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M1 muscarinic receptors are necessary for retrieval of remote context fear memory
Physiology & Behavior 169 (2017) 202–207
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M1 muscarinic receptors are necessary for retrieval of remote context fear memory Rafael Rodisanski Patricio 1, Juliana Carlota Kramer Soares ⁎,1, Maria Gabriela Menezes Oliveira Departamento de Psicobiologia, Universidade Federal de São Paulo - UNIFESP, São Paulo, SP, Brazil
H I G H L I G H T S • • • •
Differential role of muscarinic receptors on recent and remote memories retrieval Dicyclomine impairs the retrieval of recent and remote contextual fear conditioning. Dicyclomine impairs only retrieval of remote tone fear conditioning. The M1 muscarinic receptors are not essential for retrieval of inhibitory avoidance.
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Article history: Received 4 August 2016 Received in revised form 17 October 2016 Accepted 2 December 2016 Available online 07 December 2016 Keywords: Fear conditioning Inhibitory avoidance Memory retrieval Remote memory M1 receptor Acetylcholine
a b s t r a c t Several studies have investigated the transition of consolidation of recent memory to remote memory in aversively motivated tasks, such as contextual fear conditioning (CFC) and inhibitory avoidance (IA). However, the mechanisms that serve the retrieval of remote memories, has not yet been fully understood. Some evidences suggest that the central cholinergic system appears be involved in the modulation of these processes. Therefore, the present study aimed to investigate the effects of a pre-test administration of dicyclomine, a high-affinity M1 muscarinic receptor antagonist, on the retrieval of remote memories in fear conditioning and IA tasks. Male Wistar rats were trained, and after 1 or 28 days, the rats received dicyclomine (16 or 32 mg/kg, intraperitoneally, i.p.) and were tested in CFC, tone fear conditioning (TFC) and IA tasks. At both time intervals, 32 mg/kg dicyclomine induced impairment of CFC. In TFC task only the performance of the rats 28 days after training was impaired. The IA task was not affected in any of the studied intervals. These findings suggest a differential contribution of muscarinic receptors on recent and remote memories retrieval revealing a more generalized role in remote memory. © 2016 Elsevier Inc. All rights reserved.
1. Introduction The ability to recall and use remotely learned information is critical to survival for humans and animals, for example allowing to avoid dangerous places in the future. Thus understanding how remote emotional memories are maintained in the brain may provide important evidence about how memories for unpleasant or noxious experienced events in general are represented. Traumatic memories constitute case in which remote aversive learning impairs the quality of life of individuals. Anxiety disorders such as post-traumatic stress are characterized by retrieval of intrusive memories related to the trauma event that persist for long term after the experience [1]. The difficulty of treatment of post traumatic disorders is known [2] possibly because remote memories are ⁎ Corresponding author at: Departamento de Psicobiologia, Universidade Federal de São Paulo – UNIFESP, Rua Botucatu, 862 1° Andar, Sao Paulo, SP 04024-002, Brazil. E-mail address: [email protected] (J.C.K. Soares). 1 These authors contributed equally to this manuscript.
http://dx.doi.org/10.1016/j.physbeh.2016.12.008 0031-9384/© 2016 Elsevier Inc. All rights reserved.
more stable than recent memories [3,4]. Therefore, it is important to search the neurobiological faces of remote memories. Although much has been learned about transition of consolidation of recent memory to remote memory, the mechanisms that serve the retrieval of remote memories have not yet been fully understood. Animal models of fear conditioning are widely used in the investigation of the neurobiological mechanisms of aversive memory and have received considerable attention in the last few decades [5,6]. Generally, in this paradigm a discrete conditioned stimulus (CS, typically a tone) is paired with an aversive unconditioned stimulus (US, typically a footshock) in a given context. After the stimulus pairing, the rat displays a fear response to both the tone and the training context [7,8]. Concerning the neural circuits involved, the amygdala plays a key role in both the contextual fear conditioning (CFC) and tone fear conditioning (TFC) tasks. The hippocampus was seen to exert influence only on CFC [9]. On the other hand, several studies indicated the hippocampus has a time-dependent role and performs a transient function in memory storage [10]. Indeed, Kim & Fanselow [11] showed that electrolytic
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lesions of the hippocampus 1 day after training, but not 28 days after, abolished contextual fear memories in rats. After this influential work, other papers have showed that damage to the hippocampus preferentially disrupts recent memories while sparing remote memories using similar times intervals between training and test [12,13]. According to this view, consolidation of recent memories is dependent on activity of the hippocampus whereas the remote memories involve increased activity in a network of cortical sites [10,14]. However, there is also evidence of a permanent role of the hippocampus in contextual memory [15–17]. Inhibitory avoidance (IA) is another behavioral task widely used in the study of the neurobiology of memory associated with emotional and traumatic events [18]. Similar to fear conditioning, the rat is subjected to an aversive experience (footshock) in a given context. However, in this task the rat's response of entering into dark chamber is punished by a footshock and then the animal avoids reentering that chamber. Moreover, IA is also subjected to interference from lesions in the amygdala [19] and hippocampus [20]. Several studies have shown the involvement of the central cholinergic system in the modulation of memory processes in aversively motivated tasks, including CFC, TFA and IA (for a review see [21]). Experiments employing pharmacological manipulations suggest that the administration of muscarinic cholinergic receptor antagonists impair this kind of learning [22–24]. Studies that used dicyclomine, a M1 muscarinic receptor antagonist, with higher affinity for M1 neuronal receptors [25–27] have reported the critical role of M1 subtype muscarinic receptors in these processes [28–31]. Studies from our lab showed that the pre-training administration of dicyclomine impaired the performance of rats in both CFC and IA tasks, sparing TFC, which suggested that this antagonist exerts its effects on hippocampal-dependent tasks without modifying performance in similar tasks hippocampal-independent [28]. Subsequently, it was indicated that dicyclomine does not modify the consolidation of either CFC or IA, which showed a specific involvement of these receptors in the acquisition of the tasks [29]. In addition, it has been showed that the M1 receptor subtype predominates in the cerebral cortex, hippocampus and amygdala [32,33], areas related with the transition of recent memory to remote memory. Particularly regarding the role of M1 receptors in the retrieval of recent memory, the pre-test administration of intermediate doses of dicyclomine led to impairment in CFC but did not alter TFC or IA, suggesting that retrieval in these tasks was mediated by different neurochemical mechanisms [30]. Nevertheless, the effects of the administration of this antagonist on the retrieval of remote memory in the CFC task, which is putatively independent from hippocampal functional integrity and its effects on TFC and IA tasks cannot be inferred from recent memory studies. Therefore, the aim of the present study was to investigate the effects of dicyclomine, an antagonist with a high affinity for M1 receptors, on remote memory retrieval of CFC, TFC and IA tasks.
2. Materials and methods 2.1. Rats In this study were used 193 male Wistar rats (three- to four-month old) from the Center for the Development of Experimental Models for Medicine and Biology (Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia – CEDEME) of the Universidade Federal de São Paulo – UNIFESP. The rats were maintained under a controlled temperature (23 ± 2 °C) and 12-h light/dark cycle (lights on at 7:00 A.M.). Rat chow and water were provided ad libitum. This study was approved by the institution's Research Ethics Committee (Comitê de Ética em Pesquisa, CEP-UNIFESP #0073/09) and followed the international guidelines for rat use and care. Every effort was conducted to minimize the number of rats used.
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2.2. Drug Dicyclomine hydrochloride (Sigma) was diluted in 0.9% saline solution and injected intraperitoneally (i.p.) at a volume of 1 ml/kg. The doses were 16 and 32 mg/kg. The drug was prepared on the day of testing and maintained in a water bath at 30 °C to avoid salt precipitation. The control group rats only received a 0.9% saline solution. The doses in this study were based on previous studies, which reported the absence of effects of 2 and 8 mg/kg doses of dicyclomine in the acquisition of CFC and IA tasks. Additionally, 16 and 32 mg/kg doses induced an amnestic effect [28,30]. 2.3. Apparatus The avoidance apparatus was used for both the CFC and IA tasks. This apparatus consisted of 2 acrylic boxes measuring 22 × 21 × 22 cm connected by a guillotine door. The walls of the safe box or chamber were white, thus generating a light environment, whereas black walls in the other chamber generated a dark environment, in which the rats received the shocks. Both chamber tops were composed of clear acrylic. The floor consisted of a metal grid (0.4-cm diameter rods placed 1.2 cm apart) connected to an electric shock generator (AVS – Projetos Especiais), which produced a 0.6 mA electric current that lasted 1 s and caused a footshock. A test chamber was used for the TFC test. This apparatus consisted of a cylindrical white acrylic container that measured 35 cm in diameter × 60 cm high. Each apparatus was maintained in a different room. A buzzer outside of the IA apparatus or the test chamber produced a 60-dB tone, which was used as a CS. 2.4. Behavioral procedures In early studies we use only one context-shock pairings, which by itself could influence the remote memory retrieval [30]. Then, to compare the effects of the administration of an M1antagonist (dicyclomine) on the retrieval memory when five context-shock pairings are used, similar parameters were used in retrieval of recent and remote memory tasks. Each behavioral procedure was performed on different control and experimental groups. Prior to the behavioral procedures, the experimenter individually handled each rat for 2–3 min over 5 days for habituation. 2.4.1. Contextual fear conditioning The CFC task was performed over 2 days. On the first day (training), each rat was placed directly in the dark chamber of the avoidance apparatus. The access door to the light chamber was closed. After 2 min, the rat received 5 0.6-mA footshocks (US) over 1 s in 30-second-intervals. The rats were removed from the apparatus 1 min after the final shock. The CFC test was performed 1 day after training to assess recent memory. Other groups of animals were tested 28 days after training to assess remote memory. In the test session, the rats were randomly allocated to 3 groups (n = 8–12 per group), which received pharmacological treatment with saline or dicyclomine (16 or 32 mg/kg). Thirty minutes after the injection, each rat was placed into the identical training context, i.e., directly placed into the dark chamber of the avoidance apparatus. The access door to the light chamber remained closed. No footshocks were administered. The freezing time, which was defined as complete body immobility without whisker movement or sniffing activity [7], was recorded for 5 min. The freezing time was scored manually with a chronometer off-line from video-recordings by an experienced observer blinded to the treatment conditions. 2.4.2. Tone fear conditioning This paradigm was also performed over 2 days. On the first day (training), each rat was placed directly in the dark chamber of the avoidance apparatus. The access door to the light chamber was closed.
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After 2 min, a tone (CS) was produced for 5 s, and a 0.6-mA-intensity footshock was administered in the final second for 1 s (US) and ceased with the tone. The tone-shock pairing was repeated 5 times with an interval of 30 s between each presentation. One minute after the final shock, the rats were removed from the apparatus. The TFC test was performed 1 or 28 days after training in different rat groups. On the test day, the rats were assigned to 3 groups (n = 11–12 per group), which received pharmacological treatment with saline or dicyclomine (16 or 32 mg/kg). Thirty minutes after the injection, each rat was placed in the test chamber (a new context) for 5 min. By the end of the second minute of exposure, the CS was administered 5 times over 30 s. The freezing time was measured and recorded during the initial 2 min (before tone presentation) and final 3 min (after tone presentation). 2.4.3. Inhibitory avoidance On training day, each rat was placed in the light (safe) chamber of the apparatus with the access door to the dark chamber closed. After 10 s, the door was opened. When the rats entered the dark chamber with 4 paws, the door was closed and they received 5 0.6-mA footshocks for 1 s in 30-second intervals. The time (latency) that the rat took to enter the dark chamber with 4 paws was registered. Thirty seconds after the final footshock, the rats were removed from the apparatus. The test session was performed 1 or 28 days after the training in different rat groups. During testing, the rats were allocated to 3 groups (n = 12 per group), which received pharmacological treatment of saline or dicyclomine (16 or 32 mg/kg). Thirty minutes after injection, each rat was again placed in the light chamber of the avoidance apparatus. After 10 s, the door was opened, and the time it took for each rat to enter the dark chamber with 4 paws was recorded (test latency). A latency of 300 s was attributed in the cases in which the rats did not cross the door within 300 s. No footshock was administered during testing. 2.5. Statistical analyses The mean values of freezing time per minute of the fear conditioning tests were analyzed using two-way analyses of variance (ANOVAs) [Factor 1: Treatment (saline × dicyclomine); Factor 2: Minute (5 min)] with repeated measures for Factor 2. The latency means from the IA test were analyzed using two-way ANOVA [Factor 1: Treatment (saline × dicyclomine); Factor 2: Session (training × test)]. These analyses were followed by Tukey's multiple comparison test when necessary. A pvalue b0.05 was considered significant.
dicyclomine was not significantly different from the other groups. The Minute × Treatment interaction was not significant [F(8,128) = 1.11; p = 0.36]. Thus, these results suggested impairment in the retrieval of recent (Fig. 1A) and remote (Fig. 1B) memories of CFC after the administration of 32 mg/kg dicyclomine. 3.2. The effects of the pre-test administration of dicyclomine on the retrieval of recent and remote memories of TFC The Fig. 2A showed the results for recent memories of TFC. The statistical analysis showed significant effects for Minute [F(4,124) = 77.43; p b 0.01]. Tukey's test showed that all groups exhibited higher freezing times after tone presentation (p b 0.01). Neither the Treatment factor [F(2,31) = 1.41; p = 0.26] nor the Minute × Treatment interaction was significant [F(8,124) = 0.82; p = 0.58]. Regarding the remote memory of the task (Fig. 2B), a two-way ANOVA showed significant differences for Minute [F(4,104) = 82.16; p b 0.01]. Tukey's test showed that all groups increased their freezing times after tone administration (p b 0.01). The Treatment factor [F(2,26) = 6,59; p b 0.01] was also significant. Tukey's test showed that rats treated with 32 mg/kg dicyclomine had reduced freezing times compared to those in the control group (p b 0.01). The group that received 16 mg/kg dicyclomine was not significantly different from control group (p = 0.054). The Minute × Treatment interaction [F(8,104) = 1.89; p = 0.07] was not significant. 3.3. The effects of the pre-test administration of dicyclomine on the retrieval of recent and remote memories of IA Concerning the retrieval of recent memories for the IA task (Fig. 3A), a two-way ANOVA showed significant effects for Session [F(1,33) = 70.88; p b 0.01]. Tukey's test showed that all groups had higher latencies to enter the dark chamber on the test day (p b 0.01). The Treatment factor [F(2,33) = 0.07; p = 0.93] and Session × Treatment interaction [F(2,33) = 0.09; p = 0.92] were not significant. For the remote memory of IA (Fig. 3B), a two-way ANOVA showed a significant effect for Session [F(1,30) = 144.02; p b 0.01]. Tukey's test showed that all groups had an increased latency to enter the dark chamber during the test session (p b 0.01). Neither the Treatment factor [F(2,30) = 2.79; p = 0.08] nor the Session × Treatment interaction was significant [F(2,30) = 2.25; p = 0.12]. 4. Discussion
3. Results 3.1. The effects of the pre-test administration of dicyclomine on the retrieval of recent and remote memories of CFC Concerning the retrieval of recent memories of CFC, a two-way ANOVA showed a significant effect of the Minute [F(4,92) = 6.33; p b 0.01] and Treatment [F(2,23) = 22.07; p b 0.01] factors. The posthoc Tukey's test showed that the group treated with 32 mg/kg dicyclomine exhibited a significant reduction in freezing time compared to the other 2 groups (p b 0.01 in both cases). The rats treated with 16 mg/kg dicyclomine were not significantly different from the saline group. The Minute × Treatment interaction was also significant [F(8,92) = 2.89; p b 0.01]. Tukey's test reveal that group treated with 32 mg/kg of dicyclomine exhibited lower freezing time from the second minute onwards (p b 0.05 for second minute compared to 16 mg/kg of dicyclomine and p b 0.01 for all other minutes). A two-way ANOVA for the retrieval of remote memories of CFC showed significant effects for Minute [F(4,128) = 6.11; p b 0.01] and Treatment [F(2,32) = 4.32; p b 0.05]. Tukey's test showed that rats treated with 32 mg/kg dicyclomine had reduced freezing times compared to those in the control group (p b 0.05). The group that received 16 mg/kg
Memory is not immediately and definitely formed at the moment it is acquired; gradual reorganization and stabilization processes occur during its storage [4]. As the consolidation of hippocampal-dependent memories occur, there is a progressive decrease in the participation of this structure, and a memory system is formed independent of the hippocampus. However, memory is not literally transferred from the hippocampus to other regions, as the hippocampus actively interacts with neocortical sites, in which cortico-cortical connections are gradually developed and become responsible for the storage and retrieval of remote memories [10,14]. Considering the influence of the cholinergic system on the modulation of mnemonic processes, the present study analyzed the role of the M1 muscarinic receptors in the retrieval process of recent and remote memories of aversively motivated tasks. To our knowledge, this is the first report that successfully shows the contribution of muscarinic cholinergic system in the retrieval of remote fear memories. The results showed that systemic pre-test administration of dicyclomine, a M1-selective muscarinic receptor antagonist, impaired the retrieval of the CFC task. Such an effect, detected by the reduction in the time of freezing of the rats during the test session, occurred when the training-test interval was 1 day or 28 days. The dose of 32 mg/Kg also impaired the performance of the rats during retrieval of
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Fig. 1. Freezing time per minute (mean ± SEM) during contextual fear conditioning test. A. Recent memory (test 1 day after training). B. Remote memory (test 28 days after raining). The retrieval of recent and remote memories of CFC was impaired by treatment with 32 mg/kg of dicyclomine. “Sal”, saline; “Dic16”, dicyclomine 16 mg/kg; “Dic32”, dicyclomine 32 mg/kg. The number of animals per group is shown in parentheses. *p b 0.01 compared to Sal group (two-way ANOVA followed by Tukey test).
remote memory in TFC task. The IA task was not affected in any of the studied intervals. Previous studies in our lab suggested an involvement of the M1 receptors in the retrieval of recent memory of the CFC task because the pre-test administration of 16 and 32 mg/kg of dicyclomine impaired performance of animals [30]. In the present study, we use a protocol with 5 context-shocks pairings (to produce a persistent memory in order to investigate the effect of the drug on retrieval of the remote memory). In this case, the pre-test dicyclomine administration at dose of 16 mg/kg was not able to impair the performance of the animals. On the other hand, the administration of a higher dose (32 mg/kg) affected the conditioned freezing response to the context. In the present study, the administration of 32 mg/kg dicyclomine impaired the retrieval of recent and remote memories of CFC. Considering the current hypothesis that the hippocampus plays a transient role in memory storage and that as a new memory is consolidated it becomes dependent of other brain regions, possibly the cerebral cortex [10,34], and that M1-subtype muscarinic receptors are predominantly distributed in the cortex, hippocampus and amygdala in rats [32,33], it is not possible to determine which structures are critical for the retrieval of recent and remote memories of tasks based on a pharmacological approach alone. In any case, the activation of M1-subtype muscarinic receptors seems to be critical for both recent and remote retrieval processes. Although the TFC task had some similarities to CFC (for example, using shock as a US and the evaluation of freezing time as a conditioned response), this task was not affected by the pre-test administration of dicyclomine when the test was performed 1 day after the training. Thus, this drug specifically induced impairment in an aversively motivated hippocampal-dependent task (CFC), while sparing the performance of rats in a similar, but hippocampal-independent, task (TFC). Although both tasks are amygdala-dependent [9] and M1 receptors are observed in this region [32,33], the administration of dicyclomine distinctly affected the performance of the two tasks during recent
memory. One possibility is that the hippocampus is more susceptible to manipulations of the cholinergic system than the amygdala. The administration of dicyclomine at a dose of 32 mg/kg impaired the retrieval of remote memory of TFC. Interestingly, this task was affected by the drug only when the test was performed 28 days after training, suggesting that a change in the modulatory role of M1 receptors on retrieval after a certain time interval is needed to acquire greater relevance in remote memory. In the similar manner, that it is supposed to occur in CFC, the neurobiological substrate involved with the retrieval of recent memory appears to be different than involved in remote memory retrieval of TFC, being more sensitive to changes in muscarinic system mediated by M1 receptors. Consistent with this hypothesis, it was shown that lesions in the secondary auditory cortex of rats impaired only the remote memory retrieval of TFC, sparing recent memory [35]. In another study, the number of Zif268-positive neurons increased following remote memory, but not recent memory retrieval of TFC in the thalamus and auditory cortex [36]. It is possible to assume that this change in brain activity pattern also would be reflected in a change in modulation performed by M1 receptors, so that their activation is more relevant some long time after training. Although not possible to identify the involved brain structures, the pharmacological approach used in this study showed that the activation of the M1 receptors is important only for the remote memory retrieval of TFC. IA is similar to CFC in some aspects: the rat is subjected to an aversive experience (footshock) in a given context. In addition, IA is also sensitive to lesions in the amygdala [19] and hippocampus [20,37,]. Conversely, as the shock was applied only after the rats' response, it can be interpreted as a punishment, an element characteristic of operant or instrumental conditioning. Moreover, the neuronal mechanisms involved in the performance during these paradigms are showed to be partially distinct [21]. In general, aversive conditions that require an instrumental response are less dependent on basolateral amygdaloid nuclei [38]. Thus, investigating the effects of pharmacological manipulations on both tasks, under similar experimental conditions, can be useful
Fig. 2. Freezing time per minute (mean ± SEM) during tone fear conditioning test. A. Recent memory (test 1 day after training). B. Remote memory (test 28 days after training). Note that the treatment with 32 mg/kg of dicyclomine significantly impaired only the retrieval of remote memories of TFC relative to saline control group. “Sal”, saline; “Dic16”, dicyclomine 16 mg/kg; “Dic32”, dicyclomine 32 mg/kg. The number of animals per group is shown in parentheses. *p b 0.01 compared to Sal group (two-way ANOVA followed by Tukey test).
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Fig. 3. Latency (mean ± SEM) to cross to black compartment during training and test of the inhibitory avoidance task. A. Recent memory (test 1 day after training). B. Remote memory (test 28 days after training). The treatment did not modify the performance of animals. All group learned the task. “Sal”, saline; “Dic16”, dicyclomine 16 mg/kg; “Dic32”, dicyclomine 32 mg/kg. The number of animals per group is shown in parentheses.
for understanding the overall influence of cholinergic transmission on memory and learning. The pre-test administration of both doses of dicyclomine did not interfere with the performance of the rats during the retrieval of recent memory of IA. Despite the putative importance of the hippocampus in the performance of this task, the absence of effect could be due to the involvement of other brain structures related to the instrumental components inherent to IA, such as the striatum. However, controversial results have been reported concerning the participation of the cholinergic system in the retrieval of this behavioral paradigm. Whereas some studies have described deficits induced by the systemic administration of scopolamine a non-selective muscarinic antagonist [39], or of M1-selective muscarinic receptor antagonists, such as biperiden and trihexyphenidyl [40], other studies failed to report such deficits [29,30, 41,42]. As previously discussed in Soares et al. [30], these controversial results could be explained by differences between IA procedures adopted in those studies. Studies that observed impairment in retrieval of IA task, often used a step-down procedure, independently of muscarinic antagonist used [40,43]. On the other hand, the studies that did not find impairment have used a step-through procedure [24,29,30,41,42, 44]. Relative to the effects of dicyclomine on retrieval of remote memories (training-test interval of 28 days) of the IA task, no alteration was detected in the performance of the rats because of pharmacological treatment. Thus, the retrieval of both recent and remote memories of this behavioral paradigm was independent of the activation of the M1-subtype muscarinic receptors. Some studies have indicated the involvement of other neurotransmitter systems in the performance of this task, such as AMPA-type glutamate receptors in the amygdala related to the retrieval of recent memories and AMPA receptors in the medial prefrontal cortex related to remote memory [45]. Dicyclomine shows a higher affinity for M1 and M3 and low affinity for M2 receptors [26,27]. So it is not possible to rule out the possibility that the effect of dicyclomine is due in part to the combined inactivation of M1 and M3 receptors. Nevertheless, other studies suggest that the cerebral cortex, hippocampus and amygdala of the rat contain a mixed population of M1 and M3 receptors [32,33]. As both types of receptors mediate excitatory responses, this would explain the effect of the antagonist dicyclomine on learning and memory. Indeed, M3-muscarinic receptor knockout mice show a deficit in CFC, suggesting that M3 receptors are also important memory process [46]. However, a recent study showed that systemic administration or administration directly into basolateral amygdala of cevimeline, a new M1 selective agonist, caused an increase in freezing in response to tone [47]. Furthermore, the same study showed that knockout mice to M1 receptors injected with cevimeline did not exhibit the increase in freezing response to tone, suggesting that other muscarinic receptors, such as M3 would not be needed to promote memory consolidation and that the consolidation enhancement observed in response to administration of cevimeline is mediated specifically by M1 receptors. In the same way, the pre-training chronic administration of EUK1001, another M1
agonist, appears to improve the memory to CFC, TFC and IA suggesting a general effect on cognition [48]. Although these reports did not to assess the effects of these drugs directly on memory retrieval, taken together, these findings strengthen the main role M1 muscarinic receptors on learning and memory processes. Nevertheless, a more complex view of M1 muscarinic receptors in memory processing has been suggested. M1 mutant mice showed no evidence of increase or change in activity or shock reactivity, but showed enhanced CFC and normal TFC responses [49]. These M1 mutant mice would have a cortical memory dysfunction or impaired hippocampal – cortical interaction that would be specific to remote memory, which appears in accordance to results obtained in the present study regarding impairment of remote memory to contextual and tone conditioning. Previous results from our lab showed that dicyclomine, at the dose of 32 mg/kg, did not affect the ambulation of rats in the open field task [29]. Moreover, if dicyclomine had increased the locomotor activity impairment on the IA task would also be expected, because the animals tend to move to the dark compartment of the apparatus. Therefore the effects of dicyclomine likely not were caused by locomotion or motivational factors. Collectively, our data indicated that M1 muscarinic receptors are necessary for the retrieval of both recent and remote memories of contextual fear conditioning. In the present study, the remote memory of tone fear conditioning was also impaired. This was a surprising finding and suggests a differential contribution of muscarinic receptors on recent and remote memories revealing a more generalized role in remote memory. Acknowledgments This study was financially supported by grant 2009/12871-0, São Paulo Research Foundation (FAPESP); the Research Incentive Fund Association (Associação Fundo de Incentivo à Pesquisa – AFIP), the Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES), the National Council of Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq). The authors would like to thank Jose Bernardo Costa for technical assistance during the experiments. References [1] L.H. Tsai, J. Gräff, On the resilience of remote traumatic memories against exposure therapy-mediated attenuation, EMBO Rep. 15 (8) (2014) 853–861. [2] M.C. Kearns, K.J. Ressler, D. Zatzick, B.O. Rothbaum, Early interventions for PTSD: a review, Depress. Anxiety 29 (10) (2012) 833–842. [3] M.H. Milekic, C.M. Alberini, Temporally graded requirement for protein synthesis following memory reactivation, Neuron 36 (3) (2002) 521–525. [4] Y. Dudai, The neurobiology of consolidations, or, how stable is the engram? Annu. Rev. Psychol. 55 (2004) 51–86. [5] M. Fendt, M.S. Fanselow, The neuroanatomical and neurochemical basis of conditioned fear, Neurosci. Biobehav. Rev. 23 (5) (1999) 743–760.
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Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
M1 muscarinic receptors are necessary for retrieval of remote context fear memory Rafael Rodisanski Patricio 1, Juliana Carlota Kramer Soares ⁎,1, Maria Gabriela Menezes Oliveira Departamento de Psicobiologia, Universidade Federal de São Paulo - UNIFESP, São Paulo, SP, Brazil
H I G H L I G H T S • • • •
Differential role of muscarinic receptors on recent and remote memories retrieval Dicyclomine impairs the retrieval of recent and remote contextual fear conditioning. Dicyclomine impairs only retrieval of remote tone fear conditioning. The M1 muscarinic receptors are not essential for retrieval of inhibitory avoidance.
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Article history: Received 4 August 2016 Received in revised form 17 October 2016 Accepted 2 December 2016 Available online 07 December 2016 Keywords: Fear conditioning Inhibitory avoidance Memory retrieval Remote memory M1 receptor Acetylcholine
a b s t r a c t Several studies have investigated the transition of consolidation of recent memory to remote memory in aversively motivated tasks, such as contextual fear conditioning (CFC) and inhibitory avoidance (IA). However, the mechanisms that serve the retrieval of remote memories, has not yet been fully understood. Some evidences suggest that the central cholinergic system appears be involved in the modulation of these processes. Therefore, the present study aimed to investigate the effects of a pre-test administration of dicyclomine, a high-affinity M1 muscarinic receptor antagonist, on the retrieval of remote memories in fear conditioning and IA tasks. Male Wistar rats were trained, and after 1 or 28 days, the rats received dicyclomine (16 or 32 mg/kg, intraperitoneally, i.p.) and were tested in CFC, tone fear conditioning (TFC) and IA tasks. At both time intervals, 32 mg/kg dicyclomine induced impairment of CFC. In TFC task only the performance of the rats 28 days after training was impaired. The IA task was not affected in any of the studied intervals. These findings suggest a differential contribution of muscarinic receptors on recent and remote memories retrieval revealing a more generalized role in remote memory. © 2016 Elsevier Inc. All rights reserved.
1. Introduction The ability to recall and use remotely learned information is critical to survival for humans and animals, for example allowing to avoid dangerous places in the future. Thus understanding how remote emotional memories are maintained in the brain may provide important evidence about how memories for unpleasant or noxious experienced events in general are represented. Traumatic memories constitute case in which remote aversive learning impairs the quality of life of individuals. Anxiety disorders such as post-traumatic stress are characterized by retrieval of intrusive memories related to the trauma event that persist for long term after the experience [1]. The difficulty of treatment of post traumatic disorders is known [2] possibly because remote memories are ⁎ Corresponding author at: Departamento de Psicobiologia, Universidade Federal de São Paulo – UNIFESP, Rua Botucatu, 862 1° Andar, Sao Paulo, SP 04024-002, Brazil. E-mail address: [email protected] (J.C.K. Soares). 1 These authors contributed equally to this manuscript.
http://dx.doi.org/10.1016/j.physbeh.2016.12.008 0031-9384/© 2016 Elsevier Inc. All rights reserved.
more stable than recent memories [3,4]. Therefore, it is important to search the neurobiological faces of remote memories. Although much has been learned about transition of consolidation of recent memory to remote memory, the mechanisms that serve the retrieval of remote memories have not yet been fully understood. Animal models of fear conditioning are widely used in the investigation of the neurobiological mechanisms of aversive memory and have received considerable attention in the last few decades [5,6]. Generally, in this paradigm a discrete conditioned stimulus (CS, typically a tone) is paired with an aversive unconditioned stimulus (US, typically a footshock) in a given context. After the stimulus pairing, the rat displays a fear response to both the tone and the training context [7,8]. Concerning the neural circuits involved, the amygdala plays a key role in both the contextual fear conditioning (CFC) and tone fear conditioning (TFC) tasks. The hippocampus was seen to exert influence only on CFC [9]. On the other hand, several studies indicated the hippocampus has a time-dependent role and performs a transient function in memory storage [10]. Indeed, Kim & Fanselow [11] showed that electrolytic
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lesions of the hippocampus 1 day after training, but not 28 days after, abolished contextual fear memories in rats. After this influential work, other papers have showed that damage to the hippocampus preferentially disrupts recent memories while sparing remote memories using similar times intervals between training and test [12,13]. According to this view, consolidation of recent memories is dependent on activity of the hippocampus whereas the remote memories involve increased activity in a network of cortical sites [10,14]. However, there is also evidence of a permanent role of the hippocampus in contextual memory [15–17]. Inhibitory avoidance (IA) is another behavioral task widely used in the study of the neurobiology of memory associated with emotional and traumatic events [18]. Similar to fear conditioning, the rat is subjected to an aversive experience (footshock) in a given context. However, in this task the rat's response of entering into dark chamber is punished by a footshock and then the animal avoids reentering that chamber. Moreover, IA is also subjected to interference from lesions in the amygdala [19] and hippocampus [20]. Several studies have shown the involvement of the central cholinergic system in the modulation of memory processes in aversively motivated tasks, including CFC, TFA and IA (for a review see [21]). Experiments employing pharmacological manipulations suggest that the administration of muscarinic cholinergic receptor antagonists impair this kind of learning [22–24]. Studies that used dicyclomine, a M1 muscarinic receptor antagonist, with higher affinity for M1 neuronal receptors [25–27] have reported the critical role of M1 subtype muscarinic receptors in these processes [28–31]. Studies from our lab showed that the pre-training administration of dicyclomine impaired the performance of rats in both CFC and IA tasks, sparing TFC, which suggested that this antagonist exerts its effects on hippocampal-dependent tasks without modifying performance in similar tasks hippocampal-independent [28]. Subsequently, it was indicated that dicyclomine does not modify the consolidation of either CFC or IA, which showed a specific involvement of these receptors in the acquisition of the tasks [29]. In addition, it has been showed that the M1 receptor subtype predominates in the cerebral cortex, hippocampus and amygdala [32,33], areas related with the transition of recent memory to remote memory. Particularly regarding the role of M1 receptors in the retrieval of recent memory, the pre-test administration of intermediate doses of dicyclomine led to impairment in CFC but did not alter TFC or IA, suggesting that retrieval in these tasks was mediated by different neurochemical mechanisms [30]. Nevertheless, the effects of the administration of this antagonist on the retrieval of remote memory in the CFC task, which is putatively independent from hippocampal functional integrity and its effects on TFC and IA tasks cannot be inferred from recent memory studies. Therefore, the aim of the present study was to investigate the effects of dicyclomine, an antagonist with a high affinity for M1 receptors, on remote memory retrieval of CFC, TFC and IA tasks.
2. Materials and methods 2.1. Rats In this study were used 193 male Wistar rats (three- to four-month old) from the Center for the Development of Experimental Models for Medicine and Biology (Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia – CEDEME) of the Universidade Federal de São Paulo – UNIFESP. The rats were maintained under a controlled temperature (23 ± 2 °C) and 12-h light/dark cycle (lights on at 7:00 A.M.). Rat chow and water were provided ad libitum. This study was approved by the institution's Research Ethics Committee (Comitê de Ética em Pesquisa, CEP-UNIFESP #0073/09) and followed the international guidelines for rat use and care. Every effort was conducted to minimize the number of rats used.
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2.2. Drug Dicyclomine hydrochloride (Sigma) was diluted in 0.9% saline solution and injected intraperitoneally (i.p.) at a volume of 1 ml/kg. The doses were 16 and 32 mg/kg. The drug was prepared on the day of testing and maintained in a water bath at 30 °C to avoid salt precipitation. The control group rats only received a 0.9% saline solution. The doses in this study were based on previous studies, which reported the absence of effects of 2 and 8 mg/kg doses of dicyclomine in the acquisition of CFC and IA tasks. Additionally, 16 and 32 mg/kg doses induced an amnestic effect [28,30]. 2.3. Apparatus The avoidance apparatus was used for both the CFC and IA tasks. This apparatus consisted of 2 acrylic boxes measuring 22 × 21 × 22 cm connected by a guillotine door. The walls of the safe box or chamber were white, thus generating a light environment, whereas black walls in the other chamber generated a dark environment, in which the rats received the shocks. Both chamber tops were composed of clear acrylic. The floor consisted of a metal grid (0.4-cm diameter rods placed 1.2 cm apart) connected to an electric shock generator (AVS – Projetos Especiais), which produced a 0.6 mA electric current that lasted 1 s and caused a footshock. A test chamber was used for the TFC test. This apparatus consisted of a cylindrical white acrylic container that measured 35 cm in diameter × 60 cm high. Each apparatus was maintained in a different room. A buzzer outside of the IA apparatus or the test chamber produced a 60-dB tone, which was used as a CS. 2.4. Behavioral procedures In early studies we use only one context-shock pairings, which by itself could influence the remote memory retrieval [30]. Then, to compare the effects of the administration of an M1antagonist (dicyclomine) on the retrieval memory when five context-shock pairings are used, similar parameters were used in retrieval of recent and remote memory tasks. Each behavioral procedure was performed on different control and experimental groups. Prior to the behavioral procedures, the experimenter individually handled each rat for 2–3 min over 5 days for habituation. 2.4.1. Contextual fear conditioning The CFC task was performed over 2 days. On the first day (training), each rat was placed directly in the dark chamber of the avoidance apparatus. The access door to the light chamber was closed. After 2 min, the rat received 5 0.6-mA footshocks (US) over 1 s in 30-second-intervals. The rats were removed from the apparatus 1 min after the final shock. The CFC test was performed 1 day after training to assess recent memory. Other groups of animals were tested 28 days after training to assess remote memory. In the test session, the rats were randomly allocated to 3 groups (n = 8–12 per group), which received pharmacological treatment with saline or dicyclomine (16 or 32 mg/kg). Thirty minutes after the injection, each rat was placed into the identical training context, i.e., directly placed into the dark chamber of the avoidance apparatus. The access door to the light chamber remained closed. No footshocks were administered. The freezing time, which was defined as complete body immobility without whisker movement or sniffing activity [7], was recorded for 5 min. The freezing time was scored manually with a chronometer off-line from video-recordings by an experienced observer blinded to the treatment conditions. 2.4.2. Tone fear conditioning This paradigm was also performed over 2 days. On the first day (training), each rat was placed directly in the dark chamber of the avoidance apparatus. The access door to the light chamber was closed.
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After 2 min, a tone (CS) was produced for 5 s, and a 0.6-mA-intensity footshock was administered in the final second for 1 s (US) and ceased with the tone. The tone-shock pairing was repeated 5 times with an interval of 30 s between each presentation. One minute after the final shock, the rats were removed from the apparatus. The TFC test was performed 1 or 28 days after training in different rat groups. On the test day, the rats were assigned to 3 groups (n = 11–12 per group), which received pharmacological treatment with saline or dicyclomine (16 or 32 mg/kg). Thirty minutes after the injection, each rat was placed in the test chamber (a new context) for 5 min. By the end of the second minute of exposure, the CS was administered 5 times over 30 s. The freezing time was measured and recorded during the initial 2 min (before tone presentation) and final 3 min (after tone presentation). 2.4.3. Inhibitory avoidance On training day, each rat was placed in the light (safe) chamber of the apparatus with the access door to the dark chamber closed. After 10 s, the door was opened. When the rats entered the dark chamber with 4 paws, the door was closed and they received 5 0.6-mA footshocks for 1 s in 30-second intervals. The time (latency) that the rat took to enter the dark chamber with 4 paws was registered. Thirty seconds after the final footshock, the rats were removed from the apparatus. The test session was performed 1 or 28 days after the training in different rat groups. During testing, the rats were allocated to 3 groups (n = 12 per group), which received pharmacological treatment of saline or dicyclomine (16 or 32 mg/kg). Thirty minutes after injection, each rat was again placed in the light chamber of the avoidance apparatus. After 10 s, the door was opened, and the time it took for each rat to enter the dark chamber with 4 paws was recorded (test latency). A latency of 300 s was attributed in the cases in which the rats did not cross the door within 300 s. No footshock was administered during testing. 2.5. Statistical analyses The mean values of freezing time per minute of the fear conditioning tests were analyzed using two-way analyses of variance (ANOVAs) [Factor 1: Treatment (saline × dicyclomine); Factor 2: Minute (5 min)] with repeated measures for Factor 2. The latency means from the IA test were analyzed using two-way ANOVA [Factor 1: Treatment (saline × dicyclomine); Factor 2: Session (training × test)]. These analyses were followed by Tukey's multiple comparison test when necessary. A pvalue b0.05 was considered significant.
dicyclomine was not significantly different from the other groups. The Minute × Treatment interaction was not significant [F(8,128) = 1.11; p = 0.36]. Thus, these results suggested impairment in the retrieval of recent (Fig. 1A) and remote (Fig. 1B) memories of CFC after the administration of 32 mg/kg dicyclomine. 3.2. The effects of the pre-test administration of dicyclomine on the retrieval of recent and remote memories of TFC The Fig. 2A showed the results for recent memories of TFC. The statistical analysis showed significant effects for Minute [F(4,124) = 77.43; p b 0.01]. Tukey's test showed that all groups exhibited higher freezing times after tone presentation (p b 0.01). Neither the Treatment factor [F(2,31) = 1.41; p = 0.26] nor the Minute × Treatment interaction was significant [F(8,124) = 0.82; p = 0.58]. Regarding the remote memory of the task (Fig. 2B), a two-way ANOVA showed significant differences for Minute [F(4,104) = 82.16; p b 0.01]. Tukey's test showed that all groups increased their freezing times after tone administration (p b 0.01). The Treatment factor [F(2,26) = 6,59; p b 0.01] was also significant. Tukey's test showed that rats treated with 32 mg/kg dicyclomine had reduced freezing times compared to those in the control group (p b 0.01). The group that received 16 mg/kg dicyclomine was not significantly different from control group (p = 0.054). The Minute × Treatment interaction [F(8,104) = 1.89; p = 0.07] was not significant. 3.3. The effects of the pre-test administration of dicyclomine on the retrieval of recent and remote memories of IA Concerning the retrieval of recent memories for the IA task (Fig. 3A), a two-way ANOVA showed significant effects for Session [F(1,33) = 70.88; p b 0.01]. Tukey's test showed that all groups had higher latencies to enter the dark chamber on the test day (p b 0.01). The Treatment factor [F(2,33) = 0.07; p = 0.93] and Session × Treatment interaction [F(2,33) = 0.09; p = 0.92] were not significant. For the remote memory of IA (Fig. 3B), a two-way ANOVA showed a significant effect for Session [F(1,30) = 144.02; p b 0.01]. Tukey's test showed that all groups had an increased latency to enter the dark chamber during the test session (p b 0.01). Neither the Treatment factor [F(2,30) = 2.79; p = 0.08] nor the Session × Treatment interaction was significant [F(2,30) = 2.25; p = 0.12]. 4. Discussion
3. Results 3.1. The effects of the pre-test administration of dicyclomine on the retrieval of recent and remote memories of CFC Concerning the retrieval of recent memories of CFC, a two-way ANOVA showed a significant effect of the Minute [F(4,92) = 6.33; p b 0.01] and Treatment [F(2,23) = 22.07; p b 0.01] factors. The posthoc Tukey's test showed that the group treated with 32 mg/kg dicyclomine exhibited a significant reduction in freezing time compared to the other 2 groups (p b 0.01 in both cases). The rats treated with 16 mg/kg dicyclomine were not significantly different from the saline group. The Minute × Treatment interaction was also significant [F(8,92) = 2.89; p b 0.01]. Tukey's test reveal that group treated with 32 mg/kg of dicyclomine exhibited lower freezing time from the second minute onwards (p b 0.05 for second minute compared to 16 mg/kg of dicyclomine and p b 0.01 for all other minutes). A two-way ANOVA for the retrieval of remote memories of CFC showed significant effects for Minute [F(4,128) = 6.11; p b 0.01] and Treatment [F(2,32) = 4.32; p b 0.05]. Tukey's test showed that rats treated with 32 mg/kg dicyclomine had reduced freezing times compared to those in the control group (p b 0.05). The group that received 16 mg/kg
Memory is not immediately and definitely formed at the moment it is acquired; gradual reorganization and stabilization processes occur during its storage [4]. As the consolidation of hippocampal-dependent memories occur, there is a progressive decrease in the participation of this structure, and a memory system is formed independent of the hippocampus. However, memory is not literally transferred from the hippocampus to other regions, as the hippocampus actively interacts with neocortical sites, in which cortico-cortical connections are gradually developed and become responsible for the storage and retrieval of remote memories [10,14]. Considering the influence of the cholinergic system on the modulation of mnemonic processes, the present study analyzed the role of the M1 muscarinic receptors in the retrieval process of recent and remote memories of aversively motivated tasks. To our knowledge, this is the first report that successfully shows the contribution of muscarinic cholinergic system in the retrieval of remote fear memories. The results showed that systemic pre-test administration of dicyclomine, a M1-selective muscarinic receptor antagonist, impaired the retrieval of the CFC task. Such an effect, detected by the reduction in the time of freezing of the rats during the test session, occurred when the training-test interval was 1 day or 28 days. The dose of 32 mg/Kg also impaired the performance of the rats during retrieval of
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Fig. 1. Freezing time per minute (mean ± SEM) during contextual fear conditioning test. A. Recent memory (test 1 day after training). B. Remote memory (test 28 days after raining). The retrieval of recent and remote memories of CFC was impaired by treatment with 32 mg/kg of dicyclomine. “Sal”, saline; “Dic16”, dicyclomine 16 mg/kg; “Dic32”, dicyclomine 32 mg/kg. The number of animals per group is shown in parentheses. *p b 0.01 compared to Sal group (two-way ANOVA followed by Tukey test).
remote memory in TFC task. The IA task was not affected in any of the studied intervals. Previous studies in our lab suggested an involvement of the M1 receptors in the retrieval of recent memory of the CFC task because the pre-test administration of 16 and 32 mg/kg of dicyclomine impaired performance of animals [30]. In the present study, we use a protocol with 5 context-shocks pairings (to produce a persistent memory in order to investigate the effect of the drug on retrieval of the remote memory). In this case, the pre-test dicyclomine administration at dose of 16 mg/kg was not able to impair the performance of the animals. On the other hand, the administration of a higher dose (32 mg/kg) affected the conditioned freezing response to the context. In the present study, the administration of 32 mg/kg dicyclomine impaired the retrieval of recent and remote memories of CFC. Considering the current hypothesis that the hippocampus plays a transient role in memory storage and that as a new memory is consolidated it becomes dependent of other brain regions, possibly the cerebral cortex [10,34], and that M1-subtype muscarinic receptors are predominantly distributed in the cortex, hippocampus and amygdala in rats [32,33], it is not possible to determine which structures are critical for the retrieval of recent and remote memories of tasks based on a pharmacological approach alone. In any case, the activation of M1-subtype muscarinic receptors seems to be critical for both recent and remote retrieval processes. Although the TFC task had some similarities to CFC (for example, using shock as a US and the evaluation of freezing time as a conditioned response), this task was not affected by the pre-test administration of dicyclomine when the test was performed 1 day after the training. Thus, this drug specifically induced impairment in an aversively motivated hippocampal-dependent task (CFC), while sparing the performance of rats in a similar, but hippocampal-independent, task (TFC). Although both tasks are amygdala-dependent [9] and M1 receptors are observed in this region [32,33], the administration of dicyclomine distinctly affected the performance of the two tasks during recent
memory. One possibility is that the hippocampus is more susceptible to manipulations of the cholinergic system than the amygdala. The administration of dicyclomine at a dose of 32 mg/kg impaired the retrieval of remote memory of TFC. Interestingly, this task was affected by the drug only when the test was performed 28 days after training, suggesting that a change in the modulatory role of M1 receptors on retrieval after a certain time interval is needed to acquire greater relevance in remote memory. In the similar manner, that it is supposed to occur in CFC, the neurobiological substrate involved with the retrieval of recent memory appears to be different than involved in remote memory retrieval of TFC, being more sensitive to changes in muscarinic system mediated by M1 receptors. Consistent with this hypothesis, it was shown that lesions in the secondary auditory cortex of rats impaired only the remote memory retrieval of TFC, sparing recent memory [35]. In another study, the number of Zif268-positive neurons increased following remote memory, but not recent memory retrieval of TFC in the thalamus and auditory cortex [36]. It is possible to assume that this change in brain activity pattern also would be reflected in a change in modulation performed by M1 receptors, so that their activation is more relevant some long time after training. Although not possible to identify the involved brain structures, the pharmacological approach used in this study showed that the activation of the M1 receptors is important only for the remote memory retrieval of TFC. IA is similar to CFC in some aspects: the rat is subjected to an aversive experience (footshock) in a given context. In addition, IA is also sensitive to lesions in the amygdala [19] and hippocampus [20,37,]. Conversely, as the shock was applied only after the rats' response, it can be interpreted as a punishment, an element characteristic of operant or instrumental conditioning. Moreover, the neuronal mechanisms involved in the performance during these paradigms are showed to be partially distinct [21]. In general, aversive conditions that require an instrumental response are less dependent on basolateral amygdaloid nuclei [38]. Thus, investigating the effects of pharmacological manipulations on both tasks, under similar experimental conditions, can be useful
Fig. 2. Freezing time per minute (mean ± SEM) during tone fear conditioning test. A. Recent memory (test 1 day after training). B. Remote memory (test 28 days after training). Note that the treatment with 32 mg/kg of dicyclomine significantly impaired only the retrieval of remote memories of TFC relative to saline control group. “Sal”, saline; “Dic16”, dicyclomine 16 mg/kg; “Dic32”, dicyclomine 32 mg/kg. The number of animals per group is shown in parentheses. *p b 0.01 compared to Sal group (two-way ANOVA followed by Tukey test).
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Fig. 3. Latency (mean ± SEM) to cross to black compartment during training and test of the inhibitory avoidance task. A. Recent memory (test 1 day after training). B. Remote memory (test 28 days after training). The treatment did not modify the performance of animals. All group learned the task. “Sal”, saline; “Dic16”, dicyclomine 16 mg/kg; “Dic32”, dicyclomine 32 mg/kg. The number of animals per group is shown in parentheses.
for understanding the overall influence of cholinergic transmission on memory and learning. The pre-test administration of both doses of dicyclomine did not interfere with the performance of the rats during the retrieval of recent memory of IA. Despite the putative importance of the hippocampus in the performance of this task, the absence of effect could be due to the involvement of other brain structures related to the instrumental components inherent to IA, such as the striatum. However, controversial results have been reported concerning the participation of the cholinergic system in the retrieval of this behavioral paradigm. Whereas some studies have described deficits induced by the systemic administration of scopolamine a non-selective muscarinic antagonist [39], or of M1-selective muscarinic receptor antagonists, such as biperiden and trihexyphenidyl [40], other studies failed to report such deficits [29,30, 41,42]. As previously discussed in Soares et al. [30], these controversial results could be explained by differences between IA procedures adopted in those studies. Studies that observed impairment in retrieval of IA task, often used a step-down procedure, independently of muscarinic antagonist used [40,43]. On the other hand, the studies that did not find impairment have used a step-through procedure [24,29,30,41,42, 44]. Relative to the effects of dicyclomine on retrieval of remote memories (training-test interval of 28 days) of the IA task, no alteration was detected in the performance of the rats because of pharmacological treatment. Thus, the retrieval of both recent and remote memories of this behavioral paradigm was independent of the activation of the M1-subtype muscarinic receptors. Some studies have indicated the involvement of other neurotransmitter systems in the performance of this task, such as AMPA-type glutamate receptors in the amygdala related to the retrieval of recent memories and AMPA receptors in the medial prefrontal cortex related to remote memory [45]. Dicyclomine shows a higher affinity for M1 and M3 and low affinity for M2 receptors [26,27]. So it is not possible to rule out the possibility that the effect of dicyclomine is due in part to the combined inactivation of M1 and M3 receptors. Nevertheless, other studies suggest that the cerebral cortex, hippocampus and amygdala of the rat contain a mixed population of M1 and M3 receptors [32,33]. As both types of receptors mediate excitatory responses, this would explain the effect of the antagonist dicyclomine on learning and memory. Indeed, M3-muscarinic receptor knockout mice show a deficit in CFC, suggesting that M3 receptors are also important memory process [46]. However, a recent study showed that systemic administration or administration directly into basolateral amygdala of cevimeline, a new M1 selective agonist, caused an increase in freezing in response to tone [47]. Furthermore, the same study showed that knockout mice to M1 receptors injected with cevimeline did not exhibit the increase in freezing response to tone, suggesting that other muscarinic receptors, such as M3 would not be needed to promote memory consolidation and that the consolidation enhancement observed in response to administration of cevimeline is mediated specifically by M1 receptors. In the same way, the pre-training chronic administration of EUK1001, another M1
agonist, appears to improve the memory to CFC, TFC and IA suggesting a general effect on cognition [48]. Although these reports did not to assess the effects of these drugs directly on memory retrieval, taken together, these findings strengthen the main role M1 muscarinic receptors on learning and memory processes. Nevertheless, a more complex view of M1 muscarinic receptors in memory processing has been suggested. M1 mutant mice showed no evidence of increase or change in activity or shock reactivity, but showed enhanced CFC and normal TFC responses [49]. These M1 mutant mice would have a cortical memory dysfunction or impaired hippocampal – cortical interaction that would be specific to remote memory, which appears in accordance to results obtained in the present study regarding impairment of remote memory to contextual and tone conditioning. Previous results from our lab showed that dicyclomine, at the dose of 32 mg/kg, did not affect the ambulation of rats in the open field task [29]. Moreover, if dicyclomine had increased the locomotor activity impairment on the IA task would also be expected, because the animals tend to move to the dark compartment of the apparatus. Therefore the effects of dicyclomine likely not were caused by locomotion or motivational factors. Collectively, our data indicated that M1 muscarinic receptors are necessary for the retrieval of both recent and remote memories of contextual fear conditioning. In the present study, the remote memory of tone fear conditioning was also impaired. This was a surprising finding and suggests a differential contribution of muscarinic receptors on recent and remote memories revealing a more generalized role in remote memory. Acknowledgments This study was financially supported by grant 2009/12871-0, São Paulo Research Foundation (FAPESP); the Research Incentive Fund Association (Associação Fundo de Incentivo à Pesquisa – AFIP), the Coordination for the Improvement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES), the National Council of Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq). The authors would like to thank Jose Bernardo Costa for technical assistance during the experiments. References [1] L.H. Tsai, J. Gräff, On the resilience of remote traumatic memories against exposure therapy-mediated attenuation, EMBO Rep. 15 (8) (2014) 853–861. [2] M.C. Kearns, K.J. Ressler, D. Zatzick, B.O. Rothbaum, Early interventions for PTSD: a review, Depress. Anxiety 29 (10) (2012) 833–842. [3] M.H. Milekic, C.M. Alberini, Temporally graded requirement for protein synthesis following memory reactivation, Neuron 36 (3) (2002) 521–525. [4] Y. Dudai, The neurobiology of consolidations, or, how stable is the engram? Annu. Rev. Psychol. 55 (2004) 51–86. [5] M. Fendt, M.S. Fanselow, The neuroanatomical and neurochemical basis of conditioned fear, Neurosci. Biobehav. Rev. 23 (5) (1999) 743–760.
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