Novelty exposure overcomes foot shock-induced spatial-memory impairment by processes of synaptic-tagging in rats.
ABSTRACT Novelty processing can transform short-term into long-term memory. We propose that this memory-reinforcing effect of novelty could be explained by mechanisms outlined in the "synaptic tagging hypothesis." Initial short-term memory is sustained by a transient plasticity change at activated synapses and sets synaptic tags. These tags are later able to capture and process the plasticity-related proteins (PRPs), which are required to transform a short-term synaptic change into a long-term one. Novelty is involved in inducing the synthesis of PRPs [Moncada D, et al. (2011) Proc Natl Acad Sci USA 108:12937-12936], which are then captured by the tagged synapses, consolidating memory. In contrast to novelty, stress can impair learning, memory, and synaptic plasticity. Here, we address questions as to whether novelty-induced PRPs are able to prevent the loss of memory caused by stress and if the latter would not interact with the tag-setting process. We used water-maze (WM) training as a spatial learning paradigm to test our hypothesis. Stress was induced by a strong foot shock (FS; 5 × 1 mA, 2 s) applied 5 min after WM training. Our data show that FS reduced long-term but not short-term memory in the WM paradigm. This negative effect on memory consolidation was time- and training-dependent. Interestingly, novelty exposure prevented the stress-induced memory loss of the spatial task and increased BDNF and Arc expression. This rescuing effect was blocked by anisomycin, suggesting that WM-tagged synapses were not reset by FS and were thus able to capture the novelty-induced PRPs, re-establishing FS-impaired long-term memory.
Article: Chapter 7 ‘Synaptic tagging’ and ‘cross-tagging’ and related associative reinforcement processes of functional plasticity as the cellular basis for memory formation[show abstract] [hide abstract]
ABSTRACT: We focus on new properties of cellular and network processes of memory formation involving ‘synaptic tagging’ and ‘cross-tagging’ during long-term potentiation (LTP) and long-term depression (LTD) as well as associative heterosynaptic interactions, the latter of which are characterized by a time-window of about 1 h. About 20 years ago we showed for the first time that the maintenance of LTP, like memory storage, depends on intact protein synthesis and thus consists of at least two temporal phases. Later, similar properties for LTD were shown by our own and other laboratories. Here we describe the requirements for the induction of the transient early-LTP/LTD and of the protein synthesis-dependent late-LTP/LTD. Late-LTP/LTD depend on the associative activation of heterosynaptic inputs, i.e. the synergistic activation of glutamatergic and modulatory reinforcing inputs within specific, effective time-windows during their induction. The induction of late-LTP/LTD is characterized by novel, late-associative properties such as ‘synaptic tagging’, ‘cross-tagging’ and ‘late-associative reinforcement’. All of these phenomena require the associative setting of synaptic tags as well as the availability of plasticity-related proteins (PRPs) and they are restricted to functional dendritic compartments, in general. ‘Synaptic tagging’ guarantees input specificity, ‘cross-tagging’ determines the interaction between LTP and LTD in a neuron, and thus both are required for the specific processing of afferent signals for the establishment of late-LTP/LTD. ‘Late-associative reinforcement’ describes a process where early-LTP/LTD by the co-activation of modulatory inputs can be transformed into late-LTP/LTD in activated synapses where a tag is set. Recent experiments in the freely moving rat revealed a number of modulatory brain structures involved in the transformation of early-plasticity events into long-lasting ones. Further to this, we have characterized time-windows and activation patterns to be effective in the reinforcement process. Studies using a combined electrophysiological and behavioural approach revealed the physiological relevance of these reinforcement processes, which is also supported by fMRI studies in humans, which led to the hypothesis outlined here on cellular and system memory-formation by late-associative heterosynaptic interactions at the cellular level during functional plasticity events.Progress in Brain Research.
[show abstract] [hide abstract]
ABSTRACT: The basolateral region of the amygdala (BLA) plays a crucial role in making significant experiences memorable. There is extensive evidence that stress hormones and other neuromodulatory systems activated by arousing training experiences converge in regulating noradrenaline-receptor activity within the BLA. Such activation of the BLA modulates memory consolidation via BLA projections to many brain regions involved in consolidating lasting memory, including the hippocampus, caudate nucleus, nucleus basalis and cortex. Investigation of the involvement of BLA projections to other brain regions is essential for understanding influences of the amygdala on different aspects and forms of memory.Trends in Neurosciences 10/2002; 25(9):456. · 14.23 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: The antibiotic, puromycin, caused loss of memory of avoidance discrimination learning in mice when injected intracerebrally. Bilateral injections of puromycin involving the hippocampi and adjacent temporal cortices caused loss of short-term memory; consistent loss of longer-term memory required injections involving, in addition, most of the remaining cortices. Spread of the effective memory trace from the temporal-hippocampal areas to wide areas of the cortices appears to require 3 to 6 days, depending upon the individual animal. Recent reversal learning was lost while longer-term initial learning was retained after bilateral injections into the hippocampal-temporal areas.Science 08/1963; 141(3575):57-9. · 31.20 Impact Factor
Novelty exposure overcomes foot shock-induced
spatial-memory impairment by processes
of synaptic-tagging in rats
William Almaguer-Meliana, Jorge Bergado-Rosadoa, Nancy Pavón-Fuentesb, Esteban Alberti-Amadorc,
Daymara Mercerón-Martínezd, and Julietta U. Freye,f,1
aDepartamento de Neurofisiología Experimental,bDepartamento de Neuroinmunología, andcDepartamento de Neurobiología, Centro Internacional de
Restauración Neurológica, Havana 11300, Cuba;dDpto de Neuroestimulación, Centro de Neurociencias de Cuba, Havana 11300, Cuba;eMedical Faculty,
Otto-von-Guericke University, Magdeburg D-39120, Germany; andfDepartment of Neurophysiology, Leibniz-Institute for Neurobiology, Magdeburg
Edited by Ivan Izquierdo, Centro de Memoria, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil, and approved December 5, 2011
(received for review August 30, 2011)
We propose that this memory-reinforcing effect of novelty could be
explained by mechanisms outlined in the “synaptic tagging hypoth-
esis.” Initial short-term memory is sustained by a transient plasticity
change at activated synapses and sets synaptic tags. These tags are
which are required to transform a short-term synaptic change into
[Moncada D, et al. (2011) Proc Natl Acad Sci USA 108:12937–12936],
ory. In contrast to novelty, stress can impair learning, memory, and
inducedPRPs are able topreventthelossofmemory causedbystress
and if the latter would not interact with the tag-setting process. We
our hypothesis. Stress was induced by a strong foot shock (FS; 5 × 1
mA, 2 s) applied 5 min after WM training. Our data show that FS
reduced long-term but not short-term memory in the WM paradigm.
dependent. Interestingly, novelty exposure prevented the stress-in-
duced memory loss of the spatial task and increased BDNF and Arc
expression. This rescuing effect was blocked by anisomycin, suggest-
ing that WM-tagged synapses were not reset by FS and were thus
memory rescue|memory reinforcement|novelty exploration
integrate various stimuli. Memory formation includes widespread
brain regions and consists of sequential and parallel events that
define memory phases (1–4). Underlying mechanisms are based on
the activation of preexisting proteins during an early, labile phase
(short-term memory, STM) and the synthesis of new proteins for
a late, stable phase (long-term memory, LTM) (1, 5). These cellular
processes can be activated by a single experience or by two in-
be involved in long-term potentiation (LTP), a model of synaptic
plasticity and cellular memory. Thus, motivation and emotion can
late-LTP (L-LTP), a phenomenon known as behavioral LTP-re-
inforcement (3). Novelty exploration in an open field (OF) can also
effectively reinforce LTP (6). Behavioral LTP-reinforcement is
protein synthesis-dependent (7) and basolateral amygdala-stimula-
tion mimics the reinforcing effects of distinct behavioral stimuli (8,
9). LTP reinforcement by behavioral stimuli has been explained by
the synaptic-tagginghypothesis (STH) (3).According to this theory,
efficacy, but can also set tags at activated synapses, which can
earning and memory are important to survive in a changing
environment and use a limited, predetermined neural subset to
LTP. At the behavioral level, it was shown that STM can be pro-
longed into LTM if, shortly before or after training, animals ex-
plored a novel environment (10, 11). Thisnovelty effect on memory
propose that training induces “learning tags” that capture the pro-
teins required for memory consolidation, and novelty exploration
induces the required PRP-synthesis.
Recently, we investigated how different combinations of LTP-
and long-term depression (LTD)-inducing paradigms interact at
the same synapses (12). After LTP-depotentiation, using low-fre-
temporarily refractory to novel LTP-induction, obviously compet-
ing with LTD-processes. If a behavioral reinforcing stimulus was
applied during that time, no LTP-reinforcement was seen, sug-
gesting that depotentiation also resets synaptic LTP-tags (13), al-
though PRPs were made available by the behavioral stimulus. If E-
LTP was induced beyond the refractory period, the formerly ap-
plied behavioral stimulus was able to reinforce E-LTP into L-LTP.
affective modulation can influence the final outcome (12).
Here we expanded our work to the organism level. We in-
vestigated if memory can be recovered after being disrupted by
foot shock (FS), using different temporal patterns of a combi-
nation of training, FS, and exploration. The cellular mechanisms
involved were also studied blocking protein synthesis and eval-
uating the expression of PRP-associated gene candidates.
Water-Maze Training. First, we measured the 24-h retention of the
spatial memory trace after four training trials in the water maze
(WM). Fig. 1A shows the performance during training and re-
tention (control, n = 24). Average latency during the first trial in
the retention test was 33.57 ± 4.99 s, a value that appears to be
convenient to demonstrate effects of memory-impairing or -fa-
cilitating paradigms. The animals also learned during the second
session 24 h later, as can be seen in the figure. Therefore, to
evaluate retention we used only the latencies in the first trial of
the second session to evaluate retention (filled circle in Fig. 1A)
under different conditions and manipulations.
Author contributions: W.A.-M., J.B.-R., and J.U.F. designed research; W.A.-M., J.B.-R., N.P.-F.,
E.A.-A., and D.M.-M. performed research; W.A.-M. and J.B.-R. analyzed data; and W.A.-M.,
J.B.-R., and J.U.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 17, 2012
| vol. 109
| no. 3
FS Impairs Retention in the WM in a Time-Dependent Manner. Next,
we tested the effects of an electrical FS, applied 5 min after
training, to interfere with memory retention at 24 h (Fig. 1) (trial
2.1; WM+FS, n = 15).
Fig. 1B shows that FS impaired memory retention at 24 h, as
expressed by the higher escape latency. The memory-impairing
effect is the same when FS was applied after a 30-s rest period in
the punishment box, or when it was applied immediately after
entering the box. The ANOVA (*F = 4.32) showed differences
among groups. The post hoc Duncan’s test showed differences
between the control animals and both FS groups, but no differ-
ences between the two FS groups.
The impairing effects of FS were dependent on the timing be-
retention 5 min after FS (MWM+FS+5 min, n = 10), their per-
formance was similar to the control group, as shown in Fig. 2A
(WM+10 min, n = 10; Student t test, t = −1.03). Application
affect the escape latency (Student t test, t = −1.43), suggesting that
the memory trace was not labile at that time.
Finally, when WM training was prolonged to eight trials (8
Trials+FS, n = 12) (Fig. 2C), no impairing effects of FS on
memory were observed. There were no significant differences
when latencies were compared with the control group (8 Trials,
n = 10; Student t test, t = 1.75).
Novelty Exploration Prevents the Memory Impairment by FS. We
then assessed if novelty exploration was able to re-establish mem-
ory,which was impairedby FS.Exploration ofa novel environment
(for 3 min) 15 min before or after WM training reduced the escape
latency to control levels in the first trial of the retention test, sug-
gesting a recovery of the FS-impaired memory (Fig. 3A). Latencies
to escape from water did not significantly differ between control
group and the FS groups that explored the OF in temporal vicinity
to training (15 min after, WM+FS+OF, n = 15; or 15 min before,
only the FS. The ability of OF exploration to protect or recover
memory was time-dependent. OF exploration was unable to re-
received only the FS, but was significantly different from control
animals and both OF exploring groups with 15-min intervals (one-
way ANOVA, *F = 3.22, followed by a post hoc Duncan’s test).
The exploration of a novel OF was able to promote memory
retention also in the absence of the FS. Fig. 3B shows that OF
exploration reduced the escape latency evaluated 4 d after
training (WM+4 d, n = 10 vs. WM+OF+4 d, n = 12). Note,
however, that retention at 24 h did not differ in OF-exposed and
control animals (Control vs. WM+OF), suggesting that OF ex-
ploration prolonged the memory trace (one-way ANOVA, *F =
3.09, post hoc Duncan’s test).
To confirm that the effect was caused by novelty exploration in
the OF scenery, we habituated a group of animals in the OF ap-
paratus 24 h before the training. Animals were exposed four times
to 3-min OF exploration with 5-min intertrial intervals (OF+24 h
1.1 1.2 1.3 1.4 2.1 2.2 2.3 2.4
Control, N= 24
produce appreciable learning, which was maintained up to 24 h after train-
ing. Data presented show the averaged escape latency mean and SEM of
each trial per day. Trials are identified by two numbers: training day (1 or 2)
and trial (1–4). Trial 2.1 (filled circle) was used as a retrieval test in all of the
following experiments. (B) WM training and FS. FS application 5 min after the
fourth trial interfered with memory at the second day. Two FS groups were
tested, one (WM+FS, n = 15) in which the animals remained in the box 30 s
before FS application, and another in which the animals received FS imme-
diately after entering the box (WM+FS no context, n = 8) to evaluate the
possible influence of contextual learning on the FS effect. Escape latencies
increased in both FS groups in the retrieval test 24 h later. (Upper) The ex-
perimental design for each group is presented (shaded boxes: training; R:
time for retrieval, FS: time for FS). Means and SEM are shown. F values from
ANOVA are given, and the post hoc Duncan’s test results indicated by low-
ercase letters (a and b).
WM training and the effect of FS. (A) WM training was able to
5 10min 5 10min5 10min
FS FS FS
8 Trials8 Trials+FS
because the escape latency was similar to the control group when the retrieval test was performed 5 min later. Data for the group tested at 10 min after
finishing the training (WM+10 min) and animals that received FS 5 min after the training and tested 5 min thereafter (WM+FS+5 min) are presented. (B) If
application of FS was delayed for 5 h after finishing the fourth trial, no interference with memory was observed because the escape latency was similar to the
control group in the retrieval test 24 h later. The control group (Control) and animals with FS 5 h after the training (WM+5 h+FS) are shown. (C) FS after the
eighth trial. FS application 5 min after eight training trials did not interfere with memory because the escape latency was similar to the eight-trial group in the
retrieval test 24 h later. The control eight-trial group (8 Trials) and animals that received FS 5 min after the training (8WM+FS) are shown. (Upper) The
experimental design for each group is presented (shaded boxes: training; R: time for retrieval, FS: time for FS). Means and SEM are shown. F values from
ANOVA are given, NS, no significant differences.
Temporal and training-dependent constrains to FS effects on memory. (A) FS application 5 min after the fourth trial did not interfere with STM
| www.pnas.org/cgi/doi/10.1073/pnas.1114198109Almaguer-Melian et al.
+WM+FS+OF, n = 10). Our data show (Fig. 3C) that the escape
latency was significantly higher compared with the control group,
indicating that the protective effect of OF exploration on memory
disruption by FS was lost when the novelty value of the arena was
reduced by previous habituation (one-way ANOVA, *F = 3.72,
post hoc Duncan’s test). Moreover, if the animals were habituated
to one OF arena before training and placed to explore a new OF
arena after training and FS (OF+24 h+WM+FS+novel OF,
n = 10), the animals showed an escape latency comparable to the
control group (Fig. 3C).
Novelty Exploration Effect on Memory Was Protein Synthesis-
Dependent. We also addressed the question if the effect of nov-
elty exploration in the OF on memory was protein synthesis-de-
pendent. Fig. 4A shows that anisomycin, a reversible blocker of
protein synthesis, injected 15 min after WM training (WM+Aniso,
n = 8) impairs retention at 24 h. In the same way, treatment after
training, FS, and novelty exploration (WM+FS+OF+Aniso, n =
13) abolishes therecovery ofmemory by OF, supporting thetheory
that the effect of novelty exploration on memory was mediated by
protein synthesis. Both groups differed significantly from the saline
control (NaCl, n = 10; ANOVA *F = 12.78).
Novelty Exploration Induces Gene Expression. Finally, we studied
the possible participation of Arc and BDNF gene expression, as
potential molecular players in the mechanisms of memory rescue
by OF exploration. The findings are shown in Fig. 4B. Arc and
BDNF gene expression was increased in the hippocampus of all
of the animals that performed a spontaneous exploration in the
OF for 3 min (OF, n = 6) compared with naive animals not
exposed to OF (Naive, n = 6).
Exploration of a novel environment in OF is able to convert
a STM into a LTM trace (11). We have now studied if it could
also recover the spatial-memory impairment caused by FS in
a spatial-memory model. We show that four trials in the WM
were sufficient for rats to learn to find the platform during
training. This LTM trace was preserved for 24 h. FS impaired the
consolidation of spatial LTM in the WM but not STM. This
FS FS FS
24h24h 24h5h5h 5h
15min15min 15min4 days 4 days4 days
R R R
5 15min 5 15min5 15min-24h-24h -24h24h 24h 24h
new-OF new-OF new-OF
spatial training in the WM was able to protect memory from the FS-impairing effects because both groups (WM+FS+OF; OF+WM+FS) were similar to the
control group, but different from the WM+FS group. This effect was time-dependent because OF 5 h later (WM+FS+5 h+OF) did not benefit memory recovery.
(B) Spatial memory could be stabilized or prolonged for up to 4 d (WM+OF+4 days; statistically different from the WM+4 days group; and similar to control
group) by novelty exploration. (C) Previous habituation to OF did not produce a memory recovery effect when the animals were put into the same OF 15 min
after the training (OF+24 h+WM+FS+OF). However, if the second OF was changed by a new one, the memory-preserving effect of exploration was re-
established (OF+24 h+WM+FS+novel OF). (Right) The experimental design for each group is presented (shaded boxes: training; R: time for retrieval, FS: time
of FS). Means and SEM are shown. F-values from ANOVA are given; lowercase letters identify similar and different values after the Duncan’s test.
Effect of exploration of novel environment on memory affected by FS. (A) Novelty scenery exploration in the OF 10 min after FS or 15 min before
Almaguer-Melian et al.PNAS
| January 17, 2012
| vol. 109
| no. 3
impairment was dependent on the timing between WM training
and FS, as well as the training intensity. When memory was
measured 5 min after FS, the animals performed similar to the
control group; however, LTM was prevented at 24 h. FS appli-
cation 5 h after training did not affect LTM measured 24 after
training. Similarly, FS application after a more intense training
(eight trials) was not able to block memory at 24 h. FS is
a stressful event, and stress impairs spatial memory and related
cellular processes, such as LTP (14, 15). An alternative expla-
nation would be that impairment was produced by a concurrent
memory process of contextual fear-conditioning initiated by ex-
posure and punishment in the FS box. Rosen et al. (16) have
shown that applying FS immediately after placing the animals in
the box prevents contextual fear conditioning. Using this ap-
proach, we found that our results showed no significant change in
the impairing effect of FS, reducing the likelihood of the mem-
ory-competition hypothesis and favoring the stressful effects of
FS as the causal factor for the spatial-memory impairment.
Our results agree with the consolidation theory (17). FS
shortly after training interfered with consolidation of LTM, al-
though it did not affect short-term storage mechanisms. When
FS was applied hours later, LTM was already established and not
affected by FS anymore (18, 19). Prolonged training (eight trials)
created a stronger memory trace. The longer training probably
allowed some consolidation during the prolonged time of train-
ing, finally being refractory to FS. It does not seem likely that FS
affected retrieval 24 h later by mechanisms different from
impairing memory consolidation. FS can directly affect retrieval
of a consolidated memory trace only in a short period (less than
30 min) but not after 4 h (20).
Our central question was whether exposing the animal to
a novel environment, before or after training and FS, was able to
prevent the memory-impairing effect of FS. Novelty exploration
in an OF is effective to enhance memory and hippocampal LTP
(6, 10, 11). We now show that novelty exploration was also able
to protect memory from FS-induced impairments. Exploration of
a novel environment 15 min before or after training reduced the
escape latency to control levels in the first trial of the retention
test, despite the application of FS after training. Furthermore,
the main factor modulating memory consolidation seemed to be
novelty and not simply exploration or movement, because explo-
protective effect. If the familiar OF box was exchanged by a dif-
ferent one (color, ground, cues), the protective effect reappeared.
Interestingly, novelty exploration did not improve memory per-
formance at 24 h in the retention test (in animals without FS),
suggesting that it modulates the mechanisms involved in the sta-
bilization of the memory trace, but not those related to the acqui-
sition or encoding. Novelty can increase dopamine activity in the
hippocampus and the medial prefrontal cortex (21). It has been
proposed that the ventral tegmental area-hippocampus projection
acts as a novelty-detection mechanism to reinforce the storage of
relevant spatial information within the hippocampus (22, 23). We
have confirmed that memory consolidation in the WM is protein
synthesis-dependent, and provided evidence that the effect of
novelty exploration on memory consolidation was protein synthe-
sis-dependent because application of anisomycin disrupted the
protective effect of novelty.
Hippocampal Arc and BDNF gene expression was also in-
creased in animals that spontaneously explored a novel OF for 3
min, which is supported by previous data of increased Arc ex-
pression in CA1 (24). Furthermore, Arc-knockout mice failed to
develop long-term plasticity and LTM for implicit and explicit
learning tasks, despite intact STM, suggesting that Arc is critical
for the consolidation of an enduring plasticity and memory
storage (25). Additionally, spatial learning increased Arc-mRNA
levels in the hippocampus and entorhinal cortex (26), but its
inhibition by intrahippocampal infusions of antisense Arc-
mRNA impaired L-LTP without affecting its induction, and
impaired consolidation of spatial LTM without affecting acqui-
sition or STM (27). Thus, Arc seems to play a basic function in
the stabilization of activity-dependent hippocampal plasticity
This increase in Arc-expression could be a consequence of an
increased BDNF production. It has been demonstrated that
BDNF can trigger Arc-expression via the Ras-Raf-MAPK sig-
naling pathway through ERK (29, 30). In the dorsal hippocam-
pus, BDNF-mRNA expression can be induced by novelty
exploration triggered by NMDA-receptor-dependent mecha-
nisms (31), likely in the same neuronal population involved in
spatial memory acquisition. Similarly, some of the known effects
of BDNF on LTP (32, 33) and memory stabilization (34–36)
could require Arc activation. It was also reported that BDNF
infusion can prevent LTP and spatial memory impairments in the
WM provoked by chronic immobilization stress (37). We pro-
pose that the protective effect of novelty exploration on memory
could be mediated by an increased BDNF expression and the
subsequent expression of Arc and other PRPs, which are cap-
tured by tagged synapses. Alternatively, the negative effects of
FS on retention can be the result of a reduction in the BDNF
expression induced by WM training (38, 39).
Our data can be explained in terms of the STH (3, 40, 41). At
the cellular level, it was shown that electrical or behavioral ac-
tivation of neuromodulatory structures, such as the basolateral
amygdala (8, 42) or the medial septum (43), within an effective
associative time window can reinforce E-LTP into L-LTP. It has
5 15min 5 15min
Naive, N= 6
OF, N= 6
normalized to b-actin (%)
(A) Application of anisomycin immediately after exploration the OF (WM+FS+
OF+Aniso) prevented the memory-preserving effect of OF exploration com-
pared with controls (NaCl). When anisomycin was applied 15 min after training
in theabsence of FSorOFa similar result was obtained,confirmingthe amnesic
effect of protein synthesis inhibition. (Right) The experimental design for each
group is presented (abbreviations as in Fig. 3). Means and SEM are shown. F-
values from ANOVA are given; lowercase letters identify similar and different
values after the Duncan’s test. (B) Semiquantitative RT-PCR allowed the iden-
tification and verificationof anincrease ingene expressionfor Arcand BDNF in
those animals that were exposed to novelty scenery for 3-min spontaneous
for explorer animals (OF) for three genes are shown: Arc and BDNF as imme-
diate-early genes, and β-actin as control.
Protein synthesis-dependency and gene expression by OF exploration.
| www.pnas.org/cgi/doi/10.1073/pnas.1114198109Almaguer-Melian et al.
been demonstrated that all of these forms of synaptic memory
consolidation require PRP-synthesis (7, 8, 43). We have sug-
gested that LTP reinforcement can also be explained by the STH
(3); that is, E-LTP induction sets synaptic tags at glutamatergic
inputs, which can capture PRPs synthesized by activation of
neuromodulatory inputs within a specific time window, resulting
in L-LTP. WM training induces plasticity changes in neural cir-
cuits (involving the hippocampus), which become consolidated
when PRPs are captured at tagged synapses. Novelty by itself can
activate cellular processes similar to the ones involved in WM
learning, donating PRPs synthesized under novelty exploration
(44). This coactivation of neuromodulatory systems is only ef-
fective if it occurs in a short temporal frame on the same neu-
ronal population bearing active synaptic tags. FS disrupted WM-
LTM consolidation, which could be rescued by subsequent OF,
suggesting that FS interfered with molecular cascades re-
sponsible for the activation of gene transduction and translation,
but not with tag-setting.
Memory formation, like LTP, is a time-dependent, multiphasic
process (17, 45) sustained by sequential and parallel mechanisms
(1, 2, 46). Thus, one can block STM without affecting LTM (2).
To shed light into these processes, we have recently investigated
whether LTP can be rescued from depotentiation by amygdala
stimulation or by behavioral reinforcement (12). We hypothe-
sized, that if tags survive depotentiation, and PRPs can be do-
nated by amygdala activation or behavioral stimuli, the
potentiated state could be recovered. However, we could not
restore LTP at depotentiated synapses, supporting our hypoth-
esis that depotentiation resets synaptic tags (13). In the present
study however, we have shown that FS does not reset tags. WM
training induced a limited burst of PRP synthesis, enough to
maintain memory for up to 24 h. However, the result of the
whole process was fragile or suboptimal and was disrupted by FS.
WM-induced synaptic tags, however, survived and remained able
to capture PRPs induced by novelty. When animals explore
a novel environment, an increased dopamine release in the
hippocampus has been observed (47), with several potential
effects [e.g., affecting Arc expression (48) and activating protein
synthesis via PKA-dependent mechanisms (49–51)]. PRPs, trig-
gered by dopamine, could then be captured by “surviving” tags.
In addition, one has to keep in mind that memory consolidation
involves not only the hippocampus, but also other brain struc-
tures, such as the prefrontal cortex. Interestingly, novelty also
raises dopamine in the prefrontal cortex (21).
Our results show that memory depends on complex inter-
actions between cellular events modulated by inputs of different
origin and neurochemical signature. Understanding these inter-
actions can be an important contribution to develop methods of
reinforcing and improving memory in persons affected by de-
mentia and other memory-impairing conditions.
Materials and Methods
Two-month-old (250–300 g) male Wistar rats were used. The animals were
obtained from professional breeders (CENPALAB) and housed in translucent
plastic cages (five animals per cage) under controlled environmental con-
ditions (23 °C, 50% relative humidity, 12-h light-dark cycle), with free access
to water and food (Rat Chow; CENPALAB) throughout the experiment. All
efforts were made to minimize the number of animals used and their suf-
fering. The experimental protocols for this study followed the National
Guidelines for the Care and Use of Laboratory Animals, Cuba, and were
approved by the institutional Bioethics Committee of Centro Internacional
de Restauración Neurológica (CIREN), Cuba.
Behavioral Training. The animals were trained in the WM for four consecutive
trials, during which they could search (60 s maximum) for a hidden platform
training (10 min, 24 h, or 4 d). This weak version of a WM protocol was used
with the intention of creating a labile memory trace (52), which could be
interfered by FS. The apparatus consisted of a circular swimming pool
(diameter of 1.50 m, filled with water at 22 °C) marked by four virtual
equispaced points named N, S, E, and W, respectively. Animals were released
from one of these positions each time in a previously selected random se-
quence (S, W, N, and E). The start point for retrieval was W. In some initial
experiments, we used an eight-trial protocol with a different sequence of
starting points; at the retrieval test the release position was E. The behavior
of the animals was captured by a television camera connected to a personal
computer. The data were collected and processed using the SMART program
(Panlab, v 2.0). Several behavioral variables were measured (velocity, path
length, and time at walls) but escape latency (in seconds) was finally selected
as a reliable measure of learning and memory.
Blocking Memory by FS. To interfere with the memory process we applied an
electrical FS, which consisted of five shocks at 10-s intervals (1 mA for 2 s). The
animals were placed in a 25 × 30-cm training cage (Coulbourn Instruments)
for 30 s and then the FS was applied. After the FS, the animals were placed
back into their home cages.
Preserving Memory by Spontaneous Exploration in Novelty Scenery. Ten
blue walls and floor), which was a completely novel environment for them. To
evaluate the effect of novelty, some animals were previously habituated to the
OF by placing them into the arena for four times (5 min between trials) or
placed in an alternative OF of the same dimensions and shape but different in
the color of the walls and floor (brown and white, respectively) and printed
Times New Roman capitals (M, O, X, and Z, size 650 points in black) attached to
each of the walls.
Surgery. A group of animals was implanted with bilateral intrahippocampal
cannulas to allow the injection of substances. The animals were anes-
thetized with chloral hydrate (400 mg/kg, i.p.) and fixed in a stereotaxic
frame (David Kopf). Bilaterally, guide cannulas were placed into the hip-
pocampus (anteroposterior = −4.0; mediolateral = ±4.0, and dorsoventral =
−3.0 mm from Bregma) (10). Additionally, three miniscrews were secured
on the skull and the implant was covered with dental acrylic. Water and
food remained ad libitum through all of the experiments. At the end of this
experiment, animals were killed and the brains processed for conventional
histology to confirm the location of the cannulas. Only data from animals
with a correct cannula position were analyzed. Two animals were rejected
for this reason.
Protein Synthesis Inhibition. To study the requirement of new protein syn-
thesis for memory rescue by novelty exploration, anisomycin was applied
shortly after OF (Sigma) to a group of cannulated rats. Eighty micrograms of
anisomycin were bilaterally injected into the CA1 region (0.8 μL each site)
dissolved in HCl, diluted in saline solution, and adjusted to a pH of 7.4 with
Arc and BDNF Expression by Novelty Scenery Exploration. Gene-expression
assays were performed for Arc and BDNF using β-actin as control. Following
decapitation (30 min after OF in the case of exploring animals), hippocampi
were quickly dissected and total RNA from the dorsal hippocampus was sepa-
RNA and 0.5 μg oligo(dT) (Invitrogen) were mixed. The mixture was heated to
70 °C for 10 min. First-strand buffer, 0.1 M DTT, and 25 mM dNTPs were added
reverse-transcriptase was added and the mixture was incubated at 42 °C for 50
min. The reaction was inactivated by heating at 75 °C for 15 min. PCR reactions
were carried out using 2 μL ofcDNA mixed with 25mM dNTPs,50 pMol of each
conditions were: 94 °C for 3 min; 40 cycles for 1 min at 94 °C, annealing tem-
perature for 1 min, 72 °C for 1 min and 72 °C for 5 min (for β-actin and BDNF),
1 min at 60 °C for Arc.
the amplified products were as follows: BDNF, forward and reverse primer
sequences (5′ to 3′): forward: TTGGCCTACCCAGCTG TGCGGAC/reverse: CTC-
TTCGATCACGTGCTCAAAAGTG (annealing temperature 60 °C, product length
(60 °C, 472 bp); β-actin, ATTTGGCACCACACTTTCTACA/TCACGCACGATTTCCC-
TCTCAG (51 °C, 379 bp). β-Actin was used as an endogenous control. DNA
products were electrophoresed on 1.2% agarose gels at 100 mV and visualized
with ethidium bromide. PCR products were separated and documented by
digital imaging (29).
Almaguer-Melian et al.PNAS
| January 17, 2012
| vol. 109
| no. 3
A base pair-long product for Arc and BDNF were amplified by semi-
quantitative PCR with specific primers.
For the semiquantitative analysis, we used the free online program ImageJ
(Version 1.44; Wayne Rasband, National Institute of Health; http://imagej.nih.
gov/ij). For this analysis we first subtracted the background activity to the
target band, and then normalized using β-actin as reference.
Experimental Groups. ThegroupsofexperimentsarepresentedasTablesS1–S5.
Statistics. A one-way ANOVA was used to compare the escape latencies
among groups. As post hoc test we used the Duncan’s multiple ranges test. A
Student t test was also used when only two groups were compared. Sig-
nificant differences were considered when P < 0.05.
ACKNOWLEDGMENTS. We thank Dr. Alain Garcia Varona for animal care,
Irenia Horruitinier for skillful help in the animal training, and Dr. Diego
Moncada for his useful advice on how to administer anisomycin.
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