EFFECTS OF OLANZAPINE ON OLFACTORY DELAYED MATCHING-TO-SAMPLE IN

RATS

Timothy W. Lefever

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

Julian Keith Ray Pitts

Kate Bruce Mark Galizio

Chair

Accepted By

Dean, Graduate School

ii

TABLE OF CONTENTS

ABSTRACT .................................................................................................................. iii

LIST OF TABLES ......................................................................................................... iv

LIST OF FIGURES ........................................................................................................ v

INTRODUCTION .......................................................................................................... 1

EXPERIMENT 1 METHODS ....................................................................................... 15

EXPERIMENT 1 RESULTS & DISCUSSION ............................................................. 23

EXPERIMENT 2 .......................................................................................................... 33

EXPERIMENT 2 METHODS ....................................................................................... 34

EXPERIMENT 2 RESULTS & DISCUSSION ............................................................. 37

GENERAL DISCUSSION ............................................................................................ 50

LITERATURE CITED ................................................................................................. 55

iii

ABSTRACT

Recent research has suggested that the antipsychotic olanzapine (Zyprexa) may have

cognitive enhancing effects. However, most of these reports come from clinical research with

schizophrenics, so it may be that the ability of olanzapine to enhance cognition is an indirect

result of symptom alleviation of schizophrenia. The delayed match-to-sample procedure has

been used to measure working memory and cognitive abilities in human and non-human research

for many years and an olfactory version of the task was developed in the present study. Rats

(n=9) were trained to match/non-match-to-sample using olfactory stimuli and effects of delays

and olanzapine were assessed. Increasing delays resulted in a corresponding decrease in

accuracy, that is, a delay gradient was found. Administration of olanzapine had no effect on

accuracy on fixed delays of 2-30s, except to suppress overall behavior at the highest doses. A

titrating delay procedure was implemented to remove any ceiling effects in performance seen

with the fixed delays. The titrating delay procedure revealed that olanzapine (0.1 & 0.3 mg/kg)

produced a significant increase in criterion and mean delays when given, however only for rats

trained to NMTS. Taken together these results indicate that 1) delay gradients can be obtained

using olfactory stimuli with rats trained to match/non-match-to-sample, 2) olanzapine did not

improve performance in a fixed DM/NMTS, 3) rats can perform a titrating delay task and

olanzapine increased performance with this methodology. This suggests that a value of the

titrating DNMTS procedure relative to fixed-delay procedures is increased sensitivity to drug

effects.

iv

LIST OF TABLES

Table Page

1. Representation of past research with DM/NMTS procedures and antipsychotics ....... 13

2. List of household spices used for olfactory stimuli .................................................... 19

3. Double Bait Data: Experiment 1 ................................................................................ 28

4. Trace Control Data: Experiment 2 ............................................................................. 40

v

LIST OF FIGURES

Figure Page

1. Photographs of apparatus........................................................................................... 17

2. Group forgetting curve .............................................................................................. 24

3. MTS and NMTS effects of training on delay accuracy............................................... 26

4. Group delay accuracy olanzapine .............................................................................. 29

5. Individual delay accuracy olanzapine ........................................................................ 31

6. Group criterion delays ............................................................................................... 38

7. Group accuracy for delay and non-delay trials ........................................................... 42

8. Individual accuracy ................................................................................................... 44

9. Group criterion, longest and mean delays; effects of olanzapine ................................ 47

10. Individual criterion, longest and mean delays; effects of olanzapine ........................ 49

Introduction

Schizophrenia is a debilitating brain disorder characterized by problems with cognition,

emotion, language, memory and perception (Meltzer & McGurk, 1999). Drug treatments for

schizophrenia have been divided into two categories, typical, or first generation antipsychotic

drugs and, atypical, or second generation. Typical antipsychotic drug treatments were developed

in the early 1950’s and are effective in treating the positive symptoms of schizophrenia. Positive

symptoms of schizophrenia can be thought of as ―extra‖ effects, those behaviors not normally

seen or problematic behaviors increasing in frequency that can range from hallucinations to

delusions to thought/movement disorders and depression (DSM-IVR- American Psychological

Association, 1997). Among the first and most popular typical antipsychotic drugs were

chlorpromazine, haloperidol, perphenazine, and fluphenazine. These compounds all have a high

binding affinity for dopamine subtype 2 receptors (D2), but in addition to suppressing positive

symptoms, all create unwanted extrapyramidal side effects such as tremors, restlessness and even

anxiety (Bardin, Auclair, Kelven, Prinssen, Koek, Newman-Tancredi, & Depoortere, 2007). For

example, haloperidol is the prototype ―typical‖ antipsychotic. It has a high binding affinity for D2

receptors and relatively low affinities for any other receptor. While a highly effective treatment

for the positive schizophrenic symptoms, it often produces extrapyramidal side effects, and today

its use is limited to the acute treatment of psychotic episodes or delusional behavior.

In the 1990’s the atypical or second generation, antipsychotic drugs were developed and

it was discovered that they could treat not only positive, but also, to some extent, negative

symptoms and rarely caused any of the extrapyramidal side effects characteristic of the older

typical antipsychotics (Bardin et al., 2007). Negative symptoms refer to a loss or diminishing of

normal functioning, such as difficulty sleeping, depression, problems speaking or making plans,

2

and difficulty staying on task or concentrating (DSM-IVR- American Psychological Association,

1997). This advance in treating the negative symptoms is generally thought to be related to the

mixed serotonergic and dopaminergic action of these popular second generation treatments

which include clozapine, risperidone, olanzapine, quetiapine, sertindole, and ziprasidone

(Miyamoto et al. 2005; Keefe, Silva, Perkins & Lieberman, 1999). However, these mixed

dopaminergic-serotonergic actions also produce side effects including weight gain, metabolic

changes and in some cases agranulocytosis (a loss of white blood cells). All currently prescribed

antipsychotics still mainly function by antagonizing D2 receptors; however, many other

neurotransmitter systems are affected by antipsychotics (e.g., cholinergic, muscarinic,

adrenergic, histaminergic and serotonergic, see Miyamoto et al., 2005; Keefe et al., 1999).

Many of the negative symptoms associated with schizophrenia are thought to involve

cognitive impairment (Meltzer & McGurk, 1999; Purdon, 1999). Most of the studies examining

these cognitive impairments use a battery of cognitive tests such as the Controlled Oral Word

Association Test (COWA) for verbal fluency, the Digit Symbol Substitution Test (DSST) for

visuomotor tracking, the Wisconsin Card Sort Test (WCST) for executive function, the

California Verbal Learning Test (CVLT), and the Verbal List Learning-Delayed Recall (VLL-

DR) for testing verbal learning and memory (Meltzer & McGurk, 1999; Purdon, 1999; Keefe et

al., 1999). Schizophrenics generally show deficits on these tests and treatment with typical

antipsychotic medication fails to improve performance. However, several studies have

documented improvements after treatment with atypical antipsychotic drugs. Kern, Green,

Marshall, Wirshing, Wirshing, McGurk, Marder, & Mintz (1999) found that schizophrenics

treated with risperidone, compared to those treated with haloperidol, increased the speed of

acquisition of verbal information on the California Verbal Learning Test (CVLT), a test designed

3

to examine and determine learning and memory abilities through verbal information. Patients

treated with risperidone improved a full standard deviation from baseline scores, but haloperidol

treated patients only improved roughly .25 standard deviation from the baseline. These findings

indicated that risperidone could ameliorate some of the cognitive (verbal) deficits

schizophrenics’ experience, though not enough to normalize their abilities to perform this

particular task.

Similarly, Bilder et al. (2002) found that both olanzapine and risperidone improved

global neurocognitive functioning (memory, attention, and motor skills) in schizophrenics given

15 various neurocognitive tasks to complete, (e.g., Wisconsin Card Sorting test, Boston

Diagnostic Aphasia, Hopkins Verbal Learning, and the Wechsler Adult Intelligence Scale-

Revised-WAIS-R). More specifically, olanzapine significantly improved processing speed,

attention, and executive and perceptual organization, whereas risperidone had strong effects

improving declarative verbal learning and memory. In contrast, haloperidol had no effect on

cognitive functioning. Many of the tasks were also administered with delays between choices or

presentation of stimuli to further evaluate the effects of these compounds on working memory.

Bilder et al. noted that the efficacy of each compound to affect cognition varied as a function of

the task, the dose given, or the duration of the treatment before testing. In most cases longer

periods of treatment with any of the antipsychotics diminished any neurocognitive enhancement

seen earlier in treatment.

As the body of cognitive-enhancing effects of atypical antipsychotics grew, Meltzer and

McGurk (1999), in a review paper, concluded that clozapine, risperidone, and olanzapine

improved cognitive function in schizophrenics. More specifically, clozapine showed robust

effects improving verbal fluency and attention; risperidone improved perceptual/motor

4

processing, reaction time, executive functioning, working memory, verbal learning and memory

and motor function; and olanzapine had significant effects on reaction time, executive function,

verbal learning and memory, and verbal fluency. However, as previously noted, the level to

which these compounds ―enhance cognition‖ appears to depend in complex ways on the

particular task and procedural parameters. Another concern is whether the observed

improvement of cognitive function involves a direct action or whether the effects seen reflect the

indirect effects of an alleviation of schizophrenic symptoms which may allow for better

performance on these tasks.

It is difficult to address such concerns with patients in a clinical setting, thus, in order to

further the investigation of the effects of atypical antipsychotics on cognitive functioning, animal

models are necessary. One example of a cognitive ability that has been studied in animals is

working memory. While there are several tasks available for testing working memory, one of

the most reliable and often used in both human and non-human research is the delayed match-to-

sample (DMTS) procedure (Blough, 1954; van Hest & Steckler, 1996).

A typical DMTS procedure consists of the presentation of a sample stimulus, followed by

the presentation of two or more comparison stimuli (one matching the sample) with varying

delays separating sample and comparison presentations (van Hest & Steckler, 1996). In DMTS,

responding to comparisons that are physically identical to the sample are reinforced. Depending

on the species, number of stimuli, and modality, overall accuracy can vary widely depending on

specific cognitive effects of this task. One aspect, however, remains constant: as the delay

between the sample and comparisons increases, the accuracy to match to the sample decreases.

This negatively accelerated function is often referred to as the forgetting curve (van Hest &

Steckler, 1996; White, 1985). This decreased ability of the subject to respond accurately as

5

delays increase is thought to be a model of human working memory, which is also characterized

by limited duration of accurate stimulus control (Wolff & Leander 2003; Vigouroux, Elaabouby,

& Gervais, 1992; Wixted, 1989).

In an early example of the procedure, Blough (1959) examined the delayed matching task

using four pigeons in an operant setting. The chambers were comprised of response keys in a

row on one wall of the chamber. Sample stimuli were presented on the center key and

comparison stimuli were presented on the left and right keys. Training began when the center

key was lit with either a steady white light or a flashing white light; after the pigeons responded

to the sample, the light turned off and the comparisons would light up either with the blinking

white light or the steady one. If the pigeon responded to the key that matched the sample, access

to food was presented as reinforcement, incorrect responses resulted in no food. After this initial

training, delays were introduced between the presentation of the sample stimulus and the

presentation of the comparison stimuli, 0, 1, 2 or 5 s. While two of the birds exhibited a decrease

in accuracy as the delays increased, the others maintained high accuracies of responding up to

10-s delays. Blough postulated that these inconsistencies in responding were due to the differing

mediating behaviors employed by individual birds. However, Blough did effectively produce

one of the first forgetting functions or curves in non-humans: as delays increased, accuracy

decreased.

DMTS procedures have also been widely used to assess drug effects on working memory.

Steckler et al (1998) reviewed this literature and noted that while there is extensive research with

many drugs and the DMTS procedure, very few studies and/or drugs produce delay-dependent

effects, that is, effects on accuracy seen only at some delays rather than a change in overall

performance. In other words, a delay-dependent effect would show more pronounced drug

6

effects at some delays but not at the performance or baseline trials, a selective effect. In contrast,

delay-independent effects would be effects of a drug on all delay and baseline trials, a non-

selective effect. To reiterate Steckler et al. found that the majority of DMTS studies using drugs

found delay-independent effects, and those few studies showing delay-dependent effects were

not consistent for a particular drug or task. However, despite the fact that few drugs and studies

demonstrate delay-dependent drug effects at doses which do not affect rate of responding or

behavior, many researchers continue to believe that the DMTS procedure is a viable approach for

detecting drug effects on working memory.

Previous research has examined the effects of typical antipsychotic drugs in DMTS as

well. Watson and Blampied (1989) investigated the effects of chlorpromazine, a typical

antipsychotic, on accuracy in a DMTS task in pigeons. The pigeons were trained in an operant

chamber with three response keys; the center key was always the sample and the left and right

keys were the comparisons. When the trial started, the sample key was lit either green or red.

After the pigeon made five responses to the sample, the sample key was turned off and the

comparisons were lit. By making a response to the key that matched the same color as the

sample, the pigeon would gain reinforcement via access to a food hopper; incorrect responses

resulted in a 3-s timeout followed by the start of a new trial. After responding at 90 to 95%

accuracy the pigeons were studied with delayed matching: delays of 0, 1, 2, 4, 8, and 16s were

randomly introduced between the end of the presentation of the sample stimulus and the

beginning of the comparison stimuli. Watson and Blampied found that as delays increased,

accuracy decreased. The 0 to 2-s delays were at baseline levels, but by the 16-s delay, accuracy

for the group declined to roughly 70%. When chlorpromazine was administered prior to

sessions, a delay-independent effect was seen; in other words as the dose increased, accuracy

7

decreased at all of the delays. Because chlorpromazine disrupted behavior with and without

delays, the effects were interpreted as non-specific to working memory processes. A delay-

dependent effect would have produced a change in accuracy from baseline at some delays but

not others, which would have indicated an effect on working memory processes.

In another study with pigeons, Picker and Massie (1989) compared the effects of typical

antipsychotics (chlorpromazine, loxapine, and thiothixene), to the effects seen with atypical

antipsychotic drugs (sulpiride and clozapine) in a DMTS procedure with pigeons. Four pigeons

were trained in operant chambers consisting of three response key pecks aligned horizontally and

equidistant from one another on one wall of the chamber, a food hopper that was raised to deliver

grain reinforcement and a houselight to signal experimental events. A trial began with the center

key (the sample) illuminated either red or green. Following five responses to the center sample

key, the key was darkened and the delay interval began (0, 2, or 8 s). Following the delay the

right and left keys were illuminated either red or green and a response to the key whose color

matched the color of the sample key was reinforced with 3 s of hopper access, incorrect

responses resulted in an immediate ITI. A delay gradient was found for all of the pigeons, such

that accuracy at the 0 (mean=93%) and 2 s (mean= 88%) delays was very high but performance

at the 8 s delay was much lower (mean=68%). Typical antipsychotics chlorpromazine and

loxapine, as well as atypical clozapine, produced dose dependent, but delay-independent

decreases in accuracy, though not in similar degrees. Typical antipsychotic thiothixene had

smaller decreases and atypical sulpiride had no effect at any dose tested. Picker and Massie

concluded that the varying degree of accuracy-decreasing effects seen across neuroleptics in this

task is due to the very distinct pharmacological actions of each compound and that further

8

analysis with tasks of this type is necessary. In sum, as with the Watson and Blampied study,

only delay-independent effects of neuroleptics were found by Picker and Massie.

Gemperle, McAllister, and Olpe (2003) compared effects of typical and atypical

antipsychotics in rats using a delayed non-match-to-position paradigm. In addition to haloperidol

and clozapine, they also studied iloperidone which, unlike other atypical neuroleptic compounds,

has a high binding affinity for alpha 1 and 2C receptors sites along with the usual affinity for

5HT2A and D3. In this task, rats were placed in an operant chamber with one wall equipped with

a center food hopper/nose poke and retractable levers to either side. There were dividers

between the levers and hopper/nose poke to help control for mediating behaviors.

Mediating behaviors are strategies developed by the organism that influence how

responses are made. For example, in the case of most match-to-position/place paradigms (spatial

tasks) the rat may simply remain in the sample location during the delay period and respond to

the comparison that is presented in the same location (MTS). Most recent research has

controlled for the confound of mediating strategies by requiring a response in a different location

than the sample after it has been responded to, or having the organism wait in a different location

during delay periods.

A trial began with one of the two retractable levers being introduced into the chamber

accompanied by a stimulus light. After a single lever press, the lever was retracted and the

stimulus light extinguished. Simultaneously the light above the nose poke/hopper was

illuminated and the rat needed to make a response to the nose poke to begin the variable 64-s

delay period. After the delay period ended, the hopper light came on and another nose poke was

required to initiate the comparison phase. A non-match (NMTS) procedure was used such that

9

responses on the lever opposite the sample lever delivered a food pellet. Incorrect responses,

pressing the sample lever, were not reinforced. Drug testing criteria were defined as greater than

90% accuracy at the 0-s delay, greater than 50% correct at 16-s delay, and little to no omissions.

As with most delayed matching studies, as delays increased, accuracy decreased; with accuracy

falling from roughly 100% at 0 s to 50% at the 64-s delay. Clozapine had no significant effects

on accuracy at any dose or delay tested, but haloperidol significantly decreased accuracy

compared to vehicle at the highest dose (0.03 mg/kg) at the 64-s delay, indicating a delay-

dependent effect. In other words, haloperidol significantly decreased accuracy at the 64-s delay

as compared to vehicle while not having an effect on accuracy at shorter delays, rate of

responding or latency to respond to the sample, indicating that at the 0.03mg/kg dose and 64-s

delay haloperidol is most likely affecting working memory. Iloperidone, on the other hand,

produced delay-dependent increases in accuracy at both the 48 and 64-s delays. Both 0.03 and

0.1 mg/kg doses of iloperidone raised accuracy from approximately 60% (48 s) and 50% (64 s)

to approximately 80% and 78%, respectively. These results were interpreted as showing that the

atypical antipsychotic iloperidone enhances working memory performance. This study differed

from the pigeon studies in that delay-dependent effects were found with respect to both increases

and decreases in accuracy with two different drugs.

In another delayed non-match-to-position paradigm with rats, Didriksen (1995) also

studied typical (haloperidol, raclopride) and atypical antipsychotic (clozapine, sertindole,

risperidone) drugs as well as drugs with similar binding profiles (SCH 23390, prazosin). The

operant chamber contained three nose–poke holes on one wall with a food tray on the opposite

wall. When a trial started the center poke was illuminated and after a response to it, either the

right or left nose poke was illuminated, and a response in the illuminated poke resulted in

10

reinforcement in the hopper on the opposite wall. After reinforcement, the delay period began,

up to 9 s, followed by illumination of the center light. A center poke response illuminated both

side lights, and a response to the nose-poke hole that did not match the sample was reinforced.

Didriksen found that SCH 23390 produced delay-dependent decreases in performance at 3 and 6

s but only after the highest dose (0.02 mg/kg). In contrast, raclopride, haloperidol, clozapine,

and risperidone produced delay-independent impairments at higher doses. Sertindole and

prazosin had no effect. These results indicate that even within the class of antipsychotics many

differences can be seen across delays and doses and that further research with other tasks of

working memory is needed.

Wolff and Leander (2003) studied effects of several antipsychotic compounds

(olanzapine, risperidone, ziprasidone, clozapine and haloperidol) on a spatial version of a

delayed non-matching task. Thirty Sprague-Dawley rats were trained to navigate the alleys of an

eight arm radial maze to gain reinforcement (45mg food pellet). The training began with the rats

in the center platform and the door to all eight alleys opening simultaneously; the rats were then

allowed to freely explore the maze, gaining reinforcement when they crossed an infrared

photobeam sensor at the end of each alley. Each alley was only baited once per session and the

session lasted until the rat fully entered the entire alley and gained reinforcement or 5 min

elapsed. Errors were defined as the rat fully entering an alley that had already been visited.

Once a rat met the criterion of no more than two errors on three consecutive days, delay training

began. Delay training was different from initial training in that only four of the arms were

opened at the beginning of the session (information phase), and after the rat had traversed all

four open arms and received reinforcement in each or 5 min elapsed, it was placed back in the

center platform where a delay period began (0 min, 1 min, 30 min, 3 h, 7 h, or 24 h): (the

11

retention phase). At the conclusion of the delay period, the four previously blocked alleys were

baited and the rat was allowed to explore once again. Errors were counted as any entries into

alleys previously reinforced either before the delay or during the current exploration. After delay

data were collected for each rat, drug testing began; only the 7-h delay was used for drug testing.

Following the information phase the rats were removed from the maze and given a compound or

vehicle via oral gavage in a volume of 1 ml/kg. Initial delay data revealed that, as expected, as

the delay period increased so did the number of errors. While there was no significant difference

between the 0 min and 30-min delays on performance, there was a significant increase in errors

after a 3-h delay and an even greater increase in errors after a 7-h delay, although the number of

errors committed at the 24-h delay was not significantly different from the 7-h delay. As the

most errors occurred at the 7-h delay, this was chosen to be the delay used for drug testing.

Haloperidol, ziprasidone, and clozapine had no effect on errors at the 7-h delay; however, both

olanzapine (3.0 and 5.0 mg/kg) and risperidone (0.1 mg/kg) significantly decreased the number

of errors in a delay-dependent manner. The effects of olanzapine and risperidone can be called

delay-dependent as they had no effect on the latency to finish the task as compared to vehicle,

indicating that the effects seen were not due to an overall decrease in performance ability. It is

important to note that the compounds used in this study were administered after the information

phase and before the recall task, essentially testing the effects on retention. In contrast to Wolff

and Leander (2003), Gemperle et al. (2003) administered their compounds before the beginning

of the sessions opening the possibility that drugs can affect both acquisition and recall. Of

course, as Wolff and Leander (2003) only tested one delay (7 hr) there is no way to know what

effects may have been observed at shorter or longer delays.

12

By comparing the above studies (see Table 1), it is clear that in order to fully understand

the effects of antipsychotics on working memory in delayed matching tasks, much more research

will need to be conducted or previous research revisited. In general, all typical and most atypical

antipsychotics impair or have no effect on accurate performances. Impairment occurred most

often in a delay-independent manner. Picker and Massie (1989) and Watson and Blampied

(1989) both tested typical antipsychotics on pigeons in DMTS and found only delay-independent

impairments. However, in some cases atypical antipsychotic drugs have shown delay-dependent

improvements in performance (Gemperle et al, 2003; Wolff & Leander, 2003). While Wolff and

Leander saw an improvement in performance with olanzapine and risperidone, Didriksen found

only delay-independent impairments with risperidone. Gemperle et al. and Wolff and Leander

both found that clozapine had no effect on performance but Didriksen saw a delay-independent

decrease at similar doses with his task. Finally, the effects of haloperidol across all three studies

varied. Gemperle et al. found impairments but only at the highest dose and delay, Wolff and

Leander saw no effect of haloperidol on performance, and Didriksen found that haloperidol had

delay-independent impairments. While the results of these studies help us to decipher what the

effects of antipsychotics may play on working memory, differences between the studies in the

species, doses, routes of administration, pre-treatment times, modalities, delays, and, most

importantly, tasks are all variable and make interpreting the results collectively very difficult.

By combining the information from previous research, a consistent method of detecting

cognitive effects of atypical antipsychotics would prove very useful for treatment of

schizophrenia and improving working memory in other affected populations.

13

Table 1

Representation of Past Research Using Variations of DM/NMTS Procedures and Effects of

Antipsychotic Drugs on Performance

______________________________________________________________________________

Note. Abbreviations refer to either delay-independent (DI) or delay-dependent (DD) effects. Horizontal arrows indicate no effect to impair or

improve performance at any dose or delay tested, where as arrows pointing up and down represent improvements and impairments in

performance, respectively.

Study

Procedure

Species

Delays

Dose (mg/kg)Effect Dose (mg/kg)Effect Dose (mg/kg)Effect Dose (mg/kg)Effect Dose (mg/kg)Effect

chlorpromazine 0.5-12.5 DI ↓ 3-100 DI ↓

loxapine 0.1-10 DI ↓

thiothixene 0.03-1.7 DI ↓

haloperidol 0.003-0.03 DD↓ 0.01-0.04 DI ↓ 0.01-3 ↔

raclopride 0.02-0.08 DI ↓

sulpiride 3-300 ↔

clozapine 0.1-5.6 DI ↓ 0.1-0.3 ↔ 0.63-2.5 DI ↓ 3-10 ↔

iloperidone 0.03-0.1 DD↑

sertindole 0.04-1.25 ↔

risperidone 0.1-0.4 DI ↓ 0.003-0.3 DD↑

ziprasidone 1-10 ↔

olanzapine 1-10 DD↑

SCH 23390 0.005-0.02 DD↓

prazosin 0.5-2.0 ↔

Pigeons

0-8s

Typical Antipsychotic Drugs

Atypical Antipsychotic Drugs

Other Drugs

Gemperle 2003

DNMTP (spatial)

Rats

0-64s

Watson 1989

DMTS (non-spatial)

Pigeons

0-16s

Picker 1989

DMTS (non-spatial)

Wolff 2003

DMTP (spatial)

Rats

7hr0-9s

Rats

DNMTP (spatial)

Didriksen 1995

14

The Wolff and Leander (2003), Gemperle et al. (2003), and Didriksen (1995) studies all

employ a delayed paradigm with atypical antipsychotics that use spatial cues; i.e., match-to-

position. Recent research has indicated different processes may be involved in spatial versus

non-spatial tasks, and the stimulus modality used; olfactory, tactile, visual, auditory, etc.

(Steckler, 1998). Stimulus control by spatial stimuli may involve different neuroanatomical

systems than those using other stimuli, and these differences across both the tasks and the

modalities need to be taken into account when analyzing effects of drugs on working memory

(Steckler, 1998). In the present study, the apparatus presented olfactory stimuli and removed the

common aspect of spatial position from the task. Currently, there are no studies examining the

effects of antipsychotic drugs on a non-spatial olfactory DMTS task. Olanzapine was chosen for

investigation due to the delay-dependent improvements found in the Wolff and Leander (2003)

study. However, to expand on Wolff and Leander, more delays were examined. If olanzapine

improves DM/NMTS performance, accuracy at longer delays should improve as compared to

baseline, that is, delay-dependent improvements.

There are many benefits to using the olfactory modality in a DM/NMTS procedure.

Perhaps the biggest advantage to using this system will be the rat’s enhanced ability to learn and

discriminate olfactory cues as compared to other less salient modalities (Slotnick, 2001). Ravel,

Elaagouby and Gervais (1994) used a modified T-maze to deliver olfactory stimuli to train rats to

DMTS and found delay-dependent impairments with central administration of muscarinic

antagonist scopolamine. However, they never established a forgetting curve as rats were still

performing at 94% at the longest delay (30 s). The olfactory modality combined with the DMTS

procedure in this non-spatial system in the current experiment should produce forgetting curves

15

of cognitive abilities/working memory in the rat in a quick and accurate manner, and allow us to

observe the effects of antipsychotic drugs on these abilities.

In Experiment 1, rats were trained to M/NMTS using olfactory stimuli and, following the

development of stable levels of accuracy, fixed delays (2s, 10s, 20s, 30s) were randomly inserted

between the sample and comparison presentation. The goals of this study were to determine the

viability of this procedure to detect forgetting curves and the effects of olanzapine.

Experiment 1

Method

Subjects

Subjects were male Sprague-Dawley rats (P2, S3, S13, O12, P21), Harlan Tekland,

(Indianapolis, Indiana) approximately 90 days old at the start of the experiment. Rats were

housed individually and maintained under a 12 hr reversed light/dark cycle, lights off at 7:30am.

Subjects had free access to water, but food was restricted to maintain body weight at

approximately 85-90% of free feeding weight based on normal growth weight charts from

Harlan Tekland. The chart plots normal free feeding weights of Sprague-Dawley rats as they

grow, and by matching the age of experimental rats to this chart approximate free feeding

weights were determined; 85-90% of these free-feeding weights were calculated and used as a

guide for the rats in this experiment. Rats were fed between 13 and 18g of food daily, which

maintained motivation for experimental testing and allowed for the rats to continually gain small

amounts of weight as they aged. Subjects were tested 5 days per week and were drug naïve at the

start of the experiment.

Apparatus

16

A modified operant chamber was used as the testing apparatus (28cm x 26cm x 30cm).

The top, front and back of the operant chamber walls were Plexiglas and the two side walls and

floor grids were stainless steel (see Figure 1). The front wall of the chamber was altered to allow

plastic trays to be slid into the operant chamber, above the grid floor. The two trays (10 cm x 8

cm) used for the experiment were made of plastic. The first tray was used for the presentation of

the sample stimulus, and had a centered hole (5 cm diameter), 3.2 cm from the top and 10 cm

from the sides to hold a 2 oz opaque plastic cup, filled halfway with sand. There were four bolts

fastened to the tray in a square around the hole to create a track for the plastic scented lids to rest

in. The second tray, used for the comparison stimuli, had two holes the same size as the sample

hole placed equidistant from one another and the sides of the tray (3.2 cm from the top and 3.8

cm from the sides). These two holes also held cups with sand in them and had the same

configuration of bolts mounted to act as the guide for the comparison scented lids (see Figure 1).

The opening at the base of the chamber for the trays to slide into was covered with a clipboard

during intertrial intervals (ITIs) and delay periods. A white noise generator and speaker created

70 dB of white noise continuously throughout all sessions. 45mg sucrose pellets, Lab Diet,

served as reinforcement and were buried with tweezers 1 cm into the sand in the sample and/or

comparison cups, depending on the training/testing condition.

17

Figure 1. Testing apparatus. Top panel is the modified operant chamber with sample tray inserted. The bottom panel

is the comparison tray with the right stimulus (scented lid) partially removed.

18

Stimuli

The 7.5 x 7.5 cm scented plastic lids used as stimuli in the experiment were cut from

0.452 cm Plexiglas sheets. When not in use, the lids were placed in airtight containers with

approximately 6 oz. of powdered common household spices or aromatic oils (The Great

American Spice Co. and Rocky Mountain Spice Co—see Table 2). The lids were kept away

from direct contact with the spice and separated from one another by small pieces of plastic to

ensure that the full surface area was exposed to the scented air within the containers. The lids

were placed in the containers for a minimum of 72 hours before being used for testing to ensure

that the plastic had absorbed the scent. The lids were removed from the containers for

training/test sessions and were immediately returned when finished. Spices in the container were

changed every 2 weeks and lids washed regularly.

19

Table 2

Stimulus List

______________________________________________________________________________

Spinach

Worcestershire

Sage

Savory

Sumac

Thyme

Tomato

Turmeric

Mustard

Nutmeg

Onion

Oregano

Paprika

Rosemary

Coriander

Cumin

Dill

Garlic

Ginger

Marjoram

Beet

Caraway

Carob

Celery

Cinnamon

Clove

Great American Spice Co. Rocky Mountain Spice Co.

Purchased From

Allspice

Anise

Bay

Fennel

Lime

Raspberry

20

Procedure

MTS Training. Removal of a black clipboard in front of the chamber signaled the start of

each trial and its insertion after the trial defined the intertrial interval (ITI= 30 s). When the trial

began the sample tray was inserted and following a response (pushing the lid past the lip of the

cup), the sucrose pellet was available. The sample tray was then removed and the comparison

tray was introduced as rapidly as possible. As the procedure was not automated, the time

between the sample response and the presentation of the comparison tray varied slightly from

trial to trial, but was approximately 2 s (based on video review). Thus, the baseline schedule can

be described as a DMTS procedure with 2-s delay interval. When the rat responded to the

comparison stimulus that matched the sample, it could collect the sucrose pellet buried in the

sand. After correct responses, the comparison tray was immediately removed and the 30-s ITI

began. When the rat made an incorrect response, the comparison stimuli remained in the operant

chamber until a correct response was made. Initially rats were trained with 2-10 different

scents/stimuli. The presentation and placement of the sample and comparison stimuli was

randomized with the condition that no single scent would be used as the sample more than two

trials in a row and correct comparisons would not occur on the same side for more than two

consecutive trials as well. Each session was also balanced so the correct comparison was on the

left six times and on the right six times. During this phase and all subsequent training/testing a

session was terminated if no response was made to either sample or comparison stimuli for 2 min

on three consecutive trials. After 2-5 days of this initial training, reinforcement of responses to

the sample was slowly faded out, the number of trials per session was increased to 36 and a

change was made in the procedure such that incorrect responses resulted in the immediate

removal of the comparison tray. A correction procedure continued in effect where following the

21

ITI, trials responded to incorrectly were repeated. Criterion in this and subsequent phases was

defined as 75% or better accuracy on two consecutive days of training. After the criterion was

met with 2-10 stimuli, 7-10 new stimuli were added and then another 7-10 after criterion was

met again, until each rat was responding to a total of 18-30 different scented stimuli. Once the

stability criterion was met with the 18-30 stimuli, delays were introduced and assessed.

NMTS Training. Non-match to sample training was conducted as described above, except

responses to the comparison scent that was not identical to the sample stimuli were reinforced

and those matching the sample were not.

DM/NMTS Training. During delay training, delays of 2, 10, 20 and 30 s were introduced

between the sample response and the presentation of the comparison stimuli. To begin, only one

trial of each delay was inserted per session, then as performance stabilized two and then finally

three were added, for a total of twelve delay trials per session. The delays were presented in

semi-random fashion but each of the delay intervals occurred once within a twelve trial block of

the session. During delay periods the chamber opening was blocked with a white board to signal

the delays during delay training and testing, incorrect responses during delay trials were followed

by the next programmed trial (no correction procedure), however, a correction procedure was

still in effect for non-delay baseline trials. The ITI was still signaled by a black cover. In other

words, if an incorrect response was made the tray was immediately removed and the intertrial

interval began. The only difference between non-delay trials and 2-s delay trials was that after

the rat responded to the sample during 2-s delays the white cover was flashed over the front of

the operant chamber before the comparison tray was inserted; during non-delay trials the

comparison tray was immediately inserted. As mentioned earlier, the non-delay trials did in fact

have a delay of approximately 2 s between the removal of the sample tray and insertion of the

22

comparison tray. To meet drug testing criterion, a minimum of ten complete sessions with all

twelve delay trials was required, performance on the immediate, 2-s delay trials could not differ

from non-delay trial accuracy, and overall accuracy maintained at 75% correct or greater the day

before testing.

Drug administration. Dose effect functions for olanzapine (0.1, 0.3, 1.0 mg/kg) were

obtained. The drugs were dissolved in lactic acid and saline, followed by several minutes of

sonication to ensure drugs were in solution. The vehicle used as a control was .2 ml lactic acid

mixed with 9.8 ml saline, the same as the ratio used for drug mixing. Drugs were injected

intraperitoneally in body weight volume, 30 min prior to testing and pretreatment took place in

the home cage in the colony room. Injections were given on Tuesdays and Fridays to ensure

enough wash out time between doses/drugs. Thursday sessions served as baseline control

sessions. A minimum of two determinations of the doses of each drug and vehicle were

conducted for all subjects, doses were administered randomly.

Control Measure. After match/non-match-to-sample training was completed, a control

measure was introduced to assess pellet detection in Experiment 1. For two Wednesdays, 6 of

the 36 trials, non-delay, were double baited, i.e., both comparison cups had a sucrose pellet

buried in them. This measure allowed us to determine whether the rats were responding based on

sample stimulus control as opposed to control by the smell of the sucrose pellets in the correct

comparison cup. If they were responding to the pellets and not the olfactory stimuli we should

have seen accuracy fall to 50%; in contrast, accuracy should stay at normal levels if pellet

detection was not mediating behavior. As all rats in Experiment 2 initially began in Experiment

1 and the apparatus and stimuli were identical, double bait control procedures were only

examined in Experiment 1.

23

Data Analysis. The percent correct of responding of baseline and delay trials was plotted.

The effects were analyzed using repeated-measures ANOVA. Significant findings were analyzed

by Tukey’s HSD post-hoc test.

Experiment 1 Results & Discussion

Figure 2 shows the baseline accuracy for non-drug sessions for all rats in the fixed-delay

procedure. Baseline data were obtained from all sessions occurring on Thursdays after rats met

criterion for drug testing. The percent correct of responding is represented on the vertical axis,

and the delays in seconds are represented on the horizontal axis. As delays increased, accuracy

decreased from approximately 99% at the 2-s delay to approximately 79% at the 20 and 30-s

delays. A one-way repeated measures ANOVA revealed a significant main effect for delay,

F(3,12)=10.374, p<.001. A Tukey’s HSD post-hoc test revealed that accuracy at the 2-s delay

was significantly higher than the accuracy at the 20-s, [q.05(3,12)=19.5], and 30-s

[q.05(3,12)=18.97] delays and accuracy was significantly higher at the 10-s delays compared to

the 20-s delays [q.05(3,12)=11.21]. These results indicate a forgetting function with accuracy

declining as delays increased; however after the 20-s delay the decline in accuracy reached a

plateau and remained low for the 30-s delay.

24

Figure 2. Baseline mean percent correct accuracy for non-drug sessions (Thursdays) for all rats in Experiment 1. The

percent correct is represented on the vertical axis, and the delays in seconds are represented on the horizontal axis. Error bars show standard error of the mean.

Baseline Delays

2 10 20 30

50

60

70

80

90

100

Delays (s)

Perc

en

t C

orr

ect

25

Figure 3 shows the delay accuracy for the first- and last-five delay sessions for all rats.

Percent correct is represented on the vertical axis, and the delays in s are represented on the

horizontal axis. Data are presented separately for NMTS-trained rats (red) and MTS-trained rats

(green). The mean of the two groups is shown in the upper right panel and clearly shows that the

forgetting function was evident only in the MTS rats. The two NMTS subjects showed a much

shallower gradient with little decline from 2- to 30-s delays. For most of the rats there was no

difference between the first five delay sessions. Thus, considered separately, it is quite easy to

see that the forgetting function of Figure 2 is primarily based on the contributions of the MTS

rats. Rats trained to NMTS never really developed a forgetting function as their accuracy at the

longest delays remained very high. Only when the accuracy for all the rats was combined, as in

Figure 2, does a forgetting function appear.

26

Figure 3. MTS trained rats compared to NMTS trained rats as a function of training. Delay accuracy for the First and

Last five delay sessions for all rats in the fixed delay procedure, this is 15 determinations (delays) at each point. The

percent correct of responding is represented on the vertical axis, and the delays in seconds are represented on the

horizontal axis. Rats are divided into NMTS trained rats in red and MTS trained rats in green. The mean of the two

groups is shown in the upper right panel.

P2 NMTS

2 10 20 30

0

50

60

70

80

90

100First 15

Last 15

Delays (s)

Perc

en

t C

orr

ect

S3 NMTS

2 10 20 30

0

50

60

70

80

90

100First 15

Last 15

Delays (s)

Perc

en

t C

orr

ect

Mean Delay Analysis

2 10 20 30

0

50

60

70

80

90

100First NMTS

First MTS

Last NMTS

Last MTS

Delays (s)

Perc

en

t C

orr

ect

S13 MTS

2 10 20 30

0

50

60

70

80

90

100First 15

Last 15

Delays (s)

Perc

en

t C

orr

ect

O12 MTS

2 10 20 30

0

50

60

70

80

90

100First 15

Last 15

Delays (s)

Perc

en

t C

orr

ect

P21 MTS

2 10 20 30

0

50

60

70

80

90

100First 15

Last 15

Delays (s)

Perc

en

t C

orr

ect

27

Table 3 shows the double bait control data for each rat. The double bait trials presented

here were administered on two Wednesday sessions (six each day) and were only given on non-

delay trials. All of the rats except for P2 responded accurately 100 percent of the time, and P2

only missed one double bait trial of the twelve administered. As accuracy did not decrease

significantly during double bait trials, rats were not detecting/tracking the pellets, but rather

behavior was under the control of the matching or non-matching comparison stimulus.

28

Table 3

Double Bait Data

______________________________________________________________________________

Figure 4 shows the effects of olanzapine on accuracy. Data represent the mean for all the

rats in Experiment 1combining data from MTS and NTMS subjects. The percent correct is

represented on the vertical axis and delays in seconds on the horizontal axis. Vertical bars

represent standard error of the mean. Visual inspection of the data indicates a trend towards a

significant improvement in accuracy at the 20-s delay with both 0.1 and 0.3 mg/kg olanzapine,

but the relatively high accuracy with vehicle at the 20-s delay (~70%) and the variability due to

individual differences, limits interpretation of these effects. A two-way repeated measures

ANOVA was conducted for mean accuracy and revealed a significant main effect of delay,

F(3,12)=10.081, p<.001. The main effect of olanzapine (drug) was not significant,

F(3,12)=1.145, p=.371, and there was not a significant interaction between dose of olanzapine

and delay (drug × delay), F(9,36)=.859, p=.569. The results indicate that although visually the

graph appears to show olanzapine possibly having an effect, the variability between groups and

even individual rats (see Figure 5) prevents any significant effects from being found.

S13

100%

100%

83%

100%

100%

Subjects Percent Correct

O12

P21

P2

S3

29

Figure 4. Group data. Mean percent correct plotted as a function of delay for doses of olanzapine. The percent

correct is represented on the vertical axis and delays in seconds on the horizontal axis. Vertical bars represent

standard error of the mean. Error bars show standard error of the mean. All doses are in mg/kg.

Olanzapine Delay Data

2 10 20 30

0

50

60

70

80

90

100Veh.

0.1

0.3

1.0

Delays (s)

Pe

rce

nt

Co

rre

ct

30

Figure 5 shows the individual data for accuracy as a function of delay for olanzapine.

Accuracy is represented on the vertical axis by percent correct, and the delays in s are shown on

the horizontal axis. The top two panels show accuracies for the rats trained to NMTS and the

bottom three panels show data from those rats trained to MTS. The NMTS trained rats both

showed some evidence of enhanced mean accuracy at the longer delays under some doses of

olanzapine; however, these effects were not reliable as there was large variability in each case.

Rat P2 (NMTS) showed improved performance at the 10, 20 and 30-s delays after the .1 and 1.0

mg/kg doses, but the effects were inconsistent across determinations. Rat S3 (NMTS) showed

an improved performance at the 10, 20 and 30-s delays and this effect was dose dependent, with

improvement only occurring at the 0.1 and 0.3 mg/kg dose (performance was impaired at the 1.0

mg/kg dose).

Inspection of the individual subject data for the MTS rats also revealed some tendencies

toward improved accuracy although the effects were not consistent across delays or doses. MTS

rat S13 only showed improvements at the 20-s delay and at the 0.1 and 0.3 mg/kg doses. O12

(MTS) only showed an improvement at 10-s delay with 0.1 mg/kg, however, this rat maintained

very high accuracy at each delay for vehicle, making any increases in performances hard to see.

Finally, P21 (MTS) showed an improvement at the 30-s delay with 0.1 mg/kg. It should be

noted that the MTS trained rats showed considerable variability, and thus, as was the case with

the NMTS subjects, the mean effects were not reliable. Taken together, these individual results

indicate that this procedure may be limited by individual differences, i.e., inconsistencies in

responding across delays and doses.

31

Figure 5. Individual data. Percent correct plotted as a function of delay for olanzapine. Accuracy is represented on

the vertical axis by percent correct, and the delays in seconds are shown on the horizontal axis. The top two panels

are the rats trained to NMTS and the bottom three panels are those rats trained to MTS. Error bars are standard error

of the mean. Numbers in parentheses indicate number of determinations for that condition. All doses are in mg/kg.

P2 NMTS

2 10 20 30

0

20

40

60

80

100Veh. (4)

0.1 (3)

0.3 (4)

1.0 (3)

Delays (s)

Perc

en

t C

orr

ect

S3 NMTS

2 10 20 30

0

20

40

60

80

100Veh. (3)

0.1 (3)

0.3 (3)

1.0 (2)

Delays (s)

Perc

en

t C

orr

ect

S13 MTS

2 10 20 30

0

20

40

60

80

100Veh. (4)

0.1 (3)

0.3 (4)

1.0 (4)

Delays (s)

Perc

en

t C

orr

ect

O12 MTS

2 10 20 30

0

20

40

60

80

100Veh. (3)

0.1 (2)

0.3 (3)

1.0 (2)

Delays (s)

Perc

en

t C

orr

ect

P21 MTS

2 10 20 30

0

20

40

60

80

100Veh. (3)

0.1 (3)

0.3 (3)

1.0 (3)

Delays (s)

Perc

en

t C

orr

ect

32

As noted above, a problem with using fixed delays is that some rat’s performances even

at the longest delays may reach maximal levels, perhaps even early in training. Figure 3

demonstrates clearly that all rats began the experiment with high accuracy at all delays and

maintained performance close to those initial levels until the end of the experiment. Further, the

rats trained to NMTS were often unaffected by even the longest delay, as accuracy was above

85% for both rats at the beginning and conclusion of the experiment. In sum, the differences in

baseline performance between MTS and NMTS-trained subjects and the overall individual

differences in the delay function may have limited the ability of the fixed-delay procedure to

detect effects of olanzapine in Experiment 1.

In the DMTS procedure, a titrating method, has been utilized in the past to prevent

ceiling effects and effectively measure drug effects on individual responding (Hudzik & Wenger,

1992). In a titrating procedure the delays between sample and comparison presentation generally

begin at very short values and increase through the session as long as the animal performs

accurately. When errors are made, the delay value is decreased, and by the end of the session, a

criterion delay, or delay at which accuracy becomes roughly chance, is reached. This allows for

1) each animal in an experiment to serve as their own control and 2) reduces the possibility of

ceiling effects. Thus, with a titrating procedure each rat has its own baseline delay to compare to

drug effects and the delay reached, not accuracy, becomes the dependent variable. Further, the

removal of a ceiling allows for the examination of rats of varying abilities. In order to assess the

possibility that memory enhancing effects of olanzapine might be detected using a titrating delay

procedure, Experiment 2 replicated the olanzapine dose-effect function using an olfactory

titrating delay procedure designed for use with rats.

33

Experiment 2

In the prototype study of titrating DMTS, Hudzik and Wenger (1992) tested common

drugs of abuse with squirrel monkeys. The operant chamber contained 3 keys in a row that could

be colored either white or flashing blue, and a food hopper underneath that was illuminated green

when reinforcement was available. When the trial began, the center key was illuminated either

1) solid white, paired with white noise, or 2) oscillating blue, paired with a clicking noise. After

the monkey made 30 responses on the sample (FR30), two of the three keys were illuminated

and a response to the key that matched the sample was reinforced. The first five delays

presented were all 3 s; on the 6th and subsequent delays for the session, the delays increased by 3

s if all 5 of the previous delays resulted in correct responses, stayed at the same delay if the

monkey made 4 of 5 previous correct responses, or decreased by 3 s if fewer than 4 of 5 correct

responses were made. After the administration of a compound not only could the effect of

accuracy at shorter delays be analyzed, but failure to advance to longer delays reached under

control conditions provided an index of impairment. For example, Hudzik and Wenger found

that scopolamine and diazepam decreased MTS accuracy, but not the length of the delays. In

other words, the delay that monkeys advanced to did not differ from control, but the accuracy at

those delays was decreased. Thus, performances were only slightly impaired, as they could

respond accurately enough to reach the usual delays but not well enough to maintain accuracy at

those delays. Conversely, cocaine decreased the delays the monkeys reached, but accuracy at

those shorter delays was unaffected. Pentobarbital and methylscopolamine decreased both

accuracy and the delay reached. Nicotine, phencyclidine, caffeine, morphine, physostigmine,

and neostigmine did not affect accuracy or the length of delays. As all the compounds tested had

varying effects on either accuracy or delays reached or both, effects on working memory alone

34

are not sufficient to explain the decrease in delays reached or accuracy seen with scopolamine

and diazepam.

In Experiment 2 an olfactory titrating procedure was employed based on the procedures

utilized by Hudzik and Wenger, whereby correct responses increase the length of delay on the

subsequent trial, and incorrect responses result in a decrease in delay length on the next trial.

There are no published reports of a titrating procedure in rats, and therefore a careful analysis of

differences between match and non-match-to-sample was conducted as well a dose effect

function for olanzapine to determine whether the titrating procedure might be more sensitive to

drug effects.

Method

Subjects and apparatus

Following the completion of Experiment 1, six rats were studied under the titrating

procedure. Four rats were carried over directly from Experiment 1; P2, S3, S13 and P21. Two

additional rats, P1 and S4, were added from another experiment with identical training

procedures and parameters as the current study’s Experiment 1. However, P1 and S4 were given

a full dose response function for another drug (dizocilpine). Both were tested under baseline

conditions for two weeks after completion of the dizocilpine study before Experiment 2 began to

ensure a complete wash out of the prior drug. The apparatus was the same as that used in

Experiment 1.

Procedure

Training sessions began with two non-delay trials, followed by four 2-s delay trials. If

the rat responded correctly on the final 2-s delay trial, a 10-s delay was programmed for the next

trial. Subsequent delay trials increased by 10 s following each correct response during the

35

previous trial, and decreased by 10 s following each error until a 100-s delay was reached. At

that point, delays increased or decreased in 20-s increments. Sessions continued in this way until

a criterion delay was attained which was designed to obtain a valid index of the longest delay on

which successful performances could be maintained. The operational criterion was three correct

responses on a particular delay.

For example, the criterion delay could be reached in the following way: suppose the rat

responded correctly at a delay of 60 s and then again at 70 s which would advance the delay to

80 s. Then, suppose that an incorrect response is made at the 80-s delay, but then a correct

response is made when the delay is reduced to 70 s. At this point, the delay would advance again

to 80 s and if the rat once again makes an error at this delay, and then responded correctly at 70 s

once more, then 70 s would be the delay criterion for that session. This definition of a criterion

was used to ensure that the criterion delay reflected the longest delay at which consistently

accurate performances were obtained. Although this criterion delay index is a bit complex, it

was selected because other more obvious measures might be more likely to be influenced by

chance factors. For example, rat’s accuracy on short and non-delay trials was high, however

occasionally incorrect responses were made at shorter delays. Thus, the first error on a delay was

not used to define the criterion, because errors early in the session may result in an

underestimation of delays at which accuracy could be maintained and premature termination of

the session.

After criterion had been reached, four additional non-delay trials were presented and the

session then ended. The session could also be terminated without criterion if 1) the rat failed to

respond to either sample or comparisons after 2 min, for two trials in a row (times out), 2) made

five incorrect responses consecutively, or 3) made eight responses to the same side of the

36

comparison tray consecutively. Only one rat had sessions that terminated early, for failure to

respond after administration of 1.0 mg/kg olanzapine; this happened both times the rat received

this dose. Titration training continued until stable criterion delays were reached, less than 10%

difference between the mean criterion delay of the last five trials and the first five trials as

compared to the overall mean. Once stability was reached, dose effect functions for olanzapine

were obtained.

Data Analysis. Criterion, mean (the sum of all delays divided by the number of delay

trials) and longest (longest delay reached and responded to accurately) delays were analyzed,

along with non-delay (the six trials before and 4 trials following the titrating delay trials) and

delay accuracy (accuracy of all delay trials). Further, the differences in performance between

rats trained to match versus non-match were analyzed across all measures. A 2×3 two-factor

ANOVA with repeated measures on one factor and a one-way ANOVA were used to analyze

these measures and Tukey’s HSD and simple main effects post hoc tests were conducted for

significant findings.

Control Measure. At the conclusion of the drug testing, another control measure was

utilized to determine if the delay gradient was a result of the decrease in scent left in the operant

chamber after the sample stimulus was removed. In other words, was accuracy decreasing at the

longer delays because the rat’s ―memory of the sample scent‖ was degrading or because the rats

were using the trace scent left by the sample to respond accurately at shorter delays and as delays

increased, less trace scent was available and errors increased. To examine this problem a

procedural difference was introduced.

The change was to leave the sample stimulus in the operant chamber during all the delay

periods during titrating sessions for five consecutive sessions. After the rat responded to the

37

sample stimulus, the tray was removed and the delay period began, as described above, but when

the sample tray was removed another scented lid, the same as the sample, was placed inside the

top of the operant chamber where it was freely available to the rat. At the conclusion of the

delay period the comparison tray was slid in while the ―extra‖ sample stimulus remained at the

top of the chamber and the rat responded. Following a response, the comparison tray and the

―extra‖ lid were removed and the intertrial interval began. The logic of this control procedure

was that if the accuracy at longer delays increases, this could be due to the rats extended

exposure to the sample. However, if the same effects during delays are seen, i.e., as delays

increase accuracy decreases, the extended scent exposure was not influencing responding, rather,

the decrease in accuracy was due to the decreasing memory. The logic of this procedure was that

although the sample scent was removed from the sample position, the insertion of the lid in the

upper portion of the chamber would have assured the presence of the odor throughout the delay

period.

Results and Discussion

Figure 6 shows the individual and training condition mean criterion delays for each rat in

the titrating procedure. All points used were non-drug control days (Thursdays). The mean

criterion delay reached is shown on the vertical axis; the rats/training condition is on the

horizontal axis. Red bars show delays from rats trained to NMTS and the green bars, rats trained

to MTS. Vertical black lines show standard error of the mean. The NMTS-trained rats reached

longer criterion delays on average (63-120s) than did rats trained to MTS (25-50s), and a non-

parametric Wilcoxon W test confirmed the significance of these differences, W(6)=6.00, -1.94,

p=.050. The discrepancy between the NMTS and MTS groups in the titrating procedure

continued through the remainder of the Experiment 2 results and therefore all figures for

Experiment 2 will be divided by NMTS (red) and MTS (green).

38

Figure 6. Individual and group data. Individual and training condition mean criterion delays for each rat in the

titrating procedure, all points used were non-drug control days (Thursdays). The mean criterion delay reached is

shown on the vertical axis; the rats/training condition is on the horizontal axis. Red bars show delays from rats

trained to NMTS and the green bars, rats trained to MTS. Vertical black lines show standard error of the mean.

P2

P1

S3

NM

TS M

ean

MTS

Mea

nP21

S13 S

4

0

25

50

75

100

125

150

NMTS MTS

Rats

Cri

teri

on

Dela

ys (

s)

39

Table 4 shows the data for the trace controls conducted for the titrating procedure. The

trace controls consisted of an ―extra‖ sample lid remaining in the chamber with the rat for the

duration for the delay period for all delay trials. Trace control sessions were run for five days

consecutively following the drug study. Four rats showed longer criterion delays on trace

control trials, but one of these was a very small increase and the other three (S3, S4 and P1)

remained within the range of the standard deviation. These data provide some support for the

contention that the criterion delay does not depend on a degrading scent trace left by the sample

stimulus.

40

Table 4

Trace Control Data

______________________________________________________________________________

Note. Numbers represent mean criterion delays ± the standard deviation. Numbers in parentheses indicate the number of determinations for that

particular condition.

Subject

P2 (NMTS)

P1 (NMTS)

S3 (NMTS)

P21 (MTS)

S13 (MTS)

S4 (MTS)

70.83 ± 23.92 (12)

49.09 ± 27.73 (11)

26 ± 19.93 (15)

33.33 ± 14.97 (12)

115 ± 10 (5)

83.33 ± 11.55 (5)

104 ± 55.95 (5)

40 ± 25.50 (5)

28 ± 17.89 (5)

44 ± 11.40 (5)

Baseline Criterion Delay Trace Control Criterion Delay

120 ± 28.28 (12)

63.75 ± 14.08 (16)

41

Figure 7 shows the mean effects of olanzapine for all subjects on delay and non-delay

trial accuracy. Percent Correct is on the vertical axis and Condition is on the horizontal. Rat S4

did not respond at the 1.0 mg/kg dose, therefore the 1.0 dose is withheld from all group statistics.

Rats were better on non-delay as compared to delay trials. Also, the difference between NMTS

and MTS trained rats continued for accuracy. A 2×3 ANOVA with repeated measures on one

factor revealed a significant main effect for trial type (delay versus non-delay), F(1,10)=9.34,

p=.012, but no main effect of dose, F(2,20)=0.49, p=.619, and no significant interaction,

F(2,20)=0.38, p=.688. An analysis of the non-delay trial data revealed no main effects for

training condition (MTS versus NMTS), F(1,4)=1.23, p=.329, no main effects of dose,

F(2,8)=0.70, p=.524, and no interaction, F(2,8)=1.24, p=.339. Similarly the delay trial data

showed no main effect of training condition, F(1,4)=5.56, p=.077, no main effect of dose,

F(2,8)=0.08, p=.923, and no condition × dose interaction, F(2,8)=0.36, p=.708.

42

Figure 7. Group data. Effects of olanzapine on accuracy, for delay and non-delay trials. Percent Correct is on the

vertical axis and Condition is on the horizontal. Rat S4 did not respond at the 1.0 mg/kg dose, therefore the 1.0 dose

is withheld from all group statistics. All doses of olanzapine are in mg/kg. Error bars are standard error of the mean.

Titrating Accuracy

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Figure 8 shows the effects of olanzapine on accuracy for the individual subjects of the

titrating procedure. Percent correct is represented on the vertical axis and thedose is represented

on the horizontal. In general, all rats had high accuracy on non-delay trials and relatively lower

accuracy on delay trials regardless of drug dose. Rat S13 was something of an exception with

non-delay and delay accuracies just above chance. It could be argued that this rat’s MTS

accuracy was lost under the titrating procedure, as S13’s accuracy was 100% at the 2-s delay in

Experiment 1. Further, it is clear that MTS rats P21 and S4 showed levels of accuracy on both

delay and non-delay trials that are very similar to the NMTS trained rats.

44

Figure 8. Individual data. Effects of olanzapine on accuracy, for delay and non-delay trials. Percent Correct is the

vertical axis and the condition is the horizontal. All doses of olanzapine are in mg/kg. Error bars are standard error

of the mean.

P2 Titrating Accuracy (NMTS)

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Figure 9 shows the mean effects of olanzapine on three dependent measures that can be

used to characterize performance in this task: criterion, mean and longest delays, from left to

right respectively. For all panels the vertical axes are delay (s) and the horizontal axes are the

condition. All doses of olanzapine are in mg/kg. NMTS trained rats are represented with red

lines, MTS trained rats are represented with green lines. Rat S4 did not respond at the 1.0 mg/kg

dose, therefore the means presented for the 1.0 dose represent only two animals for the MTS

group and that dose was omitted from all group inferential statistics.

Olanzapine increased criterion delays (left panel) for the NMTS-, but not the MTS-

trained rats. A 2×3 ANOVA revealed the mean criterion delays show no main effect of dose,

F(2,8)=1.33, p=.317, or training condition (MTS vs. NMTS), F(1,4)=7.5, p=.051, but there was a

significant interaction (dose × condition), F(2,8)=5.80, p=.027. The simple main effects for

criterion delay revealed a significant effect for 0.1 mg/kg F(1,4)=8.858, p=.041, and 0.3 mg/kg

F(1,4)=8.193, p=.046, but not for vehicle, F(1,4)=1.002, p=.373. A Tukey’s post hoc indicated

the NMTS trained rats reached significantly longer criterion delays than the MTS trained rats at

the 0.1 mg/kg dose [q.05(2,4)=116.89], and 0.3 mg/kg dose [q.05(2,4)=129], but not vehicle

[q.05(2,4)=30.75].

Similar results were found for the mean delays (middle panel-the sum of all delays

divided by the number of delay trials). NMTS-trained rats reached longer mean delays than

MTS-trained rats, especially at 0.1 and 0.3 mg/kg. There was no main effect of dose,

F(2,8)=1.96, p=.202, or training condition, F(1,4)=6.98, p=.057, but there was a significant

interaction, F(2,8)=5.87, p=.026. Simple main effects for the mean delays reveal a significant

effect for 0.1 mg/kg F(1,4)=7.89, p=.048, and 0.3 mg/kg F(1,4)=8.561, p=.043, but not for

vehicle, F(1,4)=2.110, p=.220. Tukey’s post hoc indicated the NMTS trained rats reached

46

significantly longer mean delays than the MTS trained rats at the 0.1 mg/kg dose

[q.05(2,4)=70.63], and 0.3 mg/kg dose [q.05(2,4)=75.52], but not vehicle [q.05(2,4)=28.69].

Finally, the right panel shows the effects of olanzapine on the longest delay reached and

responded to accurately in a titrating session. Unlike the criterion and mean delay measures the

longest delay measure yielded no significant results. There was no main effect of dose,

F(2,8)=2.33, p=.159, no main effect of training condition , F(1,4)=5.48, p=.079, and no

interaction, F(2, 8)=4.22, p=.056.

The criterion delay measure has less variability than the longest delay measure but

slightly more than the mean; this effect also holds for the length of delays across these measures;

longest delays are longer and mean delays are shorter than the criterion delays. The difference in

variability between the measures is what prevents the longest delay measure from being

significant. However, for all three measures discussed above and displayed below, the effects

were very similar; in other words, the slope of each curve across measures is consistent. The

difference in delays between rats trained to NMTS and MTS should be noted, but perhaps more

interesting is the complete lack of effect of olanzapine at any dose for the MTS trained rats. One

would expect that if there is an increase in delay in the NMTS group there should be a

corresponding increase in the MTS group; these data suggest that there may be some

pharmacological or behavioral significance to the difference between MTS and NMTS

performances.

47

Figure 9. Group data. Effects of olanzapine on longest delay reached (left panel), criterion delay (middle panel), and

mean delay (right panel). Rats trained to NMTS are red lines, those trained to MTS green. All doses of olanzapine

are in mg/kg. Error bars are standard error of the mean.

Criterion Delay Olanzapine

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Figure 10 shows the individual data for the criterion, mean and longest delay effects of

olanzapine. All graphical explanations are the same as in Figure 9 and in general, the effects

noted in Figure 9 are evident in individual subjects. Further, as in Figure 9, the three measures

all exhibit similar slopes for each rat. P2 (NMTS) showed the most robust and consistent effect

of olanzapine at the 0.1 and 0.3 mg/kg doses. However, the other NMTS-trained rats, P1 and S3,

also increased the length of delay but with less consistency. By examining the individual

subject data in Figure 10 it is clear that the MTS rats showed no effect of olanzapine whatsoever.

49

Figure 10. Individual data. Effects of olanzapine on longest delay reached (left panel), criterion delay (middle

panel), and mean delay (right panel). Rats trained to NMTS are red lines, those trained to MTS green. All doses of

olanzapine are in mg/kg. Error bars are standard error of the mean.

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50

General Discussion

The baseline performance of rats in Experiment 1 demonstrated a clear delay gradient for

MTS rats (but not NMTS), exhibiting the characteristics of a forgetting function; as delays

increase accuracy decreases. Working memory effects in tasks of this type are often

operationally defined by delay gradients/forgetting functions (Wolff & Leander 2003; Vigouroux

et al., 1992), and thus the present task may be seen as a measure of working memory. It should

be noted that rats trained to NMTS were able to perform more accurately at all delays than those

rats trained to MTS, and therefore the NMTS-trained rats did not demonstrate baseline forgetting

functions. Olanzapine failed to produce effects at any dose or delay for either the MTS- or

NMTS-trained rats, although the NMTS-trained rats did seem to indicate a trend for

improvement at the 20 and 30-s delays at the 0.1 and 0.3 mg/kg doses. Further, the individual

differences seen between rats trained to MTS or NMTS indicated a need for a procedural change.

Experiment 2, the titrating procedure, addressed the previous concern in an attempt to detect

effects of olanzapine in a possibly more sensitive procedure.

The titrating procedure utilized in Experiment 2 was validated as consistent delays were

reached for each rat. In Experiment 2 it became apparent early on that the titrating procedure

allowed rats to reach consistent delays of over 50 s. Further, the individual differences across

groups were very clear as well; NMTS trained rats consistently reached much longer delays than

MTS trained rats. In other words, rats were capable of consistently reaching delays far beyond

those we had programmed into the fixed delays of Experiment 1, and specifically rats trained to

NMTS reached much higher delays than those rats trained to MTS. This is not all that surprising

given that the NMTS trained rats maintained a high level of accuracy at the 20 and 30-s delays in

Experiment 1 and proceeded to consistently reach criterion delays over 50 s, whereas the MTS

51

trained rats had a much lower accuracy at the 20 and 30-s delay in Experiment 1 and failed to

consistently reach criterion delays longer than 50 s. Further, in Experiment 2 olanzapine

significantly increased the criterion and mean delays reached for NMTS trained rats at the 0.1

and 0.3 mg/kg doses, but not for rats trained to MTS. This finding while perplexing does fit

nicely with the trends in performance seen in Experiment 1, in other words, in both experiments

the NMTS trained rats showed evidence of improved performance following administration of

olanzapine, whereas the MTS trained rats had inconsistent effects in Experiment 1 and no effect

in Experiment 2.

The high accuracy maintained at long delays (Experiment 1) and length of delays reached

(Experiment 2) is somewhat unexpected with rodent subjects. The majority of D(N)MTS studies

with rats employ fixed delays usually between 1 and 30 s, with rats performance at the longest

delays declining to chance levels (Gemperle et al. 2003; Didriksen, 1995; van Hest & Steckler,

1996; Steckler, 1998). However, Ravel, Elaagouby &Gervais (1994), employing olfactory

stimuli in a modified T-maze, found that rat’s performance was maintained well over 90% even

at 30-s delays. In similar visual D(N)MTS tasks with pigeons’, delays presented on average

were not longer than 16 s and with monkeys generally not more than 10 min, with accuracy

falling to chance at the longest delays (Watson and Blampied, 1989; Steckler 1992; Hudzik and

Wenger,1992; Tavares and Tomaz, 2002). However, in Experiment 1 all rats maintained

accuracy above 75% at the 30-s delays. Further, the rats in Experiment 2 reached very long

criterion delays, even longer than those delays achieved by squirrel monkeys in the Hudzik and

Wenger procedure. As there are currently no published accounts of rats in a titrating D(N)MTS

procedure, the length of delays reached in the current titrating experiment cannot be compared.

These results indicate that the current procedures and parameters allow for rats to perform

52

accurately at delays never seen, and in a non-spatial procedure. Taken with the results of the

Ravel, Elaagouby &Gervais (1994) study, the common variable in the two studies is the

olfactory modality. Perhaps the salience of using olfactory stimuli for the rat allows for

performances that until recently have yet to be seen.

The titrating procedure was also able to detect drug effects on responding that the fixed

delay procedure could not. However, this effect was again limited to the rats trained to NMTS;

although the effect was most robust in P2, both P1 and S3 showed inconsistent increases in

delays reached with both 0.1 and 0.3mg/kg olanzapine. Interestingly, although the MTS rats

maintained a lower average criterion delay, there was no effect of olanzapine at any dose in any

MTS rats to improve or impair performance.

The differences seen between MTS and NMTS trained rats in both accuracy at longer

fixed delays in Experiment 1 and the criterion delays reached in Experiment 2 are hard to

interpret. Carter and Werner (1978), in a review of the learning literature in pigeons, found that

in at least three studies there were no differences in acquisition between pigeons trained to MTS

or NMTS. Further, Tavares and Tomaz (2002) found no difference in acquisition for capuchin

monkeys trained to MTS or NMTS and also no difference in accuracy on delays, up to 600 s,

between the two groups. Finally, Nakagawa (1999) found no difference in accuracy between rats

trained to MTS or NMTS. These results are all in contrast to results shown in the current

experiment. There is some documentation citing differing effects of brain lesions between the

two procedures. Seamans, Lapish, & Durstewitz (2008) lesioned the pre-frontal cortex (PFC) of

rats after NMTS or MTS training and found that rats trained to NMTS retained their ability to

non-match at a high accuracy where as MTS trained rats were severely impaired after the same

lesions. It could be that the PFC is the brain structure that modulates ability on the MTS task and

53

NMTS is controlled elsewhere, an effect that would help to explain the differential effects of

olanzapine on the two different training procedures. Further, perhaps the olfactory modality in

the rat provides a much more sensitive analysis of the abilities of rats to MTS or NMTS, and

these effects have just never been documented.

In sum, Experiment 1 failed to enhance performance with olanzapine; however,

Experiment 2 was successful in producing an ―enhancement‖ effect with rats trained to NMTS as

exhibited by an increase in criterion and mean delays. The results of Experiment 2 add to the

findings of Wolff and Leander (2003) to increase or enhance memory performance with

olanzapine, and in this case in a non-spatial task, increasing the generalization of olanzapine to

produce an ―enhancement‖ of performance in tasks of this type with rats. However, the results

between Wolff and Leander and the current study are hard to compare as the nature of the two

studies varies so widely. For example, this study was a non-spatial, match/non-match-to-sample

study, used fixed and titrating delays and analyzed percent correct/criterion delays; the Wolff and

Leander study was a spatial, non-match-to-position study that used one 7-h delay, and only

analyzed errors. Further, there is currently no study to compare the effects found with

Experiment 2; to my knowledge this is the first successful study of rats utilizing a titrating

method of delays. However, as the Wolff and Leander study was the only study we could find

indicating an enhancement of performance with rats with olanzapine, it was chosen for

replication. It should be noted that both Wolff and Leander and Gemperle et al. (2003), were the

only studies found and referenced that showed enhancement with antipsychotic drugs, and both

were non-matching procedures. Perhaps the key to finding improvements in performance in

tasks of this type is the methodological differences between the MTS and NMTS tasks.

54

A much larger sample size of both NMTS and MTS rats in the titrating procedure would

be most helpful in elucidating the difference between these two conditions, either

pharmacologically or physiologically. The ability of the titrating procedure to detect both

improvements and impairments with the same compound makes this procedure a very valuable

tool for assessing drug effects on working memory. Further, the olfactory modality allowed for

relatively rapid acquisition of both tasks, which may prove useful for high throughput screening

of compounds. Ideally, this task could be automated and much more precise timing of

experimental events could lead to greater stimulus control and therefore less variability seen

between subjects and groups.

55

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