Noradrenergic projections from the locus coeruleus to the amygdala constrain auditory fear memory reconsolidation

Memory reconsolidation is a fundamental plasticity process in the brain that allows established memories to be changed or erased. However, certain boundary conditions limit the parameters under which memories can be made plastic. Strong memories do not destabilize, for instance, although why they are resilient is mostly unknown. Here, we extend the understanding of the mechanisms implicated in reconsolidation-resistant memories by investigating the hypothesis that specific modulatory signals shape memory formation into a state that lacks lability. We find that the activation of the noradrenaline-locus coeruleus system (NOR-LC) during strong fear memory encoding increases molecular mechanisms of stability at the expense of lability in the amygdala. Preventing the NOR-LC from modulating strong fear encoding results in the formation of memories that can undergo reconsolidation within the amygdala and thus are vulnerable to post-reactivation interference. Thus, the memory strength boundary condition on reconsolidation is set at the time of encoding by the action of the NOR-LC.


INTRODUCTION
New memories do not form instantly at the moment of an experience, but rather undergo a stabilization period during which they are gradually consolidated into a stable, long-term memory.
Later, recall may cause the memory to return to an unstable state (i.e. destabilized) where it can be modified. Importantly, the memory must undergo a re-stabilization process called reconsolidation in order to persist 1 . This process of destabilization-reconsolidation allows memories to adaptively change. Importantly, manipulations targeting the reconsolidation process can permanently alter a memory trace, by enhancing, impairing or modifying it, offering great treatment potential to clinicians [2][3][4] . For instance, one can induce the destabilization of a maladaptive memory and then block reconsolidation pharmacologically-preventing memory from returning to a stable state and leading to memory impairment 5,6 . Memory content can also be updated during reconsolidation, allowing it to be modified to a less aversive form [7][8][9][10] . However, extreme fear learning can result in pathological memories that are resistant to change through reconsolidation 5,11 , making them difficult to treat ( Figure 1).
Memories created with 10P also undergo consolidation after learning, but the resulting memory is different. Unlike memories created with 1P, recall of a 10P memory does not turn it unstable and initially exists in an unstable short-term state and in order to persist as a long-term memory (LTM), it must undergo a time-dependent stabilization processes termed consolidation. Later, recall of the LTM causes memory to destabilize and become transiently unstable. Afterwards, for memory to persist it is restabilized by reconsolidation. B) Auditory fear conditioning consisting of 10 tone-shock pairings (10P). reconsolidation does not happen. The phenomenon where reconsolidation is inhibited from being triggered is termed "boundary condition", and the reason why 10P training leads to this outcome is not defined.
In order, for reconsolidation interventions to work, memory destabilization must be triggered first.
However, retrieval does not always induce destabilization. In rodent experiments, this is particularly the case in memories generated by high intensity fear conditioning or protocols that induce asymptotic levels of learning [12][13][14][15] . For instance, we 11 and others 16 have found that while a memory acquired by a 1-shock fear conditioning session can be attenuated by reconsolidation blockade, a 10-shock protocol creates an intense fear memory that is resistant to this treatment.
This boundary condition is thought to be mediated in part by mechanisms that initialize destabilization. A well described mechanism for destabilization is GluN2B-containing NMDA receptor activation 17 , which is reduced in reconsolidation-resistant memories 11 and if blocked prevents destabilization 18 . Another important mechanism involves the expression of GluA2containing AMPA receptors, which are important for LTP maintenance 19 and have been linked with memory strength and stability 20,21 . In order for destabilization to occur, and a transient reduction of GluA2 synaptic expression is necessary [22][23][24] .
The fact that strong training leads to memories that do not reconsolidate likely reflects changes triggered at the time of memory encoding affecting molecular mechanisms implicated in destabilization, such as GluN2B and GluA2. However, the specific links between memory encoding and reconsolidation have yet to be determined. It is well-established that emotionally arousing experiences cause increased release of noradrenaline in the amygdala 25 that modulates memory formation through the activation of β-adrenergic receptors 26 . The locus coeruleus is the main source of noradrenergic projections in the brain, and although it acts on many targets, its influence in the amygdala is critical to mediate the impact of stress on memory processes 27,28 . For instance, overactivation of projections steaming from the locus coeruleus (LC) to the amygdala has been implicated in encoding traumatic memories 29 and LC neurons projecting to the amygdala promote fear learning 30 . This evidence suggests the noradrenaline-locus coeruleus system (NOR-LC) is likely contributes to the formation of maladaptive, plasticity-resistant fear memories. Here we investigated the role of the NOR-LC system on intense fear encoding and found that it promotes reconsolidation-resistance during memory consolidation.

Rats
Male Sprague Dawley rats (275-300 g at arrival; Charles River, Quebec, Canada) were housed in pairs in plastic cages in a temperature-controlled environment (21-23°C) with ad libitum access to food and water and maintained on a 12 h light/dark cycle (lights on at 7:00 A.M.). All experiments were conducted during the light phase. Each rat was handled for at least 4 days before the behavioral procedures. All procedures were approved by McGill's Animal Care Committee and complied with the Canadian Council on Animal Care guidelines.

Surgery
Animals were anesthetized with a mixture of ketamine (50 mg/ml), xylazine (3 mg/ml), and Dexdomitor (0.175 mg/ml) injected intraperitoneally. Analgesic treatment was administered subcutaneously before surgery (carprofen; 5 mg/ml). Stainless-steel 22-gauge cannulae were bilaterally implanted in the basolateral amygdala (AP, −3.0 from Bregma; ML, +/−5.1 from the midline; DV, −8.0 from the skull surface). The cannulae were kept in place by dental cement tightly fixed to the skull with three stainless-steel screws. Obturators were then inserted into the cannulae to prevent blockage. An intraperitoneal injection of Antisedan (0.5 mg/ml) was administered after surgery to reverse the anesthesia, and the animals were placed in individual cages on heating pads until they woke up. The rats were then monitored daily during a 1-week recovery period, before the beginning of the behavioral procedures.
For viral injections in the LC, animals were anesthetized as above and a 33-gauge stainless steel injector was inserted into the brain (coordinates, from bregma: A/P −9.8mm, L ± 1.3mm, D/V −7.5mm from the skull surface). The injector was attached by polyethylene tubing (Intramedic #427406) to 10 uL Hamilton syringes and driven at 0.4 μL/min by a microinfusion pump (KD Scientific; model 780220). A volume of 0.7 uL/side of pAAV-hSYN-DIO-hM4D(Gi)-mCHerry (1.5x10 13 GC/mL, Neurophotonics Centre -ULAVAL) or pAAV-hSYN-tdTomato (1.5x10 12 GC/mL, Neurophotonics Centre -ULAVAL) was infused at a rate of 0.05 uL/min and the injector was withdrawn 15 min afterwards. To target LC terminals in the BLA, 7 weeks after viral infusions stainless steel cannulae were bilaterally implanted in the BLA as described above but using the following coordinates: A/P −3.0mm, L ± 5.1mm, D/V −9.0mm from bregma.
Propranolol (10mg/mL; Sigma-Aldrich) was dissolved in sterile saline. C21 (2 μg/μL, Hello Bio) was dissolved in sterile water. The pH-value of each solution was adjusted to 7.2-7.5. Propranolol was administered intraperitoneally at a volume of 1 mL/kg 15 min before training. Anisomycin (immediately after reactivation) and C21 (5 min before training) were infused bilaterally into the amygdala using a 23 gauge injectors connected to Hamilton syringes via 20 gauge plastic tubes. A total volume of 0.5 μl per side was infused by a microinfusion pump at a rate of 0.125 μl/min.
Injectors were left in place for an additional minute to ensure proper drug diffusion. Reactivation and test: Reactivation took place in context A one day after training and entailed one 30-s tone presentation without footshock after an initial 2 min acclimation. Rats remained in the boxes for 60 s after the tone presentation. Test session was identical to the reactivation session and was always conducted 1 day after either training, reactivation or extinction sessions, depending of the experimental design.

Auditory fear conditioning task
Extinction: One day after training, rats received 20 presentations of the tones (30 s each) without any footshocks in context A. The intertone interval was of 60 s. Rats remained in the boxes for 60 s after the last tone presentation.
Digital cameras recorded the animals' behavior, and memory was evaluated by a blind experimenter measuring the time spent freezing during the tone presentation, using Freeze View software (Actimetrics). Freezing was defined as immobilization except for respiration.

Western blots
For experiments requiring Western blots, rats were anesthetized with isoflurane either 1h or 24h after test and were decapitated, and their brains were removed and frozen at −80°C until further use. The basolateral amygdala was dissected from each frozen brain in the cryostat using a neuro punch (1 mm; Fine Science Tools) and homogenized in ice-cold Tris-HCl buffer (30 mm, pH 7.4) containing 4 mm EDTA, 1 mm EGTA, and a protease inhibitor cocktail (Complete; Roche). The subcellular fractionation procedure performed was described previously (Migues et al., 2010).
Briefly, the amygdala homogenates were centrifuged at 3,000 × g for 10 min at 4°C to remove the nuclei. The supernatant was then centrifuged at 100,000 × g (Beckman Coulter) for 1 h at 4°C.
The pellets were resuspended in 50 μl of 0.5% Triton X-100 homogenization buffer and incubated for 20 min on ice, before being layered over 100 μl of 1 M sucrose solution and centrifuged at 100,000 × g for 1 h at 4°C. The layer remaining above the sucrose, which contained the extrasynaptic receptors, was collected, and the Triton X-100-insoluble material that sedimented through the sucrose layer, containing the postsynaptic densities, was resuspended in 40 μl homogenization buffer and stored at −80°C. Total protein concentration was determined by the BCA protein assay kit (Pierce).

Immunohistochemistry
To analyze c-fos expression, coronal brain slices were incubated for 1h in blocking solution at room temperature (3% NGS, 0.3% Triton X-100) and then for 20h with anti-c-fos primary rabbit antibody (1:500, 226.003; Synaptic Systems, Göttingen, Germany) for 24 hr. Sections were washed and incubated with anti-rabbit Alexa-488 secondary antibody (1:500, Jackson Immunoresearch, West Grove, PA) for 2 h at room temperature. Afterwards, sections were washed, mounted on slides and immediately coverslipped with Fluoromount-G with DAPI (Thermo Fischer). Images were examined by fluorescence microscopy (Leica DM 5000 B) and cfos positive cells were counted bilaterally from two LC coronal slices for each animal with ImageJ

Histology
To identify cannulae placements, brains were removed and post-fixed in 10% formalin-saline, 20% sucrose solution and cryo-sectioned at 50 μm thickness. The slides were examined by bright-field light microscopy (Olympus IX81) by an experimenter blind to the group assignments. Only animals with injector tips bilaterally positioned within the BLA were included in the data analysis.
Rats with extensive damage were excluded from analysis. To verify viral expression, brains were post-fixed in paraformaldehyde 4%, phosphate buffer 0.2M for 3 days followed by 10% formalinsaline, 20% sucrose solution for another 3 days and were then cryo-sectioned at 80 μm thickness.
Slices were mounted onto slides with Fluoromount-G, with DAPI (Thermo Fischer) and examined by fluorescence microscopy (Leica DM 5000 B). Only animals with somatic viral expression in the LC were included in the data analysis, and rats with extensive damage were excluded.

Statistics
We used two-tailed independent-samples t test, one-way independent, two-way independent, and two-way repeated measures ANOVA for data analysis. Tukey's post hoc tests were further used to identify the critical differences that contributed to significant interaction. Type-one error rate was set at 0.05.

R1 Replicating the behavioral effects of 1 pairing vs 10 pairings fear conditioning protocol
To study the difference between mild and strong fear memories, our first aim was to assess how they differ at the behavioral level. Animals were trained in two different auditory fear conditioning tasks: to create a mild fear memory, we trained rats with 1 tone-shock pairing (1P), whereas strong fear memories were created using 10 tone-shock pairings (10P). The next day, fear memory was assessed in a test session where one unreinforced tone was presented (Figure 2.A). Animals trained with 10 shocks displayed higher freezing to the tone than rats trained with 1 shock (Independent samples t-test t(28) = 2.308, P = 0.028).
The difference in freezing during the test did not seem large due to a ceiling effect in the 10P group. To better visualize the differences in memory strength, fear-conditioned rats underwent extinction with 20 unpaired tone presentations and were tested for extinction retention in the next day (Figure 2.B). Rats in the 10P group froze more during the entire extinction session and at test (Repeated measures ANOVA followed by Tukey's post hoc test, F1,13 =58.18, P < 0.001). Only animals trained with 1P displayed extinction acquisition, with significant fear suppression within the extinction session (1-to-5 tone vs 16-to-20 tone: 1P group, P = 0.02; 10P group, P > 0.05).
Also, extinction retention 24h later was observed only in the 1P group (1-to-5 tone vs Test: 1 shock group, P=0.03; 10 shocks group, P>0.05). Therefore, in contrast with 1P, fear memories created with 10P exhibit impaired extinction learning, indicating a considerable difference in memory strength.
Next we assessed reconsolidation in 1P and 10P memories as previously described by Wang et al.
(2009). One day after 1P or 10P training, a 1-tone test was conducted to reactivate the fear memory.
The protein synthesis inhibitor anisomycin (125µg/µl; 0.5µl per hemisphere) was infused in the basolateral amygdala (BLA) immediately after to block reconsolidation. The effectiveness of the treatment was then evaluated in a test 1 day later. Post-reactivation anisomycin impaired performance in animals trained with 1 shock but had no effect in strongly trained rats (Repeated measures ANOVA followed by Tukey's post hoc test, F1,35 =18.27, P < 0.001). This shows that retrieval rendered the 1P memory labile, necessitating reconsolidation shortly afterwards. On the other hand, retrieval did not render the 10P memory vulnerable to anisomycin, and hence it can be considered a reconsolidation-resistant memory.

R2 Quantification of synaptic plasticity molecules between reconsolidation-permissive vs resistant memories in the BLA.
We evaluated the expression of molecules implicated with synaptic plasticity, GluN2B 31 and GluA2 32 , between animals trained in the 1P and 10P protocols. Animals were fear conditioned in the 1P or 10P protocol, tested the next day, and their brains collected 1h or 24h later for western blot analysis of BLA tissue. Controls were kept in the home cages during the entire behavioral procedure (Home cage controls, HC).
The 1P group displayed an upregulation of GluN2B in the BLA postsynaptic density (PSD), indicating that the formation of a reconsolidation-permissive memory coincides with an increase Here we replicated the behavioral findings reported by Wang et al (2008). Animals were trained with either 1 tone-shock pairing (1P) or 10 tone-shock pairings (10P) A) One day after training a retention test was conducted.
Animals in the 10P group displayed higher freezing levels than those trained with a 1P (N = 15 per group). B) One day after training an extinction session was conducted with 20 tone presentations, followed by a retention test the next day. Unlike in the 1P group, 10P animals did not display extinction acquisition and retention and showed higher freezing levels at all time points (N = 7 / 8 per group). C) One day after training a reconsolidation-blockade procedure was conducted with post-reactivation infusion of anisomycin in the BLA. The next day a retention test was conducted. The reconsolidation blockade procedure was effective in disrupting fear memory only in the 1P group (N =9/10 per group). Graphs show the mean ± s.e.m. Individual values are represented with circles. * P < 0.05. in this receptor critical for reconsolidation induction. However, 10P trained rats displayed GluN2B equivalent to HC levels ( Figure 3.A, left; One-way ANOVA followed by Tukey's post hoc, F2,13 =7.34, P = 0.009). This shows that unlike 1 pairing memories that do reconsolidate, strong reconsolidation-resistant memories are formed without GluN2B upregulation.
Next, we looked at how GluA2-containing AMPARs in the BLA PSD vary between 1P reconsolidation-permissive and 10P reconsolidation-resistant memories (Figure 3.A, right). In comparison with HC animals, GluA2 levels were upregulated one day after a 1P training, but an even higher increase was found in rats trained with 10P (One-way ANOVA followed by Tukey's post hoc, F2,21 =17.98, P < 0.001). Hence, GluA2 levels increase as a function of training strength, and the high levels found in the strong training group might reflect a resistance for memory to destabilize.
Lastly, we investigated GluA2 trafficking by comparing GluA2 levels 1h or 24h after recall at both the PSD and at extrasynaptic fractions of the BLA. In rats trained with1P, synaptic GluA2 levels are increased 24h after recall but are downregulated to HC levels shortly after recall ( Overall, this set of data shows that unlike reconsolidation-permissive 1P memories, memories created by 10P pairing that are reconsolidation-resistant have attenuated plasticity, therefore explaining why reconsolidation-blockade is ineffective in strong memories.

R3 B-adrenergic receptors activation during 10P training shifts memories into a reconsolidation-resistant state.
Here, we investigated whether β-adrenergic receptors activation during strong training plays a role in the expression of pro-plasticity molecules. After showing that strong training results in Next, we assessed if blocking β-adrenergic signaling during strong training would affect memory's susceptibility to later undergo reconsolidation. Again, animals were injected with propranolol or vehicle and were submitted to the 10P fear conditioning training. Afterwards, reconsolidation blockade was conducted, and its effectiveness evaluated as described above. During reactivation, animals that received vehicle and propranolol displayed the same freezing levels, indicating that propranolol did not impair strong fear learning (P > 0.05). In animals injected with vehicle, the reconsolidation-blockade procedure with post-reactivation anisomycin had no effect on fear expression at the test (P > 0.05). Thus, the strong fear memory was reconsolidation-resistant. Pretraining propranolol treatment, however, rendered animals susceptible to post-reactivation anisomycin, resulting in amnesia at test (Figure 4.B, Two-way repeated measure ANOVA followed by Tukey's post hoc test, F1,28 =5.07, P = 0.03).
These data reveal that reconsolidation-resistance induced by 10P training requires the activation of β-adrenergic receptors during initial learning. `

R4 Projections from the Locus Coeruleus to the BLA during 10P training are critical for memories to be formed in a reconsolidation resistant state.
We hypothesized that the LC-to-BLA projection could be mediating the formation of reconsolidation-resistant memories in 10P training. First, we quantified the expression of the activity-regulated gene c-fos in the LC 90 minutes after 1P and 10P training (Fig. 5.A). We observed that 10P training caused a higher c-fos activity (Figure 5.At; Independent samples t-test t(4) = 4.35, P = 0.01), indicating that the LC is overly engaged during the formation of strong fear memories. Next, we used a chemogenetic approach to silence LC to BLA projections during strong training (Fig 5.B-C). Virus (pAAV-hSYN-DIO-hM4Di-mCHerry) expressing the inhibitory DREADD receptor hM4Di was infused in the LC. Controls were infused with viruses not expressing hM4Di (pAAV-hSYN-tdTomato). After 3 months, there was robust somatic expression in the LC and terminal expression in the BLA (Figure 5.B). To silence LC inputs in the BLA during strong fear learning, 5 minutes before 10P training we infused the DREADD agonist C21 in the BLA. One day later, rats were infused with anisomycin or vehicle in the BLA to determine if reconsolidation could occur (Figure 5.B). During reactivation, all groups exhibited similar freezing behavior indicating that silencing LC-BLA projections did not impair strong fear learning.
Control animals infused with anisomycin did not show a memory impairment on a subsequent test, indicating that reconsolidation did not occur in these animals. When the LC to BLA projections were silenced during strong memory formation, however, recall did trigger reconsolidation since anisomycin caused amnesia in these animals ( Figure 5.C; Two-way repeated measure ANOVA followed by Tukey's post hoc test, F1,27 =10.29, P = 0.003).
These results show that inputs from the LC to the BLA during fear learning promote memory formation into a fixed state that is resistant to change. If these projections are silenced, even the memory of a strong fear experience can become labile upon recall and be modulated through reconsolidation. One day after training a reconsolidation-blockade procedure was conducted with intra-BLA post-reactivation anisomycin. The next day a retention test was conducted. The reconsolidation blockade procedure was effective in disrupting fear memory only in animals infused with the hM4Di virus (N = 7 / 9 per group). Graphs show the mean ± s.e.m. Individual values are represented with circles. * P < 0.05.

DISCUSSION
Previous studies revealed that unlike mild fear memories, very strong ones do not destabilize upon recall 11,16 . Some of the mechanisms required to induce destabilization have been identified 1,31 , but not much is known regarding how severe fear experiences end up forming reconsolidationresistant memories. In the current study we investigated the hypothesis that strong memories are different than mild ones due to the action of the NOR-LC system upon encoding. Our results show that strong fear conditioning creates memories with impaired plasticity mechanisms in the amygdala that do not destabilize, and that β-adrenergic receptor activation during encoding is necessary for these outcomes. At the systems level, reconsolidation-resistant memory formation entails inputs from the LC to the BLA during strong fear learning, and if these projections are silenced, even the memory of a strong fear experience can later undergo reconsolidation. This work builds on previous studies showing that the induction of reconsolidation involves the presence of specific cellular mechanism in the amygdala, such as the activation of NMDAcontaining GluN2B receptors 11 and transient reduction of GluA2-containing AMPAR upon retrieval 23 . Here, we first recapitulated the seminal findings of Wang et al. (2009) by showing that while memories created with 1 tone-shock pairing destabilize upon recall, memories created with 10 pairings do not. Also, memories created with the 10 pairings training are much more robust and display impaired extinction learning, a hallmark of posttraumatic stress disorder 33 . When looking at GluN2B expression, we confirmed previous reports showing that strongly-trained animals display lower GluN2B levels than mildly trained ones 11,16 . However, here we included a home cage control group in our analysis which revealed that GluN2B is not actually downregulated by strong training. In fact, GluN2B is normally upregulated by fear learning, which is hampered when learning is too strong. We also looked at GluA2 expression, which is central for synaptic stability and must be endocytosed following recall for memory to destabilize and undergo reconsolidation 23 . By comparing GluA2 levels of trained animals with home cages, we found a moderate increase in rats trained with 1 pairing and a significantly higher increase in rats trained with 10 pairings, indicating increased stability of strong memories. Retrieval transiently reduced synaptic GluA2 levels to home cage levels only in the mild memory, further indicating that the strong memory was overly stable and unable to become labile. Overall, these data show that strong memories are stored in a state with different metaplastic properties that results in reconsolidationresistance.
Most studies on the boundary conditions of reconsolidation focus on the mechanisms implicated at the time of retrieval, while those at initial encoding have not receive much attention. Here we show that conditions during encoding are crucial to defining the memory's future plasticity.
Emotionally arousing experiences cause increased release of noradrenaline from the locus coeruleus to the amygdala 25 , which increases its activation 34 and promotes fear learning 30 .
Moreover, the overactivation of this system has been implicated with traumatic memory formation in humans 35,36 To investigate the role of noradrenaline signaling on the formation of reconsolidation resistant memories, we used pharmacologic and chemogenetic approaches. First, we pharmacologically blocked β-adrenergic receptors during strong fear learning and re-assessed plasticity mechanism in the BLA and reconsolidation induction. Propranolol before strong training modulated GluN2B and GluA2 towards mild training' levelsincreasing GluN2B and decreasing GluA2 synaptic expression. In agreement with the reversal of plasticity impairments, when formed under β-adrenergic receptors blockade the memory of strong training was now able to destabilize upon recall. Next we investigated the role of projections from the LC to BLA during strong fear memory formation. To this end, LC neurons were infected with the inhibitory DREADD receptor HM4Di and its projections to the BLA were silenced during strong training with the local infusion of the DREADD agonist C21. When these specific LC to BLA projections were silenced during strong training, recall was successful in triggering destabilization, and reconsolidation blockade with a protein synthesis inhibitor resulted in amnesia. Importantly, both pharmacologic and chemogenetic manipulations of noradrenaline signalling did not alter rats' freezing compared to control animals. This suggests that noradrenaline does not reduce memory strength per se. Overall, this reveals that the activation of LC-BLA pathway during severe fear learning is necessary for memories to be formed in a fixed state and do not destabilize ( Figure 6). If this pathway is silenced, however, even the memory of a very aversive event displays plasticity and can undergo destabilization upon recall. Reconsolidation is a critical biological feature to maintain memory's relevance in guiding behavior 8,37 , and reconsolidation-targeted treatments provide a unique opportunity to weaken pathological fear memories 38,39 . Nonetheless, learning strength is a parameter which acts to prevent reconsolidation from happening, therefore curbing the efficacy of reconsolidation treatments in many cases. Thus, in order to better understand and treat disorders implicated in severe fear, it is crucial to elucidate how mild and strong memories differ. The current work underscores the widely accepted-but understudied-idea that mild and strong fear memories are neurobiologically distinct. We have described how the NOR-LC system shapes memory encoding towards a maladaptive state that is resistant to change. This is achieved by triggering molecular modifications that increase memory stability at the expense of plasticity. The identification of this mechanism advances our understanding of the boundaries on reconsolidation. Moreover, this knowledge will help guide future research in unveiling why strong memories are so implacable, and how resistance to treatment can be overcome.