The Object Context-place-location Paradigm for Testing Spatial Memory in Mice

[Abstract] This protocol was originally designed to examine long- term spatial memory in PKMζ knockout ( i.e ., PKMζ -null) mice (Tsokas et al ., 2016). Our main goal was to test whether the ability of these animals to maintain previously acquired spatial information was sensitive to the type and complexity of the spatial information that needs to be remembered. Accordingly, we modified and combined into a single protocol, three novelty-preference tests, specifically the object-in-context, object-in-place and object-in-location tests, adapted from previous studies in rodents (Mumby et al ., 2002; Langston and Wood, 2010; Barker and Warburton, 2011). During the training (learning) phase of the procedure, mice are repeatedly exposed to three different environments in which they learn the spatial arrangement of an environment-specific set of non-identical objects. After this learning phase is completed, each mouse receives three different memory tests configured as environment mismatches, in which the previously learned objects-in-space configurations have been modified from the original training situation. The mismatch tests differ in their cognitive demands due to the type of spatial association that is manipulated, specifically evaluating memory for object-context and object-place associations. During each memory test, the time differential spent exploring the novel (misplaced) and familiar objects is computed as an index of novelty discrimination. This index is the behavioral measure of memory recall of the previously acquired spatial associations.


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Unlike behavioral paradigms that rely on conditioned responses, novelty-preference tasks exploit the rodent's spontaneous exploratory behavior and its innate tendency to investigate instances of change (novelty) in a familiar environment (Steckler et al., 1998a). The basic reasoning is that memory for the familiar condition is estimated by the extent to which behavior differs in response to novelty. Accordingly, the fundamental principle of a novelty-preference memory paradigm is to experimentally create a 'nonmatching' condition between the learning (or encoding) phase and the memory test, such that the animal will express its memory of the original learning experience by preferentially exploring the novel stimuli over the familiar ones.
Novelty-preference tasks are easy-to-use and offer a great versatility for investigating cognitive functions such as spatial memory in rodents (Kinnavane et al., 2015). The following protocol is inspired by previous studies that have used different spatial versions of the novelty-preference paradigm, specifically the object-in-place, object-in-context and object-location task variants (Mumby et al., 2002; Langston and Wood, 2010; Barker and Warburton, 2011). Typically, these task protocols consist of a short learning phase followed by a memory retention test, during which the original spatial configuration of the environment is modified in a specific way. In prior studies however, the different task variants have been presented separately, requiring independent groups of animals to be tested. In addition, the tests are often at short delays (minutes to hours) rather than days, as required to specifically evaluate mechanisms of long-term memory persistence. In the present experimental design each memory retention test is performed to evaluate memory lasting at least 24 h in the same animal and corresponds to a specific spatial manipulation of the original learning experience. Additionally, by varying the number of objects that are presented during the learning phase (i.e., 4 objects versus 2 objects), the following protocol allows direct manipulation of the amount of information to be remembered, not only the type of information.
Here we present the behavioral protocol that was recently used to assess the molecular mechanisms of 24-h long-term memory persistence using wild-type and PKMζ knockout mice (Tsokas et al., 2016).
The protocol was sensitive enough to reveal that the molecular mechanisms that are crucial for objectin-place associations amongst four objects require the persistent kinase PKMζ, whereas non-PKMζ dependent mechanisms are sufficient for the maintenance of object-in-context associations and objectlocation associations involving two objects. A similar dissociation between the neural mechanisms that support object-in-place and object-in-context associations is also observed at the level of the dorsal hippocampus. In particular, permanent or temporary lesion of dorsal hippocampus is sufficient to impair acquisition of object-in-place associations but not object-in-context associations ; reviewed by , with a potentially greater role of the dentate gyrus-CA3 subcircuit (Lee et al., 2005). Object-in-context associations seem to critically depend on postrhinal cortex function (Norman and Eacott, 2005) whereas object-location associations tend to be sensitive to 3 or distal and whether the role of a structure is being evaluated by tests of memory acquisition and consolidation or tests of memory retention (reviewed by . Indeed, recognition memory, a subset of which is assessed by the object context-place-location paradigm that we here describe, appears to be mediated by two extended networks of structures one including the hippocampus that is specialized for spatial recognition memory and the other including postrhinal cortex that is specialized for non-spatial/item recognition memory (reviewed by Steckler et al., 1998b).

10 non-identical objects
Note: We used a combination of plastic toys (PetCo ® , USA), flasks and jars that differ in shape and/or materials that are difficult to chew, such as strong natural rubber, Pyrex ® , polypropylene, and aluminum. These are the objects that the mice will explore. Each object was unique but had approximately equal size, and they were tall enough to prevent the mice from climbing on the objects. The footprint dimension was approximately 6 cm and the height was 17 cm. The objects must be removable and should be easily washed (see Figure 1  context contains a unique set of objects. Note that the bright lighting as captured in these pictures is only for illustration, lighting should be dim, to be optimally comfortable for the mice and allow them to see and discriminate the objects. A 10-15 lux light intensity is recommended.

Objects
The objects (4 objects/context for A and B, 2 objects for context C) are fixed on the floor with removable adhesive putty such that their edges are 5 cm away from the walls. The precise position of each object is always the same (see Notes for additional comments). To ensure that 3. The duration of the entire behavioral procedure is about 9 consecutive days. As depicted below in Figure 2, each mouse is trained and tested in the object-context-place tasks first, followed by the object-location task. Each task procedure consists of three phases: pre-training, training and retention test. pretraining session starts immediately after the animal has been placed, by hand, at the center of the box, its nose facing the south wall. ii.
Between each pretraining session, the boxes and objects are cleaned with water followed by 70% EtOH, which is allowed to dry. This is done to prevent build up of olfactory cues.
iii. At the end of the 10 min of exploration the animal is removed from the context and returned to its home cage.

Note: The order of context exposures is counterbalanced between animals within each experimental group, which was genotype in the case of Tsokas et al. (2016).
b. From Day 2 to Day 4: Training in contexts A and B The mice are allowed to explore contexts A and B, each during two 5-min trials/day, separated by a 1-h inter-trial interval. These trials allow the mice to learn the spatial arrangement of the four objects that are associated with each context.

Note: The order of each context exploration is counterbalanced between training days and
between animals within each experimental group (Table 1). Mice are given a first memory retention test that is either an object/context or an object/place mismatch test. In the object/context mismatch test, two of the four objects that had only been encountered in one context are placed in the second context, whereas in the object/place mismatch test two objects from one four-object configuration are placepermuted in the same context they had previously been encountered. The animal is allowed to explore for 3 min. The 3-min duration of the retention test session has been carefully validated. After 3 min the mice become familiar with the novel object configuration and so spatial novelty triggered by the object permutation is no longer detected by the animal. The mice will subsequently explore all the objects equally, whether or not there is a mismatch. i. Behavioral measures of memory performance Each video is analyzed offline to manually score the time the mouse is engaged in exploration of an object for each of the retention tests. Object exploration is defined as the nose of the animal being oriented toward the object at a distance of < 2 cm. Each video is therefore replayed in the Tracker software using a 2 cm wide annular mask around each object to define the object exploratory area. Within this area, animal's activity such as sniffing or touching the object with paws is counted as object exploratory activity only if the animal's nose is orientated toward the object (see Notes). Measuring object exploration is performed by an experimenter who is blind to the animal's experimental group and whether the objects have been changed. Memory performance in the three different memory tests are quantified and analyzed using a discrimination index calculated as the absolute difference in time spent exploring the changed (i.e., incorrect, misplaced or relocated) objects and the unchanged objects divided by the total time spent exploring all the objects.
As such, the index takes into account individual differences in the total amount of exploration.
Good memory retention corresponds to a positive discrimination index, which reflects that the animal was spending more time exploring the incorrect (object/context mismatch), displaced (object/place mismatch) or relocated (object/location mismatch) objects than the objects that had remained unchanged (Figure 4).

Data analysis
Memory performance is analyzed using a one-way ANOVA with repeated measures. The individual effects of the Independent Factor (Group) and the Within-Subjects Factor (Retention test), as well as the Interaction and post-hoc tests are considered significant at an alpha level of 0.05.

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To make each to-be-explored object unique and identifiable, we recommend using a variety of shapes and type of materials. The choice of the set of objects should be validated prior to the experiment by testing each configuration set with mice in a pilot study to avoid obviously biased preference to investigate one object over the others.