Part 1:Aging Mice Show Impaired Memory Updating in The Novel OUL Updating Paradigm

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Janine L. Kwapis1,2, Yasaman Alaghband1, Ashley A. Keiser1, Tri N. Dong1, Christina M. Michael1, Diane Rhee1, Guanhua Shu1, Richard T. Dang1, Dina P. Matheos1, and Marcelo A. Wood1

Memories do not persist in a permanent, static state but instead must be dynamically modified in response to new information. Although new memory formation is typically studied in a laboratory setting, most real-world associations are modifications to existing memories, particularly in the aging, experienced brain. To date, the field has lacked a simple behavioral paradigm that can measure whether original and updated information is remembered in a single test session. To address this gap, we have developed a novel memory updating paradigm, called the Objects in Updated Locations(OUL) task that is capable of assessing memory updating in a non-stressful task that is appropriate for both young and old rodents. We first show that young mice successfully remember both the original memory and the updated information in OUL. Next, we demonstrate that intrahippocampal infusion of the protein synthesis inhibitor anisomycin disrupts both the updated information and the original memory at test, suggesting that memory updating in OUL engages the original memory. To verify this, we used the Arc CatFISH technique to show that the OUL update session reactivates a largely overlapping set of neurons as the original memory. Finally, using OUL, we show that memory updating is impaired in aging, 18-m.o. mice. Together, these results demonstrate that hippocampal memory updating is impaired with aging and establish that the OUL paradigm is an effective, sensitive method of assessing memory updating in rodents.

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INTRODUCTION

Memories need to be dynamically updated to incorporate the most relevant, recent information into storage. This ability to integrate new information into existing memory is critical to allow organisms to anticipate future outcomes and adapt to new situations. Most memories are not new associations but are alterations or additions (updates) to existing memories, particularly in the aging, experienced brain. Despite its fundamental importance, the mechanisms that support memory updating are largely uncharacterized, and even less is understood about how dysregulation of these mechanisms might contribute to age-related cognitive decline. Understanding how memories are modified in response to new information is, therefore, an important step toward improving memory across the lifespan.

Memories may be updated through a process termed “reconsolidation,” in which retrieval triggers a period of lability that could allow new information to be incorporated into an existing, stable memory. The reconsolidation process consists of an initial destabilization phase marked by protein degradation followed by a restabilization phase characterized by protein synthesis [1–9]. Recent work has demonstrated that reconsolidation is only initiated when new information is presented at retrieval; when retrieval consists of only familiar information, the original memory remains stable and resistant to amnesic agents like protein synthesis inhibitors [2, 10–16]. This suggests that new information triggers memory destabilization to allow for modification. Consistent with this, numerous studies now indicate that new information presented during retrieval can alter the content of memory [2, 3, 11, 17, 18], the affective component of memory [19–22], produce persistent extinction [23–27], or even reorganize the memory at the circuit level [11, 28].

location memory task, termed the Objects in Updated Locations (OUL) paradigm. The goals of this study were twofold. First, we aimed to establish the OUL task as a new method for studying hippocampus-dependent memory updating. Second, we aimed to use this paradigm to determine whether memory updating is impaired with age.

The OUL paradigm is novel in that it is able to assess both the original memory and the updated information in a single test session. Further, OUL uses incidental learning that takes advantage of rodents’ innate preference for novelty, avoiding unnecessary stress and making it appropriate for testing age-related deficits in memory updating. Here, we first validate the OUL task, demonstrating that young mice show memory for both the original training and the memory update in the OUL test session. Next, we used two complementary methods (intra-hippocampal anisomycin injections and Arc CatFISH) to verify that the OUL update requires retrieval of the original memory, rather than forming a new, independent memory. Finally, using OUL, we demonstrate that aging mice show impairments in memory updating, suggesting that an inability to update memories may contribute to age-related cognitive decline. Together, these results demonstrate that the OUL paradigm can be used to understand the mechanisms underlying hippocampal memory updating across the lifespan.

MATERIALS AND METHODS

Mice

The subjects were young adult (2–4-months-old) or old (18–20 months old) male C57BL/6 J mice (Jackson Laboratory). Mice were housed, fed, and handled as described in the Supplemental Methods. All procedures were approved by the University of California, Irvine’s Institutional Animal Care and Use Committee and were in compliance with the National Institutes of Health guidelines.

OUR task

Following handling and habituation (see Supplemental Methods), mice were trained with two identical objects in specific locations (A1 and A2) for 1 or 3d in the habituated context. Twenty-four hours later, mice were given an update session, in which they were assigned to either the No Update condition or the Update condition. No Update mice were re-exposed to training locations A1 and A2. For Update mice, one object was moved to a new location (A3). Finally, mice were given a retention test in which they were exposed to the three objects in previously experienced locations (A1, A2, A3) and the fourth object in a novel location A4. Memory for the original training formation was inferred by comparing exploration of the novel location A4 to locations A1 and A2. Memory for the update was inferred by comparing exploration of the novel location A4 to location A3.

Cannulation surgery

Mice in Fig. 2 were implanted with chronic cannulae as previously described [37] to allow for direct hippocampal infusion of anisomycin after updating (see Supplemental Methods). Mice recovered for at least 7d before behavioral testing began. Immediately after the update session, mice were bilaterally infused with anisomycin (ANI, 125/μg/μL) or vehicle (VEH) into the dorsal hippocampus (1.0 μL/side). Cannula placements were confirmed by staining coronal slices with cresyl violet.

CatFISH

Fluorescence in situ hybridization for CatFISH was performed as previously described [38, 39] (see Supplement for details). The DIG-labeled Arc antisense riboprobe was hybridized with tissue overnight and visualized with an anti-DIG-HRP conjugate, visualized with a Cy3 substrate kit, and counterstained with DAPI.

Confocal images were collected in area CA1b of the dorsal hippocampus and the images were scored to identify the subcompartment localization of Arc to calculate a similarity score as previously described [39].

Statistical analysis

The OUL task was hand-scored to measure object exploration times as previously described for object location memory experiments [40, 41]. Statistical analyses (see Supplement) were performed using two-tailed Student’s t-tests, one-way ANOVAs, or two-way ANOVAs with Sidak-corrected t-tests. Mixed-model ANOVAs were used when one variable was a repeated measure. An α value of 0.05 was required for significance.

RESULTS

Young animals successfully show memory for both training and updating information in OUL

We first aimed to validate the OUL task as capable of assessing memory updating in young mice (Fig. 1a). Following habituation, mice were first trained to learn the locations of two identical objects in a familiar context (training session, Day 1). The following day, during the update session (Day 2), mice were split into two groups. The No Update group was re-exposed to the two familiar object locations (A1 and A2). The Update group was exposed to one familiar object location (A1) and one object moved to a new location (A3). All groups were given a test session (on Day 3) to assess the animals’ memory for both the original object locations and the updated location. Attest, mice were exposed to four identical objects: three in previously exposed locations (A1, A2, and A3) and one in a novel location (A4). Memory for the original training was assessed by comparing exploration of the novel object location (A4) to exploration of the training object locations (A1 and A2). Memory for the updated information was assessed by comparing exploration of the novel object location (A4) to exploration of the “updated” object location (A3). As mice prefer novelty, memory for either the original training session or the updated information is demonstrated by increased exploration of the object in the novel location (A4) compared to each of the other three objects (indicated by a higher score on the discrimination index (DI), see methods).

During training, mice in both groups showed similar levels of exploration of objects A1 and A2, resulting in a discrimination index (DI) near zero for both groups (Fig. 1bi; two-tailed Student’s t-test: t(17) = 0.086, p = 0.40). Total exploration levels were also similar between the No Update and Update groups during training (Fig. 1bii; two-tailed Student’s t-test: t(17) = 0.035, p = 0.97, n = 10,9).

To confirm that the original object location memory was successfully acquired, we next assessed performance during the update session (Fig. 1c). Mice in the No Update group showed an equal preference for the re-exposed locations A1 and A2, resulting in a DI near-zero (Fig. 1ci). Mice in the Update group, on the other hand, preferentially explored the moved object A3 compared to the unmoved object A1 and showed a significantly higher DI compared to the No Update group (Fig. 1ci, two-tailed Student’s t-test: t(17) = 3.57, p = 0.002). Total exploration was also significantly higher in the Update group compared to the No Update group during the update session (Fig. 1cii; two-tailed Student’s t-test: t(17) = 3.39, p = 0.004), suggesting that mice spent more time exploring the objects when a novel location was introduced. Together, these results confirm that the original object locations were learned in young mice exposed to a 10-min training session, consistent with previous reports [41, 42].

To determine whether the original memory was modified to include the updated object location (A3), mice were given a test session in which each familiar location (A1, A2, and A3) was tested against a novel object location (A4) (Fig. 1d). Raw percent exploration time for each of the four objects during the test session is shown in Supplementary Fig. 5A. Both groups showed intact memory for the original information, as both Update and No Update animals similarly preferred the novel location A4 over both original locations A1 (Fig. 1di; two-tailed Student’s t-test: t(17) = 0.31, p = 0.76) and A2 (Fig. 1dii; two-tailed Student’s t-test: t(17) = 1.66, p = 0.12). This confirms that after updating, the original information is retained. To test whether the updated information was successfully learned, we also compared exploration of the novel location A4 to the updated location A3 during the test session. Mice in the No Update group showed an equal preference for objects A3 and A4, resulting in a DI that was not significantly different from zero (Fig. 1diii; one-sample t-test compared to 0: t(9) = 1.55, p = 0.156). Mice given the update, on the other hand, preferentially explored the novel location A4 over the updated location A3 (Fig. 1diii, one-sample t-test compared to 0: t(8) = 3.42, p = 0.009; two-tailed Student’s t-test comparing Update to No Update: t(17) = 2.92, p = 0.0096), indicating that they recall the updated location. No differences in total exploration during the test session were observed between groups (Fig. 1div; two-tailed Student’s t-test: t(17) = 1.27, p = 0.22). Together, these results confirm that young animals exhibit preference behaviors consistent with a successful recall of both the original information and the updated information at test.

Fig. 1 Young mice successfully perform memory updating in the OUL task. an Experimental design. b Training session behavior. Mice show a low DI, indicating no preference for object A1 or A2 (left), and have similar levels of total object exploration (right). c Update session behavior. (i) Mice in the Update condition prefer the novel location A3 to the familiar location A1 whereas No Update mice show similar exploration of the familiar locations A1 and A2, with a DI near zero. (ii) Update mice show significantly more total object exploration than No Update mice. d Behavior during the test session. (i) Mice in both the Update and No Update group show intact memory for the original training object location A1. (ii) Both groups also show memory for original training location A2. (iii) Only the Update group shows a preference for the novel location A4 over the updated location A3; No Update mice prefer objects A3 and A4 equally. (iv) Mice show similar levels of total object exploration during the test session. Data are presented as mean ± SEM. **p < 0.01

Post-update hippocampal protein synthesis inhibition disrupts both the updated information and the original memory We next tested whether the updated information learned in the OUL task modifies the original memory or forms a new, independent association. For the OUL task to be a useful model of memory updating, the original memory needs to be retrieved and altered to incorporate the new object location information presented during the update. To test this, we used two complementary methods to examine whether the original memory is engaged by the OUL update session: anisomycin infusions and catFISH analysis.

First, we tested whether anisomycin applied after the update session would affect memory for the original training information. Numerous studies have demonstrated that post-retrieval infusion of the protein synthesis inhibitor anisomycin (ANI) can block reconsolidation under certain conditions [43, 44] at least temporarily [see 45]. Presumably, the original memory is made labile by the new information presented during retrieval but fails to properly restabilize in the absence of de novo protein synthesis, leading to a disruption of the original memory [6–9]. When no new information is presented during the retrieval trial, on the other hand, ANI infusions have no effect, as new information is required to initiate reconsolidation [6].

To determine whether the updated information in the OUL task engages the original memory, we infused anisomycin immediately after the update session and assessed whether this attenuated the original association (Fig. 2a). We infused ANI directly into the dorsal hippocampus (DH) after the update session (Fig. 2b, individual cannula placements shown in Supplementary Fig. 1A), as spatial object location memories are particularly sensitive to manipulations in this region [40, 46, 47] and this would allow us to localize the memory update and avoid off-target effects of global protein synthesis inhibition. Mice showed similar exploration of objects A1 and A2 at training (two-way ANOVA, no significant main effects or update x drug interaction) and had similar levels of total exploration (two-way ANOVA, no significant main effects or update x drug interaction) (Supplementary Fig. 1B, C). During the update session, mice in the Update condition showed a significant preference for the moving object, and mice in the No Update condition continued to show similar exploration of the objects in both familiar locations (Fig. 2ci; two- way ANOVA, significant main effect of update (F(1,29) = 42.31, p < 0.0001), but no significant effect of drug or significant interaction, n = 6,6,12,9)). No group differences were observed between animals destined to get anisomycin or vehicle and no difference in total object exploration was observed during the update (Fig. 2cii; two-way ANOVA, no significant main effects or interaction). This suggests that before anisomycin infusion, mice

Fig. 2 Disrupting protein synthesis after memory updating impairs both the original memory and the updated information in young mice. an Experimental design. b Representative cannula placement targeting area CA1 of the dorsal hippocampus. c Update session behavior. (i) Both groups of mice in the Update condition preferred the novel location A3 over the familiar location A1 whereas No Update mice show a DI near zero, indicating no preference. (ii) Mice show similar total exploration across groups. d Behavior during the test session. (i) For original training object A1, mice in the No Update condition show intact memory regardless of drug treatment. For the Update condition, anisomycin-treated mice show a significantly reduced DI for A1 compared to vehicle controls, indicating that anisomycin impaired memory for the original training information in Update animals. (ii) For original training object A2, no significant effect of anisomycin was observed in either No Update or Update animals. (iii) For the updated location A3, No Update animals showed a low DI compared to object A4, regardless of drug infusion. Update animals given anisomycin showed a significantly lower DI than vehicle controls, indicating that anisomycin impaired memory for the updated information. iv) No significant differences in total exploration were observed at the test. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001. VEH, vehicle; ANI, Aniosmycin