
nected between adjacent floor bars within each cage. Current was
defined as the root–mean–square voltage across the 1 kOhm resis-
tor where mA = 0.707  0.5  peak–to–peak voltage. Shocks were
produced by LeHigh Valley shock generators (SGS 004). The condi-
tioned stimulus for all experiments was a 3.7 s light (82 lx) posi-
tioned about 3 in. from the back of the chambers. The
presentation and sequencing of all stimuli was under the control
of the Macintosh G3 computer using custom-designed software
(The Experimenter; Glassbeads Inc.).
For the experiments in Fig. 2C, four identical sound-attenuating
cabinets were used (Startle Monitor II, Kinder Scientific, Poway,
CA). During fear conditioning, rats were placed in four
26.67  20.96  15.9 cm (depth  width  height) identical Plexi-
glas and stainless steel cages with a shock grid floor. During base-
line startle and testing sessions, rats were placed in restrainers
with a floor made of plastic and a rounded cover composed of
stainless steel rods. Both the startle and shock cages sat on top of
a load cell sensing platform on the floor of the cabinet. The load cell
interfaced with a PC and reported the output in Newton v (N). Star-
tle amplitude was defined as the maximum N that occurred during
the first 500 ms after onset of the white noise burst. Intensity of the
startle, light, and shock stimuli were identical as noted above.
2.5. Behavioral procedures
2.5.1. Baseline startle and pre-conditioning test
On two consecutive days, rats were placed into the startle
chambers and, after 5 min, presented with the first of 30 95-dB
 R.G. Parsons et al. / Neurobiology of Learning and Memory 136 (2016) 47–53 49

startle-eliciting noise bursts (30-s interstimulus interval - ISI). Rats
were removed from the chambers immediately following the final
startle trial. Rats were assigned to different treatment groups such
that the average baseline startle level for each group was similar.
Twenty-hour hours later rats were given a light pre-test session
where all rats were returned to the startle chamber and presented
with 50 startle trials [30-s interstimulus interval (ISI)]. Of the last
20 startle trials, every fourth startle trial was presented 3.2 s after
the onset of the 3.7 s light, resulting in a total of 5 light pre-
conditioning test trials. This light pre-test session was done to
evaluate the unconditioned effects of the light on startle
amplitude.
2.5.2. Conditioning trials
The next day all rats were returned to the same chamber where,
after 5 min, they received a single pairing of a 3.7 s light (82 lx)
with a 0.5 s footshock (0.4 mA) beginning 3.2 s after light onset.
All rats were removed 2 min following the light-shock (or light-
alone) trial. In the first experiment (Fig. 1), 24 h later rats were
given a second light-shock conditioning trial identical to the first.
For the second and third experiments (Fig. 2), rats were given 3
light-shock conditioning trials spaced 24 h apart. In the final exper-
iment (Fig. 3), 24 h later rats were given a light-alone retrieval trial
(described below) which was followed by a second light-shock
conditioning trial the next day.
2.5.3. Retrieval and post-conditioning test
Retrieval and testing both occurred in the same chamber used
in the previous sessions. After 5 min, the first of 30 95-dB startle-
eliciting noise bursts were presented (ISI = 30 s) to slightly habitu-
ate the startle reflex. For retrieval, rats were then given a single
light-startle test trial followed by 3 startle–alone trials and were
removed immediately after the final startle-alone trial. For testing,
they received 40 additional startle trials 10 of which occurred in

A Trial 1 Trial 2 Testing

24 H 48 H

ACSF/Rp-cAMPS
B In BLA

100
ACSF
Rp-cAMPS
% Fear Potentiated Startle

80

60
**

40

20

C 0.5 D 8 ACSF
Rp-cAMPS

0.4
6
Shock Reactivity

0.3
Activity

0.2

2
0.1

0.0 0

Fig. 1. LTM as a result of a second conditioning trial requires PKA signaling in the amygdala. A. Schematic of the conditioning and testing procedure. Rats were given two
conditioning trials separated by 24 h, with an infusion of ACSF or Rp-cAMPS into the amygdala 30 min prior to the second conditioning trial. B. Fear-potentiated startle data
(LTM) during the testing session 48 h after conditioning. Rats given the PKA inhibitor showed significantly less fear-potentiated startle compared to controls. C. Disrupting
PKA in the amygdala does not affect activity levels during the second conditioning trial or the response to foot shock (D). ⁄⁄ = p < 0.05; Error bars = ±SEM.
50 R.G. Parsons et al. / Neurobiology of Learning and Memory 136 (2016) 47–53

A Trial 1 Trial 2 Trial 3 Testing

24H 24H 48H

OR
ACSF/Rp-cAMPS
In BLA
B Pre-Trial 2 Infusion C Post-Trial 2 Infusion
150 150
% Fear Potentiated Startle

ACSF

% Fear Potentiated Startle

Rp-cAMPS

100 100

50 50

0 0

ACSF
D 0.5 E 8
Rp-cAMPS

0.4
Shock Reactivity

6
Activity

0.3
4
0.2

2
0.1

0.0 0

Fig. 2. The second conditioning trial does not destabilize the metaplastic state engaged by the first trial. A. Schematic of the conditioning and testing procedure. Rats were
given three conditioning trials separated by 24 h, with an infusion of ACSF or Rp-cAMPS into the amygdala either 30 min prior to, or immediately after, the second trial. Fear-
potentiated startle data during the testing session 48 h after conditioning in rats infused prior to (B) or immediately after (C) the second conditioning trial. Both groups of rats
showed similar levels of fear-potentiated startle during testing. Disrupting PKA in the amygdala does not affect activity levels during the second conditioning session (D) or
the response to foot shock (E). Error bars = ±SEM.

the presence of the light cue and 30 in the absence of the cue. For by 24 h and infused rats with Rp-cAMPS (N = 12) or ACSF (N = 8)
light-startle trials, the startle stimulus was presented 3.2 s after into the BLA 30 min prior to the second conditioning trial. Memory
onset of the visual CS. For startle–alone test trials, the startle stim- was tested 2 days later (Fig. 1A). Rats infused with Rp-cAMPS
uli were presented without the visual CS. Between light-startle tri- showed a significant disruption (t(1, 18) = 2.538, p < 0.05) of LTM
als there were 3 startle alone trials, and the ISI for all trials was as measured by fear potentiated startle (Fig. 1B). The memory
30 s. impairment was robust, as 1-sample t-tests indicated that the
To quantify fear potentiated startle, each rat’s mean startle mean potentiation scores for the Rp-cAMPS group was not differ-
amplitude during the baseline startle session and on the first 5 ent than zero (t(11) = 0.491, p > 0.05). Furthermore, the impair-
light test trials was determined and a percentage change score ment was not due to sensory or motor disruption from infusion
was calculated. We used the first 5 trials of testing because in some of Rp-cAMPS as t-tests revealed no difference between groups
of experiments a decrease in potentiation was observed during the (p > 0.05) in their activity levels prior to the second trial (Fig. 1C)
later test trials indicating some extinction had likely taken place. and no difference (p > 0.05) in reactivity to the foot shock during
Percent change scores were used (i.e., vs. absolute difference the second conditioning trial (Fig. 1D).
scores) because previous observations found that they remain In the following experiments rats were given 3 conditioning tri-
stable across variations in baseline startle amplitude (Walker & als, each spaced by 24 h. In one group of rats Rp-cAMPS (N = 9) or
Davis, 2002). These scores were then analyzed using Student’s t- ACSF (N = 10) was infused into the BLA prior to the second condi-
tests and ANOVA to examine differences between groups. In some tioning trial. In a second group rats (ACSF N = 14; Rp-cAMPS
analyses, we used 1-sample t-tests to assess whether a particular N = 10) were infused after the second conditioning trial. We rea-
group differed from zero. The criterion for significance was soned that if a reconsolidation-like process was engaged during
p < 0.05 (two-tailed). the second conditioning trial then infusing the PKA inhibitor either
prior to or after the second trial would disrupt the memory of the
first trial so that a third conditioning trial would be like a single
3. Results conditioning trial which would not lead to LTM using these param-
eters. In contrast, if the first trial activated a metaplastic state,
To test if PKA signaling was necessary for LTM produced by a which we know can last for several days (Parsons & Davis, 2012),
second conditioning trial, we trained rats with 2 trials separated then reconsolidation disruption should have no effect because
 R.G. Parsons et al. / Neurobiology of Learning and Memory 136 (2016) 47–53
Trial 1 Retrieval
A
24H
ACSF/Rp-cAMPS
In BLA
B
160
ACSF
Rp-cAMPS
% Fear Potentiated Startle
120
80
40
-40
Retrieval
Fig. 3. A single retrieval trial does not destabilize the metaplastic state engaged by the first trial. A. Schematic of the conditioning and testing procedure. Rats were given the
first conditioning trial followed by a single retrieval trial 24 h later. Thirty minutes prior to retrieval rats were infused with ACSF or rp-cAMPS into the amygdala. A second
conditioning trial was given the next day and memory was tested 48 h later. B. Fear-potentiated startle data during the retrieval and testing sessions. There were no
differences between groups during retrieval or testing. Error bars = ±SEM.
the third trial would still be able to trigger LTM from the metaplas-
tic state set up by the first trial. The results were inconsistent with
disruption of reconsolidation interpretation. A two-way ANOVA
with drug and time of infusions as factors showed that there was
no effect of drug (F(1, 43) = 0.001, p > 0.05) or time (F(1, 43)
= 0.001, p > 0.05) and no drug by time interaction (F(1, 43)
= 0.094, p > 0.05). Furthermore, one-sample t-tests comparing both
groups’ fear-potentiated startle scores to a theoretical mean value
of 0% confirmed that rats given either pre-conditioning (Fig. 2B) or
post-conditioning (Fig. 2C) infusions of ACSF or Rp-cAMPS showed
robust LTM during testing (ACSF t(1, 18) = 3.586, p < 0.05; Rp-
cAMPS t(1, 23) = 4.002, p < 0.05). As we observed in the previous
experiment, t-tests showed that the PKA inhibitor had no effect
on activity during baseline (p > 0.05) or reactivity to the shock in
those animals which received pre-conditioning infusions
(p > 0.05) (Fig. 2D and E).
In the final experiment rats were given a single conditioning
trial and then the next day they were given a trial during which
the CS was presented alone (i.e. retrieval trial), prior to which,
the rats were infused with Rp-cAMPS (N = 9) or ACSF (N = 10) into
the BLA (Fig. 3A). T-tests revealed no difference between groups
(p > 0.05) during the retrieval session. One-sample t-tests compar-
ing both groups’ fear-potentiated startle scores to a theoretical
mean value of 0% potentiation confirmed previous findings
(Parsons & Davis, 2012) that a single conditioning trial does not
produce LTM as assessed with this measure. The next day rats
received a second conditioning trial in which the CS was paired
with the shock, and memory was tested 2 days later. A two-way
ANOVA using drug and test session as factors uncovered a signifi-
cant main effect for test session (F(1, 38) = 5.103, p < 0.0.05) indi-
cating that LTM scores increased from the retrieval to LTM test
session. Importantly, there was no main effect of drug (F(1, 38)
= 0.204, p > 0.05) and no drug by test session interaction (F(1, 38)
= 0.589, p > 0.05) (Fig. 3B).
51
Trial 3 Testing
24H 48H
Testing
4. Discussion
The first experiment showed that blocking PKA activity in the
amygdala during the second conditioning trial prevented the abil-
ity of that trial to produce a LTM. It is unlikely this finding is the
result of a sensory or motor impairment, as the two groups did
not differ in their reaction to the shock or in their activity levels
in the 5 min prior to shock. These findings argue that the consoli-
dation of LTM induced by the second conditioning trial requires
PKA activity in the amygdala. However, there are alternative ways
to conceptualize these data. The results of several studies have led
researchers to propose that the function of reconsolidation is to
update or to incorporate new information into a memory trace
(Diaz-Mataix, Ruiz Martinez, Schafe, LeDoux, & Doyere, 2013;
Lee, 2008, 2010; Sara, 2000). Proponents of this idea might argue
that there is new information being added to the memory trace
during the second conditioning trial, so that blocking PKA during
the second trial would be targeting a reconsolidation-like process
resulting in the memory deficit apparent at testing. Experiments
2 and 3 addressed this possibility.
In Experiment 2, we infused a PKA inhibitor in the BLA in two
different groups either 30 min prior to or immediately after the
second conditioning trial. Rats were then given a third condition-
ing trial 24 h after the second conditioning trial and then tested
2 days later. Our reasoning was that if a reconsolidation-like pro-
cess was engaged during the second conditioning trial then infus-
ing the PKA inhibitor either prior to or after the second
conditioning trial would disrupt the incorporation of new informa-
tion learned on trial 2 with that learned on trial 1, and thus the
third trial would be like a single conditioning trial which would
not lead to LTM. In contrast, if the second trial produces LTM in a
manner that does not involve reconsolidation-like updating of trial
1 learning, then disrupting PKA should have no effect because the
third trial would still be able to trigger LTM from the metaplastic
52 R.G. Parsons et al. / Neurobiology of Learning and Memory 136 (2016) 47–53
state set up by the first trial. The results were not consistent with
disruption of reconsolidation interpretation. A PKA inhibitor given
prior to or immediately after the second conditioning trial had no
disruptive effect because all groups showed significant LTM when
they were tested 48 h after the third conditioning trial.
In the final experiment, we tested whether or not blocking PKA
in the amygdala would disrupt the formation of LTM if the second
trial was a simply a retrieval trial with no shock given. This design
is similar to that of many reconsolidation studies where unrein-
forced presentations of the CS often serve as reminder treatments.
During the retrieval trial neither group showed evidence of a fear
memory. This is consistent with our prior findings which showed
that a single conditioning trial does not produce a long-term fear
memory under these conditioning and test conditions (Parsons &
Davis, 2012). Results from the testing session showed that there
was no difference between groups during the testing session, and
that both groups showed significant levels of LTM. Given the
results of the experiments reported here, we think the most parsi-
monious explanation is that the second conditioning trial produces
a LTM the consolidation of which can be blocked by a PKA inhibi-
tor. Importantly, this consolidation process does not appear to alter
the metaplastic state produced by the first conditioning trial
because the deficit in memory consolidation is not apparent if a
third trial is given the next day.
While our results support the notion that LTM produced as a
result of the second conditioning trial does so in a way that is
mechanistically distinct from retrieval-based memory updating, a
couple of issues are worth discussion. First, although PKA signaling
regulates a diverse set of intracellular events from the regulation of
nuclear transcription factors (Dash, Hochner, & Kandel, 1990;
Gonzalez & Montminy, 1989) to the trafficking of AMPA receptors
(Esteban et al., 2003) and modulation of ion channels (Levitan,
1994), it is possible that the mechanism operating during the sec-
ond conditioning trial in our paradigm is retrieval-based and that
non-PKA dependent pathways may be responsible. These include,
but are not limited to, signaling pathways mediated by (Ca2+/
calmodulin-dependent protein kinase II) CaMKII, exchange protein
activated by cAMP (Epac), and the mammalian target of rapamycin
(mTOR), all of which operate independent of PKA and have been
implicated in memory formation (Ma, Abel, & Hernandez, 2009;
Parsons, Gafford, & Helmstetter, 2006; Silva, Paylor, Wehner, &
Tonegawa, 1992). However, we think it is unlikely that retrieval-
based updating of memory in our paradigm is independent of
PKA given that (1) we showed that PKA inhibition given before trial
2 disrupts LTM formation if it is not followed by a third condition-
ing trial, and (2) there is considerable evidence indicating that PKA
is involved in memory reconsolidation using a variety of other
paradigms (Arguello et al., 2014; Koh & Bernstein, 2003; Sanchez,
Quinn, Torregrossa, & Taylor, 2010; Tronson, Wiseman, Olausson,
& Taylor, 2006).
Another important consideration has to do with the question of
the extent to which retrieval induced impairments in memory
undo original learning. There are a number of ways to assess this
question including testing if memory impairments recover with
time, reinstate with reminder treatments, or show faster reacquisi-
tion upon reconditioning (i.e. savings). Some studies have shown
that retrieval-induced memory impairments reverse with time
(Lattal & Abel, 2004), while others have demonstrated long lasting
deficits (Duvarci & Nader, 2004). As this issue pertains to our
experiments, it is possible that the third conditioning trial is able
to support LTM because the PKA inhibitor given around the time
of the second conditioning trial results in only a partial disruption
of memory for the first conditioning trial. Thus, the intact memory
in our experiments might reflect savings from the first condition-
ing trial. We cannot rule out this possibility, but two pieces of evi-
dence argue against this interpretation. First, when given only two
conditioning trials, rats given the PKA inhibitor show a nearly com-
plete impairment exhibiting only 5% potentiation on average (see
Fig. 1B), a value that did not statistically differ from zero. If the sec-
ond trial engages a retrieval-dependent process, and that disrup-
tion of PKA only partially blocks this process, than you would
expect the Rp-cAMPS treated rats to only show a partial impair-
ment in this experiment. Likewise, in the two experiments where
a third trial was given, the rats that received the PKA inhibitor
showed nearly identical levels of LTM as controls. Again, if the sec-
ond conditioning trial is activating a retrieval-dependent process
that is only partially affected by PKA inhibition, than you would
expect that rats treated with Rp-cAMPS would still exhibit poorer
memory than controls. It should be noted however, researchers
have been struggling with questions regarding the nature of amne-
sia for many decades (Miller & Matzel, 2006; Squire, 2006) and our
findings certainly do not resolve these questions.
An important next step will be to determine the molecular and
cellular mechanisms supporting the metaplastic process engaged
after the first conditioning trial, and if they differ from the mecha-
nisms that support LTM formation after the second trial. Thus,
while PKA is involved in both phases of our conditioning paradigm,
the specific mechanisms supporting metaplastic priming of learn-
ing after the first conditioning trial and the production of LTM after
the second might diverge. Answering this question will require tar-
geting more specific neural processes, but the advantage our con-
ditioning paradigm has versus more traditional ones is that we
can target the mechanisms underlying either trial separately given
the long interval between trials.
Metaplasticity is thought to serve a variety of possible functions
(Hulme et al., 2013), the one most germane to our findings is that
this process is thought to prepare neural circuits for learning and
memory. The next question to answer then is what form of meta-
plasticity is engaged as a result of the first conditioning trial. One
attractive possibility is that the first conditioning trial results in
an increase neuronal excitability in the amygdala which permits
LTM formation if the second trial is presented within an appropri-
ate time window. Learning-related changes in excitability have
been observed in several studies (reviewed in Oh & Disterhoft,
2015; Zhang & Linden, 2003), including recent studies which found
that fear conditioning increases excitability in a portion of neurons
in the basolateral amygdala (Sehgal, Ehlers, & Moyer, 2014; Yiu
et al., 2014). Furthermore, one study (Moyer, Thompson, &
Disterhoft, 1996) showed that learning-related reductions in the
after hyperpolarization lasted about 7 days, similar to the extent
of time when a second trial produces LTM in our paradigm
(Parsons & Davis, 2012). However, it is also possible that rather
than neuron-wide changes in excitability, the metaplasticity
induced by the first trial is compartmentalized to specific synapses,
as has shown to be the case for some types of metaplasticity
(reviewed in Hulme et al., 2013). Our previous work (Parsons &
Davis, 2012) showed that the first conditioning trial only primed
learning if the same cue predicted shock during the second trial,
indicating a level of specificity at the neural level.
Taken together, the findings we described here draw a distinc-
tion between the production of LTM from a metaplastic state and
the retrieval-based updating of memory. How the brain acquires
information is a complex process, and we will only make progress
understanding these complexities by developing additional
approaches in the laboratory for the study of learning and memory.
Our work complements a growing line of research focused on
understanding how prior experience can affect how information
is acquired and how memories can be modified (McKenzie &
Eichenbaum, 2011; Viola, Ballarini, Martinez, & Moncada, 2014).
This approach is critical not only for fully understanding how
organisms acquire information, but also identifying the predispos-
ing factors that can affect whether something is remembered,
 R.G. Parsons et al. / Neurobiology of Learning and Memory 136 (2016) 47–53 53

which has potential translational value for both people with mem- McKenzie, S., & Eichenbaum, H. (2011). Consolidation and reconsolidation: Two
lives of memories? Neuron, 71, 224–233.
ory disorders and for healthy populations as well.
Miller, R. R., & Matzel, L. D. (2006). Retrieval failure versus memory loss in
experimental amnesia: Definitions and processes. Learning & Memory, 13,
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