Object recognition testing: Methodological considerations on exploration and discrimination measures

Abstract

The object recognition task (ORT) is a popular one-trial learning test for animals. In the current study, we investigated several methodological issues concerning the task. Data was pooled from 28 ORT studies, containing 731 male Wistar rats. We investigated the relationship between 3 common absolute- and relative discrimination measures, as well as their relation to exploratory activity. In this context, the effects of pre-experimental habituation, object familiarity, trial duration, retention interval and the amnesic drugs MK-801 and scopolamine were investigated. Our analyses showed that the ORT is very sensitive, capable of detecting subtle differences in memory (discrimination) and exploratory performance. As a consequence, it is susceptible to potential biases due to (injection) stress and side effects of drugs. Our data indicated that a minimum amount of exploration is required in the sample and test trial for stable significant discrimination performance. However, there was no relationship between the level of exploration in the sample trial and discrimination performance. In addition, the level of exploration in the test trial was positively related to the absolute discrimination measure, whereas this was not the case for relative discrimination measures, which correct for exploratory differences, making them more resistant to exploration biases. Animals appeared to remember object information over multiple test sessions. Therefore, when animals have encountered both objects in prior test sessions, the object preference observed in the test trial of 1 h retention intervals is probably due to a relative difference in familiarity between the objects in the test trial, rather than true novelty per se. Taken together, our findings suggest to take into consideration pre-experimental exposure (familiarization) to objects, habituation to treatment procedures, and the use of relative discrimination measures when using the ORT.

Introduction

Twenty-three years ago, Ennaceur and Delacour [1] introduced a new one-trial learning test for neurobiological studies. Since its introduction it has been referred to as novel object preference (NOP) test [2], novel object recognition (NOR) task [3] and object recognition task (ORT) [4]. In the present paper we use the ORT abbreviation to refer to object recognition testing. The ORT is based on the underlying principle that, in a familiar environment, laboratory rodents show an instinctive attraction towards novelty, or neotic preference [5]. In general, animals are allowed to freely explore two identical sample objects during a sample- or learning trial. After a certain delay, the animals perform a test trial, in which they are confronted with one of the sample objects and a novel one. If rodents show more interaction with the novel object during the test trial, it can be presumed that they have remembered the sample object. This novel object preference is used as an indication of memory.

Because it utilizes the natural behavior of the animals, the ORT is relatively free of stress if properly administered. Furthermore the property of one-trial learning makes it suitable for the examination of temporal aspects of memory. Over time, it has become a widely used tool for the assessment of memory functions in combination with pharmacological treatments [6], [7], [8], [9], [10] and brain lesions [11], [12], [13], [14]. The ORT is mainly used for rodent experiments, most research is done with rats and mice but the task is also suitable for testing other rodent species [15]. It has also been successfully applied using larger animal species like dogs [16], pigs [17], [18] and horses [19], making it a versatile tool suitable for inter-species comparisons.

The most essential brain structures implicated in object recognition memory of rodents, are the hippocampus and rhinal cortices, the perirhinal and in particular the postrhinal cortex [20]. Of note, the human and primate counterpart of the postrhinal cortex is the parahippocampal cortex. Although there is much evidence that the hippocampus is implicated in object recognition testing [21], [22], [23], its role in processing of the actual object information is more an integrative one, i.e. combining object and contextual information into integral episodic memories [20], [24]. Concerning object information itself, the perirhinal cortex is considered to be the most crucial structure whereas the postrhinal cortex is implicated in contextual information processing [24], [25].

Many variations on the original task have been developed over time, making it difficult to compare results between different laboratories. Differences exist in object and arena features like the size, shape and materials. Also, differences in animal strain, age, gender, and housing conditions have been reported to influence object recognition [15], [26], [27], [28], [29]. Furthermore, different test protocols are being used. For example, some studies use flexible sample trial durations, allowing all animals to reach a specific amount of object interaction [12], [30], [31], [32]. Other experiments have fixed sample trial durations and consequently, each individual animal differs in the amount of sample object exposure [1], [11], [25], [33]. Before testing starts, the animals are often familiarized or habituated and protocols differ greatly between laboratories. In some laboratories the animals are allowed to familiarize with only the apparatus [2], [11], [12], [23], others also introduce objects in the pre-experimental phase [25] or even let the animals undergo the full testing procedure, including injections [34], [35]. Even more fundamental, there is no consensus about the definition of object investigation. In the original test, object investigation was defined as directing the nose towards the object at a distance of 2 cm or less [1]. However, minimal proximities of 1 cm [11] and 4 cm [2] have also been reported. Generally, climbing and leaning on objects is not considered to be object examination [36], although some do in fact consider this to be relevant object-directed behavior [2], [37], [38].

It is also important to note that novel object preference is being analyzed and reported in various ways. Some authors calculate the absolute difference in exploration times between the novel and the sample object, resulting a difference score (DS). In others, object discrimination can also be reported in terms of the relative interaction with each object in the test trial, resulting in a percentage or investigation ratio (IR). Lastly, results of the ORT have also been reported as the relative difference score (RDS), which divides the DS by the total time spent exploring both the objects in the test trial. Theoretically, random exploration would result in a (R)DS of 0 or an IR of 0.5, a higher score indicates that an animal directed the majority of exploration towards the novel object, thereby implicating that it has remembered the sample object. Since studies generally report only one of the different discrimination measures it is difficult to directly compare them. To our knowledge, it has not yet been fully investigated, in one and the same study, how the different discrimination indices are related.

The aim of our study is to shed more light on the effects of familiarization and habituation to the procedures on the measures of exploration and discrimination in the ORT. Additionally, we want to deepen our understanding of the relationships between the different discrimination and exploration measures in the sample- and test trial. This could be helpful in determining the minimum amount of exploration required for reliable object discrimination, by this we mean the amount of exploration that represents normal exploratory behavior and allows for a meaningful statistical interpretation. We have included historical data from 28 ORT studies performed in our lab to test the potential of several drugs to reverse natural or drug-induced forgetting. To investigate the latter, 1 h retention intervals were used in combination with amnesic drugs, the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 or the muscarinic receptor antagonist scopolamine. Both drugs have proven reliably to attenuate the novel object preference in a 1 h retention delay, likely due to drug induced memory impairment [6], [39], [40]. 24 h retention intervals were used to investigate drug effects on natural forgetting, as our male Wistar rats normally do not discriminate anymore between the novel and the familiar object after such an interval [1], [8], [33], [34], [35].