When is the hippocampus involved in recognition memory

Respective groups of mice completed a third arena habituation session yoked weak AH3 group or yoked strong AH3 group. Mice were euthanized and brains dissected 90 min after training. The mean distance traveled by the mice in the AH3 group 2, The novel, but otherwise non-threatening contexts tend to elicit greater amounts of exploratory locomotor responses in rodents.

A subset of mice that received weak and strong object training was presented with a test session 24 h later. These mice were euthanized, and brains dissected 90 min after testing. Three tissue sections from the CA1 and PRh of each mouse were processed immunohistochemically to detect the neuronal expression of Arc protein.

First, to examine whether there were differences in Arc counts between the AH3 conditions, a two-factor region: CA1 vs. However, there was no significant difference in raw Arc counts within the CA1 between mice that completed a weak AH3 and those that completed a strong AH3 condition. These results suggest that exploration of the empty arena for the amount of time required of the weak AH3 condition and that for the strong AH3 condition did not differentially activate neurons of the CA1 region but did so for neurons of the PRh.

The differences observed in raw Arc counts in the PRh after the two AH3 control conditions perhaps provide some degree of justification for our choosing to normalize Arc counts by the respective AH3 condition before pursuing analyses of Arc protein expression after the two object training conditions.

Next, a three-factor condition: AH3 vs. This result suggests that some threshold duration of object exploration must be reached to up-regulate Arc protein expression in the CA1 region; 10 s of exploration of each sample object is not sufficient, while 30 s of exploration of each object is sufficient. These Holm-Sidak test results are indicated by the asterisks in Figure 4B.

This pattern of results is consistent with the interpretation of our inactivation data; that is, the consolidation of a strong object or event memory is not dependent upon neuronal activity in the PRh nor does this behavioral experience appear to drive synaptic plasticity there.

The finding that the limited amount of exploration of, or exposure to, novel objects or stimuli dictated by the weak training protocol, elicited greater numbers of Arc-positive cells in the PRh as compared to CA1 of the hippocampus, is consistent with results of prior analyses of Fos expression Wan et al. This pattern of results supports the view that neuronal activity and synaptic plasticity in CA1 is necessary for the consolidation of a strong object, or event memory.

The data are also consistent with the view that the consolidation of a weak memory for objects explored does not engage CA1 neuronal activity or drive synaptic plasticity there. For clarity, these significant results were not labeled in Figure 4B but will be fairly obvious to the reader. The differential impact of object training compared to an equivalently timed arena habituation session on the number of Arc expressing neurons in the CA1 and PRh runs counter to the argument that differences in Arc protein expression between strong and weak sample sessions are attributable to the time spent within the arena.

We conducted linear regression analyses to further examine the relationship between object training-induced Arc protein expression in the PRh and CA1 after a weak or strong sample session.

While the significant correlation between regions after weak training is interesting and consistent with the results from the three-factor ANOVA described above, it should be considered with the proviso that caution should be exercised when interpreting results of regression analyses based on fewer than 10 pairs of observations. Mice were euthanized and brains dissected 90 min after testing, and tissue sections from the CA1 were processed for Arc protein expression.

Raw counts of Arc positive cells in CA1 were normalized against the average Arc cell counts from the S2 control group.

The difference in CA1 Arc protein expression observed in the test session mice compared to that of the S2 mice likely reflects the process of discriminating the presented test session objects one being novel with the retrieved memory of the objects explored during the previous sample session.

Except for the novel object, all other stimuli present during the test session are identical to that of the S2 condition. Presumably, both the test session and the S2 session experiences promote retrieval of the memory for the previous sample session, yet the respective groups of mice use that information distinctly. Thus, the significant difference in Arc protein expression in CA1 between the two groups of mice indicates the sensitivity of immunohistochemical analysis of activity-dependent Arc expression and provides further support for a critical role of CA1 in strong object memory.

Thus, the increased expression of Arc protein in CA1 neurons after strong object memory training occurs independently of a net change in the level of Arc mRNA transcripts. This result is consistent with our immunohistochemistry results, described above, suggesting that Arc mRNA expression in CA1 was not affected by weak sample exploration, likely due to the lack of change in protein expression in CA1 following weak sample session training—given that CA1 neurons were not activated by this behavioral event.

This result supports the view that CA1 activity is not critically involved in the consolidation of weak object memory. The equivalent Arc mRNA expression levels between weak sample sessions and AH3 trained mice is in contrast to the observed significant increase in Arc protein expression in PRh neurons between similarly trained groups of mice.

The increased Arc protein expression in the PRh after weak sample session training is independent of a net change in the level of Arc mRNA transcript expression, suggesting that weak training drives an increase in translation of Arc mRNA transcripts.

Our experiments used local microinfusion of muscimol to temporarily block neuronal activity in the CA1 and PRh during object memory consolidation, and immunohistochemistry to stain for Fos and Arc proteins, and qRT-PCR to quantify Arc mRNA expression triggered by behaviors associated with the encoding, consolidation, and retrieval of object memory. It was predicted that if the amount of time the mice were permitted to explore objects during the sample session of the SOR task was increased or decreased, then the strength of the resulting memory of the objects would be altered accordingly.

Based on test session measures of discrimination ratio, we characterized the resulting memory after a limited 10 s exploration of each sample session object or 13 s of one object as a weak object memory, while that after a more extensive 30 s exploration of each sample session object or 38 s of one object as a strong object memory.

These two different sample session object exploration criteria were then used to test whether the formation of strong object memory or weak object memory differentially recruited CA1 or PRh neuronal activity. Our experiments yielded several key results. First, the bilateral inactivation of CA1 after strong, but not weak, object memory training impaired object discrimination during the test session presented 24 h later.

This result suggests that CA1 neuronal activity is essential for the consolidation of strong object memory or the memory of the event of exploring novel objects within a familiar context. The second key result was the complement: the bilateral inactivation of neuronal activity in the PRh after weak, but not strong, object memory training impaired object discrimination during the test session 24 h later. This result supports the view that PRh neuronal activity is required for the consolidation of object memory that guides discrimination based on object familiarity but is not necessary for the consolidation of stronger event memory.

Although the local microinjections of muscimol were bilateral and restricted to the respective regions, both regions extend in the septo-temporal or rostral-caudal plane. Therefore, one might caution against concluding the role of a given hippocampal or extra-hippocampal structure based on the behavioral results after functional inactivation of a restricted subset of its neurons.

For the present study, all aspects of the injection volume, rate, and respective cannula placements were consistent across the groups of mice. However, whether the bilateral injections of muscimol into the CA1 or PRh impaired test session behavior depended on the amount of time the mouse was permitted to explore objects during the sample session.

Specifically, the same degree of post-training inactivation of neuronal activity in the PRh e. Thus, the effect of inactivation appears to have been dependent on the degree of behavioral experience rather than any methodological difference.

It is also important to note that by controlling the duration of the sample session by requiring the mice to accumulate 10 s or 30 s of sample object exploration, effectively matches all mice for sample session experience.

This procedural control provides some assurance that the differences observed in subsequent test session behavior are most likely a consequence of the post-sample manipulation rather than due to sensorimotor, motivational, or attentional differences in the mice during the sample session. In parallel with the regional inactivation experiments, we analyzed brain sections acquired from an additional cohort of mice to examine the behaviorally triggered expression of Arc protein, which is an IEG marker for neuronal plasticity.

The Arc protein analyses revealed that a significantly larger ensemble of CA1 neurons was recruited as a result of the exploration of objects during the sample session of the strong memory protocol, as compared to the ensemble recruited as a result of the exploration of an empty arena during AH3. This finding, that strong object memory is supported by the activity of CA1 neurons, but not PRh neurons is consistent with the finding that the firing of individual PRh neurons is modulated by object familiarity Ahn and Lee, ; Brown and Banks, The contribution of CA1 to strong object memory reported here is also consistent with previous reports that when mice are allowed to explore each sample object for at least 30 s, the memory that is encoded for that object is dependent on an intact and fully functioning hippocampus Cohen et al.

In contrast, analyses of Arc protein revealed that an equivalent, but lower number of CA1 neurons were recruited during the sample session of the weak object memory protocol and AH3. Moreover, the Arc protein analyses revealed that the CA1 neuronal ensemble recruited by the AH3 group yoked to the strong object memory trained group was not significantly different from that of the AH3 group yoked to the weak object memory trained group.

This means that exploration of the empty familiar arena, regardless of time spent within the arena, did not differentially activate CA1 neurons.

Therefore, we can conclude that the significantly greater activation of CA1 neurons observed after the strong object memory training reflects the object exploration-induced encoding and consolidation of object memory rather than simply a consequence of exploring the familiar context. Analyses revealed no significant difference in Arc mRNA expression in the CA1 after strong object memory training compared to the strong AH3 control condition, and no significant difference in Arc mRNA expression in the PRh after weak object memory training compared to the weak AH3 control condition.

Taken together with the results from the present inactivation and Arc protein expression studies, the Arc mRNA indicates a somewhat consistent pattern of results.

In summary, the data suggest that object memory consolidation that promotes Arc protein expression does so without altering the level of Arc mRNA expression beyond that induced by the respective AH3 control condition. The pattern of results may also indicate that increases in Arc protein expression in the CA1 or PRh after strong or weak training, respectively deplete local stores of Arc mRNA, and homeostatic mechanisms are engaged to replenish basal levels of Arc mRNA.

Note that Arc protein expression in PRh was significantly higher after weak object training compared to the weak AH3 condition. Collectively, the data suggest that if object exploration continues beyond the weak training, as it would during strong object training, then there is a significant increase in Arc protein expression in CA1, perhaps triggered by PRh dependent activation of hippocampal circuits consistent with the notion of transferal of object information from PRh to the hippocampus.

One, the transfer of object information from PRh to the hippocampus requires significant translation i. This result alludes to the potential for a downregulating mechanism influencing baseline levels of Arc mRNA transcripts as a potential target underlying the recruitment of CA1 to support strong object memory Bramham et al.

This result suggests that Arc mRNA expression is decreased in the brain region not engaged by the object memory training. These results suggest that the increase in Arc protein observed in our immunohistochemical studies was not dependent on an increase in Arc mRNA expression. We acknowledge that the utilization of the biopsy punch for tissue sampling introduces variability, particularly in CA1 samples, which may include white matter and portions of the overlying cortex.

A more precise tissue sampling technique e. Collectively, these findings suggest that a sufficient degree of exploration of a novel or familiar object is necessary before synaptic plasticity is induced within the CA1 region, and thereafter CA1 neuronal activity is required to successfully consolidate the memory of the objects.

Our previous work Stackman et al. Therefore, it seems that the strong object memory-training event triggers synaptic plasticity within CA1, necessary for the consolidation and subsequent retrieval of the memory for the object exploration event, both of which are dependent on CA1 neuronal activity. It remains to be determined whether the pre-test inactivation of PRh neuronal activity affects novel object preference in mice that completed strong object exploration criteria.

However, the present data suggest a transfer of critical regional neuronal activity supporting object memory consolidation from PRh to CA1, depending on the amount of time the mice are permitted to explore the novel objects during the sample session.

Taken together with the present results, we propose that the transfer of object information from the PRh to the CA1 requires time, but also depends on a critical degree of memory load. These findings may be viewed as largely consistent with the notion that PRh is critical for object recognition based on familiarity; however, the recollection of the memory for the event of exploring novel objects within the spatial and temporal context of the testing arena requires CA1.

Furthermore, the immunohistochemical analyses of the brain tissue of mice that explored the novel objects for only 10 s each i. Limiting the amount of novel object exploration during the sample session results in a weak memory, as demonstrated by significantly lower object discrimination exhibited during the test session. Our results reveal that the consolidation of such a weak object memory does not recruit CA1 neurons. Both AH3 mice and mice that received weak object training mice engaged in similar patterns of exploratory behavior and equivalent movement during their respective sessions; however, the information acquired is likely different, yet this difference was not reflected in CA1 Arc protein or mRNA expression.

One possibility is that the CA1 region may not contribute to the encoding of object memory, whether weak or strong. Testing the specific contribution of CA1 to object memory encoding will require silencing selective populations of neurons with high temporal precision. Certainly, pharmacological tools such as local muscimol microinfusion would not permit such highly precise time locking, although chemogenetic and optogenetic tools may be effective Madisen et al.

The second possibility is that CA1 is only recruited after the mouse achieves some threshold amount of object exploration. Before reaching that object exploration threshold, the processing of memory for the objects is supported by PRh. This argument then suggests that there is some information threshold or storage buffer-like mechanism within PRh that once surpassed triggers the recruitment of CA1. A similar interpretation of mnemonic transfer from PRh to the hippocampus was stated in earlier reports Gaffan and Parker, ; Liu and Bilkey, , and more recent reports suggest that interactions between hippocampal regions and extra-hippocampal regions including the PRh are critical for encoding object-based episodic memory Vilberg and Davachi, Such a transfer mechanism would be consistent with the downregulation of Arc mRNA observed in the PRh following strong object memory training compared to that observed in mice that experienced the AH3 control condition.

Moreover, the significant increase in the count of Arc protein-positive PRh neurons of mice trained in the weak object memory protocol compared to the yoked AH3 group is consistent with the contribution of the PRh to object recognition, perhaps based on object familiarity. The finding that muscimol inactivation of PRh neurons impaired object memory encoded after limited exploration of objects, is also consistent with reports from studies of humans, nonhuman primates, and rodents, that damage to the PRh increases the rate of forgetting of recently acquired information Wiig et al.

These results are in agreement with previous findings that have shown that when mice explored sample objects for a limited amount of time in this case 10 s on each object , test session object recognition was not impaired by hippocampal lesion or inactivation Winters and Bussey, ; Winters et al. Taken together, these data imply that minimal exploration of novel objects promotes PRh neuronal activity; without recruiting CA1.

There is a limit to the interpretations one can draw from the results of traditional lesion studies related to the function of a specific brain region since the lesion technique renders the region of interest destroyed and unavailable to process incoming information.

Temporary inactivation is an alternative technique that avoids some of the pitfalls of the lesion approach. Previous reports indicate that with a strong object memory protocol, pre-sample, or pre-test, intra-CA1 muscimol impairs object discrimination during the test session De Lima et al.

The results of the present regional inactivation studies extend the evidence demonstrating a significant role for CA1 in strong object memory. Additionally, the current immunohistochemical findings demonstrate that a significantly greater number of CA1 neurons are active after a strong sample session as compared to after a weak sample session, as represented by an increase in Arc expression.

Alternatively, in a weak object memory protocol, the only inactivation of PRh led to impairments in test session object memory. P21 mice underwent the same NOR protocol except with an immediate delay under 2 min.

E Object exploration during the test phase of the NOR immediate delay task during the first 20 s of object exploration. P21 animals that underwent an immediate delay displayed preference for the novel object. To determine when mice form the ability to detect recency among objects explored at different time points, we tested mice at P16, P21, P25, P28 and P35 in the TOR task Fig.

P16, P21 and P25 mice explored both old and recent objects similarly, whereas P28 and P35 mice spent more time exploring the older object Fig. Assessing relative preference by the discrimination index revealed that P16, P21 and P25 mice did not discriminate between the older and recent objects above chance level 0.

Ontogeny of temporal order recognition memory. A Schematic of the TOR task. B Object exploration during the test phase of the TOR task. Only P28 and P35 mice spent significantly more time exploring the old object compared to the recent object. C Relative preference for the old object was calculated by a discrimination index DI dividing the time spent exploring the old object by the total object exploration time throughout the first 20 s of object interaction.

A preference for the old object was observed only at P28 and P To further test whether age-dependent changes in discrimination were specific to recognition memory, and not driven by changes in other task components such as motivation, we analyzed the object exploration times in all three tasks in the full 5 min of the test phase.

Mice spent the same amount of time engaging in object exploration in all sampled ages in the OL Fig. Surprisingly, P16 mice spent more time exploring objects in the NOR task compared to all other ages Fig. To exclude the possibility that developmental changes in the time spent exploring the objects could underlie the changes in novel object preference in NOR, we probed the relationship between total exploration time and discrimination index Fig. We found no significant correlation between total object exploration and performance in NOR in P16 mice Fig.

Total exploration time and performance were similarly not significantly correlated in the remaining ages in NOR Fig. Total object exploration across ages and tasks. D—G Correlation between total object exploration during the 5 min test phase and the discrimination index DI for all ages of the NOR task.

No correlation was found for any of the age groups suggesting that behavioral performance in NOR is not influenced by differences in object exploration. Similarly, we saw no age-dependent differences in sample phase object exploration times between ages in OL Supplementary Fig. Overall, these results suggest that any differences in object exploration time likely do not explain better performance in OL, NOR or TOR in the sampled ages.

We conducted a parallel analysis of the ontogeny of three types of recognition memory in the same mouse strain, with equivalent analysis parameters to effectively compare the relative timing of onset for each of these tasks irrespective of variations in species, strain, or animal facility. The ability to recognize changes in spatial location OL 1 h interval emerges first, at P21, followed by the ability to retain the memory of distinct object features NOR for 24 h at P25, and recognition of the recency of events TOR, 1 h interval is the last to emerge, at P These data identify precise temporal windows for the onset of differential aspects of spontaneous recognition memory in mice, an essential first step towards examining the neural correlates underlying this developmental sequence.

Most rodent spontaneous recognition memory studies have used adult animals, with a few focusing on adolescence or juvenility 30 , 33 , 39 , 40 , Studies examining spontaneous recognition memory in young rats show OL memory onset at P16 27 or P17 28 —P21 29 , 30 , 31 depending on rat strain, but see 32 , and NOR onset between P23 and P29 33 for long retention intervals 24 h as used here, and at P15 27 , 34 —P18 29 , 31 , 35 at short retention intervals up to 10 min.

Given the difference in memory load between short and long 24 h intertrial intervals for NOR, it is not surprising that the age of onset differs between these task variants. These are largely consistent with our results in showing earlier onset of OL relative to NOR at a 24 h retention interval , but suggest earlier onset of TOR in rats compared to mice P17—20 compared to P28 in our study.

Importantly, we cannot exclude a role for differences in experimental design in this discrepancy. Specifically, while in our study each task was assessed independently, in both TOR rat studies the same animals had previously undergone NOR and OL 27 , 31 , leading to different levels of habituation between tasks and the possibility of memory interference.

Additionally, both studies had a shorter delay between TOR sessions 27 , We found no age-dependent changes in total object exploration time with the exception of an increase in exploration time in P16 mice in NOR. One difference of NOR compared to the other tasks is the shorter sample phase object exploration time 20 s. It is possible that reduced opportunity for exploration at the sample phase contributed to an increase in exploration in the test phase.

Indeed, overall test phase exploration time is slightly higher in NOR compared to other tasks. However, it is unclear why this would differentially affect P16 animals. This result was particularly surprising given prior work in rats 29 and CD1 mice 16 describing reduced exploration in preweaning animals. Consistent with previous studies 16 , 41 , 43 , we also did not observe sex differences in any of our tasks.

To our knowledge, sex differences in recognition memory in mice 44 and rats 45 have mostly been reported in animals at older ages, suggesting male and female mice may perform equally in spontaneous object recognition tests within the juvenile period. What may underlie the differential onset of each of these forms of recognition memory? Lesion and pharmacological studies point to circuit specialization for the memory processes probed in each of our three recognition memory tasks 22 , with primary involvement of hippocampus 46 , 47 , 48 , 49 in OL, perirhinal cortex 49 , 50 , 51 , 52 , 53 , 54 in NOR and connections 48 , 53 , 54 , 55 , 56 between hippocampus 47 , 48 , 57 , 58 , 59 , perirhinal 53 , 54 and prefrontal cortex 53 , 60 in TOR.

One possibility is that brain region-specific maturation dictates the onset of each of these behavioral competencies. This would imply that the observed asynchrony in the onset of each behavior is mediated by differential timing of circuit maturation. Little is known about the functional maturation of brain circuits underlying recognition memory.

In terms of spatial navigation necessary for OL memory, rat head direction and place cell systems feature adult-like patterns as early as P17, with grid cells following at P21 26 , 61 , 62 , paralleling the emergence of OL.

While this level of spatial representation may be sufficient to sustain OL memory, the number of cells displaying adult-like firing continues to increase through postnatal weeks 4—5 26 , 61 , 62 , perhaps accounting for the later emergence of more complex tasks such as object-place 16 , 61 , object-place-context 63 and the use of distal visual cues 64 , 65 , Perirhinal cortex anatomical development, although not extensively studied, is comparable to other neocortical regions Interestingly, there is evidence for perirhinal requirement for NOR being delay-dependent, with lesions only impairing performance for delays of 10 min or more 51 , 52 , suggesting the earlier emergence of NOR memory for short delays 27 , 29 , 31 , 34 , 35 may be perirhinal-independent.

It is important to note that this 24 h interval for NOR differs from the 1 h intertrial interval used in the other tasks in this study, and may differ from the age of onset for 1 h NOR. Prefrontal cortex develops later than other cortical structures 25 , with cytoarchitectonic development reaching adult laminar appearance by P18 in the rat 67 , and volumetric changes stabilizing at P30 Similarly, prefrontal network activity emerges later than in sensory areas, with marked changes in hippocampus-prefrontal activity within the first postnatal weeks It is tempting to speculate that the delayed onset of TOR reflects the delayed maturation of prefrontal cortex relative to other brain structures.

The distinct temporal profile of each task further underlines the notion of memory as multifactorial, and recognition memory encompassing several underlying processes rather than being unitary.

Future work delineating the anatomical and synaptic maturation of the brain regions underlying different types of spontaneous recognition memory will be key to establishing how circuit-behavior relationships emerge in development, and how they may shape behavior across the lifespan. Date of birth was designated postnatal day P 0, with litter sizes ranging from 2 to 11 pups. Mice were assigned to 1 of 5 possible age groups depending on the recognition memory task: P16, P21, P25, P28 or P At 21 days P21 , mice were weaned and housed in same-sex littermate groups of 2—5 mice.

All experiments were conducted during the light cycle. Approximately equal numbers of females and males were used for each age group. The chamber was elevated 41 cm off the floor and a camera was mounted 75 cm above the chamber using a wall mount rack. A round magnet 35 mm diameter was glued to the base of the objects to allow for stable attachment to the chamber floor. Object designs were extensively piloted to generate objects that were 1 equal in surface area, 2 made of the same materials, and 3 for which the animals displayed no innate preference.

Object types were counterbalanced for all tasks. Mice were handled and habituated to the behavioral chamber twice a day for four consecutive days prior to the day of testing for all three recognition memory tasks.

Handling took place in the testing room with a minimum 3 h interval between handling sessions. Handling and habituation consisted of 5 min of handling followed by placement into the behavioral chamber for 4 min. All mice were ear-notched at P12 for identification purposes. To avoid confounds of repeated testing, dedicated cohorts of mice were used per age and per recognition task, such that each animal was only tested at one age and in one recognition memory task.

Pre- and post-weaning mice remained in a separate transport cage during the 1-h delay period for the OL and TOR tasks. For all tasks and phases, mice were placed into the chamber with their head facing the wall located opposite the object location. For the sample phases of all three tasks, as well as for the test phases for NOR and TOR, objects were placed in the northwest and northeast corners of the chamber, 3 cm away from each wall.

Object type and side of novel stimulus i. OL was divided into one sample phase followed by a test phase Fig. In the min sample phase, mice interacted with two copies of an identical object, after which animals were removed and placed back into their transport cage. After a 1 h delay period 70 , 71 , mice underwent a 5 min test phase in which they were placed in the chamber with the same two objects, but with one relocated to a novel location Fig. The novel location was at the opposite corner of the previous location south corner, counterbalanced for side , 3 cm away from each wall Fig.

NOR was divided into one sample phase followed by a test phase. The sample phase consisted of placing the mouse into the chamber containing two copies of a single object Fig.

The sample phase lasted until a criterion of total object exploration of 20 s was reached 72 , at which point the mouse was removed and placed back into the home cage. Following a delay period of either 24 h 72 , 73 , 74 or an immediate delay lasting less than 2 min , mice underwent a 5 min test phase where they were placed in a chamber containing both the previously encountered object and a novel object.

Mice were returned to the home cage in between all phases of the experiment. We chose a longer delay 24 h for this task because the brain circuits underlying NOR with shorter delays are not as well characterized 51 , This 24 h delay, albeit different from the delay used in OL and TOR, features robust perirhinal involvement even in instances of highly dissimilar objects Objects for both sample and test phases were positioned as described above under general procedures. Total object exploration measurements took into account the complete test phase, lasting 5 min.

TOR was divided into two sample phases followed by a test phase Fig. Sample phase 1 consisted of exposure to a set of two identical objects for 10 min in the behavioral chamber. Following approximately a 1 h inter-phase interval 49 , 75 , 76 , mice underwent sample phase 2 which consisted of 10 min in a chamber containing a second distinct set of two identical objects Fig. After another 50 min to 1 h delay period, mice underwent a 5 min test phase in which they were exposed to one copy of the object from sample phase 1 old object and one copy of the object from sample phase 2 recent object Fig.

Mice remained in a separate transport cage in between all phases of the experiment. Order of object type i. Objects for both sample and test phases were positioned as described under general procedures.

Behavior was analyzed using ANY-maze software. Exploratory activity was defined as in Ref. Sitting on top of the object while sniffing the surrounding air or chewing the object were not considered exploration. All automated scoring was extensively validated through hand-scoring by an experimenter blind to experimental conditions. Leger and colleagues 72 recommend also using the 20 s criterion of exploration time for the test phase adapted from Hyams, K.

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