Long-term Memory Storage

Neuron

Article Persistence of Long-Term Memory Storage Requires a Late Protein Synthesis- and BDNFDependent Phase in the Hippocampus Pedro Bekinschtein,1 Martı´n Cammarota,1,3 Lionel Mu¨ller Igaz,1,4 Lia R.M. Bevilaqua,3 Iva´n Izquierdo,3 and Jorge H. Medina1,2,* 1 Instituto de Biologı´a Celular y Neurociencias, Facultad de Medicina, UBA, Paraguay 2155 3 piso, Buenos Aires (C1121ABG), Argentina 2 Departamento de Fisiologı´a, Facultad de Medicina, UBA, Paraguay 2155 7 piso, Buenos Aires (C1121ABG), Argentina 3 Centro de Memoria, Instituto de Pesquisas Biome´dicas, PUCRS, Av. Ipiranga 6690, Andar 2, Porto Alegre (RS90610-000), Brasil 4 Present address: Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 3600 Spruce Street, 3rd Floor Maloney Building, Philadelphia, PA 19104, USA. *Correspondence: [email protected] DOI 10.1016/j.neuron.2006.11.025

SUMMARY

Persistence is the most characteristic attribute of long-term memory (LTM). To understand LTM, we must understand how memory traces persist over time despite the short-lived nature and rapid turnover of their molecular substrates. It is widely accepted that LTM formation is dependent upon hippocampal de novo protein synthesis and Brain-Derived Neurotrophic Factor (BDNF) signaling during or early after acquisition. Here we show that 12 hr after acquisition of a one-trial associative learning task, there is a novel protein synthesis and BDNF-dependent phase in the rat hippocampus that is critical for the persistence of LTM storage. Our findings indicate that a delayed stabilization phase is specifically required for maintenance, but not formation, of the memory trace. We propose that memory formation and memory persistence share some of the same molecular mechanisms and that recurrent rounds of consolidation-like events take place in the hippocampus for maintenance of the memory trace.

INTRODUCTION Learning is the process by which we and other organisms acquire information about the world. Memory is the storage of that information. In 1900, Muller and Pilzecker proposed that the formation of permanent memory takes time and that during this time, memory remains vulnerable to disruption (McGaugh, 1966; Muller and Pilzecker, 1900). The process of developing stable memory is referred to as consolidation. During the last 40 years, a great body of evidence has uncovered many aspects of the cellular and molecular mechanisms involved in memory

formation and consolidation (Kandel, 2001; McGaugh, 1966, 2000). Memory can be divided into at least two phases: a protein and RNA synthesis-independent phase that lasts minutes to 1–3 hr (short-term memory) and a protein and mRNA synthesis-dependent phase that lasts several hours to days, weeks, or even longer (long-term memory, or LTM) (Davis and Squire, 1984; Emptage and Carew, 1993; Izquierdo and Medina, 1998; McGaugh, 1966, 2000). Therefore, a definite property of LTM is its sensitivity to protein synthesis inhibitors. During memory formation, protein synthesis is thought to be required to transform newly learned information into stable synaptic modifications (Davis and Squire, 1984; Dudai, 1996; McGaugh, 2000). Many behavioral studies in different animal species have demonstrated that LTM requires de novo protein synthesis around the time of training or during the first few hours posttraining (Bourtchouladze et al., 1998; Davis et al., 1976; Flood et al., 1975; Ghirardi et al., 1995; Grecksch and Matthies, 1980; Igaz et al., 2002; Montarolo et al., 1986; Rosenblum et al., 1993; Schafe and LeDoux, 2000; Scharf et al., 2002). However, the involvement of macromolecular synthesis in LTM at longer time delays has not been thoroughly explored so far. Regulation of the expression of several proteins has been associated with the learning process. One molecule that has been proven to be necessary for memory formation in different learning tasks is Brain-Derived Neurotrophic Factor (BDNF), which is a small dimeric protein widely expressed in the adult mammalian brain. BDNF has been extensively implicated in synaptic plasticity and memory processing, and it has been demonstrated to be essential for the development of the protein synthesis-dependent late phase of long-term potentiation (L-LTP) (Kang et al., 1997; Korte et al., 1995; Pang et al., 2004; Patterson et al., 1996; Poo, 2001; Tyler et al., 2002), a form of synaptic plasticity thought to underlie LTM (Morris et al., 2003; Whitlock et al., 2006). Importantly, this molecule has been implicated in the modulation of synapse formation

Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc. 261

Neuron Memory Persistence and New Protein Synthesis

and dendritic spine growth in the hippocampus (Bamji et al., 2006; Tyler and Pozzo-Miller, 2001, 2003), which are processes thought to underlie memory storage. BDNF expression and activity have been shown to play a critical role in memory formation for a variety of learning tasks in different species, including humans (Egan et al., 2003; Hall et al., 2000; Johnston et al., 1999; Lee et al., 2004; Liu et al., 2004; Mizuno et al., 2000; Monteggia et al., 2004; Ou and Gean, 2006; Pezawas et al., 2004; Qiao et al., 1998; Rattiner et al., 2004; Tokuyama et al., 2000), and they have also been implicated in brain disorders that involve cognitive decline (Howells et al., 2000; Peng et al., 2005; Phillips et al., 1991; Saura et al., 2004; Tong et al., 2004; Zuccato et al., 2001). In particular, endogenous BDNF is required for LTM formation of inhibitory avoidance in the rat hippocampus and parietal cortex (Alonso et al., 2002, 2005). Little is known about the cellular and molecular processes that continue for several hours or days after learning to mediate persistence, a key feature of LTM. With the exception of a few examples (Riedel et al., 1999; Shimizu et al., 2000), most studies have evaluated the effect of pre- or posttraining treatments on LTM measured 24 hr after training. However, memories may last days, months, or even a lifetime. So, for them to become long-lasting, changes must persist after acquisition in order to render the memory trace immune to molecular turnover (Bailey et al., 2004; Dudai, 2002; McGaugh, 2000). Thus, the question of memory persistence remains central in understanding the neurobiology of learning and memory. Recent studies have started to address this issue of memory persistence, focusing mainly on the neocortex as the region of permanent memory storage (Bontempi et al., 1999; Burwell et al., 2004; Frankland et al., 2004; Maviel et al., 2004). On the other hand, few works have assessed the involvement of the hippocampus in this process (Cui et al., 2004; Riedel et al., 1999). This is probably because, based on lesion experiments, the hippocampus appears to have a temporary role in memory storage (Anagnostaras et al., 1999; Brown, 2002; Scoville and Milner, 1957; Squire, 1987; Zola-Morgan and Squire, 1990). Given that protein synthesis and BDNF signaling are crucially involved in LTM formation, and that BDNF is known to promote morphological changes at the synapse that could underlie memory maintenance (Barco et al., 2006; Lamprecht and LeDoux, 2004), we decided to investigate whether protein synthesis and BDNF-mediated signaling in the hippocampus are also required for persistence of LTM storage at a time when these processes are no longer necessary for LTM formation. Here we show for the first time (to the best of our knowledge) that de novo protein synthesis and BDNF expression and activity are engaged in the hippocampus during a restricted time window after LTM has already formed in order to ensure successful storage for memory persistence over days. We propose that memory formation and memory persistence share some of the same molecular mechanisms and that recurrent rounds of consolida-

262 Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc.

tion-like events take place in the hippocampus for maintenance of the memory trace. RESULTS To study persistence of memory storage, rats were submitted to a one-trial inhibitory avoidance training (IA) task, a hippocampus-dependent learning task (Izquierdo et al., 2006; Power et al., 2006; Taubenfeld et al., 1999; Tronel et al., 2005; Whitlock et al., 2006). In the training session a rat was placed on a platform located in the training apparatus. The animal explored the platform for a few seconds and then stepped down onto a grid where it received a 3 s mild foot-shock. During the test session, LTM (conventionally measured 24 hr after training) was assessed as the time the animal spent on the platform before stepping down. The use of protein synthesis inhibitors has been fundamental for the development of the hypothesis that macromolecular synthesis is required for LTM (Davis and Squire, 1984; Kandel, 2001), and for a long time it yielded the sole evidence for the participation of de novo protein synthesis in the learning process. In recent years this assumption has been further confirmed through the use of molecular and genetic approaches (Abel and Lattal, 2001). Nevertheless, the effect of protein synthesis inhibitors on learning has been extremely consistent among different learning tasks and species. Therefore, protein synthesis inhibitors are still the best tools to examine the ‘‘when’’ and the ‘‘where’’ of protein synthesis requirements for learning and memory. To test whether hippocampal de novo protein synthesis is required for the persistence of IA memory, we injected the protein synthesis inhibitor anisomycin (Ani) into the CA1 region of the dorsal hippocampus (Figure 1), an area of the brain extensively implicated in memory processing and known to be essential for IA LTM formation (Igaz et al., 2002; Izquierdo et al., 2006; Taubenfeld et al., 1999). Ani has been widely used to block memory formation of several learning tasks in different species (Bourtchouladze et al., 1998; Davis et al., 1976; Grecksch and Matthies, 1980; Igaz et al., 2002; Rossato et al., 2006; Santini et al., 2004; Schafe and LeDoux, 2000; Tiunova et al., 1998). It has been previously shown that targeted infusion of Ani into the dorsal hippocampus reduced the uptake and incorporation of [14C] L-leucine in hippocampal cell populations by 94%–97% 1 hr after treatment (Morris et al., 2006) Similar findings were obtained for other brain regions where protein synthesis stayed inhibited by 65%–80% for at least 3 hr (Duvarci et al., 2006; Rosenblum et al., 1993). Pretraining or 3 hr posttraining infusion of Ani (80 mg/side) caused amnesia in a retention test carried out 2 days after acquisition (Figure 2A, white bars, t = 5.1, p < 0.0001, and t = 4.38, p < 0.0001, respectively, n = 10, Student’s t test). In contrast, Ani infusion at 9, 12, or 24 hr after IA training did not affect memory retention when rats were tested 2 days after training (Figure 2A, white bars), indicating that early, but

Neuron Memory Persistence and New Protein Synthesis

Figure 1. Cannulae Placements and Drug Infusion (A) Photomicrograph of a representative Nissl-stained coronal brain section showing the infusion cannula track terminating in the CA1 region of the dorsal hippocampus. (B) Schematic representations of rat brain sections at three rostrocaudal planes (3.80, 4.30, and 4.80 from bregma) taken from the atlas of Paxinos and Watson (1986), showing, in stippling, the extension of the area reached by the infusions in the dorsal hippocampus. Reprinted from The Rat Brain in stereo taxic coordinates by Paxinos and Watson, pages 33, 35, and 37, Academic Press (1997), with permission from Elsevier.

not late, rounds of protein synthesis in rat hippocampus are required for LTM formation. In a different set of experiments, animals were treated as mentioned above, but were tested for retention at 7 instead of 2 days posttraining. As expected, when given 15 min before training or 3 hr posttraining, Ani also impaired LTM retention as tested 7 days later (Figure 2A, gray bars, t = 3.28, p = 0.0021, and t = 2.91, p = 0.0057, respectively, n = 10, Student’s t test). However, inhibition of protein synthesis 12 hr after training, which did not affect LTM as measured 2 days following the training session (see Figure 2A, white bars), impaired LTM when rats were tested at 7 days posttraining (Figure 2A, gray bars, t = 2.48, p = 0.016, n = 12). No effect was seen on retention scores when Ani was infused into the CA1 region at 9 or 24 hr after training. Another group of animals injected 12 hr posttraining and tested 4 days later showed shorter step-down latencies than control animals (Figure 2B). This suggests that Ani injection at 12 hr may cause gradual memory decay rather than a sudden decrease in memory retention at 7 days. In all experiments, animals were tested only once, so retention scores were not affected by an extinction process. To determine whether Ani infusion at 12 hr posttraining produced any alteration in the functional integrity of the dorsal hippocampus, rats received bilateral intra-CA1 infusions of Ani 12 hr after IA training, and 7 days later were trained in the hidden platform version of the Morris Water Maze (MWM), a well-

known hippocampus-dependent learning task (Morris et al., 1982). Animals injected with Ani or Vehicle (Veh) normally acquired and retained the spatial memory associated with training in the MWM (Figure 2C), indicating that Ani does not cause permanent damage to the hippocampus. Staining of the hippocampal tissue of trained animals injected with Ani or Veh 12 hr after training did not reveal any differences between Veh- and Ani-injected groups 7 days later (Figure 3A), demonstrating that the memory deficit observed at 7 days was not due to necrosis of the area infused. Since performance in the IA task could be modified by factors such as basal locomotor activity and anxiety, which can be potentially affected by Ani, we analyzed the behavior of animals that had been injected with Ani 12 hr after IA training in the open field and elevated plus maze tests. Intra-CA1 infusion of Ani 12 hr after IA training did not affect anxiety state or exploratory behavior in a novel environment and did not modify basal locomotor activity as evaluated 2 or 7 days after Ani administration (Figure 3B), strongly suggesting that the observed memory deficit was directly caused by inhibition of protein synthesis required for persistence of the memory trace. Moreover, it is not likely that the lower retention scores at 7 days were caused by modifications in performance, since Ani infusion at posttraining time points surrounding the critical period did not cause any deficit in memory retention at 7 days (see Figure 2A). To assess whether a critical period for new protein synthesis for persistence of LTM storage is also required during other forms of learning, we trained animals in a contextual fear conditioning (CFC) task, another well-known one-trial hippocampus-dependent learning paradigm (Abel et al., 1997; Anagnostaras et al., 1999; Bourtchouladze et al., 1998; Fischer et al., 2004; Lee et al., 2004). In this task, animals learn to associate a context (conditioned stimulus [CS]), in this case the training chamber, with an electric foot-shock (unconditioned stimulus [US]). Re-exposure to the context elicits a fear response known as freezing, which serves as a marker for memory (Anagnostaras et al., 1999). CFC training resulted in a robust and long-lasting fear memory, since trained animals injected with Veh showed highly significant freezing both at 2 and 7 days posttraining (Figure 2D). As expected, pretraining infusion of Ani (80 mg/side) at a dose that has been shown to block CFC memory consolidation (Barrientos et al., 2002) caused a deficit in memory retention at 2 and 7 days after training (Figure 1D, t = 4.32, p = 0.0003, n = 8, and t = 3.47, p = 0.0023, n = 8, respectively, Student’s t test). This indicates that hippocampal protein synthesis is required around training for contextual memory formation. Inhibition of protein synthesis 12 hr after CFC training did not cause any deficit in memory retention 2 days after training (Figure 2D, white bars), but produced a clear-cut memory impairment 7 days posttraining (Figure 2D, gray bars, t = 3.96, p = 0.0006, n = 8, Student’s t test), demonstrating that, as happens for IA, protein synthesis is required around 12 hr after training for persistence, but not formation, of CFC memory.

Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc. 263

Neuron Memory Persistence and New Protein Synthesis

Figure 2. Inhibition of Protein Synthesis in the Rat Hippocampus 12 hr after Training Hinders Persistence, but Not Formation, of Hippocampus-Dependent Memory (A) (Top) Schematic of the procedure used in this experiment. (Bottom) Male Wistar rats (2.5 months old, 220–250 g) were bilaterally infused with vehicle (Veh) or anisomycin (Ani) (80 mg/side) into the CA1 region of the dorsal hippocampus, 15 min before or 3, 9, 12, and 24 hr after IA training. Data are expressed as the mean (±SEM) of training (TR, black bars) or test session step-down latency at 2 days (white bars) or 7 days (gray bars) after IA training. *p < 0.05, **p < 0.01, and ***p < 0.001, versus Veh group; two-tailed Student’s t test, n = 10–12 per group. (B) (Top) Schematic of the procedure used in this experiment. (Bottom) Independent groups of animals were infused with Veh (white bars) or Ani (gray bars) 12 hr after IA training and tested 2, 4, or 7 days posttraining. Data are expressed as in (A). *p < 0.05 versus the corresponding Veh group; two-tailed Student’s t test, n = 10–12 per group. (C) (Top) Schematic of the procedure used in this experiment. (Bottom) Animals injected in the CA1 region of the dorsal hippocampus with Veh (open circles and white bars) or Ani (gray circles and gray bars) 12 hr after IA training were submitted 7 days later to the hidden platform version of the Morris Water Maze. The left panel shows mean escape latencies during the 5 days of acquisition of spatial learning. Data are presented in blocks of eight trials as the mean ± SD, p > 0.05 versus Veh in Bonferroni post hoc test after two-way ANOVA, n = 8 per group. The right panel shows the mean time

264 Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc.

Neuron Memory Persistence and New Protein Synthesis

Figure 3. Infusion of Anisomycin 12 hr after IA Training Does Not Affect Exploratory Behavior, Basal Locomotor Activity, or Anxiety, and Does Not Cause Lesion to the Hippocampus 7 Days Posttraining (A) (Top) Schematic of the procedure used in this experiment. (Bottom) Representative photomicrographs showing Nissl-stained coronal brain sections of rats infused with Veh (left) or Ani (right) 12 hr after IA training and sacrificed 7 days later. There was no evidence of cell loss or gliosis. (B) Top panels show the number of rearings (left) and crossings (right) during a 5 min open field session for animals that had received Veh (open bars) or Ani (gray bars) in the CA1 region of the dorsal hippocampus 12 hr after IA training, 2 or 7 days before. Data are expressed as mean (±SEM) number of crossings or rearings, p > 0.1 in a two-tailed Student’s t test, n = 8 per group. Bottom panels show the total number of entries (left), the time spent in open arms (middle), and the number of entries into the open arms (right) during a 5 min plus maze session for rats that had received bilateral intra-CA1 infusion of Veh (white bars) or Ani (gray bars) 2 or 7 days before; p > 0.1 in a two-tailed Student’s t test, n = 8 per group. The upper part of both panels show the schematic of the procedure used in each experiment.

Our results indicate that a delayed wave of protein synthesis in the hippocampus is required for persistence, but not formation, of LTM. This is consistent with a recent preliminary report showing that a late phase of protein synthesis is neccesary for persistence of long-term facilitation in Aplysia (Miniaci et al., 2005). BDNF is a key protein involved in long-term structural and functional modifications, including protein synthesis-dependent long-lasting changes in synaptic efficacy (Pang et al., 2004) and learning and memory (Alonso et al., 2002). Therefore, to investigate the role of BDNF in memory persistence, we first determined whether IA training is associated with changes in BDNF expression in the dorsal hippocampus at the time that protein synthesis is required for persistence of IA memory. IA training resulted in a 56% increase of BDNF levels 12 hr after acquisition (Figure 4A, t = 2.86, p = 0.02, n = 5, Student’s t test). We

did not observe any differences in BDNF immunoreactivity at 9 or 24 hr after training (Figure 4A), two time points at which Ani did not cause any memory deficit as measured at 7 days (see Figure 2A). This late training-associated increase in hippocampal BDNF protein was blocked by intra-CA1 Ani infusion (80 mg/side) 30 min prior to sacrifice (Figure 4B, F = 5.1, p < 0.05, n = 6, Ani versus Veh, Newman-Keuls test after one-way ANOVA), suggesting a possible role for BDNF in the protein synthesis-dependent mechanism involved in memory persistence. Although the experiments presented above indicate that expression of hippocampal BDNF was increased 12 hr after IA training, they do not address the question of whether endogenous BDNF is in fact required for memory persistence. We then asked if hippocampal BDNF activity was required during the same critical period of protein synthesis for persistence of LTM storage. To do that, we

spent in the target quadrant (TQ) during a 60 s probe test carried out in the absence of the escape platform 24 hr after the last training day. Data are presented in blocks of eight trials as the mean (±SD), p = 0.24, two-tailed Student’s t test, n = 8 per group. (D) (Top) Schematic of the procedure used in this experiment. (Bottom) Animals were infused with Veh or Ani (80 mg/side) into the CA1 region of the dorsal hippocampus, 15 min before or 12 hr after CFC training. Data are expressed as mean (±SEM) percentage of freezing before training (preshock, black bars) or during a memory retention test carried out 2 (white bars) or 7 (gray bars) days after CFC training.**p < 0.01 and ***p < 0.001, versus Veh group; two-tailed Student’s t test, n = 8 per group.

Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc. 265

Neuron Memory Persistence and New Protein Synthesis

Figure 4. Hippocampal BDNF Is Required at 12 hr Posttraining for Persistence, but Not Formation, of IA Memory (A) Time-restricted expression of BDNF was detected at 12 (middle), but not at 9 (left) or 24 (right), hr after IA training. The dorsal hippocampus was dissected out from naive rats (white bars) or IA trained rats (9, 12, or 24 hr posttraining, gray bars) and total homogenates were subjected to SDS-PAGE followed by western blot analysis with antibodies against BDNF or b-actin. (Top) Bars show normalized mean percentage levels respect to the naive (N) group. Data are expressed as mean ± SEM, *p < 0.05, trained (12 hr) versus naive, Student’s t test, n = 6 per group. (B) (Left) Schematic of the procedure used in this experiment. (Right) Inhibitory avoidance learning-induced increase in BDNF expression around 12 hr posttraining is abolished by a preceeding intra-CA1 infusion of Ani. IA trained rats received bilateral intra-CA1 infusions of Veh (trained, Veh) or Ani (80 mg/side) (trained, Ani) 11.5 hr posttraining. Thirty minutes later animals were killed by decapitation and the dorsal hippocampus was dissected out and utilized to prepare a total homogenate on which western blot analysis for BDNF and b-actin content was performed. In addition, two control groups were evaluated: nontrained animals (naive, N group) and nontrained animals that were infused with Ani and sacrificed 30 min afterwards (naive, N Ani group). (Top) Bars show normalized mean percentage levels with respect to the control (N) group. Data are expressed as

266 Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc.

Neuron Memory Persistence and New Protein Synthesis

neutralized endogenous BDNF biological activity by delivering function-blocking anti-BDNF antibodies into the CA1 region of the dorsal hippocampus. This approach offers a rapid and transient way to block BDNF activity and allows for a precise temporal resolution of BDNF requirements during memory formation (Alonso et al., 2002). Confirming and extending previous findings (Alonso et al., 2002), the infusion of anti-BDNF antibody (0.5 mg/side) in the CA1 region of the dorsal hippocampus 15 min before training blocked LTM retention as measured 2 or 7 days posttraining (Figure 4C, t = 3.06, p = 0.0057, and t = 3.19, p = 0.003, respectively, n = 10, Student’s t test). Mimicking the effect of Ani on memory persistence (see Figure 2A), neutralizing endogenous BDNF biological activity in the CA1 12 hr after acquisition caused amnesia at 7, but not 2, days posttraining (Figure 4C, t = 3.1, p = 0.0032, n = 10, Student’s t test). This indicates that hippocampal BDNF activity is not only required for memory formation, but also for persistence of LTM storage. No effect on LTM retention was observed at 2 or 7 days when anti-BDNF antibody was injected at 9 or 24 hr posttraining (Figure 4C, n = 9). This demonstrates that endogenous BDNF activity is required during a restricted time window around 12 hr after training for persistence of LTM storage. To give further support to the hypothesis that BDNF synthesis is required for memory persistence, we used an antisense oligonucleotide (ASO; Lee et al., 2004) to specifically block de novo BDNF expression in the hippocampus. Rats were injected with 2 nmol of biotinylated BDNF ASO and sacrificed 2 or 24 hr after injection. BDNF ASO was consistently observed in the dorsal hippocampus 2, but not 24, hr after injection (Figure 5A). The distribution and stability of ASO are consistent with other reports describing the use of intra-hippocampal oligonucleotide injections (Guzowski et al., 2000; Lee et al., 2004; Taubenfeld et al., 2001a). A single infusion of BDNF ASO (2 nmol, 1 ml/side) was sufficient to reduce hippocampal BDNF levels in nontrained animals by more than 60% 6 hr after treatment (Figure 5B, t = 4.8, p = 0.006, n = 4, Student’s t test) compared with controls infused with a BDNF scrambled missense oligonucleotide (MSO), indicating that ASO can effectively block BDNF expression in the dorsal hippocampus. The effect was reversible, since BDNF levels were completely recovered 24 hr after infusion (Figure 5B). Infusion of BDNF ASO 10 hr after IA training prevented the learning-induced increase of BDNF levels at 12 hr after training (Figure 5C, F = 5.1, p < 0.05, n = 5, trained MSO versus trained ASO, one-way ANOVA), indicating that a single injection of BDNF ASO is sufficient to block de novo BDNF expression induced by IA training. We next determined whether inhibition of BDNF protein expression

was sufficient to block memory persistence. Injection of BDNF ASO, but not BDNF MSO, 10 hr after IA training caused a severe memory impairment 7 days posttraining, but left memory intact at 2 days after acquisition (Figure 5D, t = 2.91, p = 0.007, n = 10, ASO versus MSO at 7 days, Student’s t test). Importantly, this impairment in memory retention at 7 days was rescued by infusion of human recombinant BDNF (hrBDNF; 0.25 mg/side) 12 hr after training (i.e., 2 hr after ASO injection; Figure 5D, F = 14.5, p < 0.05, n = 8, ASO+Veh versus ASO+hrBDNF, Newman-Keuls test after one-way ANOVA). Therefore, the deficit in LTM persistence does indeed reflect a loss of BDNF protein in the hippocampus and indicates that BDNF is specifically required for the maintenance of IA memory. These results mimicked those obtained by general inhibition of protein synthesis (see Figure 2A) and blockade of endogenous BDNF activity (see Figure 4C) and directly demonstrate that BDNF expression is required late after training for persistence of LTM storage. LTM lasting days, weeks, or years is associated with the growth of new synaptic connections initiated by a cellular program of gene expression (Lamprecht and LeDoux, 2004). It is known that BDNF can alter synaptic scaling and excitability of neurons over days (Rutherford et al., 1998) by modulating morphologic synapse development over the same time frame (Labelle and Leclerc, 2000). These changes include enhanced neurite outgrowth, which may result in synaptogenesis associated with learning and memory (Geinisman, 2000; Shors, 2004), and make BDNF an ideal player to be involved in memory storage. However, it is not well understood how a single molecule can exert diverse effects spanning a temporal continuum. It is likely that molecules induced by BDNF mediate downstream actions that underlie these structural changes. Therefore, we decided to explore what the downstream pathways associated with learning are that can be affected when BDNF and protein synthesis are blocked at the late critical period. Activity-regulated genes can be induced in the hippocampus with a slow time course after learning (Lee et al., 2004; Merhav et al., 2006; Taubenfeld et al., 2001b). c-Fos is an immediate early gene (IEG) and a transcription factor widely associated with synaptic activity, synaptic plasticity, and memory processing (Cammarota et al., 2000; Countryman et al., 2005; Fleischmann et al., 2003; Guzowski, 2002; He et al., 2002; Morrow et al., 1999; Worley et al., 1993; Yasoshima et al., 2006). We had previously shown that c-Fos levels increase in the hippocampus late after IA learning (Igaz et al., 2004). Given that c-Fos expression can be regulated by BDNF (Glorioso et al., 2006) and is induced by BDNF in hippocampal neurons (Alder et al.,

mean ± SEM. *p < 0.05 in Newman-Keuls test after one-way ANOVA, n = 6 per group. (Bottom) Representative western blots showing BDNF and b-actin levels. One nanogram of human recombinant BDNF (hrBDNF) was loaded as control for antibody specificity (lane five). (C) (Left) Schematic of the procedure used in this experiment. (Right) Blockade of BDNF activity at 12 hr after training hinders persistence, but not formation, of IA memory. Animals were infused with Veh or BDNF-blocking antibody (0.5 mg/side; anti-BDNF) into the CA1 region of the dorsal hippocampus 15 min before or 9, 12, or 24 hr after training. Data are expressed as mean (±SEM) of training (TR, black bars) or test session step-down latency, 2 (white bars) or 7 (gray bars) days after IA training. **p < 0.01, versus Veh group; Student’s t test, n = 10–12 per group.

Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc. 267

Neuron Memory Persistence and New Protein Synthesis

Figure 5. Intra-Hippocampal Infusion of BDNF Antisense Oligonucleotide 10 hr after IA Training Blocks Memory Retention at 7, but Not at 2, Days Posttraining (A) Biotinylated antisense oligonucleotide (ASO) distribution and relative concentrations at different times after infusion (2 nmol/ml; 1 ml/side) in the CA1 region of the dorsal hippocampus. Rats were injected and sacrificed 2 (left) or 24 (right) hr after infusion. By 2 hr, the ASO diffused throughout the dorsal hippocampus and slightly into the overlying cortex. After 24 hr, the ASO was cleared out from the hippocampus. (B) (Top) Schematic of the procedure used in this experiment. (Bottom) Infusion of BDNF ASO in the CA1 region of the dorsal hippocampus transiently decreases BDNF steady-state levels. Rats were injected with BDNF scrambled missense oligonucleotide (MSO) (white bars) or BDNF ASO (black

268 Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc.

Neuron Memory Persistence and New Protein Synthesis

2003), we asked if blocking protein synthesis or endogenous BDNF activity 12 hr after IA training could affect late c-Fos expression. We detected a learning-associated increase in c-Fos immunoreactivity in hippocampal homogenates (Figure 6A, F = 6.9, p < 0.05, n = 5, Veh versus naive, Newman-Keuls test after one-way ANOVA) that was completely abolished by Ani infusion 12 hr after training (Figure 6A, F = 6.9, p < 0.05, n = 5, Ani versus Veh, Newman-Keuls test after one-way ANOVA). Thus, inhibiting protein synthesis at a time when this inhibition would impair long-lasting LTM retention blocked the late learning-induced expression of c-Fos. Intra-CA1 administration of BDNF-blocking antibodies 12 hr posttraining (0.5 mg; 0.5 ml/side) also hindered c-Fos expression, indicating that this late c-Fos induction is dependent on BDNF activity at the time that BDNF is required for memory persistence (Figure 6C, F = 9.75, p < 0.01, n = 5, anti-BDNF versus Veh, Newman-Keuls test after one-way ANOVA). Another IEG that has been consistently implicated in synaptic plasticity and memory processing is Zif268 (a.k.a. krox24 or egr1) (Malkani et al., 2004; Worley et al., 1993) which, like c-Fos, is also a transcription factor. Expression of this gene is modulated and induced by BDNF (Alder et al., 2003; Glorioso et al., 2006). We observed a significant increase in Zif268 24 hr after IA training (Figure 6B, F = 4.07, p < 0.05, n = 5, Veh versus naive, Newman-Keuls test after one-way ANOVA). The change in Zif268 protein level was completely blocked by Ani infusion 12 hr posttraining, indicating that protein synthesis around 12 hr after IA learning is required for the late posttraining Zif268 induction (Figure 6B, F = 4.07, p < 0.05, n = 5, Ani versus Veh, Newman-Keuls test after one-way ANOVA). We next determined if Zif268 expression was also dependent on BDNF activity at 12 hr posttraining. Intra-CA1 infusion of anti-BDNF 12 hr after IA training impaired the increase in Zif268 levels occurring late after learning (Figure 6D, F = 4.1, p < 0.05, n = 5, anti-BDNF versus Veh, Newman-Keuls test after oneway ANOVA). This indicates that, like c-Fos induction, delayed expression of Zif268 also depends on BDNF

activity around 12 hr after training. These results reveal that the events triggered by BDNF in the hippocampus that are necessary for persistence of LTM storage involve, at least in part, the induction of c-Fos and Zif268, both of which, in turn, can interact with regulatory elements on a variety of effector genes. DISCUSSION The main finding of this work is that protein synthesis and BDNF in the rat hippocampus are required during a restricted time window around 12 hr after training for persistence of LTM storage, but not for memory formation. The deficit in memory persistence was observed by inhibiting hippocampal protein synthesis at 12 hr in two different one-trial learning tasks, IA and CFC, suggesting that this process may be conserved across different types of learning tasks. These results demonstrate that the hippocampus is still engaged in memory processing after LTM is already formed and that a protein synthesis- and BDNFdependent phase is required for persistence of LTM storage. We also propose that long-lasting LTM storage may be achieved by recurrent rounds of consolidationlike protein synthesis-dependent processes. One of the questions that arises from our results is how long memory consolidation lasts. Two other studies have addressed this issue. Shimizu et al. (2000) demonstrated that reactivation of CA1 NMDA receptors in the days that follow learning is indeed crucial for contextual fear LTM (Shimizu et al., 2000). In another study, Riedel and colleagues established that hippocampal neural activity is required for long-term storage of spatial memory for at least 5 days following training (Riedel et al., 1999). However, none of the mentioned studies investigated the requirement of protein synthesis in these memory processes. Our findings also indicate that a subset of newly synthesized proteins, including BDNF, plays a role in the remodeling processes involved in LTM persistence. In a recent work, Pang and colleagues demonstrated that BDNF is both necessary and sufficient for L-LTP expression in hippocampal slices (Pang et al., 2004). Indeed, application of

bars) and sacrificed 2 (left), 6 (middle), or 24 (right) hr after infusion. Animals were killed by decapitation and the dorsal hippocampus was dissected out and homogenized to perform western blot analysis of BDNF content. (Top) Bars show normalized mean percentage levels with respect to animals injected with BDNF MSO. Data are expressed as mean ± SEM. **p = 0.006, ASO versus MSO; Student’s t test, n = 4 per group. (Bottom) Representative western blots showing BDNF and b-actin levels. (C) (Top) Schematic of the procedure used in this experiment. (Bottom) Intra-CA1 BDNF ASO infusion 10 hr after IA training prevented the learningassociated increase in BDNF protein 12 hr after training. Naive or IA trained rats received bilateral intra-CA1 infusions of BDNF MSO (white bars) or BDNF ASO (gray bars). Two hours later the animals were sacrificed and the dorsal hippocampus was dissected out and used to prepare total homogenates for western blot analysis of BDNF content. (Top) Bars show the normalized mean percentage levels with respect to the naive animals injected with MSO. Data are expressed as mean ± SEM. *p < 0.05 in Newman-Keuls test after ANOVA, n = 5 per group. (Bottom) Representative western blots showing BDNF and b-actin levels. (D) (Top) Schematic of the procedure used in this experiment. (Bottom) BDNF ASO, but not BDNF MSO, infusion 10 hr after IA training hinders memory persistence at 7 days, but left memory intact 2 days, posttraining. Animals were infused with BDNF MSO (2 nmol; 1 ml/side) (white bars) or BDNF ASO (2 nmol; 1 ml/side) (gray bars) into the CA1 region of the dorsal hippocampus 10 hr after training. Data are expressed as mean (± SEM) of training (TR, black bars) or test session step-down latency, 2 or 7 days after IA training. **p < 0.01, ASO versus MSO at 7 days; Student’s t test, n = 8–10 per group. (E) (Left) Schematic of the procedure used in this experiment. (Right) Intra-CA1 infusion of hrBDNF (0.25 mg/side), but not Veh, 12 hr after IA training rescued the BDNF ASO-induced memory deficit at 7 days posttraining. Data are expressed as mean (± SEM) of training (TR, black bars) or test session step-down latency 7 days after IA training. **p < 0.01, ASO+Veh versus MSO+Veh; *p < 0.05, ASO+hrBDNF versus MSO+Veh; n = 10, Newman-Keuls test after one-way ANOVA.

Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc. 269

Neuron Memory Persistence and New Protein Synthesis

Figure 6. Intra-CA1 Infusion of Ani or Anti-BDNF 12 hr Posttraining Blocks the IA Learning-Induced Increase in c-Fos and Zif268 Levels that Occurs Late after Training (Top) IA trained animals were injected in the dorsal CA1 region with Veh (white bars) or Ani (80 mg/side) (gray bars) 12 hr after acquisition of IA and sacrificed 24 hr posttraining. The hippocampus was dissected out and homogenized to perform western blot analysis of (A) c-Fos and (B) Zif268 expression. Bars show normalized mean percentage levels with respect to the naive group. Data are expressed as mean ± SEM. *p < 0.05, trained versus naive, one-way ANOVA, n = 6 per group. (Bottom) In an independent series of experiments, IA trained animals were injected with Veh (white bars) or anti-BDNF (0.5 mg/side) (gray bars) 12 hr after acquisition of IA and sacrificed 24 hr afterwards. The hippocampus was dissected out and homogenized to carry out western blot analysis of (C) c-Fos and (D) Zif268 expression. Bars show normalized mean percentage levels with respect to the naive group. Data are expressed as mean ± SEM. **p < 0.01, *p < 0.05, trained versus naive, one-way ANOVA, n = 6 per group. The upper part of each panel shows the schematic of the procedure used in each experiment.

BDNF was shown to completely reverse the blockade of L-LTP by Ani. We and others have previously demonstrated the existence of two time windows of sensitivity to protein synthesis inhibitors and BDNF signaling during the first few hours after memory acquisition (Alonso et al., 2002; Bourtchouladze et al., 1998; Grecksch and Matthies, 1980; Igaz et al., 2002), suggesting that the effect of Ani on memory could be partially related to inhibition of BDNF synthesis. In this sense, it has been recently demonstrated that IA learning induces LTP in the hippo-

270 Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc.

campus (Whitlock et al., 2006), raising the possibility that BDNF-induced LTP is involved in memory processing for this learning task. In this work we show that blockade of hippocampal BDNF activity during a restricted time period beyond the first few hours after training affects memory persistence at the same time point than the inhibition of protein synthesis does. Furthermore, blocking BDNF expression in the hippocampus during the critical time period also causes a marked deficit in memory persistence without affecting memory formation, indicating that

Neuron Memory Persistence and New Protein Synthesis

synthesis of BDNF is critical during this late protein synthesis-dependent phase that is required for persistence of LTM storage. Synaptic remodeling and the growth of new synaptic connections have been found to accompany various forms of LTM in both invertebrates (Bailey et al., 2004) and vertebrates (Lamprecht and LeDoux, 2004; Segal, 2005). Clearly, structural changes go together with longterm behavioral modifications in Aplysia (Barco et al., 2006). Recent studies in the mammalian central nervous system have provided additional evidence that synaptic remodeling and the growth of new connections are involved in LTM storage (Lamprecht and LeDoux, 2004). BDNF induces enduring structural changes in a manner consistent with the morphological modifications thought to underlie learning and memory. In fact, BDNF signaling has been shown to enhance dendritic growth and branching (Bamji et al., 2006; McAllister et al., 1995; Tyler and Pozzo-Miller, 2003). Moreover, the axons of entorhinal and commissural fibers from TrkB/ knockout mice have, among other synaptic alterations, fewer collaterals and varicosities in the hipocampal formation (Martinez et al., 1998). Furthermore, BDNF increases the number of dendritic spines on apical dendrites of CA1 pyramidal neurons as well as the number of CA3-CA1 excitatory synapses in hippocampal slice cultures (Bamji et al., 2006; Tyler and Pozzo-Miller, 2001, 2003). BDNF overexpression also protects against stress-induced hippocampal atrophy through a mechanism involving dendritic remodeling of CA3 pyramidal neurons (Govindarajan et al., 2006). Therefore, it seems that the structural modifications induced by BDNF are compatible with several of the processes allegedly implicated in memory maintenance. Induction of BDNF around 12 hr after acquisition may initiate a cascade of molecular and cellular events that leads to the synaptic remodeling necessary for persistence of LTM storage. The events triggered by BDNF may involve, among others, the modulation of gene expression via the induction of IEGs and late-response genes, and it is also possible that this molecule regulates local translation at synapses. In fact, it has been demonstrated that BDNF can increase local protein translation initiation via multiple signaling pathways, including phosphatidylinositol-3 kinase (PI3K) and mammalian target of Rapamycin (mTOR) (Takei et al., 2004). In a study carried out by Kandel and colleagues (Martin et al., 1997), it was demonstrated that in a culture system of Aplysia sensory and motor neurons, local synaptic protein synthesis is required for the long-term maintenance of the plasticity and stabilization of the growth beyond 24 hr. Later, Si and coworkers (Si et al., 2003) established that induction of the Aplysia homolog of cytoplasmatic polyadenylation element binding protein (CPEB), a molecule capable of activating dormant mRNAs, is required for long-term maintenance of synaptic facilitation, but not for its early expression, at 24 hr. Induction of CPEB seems to be independent of transcription, but it requires new protein synthesis and is sensitive to inhibition of the mTOR and

the PI3K pathways. This observation raises the possibility that BDNF expression 12 hr after IA training induced local translation at synapses, which would be required for the long-term maintenance of synaptic changes. This putative mechanism deserves further examination. In our study, we show that two IEGs, c-Fos and Zif268, are upregulated late after IA training by protein synthesisand BDNF-dependent events involved in persistence of LTM storage. c-Fos has been widely implicated in synaptic plasticity and memory (Countryman et al., 2005; He et al., 2002; Morrow et al., 1999; Yasoshima et al., 2006), and recent works have also involved Zif268 in memory processing (Bozon et al., 2003; Bruel-Jungerman et al., 2006; Jones et al., 2001; Lee et al., 2004; Malkani et al., 2004; Valjent et al., 2006). Both genes have been shown to be induced in hippocampal neurons by BDNF treatment (Alder et al., 2003; Ring et al., 2006). Their regulation by BDNF during memory processing may explain how one molecule—BDNF—can produce such global modifications to the synapses, since binding of these transcription factors to target promoter elements can regulate the expression of a broad range of late-response genes with a variety of functions. These results indicate that an orchestrated process of translational and transcriptional events participates in the mechanisms of memory maintenance. However, is there a critical phase of hippocampal transcription involved in LTM persistence? Preliminary results from our laboratory show that persistence of LTM storage at 7 days, which depends on new protein synthesis (see Figure 2), might not be dependent on transcription at 12 hr posttraining. Pretraining intra-CA1 infusion of the transcriptional inhibitor 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB; 8 ng/side) has been previously shown to impair memory formation (Igaz et al., 2002). In contrast, infusion of DRB 12 hr posttraining at the same dose that hinders memory formation did not impair memory retention at 7 days (mean step-down latencies: 106.65 ± 10.64 [Veh] versus 90.21 ± 19.8 [DRB], n = 9, p = 0.31, Student t test). Although these findings are quite preliminary, they reveal a complex dynamic regulation of transcription and translation processes that deserves thorough examination. In fact, mRNAs that encode the proteins needed for memory persistence at 12 hr, such as BDNF, might be transcribed hours before, during the first rounds of transcription associated with LTM formation (Bernabeu et al., 1997; Igaz et al., 2002). In this context, cAMP-response element binding protein (CREB) activation, which has been consistently implicated in memory processing (Silva et al., 1998) and is considered a molecular marker for memory (Viola et al., 2000), exhibited a protracted increase after learning (Bernabeu et al., 1997; Bilang-Bleuel et al., 2002; Cammarota et al., 2000; Taubenfeld et al., 1999, 2001b; Trifilieff et al., 2006). Confirming and extending previous findings from Taubenfeld et al. (2001b), we have found that phosphorylation of CREB at Ser 133 increased at 0, 3, 6, 12, and 18–20 hr, but not at 9 hr, after training (data not shown). Thus, it seems likely that CREB activation is required at

Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc. 271

Neuron Memory Persistence and New Protein Synthesis

some point either to initiate the process of late consolidation or as readout of this process. Further experiments will be required to clarify this issue. It has been proposed that memory formation is a multiphasic process during which memory persists in an inactive state interrupted by reactivations of the trace that trigger plasticity events (Dudai, 2004; Kandel, 2001). It has also been suggested that these reactivations could be caused exogenously by retrieval, or they might occur endogenously in the course of maintenance of the memory trace (Alberini, 2005; Dudai and Eisenberg, 2004). It has even been postulated that memory reconsolidation—i.e., the restabilization of the memory trace that follows the labilization induced by its nonreinforced retrieval—could be just a manifestation of a lingering consolidation process. More extensive reactivation periods may lead to a far-reaching plasticity and a longer-lasting memory. This hypothesis predicts that there should be recurrent time windows of susceptibility to consolidation blockers over hours, days, or weeks. In this scenario, late hippocampal protein synthesis and BDNF requirement for persistence of IA memory might be interpreted as evidence for an endogenous reactivation of the trace that allows longer memory retention. At this point, a note of caution should be added: in most experiments published so far, LTM has been measured 24 hr after training. Our experiments show an amnesic effect that is evident 7 days after training, but not 2 days posttraining. This could be the case for many treatments that did not appear to be amnesic for LTM retention at only 24 hr. In a naturally behaving environment where new experiences happen frequently and in some cases overlap, a delayed critical period for memory persistence has relevant implications. For example, new learning has been shown to produce retroactive interference with encoding of recently acquired memories (Izquierdo et al., 1999; Muller and Pilzecker, 1900; Squire et al., 1975; Xu et al., 1998) when the new information is acquired during consolidation of the older memory. Memory persistence could also be modified if processes needed for encoding new information interfere with the memory trace during a critical stabilization phase. Thus, it is not only memory formation that could be modulated by experience, but also its persistence. Recent studies suggest the existence of an active process of forgetting (Sangha et al., 2005; Villarreal et al., 2002). It has yet to be determined whether memory persistence is the result of two opposite active mechanisms (one that strengthens the memory trace and another that induces its decay). In this context, inhibition of the mechanism that underlies maintenance would tip the scale in favor of a putative forgetting process. In future studies we will address the subject of memory decay, taking into account not only the mechanisms involved in maintenance, but also those involved in forgetting. In conclusion, we described a delayed stabilization phase involved specifically in persistence of LTM storage, but not in memory formation. This process is similar to

272 Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc.

what is currently referred to as memory consolidation in its requirement for protein synthesis and BDNF activity. We believe that this is one of the first steps toward unveiling the cellular and molecular mechanisms involved in memory persistence and decay. EXPERIMENTAL PROCEDURES Subjects Male Wistar rats (age, 2.5 months old; weight, 220–250 g) from our own breeding colony were used. The animals were housed in plastic cages, five to a cage, with water and food ad libitum, under a 12 hr light/dark cycle (lights on at 7:00 a.m.) at a constant temperature of 23 C. The experimental protocol for this study followed the guidelines of the USA National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Animal Care and Use Committees of the University of Buenos Aires and PUCRS. Surgery Rats were implanted under deep thionembutal anesthesia with 22-g guide cannulae in the dorsal CA1 region of the hippocampus in accordance with coordinates A 4.3, L ± 3.0, V 1.4, taken from the atlas of Paxinos and Watson (Paxinos and Watson, 1986). The cannulae were fixed to the skull with dental acrylic. Histology After the behavioral procedures, the rats received an overdose of thionembutal and were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde. Brains were removed and placed in 10% buffered formalin with 30% sucrose. Frozen sections (50 mm thickness) were stained for Nissl bodies. For analysis of oligonucleotide (ODN) spread after injection, rats were injected with 2 nmol of biotinylated BDNF antisense ODN (ASO), and 2 or 24 hr after injection, the animals were anesthetized and perfused with 4% paraformaldehyde. The brains were isolated and sliced and the ASO was detected by avidin-biotin staining (Taubenfeld et al., 2001a). Inhibitory Avoidance Training and Testing After recovery from surgery, animals were handled once a day for 2 days and then trained in inhibitory avoidance as described (Bernabeu et al., 1997). Briefly, the apparatus was a 50 3 25 3 25 cm acrylic box whose grid was a series of 1 mm caliber bronze bars spaced 1 cm apart. The left end of the floor was covered by an 8 cm wide, 5.0 cm high wood platform. During training, animals were gently placed on the platform; as they stepped down onto the grid they received a 3 s, 0.7 mA scrambled foot-shock. Rats were tested for retention at 2, 4, or 7 days after training. In the test sessions the foot-shock was omitted. In all experiments animals were tested only once. Morris Water Maze Training and Testing The water maze was a black circular pool (200 cm in diameter) conceptually divided into four equal imaginary quadrants for the purpose of data analysis. The water temperature was 21 C–23 C. Two centimeters beneath the surface of the water and hidden from the rat’s view was a black circular platform (12 cm in diameter). It had a rough surface, which allowed the rat to climb onto it easily once its presence was detected. The water maze was located in a well-lit white room with several posters and other distal visual stimuli hanging on the walls to provide spatial cues. A curtain separated the water maze room from the room where the computer setup was installed and where the animals were temporarily housed during the behavioral sessions. Training in the hidden platform (spatial) version of the MWM was carried out during 5 consecutive days as previously described (Rossato et al., 2006). On each day, rats received eight consecutive training trials during which the hidden platform was kept in a constant location. A different starting location was used on each trial, which consisted of

Neuron Memory Persistence and New Protein Synthesis

a swim followed by a 30 s platform sit. Any rat that did not find the platform within 60 s was guided to it by the experimenter. The intertrial interval (ITI) was 30 s. During the ITI, rats were carefully dried with a towel by the experimenter. Memory retention was evaluated in a 60 s probe trial carried out in the absence of the escape platform 24 hr after the last training session. Contextual Fear Conditioning The conditioning chamber was made of white plastic (20 3 23 3 20 cm) with a clear lid. The floor of the chamber consisted of 10 parallel stainless steel grid bars, each measuring 4 mm in diameter and spaced 1.5 cm apart. The grid was connected to a scrambled shocker to deliver the foot-shocks. Training consisted of placing the rat in the chamber and allowing a 3 min acclimation period (preshock period). After this period, rats received three foot-shocks (0.8 mA, 3 s duration and intershock interval of 30 s; US). They remained in the chamber for an additional 2 min (postshock period), and after this period, rats were returned to their home cages. CFC during the test session was evaluated 2 or 7 days after training by placing the rats in the training environment for 5 min. Memory was assessed and expressed as the percentage of time that rats spent freezing. Such behavior is commonly used as an index of fear in rats. An animal was considered to be freezing when it was crouching without movement of the body and the head except that associated with breathing. Open Field and Elevated Plus Maze Tests The open field was a 50 cm high, 50 cm wide, and 39 cm deep arena with black plywood walls and a brown floor divided into nine squares by black lines. Number of line crossings and rearings were measured during a 5 min test session. To evaluate their anxiety state, animals were exposed to an elevated plus maze. The total number of entries into the four arms, the number of entries, and the time spent in the open arms were recorded over a 5 min session. Drug Infusion Procedures Cannulated rats received, 15 min before training or 3, 9, 12, or 24 hr after training, bilateral 0.8 ml infusions of saline or Ani (80 mg/side, Sigma) dissolved in saline or 0.5 ml of a BDNF blocking antibody (Alonso et al., 2002) (0.5 mg/side, Chemicon). ODN (Genbiotech S.R.L.) were HPLC-purified phosphorothioate end-capped 18-mer sequences, resuspended in sterile saline to a concentration of 2 nmol/ml. Both ODNs were phosphorothioated on the three terminal bases of both 50 and 30 ends. This modification results in increased stability and less toxicity of the ODN. BDNF ASO, 50 -TCTTCCCCTTTTAATGGT-30 ; BDNF MSO, 50 -ATACTTTCTGTTCTTGCC-30 . Both ODN sequences were subjected to a BLAST search on the National Center for Biotechnology Information BLAST server using the Genbank database. ASO is specific for rat BDNF mRNA. Control MSO sequence, which included the same 18 nucleotides as the ASO but in a scrambled order, did not generate any full matches to identified gene sequences in the database. hrBDNF (Alomone labs, Israel) was prepared in sterile saline (final concentration 0.25 mg/ml). The volume infused was 1 ml/side. The entire bilateral infusion procedure took about 2 min, including 45 s for the infusions themselves, first on one side and then on the other. Histological examination of cannulae placements was performed as described previously (Bernabeu et al., 1997). Briefly, 24 hr after the end of the behavioral procedures, 0.8 ml of 4% methylene blue in saline was infused as indicated above. Animals were killed by decapitation 15 min later and the brains were stored in formalin for histological localization of the infusion sites. Infusions spread with a radius of less than 1.2 mm3, as described before, and were found to be correct (i.e., within 1.5 mm3 of the intended site) in 95% of the animals. Only data from animals with the cannulae located in the intended sites were included in the final analysis. Immunoblot Assays Tissue was homogenized in ice-chilled buffer (20 mM Tris-HCL [pH 7.4], 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 mg/ml

aprotinin, 15 mg/ml leupeptin, 10 mg/ml bacitracin, 10 mg/ml pepstatin, 15 mg/ml trypsin inhibitor, 50 mM NaF, and 1 mM sodium orthovanadate). Samples of homogenates (15 mg of protein) were subjected to SDS-PAGE (10% or 12% gels) under reducing conditions. Proteins were transferred onto PDVF membranes in transfer buffer (25 mM Tris, 192 mM glycine, 20% v/v methanol) for 2 hr at 100V and 4 C for BDNF analysis. For c-Fos and Zif268 analysis, proteins were transferred onto PVDF membranes in transfer buffer (25 mM Tris, 192 mM glycine, 10% v/v methanol) overnight at 4 C. Western blots were performed by incubating membranes first with anti-BDNF antibody (N20, 1:1000, Santa Cruz Biotechnology Inc, Santa Cruz, CA), antiFos antibody (1:2000, Santa Cruz Biotechnology Inc, Santa Cruz, CA) or anti Zif268 (1:2000, Santa Cruz Biotechnology Inc, Santa Cruz, CA), then stripped and incubated with anti-b-actin antibody (1:5000, Santa Cruz Biotechnology Inc, Santa Cruz, CA). One nanogram of recombinant human BDNF was used as a standard for western blot (hrBDNF, Alomone labs, Israel). Film densitometry analysis was performed by using an MCID Image Analysis System (version 5.02, Imaging Research Inc., St. Catharines, Ontario, Canada). Data Analysis In all behavioral experiments statistical analysis was performed by unpaired Student’s t test or one-way ANOVA followed by NewmanKeuls or Bonferroni post hoc tests, comparing mean step-down latencies, percentage of freezing, or percentage of escape latencies of the drug-treated groups and vehicle-treated groups at each time point studied. Western blot data were analyzed by unpaired two-tailed Student’s t test or one-way ANOVA followed by Newman-Keuls multiple comparison test. ACKNOWLEDGMENTS We thank Cynthia Katche, Leandro Slipczuk, Diego Moncada, Andrea Goldin, Lina Levi, and Dr. Hayde´e Viola for help with some experiments and Dr. Paula Faillace for helpful discussion of our manuscript. This work was supported by research grants from the National Agency of Scientific and Technological Promotion of Argentina (ANPCyT) to J.H.M. and M.C., and the National Research Council of Brazil (CNPq) through the National Program for Nuclei of Excellence (PRONEX) to I.I., M.C., and L.R.M.B. Received: April 3, 2006 Revised: September 20, 2006 Accepted: November 27, 2006 Published: January 17, 2007 REFERENCES Abel, T., and Lattal, K.M. (2001). Molecular mechanisms of memory acquisition, consolidation and retrieval. Curr. Opin. Neurobiol. 11, 180–187. Abel, T., Nguyen, P.V., Barad, M., Deuel, T.A., Kandel, E.R., and Bourtchouladze, R. (1997). Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory. Cell 88, 615–626. Alberini, C.M. (2005). Mechanisms of memory stabilization: are consolidation and reconsolidation similar or distinct processes? Trends Neurosci. 28, 51–56. Alder, J., Thakker-Varia, S., Bangasser, D.A., Kuroiwa, M., Plummer, M.R., Shors, T.J., and Black, I.B. (2003). Brain-derived neurotrophic factor-induced gene expression reveals novel actions of VGF in hippocampal synaptic plasticity. J. Neurosci. 23, 10800–10808. Alonso, M., Vianna, M.R., Depino, A.M., Mello e Souza, T., Pereira, P., Szapiro, G., Viola, H., Pitossi, F., Izquierdo, I., and Medina, J.H. (2002). BDNF-triggered events in the rat hippocampus are required for both short- and long-term memory formation. Hippocampus 12, 551–560.

Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc. 273

Neuron Memory Persistence and New Protein Synthesis

Alonso, M., Bekinschtein, P., Cammarota, M., Vianna, M.R., Izquierdo, I., and Medina, J.H. (2005). Endogenous BDNF is required for longterm memory formation in the rat parietal cortex. Learn. Mem. 12, 504–510. Anagnostaras, S.G., Maren, S., and Fanselow, M.S. (1999). Temporally graded retrograde amnesia of contextual fear after hippocampal damage in rats: within-subjects examination. J. Neurosci. 19, 1106– 1114. Bailey, C.H., Kandel, E.R., and Si, K. (2004). The persistence of longterm memory: a molecular approach to self-sustaining changes in learning-induced synaptic growth. Neuron 44, 49–57. Bamji, S.X., Rico, B., Kimes, N., and Reichardt, L.F. (2006). BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin-beta-catenin interactions. J. Cell Biol. 174, 289–299. Barco, A., Bailey, C.H., and Kandel, E.R. (2006). Common molecular mechanisms in explicit and implicit memory. J. Neurochem. 97, 1520–1533. Barrientos, R.M., O’Reilly, R.C., and Rudy, J.W. (2002). Memory for context is impaired by injecting anisomycin into dorsal hippocampus following context exploration. Behav. Brain Res. 134, 299–306. Bernabeu, R., Bevilaqua, L., Ardenghi, P., Bromberg, E., Schmitz, P., Bianchin, M., Izquierdo, I., and Medina, J.H. (1997). Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats. Proc. Natl. Acad. Sci. USA 94, 7041–7046. Bilang-Bleuel, A., Rech, J., De Carli, S., Holsboer, F., and Reul, J.M. (2002). Forced swimming evokes a biphasic response in CREB phosphorylation in extrahypothalamic limbic and neocortical brain structures in the rat. Eur. J. Neurosci. 15, 1048–1060. Bontempi, B., Laurent-Demir, C., Destrade, C., and Jaffard, R. (1999). Time-dependent reorganization of brain circuitry underlying long-term memory storage. Nature 400, 671–675. Bourtchouladze, R., Abel, T., Berman, N., Gordon, R., Lapidus, K., and Kandel, E.R. (1998). Different training procedures recruit either one or two critical periods for contextual memory consolidation, each of which requires protein synthesis and PKA. Learn. Mem. 5, 365–374. Bozon, B., Kelly, A., Josselyn, S.A., Silva, A.J., Davis, S., and Laroche, S. (2003). MAPK, CREB and zif268 are all required for the consolidation of recognition memory. Philos. Trans. R. Soc. Lond. B Biol. Sci. 358, 805–814. Brown, A.S. (2002). Consolidation theory and retrograde amnesia in humans. Psychon. Bull. Rev. 9, 403–425. Bruel-Jungerman, E., Davis, S., Rampon, C., and Laroche, S. (2006). Long-term potentiation enhances neurogenesis in the adult dentate gyrus. J. Neurosci. 26, 5888–5893. Burwell, R.D., Bucci, D.J., Sanborn, M.R., and Jutras, M.J. (2004). Perirhinal and postrhinal contributions to remote memory for context. J. Neurosci. 24, 11023–11028. Cammarota, M., Bevilaqua, L.R., Ardenghi, P., Paratcha, G., Levi de Stein, M., Izquierdo, I., and Medina, J.H. (2000). Learning-associated activation of nuclear MAPK, CREB and Elk-1, along with Fos production, in the rat hippocampus after a one-trial avoidance learning: abolition by NMDA receptor blockade. Brain Res. Mol. Brain Res. 76, 36–46.

Davis, H.P., Spanis, C.W., and Squire, L.R. (1976). Inhibition of cerebral protein synthesis: performance at different times after passive avoidance training. Pharmacol. Biochem. Behav. 4, 13–16. Dudai, Y. (1996). Consolidation: fragility on the road to the engram. Neuron 17, 367–370. Dudai, Y. (2002). Molecular bases of long-term memories: a question of persistence. Curr. Opin. Neurobiol. 12, 211–216. Dudai, Y. (2004). The neurobiology of consolidations, or, how stable is the engram? Annu. Rev. Psychol. 55, 51–86. Dudai, Y., and Eisenberg, M. (2004). Rites of passage of the engram: reconsolidation and the lingering consolidation hypothesis. Neuron 44, 93–100. Duvarci, S., Mamou, C.B., and Nader, K. (2006). Extinction is not a sufficient condition to prevent fear memories from undergoing reconsolidation in the basolateral amygdala. Eur. J. Neurosci. 24, 249–260. Egan, M.F., Kojima, M., Callicott, J.H., Goldberg, T.E., Kolachana, B.S., Bertolino, A., Zaitsev, E., Gold, B., Goldman, D., Dean, M., et al. (2003). The BDNF val66met polymorphism affects activitydependent secretion of BDNF and human memory and hippocampal function. Cell 112, 257–269. Emptage, N.J., and Carew, T.J. (1993). Long-term synaptic facilitation in the absence of short-term facilitation in Aplysia neurons. Science 262, 253–256. Fischer, A., Sananbenesi, F., Schrick, C., Spiess, J., and Radulovic, J. (2004). Distinct roles of hippocampal de novo protein synthesis and actin rearrangement in extinction of contextual fear. J. Neurosci. 24, 1962–1966. Fleischmann, A., Hvalby, O., Jensen, V., Strekalova, T., Zacher, C., Layer, L.E., Kvello, A., Reschke, M., Spanagel, R., Sprengel, R., et al. (2003). Impaired long-term memory and NR2A-type NMDA receptor-dependent synaptic plasticity in mice lacking c-Fos in the CNS. J. Neurosci. 23, 9116–9122. Flood, J.F., Bennett, E.L., Orme, A.E., and Rosenzweig, M.R. (1975). Effects of protein synthesis inhibition on memory for active avoidance training. Physiol. Behav. 14, 177–184. Frankland, P.W., Bontempi, B., Talton, L.E., Kaczmarek, L., and Silva, A.J. (2004). The involvement of the anterior cingulate cortex in remote contextual fear memory. Science 304, 881–883. Geinisman, Y. (2000). Structural synaptic modifications associated with hippocampal LTP and behavioral learning. Cereb. Cortex 10, 952–962. Ghirardi, M., Montarolo, P.G., and Kandel, E.R. (1995). A novel intermediate stage in the transition between short- and long-term facilitation in the sensory to motor neuron synapse of aplysia. Neuron 14, 413–420. Glorioso, C., Sabatini, M., Unger, T., Hashimoto, T., Monteggia, L.M., Lewis, D.A., and Mirnics, K. (2006). Specificity and timing of neocortical transcriptome changes in response to BDNF gene ablation during embryogenesis or adulthood. Mol. Psychiatry 11, 633–648. Govindarajan, A., Rao, B.S., Nair, D., Trinh, M., Mawjee, N., Tonegawa, S., and Chattarji, S. (2006). Transgenic brain-derived neurotrophic factor expression causes both anxiogenic and antidepressant effects. Proc. Natl. Acad. Sci. USA 103, 13208–13213.

Countryman, R.A., Kaban, N.L., and Colombo, P.J. (2005). Hippocampal c-fos is necessary for long-term memory of a socially transmitted food preference. Neurobiol. Learn. Mem. 84, 175–183.

Grecksch, G., and Matthies, H. (1980). Two sensitive periods for the amnesic effect of anisomycin. Pharmacol. Biochem. Behav. 12, 663– 665.

Cui, Z., Wang, H., Tan, Y., Zaia, K.A., Zhang, S., and Tsien, J.Z. (2004). Inducible and reversible NR1 knockout reveals crucial role of the NMDA receptor in preserving remote memories in the brain. Neuron 41, 781–793.

Guzowski, J.F. (2002). Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches. Hippocampus 12, 86–104.

Davis, H.P., and Squire, L.R. (1984). Protein synthesis and memory: a review. Psychol. Bull. 96, 518–559.

274 Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc.

Guzowski, J.F., Lyford, G.L., Stevenson, G.D., Houston, F.P., McGaugh, J.L., Worley, P.F., and Barnes, C.A. (2000). Inhibition of activity-dependent arc protein expression in the rat hippocampus

Neuron Memory Persistence and New Protein Synthesis

impairs the maintenance of long-term potentiation and the consolidation of long-term memory. J. Neurosci. 20, 3993–4001.

facilitation of aplysia sensory to motor synapses: a function for local protein synthesis in memory storage. Cell 91, 927–938.

Hall, J., Thomas, K.L., and Everitt, B.J. (2000). Rapid and selective induction of BDNF expression in the hippocampus during contextual learning. Nat. Neurosci. 3, 533–535.

Martinez, A., Alcantara, S., Borrell, V., Del Rio, J.A., Blasi, J., Otal, R., Campos, N., Boronat, A., Barbacid, M., Silos-Santiago, I., and Soriano, E. (1998). TrkB and TrkC signaling are required for maturation and synaptogenesis of hippocampal connections. J. Neurosci. 18, 7336–7350.

He, J., Yamada, K., and Nabeshima, T. (2002). A role of Fos expression in the CA3 region of the hippocampus in spatial memory formation in rats. Neuropsychopharmacology 26, 259–268. Howells, D.W., Porritt, M.J., Wong, J.Y., Batchelor, P.E., Kalnins, R., Hughes, A.J., and Donnan, G.A. (2000). Reduced BDNF mRNA expression in the Parkinson’s disease substantia nigra. Exp. Neurol. 166, 127–135. Igaz, L.M., Vianna, M.R., Medina, J.H., and Izquierdo, I. (2002). Two time periods of hippocampal mRNA synthesis are required for memory consolidation of fear-motivated learning. J. Neurosci. 22, 6781–6789. Igaz, L.M., Bekinschtein, P., Izquierdo, I., and Medina, J.H. (2004). One-trial aversive learning induces late changes in hippocampal CaMKIIalpha, Homer 1a, Syntaxin 1a and ERK2 protein levels. Brain Res. Mol. Brain Res. 132, 1–12. Izquierdo, I., and Medina, J.H. (1998). On brain lesions, the milkman and Sigmunda. Trends Neurosci. 21, 423–426. Izquierdo, I., Schroder, N., Netto, C.A., and Medina, J.H. (1999). Novelty causes time-dependent retrograde amnesia for one-trial avoidance in rats through NMDA receptor- and CaMKII-dependent mechanisms in the hippocampus. Eur. J. Neurosci. 11, 3323–3328. Izquierdo, I., Bevilaqua, L.R., Rossato, J.I., Bonini, J.S., Medina, J.H., and Cammarota, M. (2006). Different molecular cascades in different sites of the brain control memory consolidation. Trends Neurosci. 29, 496–505. Johnston, A.N., Clements, M.P., and Rose, S.P. (1999). Role of brainderived neurotrophic factor and presynaptic proteins in passive avoidance learning in day-old domestic chicks. Neuroscience 88, 1033– 1042. Jones, M.W., Errington, M.L., French, P.J., Fine, A., Bliss, T.V., Garel, S., Charnay, P., Bozon, B., Laroche, S., and Davis, S. (2001). A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories. Nat. Neurosci. 4, 289–296. Kandel, E.R. (2001). The molecular biology of memory storage: a dialogue between genes and synapses. Science 294, 1030–1038. Kang, H., Welcher, A.A., Shelton, D., and Schuman, E.M. (1997). Neurotrophins and time: different roles for TrkB signaling in hippocampal long-term potentiation. Neuron 19, 653–664. Korte, M., Carroll, P., Wolf, E., Brem, G., Thoenen, H., and Bonhoeffer, T. (1995). Hippocampal long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor. Proc. Natl. Acad. Sci. USA 92, 8856–8860. Labelle, C., and Leclerc, N. (2000). Exogenous BDNF, NT-3 and NT-4 differentially regulate neurite outgrowth in cultured hippocampal neurons. Brain Res. Dev. Brain Res. 123, 1–11. Lamprecht, R., and LeDoux, J. (2004). Structural plasticity and memory. Nat. Rev. Neurosci. 5, 45–54. Lee, J.L., Everitt, B.J., and Thomas, K.L. (2004). Independent cellular processes for hippocampal memory consolidation and reconsolidation. Science 304, 839–843. Liu, I.Y., Lyons, W.E., Mamounas, L.A., and Thompson, R.F. (2004). Brain-derived neurotrophic factor plays a critical role in contextual fear conditioning. J. Neurosci. 24, 7958–7963.

Maviel, T., Durkin, T.P., Menzaghi, F., and Bontempi, B. (2004). Sites of neocortical reorganization critical for remote spatial memory. Science 305, 96–99. McAllister, A.K., Lo, D.C., and Katz, L.C. (1995). Neurotrophins regulate dendritic growth in developing visual cortex. Neuron 15, 791–803. McGaugh, J.L. (1966). Time-dependent processes in memory storage. Science 153, 1351–1358. McGaugh, J.L. (2000). Memory–a century of consolidation. Science 287, 248–251. Merhav, M., Kuulmann-Vander, S., Elkobi, A., Jacobson-Pick, S., Karni, A., and Rosenblum, K. (2006). Behavioral interference and C/ EBPß expression in the insular-cortex reveal a prolonged time period for taste memory consolidation. Learn. Mem. 13, 571–574. Published online September 15, 2006. 10.1101/lm.282406. Miniaci, M.C., Kim, J.H., Zhu, H., Alarcon, J.M., Bailey, C.H., and Kandel, E.R. (2005). Society for Neuroscience Program No. 848.7, Washington, D.C. Mizuno, M., Yamada, K., Olariu, A., Nawa, H., and Nabeshima, T. (2000). Involvement of brain-derived neurotrophic factor in spatial memory formation and maintenance in a radial arm maze test in rats. J. Neurosci. 20, 7116–7121. Montarolo, P.G., Goelet, P., Castellucci, V.F., Morgan, J., Kandel, E.R., and Schacher, S. (1986). A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia. Science 234, 1249– 1254. Monteggia, L.M., Barrot, M., Powell, C.M., Berton, O., Galanis, V., Gemelli, T., Meuth, S., Nagy, A., Greene, R.W., and Nestler, E.J. (2004). Essential role of brain-derived neurotrophic factor in adult hippocampal function. Proc. Natl. Acad. Sci. USA 101, 10827–10832. Morris, R.G., Garrud, P., Rawlins, J.N., and O’Keefe, J. (1982). Place navigation impaired in rats with hippocampal lesions. Nature 297, 681–683. Morris, R.G., Moser, E.I., Riedel, G., Martin, S.J., Sandin, J., Day, M., and O’Carroll, C. (2003). Elements of a neurobiological theory of the hippocampus: the role of activity-dependent synaptic plasticity in memory. Philos. Trans. R. Soc. Lond. B Biol. Sci. 358, 773–786. Morris, R.G., Inglis, J., Ainge, J.A., Olverman, H.J., Tulloch, J., Dudai, Y., and Kelly, P.A. (2006). Memory reconsolidation: sensitivity of spatial memory to inhibition of protein synthesis in dorsal hippocampus during encoding and retrieval. Neuron 50, 479–489. Morrow, B.A., Elsworth, J.D., Inglis, F.M., and Roth, R.H. (1999). An antisense oligonucleotide reverses the footshock-induced expression of fos in the rat medial prefrontal cortex and the subsequent expression of conditioned fear-induced immobility. J. Neurosci. 19, 5666– 5673. Muller, G.E., and Pilzecker, A. (1900). Experimentelle Beitra¨ge zur Lehre vom Geda¨chtnis. Z. Psychol. Erga¨nzungsband 1, 1–300. Ou, L.C., and Gean, P.W. (2006). Regulation of amygdala-dependent learning by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol-3-kinase. Neuropsychopharmacology 31, 287–296.

Malkani, S., Wallace, K.J., Donley, M.P., and Rosen, J.B. (2004). An egr-1 (zif268) antisense oligodeoxynucleotide infused into the amygdala disrupts fear conditioning. Learn. Mem. 11, 617–624.

Pang, P.T., Teng, H.K., Zaitsev, E., Woo, N.T., Sakata, K., Zhen, S., Teng, K.K., Yung, W.H., Hempstead, B.L., and Lu, B. (2004). Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science 306, 487–491.

Martin, K.C., Casadio, A., Zhu, H., Yaping, E., Rose, J.C., Chen, M., Bailey, C.H., and Kandel, E.R. (1997). Synapse-specific, long-term

Patterson, S.L., Abel, T., Deuel, T.A., Martin, K.C., Rose, J.C., and Kandel, E.R. (1996). Recombinant BDNF rescues deficits in basal synaptic

Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc. 275

Neuron Memory Persistence and New Protein Synthesis

transmission and hippocampal LTP in BDNF knockout mice. Neuron 16, 1137–1145.

long-term potentiation and long-term memory by spaced training. J. Neurophysiol. 87, 2770–2777.

Paxinos, G., and Watson, C. (1986). The Rat Brain in Stereotaxic Coordinates (San Diego: Academic Press).

Scoville, W.B., and Milner, B. (1957). Loss of recent memory after bilateral hippocampal lesions. J. Neurochem. 20, 11–21.

Peng, S., Wuu, J., Mufson, E.J., and Fahnestock, M. (2005). Precursor form of brain-derived neurotrophic factor and mature brain-derived neurotrophic factor are decreased in the pre-clinical stages of Alzheimer’s disease. J. Neurochem. 93, 1412–1421.

Segal, M. (2005). Dendritic spines and long-term plasticity. Nat. Rev. Neurosci. 6, 277–284.

Pezawas, L., Verchinski, B.A., Mattay, V.S., Callicott, J.H., Kolachana, B.S., Straub, R.E., Egan, M.F., Meyer-Lindenberg, A., and Weinberger, D.R. (2004). The brain-derived neurotrophic factor val66met polymorphism and variation in human cortical morphology. J. Neurosci. 24, 10099–10102. Phillips, H.S., Hains, J.M., Armanini, M., Laramee, G.R., Johnson, S.A., and Winslow, J.W. (1991). BDNF mRNA is decreased in the hippocampus of individuals with Alzheimer’s disease. Neuron 7, 695–702. Poo, M.M. (2001). Neurotrophins as synaptic modulators. Nat. Rev. Neurosci. 2, 24–32. Power, A.E., Berlau, D.J., McGaugh, J.L., and Steward, O. (2006). Anisomycin infused into the hippocampus fails to block ‘‘reconsolidation’’ but impairs extinction: the role of re-exposure duration. Learn. Mem. 13, 27–34. Qiao, X., Chen, L., Gao, H., Bao, S., Hefti, F., Thompson, R.F., and Knusel, B. (1998). Cerebellar brain-derived neurotrophic factor-TrkB defect associated with impairment of eyeblink conditioning in Stargazer mutant mice. J. Neurosci. 18, 6990–6999. Rattiner, L.M., Davis, M., French, C.T., and Ressler, K.J. (2004). Brainderived neurotrophic factor and tyrosine kinase receptor B involvement in amygdala-dependent fear conditioning. J. Neurosci. 24, 4796–4806. Riedel, G., Micheau, J., Lam, A.G., Roloff, E.L., Martin, S.J., Bridge, H., de Hoz, L., Poeschel, B., McCulloch, J., and Morris, R.G. (1999). Reversible neural inactivation reveals hippocampal participation in several memory processes. Nat. Neurosci. 2, 898–905. Ring, R.H., Alder, J., Fennell, M., Kouranova, E., Black, I.B., and Thakker-Varia, S. (2006). Transcriptional profiling of brain-derived-neurotrophic factor-induced neuronal plasticity: a novel role for nociceptin in hippocampal neurite outgrowth. J. Neurobiol. 66, 361–377. Rosenblum, K., Meiri, N., and Dudai, Y. (1993). Taste memory: the role of protein synthesis in gustatory cortex. Behav. Neural Biol. 59, 49–56. Rossato, J.I., Bevilaqua, L.R., Medina, J.H., Izquierdo, I., and Cammarota, M. (2006). Retrieval induces hippocampal-dependent reconsolidation of spatial memory. Learn. Mem. 13, 431–440. Rutherford, L.C., Nelson, S.B., and Turrigiano, G.G. (1998). BDNF has opposite effects on the quantal amplitude of pyramidal neuron and interneuron excitatory synapses. Neuron 21, 521–530. Sangha, S., Scheibenstock, A., Martens, K., Varshney, N., Cooke, R., and Lukowiak, K. (2005). Impairing forgetting by preventing new learning and memory. Behav. Neurosci. 119, 787–796. Santini, E., Ge, H., Ren, K., Pena de Ortiz, S., and Quirk, G.J. (2004). Consolidation of fear extinction requires protein synthesis in the medial prefrontal cortex. J. Neurosci. 24, 5704–5710. Saura, C.A., Choi, S.Y., Beglopoulos, V., Malkani, S., Zhang, D., Shankaranarayana Rao, B.S., Chattarji, S., Kelleher, R.J., 3rd, Kandel, E.R., Duff, K., et al. (2004). Loss of presenilin function causes impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Neuron 42, 23–36. Schafe, G.E., and LeDoux, J.E. (2000). Memory consolidation of auditory pavlovian fear conditioning requires protein synthesis and protein kinase A in the amygdala. J. Neurosci. 20, RC96. Scharf, M.T., Woo, N.H., Lattal, K.M., Young, J.Z., Nguyen, P.V., and Abel, T. (2002). Protein synthesis is required for the enhancement of

276 Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc.

Shimizu, E., Tang, Y.P., Rampon, C., and Tsien, J.Z. (2000). NMDA receptor-dependent synaptic reinforcement as a crucial process for memory consolidation. Science 290, 1170–1174. Shors, T.J. (2004). Memory traces of trace memories: neurogenesis, synaptogenesis and awareness. Trends Neurosci. 27, 250–256. Si, K., Giustetto, M., Etkin, A., Hsu, R., Janisiewicz, A.M., Miniaci, M.C., Kim, J.H., Zhu, H., and Kandel, E.R. (2003). A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapsespecific long-term facilitation in aplysia. Cell 115, 893–904. Silva, A.J., Kogan, J.H., Frankland, P.W., and Kida, S. (1998). CREB and memory. Annu. Rev. Neurosci. 21, 127–148. Squire, L.R. (1987). The organization and neural substrates of human memory. Int. J. Neurol. 21-22, 218–222. Squire, L.R., Slater, P.C., and Chace, P.M. (1975). Retrograde amnesia: temporal gradient in very long term memory following electroconvulsive therapy. Science 187, 77–79. Takei, N., Inamura, N., Kawamura, M., Namba, H., Hara, K., Yonezawa, K., and Nawa, H. (2004). Brain-derived neurotrophic factor induces mammalian target of rapamycin-dependent local activation of translation machinery and protein synthesis in neuronal dendrites. J. Neurosci. 24, 9760–9769. Taubenfeld, S.M., Wiig, K.A., Bear, M.F., and Alberini, C.M. (1999). A molecular correlate of memory and amnesia in the hippocampus. Nat. Neurosci. 2, 309–310. Taubenfeld, S.M., Milekic, M.H., Monti, B., and Alberini, C.M. (2001a). The consolidation of new but not reactivated memory requires hippocampal C/EBPbeta. Nat. Neurosci. 4, 813–818. Taubenfeld, S.M., Wiig, K.A., Monti, B., Dolan, B., Pollonini, G., and Alberini, C.M. (2001b). Fornix-dependent induction of hippocampal CCAAT enhancer-binding protein [beta] and [delta] Co-localizes with phosphorylated cAMP response element-binding protein and accompanies long-term memory consolidation. J. Neurosci. 21, 84–91. Tiunova, A.A., Anokhin, K.V., and Rose, S.P. (1998). Two critical periods of protein and glycoprotein synthesis in memory consolidation for visual categorization learning in chicks. Learn. Mem. 4, 401–410. Tokuyama, W., Okuno, H., Hashimoto, T., Xin Li, Y., and Miyashita, Y. (2000). BDNF upregulation during declarative memory formation in monkey inferior temporal cortex. Nat. Neurosci. 3, 1134–1142. Tong, L., Balazs, R., Thornton, P.L., and Cotman, C.W. (2004). Betaamyloid peptide at sublethal concentrations downregulates brainderived neurotrophic factor functions in cultured cortical neurons. J. Neurosci. 24, 6799–6809. Trifilieff, P., Herry, C., Vanhoutte, P., Caboche, J., Desmedt, A., Riedel, G., Mons, N., and Micheau, J. (2006). Foreground contextual fear memory consolidation requires two independent phases of hippocampal ERK/CREB activation. Learn. Mem. 13, 349–358. Tronel, S., Milekic, M.H., and Alberini, C.M. (2005). Linking new information to a reactivated memory requires consolidation and not reconsolidation mechanisms. PLoS Biol. 3, e293. 10.1371/journal.pbio. 0030293. Tyler, W.J., and Pozzo-Miller, L. (2003). Miniature synaptic transmission and BDNF modulate dendritic spine growth and form in rat CA1 neurones. J. Physiol. 553, 497–509. Tyler, W.J., and Pozzo-Miller, L.D. (2001). BDNF enhances quantal neurotransmitter release and increases the number of docked vesicles at the active zones of hippocampal excitatory synapses. J. Neurosci. 21, 4249–4258.

Neuron Memory Persistence and New Protein Synthesis

Tyler, W.J., Alonso, M., Bramham, C.R., and Pozzo-Miller, L.D. (2002). From acquisition to consolidation: on the role of brain-derived neurotrophic factor signaling in hippocampal-dependent learning. Learn. Mem. 9, 224–237.

Worley, P.F., Bhat, R.V., Baraban, J.M., Erickson, C.A., McNaughton, B.L., and Barnes, C.A. (1993). Thresholds for synaptic activation of transcription factors in hippocampus: correlation with long-term enhancement. J. Neurosci. 13, 4776–4786.

Valjent, E., Aubier, B., Corbille, A.G., Brami-Cherrier, K., Caboche, J., Topilko, P., Girault, J.A., and Herve, D. (2006). Plasticity-associated gene Krox24/Zif268 is required for long-lasting behavioral effects of cocaine. J. Neurosci. 26, 4956–4960.

Xu, L., Anwyl, R., and Rowan, M.J. (1998). Spatial exploration induces a persistent reversal of long-term potentiation in rat hippocampus. Nature 394, 891–894.

Villarreal, D.M., Do, V., Haddad, E., and Derrick, B.E. (2002). NMDA receptor antagonists sustain LTP and spatial memory: active processes mediate LTP decay. Nat. Neurosci. 5, 48–52.

Yasoshima, Y., Sako, N., Senba, E., and Yamamoto, T. (2006). Acute suppression, but not chronic genetic deficiency, of c-fos gene expression impairs long-term memory in aversive taste learning. Proc. Natl. Acad. Sci. USA 103, 7106–7111.

Viola, H., Furman, M., Izquierdo, L.A., Alonso, M., Barros, D.M., de Souza, M.M., Izquierdo, I., and Medina, J.H. (2000). Phosphorylated cAMP response element-binding protein as a molecular marker of memory processing in rat hippocampus: effect of novelty. J. Neurosci. 20, RC112.

Zola-Morgan, S.M., and Squire, L.R. (1990). The primate hippocampal formation: evidence for a time-limited role in memory storage. Science 250, 288–290.

Whitlock, J.R., Heynen, A.J., Shuler, M.G., and Bear, M.F. (2006). Learning induces long-term potentiation in the hippocampus. Science 313, 1093–1097.

Zuccato, C., Ciammola, A., Rigamonti, D., Leavitt, B.R., Goffredo, D., Conti, L., MacDonald, M.E., Friedlander, R.M., Silani, V., Hayden, M.R., et al. (2001). Loss of huntingtin-mediated BDNF gene transcription in Huntington’s disease. Science 293, 493–498.

Neuron 53, 261–277, January 18, 2007 ª2007 Elsevier Inc. 277