Effects of NMDA antagonists ketamine, methoxetamine and phencyclidine on the odor span test

of working memory in rats

Michael J. Mathews1, Ralph N. Mead2 and Mark Galizio1

1Department of Psychology, UNC Wilmington

2Department of Chemistry, UNC Wilmington

Corresponding author: Mark Galizio, PhD, Department of Psychology, UNC Wilmington, 601

S. College Rd., Wilmington, NC, 28403, galizio@uncw.edu, 910-962-3813. Ralph N. Mead,

PhD, is at UNC Wilmington and Michael Mathews, MA, is now at West Virginia University.

Abstract

The present study used a 2-choice version of the odor span task to assess the effects of NMDA

antagonists on remembering multiple stimuli in rodents. The odor span task uses an incrementing

non-match to sample procedure in which responses to a new olfactory stimulus are reinforced on

each trial, whereas responses to previously presented stimuli are not. NMDA antagonists have

been associated with memory impairments in a variety of animal models, however, there are

inconsistencies across different NMDA antagonists and tasks used. The current study compared

the acute effects of phencyclidine, ketamine, and the novel NMDA antagonist methoxetamine on

responding in the odor span task and a simple discrimination control task. Phencyclidine and

methoxetamine impaired odor span accuracy at doses that did not impair simple discrimination

in most rats, however the effects of ketamine were less selective. Within-session analyses

indicated that the effects of phencyclidine and methoxetamine depended on the number of

stimuli to remember, i.e., impairment only occurred when the memory load was relatively high.

These effects of phencyclidine and methoxetamine were consistent with previous research

showing that NMDA antagonists may interfere with working memory capacity, but the basis for

the discrepant results with ketamine are unclear.

Keywords: NMDA antagonist, working memory, odor span task, ketamine, phencyclidine,

methoxetamine.

Public Significance Statement: The glutamate hypothesis of schizophrenia posits that NMDA

receptor hypofunction may underly the cognitive symptoms of the disorder. The present study

provided some support for this hypothesis in that we found that two NMDA receptor antagonists,

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phencyclidine and methoxetamine (but not ketamine), selectively reduced memory capacity as

measured by the odor span task. These results support the idea of the NMDA receptor as a

potential target for new treatments for schizophrenia.

3

Disclosures and Acknowledgements

This research was supported in part by NIDA grant DA029252, Mark Galizio, PI. Only

financial support was provided by NIDA.

All authors contributed in significant ways to the paper and all have read and approved

the ms.

None of the authors report any conflict of interest relevant to the present research.

The authors thank Katrina Gobenciong, Angela Goolsby, Chloe Myers, Danielle Panoz-Brown,

and Ashley Prichard who assisted in data collection and analysis.

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The initial impetus for the NMDA hypofunction hypothesis of schizophrenia was the

observation that NMDA antagonists like phencyclidine and ketamine produced many of the

symptoms that characterize the disorder in humans, including the full spectrum of positive,

negative and cognitive symptomatology (Merritt, McGuire & Egerton, 2013; Moghaddam &

Javitt, 2012). There has been particular interest in the translational value of the hypothesis with

respect to cognitive impairment (Gilmour et al., 2012). For example, the effects of NMDA

antagonists have been assessed in a variety of animal models of working memory. However,

results of these studies have provided mixed support for the hypothesis, and suggest that whether

impairment of working memory by NMDA antagonist is observed depends on the nature of the

task, task parameters, and the particular compound employed (Bannerman, Rawlins, & Good,

2006; Dix et al., 2010; Gastambide et al., 2013; Gilmour et al., 2009; Smith et al., 2011). One of

the more promising tasks is the Odor Span Task (OST), which was nominated as a benchmark

procedure to model working memory capacity in rodents by the CNTRICS group --Cognitive

Neuroscience Treatment Research to Improve Cognition in Schizophrenia—(Dudchenko,

Talpos, Young & Baxter, 2013).

The OST was originally developed as a way to study the neurobiology of working

memory for multiple stimuli by Dudchenko, Wood & Eichenbaum (2000). The procedure uses

an incrementing non-matching to sample task in which rats are presented with odors in a large

arena; each odor serves as a stimulus indicating reinforcer availability the first time it is

presented, but not during following presentations. For example, in the first trial, one odor is

presented (a cup with scented sand or covered with a scented lid) in the arena and digging in the

sand or lid removal produces a food reinforcer. In the second trial, two stimuli are presented, the

odor from the first trial along with a novel odor. Now the odor from Trial 1 serves as the sample

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stimulus, as such, a response to the novel odor results in reinforcement. With each subsequent

trial, a new odor is presented along with comparison stimuli from any of the previous trials and

selection of the new odor is always reinforced. Thus the number of sample stimuli to remember

increases across the session. The number of correct responses until the first error (span) is one

measure purported to reflect memory capacity in the OST and percent correct plotted as a

function of the number of odors to remember is another (Dudchenko et al., 2000; Galizio, Deal,

Hawkey & April, 2013).

There is a growing literature using the OST to analyze drug effects which is beginning to

identify the pharmacological determinants of performance in this task. Guided by the NMDA

hypofunction hypothesis of cognitive impairment in schizophrenia, much of the early work on

the behavioral pharmacology of the OST focused on NMDA antagonists. As that hypothesis

would predict, several studies have shown that these compounds impair OST performances in

rats. For example, non-competitive NMDA antagonist MK-801 impaired span length and overall

OST accuracy at doses that had no effect on a simple discrimination task that controlled for drug

effects on sensori-motor function, motivation and reference memory (Galizio, et al., 2013;

MacQueen, Bullard & Galizio, 2011). Competitive NMDA antagonist CPP produced effects that

were similarly selective to OST performance (Davies, Greba & Howland, 2013; MacQueen,

Dalrymple, Drobes & Diamond, 2016). Importantly the effects of MK-801 were shown to

depend on the number of stimuli to remember. That is, effects were minimal when the memory

load was low, but impairment of accuracy was accelerated as the number of odors to remember

increased during the session (Galizio et al., 2013; MacQueen, et al., 2011).

Other amnestic drugs have been studied in the OST, but have produced less clear

evidence of working memory impairment. Rushforth, Allison, Wonnacott, and Shoaib (2010)

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found that scopolamine decreased span length in rats at doses that did not increase latency to

respond. However, in a replication of that study that included the discrimination control

condition, scopolamine effects occurred only at doses that also impaired control performances

(Galizio et al., 2013). Benzodiazepines have produced selective impairments on OST

performances but the effects were quite different from those produced by NMDA antagonists.

For example, the effects of chlordiazepoxide were relatively weak and limited to span length

(Galizio et al., 2013). In contrast, flunitrazepam impaired OST accuracy at doses that spared

control performances, but these effects did not depend on the number of stimuli to remember

(Galizio, et al., in review). Rather, the effects of flunitrazepam appeared to involve a breakdown

of the overall non-matching task. Thus, at this point only NMDA antagonists have been shown

to impair working memory in the OST.

Although most of the current literature implicates NMDA receptor activity as a key

modulator of OST performance, a recent study found contrary results. Galizio et al. (2016)

found that the effects of the NMDA antagonist ketamine affected OST performances only at

doses that also impaired simple discrimination in 5/6 subjects. In most rats, effective doses of

ketamine produced a cessation of responding on both OST and simple discrimination tasks. The

discrepancy between findings of selective effects of MK-801 and CPP compared with the non-

selective effects of ketamine is puzzling, particularly as both MK-801 and ketamine share a

common mechanism as open channel blockers (Lodge & Mercier, 2015). Ketamine is less potent

and is a lower affinity channel blocker than MK-801 (Lodge & Mercier, 2015; Monaghan,

Irvine, Costa, Fang & Jane, 2012) which might account for the differences in their effects on

OST performance. However, a number of studies have also observed differences between the

behavioral effects of various uncompetitive NMDA antagonists in a variety of tasks (Dix et al.,

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2010; Gastambide et al., 2013, Gilmour, et al., 2009; Smith et al., 2011) and it seems important

to determine the effects of different compounds in the OST.

Thus, one purpose of the present study was to extend the analysis of NMDA antagonists

on OST performance to two additional compounds: phencyclidine (PCP) and methoxetamine

(MXE). PCP is a potent channel blocker that produces schizophrenic-like symptoms in humans

and also was one of the key compounds that originally led to the NMDA hypofunction

hypothesis of schizophrenia (Merritt et al., 2013). MXE is a designer drug that has recently

emerged as an abuse problem in the United States and Europe in recent years (Zanda, Fadda,

Chiamulera, Fratta, & Fattore, 2016). Although MXE research is in early stages, it does appear to

act as an uncompetitive NMDA channel blocker (Roth et al., 2013) and substitutes for ketamine

in drug self-administration and drug discrimination procedures (Mutti et al., 2016; Chiamulera,

Armani, Mutti, & Fattore, 2016). No research has yet assessed the effects of MXE using

working memory tasks.

A second purpose of the study was to replicate the effects of ketamine in the OST using a

more refined variation of the task. In the original version of the OST, the number of comparison

stimuli in the arena incremented along with the number of stimuli to remember (Dudchenko et

al., 2000). As this procedure results in a potential confound, our laboratory adjusted the task to

hold the number of comparison stimuli constant at five while continuing to increase the memory

load (Galizio et al., 2013; 2016; MacQueen et al., 2011). However, the presence of even five

different comparison stimuli in the arena might reduce the sensitivity of the task to working

memory effects by increasing the discrimination difficulty and the need to inhibit responding.

Perhaps potential amnestic effects of ketamine in the Galizio et al., (2016) study were masked by

other actions that led to disrupted performance due to task complexity. In order to test this

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hypothesis, the present version of the OST used a two-choice procedure in which each trial (after

the first) presents one new odor (reinforced--S+) and one previously presented negative

comparison (S-). MacQueen et al. (2016) used a two-choice OST and found it sensitive to the

effects of CPP; our hypothesis was that a two-choice OST would show higher sensitivity to

amnestic ketamine effects than the five-choice procedure.

Methods

Subjects

Sixteen male Sprague-Dawley rats between 90 and 150 days old at the onset of the study

served as subjects. Rats were housed individually in Plexiglas cages measuring 34.5 cm L x 18

cm W x 24.5 cm H and were maintained on a 12:12 reverse dark/light schedule with artificial

lighting initiated at 7 P.M. and turned off at 7 A.M. Subjects were allowed free access to water

in the home cage, but food (Purina Lab Diet 5001 Rodent Diet--PMI Nutrition International, INC

Brentwood, MO) was restricted to maintain 85% of free feeding weight. Animal care as well as

all aspects of the procedures were approved by the UNC Wilmington IACUC.

Apparatus

A circular open-field arena (94 cm in diameter) surrounded by 32 cm high metal baffling

was used for this experiment. The floor of the arena included 18 holes, spaced 13.3 cm apart in

two circular arrangements in which 60 ml plastic cups were placed (see Figure 1). Each arena

was located in a small testing room with a video camera positioned in the ceiling for digital

recording of each session. White noise (70 dB) was presented for the duration of the session.

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Stimuli

Plastic cups half full of sand were covered with plastic opaque scented lids. The lids were

scented with various odorants. These lids were odorized via storage in plastic containers

containing aromatic oils and spices as odorants (acai, allspice, anise, apple, banana, bay, black

walnut, bubblegum, caramel, caraway, carob, celery, cherry, chocolate, cinnamon, clove,

coconut, coriander, cumin, dill, fennel, fenugreek, garlic, ginger, honey, lilac, maple, marjoram,

marshmallow, mustard, nutmeg, onion, oregano, pecan, pine forest, pumpkin, root beer,

rosemary, sage, savory, spinach, sumac, thyme, watermelon) purchased from Nature’s Garden,

McCormick, Sigma Aldrich, and Great American Spice Co. Lids were suspended above oils or

spices during storage, rather than directly in the odorants, so that the concentration of odorant

was relatively consistent across lids and were stored at least 3 weeks prior to initial use.

Containers of spices and oils were refreshed every three to four weeks. To control for scent

marking on lids within session, each lid was replaced with one of the same odor every time the

scent was presented during a session.

Procedure

OST Training. Sessions were conducted 5 days per week (Monday-Friday). Subjects

were first habituated to the apparatus, and hand shaped to remove a lid from a baited stimulus

cup. Rats were then trained on an incrementing non-match-to-sample procedure. In this

procedure, a novel odorant was presented during each trial along with odorants from previous

trials. Removal of the novel scented lid resulted in food reinforcement, while removal of any

previously presented lid did not. Initially, the number of cups in the arena incremented on each

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trial, and errors (removal of a previously presented scented lid) resulted in a reset—that is, on the

following trial only one scented lid was present. After an average of 11 days, the baseline OST

schedule was implemented.

Following initial OST training, rats began training on the 2-choice OST baseline

procedure. Two features of baseline OST training were different from that of initial training.

First, errors no longer resulted in resetting the stimulus arrangement to a single stimulus. Second,

following Trial 2 the number of comparison stimuli in the arena was held constant at two (See

Fig. 1): a new baited odor each trial (+) and an unbaited odor (-) which was randomly selected

from any of the previously presented odors. Figure 1 illustrates four typical OST trials (OST1-

OST4). During the first trial (OST1) only one cup is presented, covered by a lid scented with

odor A. Upon removal of lid A (A+), a sugar pellet, which had been covered by the lid, is

available as food reinforcement (signified by the + symbol). In the second trial (OST2), two

odors are presented: scented lids A and B; as odor A has been previously presented, there is no

sugar pellet available under lid A (signified by the – symbol). Odor B is novel in this trial, thus

removal of lid B will provide access to a sugar pellet. Trial 3 (OST3) contains odors B and C,

and as B has been previously presented, removal of lid B will not produce a sugar pellet. These

three trials each use the previous positive (+) stimulus as the negative (-) stimulus in the

successive trial, however, any previously presented odor can serve as a negative stimulus. This is

illustrated in trial 4 (OST4), where odor A is presented again as a negative stimulus, with the

novel odor D. Figure 1 also contains an example of two simple discrimination control (SDC)

trials, SDC1 & SDC2, which were added once subjects met a mastery criterion: two consecutive

sessions with accuracy at 72% or greater.

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Combined OST/SDC Baseline. SDC trials (See SDC1 & SDC2; Fig. 1) consisted of a

pair of scented lids. Unlike the OST, in SDC trials the same odor (X+) was consistently baited

across all SDC trials and sessions, while the alternative odor (Y-) was never baited. This

consistency is demonstrated in figure 1: X is presented with the + symbol and Y with the –

symbol in both SDC1 & SDC2. Although the pair of odors (X & Y) differed between subjects,

each pair remained constant for each subject (see Table 1) once SDC trials were introduced, and

these odors were never presented as choices during OST trials. As task demands were similar

between the OST and SDC task, save for the increase in negative comparison stimuli (number of

stimuli to remember) in the OST, the SDC served to control for drug effects on reference

memory as well as non-mnemonic drug effects.

Initially, six SDC trials were presented following 25 OST trials. After rats responded

with 100% accuracy to the 6 SDC trials while maintaining at least 72% accuracy in the OST

during one session, the SDC trials were pseudo-randomly interspersed throughout each session

during the following sessions (As shown in Fig. 1). SDC trials were spread across the session so

that the varying effects of drugs on performance over time could be captured. Although

interspersing the SDC trials among OST trials was expected to initially cause some disruption of

the odor span task, after a few sessions subjects readily learned to discriminate between odors

signaling the SDC task versus the OST and respond accordingly.

Two additional control procedures were applied when the OST and SDC were combined.

First, a “no-bait” control procedure was added in six pseudo-randomly chosen OST trials per

session, one session per week. In these trials, neither stimulus cup contained a reinforcer, but a

sucrose pellet was placed in the S+ cup immediately following a correct response. This control

procedure allowed for verification that the odor of the lid, rather than the sucrose pellet beneath,

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controlled responding. Second, one session per week included a fresh cup control procedure.

This procedure consisted of four pseudo-randomly chosen OST trials in which the S- cup was

replaced with a cup that had not been used previously in the session. This control procedure

allowed the experimenters to examine whether trace odors from the stimulus cup, rather than the

scented lid, affected OST accuracy. Table 1 details mean percent correct across baseline, no bait,

and fresh cup trials for each rat. Several subjects (M22, M5, K10, L7, & L24) did not receive the

fresh cup procedure as it was implemented later in the experiment. Accurate responding was

maintained during fresh cup and non-baited sessions. This indicates that neither the scent of the

sugar pellet nor the scent of the cup was likely responsible for accurate responding in the OST.

The combined OST and SDC tasks constituted the drug baseline. After meeting a stability

criterion for OST accuracy, which required that the mean difference in accuracy between the

most recent five sessions and the five preceding sessions was less than 10% of the grand mean of

those ten sessions, subjects began to receive injections. The number of training sessions required

ranged from 37 to 97 with an average of 54.7 sessions (Table 1). Following this extensive

training, rats maintained stable and high levels of accuracy on both the OST and SDC task.

Drugs

All drugs were dissolved in .09% saline solution and delivered in a volume of 1ml/kg.

PCP HCl (a generous gift from the NIDA drug supply program, RTP) was administered in doses

of 1.0, 3.0, 5.6, and 10.0 mg/kg and Ketamine HCl (Sigma) in doses of 1.0, 3.0, 5.6, 10.0, 18.0,

30.0, and 40.0 mg/kg. MXE HCl (Debora Labs, Copenhagen) was administered in doses of 1.0,

3.0, 10.0, 18.0, 30.0 and 40.0 mg/kg. Because the provenance of MXE was uncertain, the

structure was verified using 1- and 2- dimensional Nuclear Magnetic Resonance (NMR)

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spectroscopy, High Resolution Mass Spectrometry (HRMS), and comparison with the data in the

current literature. NMR and LC/MS found no significant organic impurity.

Drug Protocol

Intraperitoneal injections were administered 15 minutes prior to session start three days

per week. Saline was delivered Thursdays as an injection control, while active drug was

administered on Tuesdays and Fridays. Experimenters remained blind to the dose administered.

No injections were given Mondays or Wednesdays, which served as a continuation of the

baseline. Two or three determinations of a dose were given for each rat; each dose in a set of

determinations was administered prior to the second round of doses. Some subjects received

more than one drug, either in this study or in an experiment not described here; for these animals

a two-week washout period intervened before the next drug study began.

Data Analysis

Accuracy was determined by the first response in each trial for both OST and SDC trials.

If a subject failed to respond within 2 minutes, the trial was scored as an omission; omitted trials

were not included in the percent correct calculation. Omitted trials were repeated with

presentation of only the positive stimulus, and sessions were terminated following either 3

consecutive or 6 total omissions. Consecutive correct measures included span and longest run.

Span was defined by number of correct responses made until the first error in the session,

excluding Trial 1 as there was no comparison stimulus. Longest run was determined as the

greatest number of correct consecutive responses in the session. This measure provided more

information then span, as a rat could make an error on Trial 2 resulting in a span of 1. However,

the rat could then respond correctly on trials 3-20, resulting in a longest run of 17. Latencies for

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each trial were measured in seconds from the placement of the rat in the arena until a response

was made.

Video recordings of ten randomly selected sessions were scored by a blind observer to

determine inter-rater reliability (IRR). IRR was calculated by comparing scores of the observer

with scores of the experimenter from the chosen session. The two raters agreed on 98% of the

chosen trials, which indicated a high reliability of the operational definition of the lid removal

response.

Results

Mean effects of PCP on the main dependent variables are displayed in Figure 2. The top

panel shows mean percent correct as a function of dose for the OST (closed circles) and SDC

task (open circles). Under control conditions (saline), accuracy for the OST was around 90%,

while accuracy for the SDC task was near 100%. PCP produced dose-dependent impairment in

accuracy on both tasks. At the 3.0 mg/kg dose, OST accuracy was slightly decreased and then

more clearly impaired at the 5.6 mg/kg dose. In contrast, at both of these doses, accuracy on the

SDC task was relatively unaffected, remaining above 90%. Thus, the effects of the 5.6 mg/kg

dose were selective to the OST. Percent omissions is indicated by the bars on the right axis of the

graph, representing trials in which a failure to respond to either stimulus within two minutes

occurred. At the 5.6 mg/kg dose omissions began to occur for 3 rats, while at the 10.0 mg/kg

dose behavior was grossly affected across all rats, resulting in over 70% omissions. Percent

correct data were analyzed statistically by a within-subject ANOVA with dose (4 levels—the 10

mg dose was omitted from the analysis due to the high number of omissions) and task (OST vs.

SDC) as the main variables. The effects of PCP were verified by a significant dose x task

interaction: F (3, 15)=6.95, p<.05, with main effects of dose [F(3,15)=5.16, p<.05] and task

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[F(3,15)=23.50, p<.05]. Fisher’s LSD tests revealed that the 5.6 mg/kg dose significantly

reduced OST accuracy from saline levels (p<.05) and but that SDC accuracy below SDC

accuracy (p<.05). Thus, PCP produced effects that were selective to OST performances at this

dose.

Under control conditions, mean spans (closed circles) ranged from 7-11 odors, while

longest runs (open circles) were higher at 12-15 consecutively correct responses (Fig. 2, middle

panel). Span and longest run were each statistically analyzed using one-way within-subject

ANOVAs. Both span [F (4, 20)= 5.92, p<.05] and longest run [F (4, 20)= 21.70, p<.05] were

dose-dependently decreased by PCP administration. Fisher’s LSD tests revealed span was

significantly lower at the 10.0mg/kg dose compared to saline (p<.05) and longest run was

significantly below saline levels at the 5.6 and 10.0 mg/kg dose (p<.05). Latencies to respond to

(Fig. 2, bottom panel) were analyzed statistically using within-subject ANOVAs with dose (4

levels) and task (OST vs. SDC) as the main variables. A main effect of dose was found [F (3,

15)=7.01, p<.05], but no main effect of task [F (3, 15)=1.08, p>.05], nor interaction [F (3,

15)=0.73, p>.05]. Fisher’s LSD tests indicate latencies to respond to the OST were significantly

increased at the 5.6 mg/kg dose (p<.05).

To further evaluate the selective effects of PCP, individual subject data were analyzed

(Figure 3). In these graphs mean percent correct for the OST (closed circles) and SDC task (open

circles) for each rat is plotted as a function of PCP dose. Individual drug determinations are also

plotted for the OST (stars) and SDC task (triangles). Of the six rats, P17 and N33 (bottom right)

show the most striking selective effects of PCP on OST percent correct. Both rats clearly show

selectivity at the 5.6 mg/kg dose, while P17 also shows this effect at 3.0 mg/kg. The graphs for

M5, N11, and P25 (left panels) also indicated selective effects at the 5.6 mg/kg dose, although

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there was some inter-determination variability. M10 (top right panel) was the only subject in

which no selective effects were evident. In summer, PCP selectively decreased OST accuracy as

well as span and longest run measures at the 5.6 mg/kg dose. This dose had no effect on SDC

accuracy, but did cause omissions and increased latency in some instances, while the 10.0 mg/kg

dose virtually eliminated responding in all subjects. Apart from M10, the individual subject data

support the selectivity evident in the group data.

The effects of MXE on the main dependent variables are shown in Figure 4. The top

panel shows mean percent correct as a function of dose for the OST (closed circles) and SDC

task (open circles). Under the saline control condition, accuracies were around 85% for the OST

and 95% for the SDC task. MXE dose-dependently impaired accuracy on both tasks, however

this did not occur until the 18.0 and 30.0 mg/kg doses. The data were analyzed statistically by a

within-subject ANOVA with dose (6 levels) and task (OST vs. SDC) as the main variables. A

main effect of dose was found [F (5, 25)= 5.93, p<.05]. There was not a significant main effect

for task [F (5, 25)= 3.27, p>.05], nor a significant interaction [F (5, 25)= 0.4, p>.05] Fisher’s

LSD tests revealed a significant overall difference between saline and the 30.0 mg/kg dose. The

high doses disrupted responding in both tasks, thus the effect appears non-selective. Percent

omissions (bars on right side) were also increased to around 40% at the 18.0 and 30.0 mg/kg

doses.

Mean spans (closed circles) ranged from 6-10 odors, while longest runs (open circles)

were higher at 11-16 consecutively correct responses under control conditions (Fig. 4, middle

panel). Span and longest run were individually analyzed using one-way within-subject

ANOVAs. Both span [F (5, 25)= 3.30, p<.05] and longest run [F (5, 25)= 11.30, p<.05] were

significantly decreased by MXE administration dose-dependently. Fisher’s LSD tests show span

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and longest run measures were both significantly below saline levels only at 30.0 mg/kg (p<.05).

Latencies to respond to both tasks (Fig. 4, bottom panel) were analyzed statistically using within-

subject ANOVAs with dose (6 levels) and task (OST vs. SDC) as the main variables. A

significant main effect of dose was found [F (5, 25)= 3.68, p<.05], but no main effect of task [F

(5, 25)= 0.18, p>.05] or interaction [F (5, 25)= 0.40, p>.05]. Latencies increased at 18.0 and 30.0

mg/kg high doses, however this effect was not statistically significant. To summarize, the group

data does not show a selective effect of MXE, as responding on both tasks were affected

similarly, and impairment only occurred at doses in which high rates of omissions were

observed.

The individual subject data for the rats administered MXE (Fig. 5) provide a somewhat

more complex picture of the effects of MXE on responding. Three subjects showed a clear

selective effect of MXE on OST percent correct: subjects O7, P3, and M22 (right three panels).

Subject O7 (top left panel) was unaffected by the 1.0, 3.0 and 10.0 mg/kg doses of MXE.

However, at 18.0 mg/kg dose accuracy declined on the OST while remaining at 100% for the

SDC task. P3 showed a decrease in OST accuracy across the 10.0, 18.0 and 30.0 mg/kg doses,

while accuracy on the SDC task was unaffected. Rat M22’s percent correct was decreased under

both the 18.0 and 30.0 mg/kg doses, while responding on the SDC task remained at 100%

accuracy. Two additional rats (P24 and P26) showed trends towards selective effects. In the case

of Rat P24, a small decrease in OST, but not SDC, accuracy was observed at the 10 mg/kg dose,

although there was variability across determinations. Rat P26 showed a consistent drop in OST

accuracy at the 10 and 18 mg/kg doses, but SDC was also impaired on some determinations at

these doses. Finally, Rat P18 (top left panel) showed no effects of MXE until the 18.0 and 30.0

mg/kg doses which resulted in complete omissions.

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In sum, MXE disrupted accuracies on the OST and SDC and reduced spans and longest

runs. Although the group data analysis failed to reveal evidence of selective effects on the OST,

the individual subject data for the MXE group provided some evidence of selectively in five of

the six rats. The doses at which responding was disrupted and omissions were noted were

variable across the six subjects in the MXE study. This differential sensitivity to MXE effects

may explain why the mean data appeared to be non-selective whereas individual rats in fact did

show selective effects at some doses.

The effects of KET on the main dependent variables are shown in Figure 6. The top panel

contains mean percent correct as a function of dose for the OST (closed circles) and SDC task

(open circles). Under the saline control condition, accuracies were around 90% for the OST and

95% for the SDC task. KET only impaired accuracy at the 30.0 mg/kg dose, a dose at which

increases in omissions also occurred. A within-subject ANOVA with dose (6 levels) and task

(OST vs. SDC) as the main variables was used to statistically analyze the percent correct data. A

main effect of dose was found [F (5, 25)= 6.34, p<.05]. There was no significant main effect for

task [F (5, 25)= 2.78, p>.05], nor significant interaction [F (5, 25)= 0.56 , p>.05]. The 30.0

mg/kg dose disrupted responding in both tasks, thus the effect appears non-selective. Percent

omissions (bars on right side) increased to near 40% at the 30.0 mg/kg dose.

Mean spans (closed circles) ranged from 8-16 odors, while longest runs (open circles)

were higher at 14-19 consecutively correct responses under control conditions (Fig. 6, middle

panel). Span and longest run were individually analyzed using one-way within-subject

ANOVAs. KET administration dose-dependently decreased span [F (5, 25)= 6.34, p<.05] and

longest run [F (5, 25)= 5.36, p<.05] significantly. Fisher’s LSD tests show span was significantly

below saline levels only at 30.0 mg/kg (p<.05). Response latencies (Fig. 6, bottom panel) were

19

analyzed statistically using within-subject ANOVAs with dose (6 levels) and task (OST vs.

SDC) as the main variables, but no significant effects were obtained ( p>.05). In sum, the group

data does not show a selective effect of KET, as accuracy on both tasks was maintained across

doses until the 30 mg/kg dose, at which high rates of omissions were observed in both OST and

SDC tasks.

Individual subject data for the KET group (Fig. 7) generally support the mean data. All

subjects maintained SDC accuracy similar to saline levels, and most subjects did not show

declines in OST accuracy below saline levels at any KET dose. Four animals (M5, L24, K10,

L7) were nearly unaffected by ketamine until doses were reached which caused substantial

omissions. However, one rat (P17) did show a clearly selective effect with a reliable drop in

OST, but not SDC, accuracy at the 30.0 mg/kg dose. In sum, effects of KET were generally

limited to doses that disrupted overall responding with the selective effects in Rat P17 standing

as the exception.

Although PCP selectively impaired accuracy on the OST, a within-session analysis is

needed to determine whether the effects of PCP depended on the number of odors to remember.

Figure 8 shows such an analysis comparing the selective dose (5.6 mg/kg) of PCP (open

triangles) to the saline control (closed triangles) across the OST (3 bins of 8 trials--left half of x-

axis) and the SDC trials (3 bins of 2 trials--right half of x-axis) for the five animals that showed

selective effects. Note that accuracy decreased only slightly across the session under saline

conditions. Indeed, during the final 8 trials with 17-24 odors to remember, OST accuracy

remained above 80% after saline. However, there was a sharp drop in OST accuracy as the

number of stimuli to remember increased under PCP. Accuracy was only slightly lower than

control in the first bin (1-8 odors), but dropped to about 60% correct when the memory load was

20

9-24 stimuli. ANOVA with drug (2 levels: Saline vs. PCP) and bin (1 vs. 2 vs. 3) as the main

variables confirmed these conclusions with a significant Dose X bin interaction [F (1,4)=7.10,

p<.05]. Note that accuracy on SDC trials remained high throughout the session with or without

PCP.

Within-session analysis for MXE was complicated by the individual differences in

sensitivity. Due to this variability, MXE within-session data (Fig. 9) were analyzed using a best

dose analysis which compared the means of the most selective dose for the five rats that showed

selective effects (open triangles) with saline (closed triangles) across the session. The doses used

were 10.0 (P24 & P26), 18.0 (O7), and 30.0 mg/kg (M22 & P3). The results were similar to PCP

in that there was very little decline in accuracy across the session under saline conditions, and

MXE impairment of accuracy was most evident when the memory load was fairly high (9-24

stimuli). Factorial ANOVA revealed a significant interaction of drug and bins of trials [F (1,4)=

8.73, p<.05). Accuracy on the SDC trials remained relatively high across the session.

Discussion

All three NMDA antagonists reduced span length, longest run and accuracy in a dose-

dependent fashion, but the three compounds differed in the extent to which the effects were

selective to the OST. At high doses, all three drugs disrupted SDC as well as OST responding

with frequent response omissions. At moderate doses, both PCP and MXE produced effects that

were selective to the OST and were consistent with an interpretation in terms of impaired within-

session or working memory. Ketamine effects were much less selective to the OST, and appear

to have been based on non-amnestic disruption of performance in most rats tested.

21

PCP produced the most selective effects as OST accuracy and longest run were

significantly decreased (and decreases in span length approached significance) at the 5.6 mg/kg

dose which had no effect on SDC accuracy. Three of the animals did show an increase in

omissions at this dose and response latency also increased, suggested that some general task

disruption was occurring. That said, the observed change in OST accuracy approached a 15%

decline while SDC performance remained nearly perfect at the 5.6 mg/kg dose suggesting that

the PCP effects were selective to the OST at this dose. This observation was corroborated by the

within-session analysis of PCP effects, which revealed that when memory load was 1-8 stimuli,

5.6 mg/kg PCP had little effect on accuracy compared to saline. However, as the number of

stimuli to remember increased from 9-23 stimuli, accuracy declined sharply under 5.6 mg/kg

PCP, but only a slight decline was observed in the saline conditions. In short, the effects of PCP

depended on the number of stimuli to remember.

Overall, MXE effects were similar to those of PCP, but individual rats varied

considerably in their sensitivity to the drug. Some animals showed selective effects of MXE at

18 mg/kg (O7, P3) or 30 mg/kg (M22) while both OST and SDC performance was disrupted in

the other three rats at those doses. A best-dose analysis used to detect within session effects

produced a function resembling that for PCP with very little MXE effect when the memory load

was 1-8 stimuli, but substantial impairment relative to saline as the number of stimuli to

remember increased throughout the session.

Finally, ketamine effects were somewhat different than the other compounds as it

produced very little effect on accuracy of either OST or SDC responding until high doses were

reached that disrupted both tasks and increased omissions in most rats. One rat (P17) did show a

selective effect with reduced OST but not SDC accuracy at the 30 mg/kg dose, but overall

22

ketamine effects were clearly less selective than MXE or PCP. These findings replicate those of

a previous study from our laboratory, which used a five-choice OST (Galizio et al., 2016). In that

study, as in the present one, only one out of six rats showed a selective effect of ketamine on the

OST. Thus, the limited selectivity of ketamine on OST responding in Galizio et al. (2016) was

not related to the number of comparison stimuli—rather ketamine appears to produce relatively

non-selective effects with the both the five-choice and the present two-choice versions of the

OST.

The present study was the first to determine the effects of PCP and MXE using the OST.

The effects of PCP on OST observed in the present study closely resembled previous results with

NMDA antagonists MK-801 and CPP (Davies, et al., 2013; Galizio, et al., 2013; MacQueen et

al., 2011; 2016). In those studies NMDA antagonists selectively impaired OST accuracy at

doses that did not affect other aspects of performance (e.g., SDC accuracy). One qualification

with respect to PCP is that although the 5.6 mg/kg dose reliably produced selective impairment

of OST accuracy, this dose also resulted in some global impairment (increased omissions and

response latency) in several animals. Still an interpretation of PCP disruption of OST accuracy in

terms of amnestic effects seems appropriate. Interpretation of the MXE results is more complex.

If the group mean data alone were considered, the conclusion would be the MXE did not produce

selective effects; only when individual subject dose-response functions were analyzed was

evidence of selective actions observed. Group statistical analysis using a best-dose procedure did

show selectivity and supported the observation that MXE effects depended on the number of

stimuli to remember. One caveat here is that best-dose analyses are potentially biased in the

direction of showing positive effects (Soto, Dallery, Ator, & Katz, 2013). Very little research has

23

been conducted with MXE and further studies will be needed to determine the extent to which

MXE selectively impairs within-session/working memory.

The present study provided mixed support for the hypothesis that NMDA hypofunction is

associated with reduced memory capacity. On the one hand, the selective effects and memory-

load dependent effects of PCP and MXE obtained in the present study were consistent. These

findings, combined with those of previous studies with MK-801 and CPP on OST performances,

provide strong support for the hypothesis. However, the relatively non-selective effects of

ketamine were inconsistent. Why would ketamine result in such different effects than other

NMDA antagonists? As noted previously, other researchers have observed differences between

various NMDA antagonists in a variety of behavioral tasks, and it is unclear whether these may

reflect pharmacokinetic or pharmacodynamic differences across compounds (Dix et al., 2010;

Gastambide et al., 2009; 2013, Smith et al., 2011). Using a somewhat different procedure—the

self-ordered spatial search task—to assess memory capacity, Taffe, Davis, Gutierrez & Gold

(2002) found that ketamine did produce selective impairments in rhesus monkeys that depended

on the number of stimuli to remember. Thus, it is possible that a species difference in sensitivity

to ketamine may be involved. It has also been noted that binding site affinity varies across

NMDA antagonists. Ranking the uncompetitive antagonists in terms of affinity, MK-801 > PCP

> MXE > S-(+)-KET (Wong et al., 1986; Halberstadt, Slepak, Hyun, Buell, & Powell, 2016).

This hierarchy might account for the findings of ketamine’s limited selectively on OST

performance. Perhaps some affinity threshold is required to produce selective effects on working

memory in the OST. Studies with additional compounds (e.g., lower affinity compounds such as

memantine) are needed to test such a hypothesis.

24

Finally, it should be added that although results from the OST are generally interpreted in

terms of within-session or working memory, the translational significance of such interpretations

remain to be determined. Certainly numerous studies, including the present one, have shown

functional differences between the within-session remembering involved in the OST relative to

reference memory tasks like the SDC. However, the relationship between the within-session

memory of the OST and human working memory tasks remains puzzling. Consider that human

memory span is highly limited (Gathercole, 2009). In contrast, very little decline in accuracy was

observed in the present study even as the memory load reached 16-24 odors. Indeed, the upper

limit on rat’s capacity to remember odors has not been reached with previous OST studies

showing above chance memory for 70-100 different odors (April, Bruce, & Galizio, 2013;

Bratch et al., 2016). It has been suggested that performance in the OST is best conceptualized as

a form of short-term episodic-like memory (Branch et al., 2014; Panoz-Brown, Corbin, Dalecki

et al, 2016) and if this hypothesis is confirmed, it would have implications for the interpretation

of the effects of NMDA antagonists as well.

25

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Table 1

Training, Performance Under Baseline and Control Conditions and SDC Odorants

Rat

Number of Sessions Percent Correct

OST OST/SDC Total Baseline No Bait

FreshCup Drug SDC odors (+,-)

O7 7 65 72 82.33% 93.00% 94.00% MXE Pumpkin, Honey

M22 10 27 37 96.89% 88.98% --- MXEMarshmallow, Pine Forest

P3 11 86 97 81.76% 84.00% 83.33% MXEWatermelon, Black Walnut

P18 13 24 37 78.00% 81.00% 67.00% MXE Maple, Acai

P24 7 36 43 82.00% 86.00% 89.00% MXE Caramel, Apple

P26 20 33 53 79.81% 87.00% 77.00% MXE Banana, Root beer

M10 10 54 64 92.00% 100.00% 87.50% PCP Lilac, Honeydew

N11 7 81 88 87.38% 89.00% 86.00% PCP Pecan, Pine Forest

N33 7 30 37 85.20% 90.00% 100.00% PCPMarshmallow, Pine Forest

P25 9 30 39 87.73% 88.00% 90.00% PCP Pumpkin, Honey

P17 15 31 46 79.14% 74.00% 78.00%PCP, KET Pecan, Pine Forest

M5 10 33 43 88.33% 90.00% ---PCP,KET

Chocolate, Bubblegum

K10 11 56 67 86.15% 74.35% --- KETBlack Walnut, Champagne

L7 11 24 35 93.00% 91.67% --- KET Coconut, Lavender

L24 13 28 41 89.00% 89.28% --- KET Rum, Maple

N36 16 27 43 92.00% 94.74% 81.58% KET Apple, Banana

31

Figure 1. Examples of OST and SDC trials. Each large circle represents an individual trial;

OST1-OST4 represent OST trials 1-4 while SDC1 and SDC2 represent interspersed SDC trials.

The small circles represent 18 possible stimulus locations in the apparatus. A letter represents a

specific odor in a location; letters A, B, C, D, X, & Y indicate different odor stimuli (e.g. A=

apple, B= banana). A plus sign next to a letter (e.g. A+) indicates that selection of that stimulus

(A) would produce a sugar pellet. A minus (e.g. B-) indicates that no pellet is available for

selection of that odor (B). Notice that possible negative stimuli increase as OST trials increment:

1 possible negative (A) for OST2, 2 (A, B) for OST3, 3 (A, B, C) for OST4, and so on. During all

SDC trials selection of X (X+) always produced a sugar pellet, while a selection of Y (Y-) did

not.

32

Figure 2. Means for percent correct, consecutive correct, and latency for the PCP group. Top

panel shows the effects of PCP on percent correct (circles) and omissions (bars) for the OST

(closed circles, dark bars) and SDC task (open circles, light bars). Middle panel shows span

(closed circles) and longest run (open circles). Bottom panel shows latency to first response for

OST (closed circles) and SDC task (open circles). Vertical lines indicate SEM.

33

Figure 3. Individual subject graphs show the effects of PCP on percent correct (circles) and

omissions (bars) for the OST (closed circles, dark bars) and SDC task (open circles, light bars).

Individual determinations are plotted for OST (open stars) and SDC task (open triangles).

Vertical lines indicate SEM.

34

Figure 4. Means for percent correct, consecutive correct, and latency for the MXE group. Top

panel shows the effects of MXE on percent correct (circles) and omissions (bars) for the OST

(closed circles, dark bars) and SDC task (open circles, light bars). Middle panel shows span

(closed circles) and longest run (open circles). Bottom panel shows latency to first response for

OST (closed circles) and SDC task (open circles). Vertical lines indicate SEM.

35

Figure 5. Individual subject graphs show the effects of MXE on percent correct (circles) and

omissions (bars) for the OST (closed circles, dark bars) and SDC task (open circles, light bars).

Individual determinations are plotted for OST (open stars) and SDC task (open triangles).

Vertical lines indicate SEM.

36

Figure 6. Means for percent correct, consecutive correct, and latency for the KET group. Top

panel shows the effects of KET on percent correct (circles) and omissions (bars) for the OST

(closed circles, dark bars) and SDC task (open circles, light bars). Middle panel shows span

(closed circles) and longest run (open circles). Bottom panel shows latency to first response for

OST (closed circles) and SDC task (open circles). Vertical lines indicate SEM.

37

Figure 7. Individual subject graphs show the effects of KET on percent correct (circles) and

omissions (bars) for the OST (closed circles, dark bars) and SDC task (open circles, light bars).

Individual determinations are plotted for OST (open stars) and SDC task (open triangles).

Vertical lines indicate SEM.

38

39

Figure 8. Within session graphs show the effects of 5.6 mg/kg PCP (open triangles) and Saline

(closed triangles) on OST and SDC percent correct across the session in bins of 8 for the OST

and 2 for the SDC. Vertical lines indicate SEM. Note that N = 5—P5 not included in this figure.

40

SDC Trial

Figure 9. Group within session graphs show the effects of the selective dose of MXE for each rat

(open triangles) and Saline (closed triangles) on OST percent correct across the session (bins of 8

trials) and the 6 SDC trials (bins of 2). Vertical lines indicate SEM. Note that N = 5—P18 not

included in this figure.

41