PNAS PLUS
Extinction learning, which consists of the inhibition of retrieval, can be learned without retrieval Jociane de Carvalho Myskiw1, Cristiane Regina Guerino Furini, Bianca Schmidt, Flávia Ferreira, and Ivan Izquierdo1 National Institute of Translational Neuroscience, National Research Council of Brazil, and Memory Center, Brain Institute of Rio Grande do Sul, Pontifical Catholic University of Rio Grande do Sul, 90610-000 Porto Alegre, RS, Brazil
In the present study we test the hypothesis that extinction is not a consequence of retrieval in unreinforced conditioned stimulus (CS) presentation but the mere perception of the CS in the absence of a conditioned response. Animals with cannulae implanted in the CA1 region of hippocampus were subjected to extinction of contextual fear conditioning. Muscimol infused intra-CA1 before an extinction training session of contextual fear conditioning (CFC) blocks retrieval but not consolidation of extinction measured 24 h later. Additionally, this inhibition of retrieval does not affect early persistence of extinction when tested 7 d later or its spontaneous recovery after 2 wk. Furthermore, both anisomycin, an inhibitor of ribosomal protein synthesis, and rapamycin, an inhibitor of extraribosomal protein synthesis, given into the CA1, impair extinction of CFC regardless of whether its retrieval was blocked by muscimol. Therefore, retrieval performance in the first unreinforced session is not necessary for the installation, maintenance, or spontaneous recovery of extinction of CFC.
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contextual fear conditioning fear extinction unreinforced conditioned stimulus
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| hippocampus | retrieval |
n animal experiments, retrieval can be defined as the behavioral expression of recalled memories; the actual performance of retrieval is usually thought to destabilize memories and trigger two opposite protein synthesis-dependent processes: reconsolidation (1–7) and extinction (8–13). Reconsolidation is viewed as a consequence of the labilization of consolidated memories at the time of the unreinforced retrieval, which renders them open to strengthening and updating (3–7, 14–20), whereas extinction is viewed as a form of learning to inhibit retrieval of original memory (8–12). Pavlov observed more than a century ago (8) that extinguished responses can recover spontaneously with the passage of time, which indicates that extinction does not erase memories. This was corroborated and expanded by Konorski (9) and Rescorla (10, 11). In contextual fear conditioning (CFC), animals learn to associate a context used as a conditioned stimulus (CS) and relatively mild foot shocks used as an unconditioned stimulus (US). The conditioned response (CR), usually measured, is the increase of the time spent freezing in unreinforced sessions carried out later. Most accounts consider that extinction begins in the first unreinforced retrieval session because of labilization of the memory (3, 14, 15), or by the mismatch between what the animals expect and what really happens at the time of retrieval (19), or both. However, it is always possible that, alternatively, the CS itself in the absence of a CR may trigger memory labilization (3) and make it susceptible both to extinction (13) and to its counterpart, reconsolidation (14, 20). Bermudez-Rattoni and his group showed that the performance of unreinforced retrieval is not necessary to trigger reconsolidation of conditioned taste aversion (5, 21) or of object recognition learning (6, 16). The methodology used by this group is very straightforward: it consists of the pharmacological blockade of retrieval with microinfusions of ciano-nitro-quinoxaline-dione, an antagonist of AMPA receptors (16, 21), or of the GABAA receptor agonist muscimol (Mus) (5) given into the basolateral www.pnas.org/cgi/doi/10.1073/pnas.1423465112
amygdala (BLA) for conditioned taste aversion or into the perirhinal cortex for object recognition (6). In both tasks, the drugs selectively blocked retrieval but spared reconsolidation, suggesting that the two neural processes are independent from each other (16, 21). In contrast, a glutamate NMDA receptor antagonist blocked the reconsolidation of conditioned taste aversion when given into the BLA (21) and that of object recognition when infused into the perirhinal cortex (16). In the present study we test the hypothesis that extinction is not a consequence of retrieval in unreinforced CS presentations (13, 22) but to the mere perception of the CS in the absence of a CR. This is important because, depending on the answer, the origin of both postretrieval processes (extinction and perhaps reconsolidation) will have to be searched for in sensory or sensory-motor rather than in cognitive or behavioral events, and the unstabilization of the memory being studied may depend on the former. Results Effect of Mus Given into the Hippocampus Before or After the Extinction Training Session. Animals received intra-CA1 infusions
of vehicle (Veh) or Mus (0.01 μg per side) 10 min before or immediately after the extinction training session (Ext Tr) of CFC, and 24 h later they were subjected to a 3-min extinction retention test (Ext Test) (23). As shown in Fig. 1A, animals that received Mus into the CA1 before the Ext Tr expressed less freezing behavior compared with the Veh-treated animals during the Ext Tr. However, both groups (Veh and Mus) exhibited similar levels of freezing during the extinction retention test, indicating that even in the absence of retrieval, animals that received intra-CA1 infusion of Mus were able to learn the extinction of CFC. As can be seen in Fig. 1B, animals that received Mus intraCA1 immediately after the Ext Tr expressed the same freezing behavior in the Ext Test as the Veh group, indicating that Mus does not affect the consolidation of the extinction of CFC. The findings on retrieval of the original task are in agreement with those observed by Raybuck and Lattal (24), in which Mus given into the hippocampus inhibits the retrieval of other forms of memory; with those of Bermudez-Rattoni’s group that the Significance Blockade of the retrieval of contextual fear conditioning by intrahippocampal muscimol administration does not impede extinction of the task measured up to 1 wk later, its eventual spontaneous recovery at 14 d, or its inhibition by two different protein synthesis inhibitors given into the hippocampus. These results show that extinction and retrieval are separate processes and strongly suggest that extinction is triggered or gated by the conditioned stimulus even in the absence of retrieval. Author contributions: J.d.C.M., C.R.G.F., and I.I. designed research; J.d.C.M., C.R.G.F., B.S., and F.F. performed research; J.d.C.M., C.R.G.F., B.S., and I.I. analyzed data; and J.d.C.M., C.R.G.F., and I.I. wrote the paper. The authors declare no conflict of interest. 1
To whom correspondence may be addressed. Email: [email protected] or [email protected].
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Contributed by Ivan Izquierdo, December 9, 2014 (sent for review December 1, 2014)
Fig. 1. Effect of Mus given into the hippocampus before or after the extinction training session. Animals were subjected to a CFC task. After 24 h they received intra-CA1 infusion of Veh or Mus (0.01 μg per side) 10 min before (A) or immediately after (B) the Ext Tr of CFC, and then were subjected to a 3-min Ext Test 24 hours later. (A) The retrieval of CFC was blocked by Mus, expressed as a decrease of freezing behavior during the Ext Tr compared with the Veh group, but does not prevent the extinction learning. (B) Animals that received Mus immediately after the Ext Tr expressed the same freezing behavior in the Ext Test as the Veh group. The figure shows the percentage of time spent freezing in the first 2 min of the CFC session, in the first 3 min and last 3 min of the Ext Tr, and in the Ext Test. Data are expressed as mean ± SEM (n = 10–12 animals per group). ***P < 0.001 vs. Veh group in the first 3 min of the Ext Tr, Newman–Keuls test after one-way ANOVA. (Upper) Schematic representation of the behavioral protocol used.
same drug given into other brain nuclei blocks retrieval of conditioned taste aversion (6); with those of Rosa et al. (25) on the blockade of retrieval of fear memory with Mus infused into the nucleus of the tractus solitaries; and with those of Quirk and his group infusing Mus into the basolateral amygdala or into the infra-limbic prefrontal cortex for the expression of fear extinction (26). The results, when put together, suggest a multicenter GABAA modulation of retrieval reminiscent of the multicenter GABAA modulation of consolidation suggested 20 years ago by Brioni (27). GABA is the main inhibitory neurotransmitter in the brain, and its effect on GABAA receptors is mimicked by Mus. Effect of Mus Given into the Hippocampus Before the Extinction Training Session When the Retention Test Takes Place 7 or 14 d Later. Animals received intra-CA1 infusions of Veh or Mus
(0.01 μg per side) 10 min before the Ext Tr of CFC, and 7 or 14 d later they were subjected to a 3-min Ext Test (23). As shown in Fig. 2, animals treated with Mus before the Ext Tr and subjected to the Ext Test 7 (Fig. 2A) or 14 d (Fig. 2B) later expressed levels of freezing behavior similar to those of the Veh groups, indicating that Mus does not affect the persistence of the extinction of CFC at 7 d after Ext Tr, nor its spontaneous recovery at 14 d. These results suggest that, whatever the mechanism for the early persistence of extinction is, or that of its spontaneous recovery after 2 wk, the inhibition of retrieval caused by Mus in the session in which it was given does not affect extinction persistence or spontaneous recovery. Effect of Anisomycin and Rapamycin Given into the Hippocampus After the Extinction Training Session Regardless of the Effect of Mus. Animals received intra-CA1 infusions of Veh or Mus
(0.01 μg per side) 10 min before the Ext Tr of CFC and also received Veh or anisomycin (Ani; 80 μg per side) (Fig. 3A) or rapamycin (Rapa; 5 pg per side) (Fig. 3B) immediately after that. Twenty-four hours later animals were subjected to a 3-min Ext Test (23). As shown in Fig. 3, animals treated with Veh 10 min before plus Ani (Fig. 3A) or Rapa (Fig. 3B) immediately after the Ext Tr expressed higher levels of freezing behavior than the Veh group during the Ext Test, and the same effect was seen in the group of animals that received Mus 10 min before plus Ani or Rapa immediately after the Ext Tr, indicating that even in the absence of retrieval, Ani and Rapa were able to impair the consolidation of the extinction of CFC. 2 of 4 | www.pnas.org/cgi/doi/10.1073/pnas.1423465112
Discussion Here we have demonstrated that the infusion of Mus into the CA1 region of the hippocampus before the extinction training session blocks the retrieval of CFC, as it does with that of other tasks, depending on where it is given (5, 6, 24, 25). Additionally, we showed here that the infusion of Mus intra-CA1 after the extinction training session of CFC does not disrupt the consolidation of extinction, which in relation to the participation of GABA-mediated transmission might take place predominantly elsewhere (ventromedial prefrontal cortex, basolateral amygdala; see refs. 12, 23, and 28). The intra-CA1 infusion of Ani or Rapa after the extinction training session blocks CFC extinction, as previously shown, indicating that this form of learning requires both ribosomal and mTOR (mammalian target of rapamycin)mediated protein synthesis in the hippocampus (29, 30). These findings indicate that behavioral expression during an unreinforced retrieval session is not necessary for the initiation, maintenance, or spontaneous recovery of the extinction learning, as was previously thought (14, 15). Instead, they suggest that the mere perception of the CS, at least in the absence of retrieval, could well be necessary for extinction to start or persist. Perception of the CS (in this case, the context) would not, of course, be expected to be hindered by Mus given outside the sensory pathways involved. Thus, perception of the CS seems to be far more important for the triggering of extinction learning than the behavioral response to it. Unless some other subbehavioral correlate of retrieval is found, this seems to be the simplest and most parsimonious explanation for the present results and perhaps also for those of Balderas et al. (6), Santoyo-Zedillo et al. (16), and García de la Torre et al. (21) on reconsolidation. When our results are put together with those obtained by Bermudez-Rattoni and his group in two other different tasks (5, 6, 16, 21), it seems clear that extinction and reconsolidation must be considered under a new light: perhaps not as opposite behavioral processes that can be triggered by retrieval (14) but possibly as part of a continuum or a diversity of protein synthesismediated events, some of which may indeed overlap (31), and that can be set into motion by the simple perception of the CS or CSs, or some other event other than the performance or the inhibition of performance of a behavioral response. Both extinction and reconsolidation require two sets of protein synthesis-mediated events, one ribosomal and sensitive to Ani, and the other presumably dendritic and sensitive to Rapa de Carvalho Myskiw et al.
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hippocampal pyramidal cells have been described by Frey and Morris (33, 34), and since then by others (35–40), as part of the process of synaptic tagging and capture believed to play a key role in the maintenance and interaction of memories (33–40). Materials and Methods Animals. Male Wistar rats (3 mo old, 300–330 g), purchased from Centro de Reprodução e Experimentação de Animais de Laboratorio of the Universidade Federal do Rio Grande do Sul (our regular provider), were used. Animals were housed four to a cage with free access to food and water, under a 12-h light/dark cycle (lights on at 7:00 AM). The temperature of the animals’ room was maintained at 22–24 °C. All procedures are in accordance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Bioethics Committee of the Pontifical Catholic University of Rio Grande do Sul.
PSYCHOLOGICAL AND COGNITIVE SCIENCES
Surgery. Animals were anesthetized with i.p. injections of a mixture of ketamine (75 mg/kg) and xylazine (10 mg/kg) and implanted with a 22-gauge
Fig. 2. Effect of Mus into the hippocampus before the extinction training session on the extinction retention test 7 or 14 d later. Animals were subjected to a CFC task. After 24 h they received intra-CA1 infusion of Veh or Mus (0.01 μg per side) 10 min before the Ext Tr, and were subjected to the Ext Test 7 d (A) or 14 d (B) later. Mus did not affect the persistence or spontaneous recovery of the extinction of CFC. Data are expressed as mean ± SEM (n = 8–12 animals per group). **P < 0.01; ***P < 0.001 vs. Veh group in the first 3 min of the Ext Tr, Newman–Keuls test after one-way ANOVA. (Upper) Schematic representation of the behavioral protocol used.
(30, 32). The two are indispensable for extinction and for reconsolidation, and they may be triggered or gated by the sheer perception of the CS or by other related behaviorally unexpressed consequences of memory reactivation. The protein synthesis events involved in memory formation occur in several posttraining waves and are essential for the consolidation of the several forms of learning in which they have been identified (2, 28, 30), and take place, as far as is known, in just one wave. They are also essential for the consolidation of extinction learning (29, 30). The posttraining protein synthesis processes take place in different cell regions (the mTOR apparatus and the ribosomes), so it might be necessary to postulate that they may relate to each other by mechanisms linked to the synapses whose plastic changes mediate extinction (30) and the hitherto undescribed synaptic events involved in reconsolidation (20). Such trafficking processes between synapses and protein synthesis systems in de Carvalho Myskiw et al.
Fig. 3. Effect of Ani and Rapa given into the hippocampus after the extinction training session, regardless of the effects of Mus. Animals were subjected to a CFC task. After 24 h they received intra-CA1 infusion of Veh or Mus (0.01 μg per side) 10 min before the Ext Tr plus Veh or Ani (A; 80 μg per side) or Rapa (B; 5 pg per side) immediately after, and then were subjected to a 3-min Ext Test 24 hours later. Ani and Rapa impaired the consolidation of the extinction of CFC in both groups of animals treated with Veh or Mus before the Ext Tr. Data are expressed as mean ± SEM (n = 8–12 animals per group). ***P < 0.001 vs. Veh group in the first 3 min of the Ext Tr; ##P < 0.01, ### P < 0.001 vs. Veh group in the Ext Test, Newman–Keuls test after one-way ANOVA. (Upper) Schematic representation of the behavioral protocol used.
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bilateral guide cannula 1 mm above the dorsal CA1 area of the hippocampus (anterior, −4.2 mm; lateral, ±3.0 mm; ventral, −1.8 mm) (41). Acrylic cement was used to affix the cannulae to the skull. Animals were allowed 7 d to recover from surgery before behavioral procedures. Animals were handled once daily for 3 consecutive days, and all behavioral procedures were conducted between 8:00 and 11:00 AM. CFC Apparatus. CFC was performed in the conditioning chamber (Panlab) with aluminum walls (35 × 35 × 35 cm) and a transparent plastic front lid. The floor of the chamber consists of parallel stainless-steel grid bars. The grid was connected to a device to deliver the foot-shock presentations. The conditioning chamber was placed inside a sound-attenuating box (Panlab) with a ventilating fan. The chamber was cleaned with 70% ethanol before and after each use. The percentage of the time that the animals spent freezing in the apparatus was measured automatically by a counter connected to photocells. Freezing (no visible movement except for respiration) was scored and converted into a percentage.
of the foot shocks. Twenty-four hours later, animals were placed again in the same apparatus for a 3-min extinction retention test, with no foot shocks (23). Pharmacological Treatments. For the pharmacological treatments, animals were gently restrained by hand, and an injection needle (30 gauge) was fitted tightly into the guides, extending 1 mm from the tip of the guide cannulae. The injection needle was connected to a 10-μL Hamilton microsyringe, and the infusion was performed at a rate of 0.5 μL/30 s. The microinfusion volume used was 1 μL per side into the dorsal CA1 area of the hippocampus. After the injection the needle was left in place for 1 additional minute and then carefully withdrawn and placed on the other side. The drugs and the doses used were the agonist of GABAA receptors, Mus (0.01 μg per side); the inhibitor of ribosomal translation, Ani (80 μg per side); and the inhibitor of mTOR-mediated protein synthesis, Rapa (5 pg per side) (Sigma-Aldrich). All drugs were freshly dissolved in sterile saline 0.9%.
Extinction of CFC. For the CFC, animals were placed into the conditioning chamber, and after 2 min two electrical foot shocks (0.5 mA, 2 s) were delivered with a 30-s interval between them. Animals were removed from the conditioning chamber 30 s after the last foot shock and placed back in their home cages. After 24 h, animals were placed in the same conditioning chamber for a 20-min extinction training of CFC, with the absence
Statistical Analysis. Data are presented as means ± SEs and were analyzed statistically by one-way ANOVA followed by Newman-Keuls Test using Graphpad Prism software. P < 0.05 was considered statistically significant.
1. Pérez-Cuesta LM, Maldonado H (2009) Memory reconsolidation and extinction in the crab: Mutual exclusion or coexistence? Learn Mem 16(11):714–721. 2. Myskiw JC, et al. (2008) On the participation of mTOR in recognition memory. Neurobiol Learn Mem 89(3):338–351. 3. Sara SJ (2000) Strengthening the shaky trace through retrieval. Nat Rev Neurosci 1(3): 212–213. 4. Inda MC, Muravieva EV, Alberini CM (2011) Memory retrieval and the passage of time: From reconsolidation and strengthening to extinction. J Neurosci 31(5): 1635–1643. 5. Rodriguez-Ortiz CJ, Balderas I, Garcia-DeLaTorre P, Bermudez-Rattoni F (2012) Taste aversion memory reconsolidation is independent of its retrieval. Neurobiol Learn Mem 98(3):215–219. 6. Balderas I, Rodriguez-Ortiz CJ, Bermudez-Rattoni F (2013) Retrieval and reconsolidation of object recognition memory are independent processes in the perirhinal cortex. Neuroscience 253:398–405. 7. Schiller D, et al. (2010) Preventing the return of fear in humans using reconsolidation update mechanisms. Nature 463(7277):49–53. 8. Pavlov IP, Gantt WH, Fol’bort GV (1928) Lectures on Conditioned Reflexes (International Publishers, New York). 9. Konorski J (1948) Conditioned Reflexes and Neuron Organization (Cambridge Univ Press, Cambridge, UK). 10. Rescorla RA (2001) Experimental extinction. Handbook of Contemporary Learning Theories, eds Mowrer RR, Klein SB (Erlbaum, Mahwah, NJ), pp 119–154. 11. Rescorla RA (2004) Spontaneous recovery. Learn Mem 11(5):501–509. 12. Milad MR, Quirk GJ (2012) Fear extinction as a model for translational neuroscience: Ten years of progress. Annu Rev Psychol 63:129–151. 13. Eisenhardt D, Menzel R (2007) Extinction learning, reconsolidation and the internal reinforcement hypothesis. Neurobiol Learn Mem 87(2):167–173. 14. Nader K (2003) Memory traces unbound. Trends Neurosci 26(2):65–72. 15. Nader K, Schafe GE, Le Doux JE (2000) Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature 406(6797):722–726. 16. Santoyo-Zedillo M, Rodriguez-Ortiz CJ, Chavez-Marchetta G, Bermudez-Rattoni F, Balderas I (2014) Retrieval is not necessary to trigger reconsolidation of object recognition memory in the perirhinal cortex. Learn Mem 21(9):452–456. 17. Schiller D, Kanen JW, LeDoux JE, Monfils MH, Phelps EA (2013) Extinction during reconsolidation of threat memory diminishes prefrontal cortex involvement. Proc Natl Acad Sci USA 110(50):20040–20045. 18. Garcia-Delatorre P, Rodríguez-Ortiz CJ, Balderas I, Bermúdez-Rattoni F (2010) Differential participation of temporal structures in the consolidation and reconsolidation of taste aversion extinction. Eur J Neurosci 32(6):1018–1023. 19. Pedreira ME, Pérez-Cuesta LM, Maldonado H (2004) Mismatch between what is expected and what actually occurs triggers memory reconsolidation or extinction. Learn Mem 11(5):579–585. 20. Merlo E, Milton AL, Goozée ZY, Theobald DE, Everitt BJ (2014) Reconsolidation and extinction are dissociable and mutually exclusive processes: Behavioral and molecular evidence. J Neurosci 34(7):2422–2431. 21. Garcia-Delatorre P, Pérez-Sánchez C, Guzmán-Ramos K, Bermúdez-Rattoni F (2014) Role of glutamate receptors of central and basolateral amygdala nuclei on retrieval and reconsolidation of taste aversive memory. Neurobiol Learn Mem 111:35–40.
22. Pérez-Cuesta LM, Hepp Y, Pedreira ME, Maldonado H (2007) Memory is not extinguished along with CS presentation but within a few seconds after CS-offset. Learn Mem 14(1):101–108. 23. Fiorenza NG, Rosa J, Izquierdo I, Myskiw JC (2012) Modulation of the extinction of two different fear-motivated tasks in three distinct brain areas. Behav Brain Res 232(1):210–216. 24. Raybuck JD, Lattal KM (2014) Differential effects of dorsal hippocampal inactivation on expression of recent and remote drug and fear memory. Neurosci Lett 569:1–5. 25. Rosa J, Myskiw JC, Furini CRG, Sapiras GG, Izquierdo I (2014) Fear extinction can be made state-dependent on peripheral epinephrine: Role of norepinephrine in the nucleus tractus solitarius. Neurobiol Learn Mem 113:55–61. 26. Bravo-Rivera C, Roman-Ortiz C, Brignoni-Perez E, Sotres-Bayon F, Quirk GJ (2014) Neural structures mediating expression and extinction of platform-mediated avoidance. J Neurosci 34(29):9736–9742. 27. Brioni JD (1993) Role of GABA during the multiple consolidation of memory. Drug Dev Res 28(1):3–27. 28. Santini E, Ge H, Ren K, Peña de Ortiz S, Quirk GJ (2004) Consolidation of fear extinction requires protein synthesis in the medial prefrontal cortex. J Neurosci 24(25): 5704–5710. 29. de Carvalho Myskiw J, Benetti F, Izquierdo I (2013) Behavioral tagging of extinction learning. Proc Natl Acad Sci USA 110(3):1071–1076. 30. de Carvalho Myskiw J, Furini CRG, Benetti F, Izquierdo I (2014) Hippocampal molecular mechanisms involved in the enhancement of fear extinction caused by exposure to novelty. Proc Natl Acad Sci USA 111(12):4572–4577. 31. Almeida-Corrêa S, Amaral OB (2014) Memory labilization in reconsolidation and extinction: Evidence for a common plasticity system? J Physiol Paris 108(4-6):292–306. 32. Myskiw JC, Izquierdo I, Furini CRG (2014) Modulation of the extinction of fear learning. Brain Res Bull 105:61–69. 33. Frey U, Morris RG (1997) Synaptic tagging and long-term potentiation. Nature 385(6616):533–536. 34. Frey U, Morris RG (1998) Synaptic tagging: implications for late maintenance of hippocampal long-term potentiation. Trends Neurosci 21(5):181–188. 35. Sajikumar S, Frey JU (2004) Late-associativity, synaptic tagging, and the role of dopamine during LTP and LTD. Neurobiol Learn Mem 82(1):12–25. 36. Sajikumar S, Morris RGM, Korte M (2014) Competition between recently potentiated synaptic inputs reveals a winner-take-all phase of synaptic tagging and capture. Proc Natl Acad Sci USA 111(33):12217–12221. 37. O’Donnell C, Sejnowski TJ (2014) Selective memory generalization by spatial patterning of protein synthesis. Neuron 82(2):398–412. 38. Reymann KG, Frey JU (2007) The late maintenance of hippocampal LTP: Requirements, phases, ‘synaptic tagging’, ‘late-associativity’ and implications. Neuropharmacology 52(1):24–40. 39. Redondo RL, Morris RGM (2011) Making memories last: The synaptic tagging and capture hypothesis. Nat Rev Neurosci 12(1):17–30. 40. Almaguer-Melian W, et al. (2012) Novelty exposure overcomes foot shock-induced spatial-memory impairment by processes of synaptic-tagging in rats. Proc Natl Acad Sci USA 109(3):953–958. 41. Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic Coordinates (Academic, San Diego).
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ACKNOWLEDGMENTS. This work was supported by research grants from the National Council of Research of Brazil.
de Carvalho Myskiw et al.
Extinction learning, which consists of the inhibition of retrieval, can be learned without retrieval Jociane de Carvalho Myskiw1, Cristiane Regina Guerino Furini, Bianca Schmidt, Flávia Ferreira, and Ivan Izquierdo1 National Institute of Translational Neuroscience, National Research Council of Brazil, and Memory Center, Brain Institute of Rio Grande do Sul, Pontifical Catholic University of Rio Grande do Sul, 90610-000 Porto Alegre, RS, Brazil
In the present study we test the hypothesis that extinction is not a consequence of retrieval in unreinforced conditioned stimulus (CS) presentation but the mere perception of the CS in the absence of a conditioned response. Animals with cannulae implanted in the CA1 region of hippocampus were subjected to extinction of contextual fear conditioning. Muscimol infused intra-CA1 before an extinction training session of contextual fear conditioning (CFC) blocks retrieval but not consolidation of extinction measured 24 h later. Additionally, this inhibition of retrieval does not affect early persistence of extinction when tested 7 d later or its spontaneous recovery after 2 wk. Furthermore, both anisomycin, an inhibitor of ribosomal protein synthesis, and rapamycin, an inhibitor of extraribosomal protein synthesis, given into the CA1, impair extinction of CFC regardless of whether its retrieval was blocked by muscimol. Therefore, retrieval performance in the first unreinforced session is not necessary for the installation, maintenance, or spontaneous recovery of extinction of CFC.
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contextual fear conditioning fear extinction unreinforced conditioned stimulus
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| hippocampus | retrieval |
n animal experiments, retrieval can be defined as the behavioral expression of recalled memories; the actual performance of retrieval is usually thought to destabilize memories and trigger two opposite protein synthesis-dependent processes: reconsolidation (1–7) and extinction (8–13). Reconsolidation is viewed as a consequence of the labilization of consolidated memories at the time of the unreinforced retrieval, which renders them open to strengthening and updating (3–7, 14–20), whereas extinction is viewed as a form of learning to inhibit retrieval of original memory (8–12). Pavlov observed more than a century ago (8) that extinguished responses can recover spontaneously with the passage of time, which indicates that extinction does not erase memories. This was corroborated and expanded by Konorski (9) and Rescorla (10, 11). In contextual fear conditioning (CFC), animals learn to associate a context used as a conditioned stimulus (CS) and relatively mild foot shocks used as an unconditioned stimulus (US). The conditioned response (CR), usually measured, is the increase of the time spent freezing in unreinforced sessions carried out later. Most accounts consider that extinction begins in the first unreinforced retrieval session because of labilization of the memory (3, 14, 15), or by the mismatch between what the animals expect and what really happens at the time of retrieval (19), or both. However, it is always possible that, alternatively, the CS itself in the absence of a CR may trigger memory labilization (3) and make it susceptible both to extinction (13) and to its counterpart, reconsolidation (14, 20). Bermudez-Rattoni and his group showed that the performance of unreinforced retrieval is not necessary to trigger reconsolidation of conditioned taste aversion (5, 21) or of object recognition learning (6, 16). The methodology used by this group is very straightforward: it consists of the pharmacological blockade of retrieval with microinfusions of ciano-nitro-quinoxaline-dione, an antagonist of AMPA receptors (16, 21), or of the GABAA receptor agonist muscimol (Mus) (5) given into the basolateral www.pnas.org/cgi/doi/10.1073/pnas.1423465112
amygdala (BLA) for conditioned taste aversion or into the perirhinal cortex for object recognition (6). In both tasks, the drugs selectively blocked retrieval but spared reconsolidation, suggesting that the two neural processes are independent from each other (16, 21). In contrast, a glutamate NMDA receptor antagonist blocked the reconsolidation of conditioned taste aversion when given into the BLA (21) and that of object recognition when infused into the perirhinal cortex (16). In the present study we test the hypothesis that extinction is not a consequence of retrieval in unreinforced CS presentations (13, 22) but to the mere perception of the CS in the absence of a CR. This is important because, depending on the answer, the origin of both postretrieval processes (extinction and perhaps reconsolidation) will have to be searched for in sensory or sensory-motor rather than in cognitive or behavioral events, and the unstabilization of the memory being studied may depend on the former. Results Effect of Mus Given into the Hippocampus Before or After the Extinction Training Session. Animals received intra-CA1 infusions
of vehicle (Veh) or Mus (0.01 μg per side) 10 min before or immediately after the extinction training session (Ext Tr) of CFC, and 24 h later they were subjected to a 3-min extinction retention test (Ext Test) (23). As shown in Fig. 1A, animals that received Mus into the CA1 before the Ext Tr expressed less freezing behavior compared with the Veh-treated animals during the Ext Tr. However, both groups (Veh and Mus) exhibited similar levels of freezing during the extinction retention test, indicating that even in the absence of retrieval, animals that received intra-CA1 infusion of Mus were able to learn the extinction of CFC. As can be seen in Fig. 1B, animals that received Mus intraCA1 immediately after the Ext Tr expressed the same freezing behavior in the Ext Test as the Veh group, indicating that Mus does not affect the consolidation of the extinction of CFC. The findings on retrieval of the original task are in agreement with those observed by Raybuck and Lattal (24), in which Mus given into the hippocampus inhibits the retrieval of other forms of memory; with those of Bermudez-Rattoni’s group that the Significance Blockade of the retrieval of contextual fear conditioning by intrahippocampal muscimol administration does not impede extinction of the task measured up to 1 wk later, its eventual spontaneous recovery at 14 d, or its inhibition by two different protein synthesis inhibitors given into the hippocampus. These results show that extinction and retrieval are separate processes and strongly suggest that extinction is triggered or gated by the conditioned stimulus even in the absence of retrieval. Author contributions: J.d.C.M., C.R.G.F., and I.I. designed research; J.d.C.M., C.R.G.F., B.S., and F.F. performed research; J.d.C.M., C.R.G.F., B.S., and I.I. analyzed data; and J.d.C.M., C.R.G.F., and I.I. wrote the paper. The authors declare no conflict of interest. 1
To whom correspondence may be addressed. Email: [email protected] or [email protected].
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PSYCHOLOGICAL AND COGNITIVE SCIENCES
Contributed by Ivan Izquierdo, December 9, 2014 (sent for review December 1, 2014)
Fig. 1. Effect of Mus given into the hippocampus before or after the extinction training session. Animals were subjected to a CFC task. After 24 h they received intra-CA1 infusion of Veh or Mus (0.01 μg per side) 10 min before (A) or immediately after (B) the Ext Tr of CFC, and then were subjected to a 3-min Ext Test 24 hours later. (A) The retrieval of CFC was blocked by Mus, expressed as a decrease of freezing behavior during the Ext Tr compared with the Veh group, but does not prevent the extinction learning. (B) Animals that received Mus immediately after the Ext Tr expressed the same freezing behavior in the Ext Test as the Veh group. The figure shows the percentage of time spent freezing in the first 2 min of the CFC session, in the first 3 min and last 3 min of the Ext Tr, and in the Ext Test. Data are expressed as mean ± SEM (n = 10–12 animals per group). ***P < 0.001 vs. Veh group in the first 3 min of the Ext Tr, Newman–Keuls test after one-way ANOVA. (Upper) Schematic representation of the behavioral protocol used.
same drug given into other brain nuclei blocks retrieval of conditioned taste aversion (6); with those of Rosa et al. (25) on the blockade of retrieval of fear memory with Mus infused into the nucleus of the tractus solitaries; and with those of Quirk and his group infusing Mus into the basolateral amygdala or into the infra-limbic prefrontal cortex for the expression of fear extinction (26). The results, when put together, suggest a multicenter GABAA modulation of retrieval reminiscent of the multicenter GABAA modulation of consolidation suggested 20 years ago by Brioni (27). GABA is the main inhibitory neurotransmitter in the brain, and its effect on GABAA receptors is mimicked by Mus. Effect of Mus Given into the Hippocampus Before the Extinction Training Session When the Retention Test Takes Place 7 or 14 d Later. Animals received intra-CA1 infusions of Veh or Mus
(0.01 μg per side) 10 min before the Ext Tr of CFC, and 7 or 14 d later they were subjected to a 3-min Ext Test (23). As shown in Fig. 2, animals treated with Mus before the Ext Tr and subjected to the Ext Test 7 (Fig. 2A) or 14 d (Fig. 2B) later expressed levels of freezing behavior similar to those of the Veh groups, indicating that Mus does not affect the persistence of the extinction of CFC at 7 d after Ext Tr, nor its spontaneous recovery at 14 d. These results suggest that, whatever the mechanism for the early persistence of extinction is, or that of its spontaneous recovery after 2 wk, the inhibition of retrieval caused by Mus in the session in which it was given does not affect extinction persistence or spontaneous recovery. Effect of Anisomycin and Rapamycin Given into the Hippocampus After the Extinction Training Session Regardless of the Effect of Mus. Animals received intra-CA1 infusions of Veh or Mus
(0.01 μg per side) 10 min before the Ext Tr of CFC and also received Veh or anisomycin (Ani; 80 μg per side) (Fig. 3A) or rapamycin (Rapa; 5 pg per side) (Fig. 3B) immediately after that. Twenty-four hours later animals were subjected to a 3-min Ext Test (23). As shown in Fig. 3, animals treated with Veh 10 min before plus Ani (Fig. 3A) or Rapa (Fig. 3B) immediately after the Ext Tr expressed higher levels of freezing behavior than the Veh group during the Ext Test, and the same effect was seen in the group of animals that received Mus 10 min before plus Ani or Rapa immediately after the Ext Tr, indicating that even in the absence of retrieval, Ani and Rapa were able to impair the consolidation of the extinction of CFC. 2 of 4 | www.pnas.org/cgi/doi/10.1073/pnas.1423465112
Discussion Here we have demonstrated that the infusion of Mus into the CA1 region of the hippocampus before the extinction training session blocks the retrieval of CFC, as it does with that of other tasks, depending on where it is given (5, 6, 24, 25). Additionally, we showed here that the infusion of Mus intra-CA1 after the extinction training session of CFC does not disrupt the consolidation of extinction, which in relation to the participation of GABA-mediated transmission might take place predominantly elsewhere (ventromedial prefrontal cortex, basolateral amygdala; see refs. 12, 23, and 28). The intra-CA1 infusion of Ani or Rapa after the extinction training session blocks CFC extinction, as previously shown, indicating that this form of learning requires both ribosomal and mTOR (mammalian target of rapamycin)mediated protein synthesis in the hippocampus (29, 30). These findings indicate that behavioral expression during an unreinforced retrieval session is not necessary for the initiation, maintenance, or spontaneous recovery of the extinction learning, as was previously thought (14, 15). Instead, they suggest that the mere perception of the CS, at least in the absence of retrieval, could well be necessary for extinction to start or persist. Perception of the CS (in this case, the context) would not, of course, be expected to be hindered by Mus given outside the sensory pathways involved. Thus, perception of the CS seems to be far more important for the triggering of extinction learning than the behavioral response to it. Unless some other subbehavioral correlate of retrieval is found, this seems to be the simplest and most parsimonious explanation for the present results and perhaps also for those of Balderas et al. (6), Santoyo-Zedillo et al. (16), and García de la Torre et al. (21) on reconsolidation. When our results are put together with those obtained by Bermudez-Rattoni and his group in two other different tasks (5, 6, 16, 21), it seems clear that extinction and reconsolidation must be considered under a new light: perhaps not as opposite behavioral processes that can be triggered by retrieval (14) but possibly as part of a continuum or a diversity of protein synthesismediated events, some of which may indeed overlap (31), and that can be set into motion by the simple perception of the CS or CSs, or some other event other than the performance or the inhibition of performance of a behavioral response. Both extinction and reconsolidation require two sets of protein synthesis-mediated events, one ribosomal and sensitive to Ani, and the other presumably dendritic and sensitive to Rapa de Carvalho Myskiw et al.
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hippocampal pyramidal cells have been described by Frey and Morris (33, 34), and since then by others (35–40), as part of the process of synaptic tagging and capture believed to play a key role in the maintenance and interaction of memories (33–40). Materials and Methods Animals. Male Wistar rats (3 mo old, 300–330 g), purchased from Centro de Reprodução e Experimentação de Animais de Laboratorio of the Universidade Federal do Rio Grande do Sul (our regular provider), were used. Animals were housed four to a cage with free access to food and water, under a 12-h light/dark cycle (lights on at 7:00 AM). The temperature of the animals’ room was maintained at 22–24 °C. All procedures are in accordance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Bioethics Committee of the Pontifical Catholic University of Rio Grande do Sul.
PSYCHOLOGICAL AND COGNITIVE SCIENCES
Surgery. Animals were anesthetized with i.p. injections of a mixture of ketamine (75 mg/kg) and xylazine (10 mg/kg) and implanted with a 22-gauge
Fig. 2. Effect of Mus into the hippocampus before the extinction training session on the extinction retention test 7 or 14 d later. Animals were subjected to a CFC task. After 24 h they received intra-CA1 infusion of Veh or Mus (0.01 μg per side) 10 min before the Ext Tr, and were subjected to the Ext Test 7 d (A) or 14 d (B) later. Mus did not affect the persistence or spontaneous recovery of the extinction of CFC. Data are expressed as mean ± SEM (n = 8–12 animals per group). **P < 0.01; ***P < 0.001 vs. Veh group in the first 3 min of the Ext Tr, Newman–Keuls test after one-way ANOVA. (Upper) Schematic representation of the behavioral protocol used.
(30, 32). The two are indispensable for extinction and for reconsolidation, and they may be triggered or gated by the sheer perception of the CS or by other related behaviorally unexpressed consequences of memory reactivation. The protein synthesis events involved in memory formation occur in several posttraining waves and are essential for the consolidation of the several forms of learning in which they have been identified (2, 28, 30), and take place, as far as is known, in just one wave. They are also essential for the consolidation of extinction learning (29, 30). The posttraining protein synthesis processes take place in different cell regions (the mTOR apparatus and the ribosomes), so it might be necessary to postulate that they may relate to each other by mechanisms linked to the synapses whose plastic changes mediate extinction (30) and the hitherto undescribed synaptic events involved in reconsolidation (20). Such trafficking processes between synapses and protein synthesis systems in de Carvalho Myskiw et al.
Fig. 3. Effect of Ani and Rapa given into the hippocampus after the extinction training session, regardless of the effects of Mus. Animals were subjected to a CFC task. After 24 h they received intra-CA1 infusion of Veh or Mus (0.01 μg per side) 10 min before the Ext Tr plus Veh or Ani (A; 80 μg per side) or Rapa (B; 5 pg per side) immediately after, and then were subjected to a 3-min Ext Test 24 hours later. Ani and Rapa impaired the consolidation of the extinction of CFC in both groups of animals treated with Veh or Mus before the Ext Tr. Data are expressed as mean ± SEM (n = 8–12 animals per group). ***P < 0.001 vs. Veh group in the first 3 min of the Ext Tr; ##P < 0.01, ### P < 0.001 vs. Veh group in the Ext Test, Newman–Keuls test after one-way ANOVA. (Upper) Schematic representation of the behavioral protocol used.
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bilateral guide cannula 1 mm above the dorsal CA1 area of the hippocampus (anterior, −4.2 mm; lateral, ±3.0 mm; ventral, −1.8 mm) (41). Acrylic cement was used to affix the cannulae to the skull. Animals were allowed 7 d to recover from surgery before behavioral procedures. Animals were handled once daily for 3 consecutive days, and all behavioral procedures were conducted between 8:00 and 11:00 AM. CFC Apparatus. CFC was performed in the conditioning chamber (Panlab) with aluminum walls (35 × 35 × 35 cm) and a transparent plastic front lid. The floor of the chamber consists of parallel stainless-steel grid bars. The grid was connected to a device to deliver the foot-shock presentations. The conditioning chamber was placed inside a sound-attenuating box (Panlab) with a ventilating fan. The chamber was cleaned with 70% ethanol before and after each use. The percentage of the time that the animals spent freezing in the apparatus was measured automatically by a counter connected to photocells. Freezing (no visible movement except for respiration) was scored and converted into a percentage.
of the foot shocks. Twenty-four hours later, animals were placed again in the same apparatus for a 3-min extinction retention test, with no foot shocks (23). Pharmacological Treatments. For the pharmacological treatments, animals were gently restrained by hand, and an injection needle (30 gauge) was fitted tightly into the guides, extending 1 mm from the tip of the guide cannulae. The injection needle was connected to a 10-μL Hamilton microsyringe, and the infusion was performed at a rate of 0.5 μL/30 s. The microinfusion volume used was 1 μL per side into the dorsal CA1 area of the hippocampus. After the injection the needle was left in place for 1 additional minute and then carefully withdrawn and placed on the other side. The drugs and the doses used were the agonist of GABAA receptors, Mus (0.01 μg per side); the inhibitor of ribosomal translation, Ani (80 μg per side); and the inhibitor of mTOR-mediated protein synthesis, Rapa (5 pg per side) (Sigma-Aldrich). All drugs were freshly dissolved in sterile saline 0.9%.
Extinction of CFC. For the CFC, animals were placed into the conditioning chamber, and after 2 min two electrical foot shocks (0.5 mA, 2 s) were delivered with a 30-s interval between them. Animals were removed from the conditioning chamber 30 s after the last foot shock and placed back in their home cages. After 24 h, animals were placed in the same conditioning chamber for a 20-min extinction training of CFC, with the absence
Statistical Analysis. Data are presented as means ± SEs and were analyzed statistically by one-way ANOVA followed by Newman-Keuls Test using Graphpad Prism software. P < 0.05 was considered statistically significant.
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ACKNOWLEDGMENTS. This work was supported by research grants from the National Council of Research of Brazil.
de Carvalho Myskiw et al.
