Diss. ETH No. 14968

PSYCHOSTIMULANT WITHDRAWAL AS AN ANIMAL

MODEL OF SCHIZOPHRENIA

A dissertation submitted to the

SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH

for the degree of

Doctor of Natural Science

presented by

Holger Russig

Dipl.-Biologist, Humboldt University Berlin

born April 21th, 1970

citizen of Germany

Prof. Dr. Joram Feldon, examiner

Prof. Dr. Jeffrey Gray, co-examiner

Dr. Carol Murphy, co-examiner

2003

TO MY SON JAKOB

ACKNOWLEDGEMENTS This thesis was performed at the Behavioral Neurobiology Laboratory of the Swiss Federal Institute of Technology (ETH Zurich) in Schwerzenbach, Switzerland. My best thanks are due to Prof. Dr. Joram Feldon who supervised me during the last three years and explained me how to create a poster within 2 hours. Seriously, thanks for giving me the opportunity to come in your lab, for all the scientific discussions, providing excellent research facilities, nice dinners and much more…….. I am sincerely grateful to Prof. Jeffrey Gray who supported this thesis by agreeing to co-examinate it. I am grateful to Dr. Carol Murphy for being a coexaminer and her helpful assistance during the last years. Best thanks are also due to all the technicians, Oliver Aspiron, Pascal Guela, Sepp Terluci and others for animal care, Bonnie Strehler and Jane Fotheringham for being the good mum of the laboratory, and all the people in the background they helped that the machine is going on in Schwerzenbach. Great thanks go to Peter for dealing with all my computer problems. Best thanks go to all members of the Schwerzenbach tigers, in particular Marianne for the discussions about statistic and Tilly for providing an anti-depression drawer. Special thanks are due to my Phd companions Tobias and Marie for the discussions and the great time in Marseille with the cheese and the wine in the bag pack. A lot of studies were conducted in collaboration; therefore I would like to thank Marie, Aneta, Makoto, Andre, Isabelle, Julia, Matti, Andreas, Chris, and Boris. Great thanks go to Ben and Gael for the scientific discussion of my thesis and for much more issues, which I shared with them. Ein ganz herzlicher Dank geht an meinen Sohn Jakob, der oft auf mich verzichten musste in drei Jahren escalating dose. Ganz besonders möchte ich Kathrin danken, die mich nun seit 12 Jahren begleitet und unterstützt. Danke für die Zeit die Du mir eingeräumt und die Kraft die Du mir gegeben hast diese Arbeit zu verwirklichen. Finally, warm thanks go to my family and my close friends for their continuous support throughout this period. Without their help this achievement would not have been possible.

TABLE OF CONTENTS

1

TABLE OF CONTENTS

LIST OF ABBREVIATIONS 3

ABSTRACT 5

ZUSAMMENFASSUNG 8

INTRODUCTION 11

CHAPTER 1 21

Latent inhibition, but not prepulse inhibition, is reduced during withdrawal

from an escalating dosage schedule of amphetamine

CHAPTER 2 32

Clozapine and haloperidol reinstate latent inhibition following its disruption

during amphetamine withdrawal

CHAPTER 3 46

Prepulse inhibition during withdrawal from an escalating dosage schedule of

amphetamine

CHAPTER 4 82

The acquisition, retention and reversal of spatial learning in the Morris water

maze task following withdrawal from an escalating dosage schedule of

amphetamine in Wistar rats

CHAPTER 5 114

Amphetamine withdrawal modulates FosB expression in mesolimbic

dopaminergic target nuclei: effects of different schedules of administration

CHAPTER 6 129

Amphetamine withdrawal does not produce a depressive-like state in rats as

measured by three behavioral tests

DISCUSSION 148

1 Amphetamine withdrawal as an animal model of schizophrenia 149

1. 1 Effects of amphetamine withdrawal on latent inhibition 150

1. 2 Effects of amphetamine withdrawal on prepulse inhibition 155

1. 3 Cognitive effects of amphetamine withdrawal 157

2 Amphetamine withdrawal as an animal model of depression 158

TABLE OF CONTENTS

2

3 The role of different amphetamine administration schedules on effects

during drug withdrawal 160

4 Brain changes associated with amphetamine withdrawal 161

5 Conclusions 162

APPENDIX 175

Haloperidol and clozapine antagonize amphetamine induced disruption of latent

inhibition in conditioned taste aversion

LIST OF PUBLICATIONS 196

CURRICULUM VITAE 198

LIST OF ABBREVIATIONS

3

LIST OF ABBREVIATIONS 5-HIAA: 5-hydroxy indoleacetic acid 5-HT: 5-hydroxytryptamine, serotonin AC: anterior cingulate prefrontal cortex ACTH: adrenocorticotropin hormone AMPH: amphetamine APO: apomorphine BDNF: brain-derived neurotrophic factor BLA: basolateral amygdala cAMP: cyclic adenosine monophosphate CAR: conditioned avoidance response Cdk5: cyclin-dependent kinase 5 ceA: central amygdala CER: conditioned emotional response CLZ: clozapine COND: conditioning CORT: corticosterone CREB: cyclic-AMP response-element-binding proteine CRF: corticotropin-releasing factor CS: conditioned stimulus CTA: conditioned taste aversion DA: dopamine DLS: dorsolateral striatum DMS: dorsomedial striatum DOPAC: 3,4-dihydroxyphenylacetic acid E: east EDTA: ethylenediaminetetraacetic acid ESC: escalating dose schedule FR: fixed ratio FRA: Fos-related antigen GluR2: glutamate receptor 2 HAL: haloperidol HClO4: perchlor acid HPA: hypothalamic-pituitary-adrenal axis HPLC: high performance liquid chromatography HVA: homovanillic acid i.p.: intraperitoneal ICSS: intra-cranial self-stimulation IEG: immediate-early gene IL: infralimbic prefrontal cortex INT: intermittent dose schedule ITI: inter-trial interval LI: latent inhibition LiCl: lithium chloride meA: medial amygdale

LIST OF ABBREVIATIONS

4

mPFC: medial prefrontal cortex N: north NA2EDTA: disodium ethylenediamine tetraacetate Na2S2O5: sodium metabisulfid NAC: nucleus accumbens NaCl: sodium chloride NPE: non-preexposure PBS: phosphate-buffered saline PE: preexposure PET: positron emission tomography PKA: proteine kinase A PL: prelimbic prefrontal cortex PPI: prepulse inhibition PR: progressive ratio Q: quadrant S: south SAL: saline SPF: specific-pathogen-free US: unconditioned stimulus VLS: ventrolateral striatum VMS: ventromedial striatum VTA: ventral tegmental area W: west

ABSTRACT

5

ABSTRACT

Schizophrenia is a neuropsychiatric brain disorder for which several causes have

been suggested, including genetic, environmental and neurodevelopmental influences

which can lead to altered neurotransmitter regulation. It has been suggested that the

presence of a sensitized DA circuitry is central to the disease of schizophrenia. Thus, the

disease could be understood as a case of endogenous sensitization of the

mesocorticolimbic system. A state of sensitization can be induced in animals by repeated

administration of psychostimulants, such as amphetamine (AMPH), followed by a period

of withdrawal from the drug. Such animals exhibit enhanced (sensitized) behavioral

response associated with enhanced dopamine release in the mesocorticolimbic system in

response to a drug or a stress challenge. This phenomenon is referred to as behavioral

sensitization and is suggested to be the consequence of an ongoing neuroadaptive process

initiated by, and in response to, repeated exposures to psychostimulant drugs.

The aim of the present thesis was to investigate whether such sensitization-

induced neuroadaptations can give rise to schizophrenia-related behavioral alterations.

We investigated rats during withdrawal from an escalating dosage schedule of AMPH

(three daily injections for six days, 1.0 – 5.0 mg/kg AMPH) in the absence of an explicit

drug challenge.

Firstly, we assessed the possible presence of schizophrenia-like behavioral

abnormalities in rats undergoing AMPH withdrawal in two paradigms considered to be of

specific relevance to the information processing deficits associated with schizophrenia -

latent inhibition (LI) and prepulse inhibition (PPI). Disruption of LI and PPI has been

reported in at least some subsets of schizophrenic patients. LI was found to be abolished

during the first 2 weeks of withdrawal (chapter 1, 2 and 3). On withdrawal day 4, the

neuroleptic drugs haloperidol (0.03 mg/kg) and clozapine (5 mg/kg) administered prior to

the test essentially reversed the disruptive influence of AMPH withdrawal on LI (chapter

2). The efficacy of neuroleptic treatment reported here has provided the initial support for

the idea that AMPH withdrawal might be able to mimic the critical neuroadaptive

changes in schizophrenic patients. In contrast, PPI was not affected, suggesting that the

ability of AMPH withdrawal to mimic schizophrenic symptomatology is, under these

ABSTRACT

6

circumstances, specific (chapter 3). It further suggests that LI and PPI disruption in

schizophrenic patients might represent two dissociable pathological mechanisms.

We went on to examine the possible presence of mnemonic deficits in AMPH

withdrawn rats using the Morris water-maze (chapter 4). We demonstrated that AMPH

withdrawal affected neither the acquisition nor the retention of spatial memory. However,

in the same animals we obtained evidence for enhanced reversal learning. This finding is

consistent with the view that AMPH withdrawal can enhance behavioral switching, and it

is in line with one theoretical account as to how LI can be disrupted.

Next, we attempted to characterize neurochemical and molecular correlates of the

neuroadaptive processes associated with AMPH withdrawal (chapter 5). The expression

of the transcription factor FosB, a putative indicator of neuronal changes associated with

the processes of psychostimulant withdrawal, was found to be markedly increased in the

nucleus accumbens shell and basolateral amygdala when examined on withdrawal day 4.

Post-mortem neurochemical analyses of dopamine, serotonin and their metabolites in

mesocorticolimbic brain regions, including nucleus accumbens and amygdala, did not

reveal significant effects. These results lend support for the idea that altered gene

expression patterns in the nucleus accumbens shell and basolateral amygdala might be

responsible for at least some of the behavioral consequences of AMPH withdrawal,

including the disruption of LI.

Anhedonia is a common symptom of both schizophrenia and depression. Previous

studies have shown anhedonia, but no other classes of depressive symptoms, in AMPH

withdrawn rats. We therefore, investigated AMPH withdrawn rats for behavioral

alterations related to depressive symptoms other than anhedonia. AMPH withdrawn

animals exhibit unaltered learned helplessness and immobility in the Porsolt swim test,

although they showed expression of behavioral sensitization (see chapter 6). We conclude

that although AMPH withdrawal may model anhedonia, it is ineffective in mimicking

other classes of depressive symptoms in rats.

We can conclude from these findings that withdrawal from an escalating dosage

schedule of AMPH is an animal model of selected symptoms of schizophrenia with face,

predictive and theoretically based construct validity. The model can be used to screen for

drugs with antipsychotic properties in the absence of any concurrent acute

ABSTRACT

7

pharmacological intervention. In addition, AMPH withdrawal specifically affects the

pathological mechanisms underlying LI, but not PPI, disruption. Taken together, this

model can be useful for the understanding of the biological basis of specific symptoms of

schizophrenia.

ZUSAMMENFASSUNG

8

ZUSAMMENFASSUNG

Schizophrenie ist eine psychiatrische Erkrankung des Gehirns, die zu

Verhaltensveränderungen führt. Die neuronalen Ursachen dieser Verhaltensänderungen und

deren Entstehung während der Entwicklung sind weitgehend unverstanden. Manipulierte

Tiere, die Verhaltensanomalien oder neuronale Veränderungen ähnlich denen schizophrener

Patienten zeigen, können als Tiermodell von grossem Nutzen sein, um die Ursachen der

Krankheit zu erforschen und potentielle Arzneistoffe zu entwickeln.

Symptome der Schizophrenie können bei Menschen auch ausgelöst werden, wenn

Psychostimulantien wie Amphetamin (AMPH) oder Kokain regelmässig konsumiert werden.

Bei wiederholter Einnahme dieser Substanzen können adaptive Anpassungen im Gehirn

entstehen, die zum Phänomen der Verhaltens-Sensitivierung (behavioral sensitization) führen.

Sensitivierung ist eine erhöhten lokomotorischen Aktivität ausgelöst von einer einzelnen

AMPH Gabe während des Psychostimulatienentzugs im Vergleich zur Verhaltensreaktion

wenn AMPH das erste Mal gegeben wird. Neuronal ist diese erhöhte Reaktion mit einer

gesteigerten Ausschüttung des Neurotransmitters Dopamin im Striatum (speziell im Nucleus

accumbens) verbunden und diese beruht neuronalen Anpassungen als Folge wiederholter

Psychostimulant-Gabe. Verhaltens-Sensitivierung kann auch durch Stress ausgelöst werden.

Es wird postuliert, dass ähnliche Mechanismen der Entstehung von Schizophrenie zu Grunde

liegen könnten. Ausserdem gibt es Hinweise, dass auch Schizophreniepatienten nach einer

einmaligen Gabe von AMPH mehr Dopamin im Striatum ausschütten. Dabei korreliert die

Menge an Dopamin mit der Schwere der Symptome. Diese Daten bilden die Grundlage der

endogenen Sensitivierungs-Hypothese der Schizophrenie. Ziel der vorliegenden Doktorarbeit

war es zu testen, ob Ratten während des Entzugs von einer wiederholten eskalierenden Gabe

von AMPH Verhaltensanomalien zeigen, die ähnlich denen von Schizophreniepatienten sind.

Auf dieser Basis könnte ein neues Tiermodell für die Schizophrenie-Forschung entwickelt

werden.

Nachdem wir zeigen konnten, dass das von uns genutzte AMPH Injektionsschema (3

tägliche Injektionen über 6 Tage, 1-5 mg/kg AMPH) zu Verhaltens-Sensitivierung führt,

testeten wir die Tiere ausschliesslich ohne erneute AMPH Injektion während des Entzugs, um

die Effekte zu untersuchen, die die neuronalen Anpassungen auf das Verhalten haben.

Schizophreniepatienten leiden unter Defiziten in der Informationsverarbeitung von Reizen,

die als Aufmerksamkeitsstörungen bezeichnet werden. Verschiedene Verhaltensparadigmen

ZUSAMMENFASSUNG

9

können zur Messung dieser Informationsverarbeitung sowohl beim Menschen als auch bei der

Ratte eingesetzt werden. Latente Inhibition (LI, latent inhibition) beschreibt die reduzierte

Konditionierung zu einem Stimulus, wenn dieser Stimulus vorher ohne Konsequenz exponiert

wurde. Präpuls Inhibition (PPI, prepulse inhibition) beschreibt einen Prozess, bei dem die

durch einen lauten Puls ausgelöste Schreckreaktion (startle response) reduziert wird, wenn vor

diesem Puls ein leiserer Präpuls präsentiert wird, der nicht zu einer Schreckreaktion führt.

Schizophrene Patienten zeigen Defizite in beiden Phänomenen. Während der ersten 14 Tage

des AMPH Entzugs bei Ratten fanden wir reduzierte LI, während PPI unverändert blieb. Die

LI Reduktion während des AMPH Entzugs konnte verhindert werden, wenn die Tiere vor

dem Test mit Haloperidol oder Clozapin behandelt wurden. Diese Antipsychotika werden in

der medizinischen Praxis zur Therapie schizophrener Symptome eingesetzt.

Da Schizophrenie auch mit Defiziten im räumlichen Arbeitsgedächtnis verbunden ist,

untersuchten wir weiterhin, ob diese Ratten kognitive Defizite im Wasserlabyrinth (Morris

water maze) zeigen. AMPH Entzug hatte keinen Einfluss auf räumliches Lernen und

Gedächtnis. Wir fanden ein leicht verbessertes Lernen, wenn die Zielplattform in den

gegenüberliegenden Quadranten versetzt wurde (reversal learning). Dieses Phänomen ist mit

erhöhtem „switching“ erklärbar und wird als eine theoretische Grundlage von gestörter LI

angesehen.

In weiteren Studien untersuchten wir, welche Hirnstrukturen und Neurotransmitter,

die eventuell für die gefundenen LI Defizite verantwortlich sein könnten, während des AMPH

Entzugs verändert sind. Erhöhte Expression des Transkriptionsfaktors FosB wurde in

Teilregionen der Amygdala (basolateral amygdala) und des Striatums (nucleus accumbens

shell) gefunden, konnten aber nicht mit einer Veränderung der basalen Gewebewerte der

Neurotransmitter Dopamin, Serotonin oder deren Metaboliten korreliert werden. Frühere

Studien haben ergeben, dass diese Hirnregionen an der Regulation von LI beteiligt sind.

Einige Wissenschaftler betrachten die frühe Phase des AMPH Entzugs als Tiermodell

für Depression, da in dieser Zeit Anhedonia auftreten kann, ein wichtiges Symptom von

Depression, das auch für die Schizophrenie von Bedeutung ist. Wir testeten, ob während des

AMPH Entzugs auch weitere Verhaltens-Effekte in Tests auftreten können, die depressive

Symptome wie Hilflosigkeit (learned helplessness) und Verzweiflung (behavioral despair)

widerspiegeln. Dies war nicht der Fall, und wir fanden auch keine Effekte in der Menge

freigesetzter Stress-Hormone. Wir schliessen daraus, dass unsere Behandlung neben

Anhedonia keine weiteren Symptome von Depression hervorruft.

ZUSAMMENFASSUNG

10

Aus den empirischen Befunden der vorliegenden Arbeit kann geschlossen werden,

dass Ratten, die einer wiederholten eskalierenden AMPH-Gabe ausgesetzt sind, während des

Entzugs einige (aber nicht alle) Defizite schizophrener Patienten zeigen. LI Reduktion, die

von einer veränderten Gen-Expression in der „basolateral amygdala“ und im „nucleus

accumbens shell“ herrühren könnten, wird durch eine Neuroleptika-Behandlung wieder

eliminiert. Wir schliessen daraus, dass Ratten, die einer wiederholten eskalierenden AMPH-

Gabe ausgesetzt waren, ein geeignetes Tiermodell zur Untersuchung der Neurobiologie

spezieller Symptome der Schizophrenie darstellen. Auf der Grundlage der vorgenommenen

Manipulation sollten Arzneistoffe auf ihre antipsychotischen Eigenschaften hin untersucht

werden können. Die Untersuchungen haben gezeigt, dass das hier postulierte Tiermodell für

Schizophrenie die Kriterien der „face“ und „predictive“ Validität erfüllt. Aufgrund des

theoretischen Hintergrundes des Tiermodells kann auch von der Erfüllung des Kriteriums für

die „construct“ Validität ausgegangen werden.

INTRODUCTION

11

INTRODUCTION

Schizophrenia is a debilitating mental disorder affecting approximately 1 % of the human

population irrespective of race and origin. It afflicts individuals in their early adulthood and

represents a major socioeconomic burden. Besides the florid psychotic symptoms, such as

hallucinations and delusions, schizophrenia is also characterized by a myriad of symptoms

extending from the emotional to the cognitive domains (Andreasen and Olsen 1982, Goldberg

and Gold 1995, and see DSM IV Diagnostic Manual, American Psychiatric Association, 2000).

Although it is now widely accepted that schizophrenia is a disease of the brain, its precise

biological basis remains poorly understood despite over a century of research. A number of

current theories postulate that schizophrenia involves a complex interplay among genetic,

developmental, and environmental factors (e.g., Lewis and Lieberman 2000), and a complete

understanding of the disease would necessarily require an integration of these factors.

Importantly, psychotic symptoms are not unique to schizophrenic patients. In humans, such

symptoms can be observed in otherwise healthy individuals who abuse psychostimulants

regularly (Connell 1958, Ellinwood 1967, Snyder 1973, Angrist 1994). One such

psychostimulant is amphetamine, the drug of interest in the present thesis. Amphetamine abuse

can induce psychosis in healthy individuals and exacerbate psychotic behavior in schizophrenic

patients. Pharmacologically, amphetamine is most widely known for its effect in enhancing

dopamine release in the mesocorticolimbic system (comprising the ventral tegmental area,

striatum, nucleus accumbens and medial prefrontal cortex; (Robinson and Becker 1986, Pierce

and Kalivas 1997, Vanderschuren and Kalivas 2000)). One hypothesis following from these

observations is that hyperdopaminergia in the mesocorticolimbic system might be related to the

emergence of psychotic-like symptoms, and by extension, a similar neurochemical imbalance

might also be involved in the pathogenesis of schizophrenia. This idea is known as the

“dopamine hypothesis” of schizophrenia (Snyder 1976, McKenna 1987, Carlsson 1988).

This hypothesis is further supported by the observation that symptoms of schizophrenia can

be effectively treated by drugs that block dopaminergic transmission (Creese et al. 1976).

Although many existing antipsychotic drugs clearly affect other neurotransmitter systems,

dopaminergic blockade remains one of the clearest predictors of a drug’s clinical efficacy against

INTRODUCTION

12

acute psychosis (Seeman et al. 1976). Recent modifications of the “dopamine hypothesis” also

postulate that functional imbalances in dopamine transmission can result from disturbances in

other neurotransmitter systems, such as the glutamatergic and GABAergic systems, which can

also modulate the action of the mesocorticolimbic system (Coyle 1996, Tamminga 1998,

Pearlson 2000, Benes and Barretta 2001, Carlsson et al. 2001).

Attempts to delineate causes of the postulated neurochemical imbalances in schizophrenia

have emphasized the importance of brain development and/or neurodegenerative processes

(Harrison 1997, Woods 1998, Lieberman 1999). Amongst those, Lieberman and colleagues

(1990, 1997) have developed an integrative theory in which schizophrenia is conceptualized to

consist of three distinct clinical stages: a neurodevelopmental (premorbid) stage, a neuroplastic

(prodromal, onset, and deteriorative) stage, and a neuroprogressive (deteriorative and

chronic/residual) stage. According to their account, neurodevelopment plays a critical role in

determining the functional integrity of the dopamine/mesocorticolimbic system, especially in

response to environmental stress factors. An initial neurodevelopmental defect could lead to

maladaptive changes in stress response mechanisms that eventually culminate in a functional

hyperdopaminergic state via neural plasticity, and, finally, in the emergence of psychotic

symptoms (Lieberman et al. 1990, 1997).

Central to Lieberman’s theory is the phenomenon of behavioral sensitization, which refers to

the observation that prior repeated exposures to psychostimulants enhance the response to the

same drug following a period of withdrawal. Psychostimulant-induced behavioral sensitization

has been documented in humans, although its neural basis has been extensively investigated in

animals (Robinson and Becker 1986, Strakowski et al. 1996). The sensitized response in animals

is associated with an elevated dopamine release in the nucleus accumbens, which is believed to

result from neural adaptive changes developed during repeated psychostimulant exposures and

the subsequent withdrawal period (Robinson and Becker 1986). Lieberman et al. conjecture that

plastic processes resembling those of behavioral sensitization underlie the pathogenesis of

schizophrenia. However, instead of repeated exposures to psychostimulant drugs, schizophrenic

patients are said to have developed a sensitization-like neurological state through endogenous

mechanisms intrinsic to their neurodevelopmental defects. This is known as the “endogenous

sensitization hypothesis” of schizophrenia. The idea behind endogeneous sensitization can be

readily appreciated through the concept of “cross sensitization”. Cross-sensitization refers to the

INTRODUCTION

13

observation that repeated exposures to psychostimulants can also exacerbate the response to

stress, and vice versa (Antelman et al. 1980, Prasad et al. 1998). For example, if long-term

abusers of psychostimulants are challenged with stress or a single dose of methamphetamine after

a period of abstinence, i. e. withdrawal from drug use, they may exhibit psychotic-like symptoms

similar to or even more severe than previous psychosis induced by repeated drug use (Sato et al.

1983, 1992). In support of this theory, schizophrenic patients show a sensitized psychotic

response to psychostimulant challenge without prior exposure to psychostimulants (Janowsky

and Davis 1976, van Kammen et al. 1982 a, b). Neurochemically, enhanced striatal release of

dopamine has been reported in schizophrenic patients in response to a single amphetamine

challenge. Notably, the magnitude of the enhanced dopamine release is positively correlated with

the severity of symptoms (Laruelle 2000).

According to Lieberman’s framework, animals withdrawn from repeated psychostimulant

administration can be considered as a potential model for at least some of the pathological

mechanisms underlying schizophrenia. This, however, does not represent an entirely novel

approach for modelling possible neural changes associated with schizophrenia. Indeed, the

expression of behavioral sensitization triggered by a challenge in animals has long been

suggested as a model of human psychoses (Robinson and Becker 1986). In the studies described

in the present thesis, we emphasize the distinction between the expression of behavioral

sensitization with or without administration of a drug challenge. Experiments were conducted to

characterize behavioral changes expressed during withdrawal from repeated psychostimulant

administration, i.e. in the absence of an explicit drug challenge. This allows one to examine the

behavioural consequences of the ongoing neuroadaptive changes during the induction phase

(including both repeated psychostimulant administration and the withdrawal period) of

behavioral sensitization without the influence of an acute pharmacological trigger. Few attempts

have been made in this direction until very recently by researchers who recognize that the

neuroadaptive changes underlying the induction of behavioral sensitization are of particular

relevance to Lieberman’s theory of schizophrenia (e.g., Murphy et al. 2001a, b).

It is the impetus of the present thesis to assess and to evaluate whether animals withdrawn

from repeated exposures to amphetamine exhibit specific behavioral alterations resembling those

observed in schizophrenic patients in the absence of an explicit challenge. The experiments serve

INTRODUCTION

14

as tests for the face validity of the use of amphetamine withdrawal as an animal model of

schizophrenia-related symptoms.

To this end, my colleagues and I conducted a series of experiments in rats that had been

subjected to repeated dosing regimes of amphetamine. During withdrawal, the animals were then

subjected to behavioral tests of relevance to the symptomatology of schizophrenia. In addition,

neurochemical characterizations of the mesocorticolimbic dopamine system in these animals

were carried out. These were further supplemented by biochemical assays of the hormone release

from the hypothalamic-pituitary-adrenal (HPA) axis in response to stress, and

immunohistochemical studies of the expression of immediate early genes.

Firstly, we investigated the effects of amphetamine withdrawal in two behavioural paradigms

considered to be of specific relevance to the information processing deficits associated with

schizophrenia - latent inhibition (LI) and prepulse inhibition (PPI; Weiner and Feldon 1997,

Swerdlow et al. 2000, Weiner 2000). Impairments in LI and in PPI have been documented in

schizophrenic patients (Baruch et al. 1988, Gray et al. 1992, 1995, Braff et al. 2001). Parallel

deficits in rats induced by a variety of drugs have been investigated as potential models of such

information-processing deficits (Moser et al. 2000, Geyer et al. 2001). These experiments

(described in Chapters 1 - 3) represent the first attempts to assess the effects of withdrawal from

an escalating dosage schedule of amphetamine in these two paradigms. This schedule was

selected because of its resemblance to the pattern of amphetamine abuse in humans, which can

give rise to psychotic-like behaviour (Davis and Schlemmer 1980, Angrist 1994).

Secondly, we explored the possible presence of mnemonic deficits in animals during the

withdrawal period (in Chapter 4). This was prompted by the reports of verbal and working

memory impairments observed in schizophrenic patients (Goldberg and Gold 1995). Two

independent cohorts of animals were trained in the Morris water maze task. One was trained prior

to amphetamine treatment and the other during the withdrawal period. This allowed us to

examine the effect of withdrawal on the acquisition and retention of spatial learning, respectively.

Next, as an attempt to search for neurochemical and molecular correlates of the neuroadaptive

processes associated with amphetamine withdrawal (Chapter 5), we carried out (i) postmortem

analysis of dopamine, serotonin and their metabolites in the mesocorticolimbic system, and (ii)

quantitative analysis of the regional expression of the transcription factor FosB, a putative

INTRODUCTION

15

indicator of neuronal changes associated with the processes of psychostimulant withdrawal

(Ujike 2002, Nestler et al. 1999, 2001, Nestler 2001).

Repeated administration of amphetamine is also known to affect emotional processing during

the initial period of withdrawal. One of the reported effects is anhedonia – a decrease in the

sensitivity/activity of the brain reward system (Wise 1982). This has been reported to follow

escalating dose schedules of amphetamine as evidenced by a reduction in (i) sexual drive, (ii) the

reward value of intracranial self-stimulation and (iii) reduced performance of progressive ratio

bar pressing for a sucrose reward (Lin et al. 1999, Wise and Munn 1995, Barr and Phillips 1999,

Barr et al. 1999). In view of the clinical observation that schizophrenic patients also exhibit

depressive symptoms including anhedonia (Gelder et al. 1989, Hausmann and Fleischhacker

2002, Kohler and Lallert 2002), we have tested whether a withdrawal schedule capable of

inducing a schizophrenia-like cognitive deficit (Chapters 1-3) also leads to such affective deficits.

We therefore performed a battery of tests including the paradigm of progressive ratio operant

responding for sucrose, Porsolt’s forced swim test, and a learned helplessness assay, in order to

assess whether specific schedules of amphetamine withdrawal are consistently associated with

signs of a depressive state, as indexed by anhedonia as well as other classes of depressive

symptoms (Chapter 6). This approach allowed us to explore whether amphetamine withdrawal is

associated with depressive-like symptoms together with psychotic-like symptoms.

Finally, I discuss these results in a general overview to conclude my work. This is aimed at

providing an assessment of the validity of amphetamine withdrawal as an animal model of

schizophrenia useful to test neuroleptic properties of new drugs and as a new tool for the

investigation of the neurobiology underlying schizophrenic symptoms.

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INTRODUCTION

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LATENT INHIBITION, BUT NOT PREPULSE INHIBITION, IS REDUCED

DURING WITHDRAWAL FROM AN ESCALATING DOSAGE

SCHEDULE OF AMPHETAMINE

Carol A. Murphy, Miriam Fend, Holger Russig and Joram Feldon

Behavioral Neuroscience 2001, 115: 1247-1256

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CLOZAPINE AND HALOPERIDOL REINSTATE LATENT INHIBITION

FOLLOWING ITS DISRUPTION DURING AMPHETAMINE

WITHDRAWAL

Holger Russig, Carol A. Murphy and Joram Feldon

Neuropsychopharmacology 2002, 26: 765-777

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CHAPTER 3 ------------------------------------------------------------------------------------------------------------

PREPULSE INHIBITION DURING WITHDRAWAL FROM AN

ESCALATING DOSAGE SCHEDULE OF AMPHETAMINE

Holger Russig, Carol. A. Murphy, Joram Feldon

Psychopharmacology, in press, published online 12. 11. 2002

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47

ABSTRACT

Rationale: Psychomotor stimulants can induce psychotic states in humans that

closely resemble those observed in patients with idiopathic schizophrenia. Attentional

and sensorimotor gating impairments are observed in schizophrenic patients using the

latent inhibition (LI) and prepulse inhibition (PPI) behavioral assays, respectively. Our

previous studies demonstrated that after 4 days of withdrawal from a period of

amphetamine (AMPH) administration, animals exhibit disrupted LI but normal PPI.

Objective: The aim of the present study was to test PPI in AMPH-withdrawn rats under

experimental conditions similar to those used to best demonstrate locomotor sensitization

following AMPH withdrawal. Methods: We examined the effects on PPI of 1) pairing

drug injections with PPI test-associated cues, 2) administration of a low-dose dopamine

agonist challenge, and 3) testing following longer withdrawal periods (23, 30, 60 days).

Results: Although none of these conditions revealed a disruption of PPI in AMPH-

withdrawn rats, we did observe that the acoustic startle response was reduced during a

restricted time period following AMPH withdrawal. Similar to our previous findings,

AMPH-withdrawn animals showed disrupted LI on day 16 of withdrawal, and locomotor

sensitization to a challenge injection of AMPH after 62 days of withdrawal. Conclusion:

We conclude that the effects of repeated AMPH on PPI are not modulated by the same

experimental parameters known to be important for eliciting locomotor sensitization, and

that withdrawal from the schedule of AMPH administration used in this study models

only specific cognitive dysfunctions linked to schizophrenic symptoms, since LI was

disrupted but PPI was not affected.

Keywords: startle, schizophrenia, latent inhibition, sensitization, rat

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INTRODUCTION

Administration of amphetamine (AMPH) can induce symptoms of psychosis in

humans. This outcome is most frequently associated with a chronic high-dose escalating

pattern of stimulant abuse (Davis and Schlemmer 1980; Angrist 1994). Given that

stimulant-induced psychosis in humans closely resembles the psychosis observed in

patients with idiopathic schizophrenia (Ellinwood 1967; Griffith et al. 1972; Snyder

1973), it has been suggested that similar neural adaptations could be responsible for the

development of these two phenomena. It is well known that repeated exposure to

psychostimulants, like AMPH or cocaine, induces psychomotor sensitization in

experimental animals. This phenomenon is indicated by a progressive behavioral

augmentation of responses (increased locomotion, stereotypies) to drug challenge, as well

as an enhanced release of nucleus accumbens dopamine (DA) following challenge

AMPH administration (Robinson and Becker 1986; Segal and Kuczenski 1994). It has

therefore been proposed that studies of the neural bases of psychomotor stimulant

sensitization might yield insights into the biological mechanisms responsible for the onset

of schizophrenia (Kokkinidis and Anismann 1980; Robinson and Becker 1986;

Lieberman et al. 1990).

Specifically, Lieberman and colleagues have suggested that a process of

endogenous sensitization, in which schizophrenic patients exhibit enhanced DA release in

response to AMPH challenge, is a key element in the pathology of the disease

(Lieberman et al. 1990; Lieberman et al. 1997). Recent PET imaging studies examining

competition for receptor occupancy between endogenously released DA and D2 receptor

radioligand binding have established that not only do schizophrenic patients show

enhanced striatal DA release in response to amphetamine administration (i.e. a sensitized

response), but that the DA response is positively correlated with the severity of their

positive symptoms (reviewed in Laruelle 2000). Thus, there is convincing evidence for

the involvement of sensitisation processes in the expression of the schizophrenic

phenotype. A number of studies have also established that during the acute phase of

withdrawal from high-dose amphetamine, animals often exhibit depressive-like

symptoms, in particular, anhedonia as it is indexed by decreased sensitivity to rewarding

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49

brain stimulation (Lin et al. 1999; Koob and Le Moal 2001; Weiss et al. 2001). However,

the state of anhedonia during amphetamine withdrawal is typically very transient,

persisting for up to only 3-5 days following the last administration. In comparison,

behavioural sensitization can persist even after prolonged periods of abstinence, a time

course which is perhaps more consistent with the long-term adaptations typically

associated with chronic mental illness.

Two animal models believed to reflect cognitive/attentional deficits typical of

schizophrenic patients are latent inhibition (LI) of classically conditioned responding and

prepulse inhibition (PPI) of the startle response (Weiner & Feldon 1997; Swerdlow et al.

2000). Both LI and PPI are disrupted in schizophrenic patients, and these deficits can be

restored by neuroleptic treatment (Gray et al. 1992, 1995; Baruch et al. 1988; Weiner &

Feldon 1997; Braff et al. 2001). Latent inhibition (LI) refers to the observation that

repeated exposure to a stimulus without consequence comes to impede the formation of

subsequent associations with that stimulus (Lubow 1973). Our previous studies have

shown that LI is disrupted in rats pretreated with escalating doses of AMPH during the

first two weeks of withdrawal (Murphy et al. 2001b). Moreover, the antipsychotic drugs

haloperidol and clozapine restored LI in AMPH-withdrawn rats (Russig et al. 2002).

Thus, the LI results during AMPH withdrawal are consistent with the hypothesis, based

on neuroimaging studies, that sensitized levels of DA are associated with an animal

model of schizophrenic deficits.

PPI is the phenomenon whereby moderate-intensity prepulse stimuli attenuate

startle responses to subsequent intense stimulation (Graham 1975; Braff et al. 1978;

Hoffman and Ison 1980). This phenomenon is thought to result from the activation of

central inhibitory mechanisms that gate behavioral responses to ensuing stimuli

(Swerdlow et al. 1992; Taylor et al. 1995). Acute administration of psychomotor

stimulants such as AMPH, apomorphine or selective D2 dopamine agonists disrupts PPI,

such that startle responses are less influenced by the prepulse presentations (Mansbach et

al. 1988; Peng et al. 1990; Bakshi et al. 1995; Caine et al. 1995; Taylor et al. 1995, Sills

1999). It is believed that PPI disruption may index the sensorimotor gating deficit

observed in schizophrenic patients (Braff et al. 1978; Braff and Geyer 1990) and that,

consequently, the assessment of psychomotor stimulant effects on PPI may be a valid

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animal model of sensorimotor gating disturbances in schizophrenia (Braff et al. 1992;

Swerdlow et al. 1992, 1994). However, previous studies from our laboratory showed no

effect of escalating dose AMPH treatment on PPI tested on day 4 of withdrawal (Murphy

et al. 2001b). Given the similarities in brain areas and cognitive functions implicated in

LI and PPI (Weiner & Feldon 1997; Swerdlow et al. 2000), and the clear disruptive

effects of withdrawal from this escalating dose AMPH schedule on LI, it is somewhat

surprising that AMPH withdrawal does not also affect PPI. The purpose of the present

investigation was to further explore this issue of PPI disruptibility during AMPH

withdrawal, by specifically examining the effects on PPI of experimental conditions that

are known to maximize locomotor sensitization effects.

The role of contextual cues in psychostimulant-related behavior has been

extensively studied in sensitization experiments (Robinson et al. 1998; Ohomori et al.

2000). It has been shown that under certain experimental circumstances, sensitization to

psychostimulants is only detected when repeated drug administrations were previously

paired with specific contextual cues (Hinson and Poulos 1981; Drew and Glick 1988;

Badiani et al. 1995; Robinson et al. 1998). There is also direct evidence that drug-

conditioned cues disrupt sensorimotor gating. A study with abstinent smokers indicated

that a presentation of smoking-associated stimuli reduced PPI (Hutchison et al. 1999).

Moreover, other studies have shown that PPI was reduced during withdrawal from

repeated DA agonist treatment when drug administrations were repeatedly paired with

PPI testing (Zhang et al. 1998, Martin-Iverson 1999) whereas repeated psychostimulant

treatment which was not paired with PPI testing failed to result in PPI reduction

(Mansbach et al. 1988; Druhan et al. 1998; Martinez et al. 1999; Byrnes et al. 2000;

Adams et al. 2001). These studies suggest that PPI reduction following repeated

psychostimulant administration may, like locomotor sensitization, be context-dependent

such that AMPH withdrawal-induced deficits in the PPI paradigm might only be detected

if a drug-related cue were present during test. This hypothesis was tested in the present

study by measuring PPI in AMPH-withdrawn animals that previously received drug

injections paired with an environment similar to that experienced during PPI testing.

Locomotor sensitization is always measured following the administration of a DA

agonist challenge. Swerdlow et al. (1995) previously demonstrated PPI disruption in rats

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51

with lesions of the hippocampus and prefrontal cortex following the administration of

very low doses of the direct DA agonist apomorphine (APO) whereas lesioned animals

receiving vehicle injections did not show reduced PPI. In the present study, we sought to

determine whether a PPI disruption during AMPH withdrawal might be similarly

revealed following low-dose APO and AMPH challenge injections.

Finally, it has been reported that sensitization of locomotor behavior becomes

more pronounced following longer withdrawal periods (Paulson et al. 1991; Paulson and

Robinson 1995). That is, effects of AMPH pretreatment that are not apparent during the

first few days of withdrawal have been found to emerge 1 to 2 weeks later. Therefore,

one objective of the present study was to investigate the time course of AMPH

withdrawal effects on PPI, to determine whether PPI disruption may become evident only

after an extended withdrawal period, similar to behavioural sensitization.

The present study was designed to test the hypothesis that effects of withdrawal

from an escalating dosage schedule of AMPH administration on PPI might be revealed

by experimental conditions similar to those used to best demonstrate locomotor

sensitization. These include administration of a dopamine (DA) agonist challenge, pairing

of drug administration with cues also associated with the PPI testing protocol, and testing

after withdrawal intervals longer than the one used in a previous study (4 days). In

addition, to enable a direct comparison of LI and PPI results in the same animals, we

tested these animals for latent inhibition following 16 days of withdrawal. Selected

animals were also tested for locomotor sensitization to a challenge AMPH injection

following 2 months of withdrawal.

MATERIALS AND METHODS

Animals

We used 3 batches of rats, 48 animals for experiment 1, 32 animals for

experiment 2, and 32 animals for experiments 3 and 4 (Table 1). Male Wistar rats (Zur:

WIST [HanIbm]; 250-350g) obtained from our in-house specific-pathogen-free (SPF)

breeding facility were used as subjects in these experiments. Animals were housed

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52

individually in Macrolon type III cages (48 x 27 x 20 cm) under reversed-cycle lighting

(lights on 21.00-09.00 hours) in a temperature (21±1ºC) and humidity (55±5%)

controlled animal facility. Food (Kliba 3430, Klibamühlen, Kaiseraugst CH) and water

were available ad libitum. All experiments were carried out during the dark phase of the

light-dark cycle and in agreement with Swiss cantonal regulations for animal

experimentation.

Drugs and pretreatment procedure

D-Amphetamine sulfate (Sigma Chemical Company, St. Louis, U.S.A.) was

dissolved in a 0.9% NaCl solution to obtain concentrations of 0.5, 1, 2, 3, 4 and 5 mg/ml

amphetamine (calculated as the salt). Vehicle-treated groups received 0.9% NaCl

solution. A solution of 0.03 mg/kg apomorphine (APO) was made by dissolving APO in

0.9% NaCl with 0.1% ascorbic acid. All solutions were freshly prepared and given in a

volume of 1ml/kg. AMPH and saline (SAL) were injected intraperitoneally during the

drug pretreatment period and as a challenge administration whereas APO and SAL

administered just prior to PPI testing were injected subcutaneously. During the

escalating-dose pretreatment schedule animals received three injections per day for 6

consecutive days, beginning with a 1 mg/kg dose and ending with doses of 5 mg/kg

AMPH on the sixth day of the cycle. The control group received injections of SAL

(0.9%) according to the same schedule. The dosing parameters are summarized in Table

2.

Apparatus

Prepulse inhibition apparatus. Testing was conducted in four ventilated startle

chambers (SR-LAB, San Diego Instruments, San Diego, CA), each containing a

transparent Plexiglas tube (diameter 8.2 cm, length 20 cm) mounted on a Plexiglas frame.

Noise bursts were presented via a speaker mounted 24 cm above the tube. Motion inside

the tube was detected by a piezoelectric accelerometer below the frame. The amplitude of

the whole body startle to an acoustic pulse was defined as the average of 100 1-ms

accelerometer readings collected from pulse onset.

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53

Two-Way Avoidance Apparatus. Testing was conducted in four identical shuttle

boxes (Coulbourn Instruments, Allentown, PA; model E10-16TC), each set in a

ventilated, sound and light-attenuating shell (model E10-20). The internal dimensions of

each chamber were 35 x 17 x 21.5 cm. The grid floor of the chambers was divided into

two identical compartments by an aluminium hurdle (17 cm long, 4 cm high). The barrier

was very thin to prevent animals from balancing on it and thus avoiding shock.

Footshocks were supplied to the grid floor by a constant direct current source (model E

13-14) and a scanner (model E 13-13) set at 0.5 mA intensity. During the experimental

session each chamber was illuminated by a diffuse light source (house light), mounted 19

cm above the grid floor in the center of the side walls. The conditioned stimulus (CS) was

a tone of 85 dB produced by a speaker (model E 12-02) placed behind the shuttle box on

the floor of the shell.

Apparatus for detection of locomotor activity: Sixteen stations were used. Each

station was a 25 cm wide X 40 cm long X 40 cm high compartment contained within an

individual sound-attenuating wooden cabinet. One end wall of the compartment, the

device wall, consisted of wooden panels, whereas the remaining walls were clear plastic.

The device wall contained a water bottle spout and an opening that provided access to

powdered chow in a feeding bin. A large drop-down door in the front wall of the

compartment allowed easy access to the animal. The floor of each compartment was a

black removable pan holding a thin layer of dark, absorbent, autoclaved earth. The ceiling

was open. A 4 Watt lamp (Lampi, model number 5304) outside the compartment but

within the cabinet, was used to produce a light-dark cycle. Each lamp was connected to

an appliance timer (Migros type NL24MI). A fan mounted on the wall of a cabinet

provided ventilation. A camera, centered approximately 49 cm above the compartment

floor, was mounted in the ceiling of each cabinet. The field of vision of the camera

included the entire area of the compartment in which an animal could move, and images

from this camera were used to quantify activity. The stations were located in a well-

ventilated, temperature, humidity, and sound controlled room that was used only for this

experiment. The room could be illuminated with red ceiling lights. The cameras from all

the stations were connected to a 16-channel multiplexer (Sony model YS-DX216CE)

located in an adjoining room, and the multiplexer was connected in turn to a Dell

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Computer (OptiPlex GXpro with a Pentium Pro Processor) running image analysis

software. The software was a custom-written Visual Basic Program (P. Schmid,

Laboratory of Behavioral Neuroscience ETH Zurich) that was based on an NIH Image

Analysis script. The software "grabbed" an image from each station every second and

compared this image pixel by pixel with an image obtained the second before. Each white

Wistar rat was monitored against a darker background. The percentage of pixels that went

from dark to light or from light to dark from one second to the next was quantified. This

percentage provided a measure of the magnitude of an animal's displacement or

"activity". One-second activity values ranged from 0% (no movement) to approximately

7.5%. The multiplexed images from the 16 stations could be taped simultaneously on a

single videotape with a video recorder (Sony model SVT-1000P), and the 16 images

could be viewed simultaneously on a single monitor.

Behavioral testing procedures

PPI procedure: A background noise level of 68 dB(A) was maintained throughout

each test session. A test session started with 5 min of acclimatisation, after which four

startle pulses [30 ms, 120 dB(A)] were presented. These four initial startle pulses served

to achieve a relatively stable level of startle reactivity for the remainder of the test

session, as the most rapid habituation of the startle reflex occurs within the first few

startle pulse presentations (Koch 1999). To measure PPI, six blocks of 11 trials were then

presented. The 11 trials of each block included: two “pulse alone“ trials, one “prepulse

followed by pulse” and one “prepulse alone” trial for each of four prepulse intensities,

and one “no stimulus” trial. The prepulses were broadband bursts of 20 ms duration and

an intensity of either 72, 76, 80, or 84 dB(A). Between prepulse offset and pulse onset,

there was a time interval of 80 ms. The different trial types were presented

pseudorandomly with an intertrial interval of 10-20 s (average 15 s). Each complete test

session lasted about 23 min. The percentage PPI (%PPI) induced by each prepulse

intensity was calculated as: [100 - (100 x startle amplitude on "prepulse followed by

pulse" trial) / (startle amplitude on "pulse alone" trial)].

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Active avoidance procedure: The latent inhibition procedure in the two-way active

avoidance paradigm was carried out over 3 days. Animals received two consecutive daily

sessions of preexposure to both the tone and the apparatus or only to the apparatus, and a

conditioning session on the third day.

Days 1-2: Preexposure to the apparatus or apparatus/tone CS. The preexposure

sessions took place on days 14 and 15 of withdrawal. Each non-preexposed (NPE) animal

was placed in the shuttle box with the house light on for a period of 50 min. Each

preexposed (PE) rat received 50 presentations of the tone (mean variable inter-stimulus

interval = 50 sec [range = 10 - 90 sec], duration = 10 sec). A general evaluation of each

animal’s activity level was supplied by recording the total number of crossings during the

preexposure sessions.

Day 3: Conditioning to the CS. On day 16 of withdrawal all animals were tested

for conditioned active avoidance. Each animal was placed into the shuttle box and

received 100 avoidance trials on a variable interval schedule of 50 sec, ranging from 10

to 90 sec. Each avoidance trial began with a 10-sec tone followed by a 2-sec 0.5 mA

shock, the tone remaining on with the shock. If the rat crossed the barrier to the opposite

compartment during the tone, the stimulus was terminated and no shock was delivered

(avoidance response). A crossing response during the shock terminated the tone and the

shock (escape response). If the rat failed to cross during the entire tone-shock trial, the

tone and the shock terminated after 12 sec (unfinished trial). As an additional measure of

activity, we analyzed the total number of inter-trial crossings.

Sensitization Procedure. Amphetamine-induced locomotion was assessed in 16

test boxes and testing was carried out in 3 stages. In the first stage each rat was placed in

the apparatus (withdrawal day 61) and allowed to remain there undisturbed for 14 hours.

On withdrawal day 62, rats were removed from the apparatus and injected with 0.9%

saline, and again placed into the apparatus for the second stage, consisting of 1 hour of

free exploration. Rats were then injected with 0.5 mg/kg AMPH and returned to the

apparatus for 6 hours free exploration. Activity levels were monitored for the entire

duration of each of the 3 stages.

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Experiment 1: Effects of drug-conditioned cues and AMPH withdrawal on PPI and

LI

Experiment 1. 1: Forty-eight Wistar rats were divided into 6 experimental groups

(N = 8 per group) in this experiment (see Table 1). During the drug pretreatment period, 8

SAL and 8 AMPH treated animals (SAL/TUBE and AMPH/TUBE groups) were placed

directly following each injection into transparent plexiglas tubes (diameter 10.5 cm,

length 28 cm) which were similar to PPI restraint tubes, but larger in size. The tubes were

placed within normal home cages that did not contain sawdust. Following each 20-min

exposure to the tube, animals were returned to their home-cages. The remaining 32

animals were returned to their home-cages immediately following each injection of SAL

(N=16) or AMPH (N=16). Preliminary results suggested that an injection of saline prior

to testing reduced PPI; therefore, we compared the effects of AMPH withdrawal on PPI

in animals which did (SAL/SAL and AMPH/SAL groups) and did not (SAL and AMPH

groups) receive a saline injection prior to test. On day 4 of withdrawal from AMPH, all

animals were tested for PPI in 4 squads randomized for drug pretreatment and the

different cue conditions. The SAL/SAL and the AMPH/SAL as well as the SAL/TUBE

and the AMPH/TUBE animals received a SAL injection (ip) 5 min before PPI testing was

conducted.

Experiment 1. 2: All of the animals from experiment 1. 1 were used for the APO-

challenge PPI test on day 5 of withdrawal. Half of the animals in each drug/cue condition

received 0.03 mg/kg APO (sc), and the other half received a SAL injection (sc) 5 minutes

prior to placement in the PPI apparatus (N = 4 per group).

Experiment 1. 3: All animals were subsequently tested for LI in a two-way active

avoidance paradigm as described above. The two preexposure sessions took place on

days 14 and 15 of withdrawal, and the test session was conducted on day 16 of

withdrawal. Preexposed and non-preexposed groups were counterbalanced for all

previous treatments and testing conditions (N = 12 per group).

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Experiment 2: Effects of AMPH withdrawal on the acoustic startle response and

PPI at 23-24 days of withdrawal, with and without a 0.5 mg/kg AMPH challenge

injection

Experiment 2. 1: Two groups of 16 animals each were pretreated with either

AMPH or SAL. Following each injection, all animals were returned to their home cages.

The effects of AMPH and SAL pretreatment on PPI and the acoustic startle response

were assessed following 23 days of withdrawal. Animals did not receive any injection

treatments on the test day.

Experiment 2. 2: On withdrawal day 24, half of the animals of each pretreatment

group used in experiment 2. 1 (see Table 1). 1 received an AMPH challenge injection of

0.5 mg/kg (i. p.) 5 minutes before beginning PPI/acoustic startle response testing

(SAL/AMPH and AMPH/AMPH groups, n = 8 per group). The remaining half of the

animals received a SAL injection 5 minutes before the test (SAL/SAL and AMPH/SAL

groups, n = 8 per group)

Experiment 3: Effects of 30 and 60 days withdrawal from an escalating dosage

schedule of AMPH on the acoustic startle response and PPI

Two additional groups of 16 animals each were pretreated with either AMPH or

SAL (see Table 1). Following each injection, all animals were returned to their home

cages. All rats were tested for the acoustic startle response and PPI, first on day 30, and

then again on day 60 of withdrawal. Animals did not receive any injection treatments on

the test day.

Experiment 4: AMPH sensitization of locomotor activity

At 61-62 days of withdrawal, 16 animals (8 SAL, 8 AMPH) from experiment 3

were tested for locomotor sensitization (see Table 1). Locomotor activity was measured

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during a baseline period of 14 hours, over a 1-hour period following a SAL injection, and

over a 6-hour period following a challenge injection of 0.5 mg/kg AMPH.

Table 1. Treatment conditions and withdrawal periods for animals used in experiment 1, 2, 3 and 4 (inj: injection, PPI: prepulse inhibition, LI: latent inhibition, SAL: saline; AMPH: amphetamine).

Pretreatment Withdrawal day and test

Day 4 Day 5 Day 16 Day 23 Day 24 Day 30 Day 60 Day 62 Experiment 1 SAL PPI SAL/APO inj. LI test SAL inj. + PPI + PPI AMPH PPI SAL inj. + PPI SAL/TUBE SAL inj. + PPI AMPH/TUBE SAL inj. + PPI Experiment 2 SAL PPI SAL/AMPH

AMPH inj. + PPI Experiment 3 SAL PPI PPI Locomotor and 4 AMPH sensitization

Data collection and analysis.

For all experiments post hoc comparisons were conducted using Fisher's protected

least significant difference test. Significant differences were accepted at p < 0.05. All

statistical analyses were performed with the StatView software program (Abacus

Concepts, Inc., Berkeley, CA, 1992). Experiment 1: The startle amplitude of the PPI test

on day 4 of withdrawal was analysed using a 3 x 2 x 16 ANOVA design consisting of the

factors of drug-related cue condition (none, SAL injection, TUBE and saline injection)

and drug pretreatment (SAL, AMPH) and a repeated measurements factor of 16 pulse-

alone presentations. Mean percentage PPI was analysed using a 3 x 2 x 4 ANOVA design

consisting of the same between-subjects factors and a repeated measurements factor of 4

prepulse intensities. The ANOVAs used for the PPI test on withdrawal day 5 included an

additional between-subjects factor of APO (SAL, APO) treatment. For the two

preexposure sessions in the active avoidance shuttle box, the total number of crossings

was analysed using a 2 x 2 x 2 ANOVA with two between-subjects factors of drug

pretreatment (AMPH, SAL) and preexposure (NPE, PE), and with 2 preexposure days as

a within-subjects factor. For the active avoidance conditioning session, the 100 avoidance

trials were divided into 10 blocks of 10 trials each. Percentage avoidance responses were

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analyzed using a 2 x 2 x 10 ANOVA consisting of between-subjects main factors of drug

pretreatment and preexposure and a repeated measurements factor of 10-trial blocks.

Total numbers of inter-trial crossings were analyzed as an index of activity level using

the between-subjects main factors of drug pretreatment and preexposure. Experiment 2:

Analysis of the acoustic startle response and PPI on withdrawal day 23 was similar to

Experiment 1 but with only one between-subjects factor of drug treatment (SAL,

AMPH). The analysis of acoustic startle and PPI on withdrawal day 24 also included a

between-subjects factor of drug challenge (AMPH, SAL). Experiment 3: Acoustic startle

response and PPI on withdrawal days 30 and 60 were analyzed similarly to Experiment 1

with a between-subjects factor of drug pretreatment (AMPH, SAL). Experiment 4:

Locomotor activity data were analysed using 3 separate two-way ANOVAs for the no-

drug baseline, saline and amphetamine periods, each consisting of a between-subjects

factor of drug pretreatment (SAL, AMPH) and a repeated measurements factor of 28

blocks of 30 min (baseline period), 6 blocks of 10 min (SAL period) or 36 blocks of 10

min (AMPH period).

RESULTS

Experiment 1: Effects of drug-conditioned cues and AMPH withdrawal on PPI and

LI

Experiment 1. 1 Effects of 4 days AMPH withdrawal and drug-conditioned cues on the

acoustic startle response and PPI

A significant habituation of the startle response was seen over the 16 pulse-alone

presentations in all groups (main effect of trials: F(15, 630) = 8.42, p < 0.0001).

However, there were no significant effects of either drug pretreatment or cue condition

(Fig. 1 A, B, C). The mean percentage of PPI as a function of prepulse intensity in the 6

conditions is also shown in Figure 1 (D, E, F). The ANOVA yielded a significant main

effect of prepulse intensity, F(3, 126) = 83.77, p < 0.0001, reflecting a gradual increase in

prepulse inhibition as a function of the intensity of the prepulse stimulus. However, there

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were no significant main effects or interactions involving the factors of drug pretreatment

or cue condition on percent PPI.

Figure 1. A-C) Startle responses during 16 pulse-alone trials, and D-F) prepulse inhibition (%PPI) measured in rats previously treated with amphetamine (AMPH) or saline (SAL) and assigned to one of 6 experimental conditions. Animals represented in panels A, B, D and E were returned to the home cage after each injection during the pretreatment phase and received either no injection treatment on the test day (A, D; AMPH and SAL groups) or received a saline injection before testing (B, E; AMPH/SAL and SAL/SAL groups). Animals represented in panels C and F received AMPH and SAL injections during the pretreatment phase that were paired with placement into restraint tubes similar to those used during PPI/startle testing, and also received a saline injection before testing (AMPH/TUBE and SAL/TUBE groups). Percentage of PPI was measured using a range of prepulse intensities (72, 76, 80, or 84 dB). Testing was conducted on day 4 of withdrawal. Values are means ± SEM. N = 8 per group.

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Experiment 1. 2 Effects of 5 days AMPH withdrawal, drug-conditioned cues and a 0.03

mg/kg APO challenge injection on the acoustic startle response and PPI

In Experiment 1.1, no differences in PPI or acoustic startle were seen between

those groups which received an injection prior to testing (SAL/SAL and AMPH/SAL

groups) and those animals which did not receive an injection prior to testing (SAL and

AMPH groups). Because all animals were to receive a SAL or APO injection before the

PPI test in Experiment 1.2, we collapsed the injected and non-injected control groups of

experiment 1. 1 to form a new no-cue treatment category. We analyzed the data in a 2 x 2

x 2 ANOVA design including the factors of drug pretreatment (SAL, AMPH), cue

treatment (no treatment, TUBE), and challenge treatment (SAL, APO).

Independent of any drug pretreatment or cue treatment, the animals again showed

habituation of the startle response over the 16-pulse alone presentations (F (15, 600) =

11.72, p < 0.0001, Fig. 2 A-D) but no other significant main effects or interactions. The

mean percentage PPI for all experimental groups is summarized in Figure 2 E-H. PPI was

clearly evident in all groups and characterized by an increased amount of inhibition as a

function of the intensity of the prepulse stimulus (main effect of prepulse intensity: F (3,

120) = 39.10, p < 0.0001). The analysis also revealed a significant main effect of cue

treatment (F(1, 40) = 4.54, p < 0.05), reflecting increased PPI in the TUBE animals

compared to the no-cue animals. Administration of 0.03 mg/kg APO reduced PPI overall

(main effect of APO treatment: F(1, 40) = 22.82 p < 0.0001). No other significant main

effects or interactions were detected. Thus, APO was not more effective in reducing PPI

in AMPH pretreated groups than in SAL pretreated groups.

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Figure 2. A-D) Startle responses during 16 pulse-alone trials, and E-H) percent prepulse inhibition (%PPI) measured in rats previously treated with amphetamine (AMPH) or saline (SAL) and assigned to one of 4 experimental conditions. Animals in panels A, B, E and F were returned to the home cage after each injection during the pretreatment phase and received either an injection of SAL (A,E; SAL/SAL and AMPH/SAL groups; N = 8 per group) or apomorphine (APO; B,F; SAL/APO and AMPH/APO groups; N = 8 per group) before testing. Animals in panels C, D, G and H received AMPH and SAL injections during the pretreatment phase that were paired with placement into restraint tubes similar to those used during PPI/startle testing, and received either an injection of SAL (C,G; SAL/TUBE/SAL and AMPH/TUBE/SAL groups; N = 4 per group) or APO (D,H; SAL/TUBE/APO and AMPH/TUBE/APO groups; N = 4 per group) before testing. Percent PPI was measured using a range of prepulse intensities (72, 76, 80, or 84 dB). Testing was conducted on day 5 of withdrawal. Values are means ± SEM.

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Experiment 1. 3: Effects of 16 days AMPH withdrawal on LI in the two-way active

avoidance paradigm

Preexposed (PE) and non-preexposed (NPE) groups were counterbalanced for all

previous treatments. There were no significant main effects or interactions including the

factors of cue condition or prior APO treatment on behaviors measured during either the

preexposure or conditioning sessions; therefore, the data were analyzed in a 2 x 2 design

including only the factors of drug pretreatment (SAL, AMPH) and preexposure (NPE,

PE).

Preexposure sessions: A comparison of the total number of crossings during the

two preexposure sessions revealed a near-significant tendency for greater activity on the

first preexposure day compared to the second day (F(1,44)=3.16, p=0.08; 35.5±2.3 for

session 1 versus 31.8±2.0 for session 2), suggesting habituation to the apparatus. There

were no significant outcomes involving the factors of drug treatment or preexposure (data

not shown).

Conditioning session: Preexposed rats made generally fewer avoidance responses

in comparison to NPE animals, as reflected by a significant main effect of preexposure

(F(1,44) = 7.86, p < 0.01). Our analysis also revealed a highly significant effect of blocks

(F(9,396) = 54.12, p < 0.0001) and a significant interaction of preexposure x drug x

blocks (F(9,396) = 2.19, p < 0.05). As can be seen in Figure 3, all groups acquired the

avoidance response; however, LI (i.e. significantly reduced avoidance performance in

preexposed compared to non preexposed rats) was only seen in the SAL groups (Fisher`s

posthoc: SAL NPE versus SAL PE, p = 0.017; AMPH NPE versus AMPH PE, p =

0.187). Latent inhibition was disrupted in the AMPH-pretreated rats primarily due to

increased avoidance responses in the PE group. Finally, an analysis performed on the

total number of inter-trial crossings made by animals during the test session revealed no

significant main effects or interactions involving the factors of drug pretreatment or

preexposure (data not shown).

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Figure 3. Percentage of avoidance responses made during a 100-trial test of conditioned two-way active avoidance acquisition in rats previously treated with either amphetamine (AMPH) or saline (SAL) and preexposed to either the apparatus (NPE) or to the tone and the apparatus (PE). Rats were tested 16 days after their last injection. Values are means ± SEM. N = 12 per group.

Experiment 2: Effects of AMPH withdrawal on the acoustic startle response and

PPI at 23-24 days of withdrawal, with and without a 0.5 mg/kg AMPH challenge

injection

Experiment 2. 1: Effects of 23 days AMPH withdrawal on the acoustic startle

response and PPI

All groups showed habituation of the startle response over the 16 pulse-alone

presentations (main effect of trials: F(15, 450) = 7.71; p < 0.0001, see Fig. 4 A). AMPH

pretreated animals exhibited a reduced acoustic startle response compared with the SAL

control animals, as reflected by a significant main effect of drug treatment (F(1, 30) =

6.49; p < 0.05). ANOVA of the PPI results yielded a significant main effect of prepulse

intensity, F(3, 90) = 63.43, p < 0.0001 (Fig. 4 D), reflecting a gradual increase in prepulse

inhibition as a function of intensity of the prepulse stimulus. However, there were no

significant main effects or interactions including the factor of drug pretreatment on PPI.

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Experiment 2. 2: Effects of 24 days AMPH withdrawal and a 0.5 mg/kg AMPH challenge

injection on the acoustic startle response and PPI

A highly significant main effect of 16 trials (F(15, 420) = 8.55, p < 0.0001)

reflected a habituation of the startle response over the 16 pulse-alone presentations (Fig. 4

B,C). There were no significant main effects or interactions including the factors of drug

treatment or challenge treatment. PPI again increased gradually as a function of prepulse

intensity (main effect of prepulse intensity: F(3, 84) = 32.16, p < 0.0001). No significant

main effects or interactions were detected for the factors of drug pretreatment or

challenge treatment on PPI (Fig. 4 E,F).

Figure 4. A-C) Startle responses during 16 pulse-alone trials and D-F) percent prepulse inhibition (%PPI) in rats previously treated with amphetamine (AMPH) or saline (SAL). Animals were tested on withdrawal days 23 (A, D; N = 16 per group) and then again on day 24 after a challenge injection of either SAL (B, E; N = 8 per group) or 0.5 mg/kg AMPH (D, F; N = 8 per group). Percent PPI was measured using a range of prepulse intensities (72, 76, 80, or 84 dB). Values are means ± SEM.

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Experiment 3: Effects of 30 and 60 days AMPH withdrawal on the acoustic startle

response and PPI

Withdrawal day 30: Habituation to the acoustic startle response over the 16 pulse-

alone presentations was seen in both AMPH and SAL treated animals (main effect of

trials: F(15, 450) = 4.65, p < 0.0001, see Fig. 5 A). The startle response was reduced

following 30 days of AMPH withdrawal, as reflected by a main effect of drug

pretreatment (F(1, 30) = 6.14, p < 0.05). Percent PPI increased gradually as a function of

prepulse intensity (main effect of prepulse intensity: F(3, 90) = 49.51, p < 0.0001).

However, PPI did not differ between AMPH and SAL pretreated animals, as supported

by an absence of significant main effects or interactions including the factor of drug

pretreatment (Fig. 5 C).

Withdrawal day 60: During the 16 pulse-alone presentations, the acoustic startle

response again appeared to habituate over trials in both AMPH pretreated and SAL

control rats, as reflected by a significant main effect of trials (F(15, 450) = 4.49, p <

0.0001). In contrast to our findings at 30 days of withdrawal, however, AMPH and SAL

groups showed similar degrees of startle responding (Fig. 5 B). Percent PPI increased as a

function of prepulse intensity, as supported by a significant main factor of prepulse

intensity (F(3, 90) = 42.57, p < 0.0001). Similar to our results at 30 days of withdrawal,

PPI did not differ between AMPH and SAL groups, as supported by an absence of

significant main effects or interactions including the factor of drug pretreatment (Fig. 5

D).

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Figure 5. A-B) Startle responses during 16 pulse-alone trials, and C-D) percent prepulse inhibition (%PPI) in rats previously treated with amphetamine (AMPH) or saline (SAL). Animals were tested 30 days (A, C) and 60 days (B, D) after their last injection. Percent PPI was measured using a range of prepulse intensities (72, 76, 80, or 84 dB). Values are means ± SEM. N = 16 per group.

Experiment 4: AMPH sensitization of locomotor activity

Sixteen of the animals from experiment 3. 2 were tested on days 61-62 of AMPH

withdrawal for baseline levels of locomotor activity and for locomotor responding to a

challenge injection of 0.5 mg/kg AMPH. Baseline locomotor activity decreased over the

28 half-hour bins (F(27, 378) = 40.30, p < 0.0001) with no significant differences

between SAL and AMPH pretreated rats (not all data are shown). This outcome reflected

habituation to the apparatus. As can be seen in Fig. 6, the last hour of the habituation

period was analyzed in blocks of 10 min and no significant effect of drug pretreatment

was detected. Animals showed an elevation of activity followed by a rapid decrease in

response to saline administration (main effect of blocks: F(5, 70) = 31.83; p < 0.0001,

Fig. 6), with no differences detected between SAL and AMPH pretreated rats. Both

AMPH and SAL groups showed an augmentation of locomotor activity in response to a

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0.5 mg/kg AMPH challenge administration (Fig. 6). However, AMPH-pretreated animals

exhibited significantly enhanced locomotor activity during the first 40 min in comparison

to the SAL control group. This outcome was supported by a significant interaction of

drug pretreatment x 10-min bins during this period (F(35, 490) = 1.86, p < 0.01).

Figure 6. Locomotor activity measured during an initial 16-hour habituation period (only the last hour shown), a 1- hour period following an injection of saline vehicle, and a 6-hour period following a challenge injection of 0.5 mg/kg amphetamine. Testing was conducted 61 - 62 days after the last injection in animals that had been pretreated with either amphetamine (AMPH) or saline (SAL). Values are means±SEM. N = 8 per group.

DISCUSSION

The present study was designed to test the effects of AMPH withdrawal on PPI

using experimental conditions known to be optimal for demonstrating behavioural

sensitization. Therefore, we measured PPI and the acoustic startle response 1) after 4

days of withdrawal in the presence and absence of a drug-conditioned context, 2)

following low-dose challenge injections of the DA agonists APO and AMPH, and 3) at

longer withdrawal intervals. We found no effect of AMPH withdrawal on PPI

irrespective of these experimental conditions. However, the acoustic startle response of

AMPH treated rats was reduced on days 23 and 30, but not on days 4 and 60 of

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withdrawal. Finally, consistent with our previous findings (Murphy et al. 2001b; Russig

et al. 2002), AMPH pretreated rats showed deficits in LI following 16 days of withdrawal

and pronounced sensitization of locomotor activity to an AMPH challenge injection after

2 months of withdrawal.

Given that disrupted LI is an animal model of the positive symptoms of

schizophrenia, the LI reduction found after a period of AMPH administration in this

study is consistent with the hypothesis that endogenous sensitization of DA contributes to

the expression of positive schizophrenic symptoms. Importantly, the disruption of LI in

AMPH-withdrawn animals was almost entirely due to the increased avoidance responses

of the AMPH PE compared to the SAL PE animals. We previously showed that LI was

disrupted following 4 and 13 days of withdrawal, and reduced but apparently beginning

to normalize after 28 days of withdrawal from the AMPH schedule used in this study

(Murphy et al. 2001b). Moreover, the LI disruption induced by AMPH withdrawal was

restored by acute treatment with either haloperidol or clozapine prior to avoidance

conditioning (Russig et al. 2002). In the present study, we have shown that LI is

significantly reduced after 16 days of withdrawal as well, representing a modest

extension of the time course during which this effect is observed. Given the reduced LI

previously reported in schizophrenic patients (Baruch et al. 1988; Gray et al. 1992, 1995),

the ability of repeated psychostimulant administration to produce symptoms of psychosis

(Davis and Schlemmer 1980; Angrist 1994), and the antipsychotic efficacy of haloperidol

and clozapine, we can speculate that AMPH withdrawal-mediated disruptions of LI may

reflect cognitive processes that are linked to positive psychotic symptoms.

We originally hypothesized that PPI might be similarly eliminated during AMPH

withdrawal. PPI disruption immediately following a single AMPH administration has

been demonstrated in a number of studies (Mansbach et al. 1988; Bakshi et al. 1995; Sills

1999; Geyer et al. 2001); however, previous investigations have failed to show

sensitization to the disruptive effects of repeated AMPH on PPI, whether testing was

conducted with or without a challenge AMPH injection (Mansbach et al. 1988; Druhan et

al.1998). In other studies, repeated cocaine treatment similarly had no effect on PPI

during withdrawal and in fact prevented PPI disruption following a challenge

administration of the drug (Martinez et al. 1999; Byrnes et al. 2000; Adams et al. 2001).

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In contrast, PPI was reduced by repeated DA agonist treatment when the injections were

paired with PPI testing (Zhang et al. 1998; Martin-Iverson 1999), suggesting that PPI

reductions following repeated psychostimulant administration might be revealed only in

the presence of a drug-associated context. Schulz and co-workers similarly demonstrated

that repeated dizocilpine (MK-801) produced a sensitized disruption of PPI only when

repeatedly administered in the context of startle response testing and Gordon and Rosen

(1999) showed that the acoustic startle response is enhanced during cocaine withdrawal

only if cocaine injections had been paired with prior exposure to the startle test

environment. In the present study, however, PPI was not disrupted during AMPH

withdrawal, either in animals presented with a SAL injection cue prior to testing, or in

animals in which a restraint tube context had been paired with AMPH injections during

pretreatment. The acoustic startle response during the 16 pulse-alone trials was likewise

not affected by the SAL injection or restraint tube-AMPH pairings. Our study differed

from earlier ones reporting PPI reductions after repeated DA agonist treatment (Zhang et

al. 1998; Martin-Iverson 1999) in that we exposed the rats during the pretreatment phase

only to an environment that was similar to the PPI tubes, whereas the earlier

investigations paired DA agonist administrations with both the context of PPI testing and

exposure to prepulses and startling stimuli. Our negative results in this regard suggest that

the reported influence of drug-paired startle testing in sensitising PPI reductions is not

strictly a contextual association phenomenon. Rather, these effects may result from

processes more akin to fear-potentiated startle (Davis 1986), whereby perhaps

associations with the sympathomimetic and/or anxiogenic properties of DA agonists lead

to either a potentiated or less-disruptible startle response (i.e. reduced PPI).

Swerdlow and colleagues (1995) previously demonstrated that PPI was reduced

following an APO injection in hippocampal lesioned rats which otherwise show no PPI

deficit. We similarly anticipated that administration of low-dose APO and AMPH

challenges prior to PPI testing might uncover evidence of dysfunctional sensorimotor

gating. Indeed, in the present study, a single low dose of 0.03 mg/kg APO disrupted PPI

as has been shown previously (Pouzet et al. 1999, Weiss et al. 1999, Geyer et al. 2001).

However, the effects of APO were similar in SAL- and AMPH-pretreated rats, regardless

of whether or not animals had received injections paired with a PPI tube context during

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the pretreatment phase. In addition, administration of a 0.5 mg/kg AMPH challenge, a

dose which is not normally sufficient to reduce PPI (Kinney et al. 1999; Feldon,

unpublished observations), also did not reveal a PPI disruption in AMPH-treated animals

following 24 days of withdrawal. As discussed above, these results are consistent with

those of previous investigations in which startle testing was not paired with repeated DA

agonist administration (Mansbach et al. 1988; Druhan et al. 1998). It is interesting to note

that tube-preexposed animals showed enhanced PPI during the APO challenge test

compared to the no-cue animals. This result indicates that the tube-pretreatment was not

totally ineffective in influencing PPI; however, the enhancement effect of the TUBE

condition was not seen on day 4 (first PPI test) and was not influenced by either drug

pretreatment or administration of an APO challenge injection. The reason for this PPI

enhancement effect is unclear at this time; however, it is conceivable that after the initial

PPI test, TUBE animals’ increased familiarity with the PPI test environment resulted in

more selective attention to the prepulse stimuli rather than to the context of the restraint

tube, thus increasing animals’ sensorimotor gating abilities.

We also found that extending the time course of PPI testing out to withdrawal day

60 did not reveal any disruption due to AMPH withdrawal. However, the acoustic startle

response was significantly reduced on withdrawal days 23 and 30 in AMPH pretreated

animals. In contrast, the acoustic startle response on day 4 was not significantly reduced

in AMPH-pretreated animals, consistent with previous findings (Murphy et al. 2001b),

and after 2 months the effect was again absent, indicating that the startle reduction effect

occurs within a restricted time window. Withdrawal from another psychostimulant drug,

cocaine, also induces a reduction of the acoustic startle response in rats (Gordon and

Rosen 1999; Adams et al. 2001) and chronic cocaine users similarly exhibit marked

impairments in the acoustic startle response (Efferen et al. 2000).

Since it is known that fear and anxiety increase the startle reflex (Davis 1986) and

a pleasant context conversely attenuates startle amplitude (Lang et al. 1990; Koch 1999),

it has been suggested that the startle response measurement could be a useful index of an

animal`s emotional state (Marsh et al. 1973; Koch and Schnitzler 1997). It seems unlikely

that a positive hedonic state develops which could be responsible for the reduction in

startle during AMPH withdrawal, given numerous reports of negative affect during

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psychostimulant withdrawal (Lin et al. 1999; Koob and Le Moal 2001; Weiss et al.

2001). In fact, we recently showed that animals receiving the schedule of AMPH

administration used in the present study showed an enhanced conditioned fear response

on day 4 of withdrawal (Pezze et al. 2002). The time course of this increase in

conditioned fear is likely to be a short-lived effect, given that the symptoms of anxiety

seen in both psychostimulant-withdrawn rats and newly-abstinent human addicts are

typically transient (Gawin 1991; Basso et al. 1999). Therefore, if a transiently increased

state of anxiety independently potentiated the acoustic startle response during the first

week of withdrawal, it may have effectively masked any reduction in startle present at

that time. It is possible then that the true time course of startle reduction during AMPH

withdrawal includes the entire first month of withdrawal. The return of a normal startle

response on withdrawal day 60 suggests that normalization of and/or compensation for

the etiology of reduced startle has taken place at this time. Given the fact that the

magnitude of the startle response in the absence of a CS or a prepulse is sometimes

viewed as a non-specific behavioural parameter, and that reduced startle during AMPH

withdrawal was only observed in 2 out of 4 timepoints in this study, and was not clearly

seen on day 24 even in the same animals that showed the reduction on day 23, it is

difficult to gauge the true significance of this effect at this time. Future studies will be

needed to determine the robustness of the startle reduction during AMPH withdrawal as

well as its biological underpinnings.

We predictably found evidence of locomotor sensitization to a 0.5 mg/kg AMPH

challenge following 2 months of AMPH withdrawal; in an earlier study, we similarly

demonstrated locomotor sensitization to a 1.0 mg/kg AMPH challenge after 30 days of

withdrawal from the same AMPH injection schedule (Russig et al. 2001). Increased

dopamine release in the nucleus accumbens after an AMPH challenge has been

repeatedly found in sensitized rats (Robinson and Becker 1986; but see Segal and

Kuczenski, 1992) and numerous studies have shown that both LI and PPI are disrupted

by DA agonists (Swerdlow et al. 1992; Weiner and Feldon 1997; Geyer et al. 2001).

However, basal levels of nucleus accumbens DA are reportedly reduced or unchanged

during AMPH withdrawal (Rossetti et al. 1992; Segal and Kuczenski 1992; Crippens et

al. 1993). Our laboratory has in fact shown that rats withdrawn from the AMPH schedule

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73

used in the present study showed no differences in basal DA levels, but decreased DA

efflux in the shell, and increased DA efflux in the core of the nucleus accumbens during

the expression of a conditioned fear response (Pezze et al. 2002). If an enhanced nucleus

accumbens core DA response contributes in some manner to the disruption of LI in

AMPH-withdrawn rats, then apparently it is not enough of a stimulus to elicit disrupted

PPI in these animals as well. On the other hand, reduced nucleus accumbens shell DA

responsiveness may contribute to startle reduction during AMPH withdrawal. In support

of this idea, blockade of DA receptors by acute administration of risperidone or clozapine

has been shown to decrease startle amplitude (Johansson et al. 1995; Depoortere et al.

1997). Of course, other neurotransmitter systems may also be involved in modulating the

startle response during AMPH withdrawal. In particular, there is strong evidence for

glutamatergic, noradrenergic and corticotropin-releasing factor (CRF) regulation of the

acoustic startle response (Davis 1986; Koch 1999).

Conclusions

To summarize, we report here that withdrawal from an escalating dosage schedule

of AMPH disrupted LI but left PPI intact. Manipulations of the PPI testing environment

that were intended to simulate the experimental conditions considered optimal for

demonstrating behavioural sensitization (contextual associations, presence of a DA

agonist challenge, later withdrawal time points for testing) likewise did not reveal any

increased sensitivity of AMPH-withdrawn animals to PPI disruption. Such a dissociation

between LI and PPI has been shown previously following other behavioural and

pharmacological treatments (Wilkinson et al. 1994; Feldon et al. 2000; Murphy et al.

2001a). The existence of this dissociation may be due at least in part to the suggested

involvement of different brain regions in the mediation of LI and PPI. LI has been linked

primarily to activity within the nucleus accumbens and hippocampus, whereas the

regulation of PPI is believed to occur in brain nuclei that extend from the prefrontal

cortex to the pontine tegmentum (Feldon and Weiner 1997; Koch and Schnitzler 1997).

Nevertheless, the attenuation of the startle response which we report here during AMPH

withdrawal is similar to cocaine withdrawal effects on startle that have been reported

previously in both humans and in rodents (Gordon and Rosen 1999; Efferen et al. 2000;

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74

Adams et al. 2001). Further investigations will be needed in order to clarify the neuronal

mechanisms underlying this effect as well as the functional significance of this reduction

in startle to an animal’s emotional state.

Acknowledgements

This study was supported by the Swiss Federal Institute of Technology (ETH-

Zurich, Switzerland). We would like to thank the staff of the animal facility for their care

and maintenance of the animals used in this study, Mr. Peter Schmid for his valuable

technical assistance and Mrs. Jane Fotheringham for her editorial help.

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CHAPTER 4 ------------------------------------------------------------------------------------------------------------

THE ACQUISITION, RETENTION AND REVERSAL OF SPATIAL

LEARNING IN THE MORRIS WATER MAZE TASK FOLLOWING

WITHDRAWAL FROM AN ESCALATING DOSAGE SCHEDULE

OF AMPHETAMINE IN WISTAR RATS

Holger Russig, Andre Durrer, Benjamin K. Yee, Carol A. Murphy and Joram Feldon

Neuroscience, in press

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ABSTRACT

Two experiments were carried out to evaluate the effects of amphetamine

withdrawal in rats on spatial learning in the water maze. A schedule of repeated d-

amphetamine administration lasting for six days, with 3 injections per day (1-5mg/kg,

i.p.), was employed. Experiment 1 demonstrated that amphetamine withdrawal did not

impair the acquisition of the water maze task (3rd-4th withdrawal days), but

amphetamine -withdrawn rats made more target zone visits and reached the former

location of the platform quicker than controls during the probe test (5th withdrawal day).

In Experiment 2, retention of the location of the escape platform was assessed in animals

having been pre-trained on the water maze task before treatment. On the 3rd withdrawal

day, retention of the former platform location was assessed in a probe test. Retention was

only clearly seen in the measure of annular crossings, and performance did not differ

between groups. Next, the animals were trained to escape to a new location in the water

maze on withdrawal days 4-5. A reversal effect could be discerned across the first 4

trials, as evident by the animal`s tendency to search in the former target quadrant. This

interfered with the new learning, but amphetamine-withdrawn animals appeared to

overcome it quicker than saline-treated controls. This finding is consistent with the view

that amphetamine withdrawal can enhance behavioural switching, which could be

expressed as a reduction of proactive interference during learning; and, it is in line with

our previous finding that latent inhibition is also attenuated during amphetamine

withdrawal.

Keywords: Memory, Schizophrenia, Addiction, Sensitization

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Abbreviations

AMPH, amphetamine

E, east

i.p., intraperitoneal

N, north

NaCl, sodium chloride

Q, quadrant

S, south

SAL, saline

SPF, specific-pathogen-free

W, west

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INTRODUCTION

Chronic administration of amphetamine (AMPH), an indirect dopamine agonist,

amongst other psychostimulant drugs of abuse, can lead to persistent changes in brain and

behaviour. Studies in animals undergoing withdrawal enable researchers to identify and

characterize such specific functional alterations in greater details. In the rat, a state of

withdrawal can reliably be observed in the drug-free period following repeated exposures

to AMPH.

One well-known effect of AMPH withdrawal is demonstrated in behavioural

sensitization studies, in which withdrawn subjects exhibit an enhanced behavioural

response to a single challenge of the drug compared to drug naïve subjects, and this effect

can last for at least a year (Paulson et al. 1991). However, behavioural alterations can also

be demonstrated in animals in the absence of any specific challenge, including reduced

motivation as well as specific cognitive impairments (e.g., Barr and Phillips 1999; Lin et

al. 1999, 2000; Murphy et al. 2001; Pezze et al. 2002; Russig et al. 2002). There is

evidence to suggest that changes observed in both cases are linked to altered

dopaminergic transmission in the prefrontal cortex and/or striatum (Robinson and Becker

1986; Pezze et al. 2002). Such neurochemical changes represent a form of neuro-adaptive

changes developed during repeated psychostimulants exposure. Related adaptive changes

have also been reported at the physiological, molecular and structural levels (Robinson

and Becker 1986; Nestler 2001).

The brain structures demonstrated to undergo adaptive changes developed during

repeated AMPH administration, the striatal complex, prefrontal cortices and limbic areas

have all been implicated in various forms of learning and memory (Goldman-Rakic 1987;

Baddeley 1992; Dias et al. 1996; Nestler and Aghajanian 1997; Robinson and Kolb 1997;

Berke and Hyman 2000). Therefore one would expect to observe changes in learning and

memory during AMPH withdrawal.

However, little is currently known about the effects of AMPH withdrawal on

mnemonic processing. A number of reports have investigated the effects of chronic

AMPH treatment in which the animals were being treated with AMPH during the course

of behavioural testing (e.g., daily post-training injection), or in which the animals were

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under the influence of a challenge dosage of AMPH during withdrawal. These

experiments suffer from the interpretative problem that the effects associated with

withdrawal as such cannot be readily discerned. For example, Gelowitz and coworkers

(1994) reported enhanced acquisition in a water maze task following repeated L-AMPH

administration in 10-month old rats. Under this drug regime, the acute and chronic effects

of AMPH cannot be readily distinguished in this study, as treatment continued

throughout the test phase. Thus, the reported promnesic effect of repeated AMPH

exposure might stem from the acute effects of the drug, because even post-trial AMPH

treatment has been shown to enhance memory consolidation in the water maze task

(Brown et al. 2000). At the same time, chronic administration of the psychostimulant,

cocaine, a drug that resembles AMPH pharmacologically, has been reported to impair

water maze performance in rats (Quirk et al. 2001).

Among the few studies that were specifically designed to assess the cognitive

effects of withdrawal, a clear consensus is lacking as to the direction and magnitude of

such effects. Spatial working memory performance on a delayed-alternation T-maze task

is apparently spared in rats 9 days withdrawn from a twice daily 2.5 mg/kg AMPH

administration schedule lasting for 5 days (Stefani and Moghaddam 2002). On the other

hand, improved mnemonic performance in the form of reduced perseverative errors has

also been reported by Bruto and Anisman (1983a, b) on the radial arm maze 24 hours

after the last AMPH injection in mice, although this effect could also be attributed to

enhanced response switching as such. Withdrawal from an intermittent schedule of

AMPH administrations has also been shown to facilitate the acquisition of appetitively-

motivated Pavlovian approach behaviour, indicating enhanced stimulus-reward learning

(Harmer and Phillips 1998; Taylor and Jentsch 2001), a finding that is suggestive of a

possible sensitization of the relevant dopaminergic pathways. Similarly, Pavlovian fear

conditioning is enhanced during AMPH withdrawal from the same schedule used in the

present study, an effect which is coincident with dopaminergic dysregulation in the

nucleus accumbens (Pezze et al. 2002).

The AMPH withdrawal schedule employed in the two experiments reported here

has previously been shown to disrupt a form of selective (or attentional) learning, known

as latent inhibition, in rats during withdrawal (Murphy et al. 2001; Russig et al. 2002).

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This effect can be restored with neuroleptic treatments (Russig et al. 2002) and appears to

be persistent and can be observed up to the withdrawal day 28 (Murphy et al. 2001). This

is in contrast to the anhedonia effect as demonstrated in an intracranial self-

administration paradigm, which was only detectable over the first five days of withdrawal

(Lin et al. 1999, 2000).

The present study was designed to extend the study of AMPH withdrawal on

memory and learning to one of the most commonly employed tests of spatial memory in

rodents. The acquisition, retention, and reversal learning of the Morris water maze task

were evaluated in subjects during the withdrawal period in the absence of any direct

influence of the drug. These experiments would serve to fill an important gap in the

literature on the cognitive effects of AMPH withdrawal.

In Experiment 1, acquisition of the water maze task commenced on days 3 and 4

of AMPH withdrawal, followed by a probe trial on withdrawal day 5. In Experiment 2,

the animals were trained on the water maze task prior to AMPH administration, and

retention performance was assessed on withdrawal day 3 in a probe test, followed by 2

days of reversal learning (i.e., learning to locate a novel position in the water maze).

Reversal learning is of potential interest given the possibility that the disruptive effect of

the present AMPH schedule on latent inhibition is indicative of an underlying

enhancement in behavioural switching (Weiner and Feldon 1997) and/or reduced

proactive interference (e.g., Bouton 1993). Based on this interpretation of our previous

findings, we expected to se facilitated reversal learning during withdrawal in AMPH-

treated animals relative to saline controls.

EXPERIMENTAL PROCEDURES

Subjects.

Two cohorts of Wistar rats (Experiment 1: n=24, Experiment 2: n=23; Zur: WIST

[HanIbm]; 250-350g) were obtained from our in-house specific-pathogen-free (SPF)

breeding facility. They were caged individually in Macrolon type III cages (48 x 27 x 20

cm) and housed in a temperature (21±1 ºC) and humidity (55±5%) controlled animal

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facility under a reversed light-dark cycle (lights on 0600-1800 hours). Food (Kliba 3430,

Klibamühlen, Kaiseraugst CH) and water were available ad libitum in the home cages.

Behavioural assessments were carried out in the dark phase of the light-dark cycle. All

experiments described here were approved by the Cantonal Veterinary Office, Zurich.

Drugs and pretreatment procedure.

AMPH for injection was prepared by dissolving d-AMPH sulfate (Sigma

Chemical Company, St. Louis, US) in 0.9% NaCl solution to yield a final concentration

of 1, 2, 3, 4 or 5 mg/ml (calculated as the salt). All solutions for injection were freshly

prepared and were administered via the intraperitoneal route in a volume of 1ml/kg.

AMPH or saline (SAL) was administered for six consecutive days, and this was

carried out for Experiments 1 and 2 at the same time. This enabled us to assess locomotor

sensitization in animals from both experiments at the same time (see later). All animals

received three injections per day on each of the injection days, at 06.00, 12.00 and 18.00

hours. Twelve rats were allocated to each of the AMPH-treatment group in Experiments

1 and 2. The injection regime for the escalating dosage schedule of AMPH treatment was

as follows. On day 1, the dosages for the three successive injections were 1mg/kg,

2mg/kg and 3mg/kg. On day 2, the dose injected were 4mg/kg, 5mg/kg and 5mg/kg. On

days 4 to 6, AMPH was administered at a dose of 5mg/kg at each injection time.

SAL-treated controls (Experiment 1: n=12; Experiment 2: n=11) received 0.9%

NaCl injections at the same time that AMPH-treated animals received their injections.

Morris water maze.

A circular tank measuring 2m in diameter and 0.6m high was positioned in the

middle of a well-lit testing room (3 x 4.9 x 2.9m) enriched with distal visual stimuli. The

water tank was made of fibreglass and painted black. The bottom of the maze was raised

0.45m above the room floor. At the beginning of each day, the water maze was filled

with a mixture of cold and hot tap water to a depth of 0.3m, and the water temperature

was maintained at 21°C. A stable circular platform, measuring 11cm in diameter and

painted black, was used as the escape platform. It had a rough surface which allowed the

animal to climb onto it easily once its presence was detected. The platform was

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submerged 2cm below the surface of the water and was therefore hidden from the

animals’ view. A separate white circular disk, also measuring 11cm in diameter, could be

mounted on top of the hidden platform so that it was 10cm above the water surface.

When in place, the white disk served as a distinct local cue for the location of the

otherwise hidden escape platform. Four points, equally spaced along the circumference of

the pool, were arbitrarily assigned as the cardinal points: N, E, S, W. These points served

as the starting positions at which the animals were lowered gently into the water, with

their head facing the wall of the water maze. The area of the pool was conceptually

divided into 4 quadrants (i.e., Q1 to Q4) of equal size with respect to these four starting

positions. A video camera was mounted above the centre of the water maze. It was

connected to an image analysis system (HVS Image, Hampton, UK), which in turn was

connected to a PC running the HVS maze software. The swim path of the animal was

tracked, digitized and stored for subsequent behavioural analysis using the same

software. The experimenter measured escape latency manually by operating a remote

switch connected to the PC signaling the start and the end of a trial.

Behavioural procedures - Experiment 1.

Behavioural testing commenced on the second day after the completion of drug-

or SAL pre-treatment. The animals were first trained to escape from the water to a visible

platform. This comprised 4 separate trials within a day with an inter-trial interval of 30s.

The location of the platform varied among four possible positions (43cm from the edge,

in the middle of the quadrant) among the four trials, but the same starting position for the

rat (namely, position S) was employed throughout. Rats that failed to escape to the

platform within 90s were guided to it by the experimenter. They were allowed to remain

on the platform for 15s before being placed in a waiting cage for 30s before the next trial

commenced. Upon completion of the last trial, the animals were dried with a towel and

returned to their home cages.

On the next two days (the third and fourth day of withdrawal), the rats were

trained to locate the position of the hidden platform, which was now fixed to quadrant

Q4. There were eight trials per day, with the four possible starting positions

counterbalanced among the first four, and among the last four trials. Again, animals that

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failed to escape within 90s were guided to the platform location by the experimenter and

were assigned an escape latency of 90s. They were allowed to spend 15s on the platform,

and then placed in the waiting cage for another 30s before the next trial commenced. The

following dependent measures were recorded on all trials of the water maze tasks: (a)

escape latency (time taken to reach and climb onto the escape platform, in sec), (b) swim

distance (cm), and (c) swim speed (cm/s).

A probe test was carried out on the next day (i.e, the fifth day of withdrawal), in

which the platform was removed, and the animals placed into the water maze for 60s. In

the probe test, the proportion of time and of swim distance allocated to each of the four

quadrants of the water maze was obtained. In addition, the numbers of visits to the target

platform zone (a circular zone of 11cm in diameter), and over alternative zones of

equivalent size located in the middle of other quadrants, were recorded (Janus et al. 2000;

Brown et al. 2000). A zone visit was defined as entry of both shoulders of the rat into the

region of the former platform position. This measure allowed the evaluation of spatial

place preference while controlling for alternative search strategies without place

preferences, such as circular search paths. The latency to the first visit to the target zone

was also noted.

Behavioural procedures - Experiment 2.

In Experiment 2, the animals were first trained in the visible platform task and the

hidden platform tasks as described above prior to AMPH or SAL treatment. The training

procedures were identical to those of Experiment 1, except that during acquisition

training with a hidden platform, there were 4 trials per day, and training continued for a

total of 7 days. Afterward, the animals were treated with AMPH or SAL for 6 days. They

were not tested during this period, or during the first two days of withdrawal.

On the third day of withdrawal, a probe test identical to that in Experiment 1 was

conducted.

On the fourth and fifth days of withdrawal, the animals were tested again as in the

previous acquisition phase, with the exception that the escape platform was now fixed to

quadrant Q2 (i.e., opposite to its previous position, Q4). This condition allowed us to

assess a form of reversal learning. There were again four trials per day in the reversal

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phase. At the end of the last trial of reversal training, animals were returned to the home

cages. Six hours later, they were tested in a probe trial as described before.

Assessment of locomotor sensitization.

At the end of Experiments 1 and 2, all animals were evaluated for locomotor

sensitization to a systemic AMPH challenge (1mg/kg, i.p.). This was carried out in 16

chambers (25cm x 40cm x 40cm high) made of Plexiglas, each located within a sound-

attenuating wooden cabinet. The chamber floor consisted of a layer of autoclaved garden

soil so as to provide a dark background. No illumination was provided within the test

chambers except for an infrared light source. A fan mounted on the wall of each cabinet

provided ventilation and background noise during testing. An infra-red sensitive camera

was mounted 49 cm directly above each chamber to allow assessment of locomotor

activity using an image processing programme written in Visual Basic™ (Microsoft Inc.,

US). It was developed in-house (P. Schmid, Laboratory of Behavioural Neurobiology,

ETH Zurich), and was based on the programme script of the NIH Image software. The

programme was implemented on a personal computer (Dell Computer, OptiPlex GXpro

with a Pentium Pro Processor) running the Windows™ operating system.

The digitized images captured by the cameras at successive seconds were

compared to yield a measure of locomotor activity. Each image was coded in an 8-bit

gray scale. The two images, 1 second apart, were subtracted to give the number of

corresponding pixels that differed by a gray value of 5 or above. The proportion of pixels

scored as different therefore served as a measure of an animal’s displacement and was

taken as an index of locomotor activity. Activity level at this temporal resolution

typically ranged from 0% pixel changed (i.e., total immobility) to a maximum of

approximately 7.5% (Richmond et al. 1998).

Locomotor sensitization was assayed in one third of the animals at each of the

three different time points of withdrawal: 30, 60 or 90 days after the cessation of the last

drug or SAL injection. Animals from Experiments 1 and 2 were tested together. Four

AMPH- and four SAL-treated rats from each of the experimental cohorts were tested at

each time point (except that Experiment 2 only contributed 3 SAL-treated animals at 90

days of withdrawal).

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Locomotor assessment began by placing the rats in the appropriate chamber and

allowing them to remain there for an hour. Following this baseline period, the rats were

briefly removed from the chambers and injected with SAL, before being returned to the

chambers for an additional period of one hour. Afterwards, all rats were given an

intraperitoneal injection of 1.0mg/kg AMPH and observed for the next four hours.

Data obtained from the pre-injection phase, post-SAL injection phase, and post-

AMPH injection phase were analysed separately.

Statistical Analysis.

All statistical analyses were carried out using parametric analysis of variance

(ANOVA) with the appropriate design using the statistical software Statview™ for

Windows (version 5.0.1, SAS Institute Inc., US). In the ANOVAs of the dependent

measures, percentage time and percentage swim distance per quadrant derived from the

probe tests and over the first 4 trials of reversal learning in Experiment 2, the degrees of

freedom associated with the within-subjects factor, quadrants, was reduced by 1. This

adjustment was necessary in order to take into account the loss of one degree of freedom

resulting from the fact that the sum of these dependent measures always summed to

100% in each subject. The degrees of freedom of the relevant interaction and error terms

were also adjusted accordingly.

RESULTS

Experiment 1: The acquisition of the water maze task after amphetamine

withdrawal

Visible platform task. This was carried out on the second day of withdrawal, and

all animals learned to escape from the water by climbing onto the visible platform.

Escape latency and swim distance decreased as training progressed, while swim speed

increased. Separate 2 x 4 (treatment x trials) split-plot ANOVAs on these measures all

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yielded a highly significant effect of trials [latency: F(3, 66) = 17.58, p<0.0001; swim

distance: F(3, 66) = 8.26, p < 0.0001; swim speed: [F(3, 66) = 9.49, p<0.0001]. There

were no significant effects involving treatment, except for a main effect of treatment on

swim speed [F(1, 22) = 7.44, p<0.05] due to the fact that AMPH withdrawn animals

generally swam faster than SAL controls (SAL: 26.00±0.84 cm/sec; AMPH: 30.09±0.88

cm/sec, data not shown)

Acquisition of the hidden platform task. This took place on the third and fourth

days of withdrawal. Both AMPH and SAL animals learned to locate the hidden platform

at a similar rate (Figure 1). Separate 2 x 2 x 8 (treatment x days x trials) split-plot

ANOVAs on the performance measures yielded a significant effect of days [latency: F(1,

22) = 26.93, p<0.0001; swim distance: F(1, 22) = 33.31, p<0.0001] and of trials [latency:

F(7, 154) = 13.62, p<0.0001; swim distance: F(7, 154) = 15.87, p<0.0001]. As expected,

between-trials improvement was more pronounced on the first day, yielding a significant

days x trials interaction in both performance measures [latency: F(7, 154) = 2.63, p<0.05;

distance: F(7, 154) = 3.42, p<0.005)]. A similar pattern of results was obtained in the

analysis of swim speed, which increased as training progressed. This yielded a main

effect of days [F(1, 22) = 5.81, p < 0.05] and of trials [F(7, 154) = 5.15, p < 0.0001], as

well as their interaction [F(7, 154) = 3.81, p<0.001]. Neither the main treatment effect

nor its interactions attained statistical significance in any of the dependent measures

described above.

Probe trial. The probe trial was carried out on the fifth day of withdrawal. As

depicted in Figure 2A and 2B, the animals clearly did not search equally among the four

quadrants. This gave rise to a significant effect of quadrants in the analysis of proportion

time and swim distance spent per quadrant in 2 x 4 (treatment x quadrants) split-plot

ANOVAs [%time: F(2, 44) = 17.14, p < 0.0001; %swim distance: F(2, 44) =17.85, p <

0.0001]. AMPH animals allocated most of their search time (36.80 ± 3.80 %) and swim

distance (36.10 ± 2.90 %) to the target quadrant Q 4, at a level significantly above chance

[p<0.05]. A distinct preference for the target quadrant was, however, lacking in the

control group. At the same time, both groups displayed a strong bias against the non-

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target quadrant Q1. The reason for this result is unclear at this time; however, it is not due

to a lack of environmental cues in this area or to differences in light levels. The treatment

x quadrants interaction did not attain significance. In contrast to the visible platform task,

the swim speed was not different between the AMPH and SAL groups [F(1, 22)=0.39,

p=0.54].

The measure of visits to the former platform position provided a more stringent

measure of accurate search behaviour. As illustrated in Figure 2D, both AMPH and SAL

rats made more crossings over the target zone [F(3, 66) = 27.24, p<0.0001] relative to the

control zones. Although the interaction between treatment and quadrants did not attain

significance, an analysis directly comparing target zone visits between groups in quadrant

4 revealed a near significant treatment effect [F(1, 22)=3.70, p=0.06]. This is consistent

with the observation that AMPH animals made the first target zone visit significantly

earlier than SAL controls [F(1,22)=4.28, p=0.05; Fig 2C].

Fig. 1. Mean escape latency (sec) to locate the hidden platform during acquisition training on days 3 and 4 of withdrawal for amphetamine -withdrawn (AMPH) and control (SAL) rats in Experiment 1. Values are means±SEM over blocks of 4 trials.

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Fig. 2. Probe trial performance of Experiment 1 in which subjects received acquisition training after the drug treatment. Mean percentage time (A) and swim distance (B) recorded during the probe trial on day 5 of withdrawal from an escalating dosage schedule of amphetamine (AMPH) or saline (SAL). Panel C shows the mean latency to the first visit to the target zone (the former platform position in quadrant 4) and panel D shows the number of crossings over the former platform position in comparison to numbers of crossings of similar zones in alternative quadrants. Values are means±SEM of groups in that. P-values denote comparisons between AMPH and SAL groups.

Experiment 2: The retention of the water maze task and reversal learning after

amphetamine withdrawal

Pre-treatment acquisition. All animals acquired the visible and hidden platform

tasks prior to any drug treatment as evidenced by all performance measures (all data not

shown). The animals were then divided into groups (AMPH and SAL), matched for their

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performance on the hidden platform task. Escape latencies on the last day of acquisition

were as follows: AMPH-designated animals – 15.7±2.2 sec; SAL-designated animals –

18.7±2.0 sec.

Probe trial. This took place on the third day of withdrawal, and 8 days after the

final acquisition trials of the hidden platform task completed before drug treatment.

Performance was similar between AMPH and SAL animals across all measures (Fig 3A,

B, C, D). As in Experiment 1, the animals failed to show a distinct preference for the

target quadrant (Q4) in either the measures of percentage time or of swim distance per

quadrant. There was however a significant effect of quadrants in the analysis of these two

measures [% time: F(2, 42) = 11.20, p < 0.01; % swim distance: F(2, 42) = 10.07,

p<0.01]. Further analysis revealed that this was solely due to a bias against quadrant Q1,

which was apparent in both AMPH and SAL rats, and was similar to that previously seen

in Experiment 1. Again, as in Experiment 1, a clearer preference for the target zone

emerged when the number of target zone visits was considered [F(3, 63)=7.78, p<0.001],

and the two groups were comparable on this measure as well as on the latency to the first

target zone visit. The two groups also did not differ in terms of total swim distance or

swim speed in the probe test.

Reversal Learning. Performance during the reversal learning phase, as

indicated by escape latency and swim distance, showed a clear improvement over the 8

trials (2 days) of training (Fig 4A), as confirmed by ANOVAs of the two measures which

yielded a highly significant effect of days [F(1, 21) = 36.79, p<0.0001], of trials [F(3, 63)

= 22.63, p<0.0001], and of their interaction [F(3, 63) = 10.08, p<0.0001]. The presence of

the interaction is consistent with the impression that within-day improvement was

significantly more pronounced on the first than on the second day of training (Figure 4A).

There was also an increase in swim speed from day 1 to day 2 [F(1, 21)=10.39, p<0.005],

but the two treatment groups did not differ on this measure (day 1: SAL = 23.58±0.9

cm/sec, AMPH = 25.08±0.92 cm/sec; Day 2: SAL = 29.51±0.89 cm/sec, AMPH =

26.43±0.82cm/sec). The main effect of treatment did not attain significance in either the

swim distance or escape latency measure, but the days ± trials ± treatment interaction

CHAPTER 4

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reached significance [F(3,63)=3.74, p<0.05] in the analysis of latency. Post-hoc analysis

confirmed that latencies were reduced in AMPH vs. SAL rats at 2 time points (reversal

day 1, trail 4: p=0.0075; reversal day 2, trail 2: p=0.0327). The 3-way interaction was,

however, not significant in the analysis of swim distance.

The analyses based on latency and swim distance do not allow one to evaluate

possible competition between the previous and current locations of the escape platform

over the animal’s performance. These effects would be expected to be particularly

pronounced in the early phase of reversal learning. We therefore calculated the

proportion time and proportion swim distance spent per quadrant (out of each animal’s

escape latency or swim distance, respectively) in order to examine more closely the

animal’s search pattern over the first 4 trials of reversal learning.

As shown in Figure 4B, both AMPH and SAL groups initially exhibited a distinct

preference for the former target quadrant (Q4) which gradually diminished across the first

four trials. At the same time, the preference for the new target quadrant (Q2) developed

progressively and emerged as the most preferred quadrant by the 3rd and 4th trials. This

reversal of quadrant preference was apparent in both AMPH and SAL rats, but was more

prominent in the AMPH group due to their superior performance over the controls on the

final trial. These interpretations are supported by separate ANOVAs of percent time and

swim distance spent per quadrant over the four trials yielding a main effect of quadrants

[%time: F(2, 42)=43.98, p<0.0001; %swim distance: F(2, 42)=53.48, p<0.0001], a

quadrants x trials interaction [%time: F(6, 126)=34.75, p<0.0001; %swim distance: F(2,

42)= 33.93, p<0.0001], a quadrants x treatment interaction [%time: F(2, 42)=3.066, ns;

%swim distance: F(2, 42)= 3.74, p<0.05], and a treatment x quadrants x trials interaction

[%time: F(6, 126)= 2.82, p<0.05; %swim distance: F(6, 126)=4.09, p<0.01]. A posthock

comparison between SAL and AMPH pretreated animals for both measures revealed a

significantly greater preference of AMPH-withdrawn animals for quadrant 2 in trial 4

[%time: p < 0.005; %swim distance: p < 0.005] as well as reduced preference for the

other three quadrants in the same trial [quadrant 1- %time: p=0.065; %swim distance:

p<0.01; quadrant 3- %time: p<0.005; %swim distance: p<0.005; quadrant 4- %time:

p<0.05; %swim distance: p<0.05].

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Second probe trial. The AMPH and SAL groups performed similarly on the

probe test, with both groups displaying a distinct preference for the new target quadrant

(Q2) as expressed in percent time and swim distance spent per quadrant. ANOVAs

revealed a significant effect of quadrants in both measures [%time (F(2, 42) = 30.12, p <

0.01); %swim distance: F(2, 42)=32.25, p<0.01, Fig 5A, B]. These results conform with

the analysis of target zone visits, with animals making more visits to the target [F(3,

63)=15.68, p<0.0001] than the control zones, a preference which was comparable

between groups (Fig 5D). No group differences were found in swim speed, total swim

distance, latency to the first target zone visit or number of target zone crossing (Fig 5C,

D).

Fig. 3. Probe trial performance of Experiment 2 on day 3 of withdrawal from an escalating dosage schedule of amphetamine (AMPH) or saline (SAL) pretrained prior to the drug treatment. Mean percentage time (A) swim distance (B), latency to first target zone visit (C) and number of zone visits (D) recorded over 60 sec of a probe trial. The target zone was located in quadrant 4. For further information see fig. 2. Values are means±SEM.

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Fig. 4. A. Mean escape latency (sec) to locate the hidden platform during reversal learning on days 4 and 5 of withdrawal for amphetamine-withdrawn (AMPH) and control (SAL) rats in Experiment 2. Panel B shows mean percentage time spent in the four quadrants (calculated based on individual escape latency) on each of the 4 trials of the first reversal day. The rats were previously trained to the former platform position (quadrant 4), the platform during reversal learning was located in quadrant 2. Values are means±SEM. * - p<0.05, AMPH vs. SAL.

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Fig. 5. Mean percentage time (A), swim distance (B), latency to first target zone visit (C) and number of zone visits (D) recorded during the probe trial on day 5 of withdrawal from an escalating dosage schedule of amphetamine (AMPH) or saline (SAL). Data are from animals in Experiment 2 that received acquisition before and reversal learning after the drug treatment. For further information see fig. 2. Values are means±SEM.

Locomotor sensitization to systemic amphetamine challenge

The three phases of locomotor activity assessment were analysed separately. In all

cases, activity level was subjected to an ANOVA with the between-subjects factors of

drug treatment and withdrawal periods, and the within-subjects (repeated measure) factor

of 10min bins (see Fig 6).

Pre-injection phase. There was a clear reduction of activity level over the 60min

of baseline exploration, as the animals acclimatized to the test chamber. This gave rise to

a significant main effect of 10-min bins [F(5, 205)=149.34 p<0.0001]. Neither the main

effect of treatment (AMPH vs SAL) nor of withdrawal periods attained statistical

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significance. However, there was a significant interaction between withdrawal periods

and 10-min bins [F(10, 205)=1.95, p<0.05] which was attributed to a slightly lower level

of activity in both AMPH and SAL animals tested at 30 days withdrawal during the first

bin and a slightly higher activity in both treatment groups tested at 60 days withdrawal

during the second and the third bin of activity monitoring.

Post SAL injection phase. As expected, the SAL challenge led to an initial

increase of locomotor activity followed by a gradual reduction over time [10min-bins:

F(5, 205)=59.11, p<0.0001]. The activity level after SAL injection was, however, more

pronounced in both AMPH and SAL animals tested 30 days into withdrawal, relative to

animals tested at 60 or 90 days into withdrawal. This led to the significant main effect of

withdrawal periods (F(2, 41) = 55.73, p<0.0001) and its interaction with bins [F(10,

205)=5.50, p<0.0001]. Neither the main effect of treatment (AMPH vs SAL) nor any of

its interaction terms attained statistical significance.

Post AMPH injection phase. AMPH potentiated locomotor activity in all

animals and this effect peaked between the 2nd and 4th bins after injection. This effect

was more pronounced in the AMPH than in the SAL rats, and this difference was clearest

over the first 60min. Afterwards, locomotor activity gradually returned back to the pre-

drug level. These observation are supported by the main effect of 10min-bins [F(23,

943)=90.22, p < 0.0001] and its interaction with treatment [F(23, 943)=3.24, p<0.0001].

Post-hoc comparisons at successive bins indicated that this interaction stemmed from a

difference between AMPH and SAL animals that was significant in the first four 10-min

bins. Neither the main effect of withdrawal periods nor any of its interactions attained

significance; however, posthoc comparisons between the AMPH and SAL animals

revealed the most significant differences in the 90-day group, somewhat more variable

sensitization in the 60-day group and only a brief peak of sensitized locomotor activity in

the 30-day group. An increasing degree of senitization at later withdrawal intervals is

consistent with previous findings (Paulson, Camp and Robinson, 1991).

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Fig. 6. Locomotor activity measured during an initial 1-hour habituation period, a 1-hour period following an injection of saline vehicle, and a 4-hour period following a challenge injection of 1 mg/kg amphetamine. Animals were tested at either 30, 60 or 90 days into withdrawal, as calculated by the day of the last saline (SAL) or amphetamine (AMPH) injection. + - p<0.05 30-day withdrawal groups, AMPH vs. SAL; # - p<0.05 60-day withdrawal groups, AMPH vs. SAL; * - p<0.05 90-day withdrawal groups, AMPH vs. SAL.

DISCUSSION

The present study represents the first attempt to evaluate the effects of withdrawal

following an escalating dose schedule of systemic AMPH administration on the classical

water maze procedure followed by reversal learning. The schedule of repeated AMPH

administration was effective in inducing long-lasting locomotor sensitization as

demonstrated subsequent to the two experiments. We demonstrated that this regimen of

AMPH withdrawal impaired neither the acquisition (Experiment 1) nor the retention

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(Experiment 2) of spatial memory. At the same time, animals undergoing AMPH

withdrawal appeared to show enhanced reversal learning in Experiment 2. AMPH

withdrawal also tended to enhance probe test performance in Experiment 1, but this effect

was only suggested in the measures of target zone visits and latency to the first target

zone visit. Examination of individual swim speed on the probe test in Experiment 1

excluded the possibility that this effect of AMPH withdrawal can be attributed to such a

difference. The limited size of this promnesic effect is also unlikely to be due to a ceiling

effect as there was room for further improvement in the probe test for both groups of

animals.

Moreover, the marginal effect of AMPH withdrawal on probe performance was

clearly not accompanied by an effect of a similar direction during acquisition in the

measures of escape latency and swim distance. Against such a clear absence of effect on

acquisition, the group difference in the probe test should be interpreted with caution,

especially when a comparable effect was not seen in the second probe test after reversal

learning in Experiment 2. Evidence suggestive of an enhancement of water maze

acquisition has also been obtained in rats following a prolonged period of chronic L-

AMPH pre-treatment for 4 months (Gelowitz et al. 1994). However, these authors

employed a peculiar trials-to-criterion measure to index water maze acquisition

performance in individual animals, without illustrating the results on either escape

latency or swim distance in the conventional manner. In appetitive Pavlovian

conditioning, a more consistent effect of enhancement has been reported in rats

undergoing AMPH withdrawal (Harmer and Phillips 1998; Taylor and Jentsch 2001).

Furthermore, using an AMPH withdrawal regime identical to the one employed here,

Pezze et al. (2002) reported a similar enhancement effect of withdrawal in aversive fear

conditioning. Acute AMPH has been reported to increase memory consolidation and

increase the impact of reinforcement in some learning paradigms (McGaugh et al. 1989;

Killcross et al. 1993). It remains to be seen whether the enhancing effects of AMPH

withdrawal in various learning paradigms might be similarly indicative of an underlying

improvement in mnemonic function or changes in reinforcement processing. However,

given that increased stressfulness of water maze testing (i.e. colder water temperature)

has been shown to improve platform retention (Sandi et al., 1997), it is possible that

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improved consolidation of maze learning mediated by increased emotionality or an

enhanced perception of the aversiveness of the water maze might have contributed to the

somewhat improved probe trial performance of the AMPH- withdrawn rats.

We conclude that the present AMPH regimen is best considered as having little

effect upon the acquisition of the water maze procedure employed here. This

interpretation is in line with a recent report of a lack of an effect during AMPH

withdrawal on spatial working memory (Stefani and Moghaddam 2002). The report by

Stefani and Moghaddam (2002) disagrees with earlier studies that also evaluated spatial

working memory (Bruto et al. 1983a, b). It is clear that differences in test procedure or

paradigms, AMPH treatment regimen or withdrawal period could be important factors in

explaining the divergent results in the literature on AMPH withdrawal and mnemonic

function. A systematic analysis of these factors is essential in clarifying some of the

existing contradictions.

On the other hand, the effect of AMPH withdrawal on reversal learning obtained

in Experiment 2 is clear. Here, the animals were trained prior to AMPH treatment. A

probe test was conducted on the 3rd withdrawal day, i.e., 9 days after the last day of

acquisition training. The relatively long retention period (the probe test is usually

conducted 24hrs after the last acquisition trail) might have contributed to the relatively

weak performance on this probe test, with few animals in either group showing a distinct

preference for the target quadrant. However, when reversal learning was initiated on the

following day, there was very clear evidence for retention on the first trial as indicated by

the analysis of proportion time spent per quadrant (see Fig 4B). The emergence of a clear

sign of retention on the first trial of reversal learning, instead of the probe test conducted

24hr earlier, suggests that the probe trial itself might serve as a reminder cue for the

location of the platform and/or the test procedure.

In practice, the first trial of reversal learning was almost identical to the standard

probe test, because the new location of the escape platform remained unknown to the

animals until they eventually made contact with it by chance. We therefore employed the

measure of proportion search time, which normally is adopted in the analysis of probe

performance (in which the total search time is fixed), to index performance over the four

reversal trials on this day. This provides a clear depiction of the shift from a preference

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for the former target quadrant to the new target quadrant. This shift or reversal was more

rapid in the AMPH-treated animals, and contributed to their superior performance over

the controls on the final trial of the first reversal day, which was also significant in terms

of escape latency. On the next day, the difference between groups diminished to a more

comparable level, implying that the effect of AMPH withdrawal on reversal was specific

to its early period when learning about the new location of the escape platform was most

vulnerable to interference by what had been previously learned.

The facilitation of reversal learning seen in Experiment 2 poses a direct contrast

to the recent report by Jentsch et al. (2002). These authors employed an object

discrimination reversal task in Vervet monkeys to assess the effect of withdrawal from

repeated cocaine exposure. A deficit specific to the reversal phase was reported.

However, it should be noted that the monkeys received extensive training on

discrimination and reversal prior to the reported experiments (pp.184, Jentsch et al.

2002). Hence, it is likely that the monkeys would have already acquired a learning set

(perhaps in the form of a “win-stay/lose-shift” strategy) when confronted with subsequent

discrimination-reversal problems. Indeed, a disruption of successive discrimination by

AMPH has been reported before (Ahlenius et al. 1974), which stands in contrast to the

drug’s facilitatory effect on single discrimination reversal (Weiner et al. 1986).

Hippocampal system damage is another manipulation that can facilitate reversal learning

after acquisition of the first discrimination problem, and at the same time can lead to a

deficit under a situation in which the subjects are tested on repeated reversals (Fagan and

Olton 1986). Secondly, opposite effects on reversal learning have also been associated

with acute AMPH (1mg/kg, i.p.) as a function of the amount of training on the initial

discrimination problem before reversal. Reversal learning is impaired by AMPH when

the animals are over-trained on the initial problem prior to reversal learning, but is

facilitated when reversal is initiated upon attainment of criterion performance (Weiner et

al. 1986). According to Sutherland and Mackintosh (1972), the amount of training on

discrimination problems can alter the attentional and perceptual processes underlying

discrimination learning. Hence, the apparently conflicting results between our data and

that reported by Jentsch et al. (2002) could be attributed to an important aspect of

procedural differences, which renders the latter experimental design less congenial to the

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demonstration of the simple discrimination reversal effect, and more likely to be

influenced by additional cognitive factors (see Sutherland and Mackintosh 1972;

Mackintosh 1974).

The simple reversal effect could, at least in part, be explained in terms of

proactive interference whereby what had been learned previously impedes the acquisition

or expression of new learning. There is evidence that acquisition was not affected by

AMPH withdrawal in Experiment 1, and AMPH withdrawal, if anything, tended to

enhance probe test performance, which is consistent with the interpretation that the

withdrawn rats were capable of remembering the location of the platform. Secondly,

AMPH-treated animals also performed at a comparable level relative to controls in the

first probe test of Experiment 2, and in the second probe test after reversal learning in

Experiment 2. The possibility that the facilitation of reversal learning seen in AMPH-

treated animals in Experiment 2 simply stemmed from poor learning prior to the

commencement of reversal training can therefore be excluded. If a mnemonic account is

unlikely to explain our present finding of an attenuation of the reversal effect seen during

AMPH withdrawal, what other psychological mechanisms might possibly be responsible

for it?

We have recently reported that the AMPH withdrawal schedule employed here

was effective in disrupting the development of a form of selective learning, called latent

inhibition (Murphy et al. 2001; Russig et al. 2002). Latent inhibition refers to the

observation that repeated non-reinforced exposures to a stimulus retard subsequent

learning about the predictive value of this stimulus when it is paired with other significant

events (Lubow 1973, 1989). The latent inhibition effect can be demonstrated in Pavlovian

and instrumental paradigms, and is interpreted by some as a form of proactive

interference (Kramer and Spear 1991, 1993; Bouton 1993; Escobar et al. 2002a, b).

Accordingly, the attenuation of the latent inhibition effect (i.e., stimulus pre-exposure

failed to impede subsequent learning) during AMPH withdrawal implies that the

deleterious effect of proactive interference (upon subsequent learning) was diminished in

these animals relative to controls. In both cases, this effect of AMPH withdrawal on

proactive interference expresses itself as facilitated learning.

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A similar pattern of results was also reported following an acute administration of

AMPH (1mg/kg, i.p.), which disrupts latent inhibition (Weiner et al. 1988) and enhances

reversal learning (Weiner et al. 1986; Weiner and Feldon 1986). The disrupted latent

inhibition observed in both cases could also be restored by the administration of

neuroleptic drugs (Warburton et al. 1994; Moran et al. 1996; Weiner et al. 1996; Russig

et al. 2002). The present preparation of AMPH withdrawal thus re-produces two specific

cognitive effects that have been associated with acute AMPH treatment. Although our

data were obtained in animals in the absence of any acute drug effect, similar brain

structures or neural circuitry might be implicated in acute AMPH challenge as well as

during withdrawal from the present escalating dose regimen. Weiner and colleagues

(Weiner 1990; Weiner and Feldon 1997) proposed an account which suggests that the

attenuation of latent inhibition and the facilitation of reversal learning following acute

(low doses) AMPH are due to an effect of the drug, acting on the nucleus accumbens, in

enhancing behavioural switching. This hypothesis attributes a role of switching to the

nucleus accumbens under the modulation of the ascending dopaminergic input from the

ventral tegmental area and the glutamatergic limbic inputs originating from the

hippocampal formation, entorhinal cortex, amygdala, and prefrontal cortices. It is

suggested that acute AMPH disrupts this balance between the two sets of inputs resulting

in enhanced behavioural switching (Weiner 1990; Weiner and Feldon 1997). It is

plausible that the present AMPH regimen induces persistent structural, physiological,

neurochemical and/or molecular changes in the relevant brain circuitry, which mimic the

functional imbalance produced by acute AMPH challenge.

The ability of repeated administration of psychostimulants (including AMPH,

cocaine, and PCP) to induce psychotic states in humans closely resembling those of acute

schizophrenic patients (Ellinwood 1967; Snyder 1973; Post 1975; Brady et al. 1991) has

led to the use of AMPH in animals for study of the neurobiology of schizophrenia. As

noted by Laruelle et al. (2001), endogenous sensitization in the striatal complex might

play a role in the aetiology of schizophrenia and the genesis and/or maintenance of

psychotic symptoms. The present study adds to our understanding of the behavioural and

cognitive effects during AMPH withdrawal, which would be instrumental in the

development of a holistic animal model of schizophrenia. Additional studies would be

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required to ascertain the generality of our current findings and the specific neural

mechanisms that might be responsible.

ACKNOWLEDGEMENTS

This study was supported by the Swiss Federal Institute of Technology (ETH-

Zurich, Switzerland). We would like to thank the staff of the animal facility for their care

and maintenance of the animals used in this study, Mr. Peter Schmid for his valuable

technical assistance and Mrs. Jane Fotheringham for her editorial help.

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CHAPTER 5 ------------------------------------------------------------------------------------------------------------

AMPHETAMINE WITHDRAWAL MODULATES FOSB

EXPRESSION IN MESOLIMBIC DOPAMINERGIC TARGET

NUCLEI: EFFECTS OF DIFFERENT SCHEDULES OF

ADMINISTRATION

Carol A. Murphy, Holger Russig, Marie-Astrid Pezze, Boris Ferger, Joram Feldon

Neuropharmacology, in press

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CHAPTER 6 ------------------------------------------------------------------------------------------------------------

AMPHETAMINE WITHDRAWAL DOES NOT PRODUCE A

DEPRESSIVE-LIKE STATE IN RATS AS MEASURED BY THREE

BEHAVIORAL TESTS

Holger Russig, Marie-Astrid Pezze, Nina I. Nanz-Bahr, Christopher R. Pryce, Joram

Feldon and Carol A. Murphy

Behavioral Pharmacology 2003, 14: 1 - 18

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DISCUSSION

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DISCUSSION

Animal models are important tools in the investigation of the mechanisms of human disease

and in the design and search for new treatments. This is particularly true for psychiatric and

mental disorders in which the defining features of the disease are often confined to behavioral

and cognitive levels. The collection of studies in this thesis tested whether withdrawal from an

escalating dosage schedule of amphetamine (AMPH) produces behavioral alterations in rats that

can be related to the symptomatology and neural mechanisms of schizophrenia. The theoretical

basis of this work is derived primarily from the endogenous sensitization theory of schizophrenia

(Lieberman et al. 1990, 1997, Laruelle 2000, Ujike 2002). This theory suggests that

neuroadaptive changes induced in animals by repeated psychostimulant administration and

developed during the subsequent withdrawal period might be associated with the pathological

changes observed in schizophrenic patients. The studies presented in this thesis are therefore of

critical value to the evaluation of the use of AMPH withdrawn animals as a model of

schizophrenia. Findings from specific studies of this thesis have already been discussed at the end

of each research article in the preceding chapters. In this chapter, a general discussion is provided

with a focus upon the validity of amphetamine withdrawal as a potential animal model of

schizophrenia.

Evaluation of the validity of an animal model of a given disease needs to take into account

three separate criteria (Willner 1986, 1991). An animal model is considered to possess face

validity when the experimentally induced condition (behavioral, physiological etc.) closely

mimics some aspects of the targeted disease. The criterion of predictive validity considers

whether the experimentally induced condition can be effectively treated with clinically relevant

therapies, and whether the model has the ability to discriminate between clinically effective and

ineffective interventions. Lastly, construct validity is concerned with whether there is a common

mechanistic theory (based on similar physiological, anatomical, or neurochemical mechanisms)

that can explain the abnormalities in both the patients and the manipulated animals.

The endogenous sensitization theory asserts that the brains of animals undergoing AMPH

withdrawal are in an altered state as a result of neuroadaptive changes, and that similar

neuroadaptive changes exist in the brains of schizophrenic patients which contribute to the

genesis of psychotic symptoms. In AMPH withdrawn animals, these neuroadaptive changes are

DISCUSSION

149

believed to lead to a state of sensitization. Behaviorally, sensitization is classically demonstrated

as an enhanced locomotor response or motor stereotypic response to a psychostimulant challenge

on a level greater than that associated with a single drug administration in previously drug-naive

subjects (Robinson and Becker 1986). The AMPH administration schedules studied in the present

thesis all lead to the expression of behavioral sensitization. The AMPH challenge administrations

included various doses (0.5 - 5.0mg/kg, i.p.) and the challenge tests were conducted following

different periods of withdrawal (see Chapters 3, 4, 6, unpublished observations). A point worth

noting is that withdrawal from an escalating dosage schedule of AMPH (three daily injections for

6 days, 1.0 - 5.0 mg/kg, ESC) and an intermittent dosage schedule of AMPH (one daily injection

for 6 days, 1.5 mg/kg, INT) were demonstrated to be associated with sensitized responses of a

comparable magnitude (although the effects of withdrawal from the 2 schedules on other

behavioral/cognitive measures and gene expression did differ). It remains to be tested whether the

same pattern of results might emerge with other forms of challenge, e.g., stress or drugs other

than AMPH.

Neurochemically, the expression of behavioral sensitization is, among other effects,

associated with enhanced dopamine release in some areas of the mesocorticolimbic system.

Hence, dopaminergic dysregulation in the mesocorticolimbic system constitutes a common

mechanistic account of the emergence of behavioral sensitization and the genesis of psychotic

symptoms in schizophrenic patients. Accordingly, this provides a source of construct validity for

AMPH withdrawal, and the expression of behavioral sensitization in particular, as a model of

schizophrenia (Snyder 1973, Robinson and Becker 1986, McKenna 1987, Carlsson 1988,

Laruelle 2000, Vanderschuren and Kalivas 2000).

1 AMPHETAMINE WITHDRAWAL AS AN ANIMAL MODEL OF SCHIZOPHRENIA

Given that the clinical definition of schizophrenia emphasizes symptoms that demand verbal

descriptions or expressions of the patient’s beliefs, how may one begin to assess the face validity

of an animal model of schizophrenia? In an attempt to assess the face validity of withdrawal from

an ESC schedule of AMPH as an animal model of schizophrenia, the present thesis focuses on

two behavioral phenomena – latent inhibition (LI) and prepulse inhibition (PPI). Both LI and PPI

DISCUSSION

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can be demonstrated in animals as well as in humans (Lubow, 1989, Weiner 2000, Braff et al.

2001; Swerdlow et al. 2001). Whilst LI is an example of selective learning, PPI is believed to

measure sensorimotor gating as a form of pre-attentional control. Disruptions of LI and of PPI

have been reported at least in some subsets of schizophrenic patients, and are considered as

evidence for the presence of specific information processing impairments that are central to the

symptomatology of the disease (Gray et al. 1992, 1995, Weiner 2000, Braff et al. 2001). A

satisfactory animal model of schizophrenia should therefore be expected to be show disruption of

LI and PPI, thereby providing evidence of face validity. Secondly, studies of the neural substrates

of the normal expression of LI and PPI have frequently emphasized the critical involvement of

the mesocorticolimbic system. In particular, enhancement of dopamine transmission in the

mesocorticolimbic system can disrupt LI and PPI (Moser et al. 2000, Weiner 2000, Geyer et al.

2001, Swerdlow et al. 2001). Hence, findings of PPI and/or LI disruption during AMPH

withdrawal would also constitute additional support for construct validity in the use of AMPH

withdrawn animals as a model of schizophrenia.

1. 1 EFFECTS OF AMPHETAMINE WITHDRAWAL ON LATENT INHIBITION

We consistently found that LI was disrupted during withdrawal from an ESC schedule of

AMPH, as demonstrated using an active avoidance paradigm (chapters 1-3). It should be noted

that this effect was not seen following withdrawal from an INT dosage schedule of AMPH,

which was instead associated with enhanced LI (Murphy et al. 2001a). Further discussion

concerning the opposite effects of withdrawal from the two different schedules will be given later

in this chapter. LI disruption following withdrawal from an ESC schedule of AMPH can be

reliably observed during the first two weeks of withdrawal, suggesting that changes in the brain,

which might be responsible for LI disruption, are present for a comparable period of time. We

have preliminary evidence that LI disruption following AMPH withdrawal does not last

indefinitely, however. When tested 2 months after cessation of AMPH, LI was apparently normal

(Russig et al. unpublished observations, see also Chapter 1). In contrast, expression of behavioral

sensitization can even be observed in rats one year following the last drug administration. This

suggests that the neuroadaptive changes in the brain responsible for the expression of behavioral

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151

sensitization are present for a much longer period of time, perhaps even throughout the animal’s

lifetime (Robinson and Becker 1986, Paulson et al. 1991). The differences in the time course of

these effects suggest dissociation between the neural substrates mediating the expression of

AMPH-induced locomotor behavioral sensitization and the disruption of LI during AMPH

withdrawal.

It should be noted that the LI disruption reported here was always obtained in the absence of

any specific challenge (drug or stress). Under such conditions, animals sensitized with

psychostimulants do not exhibit any hyperlocomotor activity as such (i.e., the neuroadaptive

changes involved remained behaviorally silent). In order to demonstrate sensitized

hyperlocomotor activity, a challenge in the form of a psychostimulant or stress challenge is

required. Hence, one might argue that the absence of LI disruption and the presence of AMPH-

induced behavioral sensitization demonstrated at longer withdrawal intervals are not readily

comparable due to the fact that the latter was demonstrated under the acute influence of a

psychostimulant challenge while the former was not. It would therefore be of interest to test

whether, under similar psychostimulant challenge, acute AMPH-induced LI disruption would be

sensitized or not in AMPH withdrawn animals. If not, it would extend our finding of a contrast

between the presence of locomotor behavioural sensitization (under psychostimulant challenge)

and the absence of LI disruption (under drug-free condition) at later withdrawal periods. This

would strengthen our interpretation of dissociation between the neural adaptive changes

responsible for the two phenomena.

One difficulty of such an experiment would be to distinguish between the sensitizing effect

of AMPH withdrawal and the acute effect of AMPH, because the latter is known to disrupt LI

when administered alone to drug-naive animals (for review Moser et al. 2000). Therefore, in

order to demonstrate a sensitized effect on LI disruption, one needs to employ conditions in

which the disruptive effect of acute AMPH on LI is minimal or even absent (e.g., with increased

CS duration during preexposure and conditioning, De la Casa et al. 1993). If LI is disrupted

under such conditions in AMPH withdrawn animals challenged with AMPH, then one might

conclude that behavioral sensitization is paralleled by a similar sensitization effect in terms of LI-

disruption at the later withdrawal periods.

Alternatively, one way to circumvent this technical difficulty is to evaluate whether a stress

challenge can lead to LI disruption following a more protracted withdrawal period, given that

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152

stress is also known to lead to the expression of behavioral sensitization through the mechanism

of cross sensitization (Antelman 1980, Prasad et al. 1998). The use of stress, in this respect, is of

particular relevance to the fact that the onset of schizophrenia is often triggered by stressful

events (Lewis and Lieberman 2001).

In summary, the data reported in Chapters 1-3 demonstrated that withdrawal from an ESC

schedule of AMPH could be considered as animal model of schizophrenia with face validity by

showing that it can mimic one central cognitive dysfunction in schizophrenia, namely disruption

of LI.

We went on test whether drugs that are effective in the treatment of schizophrenia would

prevent LI disruption during withdrawal from an ESC schedule of AMPH. These tests not only

serve to extend the face validity of the AMPH withdrawal model of schizophrenia, but also act as

a test of its predictive validity. We tested one typical and one atypical antipsychotic drug

(haloperidol and clozapine), and found both to be effective in reversing the LI disruption during

AMPH withdrawal, thus demonstrating the predictive validity of the AMPH withdrawal model of

schizophrenia.

Notably, haloperidol and clozapine also prevent LI disruption induced by acute

administration of AMPH (for rev. Moser et al. 2000). The disruption of LI by acute AMPH has

been associated with enhanced dopamine transmission in the mesocorticolimbic system,

particularly in the nucleus accumbens. Importantly, this effect can be prevented by the

coadministration of neuroleptic drugs with antagonist properties for D2 dopamine receptors

(Weiner 2000, Joseph et al. 2000, Moser et al. 2000). We therefore speculated in the discussion

of chapter 2 that, as in the acute AMPH preparation, enhanced dopamine transmission in the

nucleus accumbens, perhaps in the core subterritory, might be responsible for LI disruption

during AMPH withdrawal. Furthermore, we suggested that haloperidol and clozapine restore LI

by the blockade of D2 receptors in the nucleus accumbens (Gray et al. 1997, Weiner and Feldon

1997, Murphy et al. 2000, Weiner 2000, Pezze et al. 2002). These hypotheses can be tested in

future experiments employing more selective and specific manipulations. In vivo microdialysis

could also be used to test if the disruption of LI seen during AMPH withdrawal is associated with

enhanced dopamine transmission in either the nucleus accumbens shell or core. This hypothesis

also predicts that intra-accumbens infusions of selective D2 dopamine antagonists would be

effective in reversing the LI-disruption induced by AMPH withdrawal.

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153

Regardless of the precise anatomical locus of the hypothesized enhanced dopamine release

responsible for LI disruption, an outstanding question remains as to how the postulated enhanced

dopamine transmission can occur in AMPH withdrawn animals in the absence of a

pharmacological challenge. According to the concept of cross-sensitization between

psychostimulants and stress, dopamine release might be enhanced in sensitized animals in

response to the aversive stimuli employed during the LI testing procedures (Antelman 1980,

Prasad et al. 1998). Against this hypothesis, restraint stress or tone-shock pairing exposure during

AMPH withdrawal did not enhance nucleus accumbens dopamine release in AMPH sensitized

animals compared to controls (Weiss et al. 1997, Pezze et al. 2002). Human, as well as animal,

investigations have suggested that schizophrenic patients and AMPH withdrawn animals exhibit

higher basal levels of dopamine or dopamine utilization in the striatal complex (Robinson and

Camp 1987, Swerdlow et al. 1991, Abi-Dargham et al. 2000, Seeman and Kapur 2000).

However, we did not detect enhanced basal levels of dopamine in the nucleus accumbens shell or

core using a microdialysis approach during withdrawal from an ESC schedule of AMPH (Pezze

et al. 2002). Similarly, in post-mortem neurochemical analysis of structures of the

mesocorticolimbic projections, including nucleus accumbens core and shell, we did not obtain

evidence for altered basal dopamine tissue levels during AMPH withdrawal (chapter 5). In

addition, the stress hormone response as measured by blood plasma levels of adrenocorticotropic

hormone (ACTH) and corticosterone (CORT) was not altered in animals withdrawn from an ESC

schedule of AMPH in response to either swim or restraint stress (chapter 6, unpublished

observations).

In humans, LI might be also disrupted due to high levels of anxiety and this might be a

reason for LI disruption during AMPH withdrawal (Braunstein-Bercowitz et al. 2002). However,

against this view, AMPH withdrawn animals did not exhibit enhanced anxiety as measured on

the elevated plus maze (Russig et al. unpublished data). It might well be that this situation is not

anxiety-provoking enough for animals withdrawn from AMPH. As mentioned above, the

mechanism of an enhanced nucleus accumbens dopamine response during withdrawal from an

ESC schedule of AMPH in the absence of a pharmacological challenge postulated to occur in a

test session of active avoidance LI is unknown and requires further investigation.

Altered dopamine transmission in the nucleus accumbens has also been associated with

effects on locomotor activity during AMPH withdrawal (Robinson and Becker 1986). Results

DISCUSSION

154

obtained using the conditioned active avoidance LI paradigm can be confounded by effects of

AMPH withdrawal and haloperidol/clozapine treatment on locomotor activity and avoidance

learning. Such potential confounds have been already discussed in Chapters 1 and 2, where we

suggest that these effects do not account for our findings. However, for further investigation of LI

deficits during AMPH withdrawal and their restoration by drugs with neuroleptic properties

affecting locomotor activity, procedures that can minimize the influence of locomotor activity

should be employed. Two such paradigms would be conditioned emotional response and

conditioned taste aversion. A pilot study toward establishing the ESC AMPH withdrawal model

of disrupted LI in a conditioned taste aversion paradigm is presented in Appendix 2, showing that

haloperidol can restore acute AMPH induced LI disruption in this paradigm.

The observations that clozapine and haloperidol reinstate LI in AMPH withdrawn rats

parallel similar observations following LI disruption induced by acute AMPH (for rev. Moser et

al. 2000). A clear advantage of an AMPH withdrawal model compared to the acute AMPH model

is that behavioral assessments can be made during the drug-free withdrawal period, thus avoiding

the potential drug-drug interactions that characterize many other acute pharmacologically-

induced animal models of schizophrenia. Moreover, the relatively persistent nature of the deficit

in LI during AMPH withdrawal allows for flexibility in the precise timing of test drug

administration. The neuroadaptations induced by repeated AMPH treatment are more likely to

model the chronic changes found in a schizophrenic brain (as supported by the relatively

permanent nature of the expression of behavioral sensitization) than would any acute treatment.

Other animal models with a similar aim in mimicking chronic brain changes that might be

associated with the disease process of schizophrenia have also been proposed. These include the

neonatal hippocampal lesion model (Lipska and Weinberger 2000), pre-or post-weaning

manipulations or their combination (Feldon et al. 2000, Weiss et al. 2001, Weiss and Feldon

2001) and the methylazoxymethanol acetate (MAM) model of schizophrenia (Johnston et al.

1988, Talamini et al. 1998). In comparison to the AMPH withdrawal model, these models suffer

from the disadvantage that they are costly and time-consuming to perform.

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155

1. 2 EFFECTS OF AMPHETAMINE WITHDRAWAL ON PREPULSE INHIBITION

Given that LI is robustly disrupted during AMPH withdrawal, it is surprising that PPI

remains unaffected because (a) a number of manipulations that disrupt LI are also known to

disrupt PPI, and (b) within the context of an animal model of schizophrenia, deficits in both LI

and PPI have been reported in patients. Our first investigation showed that rats withdrawn from

an ESC schedule of AMPH showed normal startle response and normal PPI (chapter 1). This

extended previous reports that withdrawal from repeated intermittent psychostimulant treatment

was not associated with PPI disruption, regardless of whether the testing was carried out after a

challenge (Mansbach et al. 1988; Druhan et al. 1998; Martinez et al. 1999; Byrnes and Hammer

2000; Adams et al. 2001, Murphy et al. 2001a). However, sensitized PPI disruption following

AMPH withdrawal in response to an AMPH challenge has been reported (Zhang et al. 1998, but

see Feifel et al. 2002). One key procedural difference employed by these authors is that each

repeated AMPH administration (prior to withdrawal) was always followed by PPI testing, and

thereby the entire PPI testing procedure might have acquired conditioned properties through

repeated pairings with the acute effects of the drug.

One possibility is that, similar to the contribution of contextual variables to the expression of

behavioral sensitization, a contextual conditioning component might be critical in bringing about

a disruption of PPI (Badiani et al. 1995a, Robinson et al. 1998; Ohomori et al. 2000a). We

attempted to examine this possibility by testing animals for PPI during withdrawal from an ESC

schedule of AMPH under conditions considered best to observe the expression of

psychostimulant induced behavioral sensitization, namely through pairing drug injections with

PPI test-associated cues (Chapter 3). This was supplemented with additional experiments with

PPI testing following longer withdrawal periods (23, 30, 60 days) and with or without challenge

of a dopamine agonist (sees Table 1 in Chapter 3). PPI was not disrupted during AMPH

withdrawal in the absence of a challenge, and PPI disruption was not sensitized in withdrawn

animals after a challenge with dopamine agonists (Chapter 3). However, the critical experiment

where each repeated AMPH administrations is paired with PPI testing itself, and not just cues

associated with the PPI test, was not conducted. Therefore, it cannot be excluded that such a

stringent repeated pairing regime is essential to reveal PPI disruption during AMPH withdrawal.

However, until further evidence is available, it is cautious to conclude that PPI is not reliably

DISCUSSION

156

disrupted during AMPH withdrawal. This stands in contrast to the robust disruption of LI

discussed earlier.

The contrast between intact PPI and disrupted LI during withdrawal from an ESC schedule

of AMPH merits further discussion. This contrast reminds one that PPI and LI involve different

psychological processes even though there is evidence for the contention that they are subserved

by overlapping neural circuits (Wilkinson 1994, Ellenbroek et al. 1996, Feldon and Weiner 1997,

Feldon et al. 2000, Weiner 2000, Swerdlow et al. 2001). PPI measures early attentional gating

mechanisms, often referred to as pre-attentional processes. On the other hand, LI assesses later

stages of information processing related to attentional filtering, possibly via learned inattention or

other proactive interference (Lubow 1989, Bouton 1993, Escobar et al. 2002). LI is a form of

selective learning, whereas PPI obviously is not. In schizophrenic patients, LI is disrupted only

during the acute phase of the disease, an effect which has been attributed to the different phases

and to the neuroleptic treatment of the disease (Baruch et al. 1988, Gray et al. 1992, 1995, Gray

1998,). In contrast, PPI disruption does not distinguish between the acute and chronic phases of

the disease, although this assertion remains a matter of debate (Braff et al. 2001). In addition, PPI

deficits have also been reported in patients with Huntington`s disease, Tourette’s syndrome and

obsessive-compulsive disorder, suggesting that PPI deficits are not unique to schizophrenia

(Castellanos 1996, Swerdlow et al. 1993, 1995, Braff et al. 2001). On the other hand, there is no

evidence to date that LI disruption is present in neuropsychiatric conditions other than

schizophrenia. It may lead one to suggest that LI is more closely related to some clusters of

schizophrenic symptoms than PPI disruption might represent. Hence, the AMPH withdrawal

model would be most useful for modelling those symptoms. Further investigations of the

neurobiological mechanisms underlying this specific effect of AMPH withdrawal should advance

our understanding of the possible dissociable biological processes of symptom genesis in

schizophrenia.

Indeed, animal models that specifically affect only one of these different paradigms are very

useful for the investigation of the different neurobiological mechanisms of LI and PPI. However,

except for direct pharmacological manipulations, animal models that lead to selective disruption

of either PPI or LI (but not the other) are rare. One example is post-weaning social isolation,

which disrupts PPI but spares LI (Wilkinson et al. 1994, Feldon et al. 2000, Weiss et al. 2001,

Weiss and Feldon 2001). Social isolation together with withdrawal from an ESC schedule of

DISCUSSION

157

AMPH represents two known manipulations to date that possess such selective effects. It can be

readily appreciated that they compliment each other, and when used in conjunction (i.e.,

withdrawal from an ESC schedule of AMPH in post-weaning socially isolated animals), a more

comprehensive model with enhanced face validity could be achieved.

1. 3 COGNITIVE EFFECTS OF AMPHETAMINE WITHDRAWAL

In addition to the paradigms of LI and PPI, we have chosen to assess the face validity of the

AMPH withdrawal model of schizophrenia by evaluating its effects on mnemonic processing. To

this end, we elected to examine the acquisition and retention of a spatial memory task (Chapter

4). Schizophrenic patients exhibit a variety of cognitive impairments, including deficits in

working memory, attention, episodic memory and executive functioning including set switching

(Braver et al. 1999, Elvevag and Goldberg 2000, Kuperberg and Heckers 2000). Deficits have

been shown in both verbal and spatial working memory (Park and Holzman 1992, Servan-

Schreiber et al. 1996, Keefe et al. 1997, Stone et al. 1998, Wexler et al. 1998). Several

researchers have suggested that a deficit in working memory may be the fundamental cognitive

defect present in schizophrenia (Goldman-Rakic 1991, Cohen and Servan-Schreiber 1992,

Weinberger and Gallhofer 1997).

However, we failed to find any clear memory deficits on the Morris water maze task,

suggesting that withdrawal from an ESC schedule of AMPH is best considered as having no

effect on the acquisition and retention of spatial learning. This interpretation is in line with a

recent report of a lack of an effect during AMPH withdrawal on spatial working memory (Stefani

and Moghaddam 2002).

As already noted in the discussion of Chapter 4, there was, however, tentative evidence for

enhanced reversal learning, which might be related to the attenuation of LI following withdrawal

from the same ESC schedule of AMPH described earlier (see Weiner 1990, 2000, Weiner and

Feldon 1997). In the switching model of LI disruption proposed by Weiner and colleagues

(Weiner 1990, 2000, Weiner and Feldon 1997), it was hypothesized that enhanced switching

accounts for both LI disruption and facilitation of reversal learning. It has been demonstrated that

acute AMPH can lead to both phenomena, and the present thesis suggests that a similar pattern of

DISCUSSION

158

effects might also be associated with AMPH withdrawal (Weiner et al. 1986, 1988, Weiner and

Feldon 1986). Our present suggestion of facilitation in reversal learning during AMPH

withdrawal should be further tested using more conventional paradigms, such as the 2-choice

simultaneous discrimination procedure. In addition, it would also be interesting to assess the kind

of reversal and/or switching mechanism that is assessed by the Wisconsin Card Sorting Test in

humans. Schizophrenic patients are severely impaired on such a task (Goldberg and Weinberger

1988, 1994). This might be tested in rats by subjecting the animals to reversal from a non-

matching-to-sample rule to a matching-to-sample rule, as suggested by Joel et al. (1997).

At this juncture, our preliminary data showing that reversal learning in a spatial task was not

impaired during AMPH withdrawal may be taken to suggest another limitation to the face

validity of the AMPH withdrawal model.

2 AMPHETAMINE WITHDRAWAL AS AN ANIMAL MODEL OF DEPRESSION

Withdrawal from an ESC schedule of AMPH is also considered by some scientists to be a

potential animal model for investigating the neural bases of certain depressive symptoms (for rev.

Barr et al. 2002a). In particular, anhedonia can be observed in rats during the first few days of

AMPH withdrawal. Anhedonia is thought to reflect a decrease in the sensitivity/activity of the

brain reward system and is a symptom of both depression and schizophrenia (Wise 1982, Kohler

and Lallert 2002, Hausmann and Fleischhacker 2002). An ESC schedule of AMPH identical to

ours has previously been shown to elevate response thresholds in the ICSS paradigm, suggesting

a state of anhedonia (Lin et al. 1999). We therefore investigated animals withdrawn from the ESC

schedule of AMPH in three behavioral tests associated with depressive symptoms. We found no

effect in the Porsolt swim test, in the development of learned helplessness, or in responding for

sucrose on a progressive ratio schedule (chapter 6). These data extended the null effect previously

reported using the Porsolt swim test following withdrawal from intermittent dosages of AMPH

(Schindler et al 1994, Hedou et al. 2001, Russig et al. unpublished observations, but see

Kokkinidis et al. 1986). Given that the progressive ratio paradigm is also considered as an assay

for studying anhedonia, the null results in this test contrasted with the positive finding obtained

using the ICSS paradigm (Lin et al. 1999). With a more severe ESC AMPH administration

DISCUSSION

159

schedule, Barr and Philips (1999) were able to demonstrate a significant effect using the

progressive ratio paradigm. This may suggest that the ICSS procedure is a more sensitive test for

anhedonia. At this juncture, it is cautious to conclude that although withdrawal from an ESC

schedule of AMPH can induce anhedonia, it is not able to reliably model other classes of

depressive symptoms – including behavioral despair and helplessness.

The fact that withdrawal from an ESC schedule of AMPH can give rise to anhedonia may

further indicate that the mechanisms involved in this effect could also be related to the frequent

reports of depressive signs in schizophrenic patients (Gelder et al. 1989, Kohler and Lallert 2002,

Hausmann and Fleischhacker 2002), despite the fact that the anhedonia effect is only clearly seen

in the first five days of withdrawal. To my knowledge there are as yet no available animal models

that are able to capture both the psychotic and depressive facets of schizophrenia. Hence, this

possibility deserves further investigation.

Dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis has been shown in both

depression and schizophrenia. This is manifested as enhanced basal cortisol levels and altered

ACTH and cortisol release in response to stress (Lammers 1995, Heuser et al. 1996, Holsboer

and Barden 1996, Zobel et al. 1999, Meltzer et al. 2001). We therefore attempted to obtain

measures of HPA function in animals during withdrawal from an ESC schedule of AMPH

(Chapter 6, Russig et al. unpublished observations). It was found that, except for a slight

elevation at withdrawal day 4, basal ACTH and CORT plasma levels did not differ between

AMPH withdrawn animals and controls. The stress hormone response to swim and restraint stress

was also unaffected. However, animals withdrawn from a more severe ESC AMPH schedule than

that we previously used exhibited reduced ACTH and CORT plasma levels during the expression

of behavioural sensitization in response to an AMPH challenge compared to animals exposed to

AMPH for the first time (Russig et al. unpublished observations). Interestingly, this parallels the

blunted plasma cortisol and ACTH response following a challenge with the dopamine agonist

apomorphine in schizophrenic (but not depressive) patients (Mokrani et al. 1995, Meltzer et al.

2001). This provides additional evidence of face validity for ESC AMPH withdrawal as an

animal model of schizophrenia. This schizophrenia-like stress hormonal response is, however,

not seen during withdrawal from intermittent schedules of psychostimulant administration that

have either been shown to enhance or did not affect the levels of CORT or ACTH in response to

a stress or drug challenge (Borowsky and Kuhn 1991, Antelman et al. 1992, Levy et al. 1992,

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160

Badiani et al. 1995b, Schmidt et al 1995, 1999, Barr et al. 2002b). This again points to the

importance of schedule differences in deciding the precise pattern of behavioral consequences

during psychostimulant withdrawal.

3 THE ROLE OF DIFFERENT AMPHETAMINE ADMINISTRATION SCHEDULES ON

EFFCTS DURING DRUG WITHDRAWAL

A very important finding of the present set of experiments is that, despite inducing similar

magnitudes of behavioral sensitization, different administration schedules of AMPH can produce

different results in behavioral tests conducted during withdrawal in the absence of a drug

challenge. ESC and INT schedules of psychostimulant withdrawal lead to opposite effects on LI

(Murphy et al. 2001a, b and see Chapters 1-3). Phillips and colleagues also reported, in a series of

experiments, opposite effects of withdrawal from intermittent and escalating dosage AMPH

schedules on sexually motivated behavior. While 4 days of escalating high-dose AMPH

administration reduced sexually motivated behaviors during a subsequent withdrawal period

(Barr et al. 1999), withdrawal from daily intermittent AMPH treatment increased sexual

motivation (Fiorino and Phillips 1999a, b). However, unlike the experiments reported in the

present thesis, studies investigating effects of AMPH withdrawal, including the three separate

studies by Philips and colleagues above, suffer from numerous procedural inconsistencies,

including the use of varying escalating and intermittent schedules, and the fact that the critical

tests were not always conducted on the same withdrawal day. Data derived from their studies are

therefore susceptible to the argument that the reported difference between the schedules stemmed

from procedural differences, rather than from the fundamental difference between an escalating

and an intermittent schedule.

The divergent consequences of the two forms of withdrawal schedules are not limited to the

behavioral level. There is also evidence that withdrawal from the two forms of AMPH

administration schedules can lead to differential expression of the neurotrophic factor bFGF (also

known as FGF-2, basic fibroblast growth factor), considered to be important to the

neuroadaptations produced in response to repeated AMPH (Flores et al. 1998, 2000, Flores and

Stewart 2000a, b). We have started investigating potential neuroadaptive events at the molecular

DISCUSSION

161

level during withdrawal from an ESC, and from an INT, schedule of AMPH. These include the

investigation of the expression of immediate-early genes and DNA microarray analysis (chapter

5, unpublished observations).

4 BRAIN CHANGES ASSOCIATED WITH AMPHETAMINE WITHDRAWAL

We found enhanced levels of the transcription factor FosB in the basolateral amygdala and

the shell subregion of the nucleus accumbens during withdrawal from an ESC schedule of AMPH

(Chapter 5). The importance of these two brain regions in the expression of LI has been

highlighted by recent lesion studies in the rat – LI can be disrupted following lesions of both

areas (Weiner et al. 1996, Coutureau et al. 2001; Jongen-Relo et al. 2002, but see Weiner et al.

1995). We hypothesize that the increased FosB expression in the nucleus accumbens shell and

the basolateral amygdala might be functionally related to the LI disruption seen during

withdrawal (chapter 5).

Upregulation of transcription factors, like FosB, can induce changes in the expression of

synaptic proteins, like synaptophysin and synapsin, resulting in long-lasting plastic adaptations at

the network level (Nestler 2001, Ujike 2002, Ujike et al. 2002). These synaptic proteins are

implicated in the regulation of neurotransmitter release, and in learning and memory, and it might

be that they are therefore associated with LI disruption during AMPH withdrawal (Greengard et

al. 1993, Catsicas et al. 1994, Berke and Hyman 2000). This speculation is supported by the fact

that synaptic proteins in animals are differentially expressed in the nucleus accumbens core and

shell during AMPH withdrawal (Iwata et al. 1996, Subramaniam et al. 2001) and that

dysregulation of synaptic proteins has been reported in schizophrenic patients (Grebb and

Greengard 1990, Browning et al. 1993, Eastwood et al. 1995, Glantz and Lewis 1997,

Tcherepanov and Sokoloff 1997, Karson et al. 1999, Ohmori et al. 2000b, Ohtsuki et al. 2000,

Stober et al. 2000). As an additional support, reduced synaptophysin has been shown in the

hippocampus of isolation-reared rats, which exhibit PPI disruption and are considered as an

animal model of certain symptoms of schizophrenia (Varty et al. 1999, Weiss and Feldon 2001).

Direct evidence of increased dendritic length and enhanced spine density has also been

shown during AMPH withdrawal in the prefrontal cortex and the nucleus accumbens (Robinson

DISCUSSION

162

and Kolb 1997, 1999). Such neuroadaptive changes are believed to be involved in the

phenomenon of behavioral sensitization. However, we did not observe disruption of LI from an

ESC schedule of AMPH following two months of withdrawal, suggesting that the underlying

neurobiological mechanisms for LI disruption during AMPH withdrawal might be associated

with, but not similar to, mechanisms regulating the persistent expression of behavioral

sensitization. One of the future research directions should be directed towards the investigation of

the molecular and genetic bases that lead to schizophrenia-like behavioral alterations during

AMPH withdrawal. The present animal model could be an important tool for this aim.

5 CONCLUSIONS

The results presented in this thesis lead us to conclude that neuroadaptive changes occurring

in response to an ESC schedule of AMPH administration can lead to brain dysfunctions that

could mimic selective behavioral deficits in schizophrenic patients. These data lend support to the

endogenous sensitization hypothesis of schizophrenia, which in turn lends construct validity to

the application of AMPH withdrawal as a model of schizophrenia. As discussed above, this

model also enjoys a degree of face and predictive validity. It constitutes a model for the screening

of compounds with potential antipsychotic properties that is not dependent on surgical and/or

acute pharmacological interventions. The present thesis highlights the fact that the behavioral

effects associated with withdrawal from an escalating dose schedule of AMPH might be

particularly useful for studying certain classes of schizophrenic symptoms, and that further

investigation of the neural mechanisms involved could also enhance our understanding of the

genesis of specific psychotic symptoms.

DISCUSSION

163

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APPENDIX ---------------------------------------------------------------------------------------------------------------------

HALOPERIDOL AND CLOZAPINE ANTAGONISE AMPHETAMINE

INDUCED DISRUPTION OF LATENT INHIBITION IN CONDITIONED

TASTE AVERSION

Holger Russig, Aneta Kovacevic, Carol A. Murphy and Joram Feldon

Psychopharmacology, submitted

APPENDIX

176

ABSTRACT

Rationale. Latent inhibition (LI) describes a process by which repeated pre-exposure of a

stimulus without any consequence retards the learning of subsequent conditioned associations

with that stimulus. It is well established that LI is impaired in rats and in humans by injections of

the indirect dopamine agonist amphetamine (AMPH), and that this disruption can be prevented

by co-administration of either the typical neuroleptic haloperidol (HAL) or the atypical

neuroleptic clozapine (CLZ). Objectives. Most of what is known of the pharmacology of LI is

derived from studies using either the conditioned emotional response or the conditioned active

avoidance paradigm. The goal of the present study was to determine whether these results would

generalize to the conditioned taste aversion assay. Methods. We tested whether AMPH (0.5

mg/kg) pretreatment would disrupt LI of a conditioned aversion to sucrose, and if so, which stage

of the procedure is critical for mediating the disruption; in addition, we tested whether HAL (0.2

mg/kg) or CLZ (5.0 mg/kg) could restore such an expected LI disruption. Results. We

determined that AMPH disrupted LI when it was injected before preexposure and prior to

conditioning, but not if the rats were injected before either stage alone. When HAL or CLZ was

given 40 min before AMPH (before both preexposure and conditioning), it blocked LI disruption.

Conclusion. These results are in line with the pharmacology of LI as derived from other

conditioning paradigms. We conclude that the pharmacological regulation of LI in the CTA

paradigm is similar to what has been observed previously in the conditioned emotional response

and the conditioned active avoidance paradigms.

Keywords: Latent inhibition, Amphetamine, Haloperidol, Clozapine, Conditioned taste aversion

APPENDIX

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INTRODUCTION

Latent inhibition (LI) is the phenomenon, occurring in a variety of species

including humans and rats, whereby repeated unreinforced stimulus presentation retards

subsequent conditioning to a stimulus (Lubow 1989). LI is believed by many investigators to be

the product of learning to ignore irrelevant stimuli and, consequently, LI has been linked to the

selectivity of attentional processing (Mackintosh 1975, Lubow et al. 1981, Lubow 1989).

Disrupted LI in the rat is considered an animal model of cognitive/attentional deficits associated

with schizophrenia since it has been shown that acutely psychotic schizophrenic patients show

both reduced LI and attentional deficits. However, LI is normalized during later episodes of this

chronic disorder, possibly due to the effects of neuroleptic treatment (Baruch et al. 1988, Gray

NS et al 1992a, 1995).

The pharmacology of LI in non-human subjects has been investigated using aversively-

motivated LI procedures such as conditioned emotional response (CER), conditioned avoidance

response (CAR), conditioned taste aversion (CTA), conditioned eyeblink, and conditioned

freezing. LI can also be measured in appetitively motivated and discrimination learning

procedures, but there has been only sporadic use of these methods (reviewed in Moser et al.

2000). In the CTA paradigm a novel taste (CS, eg. sucrose) is associated with illness induced by

lithium chloride (LiCl, US), thereby reducing subsequent sucrose preference during test. LI is

demonstrated when animals preexposed (PE group) to the sucrose CS prior to CS-US pairing in

the conditioning session show less sucrose aversion during the test session compared to

nonpreexposed (NPE group) animals. In comparison to CER and CAR procedures, only one CS-

US pairing is necessary for strong conditioning and only a single preexposure to the CS induces

robust LI in the CTA test (Russig et al. unpublished observation).

In animals and humans, the indirect dopamine agonist amphetamine (AMPH) disrupts LI;

conversely, neuroleptic drugs with dopamine receptor antagonist activity, such as clozapine

(CLZ) or haloperidol (HAL), can restore AMPH-induced LI disruption and enhance LI when

given alone (Thornton et al. 1996, Kumari et al. 1999, Moser et al. 2000, Weiner 2000, Russig et

al. 2002). A range of low doses of AMPH disrupt LI in CER (see Moser et al. 2000 for a review)

and CAR paradigms (Solomon et al. 1981, Weiner et al. 1988, Bakshi et al. 1995, De Oliveira

Mora et al. 1999). Similarly, most of our knowledge about the capacity of compounds to reverse

APPENDIX

178

AMPH-induced disruption of LI is based on findings in the CER paradigm, with the exception of

two studies which used CAR to show that chlorpromazine, CLZ and HAL are able to restore LI

disruptions (Solomon et al. 1981, Russig et al. 2002). Ellenbroek et al. (1997) showed in rats that

0.25 and 0.5mg/kg AMPH disrupted LI in a CTA paradigm. However, the ability of antipsychotic

drugs such as HAL or CLZ to either restore disrupted LI or enhance normal LI has not yet been

demonstrated in the CTA paradigm.

For both AMPH and neuroleptic effects on LI, the timing of drug administration is of

critical importance in determining whether a drug effect is seen. In a typical experimental LI

protocol in CER, the preexposure, conditioning and test sessions are carried out in separate

sessions 24 hours apart. When the experiment is conducted in this manner, AMPH must be

administered before both preexposure and conditioning to disrupt LI (Weiner et al. 1984, 1988).

However, a single administration of AMPH has been found sufficient to disrupt LI in humans and

in rats if the preexposed CS duration was dramatically enhanced or the preexposure and

conditioning phases were run in the same session (Gray NS et al. 1992b, Moran et al. 1996,

Thornton et al. 1996, McAllister 1997). Based on these and other data it has been argued that for

AMPH to disrupt LI it is actually the conditioning stage which is critical (Weiner 1990, Gray et

al. 1995, Weiner and Feldon 1997). In the CTA paradigm, it was shown that AMPH disrupted LI

when it was given before each of 3 preexposure days and the conditioning stage 24 hours later

(Ellenbroek et al. 1997). Thus, it remains an open question before which stage animals must be

treated with AMPH in order to disrupt LI in the CTA paradigm. In contrast to the controversy

over the relative importance of different experimental stages to AMPH-induced LI disruption,

there is consistent evidence that the critical time for CLZ- or HAL-induced effects on LI is the

conditioning stage. The effectiveness of these drugs in LI facilitation or reversal of AMPH-

induced LI disruption was not altered by an additional injection before preexposure (Joseph et al.

1992, Peters et al. 1993, Weiner 2000, Russig et al. 2002, Trimble et al. 2002).

For LI disruption in CER and CAR, the nucleus accumbens has been suggested to be the

critical structure (Weiner and Feldon 1997, Murphy et al. 2000, Weiner 2000). However, recent

experiments measuring c-Fos and employing intracrebral AMPH infusions have suggested that

the striatum rather than the nucleus accumbens is the critical structure for LI disruption in CTA

(Ellenbroek et al. 1997, Turgeon and Riechstein 2002). The same investigation methods used in a

CER paradigm suggest a critical role of the nucleus accumbens rather than the striatum for

APPENDIX

179

disrupted LI (Solomon and Staton 1982, Sotty et al. 1996, JA Gray et al. 1997). With these

differences in mind, very little is known about possible differences in the pharmacology of LI in

CTA compared to other behavioral tests. An important step to address this issue is to investigate

effects of AMPH, HAL and CLZ in this paradigm.

Therefore, we tested in the present study if the effects of AMPH, HAL and CLZ in CTA

are comparable with the pharmacology of LI observed in other paradigms. We expected in

experiment 1 that using a CTA procedure in which a 24-hour delay took place between

preexposure, conditioning and test sessions, LI would be disrupted if 0.5 mg/kg AMPH was

administered 5 min before both the preexposure and conditioning sessions. We also anticipated

that this effect of LI disruption should not occur if AMPH was administered either only before

preexposure or only before conditioning. In addition, we expected that coadministration of 0.2

mg/kg HAL or 5.0 mg/kg CLZ prior to AMPH before both preexposure and conditioning would

block the AMPH-induced LI disruption..

MATERIAL AND METHODS

Animals

Male Wistar rats (Zur: WIST [HanIbm]; 250-350g) obtained from our in-house specific-

pathogen-free (SPF) breeding facility were used as subjects in these experiments. During the

experiments, the animals were housed individually in Macrolon type III cages (48 x 27 x 20 cm)

under a reversed light-dark cycle (lights on 18.00-06.00 hours) in a temperature (21±1°C) and

humidity (55±5%) controlled animal facility. Food (Kliba 3430, Klibamühlen, Kaiseraugst CH)

was available ad libitum in the home cages. All experiments were carried out between 8.00 a.m.

and 1.00 p.m. during the dark phase of the light-dark cycle. All procedures were in agreement

with Swiss Cantonal Veterinary Office regulations for animal experimentation.

Drugs

D-Amphetamine sulfate (Sigma Chemical Company, St. Louis, U.S.A.) was dissolved in

a 0.9% NaCl solution to obtain the dosage of 0.5 mg/kg amphetamine (calculated as the salt).

Vehicle-treated groups received 0.9% NaCl solution. Haloperidol (HAL; Janssen-Cilag, Baar,

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Switzerland) was prepared from 5 mg ampoules, in which the drug is present in 1ml of vehicle

solution containing 6mg lactic acid. This solution was subsequently diluted with saline to obtain

the required concentration of 0.2 mg/kg (final pH of 5.5). Clozapine (CLZ; Novartis,

Switzerland) was first dissolved in 0.1N HCL in 0.9% saline solution and then neutralized to pH

5.5 with Na2CO3 in a final concentration of 5.0 mg/kg. Vehicle-treated animals were

administered either HAL vehicle (0.9% saline/lactic acid, pH 5.5) or CLZ vehicle (0.1 N

HCL/0.9% saline, pH 5.5). All solutions were freshly prepared and administered intraperitoneally

in a volume of 1ml/kg. Lithium chloride (LiCl, Sigma Chemical Company, St. Louis, U.S.A,

0.14 M) was dissolved in 0.9 % NaCl and administered in a volume of 1.5 % of body weight.

CTA apparatus

Before each session animals were transferred from the home cage to a CTA test cage. The

test cages were Macrolon cages (42.5 x 26.6 x 15.0 cm) designed in such a way that two drinking

bottles could be attached and the spouts inserted through two holes in the anterior part of the

cage. The water and sucrose intake of each animal were recorded by measuring the weight of the

drinking bottles before and after each drinking session.

CTA procedure

Prior to the beginning of each experiment, animals were handled for 5 minutes each on 3

consecutive days and water deprived for the following 8 days. During the first 3 days of water

deprivation animals had access to water in the home cage for 1 hour beginning at a time between

9.00 and 10.00 a.m. On the following 2 days, animals were exposed to the CTA test cages for 30

min where water was given. On the next day, the preexposure session with 1 drinking bottle was

conducted in which animals were given either water (non-preexposed, NPE) or 5% sucrose

solution (preexposed, PE) for 30 min in the test cages. Up to this stage the drinking bottles were

switched once per exposure between the two holes in the test cages to avoid the development of

preference for one hole over the other. On the next day, all animals experienced a conditioning

session in which they were given access to 5% sucrose solution for 15 min immediately followed

by an injection of lithium chloride (LiCl, 0.14 M, 1.5% of body weight) and were placed back in

the home cage. During the next day, all animals were placed in the CTA cages and were given

access to both 5% sucrose solution and water presented in two different bottles for 30 min at the

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same time (test session). Conditioned taste aversion was assessed by calculating the percent

sucrose consumed (ml sucrose consumed x 100 / ml sucrose consumed + ml water consumed) on

the test day. LI was assessed by comparing the degree of taste aversion between PE and NPE

animals within each treatment group.

Experiment 1 Effects of 0.5 mg/kg amphetamine injected before different experimental

stages on the development of latent inhibition in a conditioned taste aversion paradigm

A dose of 0.5 mg/kg AMPH or saline was injected 5 min before the preexposure session,

conditioning session, or before both sessions. A saline injection was given before all sessions

without the AMPH treatment. The test session was conducted in a drug-free state. The dose of

0.5 mg/kg AMPH was selected because disrupted LI has been shown with this dose in the CER,

passive avoidance and CTA paradigms (Killcross et al. 1994, Ellenbroek et al. 1997, De Oliveira

Mora et al. 1999).

We excluded from analysis 12 animals that consumed less than 1.0 ml of solution during

the preexposure or conditioning session. The final number of animals in each of the 8 conditions

was: SAL/SAL NPE, n = 10; SAL/SAL PE, n = 10; AMPH/SAL NPE, n = 8; AMPH/SAL PE, n

= 9; SAL/AMPH NPE, n = 8; SAL/AMPH PE, n = 8; AMPH/AMPH NPE, n = 7; AMPH/AMPH

PE, n = 8.

Experiment 2 Effects of 0.2 mg/kg haloperidol on 0.5 mg/kg amphetamine-induced

disruption of latent inhibition of conditioned taste aversion

Experiment 1 clearly showed that LI was reduced only if AMPH was given before both

the preexposure and the conditioning sessions in the CTA paradigm. In Experiment 2, either 0.2

mg/kg HAL or vehicle were injected 40 min prior to injection of either 0.5 mg/kg AMPH or

saline, 5 min prior to the beginning of both the preexposure and conditioning sessions. During the

test session all the animals were drug-free. The dose of 0.2 mg/kg HAL was selected because

dosages between 0.1 mg/kg and 0.5 mg/kg i.p. are effective in the reversal of AMPH (1.0 and 1.5

mg/kg) induced disruption of LI (Warburton et al. 1994, Millan et al. 1998).

We excluded from the analyses 5 animals that consumed less then 1.0 ml during the preexposure

or the conditioning session. The final number of animals in each of the 8 conditions was:

SAL/SAL NPE, n = 10; SAL/SAL PE, n = 10; HAL/SAL NPE, n = 9; HAL/SAL PE, n = 9;

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SAL/AMPH NPE, n = 9; SAL/AMPH PE, n = 9; HAL/AMPH NPE, n = 10; HAL/AMPH PE, n

= 9.

Experiment 3 Effects of 5.0 mg/kg clozapine on 0.5 mg/kg amphetamine-induced disruption

of latent inhibition of conditioned taste aversion

The experimental procedures were similar to those of Experiment 2, but instead of the

typical antipsychotic drug HAL, the appropriate treatment groups received 5.0 mg/kg of the

typical antipsychotic drug CLZ. The dosage of 5.0 mg/kg CLZ was selected because dosages

between 2.0 mg/kg and 10.0 mg/kg i.p. are effective in the reversal of AMPH (1.0 and 1.5

mg/kg) induced disruption of LI measured in a CER paradigm (Moran et al. 1996, Millan et al.

1998, Weiner et al. 1996).

We excluded from the analyses 14 animals that consumed less then 1.0 ml during the

preexposure or the conditioning session. The final number of animals in each of the 8 conditions

was: SAL/SAL NPE, n = 9; SAL/SAL PE, n = 8; CLZ/SAL NPE, n = 7; CLZ/SAL PE, n = 9;

SAL/AMPH NPE, n = 8; SAL/AMPH PE, n = 9; CLZ/AMPH NPE, n = 8; CLZ/AMPH PE, n =

8.

Statistics

Statistical analysis of the data was conducted using StatView version 5.0.1. For all

measurements in experiment 1 we used a 2 x 2 x 2 ANOVA design with the three between-

subjects factors of drug treatment before preexposure (drug PE: SAL or AMPH), drug treatment

before conditioning (drug COND: SAL or AMPH) and preexposure (PE or NPE). All

measurements in experiments 2 and 3 were analyzed with 2 x 2 x 2 ANOVA designs with 3

between-subjects factors of drug treatment (SAL, AMPH), neuroleptic treatment (SAL, HAL or

CLZ) and preexposure (NPE, PE). Whenever an interaction between two main factors was

significant, the post-hoc Fisher's protected least significant difference test was applied.

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RESULTS

Experiment 1 Effects of 0.5 mg/kg amphetamine injected before different experimental

stages on the development of latent inhibition in a conditioned taste aversion paradigm

Preexposure session: Animals with access to sucrose (PE groups) consumed more

solution than animals that had access only to water, as reflected by a main effect of preexposure

(F(1, 60) = 7.19, p < 0.01; see Fig 1A). The analysis also revealed a significant drug PE x drug

COND x preexposure interaction (F(1, 60) = 4.219, p < 0.05). A posthoc analysis of this

interaction revealed that the SAL/AMPH PE group drank more than the other groups, effects

which were significant versus the SAL/AMPH NPE group (p = 0.0001), AMPH/SAL NPE group

(p = 0.0001) and AMPH/AMPH PE group (p = 0.0003). No other significant main effects or

interactions were found.

Conditioning session: Rats that had access to sucrose during the preexposure stage (PE

groups) exhibited overall more sucrose intake compared to the NPE groups, as reflected by a

significant main effect of preexposure (F(1, 60) = 4.785, p < 0.05; Fig 1 B). The analyses also

revealed a significant main effect of drug during conditioning (F(1, 60) = 29.916, p < 0.0001),

reflecting a reduced fluid intake for animals treated with AMPH before conditioning in

comparison to SAL injected controls. This effect was more pronounced in animals that received

AMPH also before preexposure, as reflected by a drug PE x drug COND interaction (F(1, 60) =

4.387, p < 0.05). There were no other significant main effects or interactions.

Test session: Overall expression of LI, as reflected by higher percent sucrose intake in PE

compared with NPE animals, was confirmed by a significant main effect of preexposure (F(1, 60)

= 13.563, p < 0.001, Fig 1C). However, the LI effect was not similar in all the treatment groups,

as reflected by a significant drug PE x preexposure interaction (F(1, 60) = 5.681, p < 0.03) and

drug COND x preexposure interaction (F(1, 60) = 7.161, p < 0.01) and significant drug PE x drug

COND x preexposure interaction (F(1, 60) = 4.314, p < 0.05). Posthoc analyses revealed

significantly more percent sucrose intake in PE compared to NPE animals (i.e., LI) in the groups

that were treated with AMPH either only before preexposure (AMPH/SAL, NPE vs. PE p =

0.0046), only before conditioning (SAL/AMPH, NPE vs. PE p = 0.0094) and in the control group

(SAL/SAL, NPE vs. PE, p < 0.0001). No significant LI was obtained in animals treated with an

AMPH injection both before preexposure and conditioning (AMPH/AMPH, group NPE vs. PE p

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184

= 0.2747). Comparing only the NPE groups of the different drug conditions AMPH/AMPH

animals exhibited more %sucrose intake than the AMPH/SAL NPE animals (p = 0.0259), but did

not differ significantly (p > 0.353) from the other NPE groups. Within the PE groups, the

AMPH/AMPH animals consumed significantly less percent sucrose compared to the SAL/AMPH

(p = 0.0285) and the SAL/SAL (p = 0.0068) groups, but did not differ significantly from the

AMPH/SAL group (p = 0.148). There was no significant main effect or interaction in the

measure of total liquid consumption (water + sucrose, Fig 1D).

Figure 1. Effects of amphetamine (AMPH) or saline (SAL) administration before different stages of an experiment measuring latent inhibition of conditioned taste aversion. A: Liquid consumption (in ml) during the preexposure session for sucrose-preexposed (PE) and non-preexposed (NPE) animals. NPE animals had access to water while PE animals received sucrose. B: Sucrose consumption (in ml) during the conditioning session. C: % sucrose intake during the test session during which all animals had access to water and sucrose. D: Total liquid consumption (in ml, sucrose + water) during the test session. The groups were injected with saline (SAL - SAL) or amphetamine (AMPH - AMPH) before both the preexposure and the conditioning sessions, with amphetamine before preexposure and saline before conditioning (AMPH - SAL) and with saline before preexposure and amphetamine before conditioning (SAL - AMPH). Values are means ±SEM.

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Experiment 2 Effects of 0.2 mg/kg haloperidol on 0.5 mg/kg amphetamine-induced

disruption of latent inhibition of conditioned taste aversion

Preexposure session. Animals with access to sucrose (PE groups) consumed more

solution compared to the NPE groups that drank water, as reflected by a highly significant main

effect of preexposure (F(1, 67) = 17.936, p < 0.0001, Fig 2A). A significant drug treatment x

preexposure interaction (F(1, 67) = 4.903, p < 0.05) reflected that this effect was more

pronounced in the SAL-treated compared to the AMPH-treated animals.

Conditioning session. During the conditioning session, all groups exhibited similar

sucrose intake and there were no significant main effects or interactions including the factors of

preexposure, drug treatment or neuroleptic treatment (Fig 2B).

Test session. The presence of LI, as indicated by higher percent sucrose intake in PE

compared with NPE animals, was supported by a highly significant main effect of preexposure

(F(1, 67) = 29.982, p < 0.0001; Fig 2C). AMPH-treated animals showed lower percent sucrose

intake on the test day compared to the controls and this effect was mainly due to a reduction in

the AMPH PE animals, as reflected by a significant main effect of drug treatment (F(1, 67) =

15.208, p < 0.0005) and a significant drug treatment x preexposure interaction (F(1, 67) = 5.274,

p < 0.03). HAL treated rats showed enhanced percent sucrose intake compared to controls (main

effect of neuroleptic treatment (F(1, 67) = 7.762, p < 0.01). Fishers posthoc comparisons between

NPE and PE animals for all conditions revealed that LI was not present in AMPH treated animals

and that the disruption was antagonized by pretreatment with HAL (NPE vs. PE: SAL/SAL p =

0.0018, HAL/SAL p = 0.0002, SAL/AMPH p = 0.7247, HAL/AMPH p = 0.0048). A comparison

of only the PE groups showed that the SAL/AMPH group showed reduced % sucrose intake

compared to the other 3 PE groups (all p < 0.0001) without a significant effect between these 3

groups. The NPE groups did not differ between the different treatment conditions in % sucrose

intake during the test session. In the analysis of overall water + sucrose intake, PE animals drank

more than NPE animals, as reflected by a significant main effect of preexposure (F(1, 67) =

8.136, p < 0.01), reflecting their increased sucrose intake (Fig 2D). There were no significant

interactions of total fluid intake with drug or neuroleptic treatment.

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Figure 2. Effects of amphetamine (AMPH), haloperidol (HAL) or saline (SAL) administration before preexposure and conditioning session in an experiment measuring latent inhibition of conditioned taste aversion. A: Liquid consumption (in ml) during the preexposure session for sucrose-preexposed (PE) and non-preexposed (NPE) animals. NPE animals had access to water while PE animals received sucrose. B: Sucrose consumption (in ml) during the conditioning session. C: % sucrose intake during the test session during which all animals had access to water and sucrose. D: Total liquid consumption (in ml, sucrose + water) during the test session. Animals were injected with saline (SAL - SAL), amphetamine (AMPH - SAL), haloperidol (SAL - HAL) or amphetamine and haloperidol (AMPH - HAL) before both preexposure and test sessions. Values are means ±SEM. Experiment 3 Effects of 5.0 mg/kg clozapine on 0.5 mg/kg amphetamine-induced disruption

of latent inhibition of conditioned taste aversion

Preexposure session. Similarly to experiments 1 and 2, animals with access to sucrose (PE

groups) consumed more solution compared to the NPE groups that drank water, as reflected by a

highly significant main effect of preexposure (F(1, 58) = 21.497, p < 0.0001, Fig 3A). In

addition, AMPH pretreated animals consumed less solution during the 30 minute preexposure

session compared to SAL pretreated animal irrespective of the neuroleptic pretreatment. This

effect was reflected by a significant main effect of drug treatment (F(1, 58) = 4.625, p < 0.05).

There were no additional significant main effects or interactions.

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Conditioning session. During the conditioning session, animals treated with AMPH

consumed less sucrose solution compared to animals that received SAL (F(1, 58) = 6.568, p <

0.05; Fig 3B). A significant main effect of neuroleptic pretreatment (F(1, 58) = 4.39, p < 0.05)

reflected the fact that animals treated with CLZ showed reduced sucrose consumption during the

conditioning session compared to vehicle treated animals. Rats that had access to sucrose during

the preexposure stage (PE groups) exhibited overall more sucrose intake compared to the NPE

groups, as reflected by a significant main effect of preexposure (F(1, 58) = 4.733, p < 0.05).

There were no significant interactions involving any of these three between subjects factors.

Test session. The overall presence of LI was indicated by the fact that PE groups showed

increased percent sucrose intake compared to NPE groups, as reflected by a highly significant

main effect of preexposure (F(1, 58) = 43.391, p < 0.0001; Fig. 3C). Animals treated with AMPH

before both the preexposure and the conditioning session showed reduced percent sucrose intake

during the test session compared to the SAL pretreated animals, as reflected by a significant main

effect of drug treatment (F(1, 58) = 4.972, p < 0.05). On the other hand, animals previously

treated with CLZ showed enhanced percent sucrose intake compared to animals treated with

vehicle and this effect was more pronounced in the PE groups compared to the NPE groups.

These effects were supported by a significant main effect of neuroleptic treatment (F(1, 58) =

11.162, p < 0.005) and a significant neuroleptic treatment x preexposure interaction (F1, 58) =

7.748, p < 0.01). Fisher`s posthoc comparisons between NPE and PE animals within all four

conditions revealed that LI was not present in AMPH treated animals and that the disruption was

antagonized by pretreatment with CLZ (NPE vs. PE: SAL/SAL p = 0.0158, CLZ/SAL p =

0.0008, SAL/AMPH p = 0.358, CLZ/AMPH p = 0.0001). A posthoc comparison of only the PE

groups for all conditions revealed a significant reduced percent sucrose intake in the SAL/AMPH

group compared to the other three groups (all p < 0.05). In addition, CLZ/SAL PE animals

exhibited enhanced percent sucrose intake compared to rats in the SAL/SAL PE condition (p =

0.0115). Similar Fisher`s posthoc comparisons restricted to only the NPE groups of all four

conditions did not revealed any significant outcome. The analysis of overall sucrose + water

intake during the test session revealed a significant main effect of neuroleptic treatment (F(1, 58)

= 6.027, p < 0.05), reflecting that animals previously treated with CLZ showed slightly enhanced

solution intake compared to SAL treated animals (Fig 3D). No other significant main effects or

interactions were obtained in the analysis of overall solution intake during the test session.

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Figure. 3: Effects of amphetamine (AMPH), clozapine (CLZ) or saline (SAL) administration before preexposure and conditioning session in an experiment measuring latent inhibition of conditioned taste aversion. A: Liquid consumption (in ml) during the preexposure session for sucrose-preexposed (PE) and non-preexposed (NPE) animals. NPE animals had access to water while PE animals received sucrose. B: Sucrose consumption (in ml) during the conditioning session. C: % sucrose intake during the test session during which all animals had access to water and sucrose. D: Total liquid consumption (in ml, sucrose + water) during the test session. Animals were injected with saline (SAL - SAL), amphetamine (AMPH - SAL), clozapine (SAL - CLZ) or amphetamine and clozapine (AMPH - CLZ) before both preexposure and test sessions. Values are means ±SEM. DISCUSSION

Experiment 1 showed that LI was disrupted in the CTA paradigm if an acute injection of

0.5 mg/kg AMPH was given before both the preexposure and the conditioning sessions. Animals

that received AMPH either only before preexposure or only before conditioning exhibited normal

LI. In experiments 2 and 3, administration of HAL or CLZ 40 min before the AMPH

administration on both the preexposure and conditioning days blocked the AMPH-induced LI

disruption.

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During the preexposure phase in all three experiments, animals that had access to sucrose

(PE groups) consumed more than animals that had only water to drink. These findings indicate

that sucrose had more rewarding properties than water in general and was therefore more readily

consumed, replicating the findings of Ellenbroek et al. (1997). During the conditioning session of

experiments 1 and 3 but not 2, animals treated with AMPH before conditioning showed reduced

sucrose intake compared to SAL-injected animals, thereby replicating findings of an AMPH-

induced reduction of drinking (Foltin et al. 1983, Shepard 1988, Velazquez-Martinez et al. 1995).

However, since the critical measure for LI is the difference between NPE and PE animals within

a treatment group, and both were similarly influenced by AMPH we do not believe that these

small and nonspecific effects confounded our results concerning LI in CTA.

In experiment 1, we clearly showed that LI in CTA can be disrupted by AMPH only if the

drug is administered before both preexposure and conditioning. A single injection given only

before preexposure or conditioning left LI intact. These results are consistent with findings

previously reported in the CER procedure (Weiner et al. 1984, 1988). Thus, in order to disrupt LI

in CER or CTA protocols in which preexposure, conditioning and test sessions are separated by

24-hour intervals, it seems to be necessary to administer AMPH before both preexposure and

conditioning. It has been suggested that processes of sensitization might be required for AMPH-

induced LI disruption in experimental designs in which 2 injections must be given (Weiner et al

1988, Gray JA et al. 1995). We are currently examining whether, similar to CER, sensitization

processes in CTA might also be responsible for AMPH-induced LI disruption.

The AMPH-induced LI disruption in all experiments was due to a significant reduction of

percent sucrose intake in the AMPH/AMPH PE group compared to the control PE animals.

However, in experiment 1 but not 2 or 3, an additional nonsignificant increase in percent sucrose

intake was observed in AMPH/AMPH NPE animals. It has been suggested that in order to obtain

clearly interpretable results, drugs inducing LI disruption should selectively increase retarded CS-

US conditioning in the treated PE group to levels expressed by the NPE group without altering

conditioning in the NPE group itself (Lubow 1989, Weiner and Feldon 1997, Weiner 2000). It is

unclear why in the first experiment the AMPH/AMPH NPE group showed somewhat increased

percent sucrose intake; nevertheless, percent sucrose intake in the AMPH/AMPH NPE group did

not differ significantly from that in the SAL/SAL NPE group. Given that elevated percent

sucrose intake in the AMPH/AMPH NPE group was seen in only one of the 3 experiments, we

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believe that the effect was due to higher variability from random sampling error. It should be

noted, moreover, that in both experiments, the animals belonging to the PE condition which was

treated with AMPH before preexposure and conditioning showed less percent sucrose

consumption compared with all other PE groups.

Based on the results obtained in experiment 1, we investigated the capacity of HAL and

CLZ injected 45 min before preexposure and conditioning to block AMPH induced LI disruption

in the CTA paradigm. To our knowledge there are no published studies in the LI literature in

which antipsychotic drugs and their influence on LI have been investigated in the CTA paradigm.

In addition, there are only a few studies investigating antipsychotic compounds in LI procedures

other than CER (Solomon et al. 1981, Weiner et al. 1987, Loskutova et al. 1990, Russig et al.

2002). Experiments 2 and 3 showed that HAL or CLZ injected before preexposure and

conditioning blocked AMPH-induced LI disruption in a CTA paradigm. The disruption was

clearly due to decreased percent sucrose intake in the SAL/AMPH PE group and HAL and CLZ

antagonized this effect by increasing the percent sucrose intake of the HAL/AMPH and

CLZ/AMPH PE groups to levels exhibited by the SAL/SAL PE groups. Interestingly, HAL/SAL

animals exhibited slightly enhanced percent sucrose intake compared to the SAL/SAL group.

This tendency was present in both the PE and the NPE groups but was most obvious in the PE

groups. Nevertheless, the magnitude of LI was similar in the SAL/SAL and the HAL/SAL

conditions, indicating that LI was not augmented by HAL in this experiment. In contrast, the

results of experiment 3 suggest that CLZ induced enhancement of LI can be observed also in the

CTA paradigm. Within experiment 3, CLZ/SAL PE animals exhibited significantly more percent

sucrose intake compared to the SAL/SAL PE group and this effect of CLZ cannot be interpreted

as general since the CLZ/SAL NPE animals did not significantly differ from the SAL/SAL NPE

group. Enhanced LI by HAL might also be detectable in future experiments using different

dosages, or if the procedural parameters were manipulated in such a way that LI in controls was

less pronounced, given indications that neuroleptic-induced LI enhancement is best seen on a

background of no LI in the controls (Weiner and Feldon 1997, Moser et al. 2000). LI in the

controls could be reduced by shortening the duration of the preexposure session, restricting

sucrose intake during preexposure, or reducing the dose of LiCL and thereby reducing the

strength of the CS-US association (Weiner 2000). Finally, the LI effects of experiments 2 and 3

cannot be due simply to an effect of the various treatments on drinking behavior per se, because

APPENDIX

191

drinking was not influenced by either the AMPH or the HAL treatment during the test session.

Moreover, although CLZ enhanced the total solution intake during the test session, the analysis

did not reveal any statistically significant interactions with the factors of drug treatment or

preexposure, suggesting that this effect cannot be responsible for the observed effects on LI in the

different drug conditions.

Theoretically, there are some potential problems of interpretation in using CTA as a

paradigm to investigate LI. AMPH effects on LI in CTA might be confounded by the fact that

AMPH itself can induce conditioned taste aversion (Miller and Miller 1983, Greenshaw and

Dourish 1984, Goudie and Newton 1985). In our experiments 1 and 2, there were no significant

differences in sucrose intake between the preexposure and the conditioning sessions in animals

that received AMPH before sucrose preexposure (AMPH PE group). Consequently, it is very

unlikely that our low dose of AMPH induced an independently conditioned taste aversion in

these rats. Another criticism regarding measurements of LI in CTA is that preexposure to the

sucrose CS in these experiments is not really without a consequence because the CS reduces

thirst in water-deprived animals and provides a rewarding taste. Therefore, it could be argued that

PE animals do not really learn to ignore the CS; in fact, the opposite is the case - they learn to

appreciate the sweet solution, a fact which perhaps strengthens the LI effect (Moser et al. 2000).

Nevertheless, LI was disrupted by a very low dose of AMPH in the present experiment. It is

interesting that the pharmacology of AMPH, HAL and CLZ in the LI CTA paradigm is very

similar to results obtained in paradigms not confounded by these theoretical criticisms, such as

the CAR and CER procedures.

Taken together, our results show that 1) LI is disrupted in the CTA paradigm if AMPH is

administered before both preexposure and conditioning and 2) this disruption can be blocked by

HAL and CLZ. These results indicate that the pharmacology of LI regulation by dopamine

agonists and antagonists is similar in the CTA paradigm to that of other tests such as CAR and

CER.

APPENDIX

192

Acknowledgements

This study was supported by the Swiss Federal Institute of Technology (ETH-Zurich,

Switzerland). We would like to thank the staff of the animal facility for their care and

maintenance of the animals used in this study, Mr. Peter Schmid for his valuable technical

assistance and Mrs. Jane Fotheringham for her editorial help.

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LIST OF PUBLICATIONS

196

LIST OF PUBLICATIONS Peer-reviewed publications: 2003 Russig H, Durrer A, Yee BK, Murphy CA, Feldon J. (2003) The acquisition, retention and

reversal of spatial learning in the Morris water maze task following withdrawal from an escalating dosage schedule of amphetamine in Wistar rats. Neuroscience, in press

Murphy CA, Russig H, Pezze MA, Ferger B, Feldon J. (2003) Amphetamine withdrawal

modulates FosB expression in mesolimbic dopaminergic target nuclei: effects of different schedules of administration. Neuropharmacology, in press

Russig H, Pezze MA, Nanz-Bahr NI, Pryce CR, Feldon J, Murphy CA (2003) Amphetamine

withdrawal does not produce a depressive-like state in rats as measured by three behavioral tests. Behav. Pharmacol 14: 1-18

Russig H, Murphy CA, Feldon J (2003) Prepulse inhibition during withdrawal from an escalating

dosage schedule of Amphetamine. Psychopharmacology, in press, published online 12. 11. 2002

2002 Lehmann J, Russig H, Feldon J and Pryce CR (2002) Effect of a single maternal separation at

different pup ages on the corticosterone stress response in adult and aged rats. Pharmacol Biochem Behav 73(1): 141-145

Russig H, Murphy CA, Feldon J (2002) Clozapine and haloperidol reinstate latent inhibition

following its disruption during amphetamine withdrawal. Neuropsychopharmacology 26: 765-777

2001 Murphy CA, Fend M, Russig H, Feldon J (2001) Latent inhibition, but not prepulse inhibition, is

reduced during withdrawal from an escalating dosage schedule of amphetamine. Behav Neurosci. 115(6): 1247-56

Weiss IC, Domeney AM, Moreau JL, Russig H, Feldon J (2001) Dissociation between the effects

of pre-weaning and/or post-weaning social isolation on prepulse inhibition and latent inhibition in adult Sprague-Dawley rats. Behav Brain Res. 121(1-2): 207-218

LIST OF PUBLICATIONS

197

1997 Frommolt, KH.; Kruchenkova, EP.; Russig, H. (1997): Individuality of territorial barking in

arctic foxes, Alopex lagopus (L. 1758). International J. of mammalian biology. 62 Suppl. 2: 66-70

Manuscripts submitted for publication: Russig H, Kovacevic A, Murphy CA, Feldon J. Haloperidol antagonizes amphetamine induced

disruption of latent inhibition in conditioned taste aversion. Submitted to Psychopharmacology

Böckler F, Russig H, Zhang W, Löber S, Hübner H, Ferger B, Gmeiner G, Feldon J. FAUC 213,

a highly selective dopamine D4 receptor full antagonist, exhibits atypical neuroleptic-like properties in behavioural and neurochemical models of schizophrenia. Submitted to

Psychopharmacology Mintz M, Russig H, Lacroix L, Feldon J. Sharing of the home base: A new social test in rats. Submitted to Behav. Pharmacol Yee BK, Russig H, Feldon J. Apomorphine-induced prepulse inhibition disruption is

paradoxically associated with enhanced prepulse reactivity. Submitted to Nature Neuroscience

Manuscripts in preparation for submission: Russig H, Pryce CR, Murphy CA, Feldon J. Reduced HPA axis response and behavioral

sensitization as a consequence of withdrawal from an escalating administration schedule of amphetamine

Russig H, Murphy CA, Feldon J. Withdrawal from repeated amphetamine administration as an

animal model of schizophrenia. (review in preparation) Murphy CA, Pezze MA, Russig H, Feldon J. C-Fos expression in the mesocorticolimbic system

during withdrawal from amphetamine, the effect of enhanced fear conditioning. Pryce CR, Rüedi-Bettschen D, Russig H, Nanz-Bahr NI, Weston A, Jongen A, Feldon J,

Mohammad A, Shu S. Double dissociation of the effects of early deprivation on the HPA system versus spatial cognition in aged male versus female rats.

198

CURRICULUM VITAE Holger Russig

Date and place of birth: 21. 4. 1970, Freital, Germany

Citizenship: German

Studies and degrees:

Oktober 1999 – December 2002: Ph. D. studies at the Swiss Federal Institute of

TechnologyZurich; Ph.D. program in neuroscience,

Neuroscience Center Zurich

September 1999 “Diplom” (M.S.) in biology; Humboldt University Berlin

September 1994 “Vordiplom” (B. S.) in biology, Humboldt University

Berlin

September 1993 “Vordiplom” (B. S.) in chemistry, Humboldt University

Berlin

October 1990 – September 1999: Studies of biology and chemistry, Humboldt University

Berlin

Diploma thesis:

“Untersuchungen zur Variabilität des Anzeigerufes beim Krallenfrosch Xenopus l. laevis“

(Individual variability of the advertisement call in Xenopus l. laevis)

Positions and employments:

From January 2003 Research assistant, Laboratory of Behavioral Neuroscience,

ETH Zurich

October 1999 – December 2002 PhD-student Laboratory of Behavioral Neurobiology, ETH

Zurich

October 1997 – October 1999 Assistant at the department of biology (sensory biology)

Humboldt-University Berlin

October 1996 – October 1997 Assistant at the department of zoology, Museum of natural

History Berlin