Early β adrenoceptor dependent time window for fear memory persistence in APPswe/PS1dE9 mice

In this study we demonstrate that 2 month old APPswe/PS1dE9 mice, a transgenic model of Alzheimer’s disease, exhibited intact short-term memory in Pavlovian hippocampal—dependent contextual fear learning task. However, their long-term memory was impaired. Intra-CA1 infusion of isoproterenol hydrochloride, the β-adrenoceptor agonist, to the ventral hippocampus of APPswe/PS1dE9 mice immediately before fear conditioning restored long-term contextual fear memory. Infusion of the β-adrenoceptor agonist + 2.5 h after fear conditioning only partially rescued the fear memory, whereas infusion at + 12 h post conditioning did not interfere with long-term memory persistence in this mouse model. Furthermore, Intra-CA1 infusion of propranolol, the β-adrenoceptor antagonist, administered immediately before conditioning to their wildtype counterpart impaired long-term fear memory, while it was ineffective when administered + 4 h and + 12 h post conditioning. Our results indicate that, long-term fear memory persistence is determined by a unique β-adrenoceptor sensitive time window between 0 and + 2.5 h upon learning acquisition, in the ventral hippocampal CA1 of APPswe/PS1dE9 mice. On the contrary, β-adrenoceptor agonist delivery to ventral hippocampal CA1 per se did not enhance innate anxiety behaviour in open field test. Thus we conclude that, activation of learning dependent early β-adrenoceptor modulation underlies and is necessary to promote long-term fear memory persistence in APPswe/PS1dE9.


Scientific Reports
| (2021) 11:870 | https://doi.org/10.1038/s41598-020-79487-5 www.nature.com/scientificreports/ hypothalamus 42,43 modulating innately anxiogenic behaviours. Therefore, distinct representation arise within the vCA1 based on the valence of the stimuli (innate vs learned) routing information via projection defined vCA1 neurons 40 . However, we still do not know the dynamics and the nature of learned fear versus innate anxiety in APPswe/PS1dE9 mice at an age where there is constitutive overexpression of APP and PS1 genes without β-amyloid deposition.
Here we applied contextual fear conditioning (cFC) and open field test (OFT) to investigate the causal role of local vH β-AR dependent signalling in modulating learned fear versus innate anxiety related behaviour. The current study primarily mapped the time course of fear expression after cFC in APP/PS1 mice. Consequently, this measure has proved useful in detecting the time dependent decline in the strength of fear memory after learning acquisition. Further experiments were designed specifically to study the role of β-AR dependent plasticity in the vH during early stages of memory consolidation, that would determine long-term fear memory persistence in 2-month-old APP/PS1 mice. We report that APP/PS1 has a local vH β-AR signalling dependent time window spanning from 0 to + 2.5 h, which is imperative for long-term fear memory persistence. Furthermore, using OFT, we demonstrate that vH β-AR agonist infusion per se does not interfere with innate anxiety responses of APP/PS1 mice.

Materials and methods
Experimental animals. The generation, care, and use of mice as well as all experimental procedures were approved by the Institutional Animal Ethics Committee of the Indian Institute of Science, Bangalore. Transgenic mice B6C3-Tg (APPswe/PS1dE9) 85Dbo/J (https ://www.jax.org/strai n/00586 4) were originally obtained from The Jackson Laboratory, and was kindly provided by Prof. Vijayalakshmi Ravindranath, Director, Centre for Brain Research, Bangalore, India 26 . Wild Type (WT) and APPswe/PS1dE9 (APP/PS1) mice were bred at the Institutional Central Animal Facility, and were housed in standard mouse cages under conventional laboratory conditions (12 h dark and 12 h light cycle, constant temperature and humidity), and were given food and water ad libitum. Behavioural experiments were performed using male APP/PS1 and WT mice. No samples were excluded from any of the experiments described herein, unless otherwise mentioned in the analysis. Animal experiments were designed and followed in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. WT and APP/PS1 mice were assigned randomly to the respective groups based on the genotype. Different litters of the same age group were taken and were divided into control and experimental groups.
Behavioural procedures. All experiments were carried out with male mice that were 55-65 days old (~ 30 g) at the beginning of the experiment. Mice were housed individually for 3 days, and were handled for 5 min everyday prior to behavioural testing. All behavioural experiments were conducted at approximately the same time during the light cycle (9:00-15:00) by one constant experimenter. 26 , the training context was rectangular in shape. Identity of the context was maintained with the presence of 2% acetic acid (vol/vol). The conditioning chamber was cleaned with 70% ethanol before and after each session. Mice were allowed to explore the apparatus for 1 min, and then received 3 foot shocks (2 s and 0.6 mA each, intershock interval 30 s). Contextual fear memory was assessed by returning the mice to the training context 24 h or one month after fear conditioning and analysing freezing during a test period of 2 min.

Contextual fear conditioning (cFC). For cFC
Open field test (OFT). Mice were released in the corner of a 45 × 45 cm open field arena (Fig. 3a). Their activity was recorded with a camera mounted on the ceiling above the center of the open field arena for 10 min. At the end of testing, mice were returned to their home cage. The arena was partitioned into outer periphery and center region, and the amount of time spent and the distance travelled were extracted using a previously validated open-source toolbox for automated phenotyping of mouse behaviour 44 .

Pharmacology in vivo.
All the local treatments were carried out with the help of cannulas (33G). Guide cannulae (26 gauge, Plastics One) were implanted to the skull with dental cement. Mice were then given 1 week to recover from surgery. 32-gauge stainless steel injectors attached to 5-μl Hamilton syringes were inserted into the guide cannulae to deliver the following drugs-Isoproterenol Hydrochloride (0.25 μg per side; Sigma Aldrich, India); ( ±)-Propranolol hydrochloride (0.5 μg per side; Sigma Aldrich, India), and 0.9% saline. Coordinates relative to bregma are as follows: vH (AP − 3.0, ML ± 2.6, DV − 3.2). A total volume of 200 nl was injected; the injector was left for another minute to allow diffusion into the tissue. Statistical analysis. Data analysis was performed using Prism 7 (GraphPad Software Inc., La Jolla, CA, USA). No statistical methods were used to predetermine sample sizes. Our sample sizes are similar to those generally employed in the field 33 . The sample size per group is mentioned in the respective figure legends. Statistical analyses were designed using the assumption of normal distribution and similar variance among groups. They were performed using unpaired two-tailed Student's t-test for paired comparisons, and two-way ANOVA followed by post hoc tests was performed when two factors were compared. Results are presented as mean ± S.E.M. The significance of the results was accepted at P < 0.05. Eta squared (η2) are reported as a measure of effect size 45  β-AR activation during acquisition or immediately post acquisition promotes long-term fear memory persistence in APP/PS1 mice. To determine whether endogenous local β-AR signalling is necessary to ensure longevity of short-term memory during and following learning, we delivered β-AR antagonist (±)-Propranolol hydrochloride to the vH CA1 (Fig. 2c) at different time points in APP/PS1 mice (Fig. 2a).

Discussion
In this study, we describe the temporal course of behavioural expression of fear memory upon cFC, to determine the dynamics of β-AR modulation and memory consolidation in the vH CA1 of 2-month old APP/PS1 mice. We demonstrate a unique β-AR dependent time window of early memory consolidation (+ 0-2.5 h) that is crucial for long-term fear memory persistence in APP/PS1 mice. Our findings are consistent with previous reports using 7-month-old APP/PS1 mice that are impaired in long-term but not in short-term contextual fear memory 9 . The APP/PS1 mouse model exhibits constitutive overexpression of APP and PS1 genes, elevated levels of toxic soluble oligomeric Aβ, and synaptic deficits are reported at 1-1.5 51 and 3.5 months of age in the cortex and hippocampus [52][53][54] . This might underlie the mild behavioural impairments observed in our study with 2-month-old APP/PS1 mice. We further demonstrate that β-AR agonist per se does not modulate the baseline innate anxiety levels of APP/PS1. Pharmacological studies using male Sprague-Dawley rats have indicated that between 0 and 6 h after a learning experience, the noradrenergic system is activated to reinforce long-term memory processing in the hippocampus 31,32 and prefrontal cortex [55][56][57] . Our results indicate the existence of a local β-AR signalling dependent early consolidation window between + 0 and 2.5 h which is required to ensure long-term fear memory www.nature.com/scientificreports/ persistence. Consistent with this notion, the late consolidation window which has been reported to be D1/5R dependent at + 12 h 33-35 was unaffected by administration of β-AR antagonist. Apart from being involved in consolidation of long-term fear memory, vH neurons also carry a representation of innately anxiogenic stimuli. Manipulation of the vH afferents or efferents are also known to directly impact anxiety-related behaviour [58][59][60] . Furthermore, anxiolytic effects of propranolol have been well documented in animal models of anxiety 61,62 . Therefore, the increased freezing to context induced by β-AR agonist may have emerged from β-AR agonist induced changes in innate anxiety levels of APP/PS1. This led us to perform open field experiment with APP/PS1 mice following vCA1 β-AR agonist infusion to investigate whether the β-AR agonist potentiated the consolidation of the fear memory trace, or enhanced innate anxiety levels to promote avoidance behaviour. Intriguingly, we found the concentration of β-AR agonist which we administered in our studies did not have a significant role in mediating an avoidance behaviour to the context in APP/PS1.
It was recently demonstrated that β2-AR expressed in astrocytes are the primary mediators of fear based contextual memory consolidation, rather than β1-AR which are primarily located in the neurons 63 . These findings indicate the differential contribution of β1-ARs and β2-ARs in mediating hippocampal memory formation and associated processes. More importantly, β2-AR dysregulation in astrocytes has been implicated in AD 64 . These findings further suggest that the β-AR agonist may facilitate strengthening of memory trace by gating synaptic plasticity in vCA1 synapses through distinct pathways for learned versus innate anxiety, based on the affective valence of the inputs.
Memory stabilization after learning also involves temporal molecular changes 65 . De novo protein synthesis occurring within 1 h or around 4 h after learning acquisition is known to be important for memory stabilization 66 . A recent study 67 demonstrated translational repression of specific genes such as neurensin-1 (Nrsn1) and Mitogen-activated protein kinase 6 (Mapk6) around 5-10 min post cFC acquisition without a change in mRNA levels. Furthermore, at later time points of 30 min and 4 h, they observed gene suppression especially of a group of genes which were positively controlled by Estrogen Receptor 1 (ESR1). Overexpressing Nrsn1 or activating ESR1 in the hippocampus post learning acquisition was sufficient to impair contextual fear memory formation. Interestingly, signalling pathways of ESRs are intertwined with those of the β-ARs 68 pointing to the possibility of functional convergence and their interdependence at around 2.5 h in the vH modulating long-term memory consolidation. However, we still do not know the mechanisms underlying these upstream regulations which might be pertinent for long-term memory persistence.
The most progressive hypothesis with regard to memory deficits in AD is the 'retrieval deficit' where the store of memory remains relatively intact during early AD and the deficits are associated with inability to access and modify this information long-term [7][8][9] . In contrast, our results support the notion that early consolidation deficits caused by absence of appropriate β-adrenergic neuromodulation may be responsible for memory deficits in early AD. Thus, β-adrenergic neuromodulation may be necessary to recruit and maintain system wide networks for long-term memory persistence in APP/PS1 mice. Accordingly, the unique time window for memory persistence uncovered in this study may represent a new therapeutic window to alter memory networks and promote better quality control of mechanisms that are required for long-term memory consolidation.