Transient cAMP elevation during systems consolidation enhances remote contextual fear memory

Journal Pre-proofs Transient cAMP elevation during systems consolidation enhances remote contextual fear memory Jaehyun Lee, Hye-Ryeon Lee, Jae-Ick Kim, Jinhee Baek, Eun-Hae Jang, Jihye Lee, Myeongwon Kim, Ro Un Lee, Somi Kim, Pojeong Park, Bong-Kiun Kaang PII: DOI: Reference:

S1074-7427(20)30015-0 https://doi.org/10.1016/j.nlm.2020.107171 YNLME 107171

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Neurobiology of Learning and Memory

Received Date: Revised Date: Accepted Date:

27 September 2019 31 December 2019 19 January 2020

Please cite this article as: Lee, J., Lee, H-R., Kim, J-I., Baek, J., Jang, E-H., Lee, J., Kim, M., Un Lee, R., Kim, S., Park, P., Kaang, B-K., Transient cAMP elevation during systems consolidation enhances remote contextual fear memory, Neurobiology of Learning and Memory (2020), doi: https://doi.org/10.1016/j.nlm.2020.107171

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Transient cAMP elevation during systems consolidation enhances remote contextual fear memory Jaehyun Lee*a,b , Hye-Ryeon Lee*b, Jae-Ick Kim*b, Jinhee Baekb, Eun-Hae Jangb, Jihye Leeb, Myeongwon Kimb, Ro Un Leeb, Somi Kimb, Pojeong Parkb, Bong-Kiun Kaang#a,b a

Interdisciplinary Program in Neuroscience, Seoul National University, 1 Gwanangno, Gwanak-gu,

Seoul 08826, Korea. b Neurobiology laboratory, Department of Biological Sciences, College of Natural Sciences, Seoul National University, Gwanangno 599, Gwanak-Gu, Seoul 08826, Korea. *These

authors contributed equally to the work; #Corresponding author.

Corresponding author: [email protected]

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Abstract Memory is stored in our brains over a temporally graded transition. With time, recently formed memories are transformed into remote memories for permanent storage; multiple brain regions, such as the hippocampus and neocortex, participate in this process. In this study, we aimed to understand the molecular mechanism of systems consolidation of memory and to investigate the brain regions that contribute to this regulation. We first carried out a contextual fear memory test using a transgenic mouse line, which expressed exogenously-derived Aplysia octopamine receptors in the forebrain region, such that, in response to octopamine treatment, cyclic adenosine monophosphate (cAMP) levels could be transiently elevated. From this experiment, we revealed that transient elevation of cAMP levels in the forebrain during systems consolidation led to an enhancement in remote fear memory and increased miniature excitatory synaptic currents in layer II/III of the anterior cingulate cortex (ACC). Furthermore, using an adeno-associated-virus-driven DREADD system, we investigated the specific regions in the forebrain that contribute to the regulation of memory transfer into long-term associations. Our results implied that transient elevation of cAMP levels was induced chemogenetically in the ACC, but not in the hippocampus, and showed a significant enhancement of remote memory. This finding suggests that neuronal activation during systems consolidation through the elevation of cAMP levels in the ACC contributes to remote memory enhancement. Keywords: Remote fear memory, Anterior cingulate cortex, Hippocampus, cAMP/PKA pathway, Memory transfer, Memory enhancement

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1. Introduction Declarative memory consolidation processes are divided into two phases as follows: recent memory, which is a time-limited memory, and remote memory, which is a long-lasting memory. Multiple brain regions, such as the hippocampus and cortical regions, are involved in cortical consolidation, the process that converts a recent memory into a remote memory (Frankland & Bontempi, 2005; Wiltgen, Brown, Talton, & Silva, 2004). Many researchers have used classical associative learning with rodents to measure recent and remote fear memory (Frankland et al., 2006; Kwon, Jhang, Kim, Lee, & Han, 2012; B. A. Silva, Burns, & Graff, 2019; Tayler, Tanaka, Reijmers, & Wiltgen, 2013). For instance, biological changes, such as higher metabolic activity (Bontempi, Laurent-Demir, Destrade, & Jaffard, 1999), increased spine density (Restivo, Vetere, Bontempi, & Ammassari-Teule, 2009), and immediate early gene (IEG) expression (Frankland, Bontempi, Talton, Kaczmarek, & Silva, 2004), were detected in the hippocampus during recent memory retrieval compared to remote memory retrieval. In contrast, the opposite pattern of these biological changes was observed in cortical regions during remote memory retrieval (Maviel, Durkin, Menzaghi, & Bontempi, 2004; Ross & Eichenbaum, 2006; Teixeira, Pomedli, Maei, Kee, & Frankland, 2006). Recent studies are providing more direct evidence for the role of each brain region in the formation of remote memory by modulating the neuronal activity of a specific region at distinct time points (Frankland & Bontempi, 2005; Goshen et al., 2011; Kitamura et al., 2017). Although previous studies have not reached a consensus with regard to the timeline of participation of each brain region or the exact mechanism for remote memory, these studies suggest that the hippocampus and the cortex play distinct but limited roles in recent and remote memory. During the memory consolidation period, the anterior cingulate cortex (ACC) increased in neural activity and remote memory measurements exhibited changes in spine morphology. Furthermore, blocking the normal function of the ACC induced memory impairment at the remote, but not at the recent time point (Ding, Teixeira, & Frankland, 2008; Einarsson & Nader, 2012; Frankland et al., 2004; Restivo et al., 2009; Teixeira et al., 2006). These biological changes confirm the critical role the ACC plays in remote memory, leading us to focus specifically on the ACC from among the various cortical 3

regions. Moreover, several studies uncovered the distinct molecular mechanisms contributing to the formation and retrieval of remote fear memory in which a number of proteins, such as CaMKII, c-fos, and PKMζ, are involved (Frankland & Bontempi, 2005; Sacco & Sacchetti, 2010; Squire & Bayley, 2007). In particular, it was shown that the cAMP/PKA pathway plays an important role not only in a cellular model of memory, such as long-term potentiation (LTP), but also in the consolidation and retrieval of fear memory (Lee et al., 2007; Y. S. Lee et al., 2009; Matynia, Kushner, & Silva, 2002; Park et al., 2014; Wong et al., 1999). These previous findings suggest that molecular changes in the cortical regions affect remote memory. However, it remains to be elucidated which brain region contributes to the memory transfer process, especially at the molecular level. To decipher these underlying molecular mechanisms, we investigated whether changes in cAMP, in either the hippocampus or cortical regions, during systems consolidation alter the formation of remote memory.

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2. Material & Methods 2.1 Animals The genetic information of Aplysia octopamine receptor transgenic (Apoa1) mice and their wild-type (WT) littermates were previously described (Isiegas et al., 2008; J. S. Lee et al., 2009). In brief, Apoa1 mice and their WT littermates were produced as bitransgenic mice, which carry the tetracyclineinducible transcriptional transactivator (tTA) and ApoA1 transgene controlled by the CaMKIIa promoter and tet-O promoter, respectively. This mouse line was bred into a C57B/6J background for more than 10 generations. Male Apoa1 mice and their male WT littermates were used in the experiment. Male C57B/6N mice used in the experiment were stereotaxically injected with the Gs-DREADD virus. All mice were co-housed in temperature-controlled (approximately 23°C) conditions with food and water provided ad libitum and with a 12-hour light-dark cycle (lights on 9:00 a.m: zeitgeber time [ZT] 0, lights off 9:00 p.m : [ZT 13]). After the experiment, the mice were returned to their home cages. This research was approved by the Institutional Animal Care and Use Committee of Seoul National University.

2.2 Plasmid and virus production AAV-CW3SL-GsDREADD-IRES-mCitrine

was

subcloned

as

GsDREADD-IRES-mCitrine

connected to a CW3SL expression cassette, which enables a larger transgene to be inserted into the adeno-associated virus (AAV). The expression was led by a CaMKIIa promoter included in the cassette, as previously described (Choi et al., 2014). Briefly, AAV1was purified from the human embryonic kidney (HEK)-293T cells that were transfected with plasmids containing the expression cassette, p5E18-RXC1, and pAd-ΔF6. 72-96 hours after the transfection, the medium containing AAV particles were harvested and purified using iodixanol gradient.

2.3 Surgery All surgeries were performed under ketamine/xylazine anesthesia and on a stereotaxic apparatus (Kopf 5

Instruments). Mice were injected with 0.5μl of AAV1-CW3SL-GsDREADD-IRES-mCitrine (1×1013 vg/ml) into the ACC or hippocampus. The following coordinates were used to target each region: ACC, AP+0.4&1.1 mm/ML±0.3 mm/DV-1.75&1.9 mm; hippocampus CA1, AP 1.7 mm/ML±1.5 mm/ DV1.7 mm; hippocampus DG, AP-2.3 mm/ML±2.0 mm/DV-2.15 mm.

2.4 Contextual fear conditioning The mice were handled by the experimenter for 3 min per day for 3 days before experimentation. In a given 180 s conditioning period, the mice were allowed to explore freely within the chamber (Coulbourn Instruments). Then, a foot shock (2s duration, 0.8 mA intensity) was administered through the floor grid. After the conditioning, the mice were returned to their home cages. Thirty-five days after the conditioning, the mice were re-exposed to the same chamber, and the freezing level was tested in the conditioned context without any foot shock. The Freeze-Frame software automatically quantified freezing behavior. Animals showing no viral expression or incorrect region targeted were excluded from the analysis.

2.5 Intraperitoneal injections An intraperitoneal (i.p.) injection was administered 14 days after the contextual fear conditioning. Octopamine (1 mg/kg in PBS, body weight) was injected into Apoa1 mice and WT littermates. Saline (control) or clozapine N-oxide (CNO) was injected into AAV-CW3SL-GsDREADD-IRES-mCitrine virus injected mice (2 mg/kg in saline, body weight). In our experiments, the two drugs (octopamine or CNO) and the saline control were injected intraperitoneally between ~ZT 2-4.

2.6 Electrophysiology recording Animals were anesthetized with isoflurane and killed by decapitation in accordance with the policy and regulation approved by the Institutional Animal Care and Use Committee at Seoul National 6

University. Coronal brain slices (300 μm) were prepared as described by Kang et al. (Kang et al., 2012) using a vibratome (Leica, VT1200S) in an ice-chilled artificial cerebrospinal solution (ACSF) that contained (mM) 124 NaCl, 2.5 KCl, 25 NaHCO3, 1 NaH2PO4, 2 MgSO4, 10 D-glucose and 2 CaCl2, saturated with 95% O2 and 5% CO2. The slices were transferred to an incubation chamber with ACSF and allowed to recover at 26–28 °C for a minimum of 1 h before recordings were made. Whole-cell recording was performed at 32°C during continuous perfusion at 3–4 ml/min with ACSF that contained picrotoxin (100 µM) and tetrodotoxin (1 µM) to isolate miniature EPSCs (mEPSCs). Pyramidal neurons were visualized with IR-DIC optics (Olympus). Internal solution for whole-cell patch clamp recording comprised (mM) 5 NaCl, 130 CsMeSO3, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na3-GTP, and 5 QX-314. The pH was adjusted to 7.2–7.3 with CsOH, and osmolarity was set to 280-290 mOsm/l. Borosilicate glass pipettes were used with a resistance of 4-6 MΩ, and experiments were only accepted for analysis if the series resistance values were <25 MΩ and varied by 20% during the course of the experiment. Signals were filtered at 10 kHz and digitized at 20 kHz using Axopatch 200B (Molecular Devices). Cells were clamped at a holding potential of -70 mV, and the peak amplitude (pA) and frequency (Hz) of mEPSCs were monitored with Clampex (Molecular Devices) and analyzed using MiniAnalysis (Synaptosoft).

2.7 Data analysis Kolmogotov-Smirnov test and unpaired t-test were used to determine statistical differences between the groups. A value of p < 0.05 was considered statistically significant. All data were presented as mean ± SEM.

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3. Results 3.1. Behavioral effects induced by transient elevation of cAMP levels in Apoa1 transgenic mice during systems consolidation Previous studies demonstrated that in Apoa1 mice, heterologous expression of Aplysia octopamine receptor is regulated by the CaMKII promoter and that when octopamine was injected into these transgenic mice, the cAMP was transiently increased only in the forebrain region and subsequent behavioral and physiological effects lasted for several hours, whereas WT littermates were unaffected (Chang et al., 2000; Isiegas et al., 2008; J. S. Lee et al., 2009; Wu et al., 2008). Using this mouse model, we first investigated whether transient elevation of cAMP in the forebrain region could affect systems consolidation. Apoa1 mice trained for contextual fear conditioning were injected intraperitoneally with octopamine and tested for any alteration in remote contextual fear memory. Since previous studies usually assessed remote memory approximately 2 weeks after the formation of memory (Frankland et al., 2004; Kitamura et al., 2017; Lesburgueres et al., 2011), we administered octopamine through intraperitoneal injection in both Apoa1 mice and WT littermates for 7 consecutive days during systems consolidation, which began 14 days after the contextual fear conditioning. Thirty-five days after the conditioning, both groups of mice underwent a remote memory retrieval test in which the mice were re-exposed to the same chamber where fear conditioning was performed (Fig. 1A). Notably, Apoa1 mice showed significant enhancement in remote fear memory with freezing levels of 50.48% compared to WT littermates freezing levels of 32.75 % (t-test; p <0.05) (Fig. 1B).

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Figure 1. Apoa1 transgenic mice display increased remote contextual fear memory. (A) Behavioral experiment scheme. (B) Remote contextual fear memory in Apoa1 mice is significantly enhanced compared to WT littermates (32.75 ± 4.41 %, N = 15 for WT; 50.48 ± 4.35 %, N = 11 for Apoa1; p < 0.05, unpaired t-test).

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3.2 Changes in synaptic transmission induced by transient elevation of cAMP concentrations in Apoa1 transgenic mice during systems consolidation The increased remote fear memory in Apoa1 transgenic mice might be associated with any alteration of synaptic transmission in the ACC. To examine this possibility, we tested spontaneous AMPARmediated synaptic transmission by measuring the amplitude and frequency of mEPSCs in pyramidal neurons of the ACC layer II/III in Apoa1 mice and WT littermates one day after memory retrieval. The amplitude of mEPSCs was unchanged in Apoa1 mice compared to WT littermates (Fig. 2A, B). Interestingly, the frequency of mEPSCs was significantly increased in Apoa1 mice (Fig. 2A, C). Cumulative probability also confirmed that the frequency of mEPSC was significantly enhanced in Apoa1 mice (Fig. 2E), while the amplitude of mEPSCs was comparable between the genotypes (Fig. 2D). These results suggest that transient elevation of cAMP in the forebrain may play an important role during systems consolidation in which recent contextual fear memory is turned into remote contextual fear memory. In addition, this strengthened synaptic activity in the ACC might be one of the cellular mechanisms underlying the enhancement of remote fear memory.

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Figure 2. Transient elevation of cAMP induced changes in synaptic signalling in the ACC layer II/III pyramidal neurons in Apoa1 transgenic mice. (A) Sample recording traces from Apoa1 mice and WT littermates (B) No difference in the amplitude of mEPSCs in the ACC layer II/III pyramidal neurons in both genotypes (13.58 ± 0.52 pA, N = 11 for WT; 14.07 ± 0.69 pA, N = 14 for Apoa1; p > 0.5, unpaired t-test). (C) The frequency of mEPSCs in the ACC layer II/III pyramidal neurons is significantly increased in Apoa1 mice (0.55 ± 0.15 Hz, N = 11 for WT; 1.65 ± 0.22 Hz, N = 14 for Apoa1; p < 0.001, unpaired t-test). (D) Cumulative probability for the amplitude of mEPSCs (Kolmogotov-Smirnov test, p > 0.8). (E) Cumulative probability for the frequency of mEPSCs (Kolmogotov-Smirnov test, p < 0.001).

3.3 Specifying the brain regions which contribute to remote memory enhancement induced by 11

transient elevation of cAMP during systems consolidation This finding indicates that cAMP and its downstream signalling molecules, such as PKA, may function to enhance the memory transfer process in the forebrain regions (A. J. Silva, Kogan, Frankland, & Kida, 1998), although the specific brain regions mediating this are unclear. In order to address this issue, we examined which brain regions are involved in memory enhancement by transiently increasing cAMP levels in a specifically targeted region. We utilized designer receptors exclusively activated by designer drugs (DREADD) chemogenetic system selectively activated when bound to CNO (Roth, 2016; Whissell, Tohyama, & Martin, 2016). CNO treatment could induce behavioral and physiological effects for several hours (Alexander et al., 2009). Gs-DREADD is CNO-sensitive designer G protein-coupled receptor (GPCR) that activate adenylate cyclase, resulting in increased cAMP production when coupled with Gs alpha subunit (Farrell et al., 2013; Guettier et al., 2009). Using Gs-DREADD, we transiently raised the cAMP levels in the ACC or hippocampus during systems consolidation. We designed an experiment that employed two groups of mice, both of which were stereotaxically injected with AAV1CW3SL-GsDREADD-IRES-mCitrine (Gs-DREADD virus), where the expression is controlled by CaMKII promoter. Using these mouse groups, we performed the behavior test, as mentioned above (Fig. 3A). One group of mice was injected in the ACC, and the other group was injected in the hippocampus CA1 and dentate gyrus (DG) (Fig. 3B, D). CNO-treated mice injected with Gs-DREADD virus in the ACC exhibited a higher level of freezing behavior than saline-treated mice during the retrieval test (Fig. 3C). However, CNO-treated mice injected with the Gs-DREADD virus in the hippocampus showed no significant difference in freezing levels compared to saline-treated mice (Fig. 3E). Taken together, these results suggest that the transient elevation of cAMP levels in the ACC modulates the observed remote memory enhancement.

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Figure 3. Transient elevation of cAMP levels in the ACC during systems consolidation induces remote memory enhancement. (A) Behavioral experiment scheme. (B) Gs-DREADD virus expression in the ACC (Scale bar = 500 μm, 4X magnification). (C) Remote contextual fear memory in the CNO group is significantly enhanced compared to the saline group (25.27 ± 3.22 %, N = 13 for saline group; 44.83 ± 5.59 %, N = 15 for CNO group; p< 0.01, unpaired t-test). (D) Gs-DREADD virus expression in the hippocampus (Scale bar: 500 μm, 4X magnification). (E) There is no significant difference in freezing levels between the CNO group and the saline group (28.01 ± 5.24 %, N = 8 for saline group; 29.21 ± 2.6 %, N = 9 for CNO group; p > 0.8, unpaired t-test). 13

4. Discussion Adenylyl cyclases (ACs), which catalyze the production of cAMP, have a critical role in contextual fear memory. Among the ten different ACs, the deletion of either AC1 or AC8 was not found to alter LTP; however, double mutant mice exhibited profound deficits in the late phase of LTP (Wong et al., 1999). In addition, Shan et al. reported a comparable finding to our result that AC1 overexpression increased remote fear memory (Shan, Chan, & Storm, 2008). It is important to note, however, that this finding has some limitations in demonstrating the mechanisms of systems consolidation because AC1 overexpression was maintained from the developmental stage onward, throughout the whole period of fear conditioning and memory retrieval. To our knowledge, there are no reports so far demonstrating the molecular mechanisms by which systems consolidation is mediated or modulated. For these reasons, we chose to use transgenic Apoa1 mice, as cAMP levels can be temporally and spatially controlled in the forebrain region (Isiegas et al., 2008) and revealed that an increase of cAMP during systems consolidation enhanced remote fear memory. Cumulatively, these results highlight a broad role of the cAMP/PKA pathway in contextual fear memory formation and retention from the recent to remote phase. Meanwhile, we also found that the enhancement in remote memory retrieval was accompanied by an increase in mEPSC frequency in the pyramidal neurons of the ACC layer II/III. The reason for this increase in mEPSC frequency is unclear. However, it is conceivable that sustained elevation of cAMP/PKA pathway during systems consolidation might affect synaptic transmission in the ACC critical for remote memory processing, and this physiological change may underlie the enhancement of systems consolidation in contextual fear memory. Strengthening this view, it has been previously documented that activation of Aplysia octopamine receptor by octopamine increased presynaptic glutamate release in vitro in pyramidal neurons from layer II/III of the ACC (Wu et al., 2008). Therefore, cAMP/PKA pathway may facilitate glutamatergic synaptic transmission in the ACC by increasing presynaptic glutamate release, and this alteration, in turn, might promote systems consolidation. The hippocampus and cortex participate in systems consolidation, the conversion of recent memories 14

into remote memories, in a time-dependent manner (Frankland & Bontempi, 2005). In this study, we revealed that a transient elevation of cAMP during systems consolidation in the ACC, but not in the hippocampus, enhances remote memory. This result is consistent with previous remote memory research that found the hippocampus to have a limited role in remote memory (Anagnostaras, Gale, & Fanselow, 2001; Kitamura et al., 2012; Squire, Genzel, Wixted, & Morris, 2015), whereas the ACC contributes to remote memory (Weible, 2013). Previously, Lesburgueres et al. suggested that pharmacological inactivation of the orbitofrontal cortex (OFC) within the early (day 0–7) and late (day 15–22) time-windows induced remote memory impairment whereas, in the hippocampus, remote memory impairment occurred only within the early stage inactivation during the post-learning period (Lesburgueres et al., 2011). Although it is unknown whether the hippocampus is still involved in remote memory retrieval, modulation of neuronal activation within the late time-window in the hippocampus does not affect remote memory. Sleep is important to the transfer of memory (Abel, Havekes, Saletin, & Walker, 2013; Born & Wilhelm, 2012; Hernandez & Abel, 2011; Klinzing, Niethard, & Born, 2019). Previous studies suggested that cAMP level increased during REM sleep, and adenylyl cyclases type 1 and 8 double knock-out mice displayed long-term memory impairment (Luo, Phan, Yang, Garelick, & Storm, 2013). Sleep deprivation impaired cAMP/PKA pathway-dependent LTP and memory through increasing phosphodiesterase activity (Vecsey et al., 2009). Transient elevation of cAMP in hippocampal excitatory neurons during sleep deprivation prevented memory impairment (Havekes et al., 2014).These results suggest that activation of the cAMP/PKA pathway contributes to sleep-dependent shifts of memory that lead to remote memory enhancement. In this study, we activated cAMP/PKA pathway a few hours per day during the early part of the light phase while the mice slept, during the time window of systems consolidation. Thus, the enhancement of remote memory effects observed in our results was limited to the activation levels of the cAMP/PKA pathways during consolidation. Possibly explaining this remote memory enhancement, the activation of cAMP/PKA pathway might induce changes in the processes that are generally required during the 15

memory consolidation, such as sleep reactivation and oscillation (Klinzing et al., 2019), and reorganizing various brain networks (Kitamura et al., 2017; Tonegawa, Morrissey, & Kitamura, 2018; Wheeler et al., 2013). On the other hand, increasing cAMP may enhance remote memory even after systems consolidation. It would be interesting to test the effects of increased cAMP on freezing behavior after the critical window of systems consolidation. Furthermore, comparing the effects of activating the cAMP/PKA pathway during or after systems consolidation might provide an essential towards understanding the nature of remote memory. Undoubtedly, further work will be required to dissect the specific role of this signalling pathway in remote memory formation.

5. Conclusion In this study, we revealed that increased cAMP levels during systems consolidation induced enhancement in remote memory and that the ACC, but not the hippocampus, contributes to this modulation. These findings shed light on a novel role of the cAMP/PKA pathway in the modulation of systems consolidation.

Competing interest declaration The authors declare that they have no competing financial interests.

Author contributions H.-R.L., J.-I.K. and S.K conducted electrophysiology experiments; M.K. subcloned plasmid; Jaehyun.L.and M.K. produced virus. H.-R.L., Jaehyun.L., J.B., E.-H.J., R.U.L, M.K. and Jihye L conducted behavioral experiments; Jaehyun.L., R.U.L., Jihye L and M.K. performed surgery. H.-R.L., Jaehyun.L.,P.P. and B.-K.K. supervised the project and wrote the manuscript.

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Acknowledgments This work was supported by the National Honor Scientist Program (NRF2012R1A3A1050385).

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Transient elevation of cAMP levels in the ACC during systems consolidation induces remote memory enhancement and leads to increased miniature excitatory synaptic currents in the ACC.

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Transient elevation of cAMP levels in the ACC during systems consolidation induces remote memory enhancement and leads to increased miniature excitatory synaptic currents in the ACC.

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