A NON-COMPETITIVE NMDA RECEPTOR ANTAGONIST IMPAIRS OLFACTORY MEMORY SPAN IN RATS. David A. MacQueen

Transcription

1 A NON-COMPETITIVE NMDA RECEPTOR ANTAGONIST IMPAIRS OLFACTORY MEMORY SPAN IN RATS David A. MacQueen A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Arts Department of Psychology University of North Carolina Wilmington 2009 Approved by Advisory Committee Dr. Julian Keith Dr. Jeffrey Toth Dr. Richard Ogle Dr. Mark Galizio Chair Accepted by Dean, Graduate School

2 TABLE OF CONTENTS ABSTRACT... iv ACKNOWLEDGEMENTS... v DEDICATION... vi LIST OF TABLES... vii LIST OF FIGURES... viii INTRODUCTION...1 Spatial Investigations of NMDA Receptor Antagonist Effects...3 Non-spatial Investigations of NMDA Receptor Antagonist Effects...33 Operational Definitions of Mnemonic Processes in Animal Models...46 The Odor Span Task...50 Application of an RAP Methodology to the Odor Span Task...54 METHOD...58 Subjects...58 Apparatus...58 Stimuli...62 Procedure...62 Pretraining...62 Initial Span Training Odor Span Task Comparison Array...67 Simple Discrimination Multiple Component...68 ii

3 Drug Phase...71 Scent Marking Control...71 Pellet Detection Control...72 RESULTS...73 Training Performance...73 Baseline Performance...81 Effects of Dizocilpine on Behavior in the Multiple Component Odor Task...87 DISCUSSION...97 REFERENCES iii

4 ABSTRACT The present study sought to evaluate a variation of the Odor Span Task developed by Dudchenko, Wood & Eichenbaum (2000) and used it to investigate the effects of the noncompetitive NMDAr antagonist, dizocilpine in rats. Rats were placed in a large arena with 18 food locations. In the initial trial of each session, one food cup marked with a distinct olfactory stimulus was present and responding to it was reinforced. Each subsequent trial added a new olfactory stimulus and responding to the new stimulus was always reinforced (non-matching). The dependent measures were number of stimuli that incremented without error (span) and overall percent correct responses. Sessions were conducted five days per week and each session included 24 trials of the Incrementing Non-Match to Sample (INMS) task as well as a performance control task involving a simple olfactory discrimination. Five male Sprague- Dawley rats received 30 or more sessions of training of the Multiple Component (INMS and Simple Discrimination) procedure and drug administration began when performances met criteria for accuracy and consistency over sessions. When the drug studies began, rats were exposed to I.P. injections prior to the testing session twice per week with testing conducted under baseline conditions the other three days. Dose effect functions were determined for dizocilpine (saline,.03,.10,.17, and.30 mg/kg). Baseline performance was characterized by mean spans (M=8.7) that were similar to those reported by Dudchenko et al. and decreasing percent correct as a function of stimulus set size. Dizocilpine produced significant impairments on span and overall accuracy at the highest doses. Dizocilpine only produced significant impairments on the INMS task at doses (.10 and.17 mg/kg) that did not impair performance of the Simple Discrimination control task. Thus, the effects of this NMDAr antagonist can be interpreted in terms of a selective impairment of mnemonic processes exclusive to the INMS task. iv

5 ACKNOWLEDGEMENTS I would like to thank Drs. Mark Galizio, Julian Keith, Jeff Toth, and Rich Ogle for their wisdom and guidance in regard to the development of this thesis and to the entire faculty of the UNCW psychology department for directing my education and career. A special thanks to Laura Bullard and Brooke Poerstel for their assistance in designing, conducting and presenting the research outlined presently. Thank you to all the students of the Galizio lab for the constant support, friendship and humor. To all my fellow students in the graduate program, thank you for making my education the most enjoyable experience of my life. To my family, thank you for all your love, understanding and for always believing in me. Lastly, thank you Andrew McCullough for the constant supply of coffee that made the past several years possible. v

6 DEDICATION I would like to dedicate this thesis to the memory of Henry Molaison, whose selfless contribution to science has been immeasurable. vi

7 LIST OF TABLES Table Page 1. Spices used as odorants in performance and INMTS components and their source company Number of sessions in each training phase for individual subjects prior to drugs Individual subject performance odors with assigned reinforcement contingency Individual subject performance and statistical analysis of pellet detection control sessions...85 vii

8 LIST OF FIGURES Figure Page 1. Photo of Open Field Apparatus (side view) Photo of Open Field Apparatus from above Diagram of Open Field Apparatus Chance percent correct performance by stimulus set size during the 24 Odor Span Task and 5-Comparison Array training phases Individual subject span for each subject across phases of training in which span was recorded (Initial Span Training, 24 Odor Span Task, 5 Comparison Array and Multiple Component). Labeled phase lines indicate phase of training. The break in each subjects plot represents simple discrimination training during which no span measure could be produced. The longest span recorded in each session is represented by the solid black line while average span during the session (only recorded during Initial Span Training which produced multiple spans during a session) was represented by a dashed line Individual subject percent correct performance for each subject across phases of training (Initial Span Training, 24 Odor Span Task, 5 Comparison Array and Multiple Component). Labeled phase lines indicate phase of training. The break in each subjects plot represents simple discrimination Training Average baseline span by subject. Each bar represents the mean span of an individual subject during baseline sessions throughout the course of the drug study. Error bars represent the standard deviation of individual subjects Average baseline percent correct on the INMTS and Simple Discrimination tasks by subject. Grey bars represent the mean baseline percent correct performance of individual subjects on the Simple Discrimination component during the course of the drug study while white bars represent mean baseline percent correct performance on the INMTS component. Error bars represent the standard deviation of individual subjects Group mean baseline performance on the INMTS task as a function of sample stimulus set size. Data are presented in blocks of four consecutive trials of the INMTS task. Error bars represent the standard error of the mean Mean span performance as a function of dizocilpine dose during the Multiple Component Incrementing Non-Match to Sample task. Asterisks indicate mean span values significantly less than mean saline span values (p<.05)...88 viii

9 11. Individual subject span as a function of dizocilpine dose during the incrementing non-match to sample task. Vertical bars around baseline points indicate standard deviation Mean percent correct performance as a function of dizocilpine dose during the incrementing non-match to sample and Simple Discrimination components. Closed circles represent mean percent correct performance of the Incremental Non-Match to Sample component of the Multiple Component task while open circles represent percent correct performance of the Simple Discrimination component. Asterisks indicate values significantly lower than the saline value Individual subject mean percent correct as a function of dizocilpine dose during the incrementing non-match to sample and Simple Discrimination components. Closed circles represent mean percent correct performance of the Incremental Non-Match to Sample component of the Multiple Component task while open circles represent percent correct performance of the Simple Discrimination component. Vertical bars around baseline points indicate standard deviation Group mean saline and dizocilpine performance on the INMTS task as a function of sample stimulus set size. Data are presented in blocks of four consecutive trials of the INMTS task. Closed circles represent performance during baseline, open circles represent performance after administration.10 mg/kg dizocilpine and triangles represent performance after administration of.17 mg/kg dizocilpine. Error bars represent the standard error of the mean...97 ix

10 INTRODUCTION Psychological research in the field of learning and memory over the past halfcentury has been influenced greatly by the work of Hebb. In his ground breaking book The Organization of Behavior (1949), Hebb called for an integration of psychophysiology and behavioral theory that could transcend the notion of a simple stimulus-response view of human behavior and investigate how neural plasticity organizes experiences to shape future responses. It seemed necessary to Hebb that exchanges at the neuronal level be sensitive not only to the nature of sensory stimuli but to the timing of inputs so that the basic structure of the brain might adapt to provide appropriate responses to both novel and repeated stimulation (1949). While this notion conflicted with many theories of the time that viewed behavior as a specific response to specific stimulation or that assumed a rigid structure of the brain, Hebb s writing spawned an industry of research that continues to expand upon the ability of man and other mammalian species to form adaptive associations based on experience. Since Hebb s time, neurological and behavioral research has indeed identified mechanisms of neural functioning with the type of plasticity that Hebb hypothesized. One of the first cellular models of memory to be proposed came from the laboratory of Kandel who studied the neural functioning of aplysia (sea snails). Through the isolation and repeated stimulation of several components of a neural circuit responsible for a behavioral reflex in aplysia, Kandel and his colleagues were able to show that habituation (the lessening of a response to a stimulus) could be demonstrated through a simple neural mechanism. As Hebb had proposed, the repeated stimulation of certain cells had caused long lasting neuronal changes that included a change in the connectivity 1

11 of the neurons and the availability of neurotransmitters. Kandel went on to show that both sensitization (an increased response to a stimulus) and classical conditioning could also be modeled in these same neural circuits, thus giving a basis for behavioral change at the neural level and substantiating the notion of neural plasticity as a mechanism of learning (Kandel, 1976). On a larger scale, researchers have also observed that the repeated stimulation of particular pathways within the hippocampus have resulted in long lasting changes in neural functioning. The hippocampus, located within the medial temporal lobe, has been long believed to be a crucial brain structure for certain types of human memory. Early interest in the hippocampus stemmed from the renowned case study of an individual known to the scientific community as Henry M. (H.M.), whose hippocampus was destroyed in a surgery performed to alleviate grand mal seizures resultant of his intractable epilepsy. Post-operatively, H.M. has been unable to commit experiences to long term memory while pre-operative memories and short term memory have generally remained intact. The peculiar memory impairment that occurred after damage to H.M. s hippocampus has been extensively investigated by researchers over the past several decades; however the role that the hippocampus plays in human memory is still hotly debated. Hence the discovery of stimulation induced plasticity in the hippocampus has been of particular interest to those seeking to characterize aspects of human learning and memory. In search of hippocampal plasticity, Lomo (1966) discovered that certain sections of the hippocampus exhibited long lived depolarization after receiving high frequency electrical trains. This phenomenon of heightened neural responsiveness following rapid 2

12 stimulation, now referred to as long term potentiation (LTP), has received a great deal of interest in recent years, and extensive research has been dedicated to documenting the physiological mechanisms that underlie LTP. In the hippocampus, LTP is mediated by the functioning of a subreceptor of the neurotransmitter glutamate that is selectively bound by the compound N-methyl-D-aspartate (NMDA). The NMDA receptor is unique in that it is voltage gated and requires significant depolarization of the surrounding membrane through the activation of other receptors, most commonly the activation of an ionotropic glutamate receptor known as AMPA, before it can be bound by glutamate or synthetic compounds. Once bound by glutamate or a similar agonist, activation of the NMDA receptor causes a cascade of events within the neuron that are believed to be responsible for both LTP and long term structural changes to the neuron (Eichenbaum, 2002). While the nuances of the cellular changes that are resultant of NMDA receptor activation are beyond the scope of this thesis, it is important to note that as an underlying mechanism of LTP, the NMDA receptor system has provided unique opportunities to investigate mammalian memory systems pharmacologically. Thus we will begin our discussion of NMDA antagonists with recent behavioral research aimed at exploring NMDA mediated mechanisms of memory. Spatial Investigations of NMDA Receptor Antagonist Effects Behavioral experimentation utilizing a pharmacological blockade of NMDA receptors or their ion channels to assess the effects of LTP blockade on learning task performance have returned mixed results. Initial studies used the NMDA antagonist 2- amino-5-phosphonopentanoate (AP5) or it s more potent enantiomer D-AP5. Both forms of AP5 are competitive antagonists in that they have a selective affinity for NMDA 3

13 receptors rather than their ion channels. Additionally, these compounds cannot penetrate the blood-brain barrier, thus they must be administered through intracranial infusion to assess CNS effects in vivo. A study by Morris, Anderson, Lynch and Baudry (1986) is credited with demonstrating the first learning impairment in rats infused with AP5. The Morris et al. study compared the performances of four groups of rats on a version of the Morris Swim Task (Morris, 1981). The procedure is widely used to assess place learning, which is believed to require intact hippocampal functioning and thus seemed an ideal task for testing the effects of NMDA blockade. Prior to testing, three groups of rats underwent a surgery that implanted a cannula into the lateral ventricle of the brain delivering D/L- AP5, L-AP5 (inactive at the NMDA receptor) or saline, over a 14 day period, (via an osmotic mini-pump implanted in their neck) while a fourth group was used as an unoperated control. Over the first five days of testing, all rats received three trials spaced one hour apart. Each trial began with the rat being placed into a circular pool (2.14m diameter) of opaque water from a randomized starting position. Hidden just below the surface of the water in either the northwest or southwest quadrant of the pool was an escape platform that would allow the rat to climb out of the water. Once on the platform, the rat was left to view his surroundings for 30s before being removed from the pool. The position of the escape platform was held constant for each rat across training sessions. After each rat had received five sessions (15 trials), all four groups showed substantially improved escape latencies, however latencies for the D/L-AP5 group were significantly longer than any of the other three groups, indicating impaired acquisition of the swim task. Rats in 4

14 the D/L-AP5 condition also showed impairment in relearning the task (under infusion) when each group received four additional days of testing (one trial per day) with the location of the platform moved to the opposite quadrant, indicating a relative deficiency in learning a new spatial position. Morris did however note that the D/L-AP5 group also took longer in learning to swim away from the walls of the pool in the first few sessions of training; a behavior that is necessary for place learning to occur in the Morris swim task. In a second procedure, Morris et al. attempted to control for sensory and motivational deficits by testing the subjects on a visual discrimination problem in the same apparatus. Each rat received 10 trials per day in the pool in which there were two platforms protruding 2 cm above the water that were visible to the rat. One platform was secure and allowed rats to exit the water while the other was designed to sink when a rat attempted to climb on. The two platforms were given differentiating markings (one was grey while the other was striped) and were placed in random locations for each trial while extra maze cues were obscured by a black curtain surrounding the pool. Each subject was tested until they approached the secure platform on nine successive trials in one session or until their mini-pump was expended. While the un-operated control group exhibited the shortest latencies, there were no significant differences between groups. These findings of this study suggested that only the dextro-enantiomer of AP5 was active in inducing a NMDA receptor blockade and that administration of AP5 caused a place-learning impairment that could not be accounted for by a sensory or motivational deficit (as evidenced by the lack of group differences in the visible platform task). The blockade of NMDA receptors by D/L-AP5 was additionally confirmed by 5

15 electrophysiological recordings taken in vivo from the hippocampus. When high frequency stimulation was administered to the perforant pathway, entering the hippocampus, only rats infused with D/L-AP5 failed to provide recordings indicative of LTP (thus illustrating the stereospecific action of AP5). The findings of the Morris (1986) study set off a torrent of research that questioned the conclusion of NMDA receptor mediated spatial learning in the swim task. The primary concern with Morris findings was that the control procedures used were not adequate for discerning a learning impairment from a more general sensorimotor deficit. A study by Cain, Saucier, Hall, Hargreaves and Boon (1996) illustrated how the impaired swim task performance of rats infused with NMDA antagonists could be attributable to deficits in a number of behavioral domains. Additionally, Cain et al. showed that the acquisition deficits attributed to NMDA antagonism could be eliminated through nonspatial pre-training in the swim task or simply through experience with the task. Their procedure consisted of pre-training a group of rats that was to receive AP5. The pretraining occurred over the course of four days and was similar to the Morris (1986) procedure (three trials per day, 4 hr. ITI) except that the platform was moved between trials and distal cues were blocked with black curtains. The purpose of this pre-training was to give the group experience with the behavioral requirements of the procedure (i.e. swimming, searching for and remaining on the platform) without the spatial navigation requirements of the task. Five days after completing the training, the trained group was given an acute administration of 10µg of AP5 via intracerebroventricular (I.C.V.) injection and tested on a version of the swim task comprised of two probe trials, ten hidden platform trials and 6

16 ten visible platform trials. Probe trials, in which there was no platform in the pool, occurred on the trial before and the trial after the ten hidden platform trials. The platform location remained constant during the hidden platform trials and the rat was entered into the pool from pseudo-randomized locations. Conversely, platform location was moved between trials during visible platform trials while the rat was entered into the pool from a constant location. The pre-trained rats were tested in addition to several groups of naïve rats who were administered either APV (10 or 30 µg), CNQX (A glutamate receptor antagonist that does not block NMDARs) at does of 2, 10 or 30µg, dimethyl sulfoxide (DMSO, the vehicle for CNQX), or saline. An additional group that did not receive any injections also served as a control. All groups were tested on the 22 trial version of the swim task and were assessed on several tasks outside of the pool. Tasks included an alley swim designed to assess swimming speed and a balance beam task to assess balance and coordination. Platform search time for pre-trained rats infused with APV was equivalent to that of the pooled controls (uninjected and saline groups) while both naïve APV groups (10 and 30µg) took significantly longer than controls on the hidden platform task. The APV30 group was also significantly delayed on the visible platform task. Of the CNQX doses tested (2, 10 and 30µg) only the CNQX30 group exhibited significantly longer platform search times, an effect that was found in both the hidden and visible platform tasks. Cain et al. argue that a deficit is observed in the naïve APV10 group and not in the pre-trained APV10 group because the drug-free non-spatial pre-training reduced the effect of APV induced sensorimotor impairments on behaviors relevant to the swim task. 7

17 The true elegance of this study is in the multiple behavioral measures that were used to illustrate behaviors responsible for task impairments in naïve drug groups. One important behavioral consideration in regards to the swim task is the prevalence of thigmotaxic swimming, the natural propensity for rats to swim along the walls of the pool where platforms are never located. This behavior is often accentuated by NMDA antagonists. Through the implementation of a video tracking system, the swim path of each subject could be analyzed in terms of the proportion of time spent swimming in the periphery of the pool and in an interior ring of the pool which contained all platform locations. A comparison across conditions revealed a significant effect of group in both peripheral and platform ring swimming. For both the APV and CNQX groups, the proportion of peripheral ring swim time increased and the proportion of platform ring swim time decreased in a dose dependent manner. The pre-trained APV group showed proportionally less peripheral swim time and proportionally greater platform ring swim time than all other groups (including controls). For all groups there was a significant strong correlation between the percentage of time spent swimming in the periphery and platform search time in the hidden platform task. Similar correlations were found in the visible platform task for both the CNQX groups and the APV groups. This analysis demonstrated that the amount of time spent swimming along the walls of the pool was positively related to search latency and that pre-trained rats spent less time swimming along the walls of the pool and thus shorter escape latencies were observed for this group. The analysis of peripheral swimming was complemented by an analysis of other swimming behaviors in the hidden platform task. It was noted both in this study and in 8

18 the earlier Morris (1986) study that subjects infused or injected with NMDA antagonists appear to have difficulty in climbing on or remaining on the platform after contact. For this reason, Cain et al. analyzed the frequency of swimovers and deflections. These were instances in which the rat came in contact with the platform but failed to climb onto the platform and continued swimming. Not surprisingly, the naïve CNQX and APV groups both showed a significantly higher number of deflections and swimovers per minute and significantly higher percentage of swimover/deflection contacts when compared with the pooled control. The pre-trained APV group performed comparably to the pooled control on both measures. In sum, Cain et al. make a compelling case for a sensorimotor account of the watermaze impairments found by Morris. Once rats learn to inhibit thigmotaxis and remain on the hidden platform, NMDA-antagonists failed to interfere with learning, even at doses that block LTP. However, a similar study by Bannerman, Good, Butcher, Ramsey and Morris (1995) reported slightly different results. After replicating the results of the Morris (1986) study, Bannerman et al. investigated the effects of both spatial and non-spatial pre-training on acquisition of the Morris swim task in a novel environment after administration of AP5. Subjects that received spatial pre-training (22 trials across 10 days) in environment 1 before administration of AP5 (30mM) were only slightly impaired relative to controls receiving artificial cerebral spinal fluid (acsf) on escape latency when tested in environment 2. The spatially pre-trained subjects showed significant decreases in latency across trials and were not significantly different from acsf controls in test quadrant search time during transfer tests conducted after the fourth and eighth trials of testing (both groups performed significantly above chance). 9

19 Additionally, both the AP5 and acsf groups performed above chance in training quadrant (the quadrant used during the spatial pre-training) search time when a transfer test was conducted in environment 1 after testing in environment 2. Non-spatial pre-training in the watermaze was accomplished by blocking distal cues of the environment through the placement of dark curtains around the watermaze and by moving the platform location on each successive trial of pre-training. Surprisingly, subjects who received non-spatial pre-training (22 trials across 10 days) in environment 1 were grossly impaired relative to controls receiving acsf in escape latency and during quadrant search time transfer tests when tested in environment 2 after administration of AP5 (30mM). Some degree of learning did however occur as evidenced by above chance performance in the final testing quadrant search time transfer test. In sum, when tested in a novel environment after administration of AP5, no learning was evidenced by subjects tested without the benefit of pre-training, a small degree of learning was evidenced by subjects who received non-spatial pre-training, and spatial pre-training produced little impairment in subsequent testing. The most surprising of the findings is the gross impairment shown by subjects who received non-spatial pre-training as it appears to be at odds with the findings of Cain et al. (1996). As Cain et al. showed fairly convincingly, non-spatial pre-training is capable of ameliorating AP5 induced impairments on the Morris swim task. Subjects in the Bannerman et al. study received more non-spatial pre-training than subjects in the Cain et al. study (22 trials across 10 days and 12 trials across 4 days, respectively), so one would expect a complete amelioration of AP5 induced impairments in the non-spatial pre-training condition of the Bannerman et al. study. 10

20 Bannerman posits (D. M. Bannerman et al., 1995; D.M. Bannerman, Rawlins, & Good, 2006) that while non-spatial pre-training may serve to ameliorate the sensorimotor effects NMDA antagonism on Morris swim task acquisition, it may also serve to train subjects to use a search strategy (rather than a spatial navigation strategy) that is difficult to overcome under the influence of an NMDA antagonist. If this is the case, it is conceivable that longer durations of non-spatial pre-training (while reducing the sensorimotor impact of the drug) create a proactive interference that is counterproductive to subsequent acquisition of the swim task under the influence of an NMDA antagonist. It is worth noting however that in the Cain et al. study, both pre-training and testing occur in the same environment while in the Bannerman et al. study two separate environments are used for pre-training and subsequent testing. The key point of the Bannerman et al. study is that spatial learning within the Morris swim task is composed of disparate cognitive tasks (e.g. discriminating visual cues, retaining and using spatial information obtained on previous trials, choosing between response strategies, etc.) that may be differentially influenced by pharmacological and training manipulations. The influence of NMDA antagonism upon these tasks is further expounded upon both in the Bannerman et al. study and the later Bannerman, Rawlins, and Good review and will be addressed as we discuss other investigations of the behavioral effects of NMDA antagonists. The aforementioned studies no doubt illustrate how difficult it can be to discern true learning effects from general performance effects that may also be influenced pharmacologically. From a behavioral perspective, learning is viewed as behavior in transition. Many of the designs discussed in the present review of the NMDA antagonist literature have utilized procedures that assess the ability of a subject to improve 11

21 performance on (acquire) a new task across time. Investigating a behavior in transition requires that researchers take particular care in designing procedures with stringent controls capable of isolating one behavior of interest from the other behaviors necessary to perform the task. The control conditions of the Morris et al. study have been questioned because they may not adequately separate spatial navigation and other demands of the task from the learning of a spatial location. In order to compare a learning task to a control task, the required tasks and the motivation to perform the tasks should be identical. This can be especially problematic for studies employing pharmacological manipulations. If the procedure requires the acquisition of a task, then subjects can not be retested on the acquisition of the same task and thus their performance must be compared to the performance of a separate group of subjects that did not receive the treatment of interest (i.e.: a between-subjects design must be used). Because this type of design requires a separate group of subjects to be used as controls, individual differences between subjects can reduce the sensitivity of the testing procedure. Even further complications arise in determining a dose-effect function, because for each dose included in the study an additional group of subjects must be tested. Thus, it is cumbersome to investigate multiple treatments (e.g. multiple doses of a drug) with a group design. The result is numerous studies comparing a single dose or several studies comparing the effects different doses. One solution to avoiding many of the aforementioned pitfalls of investigating a behavior in transition (learning) is the use of a within-subject, repeated-acquisition procedure. In the classic version of this procedure, subjects are trained to acquire a behavioral chain (e.g. a sequence of different responses) within a single session (e.g., 12

22 Boren and Devine, 1968). The reinforced behavioral chain remains constant within each session permitting the rate of acquisition of this sequence to be measured. However, different sequences are used across sessions so that acquisition can be studied on a daily basis. After sufficient training, the acquisition rate of a behavioral chain becomes stable and treatments can be administered. The rate of behavioral chain acquisition after a manipulation has been made (e.g. administration of a drug dose) can than be compared to the stable baseline rate of acquisition before the treatment. Post-treatment, subjects can be further tested to determine if the treatment caused long lasting changes in chain acquisition. Assuming the subject returns to pre-treatment baseline performance, additional treatments can be administered, and acquisition rates can be compared for multiple treatments within individual subjects. A repeated acquisition task is ideal for assessing drug effects on a behavior in transition because a single subject can be efficiently tested across a range of doses. Because the effect of multiple treatments (e.g. doses) can be compared within individual subjects, this type of procedure controls for individual differences in baseline performance and treatment effects. This is an important consideration with pharmacological manipulations given that there is considerable variability due to individual differences in responsiveness to a drug. The repeated acquisition procedure additionally allows for post-treatment assessment to determine if the treatment caused lasting changes to the acquisition of the behavior. This is also an important consideration for investigations of drug effects as some drugs are believed to have long lasting physiological effects. 13

23 A repeated-acquisition procedure alone does not allow a researcher to separate the effects of drug dose on the acquisition rate of a task from the effects of the drug on the physical requirements of the task. Often in operant tasks, an assessment of response rate is used to control for the non-associative effects of a drug. If subjects display accelerated or suppressed response rates under the effects of a substance, it is difficult to make meaningful interpretations of acquisition data. Because many drugs will affect response rate on operant tasks at a wide range of doses, studies using only response rate as a control face severe limitations in their ability to separate learning effects from the nonmnemonic effects of a drug. To separate learning (e.g. task acquisition) from performance (e.g. acute sensorimotor) effects of a drug, the repeated-acquisition procedure can be extended to include an additional task that serves to control for the non-mnemonic requirements of the task. The control task, commonly known as the performance task, can be trained concurrent to the acquisition (learning) task allowing for assessment of both mnemonic and non-mnemonic performance in each subject, within session across multiple treatments. Thompson and Moerschbaecher (1979) provided the first demonstration of the repeated acquisition/performance (RAP) task in which monkeys were administered stimulants (d-amphetamine and cocaine) and assessed on the acquisition of response chains. The acquisition task was similar to that used in Boren and Devine (1968), however each subject was additionally tested on a performance task identical to the acquisition task except that the reinforced response chain remained constant across all sessions. Both components were signaled by the use of differentiated light that indicated the task demands (new response chain or previously learned chain). 14

24 Subjects were tested at several doses of both drugs and in both the acquisition and the performance components, stimulants caused a dose dependent decrease in response rate, accuracy, and within-session error reduction. Importantly, the performance component appeared to be less sensitive to the effect of the stimulants than the acquisition component indicating that some doses impaired the acquisition without effecting performance. These doses were considered to have selective effects on the acquisition of a new behavioral chain. Keith and Galizio (1997) illustrated how the RAP procedure can be implemented in the Morris swim task to create an experimental design more sensitive to differential drug effects on place learning and general performance. Their procedure was designed so that each subject received extensive training in two place navigation tasks. In one task a new position had to be learned daily while the other task required only navigation to a familiar place. This was accomplished by training each rat in two separate pools that could be distinguished by their coloration. One pool had black walls and contained water dyed with black nontoxic paint while the other pool had white walls and contained water dyed with white paint. Additionally, both pools had two distinguishing distal cues that were visible from the pool. For each rat, one pool was designated for performance trials while the other was used for acquisition trials. In a performance trial, an escape platform was set in a location that remained constant for each subject across all sessions, while in an acquisition trial the escape platform was set in a pseudorandom location that was constant across each trial in a session but was relocated to a new position for each succeeding session. Thus, a subject was required to search for and locate the acquisition platform before distal cues could be used to navigate to the platform on subsequent trials. 15

25 Conversely, on every performance trial the platform could be located by the relative position of the distal cues of the performance pool. One of the main advantages of this procedure was that the distinguishing features of the two pools acted as discriminative stimuli that indicated the task requirement of each trial. This allowed for the use of a hidden platform in both conditions (rather than using a visible platform control), enabling a direct comparison of navigation to new and previously learned locations. This comparison could be drawn within each subject in each individual session, providing for a sensitive analysis of place learning. Rats were given 12 trials per session (6 trials in each pool) with a 2.5 min. ITI and each rat was able to achieve stable escape latencies in both conditions within 17 to 25 sessions. Performances were remarkably consistent with the average latency of each performance trial and the average of trials two through six of the acquisition component below 10 s for each subject. Similar stability emerged when each animal was analyzed in regard to the number of times they moved from one quadrant of the pool to another during a trial. Average latency for the first trial of the acquisition component was significantly higher and more variable as subjects were required to search out the location of the acquisition platform at the beginning of each new session. After achieving a stable baseline on the multiple component swim task procedure, six rats were then tested after being injected with dizocilpine, a non-competitive NMDA antagonist that readily passes the blood brain barrier and can be administered to rats via interperitoneal injection (I.P.). As a non-competitive antagonist, dizocilpine (DZP) exerts it s action by occupying NMDA ion channels rather than by binding to NMDA receptors and has been shown to block hippocampal LTP in unanesthetized rats at doses of.10 16

26 mg/kg (Gilbert & Mack, 1990). Each rat was tested under a saline treatment and three separate doses of DZP (.1,.2 and.3 mg/kg). DZP dose significantly increased latency in both the performance and acquisition components of the procedure in a dose-dependent manner; however DZP only impaired place learning at doses that also impaired performance in swimming to a previously learned position. As a means of comparison, three of the rats were also tested on doses of chlordiazepoxide (3, 10 and 30mg/kg I.P.), a benzodiazepine that has been shown to impair acquisition in the water maze (Morris swim task) without blocking hippocampal LTP (McNamara, DePape, & Skelton, 1993). While the 30 mg/kg dose of chlordiazepoxide (CDZ) clearly impaired both the performance and acquisition components of the task, only the acquisition component was significantly impaired in any of the three rats at the 10mg/kg dose. Thus the multiple component watermaze procedure illustrated that place learning deficits observed in the Morris swim task could be separated from performance deficits for CDZ, a drug that does not block LTP, but that such an effect was not found for DZP, even at doses that block LTP. The selective effect found for CDZ was consistent with previous work investigating the effect of benzodiazepines on water maze performance (McNamara & Skelton, 1991), however the DZP data was inconsistent with previous studies finding a selective effect for NMDA antagonists (Morris et al., 1986). Galizio, Keith, Mansfield and Pitts (2003) used a variation of this procedure to compare the effects of NMDA antagonists and morphine in the water maze. The procedure differed slightly from the previous Keith and Galizio (1997) study in that a single pool was used for both the performance and acquisition trials. To accomplish this, 17

27 the pool was surrounded by a series of curtains with distinctive cues that could be changed between trials. One set of curtain configurations signaled a performance trial while another set of curtains with different cues was used for acquisition trials. In addition, a video tracking system was installed above the pool to record trial latencies and the path the animal used to reach the platform. This allowed the researchers to analyze several measures of performance such as latency, swim path ratio (length of path taken to reach platform/shortest distance to platform) and swim speed. In this study, six subjects received the competitive NMDA antagonist LY (.3, 1 and 3 mg/kg I.P.) and six subjects received the non-competitive NMDA antagonist phencyclidine (.3, 1, 3 and 5.6 mg/kg I.P.). Selective effects were not found for any dose of LY studied despite the statistical sensitivity of the within subject design. Phencyclidine (PCP) produced selective effects at some doses in only three of the six subjects. These results were complemented by additional research that used the same procedure to examine the effects of morphine (1, 3, 5.6 and 10 mg/kg I.P.) and LY (.3, 1, 1.7 and 3 mg/kg I.P.) on water maze performance. This particular study found selective effects for morphine in four of five subjects at the 5.6 mg/kg dose but found no selective effects for any dose of LY (Miller, 2005). The absence of a consistently selective place learning impairment in subjects administered multiple doses of three separate NMDA antagonists (dizocilpine, LY235959, and phencyclidine) in an especially sensitive within subject, within session design, contrasts with the earlier findings of Morris (1986), however is consistent with the results of a later study by Steele and Morris (1999) that investigated the effects of D- AP5 and hippocampal lesions on within session place learning in the water maze. The 18

28 matching-to-place task used by Steele and Morris was similar to the acquisition component of the RAP water maze studies in that subjects were pre-trained to learn a new spatial location within the watermaze on each day of pre-training (four trials per day for 9 days) and then tested on within session acquisition of new locations. An important feature of the Steele and Morris matching-to-place task is that it incorporated a variable delay (15 sec., 20 min., or 2 hours) between the first and second trials of the task (with a 15 sec. ITI between trials 2 and 3, and trials 3 and 4) during both pre-training and testing. This design was used to provide a within session analysis of one trial spatial learning within the watermaze that was also sensitive to the effects of delay. A between subjects design was used to compare the performance of pre-trained subjects receiving hippocampal (plus dentate gyrus) lesions, sham lesions, and chronic I.C.V. infusion of either D-AP5 (30mM) or acsf. Additionally a within-subjects design similar to that implemented in the RAP watermaze studies (Galizio et al., 2003; Keith & Galizio, 1997) was used to study the effects of acute intrahippocampal microinfusion of D-AP5 or acsf. By subtracting the escape latency of trial 2 from the latency of trial 1, Steele and Morris were able to construct a sensitive measure of one trial spatial learning in the watermaze. Subjects showed rapid acquisition of the task during pre-training as was evidenced by improved savings (reduced escape latency) between trials 1 and 2 across the nine sessions of testing. It is important to note however that additional savings were still evidenced on trials 3 and 4. These trials were included in daily sessions to facilitate the acquisition of the new spatial location by the end of the session. Thus, the effect of one trial learning deficits could be minimized in regards to the overall acquisition of the location during the session. This is an important consideration given that the location 19

29 learned on a preceding session or the search strategy adopted by subjects could potentially interfere with subsequent learning of a new spatial location. During pre-training, there was not a significant effect of delay on one trial learning, though there was a trend towards decreased savings at the longest delay (2 hours). Subjects receiving lesions were significantly impaired relative to sham surgery controls on one trial learning irrespective of delay. Lesioned subjects did however show significant improvements in escape latency across the entirety of the session. Subjects receiving chronic I.C.V. infusion of D-AP5 showed impairments relative to controls at the 20 minute and 2 hour delays, but were unimpaired at the 15 second delay, thus evidencing a delay dependent impairment of one trial learning. Like the lesioned group, the chronic D-AP5 group did show significant improvements across the session (where 15 sec. ITIs were used between trials 2 and 3 and trials 3 and 4). The same delay dependent effect of D-AP5 was found in the within subject acute administration design using only the 15 second and 2 hour delays. These findings are significant to the interpretation of the RAP watermaze studies for several reasons. In the RAP watermaze studies, a 2.5 minute ITI was used between all trials. Steele and Morris (1999) however showed that both chronic and acute administration of the NMDA antagonist D-AP5 produced impairments at a 20 minute but not a 15 second ITI. While it is unclear at what ITI water maze impairments will become evident, it is conceivable that a 2.5 min. ITI is below the delay threshold of NMDA antagonist effects on spatial learning. Additionally, Steele and Morris found impairments in one trial learning during the delayed matching-to-sample task but clearly illustrated that improvements in escape latency occurred across the four trials of the session both 20

30 after D-AP5 administration and hippocampal lesions. All of the aforementioned RAP watermaze studies reported within session acquisition by averaging the escape latencies of trials 2 through 6 of the session and thus, deficits in one trial learning could have been potentially masked by subsequent learning during the remaining trials of the session. While the Steele and Morris study did not provide a control for the sensorimotor effects of the drug and did note some behavioral abnormalities in subjects administered D-AP5, as Bannerman, Rawlins, and Good (2006) point out, a sensorimotor account of NMDAr antagonist impairments still has a difficult time accounting for these results. It is not clear why spatially pre-trained subjects would not be impaired on the within session watermaze task at a 15 second delay but show gross impairment at a 2 hour delay in the Steele and Morris study, or additionally, why spatially pre-trained subjects would show impairments in the within session watermaze procedure but not in the across session matermaze procedure used by Bannerman et al. (1995). Rather than conclude that the effects found by Morris et al. are an artifact of an insufficient control task, it has alternatively been argued that NMDA antagonists impair the acquisition of a spatial strategy and that pre-training provides subjects with the opportunity to learn spatial strategies that are then implemented in the spatial learning task. This however does not fit well with the data presented by Steele and Morris (1999) in that spatial pre-training failed to ameliorate a one trial learning impairment at 20 minute and 2 hour delays or the data presented by Cain et al. (1996) in which non-spatial pre-training was sufficient to negate AP5 impairments. Such an account is additionally refuted by Hoh, Beiko, Boon, Weiss and Cain (1999) who demonstrated that place learning impairments were not found in rats who received the NMDA antagonist 21

31 CGS19755 (CGS) both during testing and during non-spatial pre-training in the watermaze, relative to similarly pre-trained controls. Electrophysiological recordings indicated that the CGS dose used (4.0 mg/kg I.P.) was effective in blocking hippocampal (dentate and CA1) LTP. The authors conclude that NMDA mediated LTP in the hippocampus is not necessary for learning behavioral strategies that facilitate place learning in the water-maze. However, as the Bannerman et al. (2006) review points out, the Hoh et al. (1999) study does not address whether the learning of spatial strategies is NMDA mediated, but only argues the case that the acquisition of behavioral strategies (whether spatial or non-spatial) ameliorates NMDA place learning impairments and that the acquisition of these strategies is not NMDA dependent. Other researchers have suggested that NMDA receptors are essential for the formation (but not necessarily retention) of a spatial representation of an environment rather than the acquisition of behavioral or spatial strategies (Carmanos & Shapiro, 1994; Uekita & Okaichi, 2005). The spatial representation theory posits that NMDA receptors are necessary for creating a working map of an environment, and that once this spatial map is formed, NMDA receptors are no longer necessary for solving tasks that require use of the previously constructed spatial representation. Thus it is not important that the task demands of pre-training resemble the task demands of testing but that subjects have prior experience in the environment in which they are to be tested and that the demands of the task relate to spatial aspects of the environment. Uekita and Okaichi (2005) tested this hypothesis in a series of experiments that manipulated pre-training task demands and the environment in which subjects were tested (familiar or novel) under administration of dizocilpine. In their first experiment, Uekita and Okaichi tested naïve rats on a spatial 22

32 discrimination task in the watermaze in which there were two visible escape platforms. Though both platforms appeared identical, one platform was secure and allowed for escape from the pool, while the other was unstable and would tip over if a subject attempted to mount it. The stable platform remained in a fixed location across trials for each rat, while the position of the unstable platform (and the rats starting position) was moved for each successive trial. Naïve rats who received dizocilpine (.10 mg/kg I.P.) were greatly impaired in acquiring this discrimination relative to saline controls, demonstrating a place learning impairment consistent with the findings of Morris et al. (1986). It is important to note however that like the Morris et al. study, no performance control task was used to isolate spatial learning from other behaviors necessary for performance of the task. In a second experiment, subjects received pre-training (the standard Morris place learning task) and were subsequently tested on the spatial discrimination task either in the same apparatus they received their pre-training in or in an identical apparatus located in a different room. For all subjects the secure platform of the spatial discrimination task was placed in a different location then was used for the escape platform of pre-training. The performance of rats who received dizocilpine (DZP) was comparable to that of saline controls when tested in the familiar environment; however DZP rats tested in the novel environment showed significant impairment relative to controls. Pre-training completely eliminated the place learning deficit evidenced in the spatial discrimination task of Experiment 1. Interestingly enough, pre-training failed to eliminate a spatial learning impairment in DZP rats that were tested in a different room from the one in which they were trained. Uekita and Okaichi acknowledge that less impairment was seen in the pre- 23

33 trained novel environment DZP rats than was seen in the Naïve DZP rats of the first experiment. The authors suggest that the size of the impairment may have been reduced by pre-training amelioration of the sensorimotor disturbance effect of NMDA antagonism as proposed by Cain et al. (1996). These results were considered as evidence that NMDA receptors are necessary for the formation (but not use) of a spatial representation of an environment. To investigate what elements of pre-training negated the DZP effect found in the naïve subjects of Experiment 1, Uekita and Okaichi (2005) conducted a third experiment. This time, rats were pre-trained on a water-free spatial task before being administered either DZP or saline and tested on the spatial discrimination task. The pre-training phase was designed to be spatial in nature and to occur in the same location as the subsequent spatial discrimination task but to require very different behaviors. To accomplish this, a raised floor was installed in the bottom of a watermaze, and eight food cups were spaced evenly along the wall of the watermaze. Each rat was randomly assigned a food cup location that would remain constant across all pre-training trials. On each pre-training trial, a rat was placed in the center of the dry maze and required to navigate to his assigned food cup. All of the food cups were baited with five sucrose pellets but only the subject s assigned cup provided access to the food. If the rat navigated to and looked inside a food cup other than his assigned cup, an incorrect response was scored. Though rats were required to navigate using the spatial cues of the environment, rats received no experience in swimming or climbing onto a platform. After pre-training, rats were given either DZP or saline and tested on the spatial discrimination task used in Experiments 1 and 2, in the same environment in which they 24

34 received the dry maze pre-training. DZP rats performed as well as controls in the acquisition of the spatial discrimination task across eight days (four trials per day) of testing, indicating no impairment in the acquisition of a spatial task. The results of Experiment 3 suggest that NMDA antagonist impairments on the spatial discrimination task are not caused solely by sensorimotor drug effects, which can be reduced by the pre-training of task requirements topographically similar to those required for the testing procedure. A spatial strategy hypothesis also fails to fully account for the data collected in Experiment 2, as any spatial strategies presumably learned during pre-training failed to generalize to an identical apparatus in an unfamiliar environment. It is also difficult to square the results of Uekita and Okaichi (2005) with the Hoh et al. (1999) study which showed that non-spatial pre-training eliminated the effects of NMDA mediated LTP blockade on a spatial learning task even when NMDA antagonists were administered during pre-training. Under the spatial-representation hypothesis, rats in the Hoh et al. (1999) study should have failed to create a spatial representation of the environment during pre-training (since they were administered NMDA antagonists during pre-training and the pre-training was non-spatial in nature) and should have been impaired during spatial testing, which was not the case. The results of Uekita and Okaichi (2005) are however consistent with the assertions of Bannerman et al. (1995) in that spatial pre-training ameliorated the NMDA antagonist impairments in the watermaze task, however Bannerman et al. (1995) failed to find an impairment in spatially pre-trained rats who were tested after AP5 infusion in a second watermaze located in a novel environment. 25

35 There are however key differences between the pre-training and testing tasks used by Uekita & Okaichi (2005) that provide an alternative explanation of the obtained results. In the spatial discrimination task there are several strategies that can be used to facilitate escape from the watermaze. Because the stable platform is always in fixed location, subjects could adopt the strategy of consistently navigating to the fixed platform based on distal spatial cues of the environment. Alternatively the subject can swim to the nearest available platform and attempt to mount it, moving on to the next available platform if the first platform approached does not provide escape. It is conceivable however that pre-training on the standard Morris water task and in the dry maze trains subjects to attend to and navigate using distal cues, reinforcing the fixed platform strategy that produces optimal performance on the subsequent spatial discrimination task. As such, subjects receiving these forms of pre-training may be more likely to adopt this strategy when tested on the spatial discrimination task. Additionally, the pre-training context may serve as a discriminative stimulus that occasions the use of the spatial navigation strategy. If this were the case, it would be expected that impairments on the spatial discrimination task resultant of dizocilpine would be minimized when tested in the training environment but not necessarily in a novel environment, consistent with the results of Experiments 2 and 3. The spatial representation hypothesis has however received support from studies of spatial learning outside of the watermaze. Carmanos and Shapiro (1994) used a radial arm maze to investigate the effects of dizocilpine (DZP) and APV in both familiar and novel environments. The procedure was also designed to be sensitive to differential impairments in working or reference memory. This dissociation was based on the 26

36 operational definitions of working and reference memory provided by Olton Becker and Handelmann (1979) who defined a working memory procedure by stating In a working memory procedure, stimulus information is useful for one trial of an experiment but not for subsequent trials and Working memory procedures have a major temporal component to them. As a contrast, In a reference memory procedure, information is useful for many trials and usually for the entire experiment Reference memory procedures do not have the same kind of temporal component as working memory procedures. Because the correct response to a stimulus is the same every time the stimulus is presented, the animal does not have to remember when the stimulus was last presented. More generally, Olton, Becker, and Handelmann speak of a flexible stimulus-response requirement, in which the subject is required to respond to a stimulus based upon a singular past experience, as a characteristic of a working memory procedure and document numerous studies (Becker, Walker, Olton, & O'Connel, 1978; Jarrard, 1975, 1978; Olton, Collison, & Werz, 1977; Olton & Feustle, 1979; Olton & Samuelson, 1976; Olton, Walker, & Gage, 1978; Olton & Werz, 1978; Walker & Olton, 1979) that would suggest that working memory procedures but not reference memory procedures are dependent on the hippocampus. In the Carmanos and Shapiro (1994) study, working memory and reference memory were dissociated by using separate dependent measures to analyze the performance of subjects tested in an eight arm radial maze with either all eight arms or four of the eight arms baited (with Froot Loops) during any given session. On the eight baited arms (8/8) task, a subject was placed into the center of the maze and chose an arm by completely traversing the entryway to that arm. Each choice constituted a discrete 27

37 trial with at 5-10s ITI in between trials and sessions continued until each arm had been entered. Working memory errors were operationally defined as re-entering an arm that had been previously chosen during that session. Thus optimal performance of the task would require that each arm be entered by the subject only once during a session. While this procedure would appear to be inconsistent with Olton s formal definition of working memory in that information obtained on a single trial is useful for all subsequent trials, it is consistent with the earlier radial arm maze experiments cited by Olton (1979) as working memory tasks (Olton et al., 1977; Olton & Samuelson, 1976; Walker & Olton, 1979). These earlier versions of this task were consistent with the formal definition offered by Olton as the original task was conducted in a single trial in which the rat was able to enter arms of the maze in succession rather than in discrete choice trials. The four baited arms (4/8) task assigned each rat four maze arms that would be baited on each session while the other four arms remained empty. The procedure was similar to the 8/8 task, however this task additionally measured reference memory errors, defined as entering an arm that was never baited in any session. In the 4/8 task, working memory errors were only scored when a rat re-entered an arm that was previously baited during the session. In this version of the task, optimal performance would be defined as entering each baited arm once without entering an un-baited arm during a session. Thus the procedure was sensitive to both working memory errors and reference memory errors Rats treated with DZP (.0625 mg/kg) prior to all pre-training and testing produced significantly more errors than controls (treated with saline) on the 8/8 task across 70 sessions of testing. While some improvement was seen in the DZP group during later 28

38 sessions, the trend failed to reach significance. This trend could represent the development of tolerance as the drug was administered repeatedly. Thus, DZP appeared to impair acquisition of the 8/8 task in a novel environment, though only WM learning was assessed. This conclusion is consistent with the spatial learning impairment found by Morris (1986) with AP5 and by Uekita & Okaichi (2005) with Dizocilpine (.10 mg/kg I.P.). There are however, several solutions that exist to achieve optimal performance on the 8/8 task. One potential solution is for subjects to construct a spatial representation of the environment based upon environmental stimuli (not confined to visual stimuli) and use the memory of discrete trials in conjunction with the spatial representation to arrive at an appropriate choice. Alternatively, rats could learn to proceed in a different direction on each subsequent trial, which would not require a spatial representation or memory for discrete trials. Neither strategy appears to have been adopted effectively by the naïve DZP subjects. A second group that had served as half of the saline control during the first 35 sessions was also administered DZP (.0625 mg/kg) before being switched from the 8/8 to the 4/8 task. These subjects were significantly impaired on the acquisition of the RM component of the task when compared to a control group during the course of 35 sessions, however no impairment was found in the performance of the WM task. The authors conclude that the DZP subjects previous familiarity with the environment and task (while serving as saline controls for the first 35 sessions) prevented DZP from impairing WM performance but that impairments in acquiring the new task demands (RM) are indicative of an inability to perform spatial remapping. Spatial remapping requires that subjects learn new associations (between reward and location in this 29

39 instance) within a familiar location. It is also possible that subjects were not relying on a spatial representation to complete the 8/8 pre-training task and had instead adopted a strategy of moving in a different direction (when leaving an arm proceed left) on each subsequent trial. Such a strategy would cause errors in the RM but not the WM task. When interpreting these results it should also be considered that procedural differences between the 4/8 and the 8/8 task create separate measures of WM in the two tasks. While in both tasks subjects are able to make an infinite number of WM mistakes during a session, the chance probability of errors is greater in the 8/8 task and presumably, the number of previous choices that inform a correct response is greater in the 8/8 task (eight arm choices as opposed to four). In addition, optimal performance of the 4/8 task requires fewer trials than optimal performance of the 8/8 task. Thus, it is conceivable that a WM deficit was not detected in the 4/8 task because the task was not as sensitive to an impairment. In a second experiment Carmanos and Shapiro (1994) additionally assessed the effects of APV infusion (33mM) on working memory performance in a familiar environment for rats pre-trained on the 8/8 task. Surprisingly, the APV group was impaired relative to controls receiving artificial cerebrospinal fluid (acsf), however the authors attribute this deficit to sensorimotor impairments caused by the dose. While the results of the second experiment appear to contradict the findings of the first experiment which were used to illustrate that pre-training on the 8/8 task eliminated a WM performance deficit, it is difficult to compare the two experiments because of procedural differences. The first experiment utilized systemic administration of a moderate dose of a non-competitive NMDA antagonist (DZP) while subjects in the second experiment 30

40 received a large dose (via I.C.V. infusion) of a competitive NMDA antagonist (APV). Also, the pre-trained DZP subjects in the first experiment that did not show a WM performance impairment when tested on the 4/8 procedure whereas APV subjects in the second experiment were tested on the 8/8 procedure, which could be more sensitive to a WM impairment. Lastly, subjects in the first experiment received 35 days of drug free pre-training on the 8/8 task while subjects in the second experiment received only 19 days of pre-training. These differences severely complicate a comparison of the two experiments. The authors attribute the discrepant findings primarily to the dose used in the second experiment. In a third experiment, rats were pre-trained and then tested on the 4/8 task with lower doses of APV (20 and 30 mm) in both a familiar and an unfamiliar environment. In the familiar environment, neither dose of APV produced a WM or RM impairment relative to control subjects infused with acsf. All subjects were impaired on both WM and RM performance relative to pre-operative performance with the exception of working memory performance in the APV30 group. In the novel environment, both doses of APV led to marked impairments in both the WM and RM performance measures relative to acsf controls. The control group was also impaired on the RM performance task relative to performance in the familiar environment, but not nearly as impaired as both APV groups. This is consistent with the interpretation used by Uekita & Okaichi (2005) who demonstrated an impairment in the acquisition of a spatial discrimination in a novel but not in a familiar environment and is seen to support a spatial representation hypothesis. As previously noted, this is in contrast to the findings of Bannerman et al. (1995) who did not find an impairment in pre-trained rats infused with AP5 when tested 31

41 in a novel environment. The Carmanos and Shapiro (1994) study is also only able to explain the dizocilpine induced impairment of RM acquisition in experiment one through the addition of a spatial remapping hypothesis that posits that NMDA receptors are necessary for not only creating a spatial representation of the environment but for forming associations between location and reward. In experiment one, DZP impaired acquisition of the 8/8 task (seen as a WM impairment) in naïve rats and impaired acquisition of the 4/8 task in rats pre-trained on the 8/8 task. In the 4/8 task, DZP produced deficits only on the RM measure. While the spatial representation theory would predict impaired acquisition of the 8/8 task in naïve rats, it would not predict an impairment in subjects pre-trained in the testing environment. The authors conclude that NMDA receptors are necessary for the creation of a spatial representation and for spatial remapping (forming associations between location and reward within a spatial map). The introduction of spatial remapping into the spatial representation hypothesis is problematic in that the hypothesis would presumably extend to any design in which reward location changes within a familiar environment. This addition to the spatial representation hypothesis conflicts with studies that have not found evidence for NMDA induced spatial learning impairments in procedures in which goal locations change between sessions. Several studies (Galizio et al., 2003; Keith & Galizio, 1997; Miller, 2005) found no selective effects of DZP (affecting spatial learning separately from spatial navigation) in a version of the water maze that compares within-session learning of new spatial locations to spatial navigation to a constant location in a familiar environment with extensive pre-training. The spatial remapping hypothesis would predict that performance on spatial learning of a new reward location within a spatial 32

42 representation would be impaired relative to spatial navigation to a well trained location in a familiar environment. Such an effect has not been found for a range of doses of three separate NMDA antagonists (LY235959, dizocilpine and phencyclidine). It is widely believed that NMDA antagonists uniquely disrupt spatial learning by blocking hippocampal LTP. However, the many discrepancies found between studies reviewed here investigating the effects of NMDA antagonists on spatial tasks raise serious questions regarding the notion that spatial learning depends on NMDA receptor activity. To further complicate the picture, there is considerable evidence that some nonspatial learning may be mediated through the NMDA receptor. Thus it seems prudent to compare these results with studies that have investigated the effects of NMDA receptor blockade on learning tasks that do not require spatial learning or that directly compare spatial learning to learning in other modalities. Non-spatial Investigations of NMDA Receptor Antagonist Effects Like several of the aforementioned spatial learning studies, the RAP methodology has also been applied to procedures investigating non-spatial learning and such studies have provided well controlled assessments of NMDA receptor antagonist effects. A study by Baron and Moerschbaecher (1996) assessed the effects of various glutamate receptor antagonists on the acquisition of 3 lever behavioral chains on a second order FR3 schedule (i.e. a three response chain of lever presses had to be completed three times before a reinforcer was delivered). In the acquisition component of the task, rats were trained such that one of three colored LED was illuminated at any given time to indicate what stage of the chain was currently active. For instance, during a session in which the subject was required to provide a response to the center (C) lever followed by a response 33

43 to the left (L) lever concluding with a response to the right (R) lever (a CLR pattern), A red LED was illuminated when a response to the center lever was required, a white LED was illuminated when a response to the left lever was required and an amber LED was illuminated when a response to the right lever required. The progression of LED illumination (red, white, amber) upon correct responses was held constant across all sessions, though the required response pattern (e.g. a LCR or RLC pattern) changed on each successive session. Incorrect responses led to a 2 second time out during which responses only served to reset the time out clock. After a time out, the schedule continued from the point at which the incorrect response was emitted. The stable performance of subjects trained on the daily acquisition of a behavioral chain was then compared to a second group of rats trained on a performance control task that was identical to the acquisition task with the exception that the reinforced response pattern (e.g.. CRL) remained constant across all sessions. Thus, both groups could then be tested across multiple doses of drugs and the acquisition of behavioral chains could be directly compared to the performance of the task without within session learning requirements. Subjects were trained on this task until stability emerged (a relatively consistent amount of errors were produced in daily sessions in both components and a reduction in errors within session was consistently seen in the acquisition component). Subjects were then tested across acute doses of a variety of glutamate receptor antagonists with differential pharmacodynamic actions. The effect of NMDA receptor antagonism was tested through the administration (I.P.) of the non-competitive antagonist dizocilpine ( mg/kg) and the competitive antagonist CGS ( mg/kg). Glycine site NMDA antagonism was assessed through the administration of 34

44 MDL104,653 ( mg/kg) and AMPA receptor antagonism was tested through administration of LY ( mg/kg). Sessions lasted until 60 reinforcers were delivered or until 30 minutes had elapsed and performance after drug administration was presented as percent change from a saline/vehicle baseline of within session errors and response rate. Both NMDA antagonists (dizocilpine and CGS 19755) produced dose dependent decreases in response rate in both the acquisition and performance components (decreases in response rate did not differ between components) and dose dependent increases in errors in the acquisition component. Importantly, two doses of dizocilpine (0.056, and 0.10 mg/kg) and one dose of CGS (5.6 mg/kg) produced significant increases in within session errors (relative to saline/vehicle) in the acquisition group without producing an impairment in the performance group, thus illustrating a selective effect on behavioral chain acquisition at these doses. While decreases in response rate were generally equivalent for both the performance and acquisitions groups after administration of dizocilpine and CGS 19755, the.056 mg/kg dose of dizocilpine was the only dose of either drugs that produced a selective impairment without a significant effect upon response rate. In contrast, neither the glycine site antagonist (MDL 104,653) nor the AMPA receptor antagonist (LY ) significantly impaired chain acquisition up to doses that completely suppressed responding. When analyzed across blocks of 15 chain completions (obtaining 5 reinforcers on the second order FR3 schedule) both the 5.6 mg/kg dose of CGS and.056 mg/kg dose of dizocilpine produce impairments that are characterized by more errors (relative to saline) during the first half of the session progressing to baseline levels of errors by the end of the session. The.10 mg/kg dose of 35

45 dizocilpine produced a more profound impairment across all bins with little within session error reduction. These results are consistent with a later non-spatial RAP study conducted by Pitts, Buda, Keith, Cerutti & Galzio (2006). The Pitts et al. study investigated the acquisition of positions within a 2 x 3 array of identical stimuli presented on a computer screen within an operant chamber. The monitor used within the chamber was affixed with a touch sensitive screen that allowed rats to respond to the stimuli by providing a nose poke to the location of the stimuli within the array presented on the screen. As an RAP study, there was both an acquisition and a performance component to the task. In the performance component, a 2 x 3 array of identical stimuli (e.g. green circles) was presented to subjects on each performance trial and only a response provided to one of the six identical stimuli (e.g. the upper middle stimuli) produced reinforcement via a sucrose pellet hopper located in the back of the chamber. For each performance trial, both within and across sessions, only responses to a single stimuli location (e.g. the upper middle stimulus) provided reinforcement. The acquisition component of the task was signaled by the presentation of a 2 x 3 array of identical stimuli different from those used in performance trials (e.g. white squares). In this component, responses to a single stimulus location (e.g. the lower right hand stimulus) produced reinforcement during a given session, but the stimulus location for which responses were reinforced changed on each given session. The position used for the performance component varied across rats and for each rat the performance position was never used during acquisition trials. Additionally, the stimuli used for the separate components were counterbalanced across subjects. 36

46 Incorrect responses were followed by 1.5 second time-out in which the stimulus array was darkened and a correction procedure was used such that the subject was required to continue to respond to trials within the component (acquisition/performance) until a correct response was emitted, at which point the array would switch to the alternate component. This procedure allowed for an analysis of errors per reinforcer delivered as well as response rate. Subjects were able to achieve stable performance in the multiple component RAP touch-screen procedure in an average of 71 sessions with an average of.22 and.96 errors per reinforcer on the performance and acquisition components, respectively. Asymptotic performance on the acquisition component was typically reached by the fifth or sixth reinforcer. After reaching stability, subjects received acute I.P. injections of the benzodiazepine chlordiazepoxide (1.0, 3.0, 10.0 and 17.0 mg/kg) the µ opioid receptor agonist morphine (1.0, 3.0, 5.6 and 10.0 mg/kg) and the non-competitive NMDA receptor antagonist dizocilpine (.03,.10,.18 and.30 mg/kg) and saline. The order of drug presentation was counterbalanced across subjects and each subject received between two and four determinations at each dose in a quasi-random dose order. Because subjects reached asymptotic performance on the acquisition component relatively quickly (as measured by errors per reinforcer), analyses were conducted only for the first 10 reinforcers delivered in each component (with the exclusion of the first reinforcer of the acquisition component as no response feedback was available prior to delivery of the initial reinforcer). Sessions were conducted until 40 reinforcers had been delivered or 60 minutes had elapsed. 37

47 All three drugs significantly reduced response rates in both components at the higher doses, and 2 x 4 (Component by Dose) repeated measures ANOVAs of response rate revealed no significant component by dose interactions for any of the drugs studied. Doses that produced significant decreases in response rate were excluded from subsequent analyses of errors per reinforcer which revealed selective acquisition impairments at the 3.0 and 10.0 mg/kg doses of chlordiazepoxide (CDP) and the 0.18 mg/kg dose of dizocilpine (DZP). No dose of morphine produced impairments on the acquisition component without producing a performance impairment. CDP produced selective increases in errors per reinforcer at two separate doses while DZP produced selective increases at only at a single dose, the selective increases caused by both doses of CDP were relatively minor when compared with the selective impairment produced by the selective dose of DZP (a two-fold increase in errors at the 10.0 mg/kg dose of CDP versus a five-fold increase in errors at the.18 mg/kg dose of DZP). While the.18 mg/kg dose of DZP caused the greatest increase in errors per reinforcer (relative to saline) during the first 4 reinforcers delivered, subjects appeared to be impaired across all 10 reinforcer deliveries included in the analysis. The observation that selective impairments have been evidenced after administration of NMDA antagonists in non-spatial RAP tasks (Baron & Moerschbaecher, 1996; Pitts et al., 2006) but were not produced in the spatial RAP studies (Galizio et al., 2003; Keith & Galizio, 1997; Miller, 2005) provides further evidence that NMDA receptor antagonist impairments are not the result of a learning impairment restricted to spatial information (as is put forth by the spatial representation hypothesis). RAP studies carefully control for non-mnemonic behavioral impairments 38

48 resultant of drug administration and as such, a sensorimotor account also fails to explain the selective impairments reported in the aforementioned studies. Additionally, the argument that NMDA receptor impairments are solely the result of a failure to acquire behavioral strategies relevant to task acquisition is refuted by the RAP studies in that impairments were evidenced in subjects extensively pre-trained (to stability) on all aspects of the procedure. It could be argued however that both the Baron & Moerschbaecher (1996) and Pitts et al. (2006) tasks contained spatial requirements. In both studies, response options differed only in the relative position of stimuli that could be responded to (levers in the case of Baron & Moerschbaecher and shapes presented on the touchscreen in Pitts et al.). As such, the processing of spatial information (discriminating between the relative position of stimuli) is arguably required in both tasks. There are however, other nonspatial procedures that have been developed for rats that do not require the animal to discriminate between response options based on any spatial elements, many of which make use of the rats keen sense of olfaction. Like humans, most animals preferentially attend to stimuli of certain sensory modalities to accomplish behavioral tasks. The preferential sensitivity of an organism to sensory modality can be described along a sensory hierarchy that need not be constant across all situations. As the term hierarchy implies, organisms often attend to stimuli of multiple modalities, however some modalities appear to be more salient in shaping particular behaviors. While less preferential sensory modalities can often be used to solve problems when the preferred modality is not available (or helpful), the performance of some tasks 39

49 appears to be completely dependent on one modality. This is well illustrated by the seemingly complete dependency of rats on olfaction to perform a reach-to-grasp-food task, where rats are required to reach for food located in one of several hidden locations (Hermer-Vasquez, Hermer-Vasquez, & Chapin, 2007; Wishaw & Tomie, 1989). Although this is an extreme example of the dependency of rats on olfaction, it has been well documented that rats preferentially use olfaction on a wide variety of behavioral tasks (See Slotnick, 2001). Early investigations of rat cognition using visual stimuli often drew the conclusion that rats were unable to learn complex tasks, however Jennings and Keefer (1969) demonstrated that impressive acquisition of a learning set could be achieved in rats when olfactory stimuli were used. Despite considerable evidence that rats can acquire more complex tasks and can acquire tasks with less training when olfactory stimuli are used alone or in conjunction with visual cues, the use of olfactory stimuli in learning tasks has been limited (compared to tasks using other sensory modalities) until fairly recently. The apparent hesitation to use olfactory stimuli is largely due the difficulty of controlling and presenting olfactory stimuli. While both light and sound exist along continuums (frequency, intensity), no such continuum exists for olfaction, thus, the generalization of olfactory stimuli is nearly impossible to assess. In addition, unlike auditory and visual stimuli, which can be presented and removed from an environment quickly and completely, it can be very difficult to present, remove, or measure the presence of olfactory stimuli. Despite these obstacles, it is quite possible to assess behavior in transition using olfactory stimuli. One of the earliest studies of the effects of NMDA antagonism on an olfactory non-spatial task was published shortly after the original Morris (1986) AP5 paper. 40

50 Staubli, Thibault, DiLorenzo and Lynch (1989) tested the effects of D-AP5 on rats trained on an operant procedure designed to assess performance on the acquisition and retention of olfactory discriminations. This procedure used a wedge-shaped apparatus with six choice alleys set up to deliver a small amount of water to water-deprived rats when a nose poke was made by the rat at the end of a choice alley. Each of the six alleys was designed to emit a scent through a tube, which pulled air through scented water. Each tube was equipped with an infrared diode capable of detecting a nose poke. On each trial, the rat was placed into the apparatus at the thin edge of the wedge and was allowed to enter any of the six alleys. Two distinct scents were chosen for each session and were emitted at the end of two alleys, randomly chosen for each individual trial of a session. During each training session, only nose pokes corresponding to one of the two novel odors chosen for the session were reinforced. Nose pokes made at the end of an incorrect scent alley led to termination of the trial and nose pokes to an unscented alley had no consequence. After becoming proficient in this task (e.g. the animal was able to attain at least 80% accuracy on the last 10 of 20 trials during a session) all 20 rats used in the experiment had a minipump surgically implanted in their neck that would deliver either.5 µl/hr AP5 or an equivalent volume of saline chronically into their lateral ventricles over the course of 14 days. After surgery, each rat was tested on novel olfactory discriminations with either short (2 min.) or long (10 min.) inter-trial intervals (ITIs) using either normal or low intensity (8 fold diluted) olfactory stimuli. Rats infused with D-AP5 performed as well as their saline infused controls in acquiring novel discriminations of normal intensity stimuli at 10 minute ITIs or of low intensity stimuli at 41

51 2 minute ITIs, but were impaired in discriminations of low intensity stimuli at 10 minute ITIs. Interestingly enough, while acquisition was impaired in the experimental rats during the first 15 trials of sessions (with a low intensity stimuli and a long ITI) averages on the last five trials of these sessions were comparable between the two groups and a 10 trial retention test conducted 10 days later (while still under infusion) also revealed no difference between the groups. In addition, retention tests of discriminations learned prior to surgery also yielded no differences between the experimental and control groups. These results imply that while chronic AP5 infusion impairs acquisition of novel olfactory discriminations (with long ITIs and low intensity stimuli), subjects were still able to both acquire novel and retain pre-training olfactory discriminations. The fact that the animals were able to retain discriminations that were impaired during the AP5 infusion suggests that their sense of olfaction was not impaired during administration of the drug. However, it has also been suggested that both the Staubli et al. (1989) and the earlier Morris et al. (1986) studies make compelling cases against NMDA receptor mediated learning, as there was evidence of improved task performance after administration of an NMDA receptor antagonist in both studies (Keith & Rudy, 1990). While the Staubli et al. (1989) study demonstrated an excellent way to present olfactory stimuli and assess behavior in transition and documented an NMDAr antagonist impairment in a clearly non-spatial task, less elaborate methods of presenting olfactory cues have been used effectively for the same purpose. Bunsey & Eichenbaum (1996) developed a procedure in which various household spices (e.g. oregano, paprika) are mixed with sand and presented to subjects in small plastic cups. Rats can be trained to dig in the sand to obtain buried sucrose pellets and after pre-training this behavior can be 42

52 viewed as a response to the olfactory stimuli. This training procedure has been used in various designs to study complex learning behaviors in rats. Peña, Pitts and Galizio (2006) used this method of stimulus presentation to study identity matching-to-sample (MTS) in rats. Rats were first presented with a sample stimulus (a cup filled with a mixture of sand and spice) in a modified operant chamber. Upon digging in the sample stimulus cup to receive a buried sucrose pellet, subjects were then presented with two stimulus cups. One cup contained an odor that matched the odor of sample stimulus while the other cup contained a different odor. Only the cup with the same odor as the sample stimulus contained a buried reinforcer. Subjects rapidly learned the MTS procedure and reached a within session accuracy of 90% or above in sessions. Subjects additionally maintained high levels of accuracy when novel scents were added, when both choice cups were baited, and when responses to the stimulus cup were only reinforced on half of the trials. Acquisition of the olfactory MTS procedure is significant in that MTS behavior has been difficult to achieve in rats using visual stimuli (Iverson, 1993). By using this technique of stimulus presentation, olfactory stimuli provide a unique opportunity assess complex non-spatial learning in rats. This mode of stimulus presentation was used by Galizio, Miller, Ferguson, McKinney and Pitts (2006) in an RAP adaptation of an olfactory reversal discrimination task to investigate the effects of dizocilpine as well as chlordiazepoxide and morphine in rats. The study was designed to compare performance on a previously trained olfactory discrimination to the acquisition of a new olfactory discrimination across doses of the drugs both within subject and within session. In each session, a subject received 16 trials (with a 5 second ITI) in which two 43

53 different odors were presented and the subject could choose to dig in either odor cup. One odor cup reliably contained a sucrose pellet (S+), while the other odor cup did not (S-). Acquisition of the olfactory discrimination was determined by the percent of correct choices the rat made within blocks (four trials per block) of trials. For each successive session, two new odors were used until all of the odors had been presented. After that, each S+ odor was matched with a different S- odor and the conditions were reversed (so that the S+ now served as the S- and vice versa). Thus, in each session, the subject was required to learn which odor was now the S+. This was termed the reversal discrimination or acquisition component. In addition to the reversal discrimination, each session included 8 trials in which two additional odors (never used in the reversal discrimination) were presented. In these trials one odor was always used as the S+ while the other was always the S- across all trials. This was termed the performance component of the procedure. Non-baited probe trials were conducted to ensure that subjects could not detect the scent of the sucrose pellet. The presentation position (right or left) of the scents was randomized. After being trained to stable performance on both components of the procedure, subjects were tested across doses of dizocilpine (.03,.1 and.3 mg/kg I.P.), chlordiazepoxide (1, 3, 10 and 17 mg/kg I.P.), morphine (1, 3, 5.6, 10 and 17 mg/kg I.P.), and saline. The order in which each subject received the active drugs was assigned randomly as was the order in which the subject received doses. Morphine produced a dose dependent impairment in both the acquisition and performance components of the procedure but at no dose was acquisition impaired without a significant performance impairment. Similarly, dizocilpine produced dose dependent impairments in both 44

54 acquisition and performance with no dose providing a selective impairment. In contrast, chlordiazepoxide produced impairment in the acquisition task at the 10.0 mg/kg without impairment of the performance task. As was the case with the earlier spatial study using the RAP adaptation of the Morris swim task (Keith & Galizio, 1997), a benzodiazepine (chlordiazepoxide) but not an NMDA antagonist produced a selective learning impairment. Thus, while spatial RAP studies (Galizio et al., 2003; Keith & Galizio, 1997) have repeatedly failed to find selective impairments after administration of a range of NMDA antagonists, results from non-spatial RAP (Baron & Moerschbaecher, 1996; Galizio et al., 2006; Pitts et al., 2006) designs are mixed. That selective effects have been in found in non-spatial but not in spatial RAP studies suggests that spatial theories of NMDAr antagonist effects (e.g. the spatial representation and spatial strategy hypotheses) are insufficient to describe the full range of impairments observed in the literature. Additionally, while Cain et al. (Cain, Saucier, & Boon, 1997; Cain et al., 1996) have demonstrated that NMDA antagonists can cause sensorimotor impairments that disrupt task acquisition/performance, the aforementioned RAP designs carefully control for sensorimotor impairment and thus sensorimotor effects are not able to account for the discrepancies either. Steele and Morris (1999) provide convincing evidence that the effect of NMDAr antagonists on learning tasks are delay dependent, however the delay intervals used in all of the non-spatial RAP studies are relatively short and variable to some degree, thus it is not immediately apparent how delay dependency explains the discrepancies either. 45

55 Operational Definitions of Mnemonic Processes in Animal Models While Steele and Morris were conservative in their discussion of how one trial versus multiple trial learning of a spatial location relates to theoretical divisions of memory processes, the Bannerman, Rawlins, and Good (2006) review uses the results of this study to support differentiating procedures by their ability to dissociate working memory from reference memory. Bannerman et al. adjust Olton s operational definition of a working memory procedure to include a flexible memory system in which conditional, trial-specific information is used to select between response options that are variably correct or incorrect, and which is dependent on the septo-hippocampal system. As such, the standard watermaze procedure in which a single spatial location is acquired across many trials (or sessions) could be considered a spatial reference memory task, as trial specific information is not necessary to provide the correct response (direction in which to swim). In contrast, the Steele and Morris (1999) analysis of one trial learning of spatial locations within the watermaze would be considered an analysis of the working memory component of the task as the subject is required select the appropriate response (direction in which to swim) based upon on information from a specific trial (trial 1 of the current session). Trials 3 and 4 in the Steele and Morris represent a reference memory component of the task and as was shown, subjects who received hippocampal lesions as well as subjects who were administered D-AP5 evidenced learning across these trials. Viewed in this sense, the acquisition component of the RAP watermaze task can also be seen as both a working memory and reference memory procedure with a working memory requirement between the 1 st and 2 nd trials of any session (at a delay of 2.5 min.) and a reference memory requirement across the 6 trials of a session. 46

56 The working memory hypothesis of NMDA antagonist impairments as proposed by Bannerman, Rawlins and Good (2006) provides an interesting framework for the interpretation of the seemingly conflicting studies discussed presently. The lack of a selective impairment on the acquisition of olfactory discriminations after administration of dizocilpine as was illustrated across multiple doses in the Galizio et al. (2006) study is consistent with the Staubli et. al study (1989) that found no impairment in olfactory discrimination acquisition with short inter-trial intervals (less than 2 minutes). By definition, the acquisition of olfactory discriminations across many trials within a session is a reference memory task and would only be aided by working memory during the initial trials of a session. As Steele and Morris (1999) have shown, one trial learning of a spatial position within the watermaze after administration of an NMDA antagonist is only impaired at significant delays. Thus, under a working memory hypothesis of NMDA receptor antagonism, impairment on the initial trials of the session would not be expected at a 5 sec. ITI, as is consistent with the data presented in the Galizio et al. (2006) study. The working memory hypothesis is supported in part by the Carmanos and Shapiro (1994) investigation of working and reference memory in an eight arm radial arm maze in which dizocilpine impaired naïve rats in the performance of the 8/8 baited arms task in a novel environment (across 70 sessions of testing), and APV impaired pre-trained rats on the 8/8 task in a familiar environment as well as on the 4/8 baited arms task in a novel environment. Indirect evidence is also provided by the spatial discrimination watermaze study by Uekita & Okaichi (2005) in which naïve rats receiving dizocilpine were impaired relative to controls in a task that required subjects to use information from 47

57 discrete unique trials (platforms located in a variety of positions do not provide escape) to facilitate the acquisition of a reference memory task (swim towards the secure platform). While all of the direct evidence for working memory impairments in rats as a result of NMDA receptor blockade comes from spatial studies, the working memory hypothesis also provides a unique interpretation of the effects of NMDA antagonists on non-spatial tasks. All of the non-spatial tasks previously reviewed would be considered reference memory tasks, however would arguably be facilitated also by working memory during early trials. The working memory hypothesis predicts that while these tasks will be learned by rats across extended testing, impairments will be evidenced during the early trials of testing given that there is sufficient delay between the presentation and testing of trial unique information. Such an effect is illustrated in the Staubli et al. (1989) study which showed an impairment in the acquisition of olfactory discriminations in rats infused with D-AP5 only during the initial trials of testing when a long ITI (10 min.) was implemented. It is important to note however, that this effect only occurred when the saliency of the olfactory stimuli was comparatively low. Early learning impairments (within the first four reinforcer presentations) also characterized the selective effects of dizocilpine on the RAP visual discrimination touch-screen task used by Pitts et al. (2006). The selective impairments of the.056 mg/kg dizocilpine dose and of the 5.6 mg/kg CGS dose on chain acquisition in the RAP procedure used by Baron and Moerschbaecher (1996) were also only seen during the early bins of testing, with performance equivalent to that produced during saline control sessions evidenced in the final bins of testing. It is important to note that this trend was not seen in the selective effect of the.10 mg/kg dose 48

58 of dizocilpine which caused gross impairment during all bins of testing, though unlike lower doses, this dose also significantly decreased response rate. These observations are of course very general as none of the aforementioned nonspatial studies specifically assessed the effects of NMDA receptor blockade on a procedure that required the retention of trial specific information. Additionally, there is considerable variability within the non-spatial literature in regard to the testing conditions that are sufficient to evidence a learning impairment as a result of NMDA receptor blockade and the degree of impairment produced. If the NMDA receptor is indeed critical to the functioning of working memory in the rat as Bannerman et al. (2006) suggests, then rats receiving a sufficient dose of an NMDA antagonist should be impaired in a non-spatial task in which accurate performance is consistently dependent upon the retention of information presented during a single trial. To avoid some of the pitfalls that have complicated the interpretation of previous studies of NMDA receptor blockade, a non-spatial investigation of NMDA effects on the retention of trial specific information must meet several criteria. First, there must be an appropriate control for separating the sensorimotor effects of the drug from an effect upon the choices that require the use of trial specific information. As discussed previously, a sensible solution to this problem is the implementation of an RAP design in which a control task with similar behavioral demands but no mnemonic requirements is trained concurrently with the task of interest. Additionally, the spatial position of stimuli should not relate to the contingencies associated with responding. While the Baron and Moerschbaecher (1996) and Pitts et al. (2006) studies are used as examples of non-spatial investigations of the effects of NMDA antagonists (because the animal is not navigating 49

59 through space or making use of distal cues), in both studies response options only vary based upon their relative spatial position to one another which could be considered a spatial element of the task. A final consideration is the stimuli used to control behavior. It should be clear what stimuli are guiding behaviors and discrimination of the stimuli must be unimpaired by the pharmacological manipulation. The Odor Span Task A procedure developed recently by Dudchenko, Wood and Eichenbaum (2000) has particular potential for achieving these goals. Dudchenko et al. have developed a procedure using olfactory stimuli that was designed to assess performance as a function of what they term memory load within rats. Referred to as the odor span task, this procedure is believed to require processes similar to those assessed by the digit span task, commonly used as test of working memory for humans. In the digit span task, subjects are required to repeat ordered strings of numbers that get progressively longer. Humans reliably provide accurate performance on this task with up to seven (plus or minus two) digits in a string, but fail to accurately repeat strings containing a greater number of digits. Training of the procedure began with shaping rats to dig in cups of scented sand for pieces of Froot Loop cereal. Subjects were then introduced to an odor non-match-tosample (NMTS) task. In this procedure, the sample stimulus consisted of an odor cup that was affixed (with Velcro) to one of 24 positions evenly distributed along the perimeter of a square Plexiglas platform. On the sample trial, rats were placed on the platform and allowed to explore until they dug in the sample cup and consumed the reinforcer. The rat was then removed from the platform and the sample cup was moved 50

60 to a different location on the platform and a second stimulus cup (baited) was placed at a random location on the perimeter of the platform. The rat was placed back upon the platform and a choice was scored when the rat dug in a stimulus cup. Subjects received trials per day using scents randomly selected from a list of 25 household spices (e.g. paprika, nutmeg, cumin). Subjects were then moved to the odor span task which differed from the odor NMTS procedure in that after the first choice trial, both scents were relocated and a third scent (baited) was added in a random location. After each trial in which a correct choice was scored, odor cups were relocated and a new odor cup (baited) was added to the apparatus in a random location. When an incorrect choice was made, all stimuli were removed from the apparatus and the procedure began again with a new collection of odors. On each day of training, subjects received as many trials as could be completed during a minute period. For each string of consecutive correct choices, memory span was defined as the number of correct choices minus one (because an incorrect response cannot be made on the first trial). After stability was achieved on this task (as evidenced by two consecutive days with an average span of 5) a correction procedure was implemented such that after an incorrect response, subjects were permitted to continue until a correct response was scored. During this phase of training the number of stimuli continued to increase to a maximum of 12 odors (allowing for a maximum span of 11). Thus on each trial of the 12-odor span task, subjects were required to discriminate the novel odor from the comparisons based on identifying and withholding responses to odors presented previously within the session during a single reinforced trial. 51

61 After 7 days of training on the 12-odor span task, a probe session was conducted in which stimulus cups were never baited and a reinforcer was placed in the stimulus cup only after the subject made a correct response. Mean span during the non-baited probe session did not differ from the average of each subjects median span during the 7 days of training, indicating that subjects were not responding to the scent of the buried reinforcer. To assess whether rats were scent marking stimulus cups and avoiding scent marked cups on subsequent trials, another probe session was conducted in which all of the stimulus cups were replaced with new stimulus cups (containing the same odors) on trials 5 and 9. On this probe session mean span was significantly lower than the average median span of subjects during training. The authors however, qualify this result with the observation that only two of the rats made their first error on trial 5, and only two made their first error on trial 9 while the remaining 12 subjects did not make their first error on a trial directly after the cups were changed out. Percent correct for either probe trial was not reported. After the completing both probe trials, subjects were matched for performance and assigned to receive either ibotenic acid lesions of the hippocampus or a sham surgery without lesioning. After being retrained on the NMS procedure, subjects were tested for an additional 7 days on the 12-Odor Task. Groups did not differ on the span measure or in percent correct (percent correct was calculated excluding trial 1). Across both groups, percent correct was correlated with span length and subjects performed better than chance at each span length. Probe sessions were again conducted after the 7 th day of testing. Average span on non-baited probes did not differ significantly between groups and when combined, both groups did not significantly differ from performance on the 7 th day of 52

62 testing. Differences were also not reported during the cup change probe. Because both groups performed remarkably well on the 12-Odor Task, subjects were additionally tested on a 25-Odor Span task to ensure that the lack of an effect between groups was not due to a ceiling effect. Span length (number of comparisons present minus 1) measured in blocks of 5, was once again correlated with the number of correct responses, however there was no main effect of group or a group by span length interaction. These results suggest that within session recognition of olfactory stimuli is sensitive to memory load, but is not hippocampally dependent. The Odor Span Task developed by Dudchenko, Wood and Eichenbaum (2000) presents a unique non-spatial procedure for assessing the effect of stimulus set size (the number of stimuli that can serve as potential comparisons) on NMTS performance in rats. The results of Dudchenko et al. study suggest that the performance of rats in this type of task is dependent upon the amount information that is needed to select between response options (stimulus set size). As we have discussed, Steele and Morris (1999) have demonstrated the importance of delay in the interpretation of NMDAr antagonist effects on behavioral assays. Delay however, does not appear sufficient to account for the discrepancies observed between well controlled investigations of the effect of NMDAr antagonists on non-spatial tasks. Arguably, the discrepant studies mentioned also differ greatly in the amount of information that must be acquired/retained by subjects during the session to provide accurate responding, though a direct comparison of the requirements of the various tasks can not be made. With the implementation of proper controls, the Dudchenko et al. procedure may provide an opportunity to explore the impact of stimulus set size on NMDAr antagonist effects. Based upon the baseline 53

63 performance of subjects during the 25 odor span reported by Dudchenko et al., the odor span task should produce a strong baseline with which to assess the effects of drugs. Additionally, the Dudchenko et al. procedure speaks directly to the Bannerman et al. working memory hypothesis as each trial is essentially a choice for which accurate responding is dependent on information obtained during discrete and independent trials (responses to each odor used in the procedure are only reinforced once during any given session). Under the Bannerman et al. hypothesis, robust impairments should be observed in this procedure after administration of an NMDA antagonist. Application of an RAP Methodology to the Odor Span Task The present study aims to adapt the Dudchenko et al. procedure for use in behavioral pharmacology and to investigate the role of the NMDA antagonist dizocilpine on olfactory span. To ensure that performance of the task is indeed dependent upon on the manipulation of stimulus set size, several adjustments will be made to the original procedure to control for extraneous variables. To improve the objectivity of experimenter scoring of responses, the present study will present olfactory stimuli by covering the stimulus cups with scented lids and defining a response as any displacement of the lid by the rat during a trial. This response is more easily observed by the experimenter during the procedure than a digging response and allows for verification of the scoring post-trial. Additionally, this response can be easily viewed from video footage and will allow the experimenter to videotape sessions so that a second experimenter (blind to the active contingency) can score responses and subsequently determine inter-rater reliability. 54

64 The use of scented lids will also help to obscure the reinforcer (a sugar pellet) from view and prevent subjects from detecting the odor of the reinforcer located in the stimulus cup. Probe trials in which none of the stimulus cups presented contain a reinforcer will be conducted frequently to ensure that responses are not aided by the presence of the reinforcer in the stimulus cup. Because the results of the cup change probes conducted in the Dudchenko et al. procedure suggested that performance could potentially be guided scent marks left on the scented stimuli during earlier trials of the task, the present study will control for scent marking by replacing the scented lids after each trial, with fresh lids presenting the same odor. While Dudchenko et al. concluded that performance of the of olfactory span task was dependent on stimulus set size, the manipulation of stimulus set size in the original procedure was confounded with the number of stimuli presented. In each successive trial of the task, both stimulus set size and the number of stimuli presented was increased. As such, the observed impairments may have been dependent on the number of comparison stimuli presented during the trial rather than the number of stimuli that had been presented during the session. To eliminate this confound, the number of comparison stimuli used during any trial will be limited to five stimuli in the present study (one novel comparison odor and four comparison odors chosen from the pool of stimuli previously presented during the session). Through the implementation of this control, the experimenter can assure that any observed impairments are reflective of the planned manipulation of stimulus set size (the number of odors that could serve as potential comparisons) rather than the number of comparisons presented during a given trial. 55

65 These controls will allow the experimenter to assess the Dudchenko et al. model of stimulus set size dependent performance on the olfactory span task and assess the roles of scent marking, pellet detection and number of comparisons in task performance. In addition to verifying the effect of stimulus set size previously observed, the procedure will be made suitable for the analysis of drug effects by introducing a control condition based on RAP procedures. This will be accomplished by including a performance task to measure behavioral aspects of the task that are required in all training and testing. The performance component will comprise of a previously learned olfactory discrimination that will remain constant across all training and testing sessions. The olfactory stimuli used in the performance component will not be used in acquisition component and will serve as the discriminative stimuli signaling the active component. This task requires subjects to be able to navigate the apparatus, discriminate olfactory cues and provide the same response scored in the olfactory span procedure however, places a low demand upon stimulus set size. Thus, this task will provide a control measure for sensorimotor effects of the drug that could potentially impact behaviors necessary to complete the task that are independent of the stimulus set size manipulation. Additionally, as the odors used in the olfactory discrimination performance control are presented repeatedly with the response contingencies held constant, the task would be considered a reference memory task under the Bannerman et al. definition and is not predicted to be affected by NMDAr antagonism under the working memory hypothesis. Selective impairments on the RAP adaptation of the olfactory span task, as evidenced by impaired performance of the olfactory span task without concurrent impairment of the performance task would provide support for the Bannerman et al. 56

66 hypothesis and will allow for analysis of the impact of stimulus set size on NMDAr antagonists impairments. It is expected that the Dudchenko et al. model of stimulus set size dependent performance of the olfactory span task will be confirmed in the present paradigm and illustrated by a decline in individual and group percent correct performance across increasing stimulus set sizes. Additionally, it is expected that systemic administration of dizocilpine will produce gross impairments characterized by reduced span and percent correct performance relative to baseline. The degree to which percent correct performance decreases across stimulus set size after administration of dizocilpine will determine the role of NMDAr antagonist effects on this variable when compared with the observed dependency of percent correct performance on stimulus set size at baseline. 57

67 Subjects METHOD Subjects were five male Holtzman Sprague-Dawley albino rats that were 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 hour light-dark cycle. All subjects were given continuous access to water in their home cage and food access was restricted to approximately 17g of rat chow per day. Food rations were delivered once per day between 30 and 60 minutes after testing and were adjusted to maintain each subject at a stable weight. Apparatus All testing was conducted in an open-field apparatus constructed from a circular particle board table 29.2cm tall and 94cm in diameter (See Figures 1 and 2). The table top was connected to the base of the table through a dolly mechanism, allowing the top to rotate 360º. The surface of the table was surrounded by a 32cm high wall of sheet metal baffling, supported at the top by a thin ring of plastic piping. The table top contained 18 holes, 5.5 cm in diameter. Twelve holes were evenly spaced in an outer ring, 2.5cm from the wall surrounding the surface of the table. Six holes were evenly spaced in an inner ring, 21.5cm from the apparatus wall. Hole positions were labeled 1 through 18 with holes in the outer ring labeled 1 through 12 and holes in the inner ring labeled 13 through 18 (See Figure 3). Plastic cups (2 oz.) were placed in each hole during a trial. Sessions were also recorded on a web cam (Logitech, inc.) using Windows movie maker (Microsoft Inc.) such that each session could be reviewed to determine the inter-rater reliability of data. 58

68 Figure 1: Photo of Open Field Apparatus (side view) 59