Hi,

I forgot one more thing. In general, you do this kind of tests to measure learning and memory functions and rely on particular parameters for your conlcusions. In this case you rely on time spent exploring the new (or displaced) object compared to the other object(s). But in general any change in the behavior of the animal during the "test" trial (i.e., when one object is displayed) might also be informative. For instance, we regulartly observe that some mice also spend more time at the location where the displaced object was during the "exposure" trial. This makes sense because in the object location memory task mice detect a spatiall rearrengment that involves both locations in the maze (old and new position of the diplaced object). Also, what we see is that animlas also display enhanced general exploration in the arena (e.g., distance moved, rearing) as well as enhanced self grooming (sometime indicative of arousal) during a test trial in which an object is displaced compare to a "test" trial in which the areana looks teh same as during the "exposure" trial. In colcusion, preference for the displaced (new) object should not be the only parameter you look at!

Best,

Fabio

Dear Artemis,

your assumption is right. Total time spent at the object is, indeed, very important to assess whether explorative behavior and/or anxiety-like behavior are affected. This is very important because the new object recognition test is based on the assumption that intrinsic novelty-seeking of mice (and rats) drives them to spend more time investigating an unfamiliar/novel stimulus versus a familiar one. While the assumption is in general correct, it does not always work with all individuals. Some of them might have high levels of state anxiety (or low levels of novelty-seeking) and thereby even avoid novel stimuli that you might erroneously interpret as altered cognitive function. Also, a decreased time spent investigating the object could also result when animals have attention deficits or are hyperactive (in which case animals should display enhanced locomotion and decrease investigation of the objects as well as decreased general explorative behaviors as for instance rearing). Moreover, total time at object is relevant in case you use a preference index calculated as percentage of time at the new object relative to time spent at all objects. In fact this index makes sense when mice spend more or less the same time at the objetcs. I make an example to explain it: 60% preference may indicate for instance 60 s at new object versus 40 s at familiar object  but also, e.g., 6 s at new object and 4 s at old object. Indeed, a difference of 2 seconds between the two subjects is surely not as relevant as a difference of 20 seconds. As usual in behavioral biology, there is NEVER a simple interpretation of the behavior of the animals in a given task. You thus need the most complete behavioral analysis to test all hypotheses with the hope that you end with the correct interpretation. You do not really need fancy tests for explorative behavior or anxiety. Already the parameters that you can collect in the new object detection test can help you (see my examples above). In case you did a familiarization to the arena without the object, you can use it to measure parameters distance moved, thigmotactic behavior (e.g., distance to the wall, time in border, time in center), rearing, self grooming. Defecation and urination also give insight to the state of anxiety and arousal of the animals. In case you are interested, I wrote a review discussing this topic (Morellini, 2013. Spatial memory tasks in rodents: What do they model? Cell and Tissue Research 354). But there are also other easy tests for anxiety for which you do not really need fancy equipment (I assume you already have the possibility to record videos and eventually even automatically track the animals in a given arena). For instance, we use also a so called "New Cage/ new Object" test to measure novelty seeking and anxiety in mice while there are in a home cage (Morellini et al., 2010. Cerebral Cortex 20:2712-27; Morellini and Scahchner, 2006, Eur J Neurosci 23:1255-68). Also, emergence latency in the free choice open field test is extremely indicative of state anxiety in mice (Jakovcevski et al., 2008, Genes Brain and Behavior 7:235-43). Hope I was of some help. Let me know in case you have questions.

Best regards ,

 Fabio

Hi,

I forgot one more thing. In general, you do this kind of tests to measure learning and memory functions and rely on particular parameters for your conlcusions. In this case you rely on time spent exploring the new (or displaced) object compared to the other object(s). But in general any change in the behavior of the animal during the "test" trial (i.e., when one object is displayed) might also be informative. For instance, we regulartly observe that some mice also spend more time at the location where the displaced object was during the "exposure" trial. This makes sense because in the object location memory task mice detect a spatiall rearrengment that involves both locations in the maze (old and new position of the diplaced object). Also, what we see is that animlas also display enhanced general exploration in the arena (e.g., distance moved, rearing) as well as enhanced self grooming (sometime indicative of arousal) during a test trial in which an object is displaced compare to a "test" trial in which the areana looks teh same as during the "exposure" trial. In colcusion, preference for the displaced (new) object should not be the only parameter you look at!

Best,

Fabio

Hi, Artemis

I think Fabio explained it very well and I agree with him.

You should note that no memory test actually measures memory! What you measure is times and distances. With that, you must pay attention to additional control measurements to be sure that you are actually testing what you intended originally, without jumping to conclusions.

If a "control measure" like total exploration time varies significantly between groups, you cannot make assumptions about memory. As you and Fabio referred, other cognitive or emotional chances seem to be in place that affect your data. That means you need to use a different test or adapt the protocol of OLT for your treatment/strain (it is normal to optimize a test depending on testing conditions, not everything that works on papers will work for you exactly the same).

If the problem is indeed anxiety, you might be able to test it on an open field. Fabio explained some parameters you can measure. If you have an anxiety-specific test available (e.g., elevated plus maze) that is way preferable in my opinion (it is variable, but more consistent). I usually do it as routine for all my memory-related tests.

To avoid the influence of anxiety in memory-related test you might use another test like the two-trial Y maze. In this test, the closed space within the walls is hardly anxiogenic and might help. I had some problems testing C57BL/6 mice on the obect location test and object recognition tests, but they behave much better on the Y maze apparatus.

Not having many options available, you can adapt the object location protocol. More exposures to the open field arena and/or more exposures to the familiar objects before the test might help. But find the right balance: too many exposures might lower exploration too much. The size of the arena is also important. Using a smaller arena might help: it is less anxyogenic and there is less novelty around, so the animals will focus on the objects. (beware they must have enough space to walk around and between objects to make their choice clear).

I hope I added some useful tips. Feel free to keep asking and good luck!

Nuno