New behavioral protocols to extend our knowledge of rodent object recognition memory

Animals often show an innate preference for novelty. This preference facilitates spontaneous exploration tasks of novelty discrimination (recognition memory). In response to limitations with standard spontaneous object recognition procedures for rodents, a new task (“bow-tie maze”) was devised. This task combines features of delayed nonmatching-to-sample with spontaneous exploration. The present study explored aspects of object recognition in the bow-tie maze not amenable to standard procedures. Two rat strains (Lister Hooded, Dark Agouti) displayed very reliable object recognition in both the light and dark, with the Lister Hooded strain showing superior performance (Experiment 1). These findings reveal the potential contribution of tactile and odor cues in object recognition. As the bow-tie maze task permits multiple trials within a session, it was possible to derive forgetting curves both within-session and between-sessions (Experiment 1). In Experiment 2, rats with hippocampal or fornix lesions performed at normal levels on the basic version of the recognition task, contrasting with the marked deficits previously seen after perirhinal cortex lesions. Next, the training protocol was adapted (Experiment 3), and this modified version was used successfully with mice (Experiment 4). The overall findings demonstrate the efficacy of this new behavioral task and advance our understanding of object recognition.Understanding the neural basis of recognition memory, the ability to discriminate whether a stimulus is novel or familiar, is heavily reliant on animal research. Here, advances have been closely tied to the introduction of new behavioral tests. The preeminent example concerns one-trial tests of recognition memory for monkeys using delayed nonmatching-to-sample (Mishkin and Delacour 1975). These tasks reward the natural preference that monkeys have for selecting novel items and permit multiple recognition trials within a single session. These features make the task relatively easy to train and then maximize findings from small group sizes. Although rat tasks closely based on delayed nonmatching-to-sample have been devised (Aggleton 1985; Mumby et al. 1990; Steckler et al. 1998; Prusky et al. 2004), they are very rarely employed as they are difficult to train and performance levels are unreliable.Almost all studies of rodent recognition memory now employ the spontaneous object recognition test and its direct variants (Ennaceur and Delacour 1988; Dix and Aggleton 1999; Winters et al. 2008). These tasks again take advantage of an innate preference for novel items, but this preference is displayed by spending more time exploring novel than familiar stimuli. In the standard version of the task, a rodent (rat or mouse) is placed in an arena containing two identical objects and then freely allowed to explore these objects for several minutes (“sample” phase). After a delay, the rodent is placed back in the arena (“test” phase), which now contains one familiar object (a copy of the sample phase objects) and a novel object. Recognition is signified by greater exploration of the novel object. Because the task measures spontaneous behavior, it requires minimal pretraining, but for the same reason it is prone to considerable variance. Unlike delayed nonmatching-to-sample, each trial (sample plus test phase) takes many minutes, and so only one recognition trial is normally given per session. Advantages are that proactive interference between objects is minimized, and the one-trial design lends itself to episodic-like tests of memory (Dere et al. 2005; Good et al. 2007). Disadvantages include the fact that data accumulation, with appropriate counterbalancing, is slow.To address these limitations, a new object recognition test using a “bow-tie maze” (Fig. 1A) has been developed for rats (Albasser et al. 2010). This test combines features of delayed nonmatching-to-sample with spontaneous object preference: It permits multiple trials per session, but the measure of recognition comes from the preferential exploration of novelty. The rat is first placed in one end of a bow-tie–shaped maze that contains a single object (object A; Fig. 1B). After a minute, the rat is allowed to run to the other end of the maze where there are two dissimilar objects (A and B; Fig. 1B). Object A is familiar as it is identical to the object previously explored, while object B is novel. Consequently, a rat will typically prefer to explore object B. On the next trial, a minute later, the rat shuttles back to the initial start point, but this time encounters objects B and C. Object B is now familiar, while object C is novel. The next trial, 1 min later, is between object C (now familiar) and object D (novel), and so on. A food reward placed under every object promotes shuttling back and forth within the maze, and encourages interaction with the objects. Unlike delayed nonmatching-to-sample, the food reward is not contingent on first selecting the novel object.Open in a separate windowFigure 1.(A) Schematic of the bow-tie maze. A sliding door separates the two ends of the maze in which two objects are placed. (B) General procedure showing the presentation order of the objects in the standard object recognition task. All objects are rewarded (+). (Arrow) Rat movements. (Black print) Novel objects, (gray print) familiar objects.The present study used the bow-tie maze to explore recognition memory on four fronts. In Experiment 1, recognition in the light and recognition in the dark were compared to help determine the cues available to detect object familiarity. It is known that rats can perform recognition tasks when solely reliant on visual cues (Aggleton 1985; Bartko et al. 2007; Winters and Reid 2010), but object recognition based on other modalities remains largely unexplored (Winters and Reid 2010). Potential cues for recognition include odor differences and tactile information. It is known that rats can discriminate novel from familiar olfactory cues (Otto and Eichenbaum 1992; Kesner et al. 2002; Fortin et al. 2004; Wolff et al. 2006), while tactile (e.g., vibrissae) cues can be used to distinguish surfaces (e.g., Birrell and Brown 2000). The bow-tie maze is ideal for studying object recognition in the dark as the rats are rewarded for visiting items in set locations (to receive food rewards), and so should readily approach the objects. In contrast, running the standard spontaneous object recognition test (in an arena) in the dark would be problematic as it is not clear how the rats would first appreciate the presence of the to-be-discriminated objects.An additional goal of Experiment 1 was to determine how readily the bow-tie maze could be used to compile within-session and between-session forgetting curves. A limitation with the standard spontaneous object task is that with only one trial per session it can be very time consuming to create forgetting curves, while within-session forgetting curves for individual animals are not feasible. These shortcomings create limitations when examining manipulations thought to affect memory. The possibility of deriving within-session forgetting curves is particularly appealing as: (1) it minimized the impact of those factors that introduce variance when performance is compared across sessions, and (2) the animal need not be removed from the maze, which could additionally disrupt performance, e.g., by increasing stress. A further component of Experiment 1 manipulated object memory strength by presenting objects either once (“single”) or six times (“repeated”). Recognition was tested after a 3-h delay with the twin goals of determining whether repeated presentation would aid performance and whether these performance levels would be sufficiently above chance so that they could be used to examine factors involved in longer term memory.At the same time, Experiment 1 provided the opportunity to compare two rat strains, Dark Agouti (DA) and Lister Hooded (LH). Previous studies suggest that the Dark Agouti strain might be particularly good at visual recognition tasks (Aggleton 1996), though others have argued that this strain has aberrant behavioral properties, including higher anxiety and higher levels of inappropriate nonspatial behaviors in spatial learning tasks (Mechan et al. 2002; Harker and Whishaw 2004; but see Aggleton and Vann 2004).Experiment 2 examined the ability of rats with either hippocampal or fornix lesions to perform object recognition in the bow-tie maze. There is a longstanding debate over the impact of hippocampal damage on recognition memory, with mixed findings coming from spontaneous object recognition tests (Clark et al. 2000; Mumby 2001; Gaskin et al. 2003; Winters et al. 2008). The bow-tie maze should prove informative as numerous trials can be run to assess the impact of selective brain lesions. Although in this initial study only short retention delays were examined, these same delays and conditions are highly sensitive to perirhinal cortex lesions (Aggleton et al. 2010; Horne et al. 2010), a brain region regarded as vital for recognition memory (Brown and Aggleton 2001). In Experiment 3 the training protocol changed so that objects did not cover food rewards. Rather, a single food reward was always placed between the test objects. This modification was examined because: (1) it would preclude any exploration scores that were simply derived from attempts to move the test objects in order to uncover the food reward, and (2) it would introduce a task variant that might be amenable to small rodents not able to move objects. Accordingly, Experiment 4 examined the performance of mice (strain C57Bl/6) on a test of object recognition based on the modified version of the bow-tie maze from Experiment 3.