A new paradigm for investigating temporal order memory shows higher order associations are present in recent but not in remote retrieval.

Memory of a sequence of distinct events requires encoding the temporal order as well as the intervals that separates these events. In this study, using order-place association task where the animal learns to associate the location of the food pellet to the order of entry into the event arena, we probe the nature of temporal order memory in mice. In our task, individual trials become distinct events, as the animal is trained to form a unique association between entry order and a correct location. The inter-trial intervals (> 30 min) are chosen deliberately to minimize the inputs from working memory. We develop this paradigm initially using four order-place associates and later extend it to five paired associates. Our results show that animals not only acquire these explicit (entry order to place) associations but also higher order associations that can only be inferred implicitly (temporal relation between the events) from the temporal order of these events. As an indicator of such higher order learning during the probe trial, the mice exhibit predominantly prospective errors that decline proportionally with temporal distance. On the other hand, prior to acquiring the sequence, the retrospective errors are dominant. In addition, we also tested the nature of such acquisitions when temporal order CS is presented along with flavored pellet as a compound stimulus comprising of order and flavor both simultaneously being paired with location. Results from these experiments indicate that the animal learns both order-place and flavor-place associations. Comparing with pure order-place training, we find that the additional flavor stimulus in a compound training paradigm did not interfere with the ability of the animals to acquire the order-place associations. When tested remotely, pure order-place associations could be retrieved only after a reminder training. Further higher order associations representing the temporal relationship between the events is markedly absent in the remote time.