Effects of dizocilpine (MK801) on olfactory span in rats

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Abstract

NMDA receptor antagonists interfere with learning and memory in some tasks, but not others. Some recent accounts have suggested that tasks placing demands on working memory are those most likely to be affected, and the present study tested this hypothesis. The purpose of the study was to adapt a recently developed procedure designed to test working memory capacity, the olfactory memory span task, for use in behavioral pharmacology and to then determine the effects of the NMDA receptor antagonist, dizocilpine (MK801) on performance in this task. Rats were trained in a non-match-to-sample procedure under conditions in which they had to remember an increasing number of olfactory stimuli as the session progressed. Simple olfactory discrimination trials were interspersed to provide a performance control. Effects of dizocilpine (.03, .10, .17, .3 mg/kg) were determined after stable performances were obtained. Rats were able to sustain stable performances on both the span and simple discrimination tasks with average spans of about 10 items. Accuracy declined as the number of stimuli to remember increased, and dizocilpine impaired accuracy in a dose-dependent and memory-load dependent fashion. The finding that the effects of dizocilpine interacted with the number of stimuli to remember is generally consistent with hypotheses linking NMDA receptors and working memory processes.

Research highlights

► The olfactory span task was used as a measure of working memory in rodents. ► Accuracy decreased as the number of stimuli to remember increased. ► NMDA antagonist, dizocilpine (MK801) decreased accuracy at doses that did not interfere with other aspects of performance. ► Dizocilpine effects were more pronounced as the memory load increased.

Introduction

The findings of Morris and his colleagues (Morris, 1989, Morris et al., 1986) that NMDA antagonists impaired spatial learning in the Morris Swim Task (MST) at doses that blocked long-term potentiation (LTP) in the hippocampus provided the first pharmacological support for the now widely-accepted hypothesis that some forms of learning are mediated by LTP-like activity. However, questions were quickly raised about Morris’ interpretations because NMDA receptor antagonists produce a host of behavioral impairments that are not specific to learning and memory. Thus, impairment in the MST may reflect processes other than spatial learning, e.g., sensorimotor or motivational effects (Cain et al., 1996, Keith and Rudy, 1990). In support of a non-mnemonic account of the Morris findings, pretraining experience in the MST abolishes the ability of NMDA antagonists to interfere with new spatial learning except at very high doses that also produce motor impairments (Bannerman et al., 1995, Saucier and Cain, 1995). Further, in a repeated acquisition task in the MST, learning of a new spatial location is impaired only at relatively high doses of NMDA antagonists that also interfere with the ability to swim to a previously learned location (Galizio et al., 2003, Keith and Galizio, 1997). However, Steele and Morris (1999) observed NMDA antagonist impairments using a procedure in which rats learned to swim to a new platform location each session, but only when the delay between the first and second trials was relatively long (20 min and 2 h); no impairment was observed at shorter delays (15 s) comparable to those used in the other repeated acquisition studies. Bannerman, Rawlins, and Good (2006) reviewed the literature on NMDA antagonists and learning and concluded that NMDA effects are primarily manifested in tasks that place demands on working memory, for example, Steele and Morris (1999) in the case of spatial learning, but also in studies of non-spatial learning (e.g., Schmitt et al., 2005, Tonkiss and Rawlins, 1991). Bannerman et al. hypothesize that NMDA receptor activity is required for learning that involves certain working memory processes (i.e., those involving single trial learning and rapid selection of conditional information).

Operational definitions of working memory procedures for non-humans typically require that stimuli be presented during only a single learning trial and are only relevant for controlling behavior during a single trial or session (Bannerman et al., 2006, Dudchenko, 2004, Olton et al., 1979). This is in contrast to definitions used in human research in which working memory is described in terms of a short term store of limited capacity requiring controlled attention (Baddeley, 2003, Saults and Cowan, 2007). Although the capacity limits of working memory in humans are still disputed (Cowan, 2001, Miller, 1956), the issue has received very little attention in non-human animals. As a result, a dearth of procedures is available for studying working memory capacity in rodents.

However, the olfactory span task (OST) for rodents (Dudchenko, Wood, & Eichenbaum, 2000) incorporates manipulations of memory load into a single-session learning paradigm. The procedure involves the presentation of a single olfactory stimulus (a cup of scented sand) in an arena. Responses to this stimulus (digging) are reinforced through the retrieval of a food reward buried within the scented sand. Following a response to the stimulus, the rat was removed from the arena and the stimulus cup was moved to a random location. A second stimulus cup scented with a different odor is then baited with a food reward and placed in a random position in the arena. The rat is free to respond to either of the scented stimuli present, but only responses to the novel stimulus produce a food reward. On the third trial, the two previously presented olfactory stimuli are moved to new positions and a third odor is introduced. Once again, only responses to the novel odor are reinforced. The procedure is continued in this fashion with the introduction of a novel olfactory stimulus on each trial until up to 24 stimuli are present. Thus the procedure can be viewed as a non-match-to-sample task in which each stimulus serves as a sample during its initial presentation and as a comparison stimulus in each additional trial. Further, it might be best described as an incrementing non-match-to-sample task as the number of comparison stimuli increases on each successive trial.

To assess performance on the OST, Dudchenko et al. recorded span for each session as well as overall accuracy. Span was defined as the number of consecutive correct choices minus one (because there are no stimuli to remember on the first trial). The spans averaged about eight stimuli and there was an inverse relationship between performance and the number of stimuli to be remembered. Thus, accuracy decreased as the memory load increased. These findings provide some validation of the OST and the task has shown promise for investigating the neurobiological determinants of working memory capacity. For example, OST performance is transiently disrupted by lesions of the basal forebrain cholinergic system (Turchi & Sarter, 2000). The procedure has also been successfully adapted for the testing of mice (Young, Kerr, et al., 2007); performance decrements have been observed in human amyloid over-expressing (Young, Sharkey, & Finlayson, 2009) and α7-nicotinic cholinergic receptor knockout mice (Young, Crawford, et al., 2007); increases in span are produced by nicotinic agonists (Rushforth, Allison, Wonnacott, & Shoaib, 2010). These effects were generally interpreted in terms of working memory capacity, however, alternative interpretations of some outcomes in the OST are possible. First, control procedures that allow separation of an effect on processes specific to remembering from the disruption of more general processes (e.g. motivation, perception, psychomotor ability) have not always been present. Second, as the number of stimuli to remember is incremented in the OST task, the number of comparison stimuli in the arena also increases. Thus, on any given trial the number of stimuli to remember (memory load) is inherently confounded with the number of comparisons the animal must choose among. Finally, stringent controls are needed to assure that stimulus control is based on the stimulus odors and not the scent of the food reward or odor trails left in the arena. In the present study, we first developed an adaptation of the OST procedure for use in behavioral pharmacology by including within-session controls for the above issues. In view of the hypothesis that NMDA receptors contribute to mechanisms supporting working memory (c.f., Bannerman et al., 2006), we investigated the effects of the NMDA antagonist dizocilpine (MK801) on OST performance.

Section snippets

Subjects

Subjects were five male Holtzman Sprague–Dawley albino rats between 90 and 150 days old at the start of testing. All rats were housed individually in a temperature and humidity regulated vivarium operating on a 12 h light–dark cycle. All subjects were given continuous access to water in their home cage and food access was restricted such that animals were kept at approximately 85% of their free-feeding weight.

Apparatus

All testing was conducted in an open-field apparatus constructed from a circular table

Results

Subjects required an average of 61 sessions to meet criteria on all training phases of the experiment required prior to drug administration. Fig. 2 shows mean span (black bars) and overall accuracy (white bars) obtained during the baseline training conditions. Mean spans were slightly above 10 items in the 5-Comparison Phase of the study and were unchanged when simple discrimination trials were interspersed within the session (5-Comparison with SD Phase). Accuracy on the span task was high

Discussion

The present study replicates and extends the findings of Dudchenko et al. (2000) and others (Rushforth et al., 2010, Young et al., 2009) that the OST can provide a sensitive, within-session measure of the effects of increasing the number of stimuli to remember on delayed-matching to sample performance. Average olfactory spans and within-session declines in accuracy as the memory load increased in the present study were comparable to those observed in previous studies with this procedure.

Acknowledgments

The research was supported in part by DA029252 to Mark Galizio. This study formed part of a thesis submitted by Dave A. MacQueen, who is now a doctoral student at the University of South Florida, to fulfill requirements for the Masters degree at the University of North Carolina Wilmington. The authors thank Chris Greenwell, Thea Guze, and L. Brooke Poerstal for assistance in the development of the procedures and data collection and Patrick McKinney and Nicholas Hahn, who designed and built the

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