Relevance of synaptic tagging and capture to the persistence of long-term potentiation and everyday spatial

Szu-Han Wang, Roger L. Redondo, and Richard G. M. Morris1

Centre for Cognitive and Neural Systems, Neuroscience, University of Edinburgh, Edinburgh EH8 9JZ, United Kingdom

Edited* by Richard D. Palmiter, University of Washington, Seattle, WA, and approved September 21, 2010 (received for review June 22, 2010)

Memory for inconsequential events fades, unless these happen generally forgotten each day. This is a closer analogy to everyday before or after other novel or surprising events. However, our memory in humans than many current behavioral tasks studied in understanding of the neurobiological mechanisms of novelty- animals, and is likely subserved by temporary engrams mediated by enhanced memory persistence is mainly restricted to aversive or distributed associative potentiation in the hippocampus. fear-associated . We now outline an “everyday appetitive” Interestingly, unrelated novelty or surprise may stabilize the behavioral model to examine whether and how unrelated novelty persistence of memories, even for inconsequential events that facilitates the persistence of spatial memory coupled to parallel elec- are normally forgotten. An example of this is the curious halo of “fl ” trophysiological studies of the persistence of long-term potentiation incidental memories surrounding ashbulb memories (10, 11), (LTP). Across successive days, rats were given one trial per day to find such as what happened to us on the occasion of momentous food in different places and later had to recall that day’s location. events such as the terrorist attacks in 2001 in the 9/11 tragedy. It This task is both hippocampus and NMDA receptor dependent. has recently been reported that memory for inhibitory avoidance First, encoding with low reward induced place memory that decayed by rats can also persist longer if exploration in a novel environ- ment occurs around the time of learning (12). Similar findings over 24 h; in parallel, weak tetanization of CA1 in brain have been shown for contextual fear conditioning, spatial object slices induced early-LTP fading to baseline. Second, novelty explora- recognition and taste memory (13, 14). tion scheduled 30 min after this weak encoding resulted in persistent To further explore the relevance of STC, we conducted parallel NEUROSCIENCE place memory; similarly, strong tetanization—analogous to novelty — behavioral and physiological studies. We occasionally scheduled both induced late-LTP and rescued early- into late-LTP on an inde- brief unrelated novelty exploration to evaluate its impact on pendent but convergent pathway. Third, hippocampal dopamine memory of individual subjects across successive conditions. Nov- D1/D5 receptor blockade or synthesis inhibition within 15 elty exploration is known increase the firing of VTA dopamine min of exploration prevented persistent place memory and blocked (15), hippocampal dopamine release (16) and the tran- late-LTP. Fourth, symmetrically, when spatial memory was encoded scription of a number of activity-related genes (17) and so, like using strong reward, this memory persisted for 24 h unless encoding strong tetanization, may induce the PRPs that the STC hypothesis occurred under hippocampal D1/D5 receptor blockade. Novelty ex- asserts as critical for memory persistence. The continuity of ex- ploration before this encoding rescued the drug-induced memory perience, memory and then forgetting through the day is similar to impairment. Parallel effects were observed in LTP. These findings what happens with activity-dependent synaptic plasticity in vivo can be explained by the synaptic tagging and capture hypothesis. that is presumably triggered frequently (18) and for which protein synthesis-independent and protein synthesis-dependent mecha- hippocampus | flashbulb memory | synaptic plasticity | dopamine | protein nisms are variously engaged (19). This commonality is the basis of synthesis our conducting parallel studies. Results eurobiological theories of long-term memory (LTM) assert Nthat strong events are remembered better than weak because The rats were trained in an event arena to dig for food pellets that they alone trigger “consolidation.” However, the memory of ap- they carried back to the start boxes to eat (Fig. S1). The regular parently unimportant things is an intriguing challenge to these daily training consisted of a memory encoding trial and a later re- accounts—particularly when these occur in association with sur- trieval choice trial. In encoding, the start box door opened allowing prising or emotionally significant events. The synaptic tagging and entry into the arena containing a single sandwell (with one or three capture (STC) hypothesis of protein synthesis-dependent long- food pellets) at a changing place each day. This trial constituted an term potentiation (1, 2) may offer an explanation of this associative opportunity to incidentally encode where food was available on – process, based on the idea that the neural mechanisms of initial that day. In retrieval, 30 40 min after encoding, the same sandwell long-term potentiation (LTP) expression (potentiation and tag- again contained food (three pellets), but there were now four other ging) can be dissociated from those regulating the availability of nonrewarded sandwells available (exacting controls for olfactory plasticity-related (PRPs) that stabilize synaptic change. cues are given in SI Materials and Methods). The animal could use Thus, weakly induced LTP that is normally transient is sustained one-trial place memory from the earlier encoding trial to retrieve fi because PRPs associated with strong LTP on a separate pathway ef ciently. The main study began once the animals were routinely are captured by the synaptic tags set on the weakly tetanized making one error or fewer per retrieval trial. There followed “ ” pathway. As synaptic plasticity may be one component of the neural a series of separate conditions conducted over 6 months con- mechanisms of information storage (3–6), the persistence of memory should parallel the persistence of synaptic potentiation (7). An important characteristic of ‘everyday memory’ is that we Author contributions: S.-H.W., R.L.R., and R.G.M.M. designed research; S.-H.W. and R.L.R. retain incidental information within LTM for only a short period, performed research; S.-H.W. and R.L.R. contributed new reagents/analytic tools; S.-H.W., rarely creating an enduring memory (e.g., where we parked our car R.L.R., and R.G.M.M. analyzed data; and S.-H.W., R.L.R., and R.G.M.M. wrote the paper. when out shopping). To model this kind of episodic-like memory, The authors declare no conflict of interest. we have developed an analogous one-trial spatial memory task in *This Direct Submission article had a prearranged editor. an ‘event arena’ that depends on synaptic transmission and plas- 1To whom correspondence should be addressed. E-mail: [email protected]. ticity in the dorsal hippocampus (8, 9). The protocol continues This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. everyday for months, with a new spatial memory encoded and 1073/pnas.1008638107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1008638107 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 sisting of analogous electrophysiological and behavioral compo- Condition 2: Novelty-Enhanced Persistence of Weak Memory. We nents. Performance stability is shown in Fig. S2. then investigated whether unexpected novelty exploration would affect the persistence of memory. In the parallel electrophysiology Condition 1: Decay of Early-LTP (E-LTP) and Weak Memory. Electro- study, we weakly tetanized one pathway (S1) and, 30 min later, physiological studies of synaptic tagging and capture (STC) were applied strong tetanization (“novelty”) to a separate pathway, S2 conducted in hippocampal brain slices using the methods of (Fig. 2 A and B). This is theoretically appropriate as behavioral Redondo et al. (20). We first examined the induction of LTP by novelty and strong tetanization both up-regulate immediate early weak tetanization in brain slices (Fig. 1 A and B). This caused LTP genes, necessary for PRP synthesis/availability; we return to on an input pathway to CA1 at 30 min posttetanus that declined to a justification of this in the discussion. Strong tetanization not only baseline over 10 h with statistically significant decay over this period. led to 10-h LTP in S2, i.e., late-LTP (L-LTP), but also rescued the The analogous “event arena” study evaluated retention, at 30 transient decay of E-LTP on S1, converting it to nondecaying L- min or 24 h, of one-trial place memory rewarded by one food LTP. This replicates a key phenomenon of STC (21). pellet only at encoding (weak encoding). We used a memory In the behavioral experiments, the same animals used in probe test in which five nonrewarded sandwells were presented condition 1 (above) were given a weak encoding trial followed by for 60 s with time spent digging at each sandwell recorded. The a probe test 24 h later, with or without (in counterbalanced or- order of the 30-min and the 24-h encoding-probe test pairs was der) the opportunity for 5-min novelty exploration in a box counterbalanced, interleaved by 1 regular training day. This in- placed within the arena 30 min after encoding (Fig. S1C and Fig. terleaved training, used throughout the study, was essential to 2C). Without exploration, there was the usual indifferent place sustain digging at the sandwells across days, as probe tests con- memory, whereas with exploration, memory was persistent (Fig. fi stituted extinction trials. There was good memory at 30 min but 2 D and E). Taken together, these ndings show that strong not at 24 h, with significant decay between 30 min and 24 h (Fig. tetanization/novelty can enhance the persistence of LTP/memory 1D). Of note is the directly analogous “pattern” of the electro- for an unrelated pathway/experience. physiological and behavioral bar graphs in Fig. 1 B and D. Given that this is a within-subjects procedure in which numerous tests Condition 3: Timing, Location, and Measurement of Novelty Exploration. are to be conducted on individual animals, Fig. 1E plots the To test whether there is a critical time window for novelty explo- ration, the 5-min exploration was either arranged to be 30 min performance of the 16 individual animals in each test.

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% 13 15 % 20 10 14 0 0 16 15 1p, 24 h 1p+novel box, 24 h 1p, 24 h 1p+novel box, 24 h 0 0 16 1p, 30 min 1p, 24 h 1p, 30 min 1p, 24 h Fig. 2. The “weak-before-strong” phenomenon in synaptic potentiation and Fig. 1. Time-dependent retention of synaptic potentiation and memory. (A) memory persistence. (A) WTET at one pathway (S1) elicited persistent LTP A weak tetanus (WTET) at one pathway (S1) elicits LTP at 30 min but not 10 h when a strong tetanus (STET) was applied to an independent pathway (S2) (n = 7). (B) Summary bar graph shows E-LTP, at 30 min and no L-LTP, at 10 h of shortly afterward (n = 7). (B) Potentiation at 10 h shows no L-LTP for a weakly this weakly tetanized pathway (open bars) and control pathway stability stimulated pathway (Left; from Fig. 1B, Right) but persistent L-LTP when WTET

(filled bars) (WTET S1 vs. control at E-LTP, t12 = 8.7, P < 0.01, at L-LTP, t12 = 0.4, was coupled with STET (WTET S1 in Weak+Strong > Weak only: t12 = 2.49, P < P > 0.05; WTET S1 E-LTP vs. L-LTP, t6 = 3.9, P < 0.01). The asterisk indicates 0.05; In Weak+Strong, WTET S1 > control, t12 = 3.08, P < 0.01). (C) Rats dug in significant difference from control pathway (∼100%, P < 0.05); surrounding one open well for one pellet. Exploration in a novel box (green grid square) gray area indicates electrophysiology. (C) Rats dug in one open well (filled 30 min after encoding. Nonrewarded probe trial with five open wells was pink circle) for one food pellet followed by a nonrewarded probe trial with conducted 24 h after encoding. (D) Spatial memory of one-pellet (1p) encod- five open wells (open pink circles) at 30 min or 24 h (n = 16). (D) Spatial ing, at 24-h delay, was enhanced by exploration in a novel box (No novel box:

memory of one-pellet (1p) encoding at 30 min but not 24 h (correct > wrong correct ∼ wrong digging, t15 < 1, P > 0.5; with novel box: correct > wrong digging at 30 min, t15 = 4.5, P < 0.001; at 24 h, t15 < 1, P > 0.5; 2 × 2 ANOVA, digging, t15 = 4.35, P < 0.001; 2 × 2 ANOVA, F1,15 = 6.94, P < 0.05). (E) Perfor- F1,15 = 15.48, P < 0.005). The asterisk indicates significant differences between mance during probe tests after one-pellet encoding or one-pellet encoding + correct and wrong digging (P < 0.05). (E) Individual performance at 30 min exploration. Data are means (±1 SEM in A, B, and D). Asterisks indicate sig- and 24 h. Data are mean (±1SEMinA, B, and D). Gray dashed line (in B and D) nificant differences between adjacent open and filled bars. Gray dashed line indicates the baseline or chance level. (in B and D) indicates baseline or chance level.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1008638107 Wang et al. Downloaded by guest on October 1, 2021 after encoding or delayed to 6 h later. To maintain novelty, dif- A B ferent substrates (e.g., mesh wire, small pebbles, etc) were placed

250 )enilesabfo%(epolsPSPEf 150 ) S1 enile on the floor of the box (Fig. S1C). Delaying the novelty by 6 h Control 200 140 * s

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o 150 place memory at 24 h (Fig. S3A). Second, we found that explora- % (epol 120 tion in a familiar context was not sufficient to induce the memory 100 110 fi s S2 persistence (Fig. S3B). Third, we con rmed that the animals re- PSPEf 50 S1 S2 WTET STET S1 100 membered the novel experience itself by showing locomotor ha- SCH 23390 Time (h) Control fi 0 90 bituation when a speci c box and substrate was given twice (Fig. -2 0 2 4 6 8 10 Weak+Strong Weak+Strong 10 h SCH S3C). Fourth, we established that an allocentric spatial strategy C D was used to perform the task (Fig. S4). 150

250 )enil )enilesabfo%(epolsPSPEf S1 Control Condition 4: Pharmacological Blockade of Novelty-Induced Persistence e 140

200 sabfo%(epol * of Memory. We then addressed whether novelty-induced persis- 130 150 tence of memory is mechanistically similar to STC in brain slices. A 120 100 key idea is that dopamine activation of D1/D5 receptors is critical 110 S2 s for the PRP synthesis necessary for L-LTP (22), the origin of these 50 S1 S2 PSPEf WTET STET S1 100 fibers coactivated by the strong tetanus being the VTA the dopa- Anisomycin Time (h) Control 0 90 mine neurons of which can be activated by novelty (2, 15, 23). In -2 0 2 4 6 8 10 Weak+Strong Weak+Strong 10 h Aniso brain slices (Fig. 3 A and B), we extend previous observations (24) with the finding that the rescue of persistent potentiation after

24 h weak tetanization of a pathway was not observed if subsequent Encoding Probe test E 30 min Novel 23.5 h strong tetanization was conducted in the presence of SCH23390. 60 1 pellet box Correct Veh/SCH23390 * Wrong Thus, D1/D5 receptors are critical for induction of the persistence 50 that characterizes L-LTP. In addition, we confirmed that anisomy- cin blocks L-LTP (Fig. 3 C and D). Maintenance of potentiation of 40 e

m the strongly tetanized pathway was impaired by anisomycin, but also it g n

i 30 there was no potentiation in the weakly tetanized pathway at 10 h. g g NEUROSCIENCE i

df The parallel behavioral studies continued with the now can- 20 o nulas-implanted rats which, with further interspersed training % 10 days, were given weak place memory encoding days that were either with or without later novelty exploration conducted under 0 fl Veh SCH Veh SCH the in uence of SCH23390 or vehicle (n = 11, due to blocked No novel box Novel box cannulas in two rats). We observed no 24-h memory with weak E, Encoding 30 min Novel 23.5 h encoding after either vehicle or SCH23390 infusions (Fig. 3 Probe test F 1 pellet box Left). Persistent place memory over 24 h was seen when weak (or 6h ) Correct 60 Veh /Anisomycin encoding was followed by novelty exploration, but this was * Wrong 50 * * blocked by intrahippocampal SCH23390 during exploration (Fig. 3E, Right). The behavioral tests conducted with anisomycin (n = emitgniggidfo% 40 8) are presented here but, in practice, were done toward the end 30 of all other experimentation because of the potential toxicity of this drug (Table S1). Intrahippocampal infusion of anisomycin 20 immediately after novelty exploration blocked the rescue of 24-h 10 memory by novelty after weak encoding of place memory (Fig. 3F, Left). Delaying the infusion by 6 h allowed rescue of 24-h memory 0 Veh Aniso Veh Aniso to occur normally (Fig. 3F, Right). These findings suggest that the Immediate infusion Delayed infusion critical time window for the PRP synthesis and/or local availability Fig. 3. The role of hippocampal D1/D5 receptors and protein synthesis in is within 6 h of exploration. facilitation of memory persistence in the weak-before-strong model. (A) SCH23390 (10 μM) at STET of pathway S2 blocked S2 LTP persistence at 10 h. Condition 5: Rescue of D1/D5-Dependent Memory Through Novelty An independent pathway (S1) with WTET before both drug infusion and STET Exploration. In classical studies of sensitization in Aplysia, it has of S2 also failed to maintain S1 potentiation for 10 h (n = 6). (B) Summary bar been well established that a single exposure to neuromodulation graph shows L-LTP of a WTET (in Weak+Strong) pathway (from Fig. 2B, Right) by 5HT induces short-term forms of presynaptic facilitation and and impaired L-LTP of the WTET (in Weak+Strong) pathway when STET was behavioral sensitization, whereas multiple exposures result in applied under SCH23390 (at 10 h: WTET S1 vs. control, t10= 1.3, P = 0.22; WTET long-term changes (25, 26). Similarly, multiple trials of training < S1 in Weak+Strong alone vs. in Weak+Strong+SCH23390, t11 = 2.26, P 0.05). give rise to more persistent memory in many tasks, e.g., sensiti- (C) Anisomycin (25 μM) present during STET of pathway S2 blocked LTP zation in Aplysia (27) and fear conditioning in rats (28). Changing maintenance at 10 h. An independent pathway (S1) given WTET before both to strong encoding in the event arena (three-pellet reward instead drug infusion and STET in S2 also failed to maintain LTP (n = 7). (D) Summary of one pellet) should induce 24-h memory, which it did in an bar graphs shows L-LTP of a WTET pathway (in Weak+Strong, from Fig. 2B, NMDA- receptor–dependent manner (Fig. S5). The persistence Right) and impaired L-LTP of WTET pathway when STET to the other pathway

was under anisomycin (at 10 h: WTET S1 vs. control, t12 < 1, P = 0.68; WTET S1 in Weak+Strong alone vs. in Weak+Strong+Aniso, t12 = 2.48, P < 0.05). (E and F)(Top) Summary of behavioral procedures. (E) Without novelty, hippo- retention unless novelty was followed immediately by hippocampal aniso-

campal infusions of vehicle (Veh) or D1/D5 receptor antagonist, SCH23390 (1 mycin (125 μg/μL. Left: correct > wrong digging in Veh t7 = 4.74, P < 0.01; in μ μ g/ L) did not affect performance (Left: similar chance level correct and Ani, t7 < 1, P > 0.5; 2 × 2 ANOVA, F1,7 = 10.29, P < 0.01). In contrast, a 6-h– wrong digging at 24 h, P > 0.5; 2 × 2 ANOVA, F1,10 < 1). Exploration in a novel delayed anisomycin infusion did not block novelty-enhanced memory (Right: box 30 min after one-pellet encoding enhanced memory persistence (Mid- correct > wrong digging in Veh, t7 = 2.66, P < 0.05; in Ani, t7 = 3.82, P < 0.01; > < right: correct wrong digging, t10 = 4.05, P 0.01). This was impaired by 2 × 2 ANOVA, F1,7 < 1, P > 0.5). Data are mean ±1 SEM. Asterisks indicate SCH23390 infusion before exploration (Right: 2 × 2 ANOVA F1,10 = 9.95, P < significant differences between adjacent open and filled bars. Gray dashed 0.01). (F) Novelty exploration after one pellet encoding facilitated memory line in B, D, E, and F indicates baseline or chance level.

Wang et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 of this strong memory encoding was sensitive to D1/D5 receptor An electrophysiological analogy is the frequent use of multiple blockade with a 3.3- but not a 1-μg/μL infusion of SCH23390 trains of high-frequency tetanization in experiments on L-LTP. (Fig. 4D). Such tetanization of one pathway in the presence of SCH23390 We then investigated whether this forgetting of strongly blocked L-LTP. However, when this was preceded by strong tet- encoded place memory could be rescued by prior novelty. To anization of second independent pathway before application of avoid the drug having an effect on the downstream effects of SCH23390 (Fig. 4 A and B), both pathways maintained LTP for 10 novelty, it was necessary to present the novelty before the memory h. This “strong-before-strong” protocol indicates that SCH23390 encoding (Fig. 4C). On the critical tests, the animals were given blocks LTP maintenance by blocking the synthesis of PRPs rather exploration of the novel box 1 h before “strong” place memory than affecting the setting of synaptic tags (20, 24). encoding with, 15 min before the memory-encoding trial, intra- hippocampal infusions of SCH23390 (3.3 μg/μL) or vehicle. Condition 6: Meta-Analysis of Good, Poor, and Rescued Memory Memory was measured, as usual, with a nonrewarded probe test Conditions. Our within-subjects design offers the unique opportu- 24 h after encoding (Fig. 4C). Remarkably, memory over 24 h was nity to compare individual subjects across conditions and so to not only observed after the preencoding vehicle infusions (Fig. provide further tests of the relevance of the STC theory to mem- 4E, Left), but also after preencoding SCH23390 infusions (Fig. ory. The entire study was conducted over 6 mo; we have noted that 4E, Right). As the STC hypothesis uniquely predicts, dopamine- not all animals completed all procedures of the full study, but it was dependent memory encoded in the presence of D1/D5 receptor still possible to pool the probe test data into a “meta-analysis” that blockade is rescued by prior novelty. offered the following three categories: (i) the average performance of individual animals in that set of training protocols in which good memory was predicted, such as the 24-h memory of a strong, three- pellet encoding trial and 30-min memory of a weak, one-pellet A B trial; (ii) those in which poor memory was predicted, such as 24-h 250 170 memory after weak encoding; and (iii) those in which memory * S1 160 * Control 200 rescue was predicted, such as 24-h memory after weak encoding 150 followed by novelty exploration (full compilation of conditions in 150 140 130 Table S2). We found that 14 of the 16 animals complied with our 100 120 prediction in showing a good–poor–rescued “V-shaped” function 2 STET STET S2 110 χ < 50 S1 S2 ( =9,P 0.005; overall ANOVA of the three categories, F2,30 = S1 100 < fl fEPSP (% slope of baseline) SCH 23390 Time (h) 22.73, P 0.001; the quadratic component, re ecting this V-shape, Control fEPSP slope baseline) of (% 0 90 -2 0 2 4 6 8 10 Strong+Strong Strong+Strong was highly significant, F1,15 = 49.88, P < 0.001) (Fig. 5). In addi- 10 h SCH tion, we analyzed whether there would be any correlation between the levels of performance of individual subjects and their sensiti- fi Encoding (3 pellets) Probe test vity to novelty. Although the STC hypothesis makes no speci c C 24 h No novel box (D) predictions, we nonetheless observed that the relative decline in performance between the good and poor categories was correlated with the relative increase in memory persistence between the poor and the rescued categories (Fig. S6). 1 h 24 h Discussion Novel box (E) Incidental memory occurs automatically in the course of our day- to-day stream of activities and is sometimes vital for their suc- D E cessful accomplishment. Although such inconsequential informa- Novel Encoding Probe Encoding 24 h Probe 24 h 3 pellets test box 3 pellets test tion enters LTM, there is little value in retaining it for any length Veh/SCH23390 Veh/SCH23390 time—leading to one of the beneficial “seven sins” of memory, namely, transience (29). However, if something surprising hap- 60 * 60 Correct * * Correct pens, not only do we remember the surprise itself, but it may in- * Wrong Wrong 50 50 duce (or appear to induce) better memory of surrounding events. 40 40 We have developed an everyday, one-trial, allocentric spatial 30 30 memory paradigm in rats, and have established that closely timed 20 20 % of digging time % % of digging time % but unrelated novelty can extend the persistence of weakly enco- 10 10 ded place memory. As the STC theory of L-LTP (1) offers a unique 0 0 explanation of this remarkable phenomenon, it was essential to Veh Sch(low) Sch(high) VehSch(high) conduct parallel, in vitro electrophysiological studies mimicking Fig. 4. Novelty exploration rescues the memory impairment induced by our behavioral experiments. These confirmed the rescue of weakly hippocampal D1/D5 receptor blockade. (A) When strong tetanization (STET) induced LTP by strong tetanization of another pathway and that of S2 occurred under SCH23390 after STET of another independent pathway strong tetanization rescues persistent LTP induced on another (S1), L- LTP was present on both pathways (n = 5). (B) Summary at 10 h shows pathway in the presence of SCH23390 (24), and revealed the SCH23390 failed to block L-LTP in S2 when coupled with predrug STET in S1 findings that rescue of L-LTP on a weakly tetanized pathway by > < < (S2 control in vehicle: t8 = 4.1, P 0.01 and SCH23390: t8 = 6.5, P 0.01; and subsequent strong tetanization is sensitive to both SCH23390 and > no difference between conditions: t8 = 0.8, P 0.05). (C) Exploration anisomycin. Our behavioral data directly parallel these LTP find- sometimes scheduled 1 h before three-pellet encoding that during or ings: (i) intrahippocampal blockade of D1/D5R receptors and in- without hippocampal D1/D5 receptor blockade. (D) Retention at 24 h of hibition of protein synthesis at the time of novelty exploration three-pellet encoding was impaired by a higher dose of SCH23390 (3.3 μg/μl) prevented novelty from enhancing memory persistence; (ii) strong (correct = wrong digging: t8 < 1, P > 0.3), but not 1 μg/μl (correct > wrong digging t = 3.2, P 0.01) (2 × 3 ANOVA (correct/wrong digging by Veh/SCH encoding of the daily place memory enabled persistence over 24 h, 8 and the blockade of 24 h memory by the D1/D5 antagonist low dose/SCH high dose) F2,16 = 4.59, P < 0.05). (E) Novelty exploration 1 h before encoding completely rescued 24-h memory despite encoding during SCH23390 could be rescued by novelty exploration shortly before

3.3 μg/μl dose of SCH23390 (similar correct digging in Veh vs. SCH, F1,8 < 1; training; and (iii) a meta-analysis of good, poor, and rescued correct > wrong digging; Veh: t8 = 5.27, P < 0.001; SCH: t8 = 4.94, P < 0.01). memory revealed that the pattern shown by 14 of the 16 individual Data are means ± 1 SEM. Asterisks indicate significant differences between subjects complies with predictions of the STC hypothesis relevant to adjacent open and filled bars. The grey dashed line indicates baseline or behavior. Our unusual use of long-time course experiments on LTP chance level. in vitro (most L-LTP studies end at ∼3 h) reveals parallels between

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1008638107 Wang et al. Downloaded by guest on October 1, 2021 1 the physiological and behavioral domains, and offers further sup- 80 2 port for the synaptic plasticity and memory hypothesis (5). 3 4 5 60 6 Behavioral Model of Everyday Memory. At a time when striking 7 8 observations in molecular biology are having an impact on 40 9 10 neuroscience, it is important to recognize the value of innovative 11 behavioral paradigms to realize a deep understanding of in- 12 % of correct20 digging 13 formation processing in the brain. Standard tasks involving fear 14 15 conditioning, inhibitory avoidance, and many others remain ana- 16 0 lytically useful, but they do not capture a key aspect of how Good Poor Rescued memory functions with respect to the continuity of experience. The “discrete” nature of so many protocols has misled us into thinking Fig. 5. Meta-analysis of individual animal data. Individual performance there is an inexorable sequence of encoding, storage and then shows V-shaped pattern of 24-h memory across the good, poor, and rescued consolidation for all aspects of LTM. In our new event arena, the categories. Examples are as follows: strong encoding that renders memory animal learns new information each day but does so against persistence (good); weak encoding that renders forgetting (poor); and con- a background of other events happening before or after, such as ditions in which novelty exploration enhances poor memory persistence (res- unexpected novelty, a phenomenon analogous to inducing weak cued). Of the 16 animals, 14 displayed a V-shaped function (described in text). LTP around the same time as strong tetanization of independent afferents. The persistence of memory and of synaptic potentiation ways: First, variation in reward magnitude (one vs. three pellets) is dramatically affected. This protocol is unique in being charac- affected memory persistence in a D1/D5-receptor–dependent terized by the continuity of daily one-trial learning coupled to manner. A relatively high dose of SCH23390 was required to occasional neuromodulatory events. block 24-h memory of spatial location after three pellets. Second, One limitation of human studies of “flashbulb memory” has novelty exploration after one-pellet memory encoding induced been the difficulty of establishing whether enhanced memory for LTM, and this could be blocked by a low dose of the antagonist. associated inconsequential events is due to repeated recollection When LTM after three-pellet encoding was blocked by a high-dose with friends (30) or to neural activity around the time of memory of SCH23390 at encoding, LTM could be rescued by 5-min novelty encoding. Because surprising events such as the 9/11 tragedy exploration. These data imply that the PRPs required for memory cannot be predicted, human studies are necessarily restricted to persistence can be supplied by other neural events happening

memory retrieval and cannot study the encoding process. Building within the novelty time window. NEUROSCIENCE an animal model of flashbulb memory is fraught with uncertain Our data, together with those of others (11, 12), therefore pro- assumptions, but the value of even limited animal work is that vide strong evidence that STC is relevant to memory. Behavioral prospective studies can be designed and thus memory encoding data that are potentially discrepant with this idea showed that prior investigated. Our animal model, together with previous studies exposure to a novel taste was insufficient to rescue an impairment in (12, 13), establishes that repeated memory retrieval is not neces- latent inhibition for conditioned taste aversion caused by the in- sary for this rescue of incidental memory in animals. It follows that hibition of protein synthesis in the gustatory cortex (14). This could neural activity at or around encoding is critical. Furthermore, our be because the unknown PRPs up-regulated by the novel taste may use of a within-subjects design with repeated experiences across not have lasted long enough for the learning taste to capture days revealed a consistency in individual subjects across conditions 100 min later, or because the novel and learning tastes engage only in which good, poor, and rescued memory was predicted. This partially overlapping populations of neurons (43). behavioral protocol will therefore be ideal, in the way that cross- sectional paradigms are not, for detailed analysis of underlying Alternative Account of Novelty-Induced Memory Enhancement. A molecular events using inducible gene targeting or optogenetics possible alternative account of our data might be in terms of (31, 32). We are presently adapting the event arena task for mice. a novelty-associated modulation of memory consolidation (44). Memory modulation theory proposes that, during postlearn- Theoretical Account in Terms of STC. Novelty triggers dopamine ing consolidation, neurotransmission in the amygdala (e.g., via neuromodulation of synaptic plasticity in the hippocampus (15, 33). β-adrenergic receptors) or stress hormones (e.g., via glucocorticoid The synergistic role of dopamine and NMDA receptors for per- sistent LTP points to the necessity of D1/D5 receptor activation for receptors) can modulate the persistence of declarative memory in the availability of PRPs (23). Novelty exploration up-regulates im- other brain areas including the hippocampus (45, 46). This theory mediate early genes (17, 34, 35), and consequently the synthesis and might be extended to include novelty and thus, although developed distribution of PRPs. Our results suggest that this time window is at in the context of emotional learning protocols, might still be rel- least between 1 h before and 30 min after encoding, consistent with evant to our data. A key difference between this and our STC other studies (12). approach is that it does not require the concept of synaptic tags (1). We recognize that novelty exploration may up-regulate neu- The novelty-enhanced memory persistence echoes studies in fi freely moving animals revealing that novelty exploration during rotransmission in the amygdala suf cient to modulate the con- and after LTP induction promotes the persistence of synaptic solidation of memory encoded elsewhere (e.g., in hippocampus). potentiation (36–38). In addition, Li et al. have separately shown However, memory modulation theory does not predict two key that novelty exploration lowers the threshold for LTP induction features of our data that may be better explained by STC theory. occurring 5 min later (39). However, the mechanisms for facili- First, intrahippocampal SCH23390 during novelty exploration tating induction are likely to differ from those for enhancing per- blocked the persistence of place memory. As dopamine fibers to sistence. This is because the facilitation of LTP induction by the hippocampus originate in the VTA, not the amygdala, it is novelty fails to occur when novelty is presented 30 min before LTP more natural to see our data as fitting the Lisman and Grace induction but the enhancement of LTP persistence still occurs theory of novelty-associated dopamine neuromodulation (15). (38). Moreover, the effect of novelty on LTP persistence is also Second, when exploration was scheduled to occur before memory seen when novelty is scheduled after LTP induction (37). encoding, a rescue of memory blocked by SCH23390 was still Dopamine activity has also been shown to be critical in observed. Given the emphasis on postencoding events in the several instances of reward learning (40). Previous work from our modulation account (44), it cannot coherently be extended to ex- laboratory has shown that, whereas LTM is impaired, short-term plain events occurring before memory encoding. We therefore spatial memory (20–30 min) is unaffected by hippocampal D1/D5 reemphasize the symmetry feature of STC theory with respect to receptor blockade during encoding in both the watermaze (41) and whether the up-regulation of PRPs occurs before or after the event event arena (42). Our data from the current study add to this in two associated with the setting of synaptic tags (47).

Wang et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 Conclusion Materials and Methods Through conjoint use of directly analogous physiological and For full details, see SI Materials and Methods. behavioral studies, we have revealed a similarity between STC and what Moncada and Viola (11) have aptly called “behavioral Subjects, Infusions, and Procedures. Adult male Lister Hooded rats were used tagging” at both the phenomenonological level (Figs. 1 and 2 and for behavior studies and Wistar rats were used for electrophysiology. All fi Fig. S3) and the level of biological mechanisms (Figs. 3 and 4). procedures followed the UK Home Of ce regulations. Rats were chronically fl implanted with cannulas aiming at the dorsal hippocampus (Fig. S7). Behavioral tagging will in uence theories of memory processing SCH23390 (1 μg/μlor3.3μg/μl), Anisomycin (125 μg/μl), D-AP5 (5.9 μg/μl) or because animal experiments in which individual training expe- vehicle were infused at 0.25 μl/min (by R.L.R. and blind to S.-H.W., who ran riences occur in isolation are artificial. Events, and the cognitive the behavior). Training consisted of habituation, pretraining, and one-trial processes set in train by their encoding as memory traces, take spatial task (Fig. S2). The six conditions, described in the main text, were place seamlessly against a background of other neural events that then conducted (Tables S1 and S2). may affect the fate of such memories via the interplay of con- solidation mechanisms. Our unique everyday memory task cap- ACKNOWLEDGMENTS. We thank Patrick Spooner for event arena construc- tures this essential and ubiquitous feature of memory, and will tion and software, and Kat Berry, Olivia Haggis, Hania Koever, Tom Miller, and Zoe Richmond for pilot studies. This work was supported by the Medical enable further investigation of the diverse determinants and Research Council (United Kingdom), the Human Frontier Science Program, triggers of the neural mechanisms of memory consolidation. and Volkswagen Stiftung.

1. Frey U, Morris RGM (1997) Synaptic tagging and long-term potentiation. Nature 385: 26. Martin KC, et al. (1997) -specific, long-term facilitation of aplysia sensory to 533–536. motor synapses: A function for local protein synthesis in memory storage. Cell 91: 2. Frey U, Morris RGM (1998b) Synaptic tagging: Implications for late maintenance of 927–938. hippocampal long-term potentiation. Trends Neurosci 21:181–188. 27. Frost WN, Castellucci VF, Hawkins RD, Kandel ER (1985) Monosynaptic connections 3. Bliss TVP, Collingridge GL (1993) A synaptic model of memory: Long-term potentiation in made by the sensory neurons of the gill- and siphon-withdrawal reflex in Aplysia the hippocampus. Nature 361:31–39. participate in the storage of long-term memory for sensitization. Proc Natl Acad Sci 4. Malinow R, Malenka RC (2002) AMPA receptor trafficking and synaptic plasticity. USA 82:8266–8269. Annu Rev Neurosci 25:103–126. 28. Wang SH, de Oliveira Alvares L, Nader K (2009) Cellular and systems mechanisms of 5. Martin SJ, Grimwood PD, Morris RGM (2000) Synaptic plasticity and memory: An memory strength as a constraint on auditory fear reconsolidation. Nat Neurosci 12: – evaluation of the hypothesis. Annu Rev Neurosci 23:649–711. 905 912. fl 6. McNaughton BL, Morris RGM (1987) Hippocampal synaptic enhancement and 29. Schacter DL (2001) The Seven Sins of Memory (Houghton Mif in, New York), pp 1–272. information storage within a distributed memory system. Trends Neurosci 10:408–415. 30. Neisser U (1982) Snapshots or Benchmarks? Memory Observed: Remembering in 7. Barnes CA (1979) Memory deficits associated with senescence: A neurophysiological Natural Contexts, ed Neisser U (Freeman, San Francisco). and behavioral study in the rat. J Comp Physiol Psychol 93:74–104. 31. Gradinaru V, et al. (2010) Molecular and cellular approaches for diversifying and 8. Bast T, da Silva BM, Morris RGM (2005) Distinct contributions of hippocampal NMDA extending optogenetics. Cell 141:154–165. and AMPA receptors to encoding and retrieval of one-trial place memory. J Neurosci 32. Mayford M, Kandel ER (1999) Genetic approaches to memory storage. Trends Genet 25:5845–5856. 15:463–470. 9. Day M, Langston R, Morris RGM (2003) Glutamate-receptor-mediated encoding and 33. Gasbarri A, Verney C, Innocenzi R, Campana E, Pacitti C (1994) Mesolimbic – retrieval of paired-associate learning. Nature 424:205 209. dopaminergic neurons innervating the hippocampal formation in the rat: A – 10. Brown R, Kulik J (1977) Flashbulb memories. Cognition 5:73 99. combined retrograde tracing and immunohistochemical study. Brain Res 668:71–79. 11. Stratton GM (1919) Retroactive hypermnesia and other emotional effects on memory. 34. Guzowski JF, et al. (2006) Recent behavioral history modifies coupling between cell – Psychol Rev 26:474 486. activity and Arc gene transcription in hippocampal CA1 neurons. Proc Natl Acad Sci 12. Moncada D, Viola H (2007) Induction of long-term memory by exposure to novelty USA 103:1077–1082. – requires protein synthesis: Evidence for a behavioral tagging. J Neurosci 27:7476 7481. 35. Vazdarjanova A, McNaughton BL, Barnes CA, Worley PF, Guzowski JF (2002) 13. BallariniF, MoncadaD, Martinez MC, AlenN, Viola H (2009) Behavioral tagging is a general Experience-dependent coincident expression of the effector immediate-early genes mechanism of long-term memory formation. Proc Natl Acad Sci USA 106:14599–14604. arc and Homer 1a in hippocampal and neocortical neuronal networks. J Neurosci 22: 14. Merhav M, Rosenblum K (2008) Facilitation of taste memory acquisition by experiencing 10067–10071. previous novel taste is protein-synthesis dependent. Learn Mem 15:501–507. 36. Davis CD, Jones FL, Derrick BE (2004) Novel environments enhance the induction and 15. Lisman JE, Grace AA (2005) The hippocampal-VTA loop: Controlling the entry of maintenance of long-term potentiation in the dentate gyrus. J Neurosci 24: information into long-term memory. Neuron 46:703–713. 6497–6506. 16. Ihalainen JA, Riekkinen P, Jr, Feenstra MG (1999) Comparison of dopamine and 37. Kemp A, Manahan-Vaughan D (2004) Hippocampal long-term depression and long- noradrenaline release in mouse prefrontal cortex, striatum and hippocampus using term potentiation encode different aspects of novelty acquisition. Proc Natl Acad Sci microdialysis. Neurosci Lett 277:71–74. USA 101:8192–8197. 17. Guzowski JF, McNaughton BL, Barnes CA, Worley PF (1999) Environment-specific 38. Straube T, Korz V, Balschun D, Frey JU (2003) Requirement of beta-adrenergic expression of the immediate-early gene Arc in hippocampal neuronal ensembles. Nat receptor activation and protein synthesis for LTP-reinforcement by novelty in rat – Neurosci 2:1120–1124. dentate gyrus. J Physiol 552:953 960. 18. Marr D (1971) Simple memory: A theory for archicortex. Philos Trans R Soc Lond B Biol 39. Li S, Cullen WK, Anwyl R, Rowan MJ (2003) Dopamine-dependent facilitation of LTP – Sci 262:23–81. induction in hippocampal CA1 by exposure to spatial novelty. Nat Neurosci 6:526 531. fi 19. Kelleher RJ, 3rd, Govindarajan A, Tonegawa S (2004) Translational regulatory mechanisms 40. Zweifel LS, et al. (2009) Disruption of NMDAR-dependent burst ring by dopamine in persistent forms of synaptic plasticity. Neuron 44:59–73. neurons provides selective assessment of phasic dopamine-dependent behavior. Proc – 20. Redondo RL, et al. (2010) Synaptic tagging and capture: Differential role of distinct Natl Acad Sci USA 106:7281 7288. 41. O’Carroll CM, Martin SJ, Sandin J, Frenguelli B, Morris RGM (2006) Dopaminergic calcium/calmodulin kinases in protein synthesis-dependent long-term potentiation. modulation of the persistence of one-trial hippocampus-dependent memory. Learn J Neurosci 30:4981–4989. Mem 13:760–769. 21. Frey U, Morris RGM (1998a) Weak before strong: Dissociating synaptic tagging and 42. Bethus I, Tse D, Morris RGM (2010) Dopamine and memory: Modulation of the plasticity-factor accounts of late-LTP. Neuropharmacology 37:545–552. persistence of memory for novel hippocampal NMDA receptor-dependent paired 22. Frey U, Matthies H, Reymann KG, Matthies H (1991) The effect of dopaminergic D1 associates. J Neurosci 30:1610–1618. receptor blockade during tetanization on the expression of long-term potentiation in 43. Wang SH, Morris RGM (2010) Hippocampal-neocortical interactions in memory – the rat CA1 region in vitro. Neurosci Lett 129:111 114. formation, consolidation, and reconsolidation. Annu Rev Psych 61:49–79, C41–C44. 23. Navakkode S, Sajikumar S, Frey JU (2007) Synergistic requirements for the induction 44. McGaugh JL (2000) Memory—a century of consolidation. Science 287:248–251. of dopaminergic D1/D5-receptor-mediated LTP in hippocampal slices of rat CA1 in 45. Cahill L, McGaugh JL (1998) Mechanisms of emotional arousal and lasting declarative vitro. Neuropharmacology 52:1547–1554. memory. Trends Neurosci 21:294–299. 24. Sajikumar S, Frey JU (2004) Late-associativity, synaptic tagging, and the role of 46. Gold PE, McGaugh JL (1975) A single-trace, two-process view of memory storage dopamine during LTP and LTD. Neurobiol Learn Mem 82:12–25. processes. Short-Term Memory, eds Deutsch D, Deutsch JA (Academic, New York), pp 25. Clark GA, Kandel ER (1993) Induction of long-term facilitation in Aplysia sensory 355–378. neurons by local application of serotonin to remote synapses. Proc Natl Acad Sci USA 47. Viosca J, Jancic D, López-Atalaya JP, Benito E (2007) Hunting for synaptic tagging and 90:11411–11415. capture in memory formation. J Neurosci 27:12761–12763.

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