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Procedural Learning and Cognitive Flexibility in a Mouse Model of Restricted, Repetitive Behavior in Neurodevelopmental ...

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PAGE 1

1 PROCEDURAL LEARNING AND COGNITIVE FLEXIBILITY IN A MOUSE MODEL OF RESTRICTED, REPETITIVE BEHAVIOR IN NEURODEVELOPMENTAL DISORDERS By YOKO TANIMURA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 by Yoko Tanimura

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3 To my parents, Nobuo and Reiko Tanimura, and to my sister, Chie Sato, whose help and support made it possible for me to pursue a career in science.

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4 ACKNOWLEDGMENTS I would like to express my sin cere thanks to my adviser, Dr. Mark H. Lewis, for the dedicated guidance and continuous help throughout my thesis wor k. I would also like to thank my committee members, Dr. Darragh Devine and Dr. Timothy Vollmer, for their contributions. I acknowledge and thank Ms. Bonnie I. McLaurin, our laboratory manager, for all the help throughout the course of my work with animals, and Dr. Mark Yang for th e help with analyzing the data collected for the thesis. Finally I would like to thank my friends in Gainesville, especially the ladies in the Behavioral Neuroscience program, as well as those in Japan for encouraging me to finish this work and to continue purs uing a career in science.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 INTRODUCTION..................................................................................................................11 Restricted, Repetitive Behaviors in Neurodevelopmental Disorders.....................................11 Animal Models of Restrict ed, Repetitive Behaviors..............................................................12 Stereotypy Associated with Ce ntral Nervous System Insults.........................................12 Pharmacologically Induced Stereotypy...........................................................................14 Stereotypy Associated with Environmental Restriction..................................................14 Deer Mouse Model of Repetitive Behaviors..........................................................................16 Effects of Environmental Enrichment....................................................................................17 Cortico-Basal Ganglia Circuitr y and Repetitive Behaviors....................................................18 Cortico-Basal Ganglia Circuitry and Cognition.....................................................................21 Cortico-Basal Ganglia Circuitry and Cognitive Flexibility....................................................23 Cortico-Basal Ganglia Circuitry and Environmental Enrichment..........................................25 Aim............................................................................................................................ .............27 2 MATERIALS AND METHODS...........................................................................................28 Subjects....................................................................................................................... ............28 Housing Conditions............................................................................................................. ...28 Stereotypy Assessment.......................................................................................................... .29 Apparatus...................................................................................................................... ..........30 Pretest Assessment............................................................................................................. .....30 Cognitive Assessment........................................................................................................... ..31 Procedural Learning........................................................................................................31 Reversal Learning............................................................................................................32 Data Analysis.................................................................................................................. ........34 3 RESULTS........................................................................................................................ .......35 Stereotypy Assessment.......................................................................................................... .35 Cognitive Assessment........................................................................................................... ..36 Effects of Housing Conditions: Enriched vs. Standard Caging..............................................36 Procedural Learning........................................................................................................36 Reversal Learning............................................................................................................38

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6 Effects of Stereotypy Levels...................................................................................................39 Procedural Learning........................................................................................................39 Reversal Learning............................................................................................................41 4 DISCUSSION..................................................................................................................... ....58 Effects of Housing Conditions on Stereotypy........................................................................59 Effects of Housing Conditions on Procedural Learning.........................................................59 Effects of Housing Conditions on Reversal Learning............................................................61 Effects of Stereotypy Levels on Procedural Learning............................................................62 Effects of Stereotypy Levels on Reversal Learning...............................................................63 Modeling a Wider Range of Rest ricted, Repetitive Behaviors...............................................66 LIST OF REFERENCES............................................................................................................. ..68 BIOGRAPHICAL SKETCH.........................................................................................................80

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7 LIST OF TABLES Table page 3-1 The frequency of repetitive vertical ju mps and the number of deer mice in four stereotypy groups.............................................................................................................. .36

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8 LIST OF FIGURES Figure page 2-1 Representative swimming paths and responses.................................................................33 3-1 Effects of environmental enrichment on st ereotypy (vertical jumping and backward somersaulting)................................................................................................................. ...44 3-2 Average stereotypy scores in four stereotypy groups........................................................44 3-3 Latency to reach the platform. A) Effect s of housing conditions. B) Effects of stereotypy levels.............................................................................................................. ...45 3-4 Distance traveled. A) Effects of housing condi tions. B) Effects of stereotypy levels.......46 3-5 Velocity. A) Effects of housing conditi ons. B) Effects of stereotypy levels.....................47 3-6 The number of first correct arm entries. A) Effects of housing conditions. B) Effects of stereotypy levels........................................................................................................... .48 3-7 The number of correct respons es. A) Effects of housing conditions. B) Effects of stereotypy levels.............................................................................................................. ...49 3-8 The number of error respons es. A) Effects of housing conditions. B) Effects of stereotypy levels.............................................................................................................. ...50 3-9 The total number of error re sponses made in the procedur al and reversal phases. A) Effect of housing conditions. B) Effect of stereotypy levels.............................................51 3-10 The proportion of error responses made in the procedural and reversal phase. A) Effects of housing conditions. B) Effects of stereotypy levels..........................................52 3-11 Probability of making a correct response in the first 24 trials of the reversal phase. A) Effects of housing conditions. B). Effects of stereotypy levels.........................................53 3-12 Probability of making an error response in th e first 24 trials of the reversal phase. A) Effects of housing conditions. B) Effects of stereotypy levels..........................................54 3-13 Regression of the total number of error res ponses in the reversal phase by individual stereotypy score............................................................................................................... ..55 3-14 Days required to reach the criterion. A) E ffects of housing conditions. B) Effects of stereotypy levels.............................................................................................................. ...56 3-15 Effects of housing conditions on trials to the cr iterion. A) Trials to the procedural criterion. B) Trials to the reversal criterion.......................................................................57

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PROCEDURAL LEARNING AND COGNITIVE FLEXIBILITY IN A MOUSE MODEL OF RESTRICTED, REPETITIVE BEHAVIOR IN NEURODEVELOPMENTAL DISORDERS By Yoko Tanimura December 2006 Chair: Mark H. Lewis Major Department: Psychology Restricted, repetitive beha viors (e.g., stereotypies, compulsions, rituals) in neurodevelopmental disorders have been linked to alterations in cortical-basal ganglia circuitry. Restricted, repetitive behavior ha s been shown to be associated specifically with deficits in cognitive flexibility. Cognitive processes mediat ed by this circuitry (e .g., procedural learning, executive function) are likely to be impaired in individuals exhibiting high rates of repetitive behavior. To test this hypothesis, we assessed both procedural learni ng and cognitive flexibility (reversal learning) using a T-maze task in deer mice exhibiting various rates of repetitive behavior (vertical jumping and backward some rsaulting). These mice exhibited high rates of stereotypy when reared in standa rd rodent cages, and such beha vior was significantly prevented by housing them in larger more complex environments. The results showed that mice reared in comple x environments exhibited significantly better procedural and reversal learni ng than standard caged mice. T hus, early experience associated with the prevention and attenuation of stereotypy was associated with bette r striatally mediated learning and cognitive flexibility. In addition, mice exhibiting high ra tes of stereotypy (especially

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10 backward somersaulting) performed most poorly in the reversal phase. The results indicate that the expression of repetitive motor behavior is associated with perseverative behavior in a learning and memory task. Our finding enhances th e applicability of the deer mouse model of spontaneous stereotypy to the wide range of re stricted, repetitive be havior (e.g., rituals, insistence on sameness) typical of neurodevelopmental disorders.

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11 CHAPTER 1 INTRODUCTION Restricted, Repetitive Behaviors in Neurodevelopmental Disorders Restricted, repetitive behavi ors encompass a range of abnormal behaviors, which are common behavioral phenotypes of several neurodev elopmental disorders. In autism, repetitive behaviors are expressed as motor stereotypies, repetitive manipulation of objects, and echolalia as well as more complex behaviors such as co mpulsions, rituals, insistence on sameness, and narrow and circumscribed interests (Bodfish et al., 2000; Lewis & Bodfish, 1998; Turner, 1999). This wide range of repetitive behaviors is also ty pical of individuals with more severe forms of mental retardation (Bodfish et al ., 2000; Lewis & Baumeister, 1982). Not surprisingly, motor stereotypies, or si mple repetitive, often rhythmic motor movements (e.g., hand flipping, body rocking), appear relatively early in development. More complex repetitive behaviors (e.g., complex moto r sequences and repetitive use of words) emerge gradually with age (Militerni et al., 2002; Mooney et al., 2006). Complex repetitive behaviors seem to involve cognitive as well as motor components, which are often accompanied by some set of rules or a just right cr iterion for completion (e.g., rituals and object attachments). This cognitive rigidity may be a component of a broader pr ofile of deficits in executive function, which has been widely repo rted in individuals with autism (Pennington & Ozonoff, 1996). The association between executive function, particularly cognitive flexibility, and repetitive behaviors is now bei ng investigated (Lopez et al., 2005). Although a wide range of rest ricted, repetitive behavior is characteristic of neurodevelopmental disorders, specific repeti tive behaviors are th e typical behavioral phenotypes of certain genetic disorders. Two exam ples include repetitive self-biting of lips or digits in Lesch-Nyhan disease (Cauwels & Ma rtens, 2005) and skin picking in Prader-Willi

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12 syndrome (Symons et al., 1999). Other psychiatri c and neurological disorders may also be associated with specific forms of repetitive behaviors. Two obvious examples include childhood onset obsessive-compulsive disorder (OCD) (re petitive checking or washing) and Tourette syndrome (vocal tics) (Cath et al., 2001; Muller et al., 1997). In general, restricted, repetitive behavior s are considered abnormal and clinically significant because they are stigmatizing, preclu de or disrupt goal-directed actions, limit interaction with the environment, and on occas ion, are self-injurious. The pathophysiology of these behaviors has received limited clinical study, alt hough several neuroimaging and pharmacological challenge studies have been pub lished (Hollander et al., 2005; Malone et al., 2005). A more complete understanding of neur obiological perturbati ons responsible for repetitive behavior disorders w ould greatly facilitate the devel opment of treatment options for patients. Such an understanding will re quire use of valid animal models. Animal Models of Restrict ed, Repetitive Behaviors To study the underlying neurobiologi cal basis of repetitive behavi ors, several categories of relevant animal models are available. These cate gories include stereotypy associated with central nervous system (CNS) insults, pharmacologically induced stereotypy, and stereotypy induced by rearing animals in restricted environments (L ewis et al., 2006). Although these models should ideally reflect the wide range of repetitiv e behaviors displayed in individuals with neurodevelopmental disorders, they tend to focu s largely on motor stereotypy. This is because stereotypy is observed relatively easily, whereas it is much more challenging to assess more complex behavior that may be considered models of rituals or insistence on sameness in animals. Stereotypy Associated with Ce ntral Nervous System Insults Recent advances in genomics, particularly th e ability to knock out specific genes, have provided important information about the etiology of neuropsychiat ric disorders. Of particular

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13 interest is that some of thes e gene-manipulated animals demons trate repetitive behaviors, which resemble the repetitive behavior symptoms in clinical populations. For example, a loss of function mutation in the methyl-CpG binding protein 2 (MeCP2) gene is known as a major cause of Rett syndrom e, and MeCP2 abnormalities are also reported in individuals with mental retard ation and autism (Carney et al ., 2003; Meloni et al., 2000). Compared to control animals, mice expressing tr uncated MeCP2 protein display more repetitive forepaw movements, which resemble hand ster eotypies (hand wringi ng and waving) commonly engaged in by patients with Rett syndrome (M oretti et al., 2005; Shahbazian et al., 2002). Excessive grooming resulting in hair removal and tissue da mage has been reported in Hoxb8 homozygous knockout mice (Greer & Capecchi, 2002). Expression of the Hoxb8 gene is found in the orbitofrontal and anterior cingulat e cortices, and caudate nucleus, which are the brain regions comprising OCD circuitry (G raybiel & Rauch, 2000). Excessive grooming in Hoxb8 knockout mice has particular resemblance to trichotillomania, an OC spectrum disorder. Similarly, a significantly increase d number of brief head or body jerks is found in D1CT-7 transgenic mice expressing a neuropotentiati ng transgene in cortico-limbic glutamatergic neurons, which seems to be overactivated in patients with OCD and Tourettes syndrome (Nordstrom & Burton, 2002). Such behavior is suppressed by clonidine, an alpha-2 adrenergic agonist that is commonly used to reduce tics in c linical population. In addition to CNS insults via gene manipulatio ns, prenatal exposure to environmental risk factors has been shown to induce similar behavior al phenotypes to those seen in patients with neurodevelopmental disorders. Pren atal exposure to valproic acid (VPA) has been advanced as a potential etiological factor for autistic symp toms (Folstein & Rosen-Sheidley, 2001; Keller & Persico, 2003). Exposure of this antiepileptic dr ug to rats on day 12.5 of gestation produced

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14 neuropathological abnormalities similar to those reported in autistic individuals (e.g., altered sensitivities to tactile stimuli, diminished pre pulse inhibition, decreased social behaviors, and hyperactivity including stereo typy-like behaviors) (Schneider & Przewlocki, 2005). Pharmacologically Induced Stereotypy Much of what we know about the neurobiolog ical basis of motor stereotypy comes from investigations using pharmacol ogical models (Lewis & Baum eister, 1982; Lewis & Bodfish, 1998). These models have a direct human analogue as clinically abnorma l repetitive behaviors were described in human amphetamine abuser s as early as the la te 1960s (Ellinwood, 1967; Kramer et al., 1967). Such phenomena were also demonstrated in several species of animals treated with amphetamine (Ra ndrup & Munkvad, 1965). Subsequen tly, a wide range of other pharmacological agents (e.g., phenm etrazine, L-dopa, morphine, me thylphenidate, pemoline, and monoamine oxidase inhibitors) that affect the nigro-striatal dopa minergic system was shown to induce stereotypy in rats (Fog, 1972). Systematic investigation of anatomical a nd neurochemical mechanisms underlying druginduced repetitive behavior implicated cortico-ba sal ganglia circuitry in the execution of these movements. Intrastriatal and systemic administ ration of direct and in direct dopamine agonists (e.g., amphetamine, apomorphine, and cocaine) as well as opiate agonists and NMDA receptor antagonists (e.g., MK-801 and PCP) have consis tently induced motor stereotypy (Ernst & Smelik, 1966; Iwamoto & Way, 1977; Lewis et al ., 1990; Segal et al., 19 95; Vandebroek et al., 1998; Vandebroek & Odberg, 1997). Cortico-basal ga nglia circuitry will be discussed more in detail in a later section. Stereotypy Associated with Environmental Restriction Repetitive motor behaviors not requiring pharmacological induc tion are frequently reported in animals housed in zoo, farm, and laboratory environments (Mason, 1991). Examples

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15 of these species-specific ster eotypic behaviors include pacing route-tracing, and feather-picking in parrots and tits (Garner et al., 2003a; Garn er et al., 2003b; Jenkins 2001; Meehan et al., 2004), bar-mouthing, vertical jumping, and back ward somersaulting in rodents (Garner & Mason, 2002; Powell et al., 1999; Vandebroek & Odberg, 1997), pacing, body-rocking, tailbiting, and self-injurious behavi or in rhesus monkeys (Lutz et al., 2003; Taylor et al., 2005), pacing, somersaulting, and over-grooming in pr osimians (Tarou et al., 2005), crib-biting, boxwalking, and head-shaking in horses (Bach mann et al., 2003; McGreevy et al., 1995), regurgitation and tongue f licking in pandas (Swaisgood et al., 2005), and head-twirling in minks (Mason, 1993). Since animals in the wild do not engage in such abnormal behaviors, stereotypy seems to develop as a consequence of animals respons es to restricted environments, which limit expression of species-typical be haviors. Conversely, alleviation of environmental deprivation seems an effective means to reduce rates of spontaneous stereotypy (Garner et al., 2003a; Meehan et al., 2004; Powell et al., 2000; Sw aisgood et al., 2005; Tu rner et al., 2002). Importantly, motor stereotypy associated w ith restricted environments has been demonstrated to be qualitatively different from pharmacologically induced stereotypy in the same species. For example, deer mice develop focused vertical jumping and backward somersaulting as a consequence of restricted housing, whereas they exhibited excessive gnawing, rearing, and locomotion following administration of apomorphine (Presti et al., 2004; Presti et al., 2002). Similarly, although repetitive jumping is commonly displayed in captive bank voles, NMDA receptor antagonist MK-801 and apomorphine elevated repetitive licking, sniffing, and locomotion instead (Vandebroek et al., 1998; Vandebroek & Odberg, 1997). These findings

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16 suggest the limitations of drug-induced stereotypy to model repetitive behavioral phenotypes of neurodevelopmental disorders. Motor abnormalities associated with environmenta l restriction are strikingly similar to the phenomena (spontaneous, repetiti ve, topologically unvarying, f unctionless, and apparently purposeless) in neurodevelopmental disorders. These individuals are likely to experience environmental restriction early in life as a func tion of their handicap. Thus, it is possible that similar neurodevelopmental pertur bations spontaneously occur in stereotypic animals reared in restricted environments and individuals with neurodevelopmental disorders. Studying the neurobiological mechanisms of stereotypy usi ng a model associated with environmental restriction will advance our knowle dge of the development of re stricted, repetitive behavior associated with neurodevelopmental disorders. To this end, we have adopted a mouse model of stereotypy induced by environmental restriction. This model utilizes a species that can be studied in the laboratory and whose stereotypy is s pontaneous, persistent, and occurs early in development. Deer Mouse Model of Repetitive Behaviors In our laboratory, we employ deer mice ( Peromyscus maniculatus ) as a model of repetitive behavior disorders. These animals exhibit high ra tes of repetitive vertical jumping or backward somersaulting when housed under standard labora tory conditions (Powell et al., 1999). These behaviors emerge as early as weaning and persis t for a prolonged period of time. Exposure to a larger, more complex environment (environmen tal enrichment or EE) following weaning significantly attenuates the de velopment of these behaviors (Powell et al., 2000). After two months of EE following weaning, approximate ly 80 percent of mice show low rates of stereotyped behaviors, whereas cl ose to 80 percent of mice reared in standard cages (SC) show

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17 high rates of stereotyped behavior Enrichment later in life is al so beneficial, yet to a lesser degree (Hadley et al., 20 06; Powell et al., 2000). Effects of Environmental Enrichment The significance of environmental stimulati on on behavior was firs t recognized through the pioneering work of Hebb (1949) He reported that rats he br ought home from his laboratory to keep as pets were better at solving cognitive ta sks than rats raised in the laboratory. Since this early observation, EE has been used to demonstrat e behavioral and brain plasticity in a large number of studies using a vari ety of behavioral and biologica l measures or endpoints (Lewis, 2004; van Praag et al., 2000). It should be note d, however, that EE is a relative term and that such experimental configurations cannot approx imate the complexity an d variability of the animals natural habitat. EE may also have beneficial effects in preven ting or attenuating the effects of CNS insult. A higher educational background, cognitively cha llenging occupations, and high socioeconomic status were found to lower the risk of Alzheime rs disease and cognitive impairment associated with age (Moceri et al., 2001; S nowdon et al., 1996; Stern et al., 1994; White et al., 1994). In a transgenic mouse model of Alzheimers disease, EE prevented overproduction of amyloid-beta protein and learning impairments associated w ith amyloid deposits (Jankowsky et al., 2005). Furthermore, the neuroprotective role of EE was found in a 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) mouse model of Parkin sons disease (Faherty et al., 2005), where EE significantly attenuated the death of dopaminergic neurons in the s ubstantia nigra pars compacta (SNpc). As another example, transgenic mice expressing human huntingtin transgene, a mouse model of Huntingtons disease, showed amelio ration of motor abnormalities following EE. In addition, EE rescued molecular alterations caused by transcriptional dysregul ation in these mice, such as reduction in brain deri ved neurotrophic factor (BDNF) in the hippocampus and striatum,

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18 and dopamine and cAMP-regulated phosphoprotein 32 kD a (DARPP-32) deficits in the anterior cortex (Spires et al., 2004). Of particular interest are behavioral modifications in animal models of neurodevelopmental disorders asso ciated with repetitive behavior A recent study showed that mice with fragile X mental retardation 1 gene mu tation, an animal model of Fragile X syndrome, displayed hyperactivity, cogniti ve alterations, and immature dendritic and spine morphology when housed in standard rodent cages, wher eas enriched experiences normalized mutationassociated cognitive and morphological impairme nts (Restivo et al., 2005). Similarly, VPAtreated rats, modeling autism, exhibited stereotypy-like beha viors under standard laboratory conditions, whereas VPA rats under EE conditions al leviated repetitive behaviors as well as other parameters charact eristic of autism (Schneider et al., 2006). As suggested previously, our lab has dem onstrated that EE markedly attenuates the development of spontaneous stereotypy in deer mice. We have also examined the neurobiological correlates of th is experientially based attenua tion. Specifically we found elevated metabolic activity and dendritic spine density in the motor cortex and dorsolateral striatum in EE mice exhibiting diminished levels of stereotypy (Turner et al ., 2003; Turner et al., 2002). Moreover, BDNF in the striatum was elevat ed in these enriched low-stereotypy mice (Turner & Lewis, 2003). Thus, it is reasonable to hypothesize that altered activity within this circuitry is responsible for the expressi on of repetitive behavior in deer mice. Cortico-Basal Ganglia Circuitry and Repetitive Behaviors The basal ganglia are a group of subcortical nuclei that regulate execution of motor and cognitive programs. They receive inputs from all areas of the neocortex except the primary visual and primary auditory cortices. The motor ci rcuit that is hypothesized to be responsible for the expression of stereotypy orig inates from the primary moto r cortex and premotor area.

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19 Glutamatergic neurons from these cortical regions enter the input nucleus of the basal ganglia (dorsolateral striatum) and the output neurons fr om the basal ganglia re gulate activation of thalamocortical neurons. These neurons terminat e at the somatosensory cortex, primary motor cortex, and supplementary motor area, providi ng positive feedback to ongoing motor programs in the primary motor cortex (Herrero et al., 2002; Parent & Hazrati, 1995). There are two pathways that travel through th e basal ganglia system: the direct pathway and indirect pathway. The direct pathway consists of GABAergic medium spiny neurons directly projecting from the dorsolateral st riatum to the output nucleus of the basal ganglia, the globus pallidus internal (GPi) and substantia nigra pars reticulata (SNpr) (striatonigral neurons). These neurons selectively express D1 dopamine receptors that are co-localized with glutamate receptors. They contain the neuropeptides dynor phin and substance P (Steiner & Gerfen, 1998). The indirect pathway consists of GABAergic st riatal medium spiny neurons (striatopallidal neurons) projecting to the globus pallidus external (GPe), GABAergic neurons in the GPe projecting to the subthalamic nucleus (STN), and finally gl utamatergic neurons sending excitatory projection to the GPi and SNpr. The striatopallidal medium spiny neurons express D2 receptors, and the neuropeptide enkephalin (Steiner & Gerfen, 1998). Normal dopaminergic innervation in the stri atum plays a crucial role in execution of movements. D1 receptors in the striatonigral neurons ar e positively coupled to adenylyl cyclase (Missale et al., 1998). Therefore, dopamine acts to amplify the glutamatergic corticostriatal inputs, resulting in increased GABAergic inhi bition of the GPi and SNpr. In contrast, D2 receptors in the striatopallidal neurons are negativel y coupled to adenylyl cyclase (Missale et al., 1998). Via D2 receptors, dopamine acts to diminish th e corticostriatal inputs, resulting in decreased inhibition of the GPe, hence causing this nucleus to exert even more inhibitory control

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20 over the STN. This increased inhibition of the STN removes its excitatory influence on the GPi and SNpr. Thus activation of D1 and D2 receptors both removes inhibitory tone of the basal ganglia output neurons, ultimately resulting in di sinhibition of the thalamocortical neurons to provide positive feedback to motor programs. Abnormal execution of movements is evident when the balance betw een the direct and indirect pathway is disrupted. It is postulated that stereotypi c behavior is expressed as a consequence of a relative increase in striatonigral tone (Graybiel et al., 2000) This hypothesis is supported by the finding that transgenic mice i nducibly overexpressing the transcription factor FosB selectively in dynorphin-containing striat onigral neurons exhibit increased daily wheel running, whereas such behavior was signi ficantly reduced in mice overexpressing FosB in enkephalin-containing striatopallidal neurons (Werme et al., 2002). To investigate alterations in cortico-basal ganglia circuitry in deer mice exhibiting spontaneous stereotypy, we ad ministered the selective D1 antagonist SCH23390 and the NMDA antagonist MK-801 intrastriatally. These comp ounds both blocked spontaneous stereotypy selectively (Presti et al ., 2003). In contrast, the D2 receptor antagonist raclopride, unexpectedly failed to reduce stereotypy (Prest i et al., 2004). Finally, the mixed agonist apomorphine failed to increase spontaneous stereotypies in deer mice wh en administered intrastriatally (Presti et al., 2002). In addition, we assessed neuronal activities of the direct and indirect pathways by measuring neuropeptides dynorphin and enkepha lin respectively. We found significantly decreased leu-enkephalin conten t and significantly increased [dynorphin]/[enkephalin] content ratios in high-stereotypy mice relative to lowstereotypy mice. Moreover, we saw a significant negative correlation between striat al enkephalin content and freque ncy of stereotypy as well as a

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21 significant positive correlation between the [dynor phin]/[enkephalin] content ratio and frequency of stereotypy in these mice (Presti & Lewis, 2005). Another line of evidence for potential ne urobiological perturba tions responsible for repetitive behaviors comes from neuroimagi ng studies in clinical populations exhibiting behaviors of interest. Increased right caudate volume was noted in i ndividuals with autism spectrum disorders, which was positively correlated with rates of repetitive behaviors, especially scores of higher-order compone nts (Hollander et al., 2005), such as compulsions, rituals, and difficulties with minor change, and complex moto r mannerisms (Sears et al., 1999). Patients with trichotillomania displaying repetiti ve hair-pulling had significantly reduced volumes of the left putamen (O'Sullivan et al., 1997). Moreover, tics in Tourette syndrome were inversely correlated with changes of blood flow and oxygen concentr ation in the basal ga nglia and thalamus (Peterson et al., 1998). These studies provide evidence fo r alterations of cort ico-basal ganglia circuitry in animals and individuals associated with repetitive beha viors. To further suppor t alterations in this circuitry in stereotypic animals, we wished to assess other non-stereotyped behaviors, which require the intact basal ganglia system for normal function. We focused on cognitive behaviors including striatally mediated pr ocedural learning a nd cognitive flexibility, which require intact fronto-basal ganglia circuitry. Cortico-Basal Ganglia Circuitry and Cognition The role of the striatum in mnemonic functi on, which is distinct from the hippocampally dependent system, is increasingly appreciate d. A number of studies demonstrated double dissociation of two memory systems; the hippo campus mediates spatial, allocentric memory, which relies on stimulus-stimulus relationshi ps or Tolmanian cognitive mapping strategy, whereas the striatum plays an im portant role in motor, egocentr ic memory, which in particular

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22 emphasizes acquisition of Hullian stimulus-respons e (S-R) relationships (Packard & Knowlton, 2002; White & McDonald, 2002; Yin & Knowlton, 2006). This dissociation of relatively independent memory systems is supported by the findings that localized lesions of fimbria-fornix, a major input-output pathway of the hippocampus, impaired spatial memory, whereas caudate lesion s were associated with poor performance in discrimination learning tasks, using spatial and cue versions of Morris water maze tasks (McDonald & White, 1994; Packard & McGaugh, 1992) or win-shift and win-stay radial-arm maze tasks (Kesner et al., 1993; Packard & White, 1991) respectively. Different biochemical processing associated wi th these systems also provides evidence for the mnemonic dissociation. For example, calci um-sensitive adenylyl cyclase activity was increased to the greatest degr ee in the hippocampus after spa tial learning, whereas calciuminsensitive adenylyl cyclase was enhanced in seve ral areas of the brain, es pecially the striatum, following procedural learning (Guillou et al., 1999). Furthermore, immediately after maze traini ng that assessed either place or response learning, both phosphorylated CREB (pCREB) and cfos expression were increased in either group of animals, which experienced place or response learning. After one hour, sustained pCREB and c-fos immunoreactivity was observed in the hippocampus (the dentate gyrus, CA1, and CA3) following place learni ng, whereas pCREB activity was sustained in the striatum (dorsolateral and dorsomedial) following respon se learning (Colombo et al., 2003). CREB is widely known to contribute to memory consol idation and dynamic modulation of synaptic strength. Thus, task acquisition seems to require initial activation of multiple brain regions, but the region responsible for acquisition of the task later undergoes greater ac tivation to consolidate the learned strategy. Additionall y, CREB mutation impaired pe rformance in several tasks

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23 associated with the dorsal striatum and altered corticostriatal long-term potentiation (LTP) and depression (LTD) (Pittenger et al., 2006), supporting th e involvement of this subcortical area in learning, which is distinct from the hippocampally mediated learning. This change in neural acti vation with learning is also supported electrophy siologically. Graybiel and colleagues (Barne s et al., 2005; Jog et al., 1999) demonstrated that many of dorsolateral striatal neurons we re activated shortly after a di scriminative auditory stimulus, which indexed the location of a reward, in th e simple discrimination T-maze task at the beginning of training. However, as rats learned the S-R continge ncy, the neuronal firings at the beginning (trial start) and end (re ward) of the maze were enhanced and the firings at the tone and turn were substantially attenuated. This shift in neuronal firings associated with procedural learning was largely reversed during extinction learning. In humans, functional magnetic resonance imag ing (fMRI) studies prov ided evidence for the involvement of the striatum in probabilistic classification task, which requires non-motor procedural memory (Poldrack et al., 1999). Patients with the ba sal ganglia pathology, such as Huntingtons (Knopman & Nissen, 1991; Sprengelme yer et al., 1995) and Parkinsons diseases (Allain et al., 1995; Krebs et al., 2001), present a variety of cognitive impairments attributable to the striatal memory system. Although some eviden ce exists (Mostofsky et al., 2000), not much is known regarding S-R learning defi cits in individuals with ne urodevelopmental disorders who exhibit restricted, re petitive behaviors. Cortico-Basal Ganglia Circuitry and Cognitive Flexibility Other domains of cognitive function, which requi re intact cortico-basal ganglia circuitry, include executive function. Executive function is an umbrella term for cognitive processes such as attention, planning, working memory, and cogn itive flexibility, which require higher-order mental acts. The disturbances of these processe s are reported in many psyc hiatric disorders such

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24 as schizophrenia and autism (Pennington & Ozonoff, 1996). According to recent human and animal studies, these processes are largely me diated by the frontal cortex, which has strong connections with the striatum in series of front o-striatal loops (Dalley et al., 2004; Heyder et al., 2004). Of most interest to us among these subc omponents of executive function is cognitive flexibility. Damage in subregions of the frontal cortex (dorsolateral prefrontal cortex and orbitofrontal cortex) causes a difficulty in shif ting a response or switchi ng strategies when S-R contingency changes. Thus, patients with front al damage often repetitively respond to the previously relevant, however no l onger associated, stimuli. This perseverative behavior pattern resembles simple motor stereotyped behaviors wh ere both are inappropriat ely repeated and rigid (Lewis & Bodfish, 1998). Hence, perturbations w ithin the striatum could dysregulate not only cortical-striatal loop inducing repe titive motor behavior, but also fronto-striatal loops impairing cognitive flexibility. To support this hypothesis, recent investiga tions presented eviden ce for the link between stereotypy associated with restri cted environment and cognitive ri gidity. For example, blue and marsh tits exhibiting repetitive route-tracing stereo typy showed a perseverative response pattern in a gambling task (Garner et al., 2003a; Garner et al., 2003b), which has been used for cognitive assessment in schizophrenia (Frith & Done, 1983) and OCD (Cavallaro et al., 2003). In this task, subjects are instructed to sear ch for the rule governing the rewa rd presentation contingent on a particular stimulus, when in fact there is no ru le. Therefore their sequenc es of responses reveal repetition of a specific response or repetitive pattern of responses. A similar correlation was also found between cag e stereotypies and reversal (where the reinforcer was switched and associated to th e opposing stimulus) or extinction (where the

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25 reinforcer no longer existed) of S-R learning in bank voles (Garner & Mason, 2002; Garner et al., 2003a) and bears (Vickery & Mason, 2005). Moreover, this relationship was recently demonstrated by Lopez and colleagues (2005) in clinical populations. Scores on the executive function task that indexed cognitive rigidity were positively correlate d with severity of restricted, repetitive behavior in autist ic adults. This relationship between motor and perseverative responses in the California Tra il Making Test is of interest to many researchers, because executive function deficits have been recognized in autistic individuals relatively independently of repetitive motor behavior. Therefore, we wish ed to demonstrate such an association between stereotypy and cognitive flexibil ity in our deer mice model. Cortico-Basal Ganglia Circuitry and Environmental Enrichment Evidence consistently supports that exposure to EE improves performance in various cognitive tasks, which are at least partially dependent on the hippocampus (e.g., go/no go task, object recognition task, contextual fear conditio ning, spatial version of Morris water maze, and open field test) (Duffy et al., 2001; Lambert et al ., 2005; Leggio et al., 2005; Park et al., 1992; Pham et al., 1999; Woodcock & Richardson, 2000). EE has also been shown to attenuate impa ired learning systems associated with hippocampal (Will et al., 1983) and fimbria-forn ix lesions (van Rijzingen et al., 1997), immunolesioned basal forebrain cholinergic sy stem (Paban et al., 2005), mutation in CA1specific NMDA receptor 1 subunit (Rampon et al., 2000) traumatic brain injury (Dahlqvist et al., 2004; Gobbo & O'Mara, 2004; Rutten et al., 2002; Wagner et al., 2002), high fat diet (Winocur & Greenwood, 1999), and age (Frick & Fernandez, 2003; Frick et al., 2003). Biochemical indices of neural pl asticity associated with learni ng improvement include increased levels of neurotrophic factor s (Pham et al., 1999), alteration of the cAMP-dependent protein

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26 kinase dependence of LTP (Duffy et al ., 2001), and increased phosphorylation of CREB (Williams et al., 2001; Young et al., 1999) in the hippocampus. Moreover, adult neurogenesis in the hippo campus is enhanced following EE. BruelJungerman and colleagues (2005) demonstrated th at these new neurons actually contribute to hippocampally mediated memory improvement a ssociated with EE. Mice were housed either under EE or standard laboratory conditions, and ha lf of EE mice were trea ted with saline and the other half were treated with antimitotic methylazoxymethanol ace tate (MAM), which prevented further cell division. There was significant elevation in 5-br omo-2-deoxyuridine (BrdU)positive new born cells in EE nave mice compar ed to EE MAM mice and mice from standard cages, implicating EE-dependent increase in neur ogenesis in the dentate gyrus. Moreover, EE nave mice showed statistically better long-term recognition memory than EE MAM-treated mice, providing the first direct evidence for a dult neurogenesis in the hippocampus following EE and their functional participat ion in memory enhancement. Thus, the role of EE on hippocampally me diated learning and memory has been extensively studied behaviorally and neurobi ologically. Fewer attempts to understand the efficacy of EE on striatum-mediated learning have been made (Frick & Fernandez, 2003; Frick et al., 2003; Schrijver & Wurbel, 2001). Frick an d Fernandez (2003) demonstrated that agerelated memory impairment was rescued following EE in the spatial version of Morris water maze, but not in the cued version of Morris wate r maze, which assesses procedural learning. An increase in synaptophysin was evident in the hi ppocampus and frontopariet al cortex in EE mice, but no such assessment was done for the striatum Similarly, other studies failed to show procedural learning enhancement following exposure to EE. Therefore, we wish to evaluate carefully EE effect on striatum-mediate d procedural learning in deer mice.

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27 Conversely, studies support the effect of EE on cognitive flexibility. Isolation housing impaired reversal learning (J ones et al., 1991; Schrijver et al ., 2004) and extradimensional setshifting (Schrijver & Wurbel, 2001), isolated an imals showing more pe rseverative responses than their socially reared counterparts. Ye t, effects of other EE components (e.g., cognitive stimulation and exercise) and the neurobiological alterations associated with cognitive rigidity have not been studied. Thus we wished to assess the effect of EE on cognitive flexibility in deer mice. Aim Restricted, repetitive behavior s are clinically significant for several neurodevelopmental disorders. Studies using pharmacologically induced animal models of stereotypy as well as our deer mice model associated with restricted enviro nment suggest that the re latively increased tone of the direct pathway compared to the indirect pathway is re sponsible for the expression of repetitive behaviors. To furthe r confirm the alteration in the basal ganglia system in animals displaying high rates of ster eotypy, we hypothesized that ot her non-stereotypy behaviors mediated by this brain region, such as procedur al learning and cognitive flexibility, are also impaired in these animals. To investigate these hypotheses, the present e xperiment examined the relationship between spontaneously emitted stereo typy and these cognitive processes as well as effects of EE on them usi ng the water-filled T-maze.

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28 CHAPTER 2 MATERIALS AND METHODS Subjects As we have previously demonstrated, both male and female Peromyscus maniculatus (deer mice) develop high rates of persistent, spontane ously emitted stereotypy consisting of repetitive vertical jumping and backward somersaulting wh en housed under standard laboratory conditions. In most mice, these behaviors can be markedly attenuated by environmental enrichment. Fiftynine mice were originally designated for this ex periment, but during the period from weaning to the completion of the reversal phase, four mice died for unknow n reasons and three mice were excluded. Data from 52 mice (23 male and 29 female ; 3-7 months old) were used for analysis. Subjects were obtained from the breeding colony maintained in our laboratory, and at weaning were randomly assigned to either standard cag e (SC) or enriched e nvironment (EE) housing conditions. Housing Conditions Rodent chow and water were available ad li bitum, and Cockatiel vita seed was provided three times each week. The housing room was maintained at 20-25 and 50-70 % humidity. Subjects were maintained on a 16:8-h light/dark cycl e, with lights off at 10:00pm. All procedures were performed in accordance with the guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. Mice assigned to EE were group-caged (6 same sex mice/cage) in large dog kennels (122 x 81 x 89 cm) consisting of two extra levels construc ted of galvanized wire mesh and connected by ramps of the same material. Bedding, a running wh eel, shelters (similar opaque, concave object), and various other objects (habitra il tubes, plastic toys, and mesh structures for climbing) were

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29 placed in each kennel prior to introducing the mice. One oz. of Cockatiel vita seed was scattered throughout the kennel three times each week to encourage foraging behavior. A running wheel remained undisturbed in the kennel, but other obj ects (except those in which mice were hiding) were removed and replaced with clean novel objects on a weekly basis. For the SC condition, mice were caged (2-3 sa me sex mice/cage) in standard laboratory rodent cages (48 x 27 x 15 cm). A half oz. of Cock atiel vita seed was place d at one corner of the cage three times each week on the same schedule as the enriched cages in order to counterbalance any nutrition effects with the EE mice. Stereotypy Assessment After being housed in their respective cagi ng conditions for 60-180 days, the mice were tested for rates of stereotyped behavior. Prior to cognitive assessment, mice from both SC and EE conditions were tested for rates of stereotypy using a modified automated photocell detection apparatus obtained from Columbus Instruments (Columbus, CO). This equipment uses photocells located 13.5 cm above the floor, to qu antify the number of interruptions made by repetitive jumps or backward somersaults of a mouse during a given time period. The session consisted of the ei ght hours of the dark cycle for two consecutive days. Any single testing day involved three mice from the EE housing and two or three mice from the SC housing sharing a common weaning date. The te sting protocol involve d removing mice from their home cages and placing them singly in tes ting cages (22 x 15 x 28 cm) made of Plexiglas prior to the beginning of the dark cycle. Food and water were pr ovided, and the mice were left undisturbed for two to three hours fo r habituation to the new cage a nd recovery from the stress of handling.

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30 All sessions were digitally vi deo recorded for further identif ication of behavior phenotypes and accuracy of the automated counters. Each animal received a stereotypy score that represented the average st ereotypy frequency per hour. Apparatus The testing apparatus consisted of a T-maze c onstructed from clear Plexiglas. Each arm measured 7.5 x 32 x 18 cm. The platform (7.5 x 7.5 x 5 cm) made of the same Plexiglas material was placed at the end of one arm. The maze wa s placed in a black plastic tank (85 cm in diameter, 36 cm in height) and a white nylon cu rtain (85 cm in diameter, 60 cm in height) surrounded the T-maze. Prior to cognitive testing, the T-maze was filled with warm tap water (25 ) to a depth of 5.8 cm so that the clear platform was submerged 8 mm from the surface of the water. In order to keep the temperature constant, the water was meas ured every eight trials and hot tap water was added as needed. Non-toxic white paint wa s added to the water to ensure opacity. A digital video camera was mounted 120 cm above the T-maze to make recordings of swim trials. The camera was connected to a com puter in the same room and used to measure mices escape latencies and tr ack swimming paths by Ethovision video tracking system (Noldus Information Technology, the Netherlands). Pretest Assessment Mice were tested in the visible-platform vers ion of Morris water maze task to detect any motor or sensory abnormalities. The black tank was filled with warm tap water (25-28 ) and the white plastic platform (10 x 10 cm) was placed in the tank, 5 mm above the surface of water. Mice were released from the edge of the tank a nd allowed to swim to the platform for up to 60 seconds. This was repeated four times and the lo cation of the platform and the releasing point

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31 were varied in every trial with an intertrial interval of approximately 15 minutes. During the intertrial interval, the mice were returned to their cage in an adjoining room, towel dried and placed underneath a heating lamp. Mices swi mming patterns were analyzed. Those mice exhibiting apparent motor and sensory impair ments were excluded from further cognitive assessment (n=3). On the following day, mice were exposed to th e T-maze, which was placed in the tank and filled with warm opaque water without the platform. Mice were released from the start arm facing to the wall and allowed to explore the Tmaze for 30 seconds. This was repeated six times with intertrial interval of approximately 15 mi nutes. The swim paths were digitally recorded to determine the existence of turning bias in each animal. Mice were determin ed to have a turning bias, when they made more than fi ve first entries to the same arm. Cognitive Assessment Procedural Learning On the day following T-maze habituation, mice were tested for procedural memory in the water-filled T-maze with the invisible platform. E ach training session consisted of eight trials. The platform was placed at the e nd of the east or west arm. The location of the platform was pseudorandomly determined among mice except for those animals exhibiting a turning bias. For those cases (n=6), the platform was positioned in the non-preferred arms. Four extra-maze cues (different color, geometric shapes, approximately 15 x 15 cm in diameter) were attached to the surrounding nylon curtain and their locations were randomly changed every trial. A trial began by placing a mouse at the end of the start arm (facing the wall) and ended when the mouse climbed onto the platform, or after 60 seconds had elapsed. If the mouse did not escape after 60 seconds, it was gently guided to the platform, placed on the platform, and removed from the maze. For each mouse there wa s a delay of approximately 15 minutes between

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32 successive trials within a session. During the delay, the remaining mice were run on the same trial. Thus, the intertrial interval varied slightly according to the mices level of performance but was approximately equal for all mice within each trial. Completion of a trial was defi ned as spending two seconds on the platform. If a mouse reached the platform but jumped out to the wate r again, the trial was continued until the mouse reached the platform again and spent two seconds there. Each mouse was left on the platform for 15 seconds and then returned to the home cage, located in an adjoining room. A correct response was defined as reaching th e platform without entry into the opposite arm (Figure 2-1). An incorrect response was scored when the mouse entered the arm not containing the platform. When an animal swam a ll the way to the end ( 7.5 x 7.5 cm) of the arm not containing the platform, the trial was recorded as an error response. The criterion for the procedural phase was set as seven or eight corre ct responses per session for three consecutive sessions. Reversal Learning A reversal-learning task was initiated the day after the animal met criterion for successful completion of the procedural task. For this ta sk, the same T-maze was employed except that the platform was placed at the end of the opposite arm (East West, West East) so that mice needed to inhibit the previously relevant response and learn the new stimulus-response association. The criterion for this task was also set as seven or more correct responses for three consecutive days.

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33 A B C Figure 2-1. Representative swimming paths a nd responses: A response in a single trial was defined as either a correct, incorrect, or error response. Th e shaded area at the end of east arm represents a submerged platform The corresponding area of the opposite arm represents the error zone. A) A correct response: completion of a trial without entry to the incorrect arm. B) An incorrect response: completion of a trial with entry to the incorrect arm. C) An error response: completion of a trial wi th entry to the error zone at the end of the incorrect arm.

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34 Data Analysis A stereotypy score was calculated for each anim al and a Students t test was performed to determine the effects of diffe rential housing on stereotypy ex pression (Sigmastat, Systat Software, CA). Based on the stereotypy score, groups of mice were constituted according to different rates of stereotypy expression. An analysis of variance (ANO VA) for repeated measures was performed to determine the effect of housing condition and stereotypy levels on indices of cognitive performance including latency, distance traveled, velocity, and the prob ability of making a corr ect or error response. Further post hoc pairwise comparison tests (T ukey) were conducted to assess differences between stereotypy groups where appropriate. Moreover, a Student s t test for housing condition and a one-way ANOVA for stereotypy groups were performed for the days required to reach the criterion. In addition, individual stereotypy scores were regresse d against the number of correct or error responses made. A p-value of .05 or less was adopted for assigning statistical significance.

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35 CHAPTER 3 RESULTS Stereotypy Assessment Stereotypy scores as measured by the automa ted photocell detection apparatus ranged from 38 to 2394 responses per hour for all animals. There was a significant housing effect on stereotypy score ( t (50)=4.1, p <0.001) with EE mice (n=25) exhibiti ng significantly lower rates of stereotypy than SC mice (n=27) (Figure 3-1). When a small number of mice exhibiting backward somersaulting, all of which were in the SC condition, were excluded from the analysis, a significant difference in housing conditions was still found ( t (41)=4.3, p =0.001). To group mice according to rates of stereotypy, we first separated mice displaying repetitive backward somersaulting (B) for the fo llowing reasons. First, this qualitatively distinct behavior takes more time to complete a single response than does a vertical jump. Thus, the frequency of backward somersaulting is typically less than that observed for vertical jumping. Second, all of the mice exhibiting backward so mersaulting (n=9) were in the SC condition. Finally as shown in the Cognitive Assessment section below, this group of mice showed distinct cognitive abnormalities. The remaining mice showing vertical jumping were categorized according to whether their frequency of jumping fell into the upper, middle, or lower third of the frequency range: high (H, more than 761 counts per hour, n=15), middle (M between 334 to 760, n=14), and low rate of jumping (L, less than 334, n=14). The number of mice in each housing condition so categorized is depicted in Table 3-1. The average stereot ypy score per hour for each group is depicted in Figure 3-2. We were not able to assess the inte raction between stereot ypy levels and housing conditions due to the small number of mice in th e enriched housing hi gh stereotypy group and the standard cage low and middle stereot ypy group. According to photocell interruption

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36 frequencies obtained for mice e xhibiting backward somersaulting, five mice would have been categorized as high stereotypy a nd two would have been categorized as middle and low each. Table 3-1. The frequency of repetitive vertical jumps and the number of deer mice in four stereotypy groups. Stereotypy Group Stereotypy Score # of SC Mice # of EE Mice High rates of jumping 761 < 12 3 Middle rates of jumping 334 760 4 10 Low rates of jumping < 333 2 12 Backward somersaulting (255-1268) 9 0 Cognitive Assessment Since some mice reached criterion on the third day of training for both the procedural learning and reversal lear ning experiments, we compared the average trial latencies, distance traveled, and swim speed as well as the number of first correct arm entr ies, correct responses, and error responses within a session for the first three days of the procedural (P1, P2, P3) and reversal (R1, R2, R3) experiments. Thus, in or der to examine the effects of housing condition, we conducted 2 (housing condition) by 3 (days) ANOVAs with repeated measures on the second factor. To assess the effects of level of stereotypy on c ognitive function, we conducted 4 (stereotypy group) by 3 (days) ANOVAs with repeat ed measures on the second factor. For all dependent variables assessed, th ere were no differences in se x or age, although there was a tendency for females and for younger mice (3 months of age) to perform better than males and older mice (7 months of age). Effects of Housing Conditions: Enriched vs. Standard Caging Procedural Learning Significant main effects of housing condition we re found on latency to reach the platform ( F (1, 100)=11.3, p =0.001) (Figure 3-3A), distance traveled ( F (1, 100)=12.0, p =0.001) (Figure 34A), velocity ( F (1, 100)=20.5, p <0.001) (Figure 3-5A), the number of first correct entries ( F (1,

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37 100)=5.7, p =0.020) (Figure 3-6A), and the number of correct ( F (1, 100)=9.0, p =0.004) (Figure 3-7A) and error responses ( F (1, 100)=4.3, p =0.044) (Figure 3-8A). With respect to all of these variables, EE mice performed better than SC mice. There were also significant main effects of time for all the variables measured: latency ( F (2, 100)= 35.6, p <0.001), distance traveled ( F (2, 100)=45.6, p <0.001), velocity ( F (2, 100)=35.0, p <0.001), the number of fi rst correct entries ( F (2, 100)=22.2 p <0.001), and the number of correct ( F (2, 100)=42.2, p <0.001) and error responses ( F (2, 100)=32.1, p <0.001). No statistically significant inte raction was found for latency ( F (2, 100)=2.8, p =0.065), distance traveled ( F (2, 100)=1.9, p =0.155), velocity ( F (2, 100)=0.3, p =0.739), the number of first correct entries ( F (2, 100)=1.3, p =0.272), and the number of correct responses ( F (2, 100)=2.2, p =0.114). A statistically significant inte raction was found, however, for error responses ( F (2, 100)=3.6, p =0.031). Specifically, EE and SC mi ce made a similar number of error responses in P1, but signi ficant differences between thes e groups were observed in P2 ( p =0.008) and P3 ( p =0.043). Analysis of the first three days of procedur al learning does not make use of all the data. Thus, the number of error res ponses across all sessions was calc ulated. A significant difference in housing condition was found for the number of error responses where SC mice made significantly more error responses than EE mice ( t (50)=-2.3, p =0.025) (Figure 3-9A). However, the proportion of error responses (the total numbe r of error responses divided by the total number of responses in the procedural learning experi ment) didnt differ between housing conditions ( t (50)=1.1, p =0.263) (Figure 3-10A). Finally a main effect of housing condition was found for days required to reach the criterion, which ranged from 3 to 11 days ( t (50)=2.6, p =0.012) (Figure 3-14A). In this, as in all

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38 other measures, mice housed in an enriched e nvironment performed better than mice housed in standard cages in th is procedural task. In addition, the proportion of animals meeting criterion across totals was calculated by survival analysis using the Cox proportional ha zard model. There was a significant difference ( p =0.024), such that a greater proportion of EE mice met criterion in fewer trials than did SC mice (Figure 3-15A). Reversal Learning A main effect of housing condition on latency to reach the platform was also observed in the reversal phase ( F (1, 100)=4.2, p =0.046) (Figure 3-3A). As seen in the procedural learning phase, significant differences between EE and SC mice were also found for the other dependent measures examined: distance traveled ( F (1, 100)=5.8, p =0.020) (Figure 3-4A), velocity ( F (1, 100)=6.9, p =0.012) (Figure 3-5A), the number of first correct arm entries ( F (1, 100)=8.0, p =0.007) (Figure 3-6A), and the number of correct ( F (1, 100)=9.5, p =0.003) (Figure 3-7A) and error responses ( F (1, 100)=13.8, p <0.001) (Figure 3-8A). A main effect of time was found on all measures employed: latency ( F (2, 100)=42.3, p <0.001), distance traveled ( F (2, 100)=75.2, p <0.001), velocity ( F (2, 100)=4.9, p =0.010), the number of first correct arm entries ( F (2, 100)=48.9, p <0.001), and the number of correct ( F (2)=53.8, p <0.001) and error responses( F (2, 100)=87.0, p <0.001). Specifically, EE mice swam significantly faster on all R1-3 days ( p =0.013, p =0.024, and p =0.027 respectivel y), and made more correct and fewer error responses in R1 ( p =0.013, p =0.010 respectively) and R2 ( p <0.001, p <0.001 respectively) than SC mice. There were no statistically si gnificant interactions for any of these measures. In addition, EE mice made significantly fewer er ror responses compared to SC mice across all sessions in the reversal phase (t (50)=-3.4, p =0.001) (Figure 3-9A). Similarly, SC mice

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39 exhibited a significantly greater proportion of error respons es compared to EE mice ( t (50)=3.6, p <0.001) (Figure 3-10A). Figure 3-11A shows the proportion of correct responses in the firs t 24 trials of the reversal phase. In the first trial of the reversal phase (r1 ), almost all the mice failed to make a correct response. Yet, as the trials progressed, EE mice quickly learned the new S-R contingency. By the end of the first session (r8), th e proportion of correct response in SC mice was below chance level (approximately 25%), whereas the propor tion of correct responses for EE mice was approximately 60%. Similarly, the proportion of error responses during reversal learning was higher for SC mice compared to EE mice in r1-2 4 (Figure 3-12A). Finally, there was a main effect of housing on days to reach criterion ( t (50)=2.2, p =0.033) (Figure 3-14A), with mice housed in the EE condition performing better th an mice housed in the SC condition in the reversal task. In addition, the proportion of animals meeting criterion across totals was calculated for survival analysis by the Cox proportional hazar d model. There was a significant difference ( p =0.021), such that a greater proportion of EE mice met criterion in fewer trials than did SC mice (Figure 3-15B). Effects of Stereotypy Levels Procedural Learning A main effect of stereotypy level was found for latency ( F (3, 96)=3.0, p =0.041) (Figure 33B), distance traveled ( F (3, 96)=6.9, p <0.001) (Figure 3-4B), and velocity ( F (3, 96)=3.3, p =0.029) (Figure 3-5B). Specifically mice in the backward somersaulting group spent a significantly longer duration of time in the maze than the low stereotypy group ( p =0.029). The differences in latency between these groups were significant for the first (P1) ( p =0.037) and second (P2) ( p =0.003) days. By day three (P3) all st ereotypy groups took roughly equal amounts

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40 of time for trial completion. Si milarly, mice in the backward somersaulting group traveled the longest distance to reach the platform comp ared to any other groups (L, M, and H) ( p <0.001, p =0.003, p =0.002 respectively) (Figure 3-4B). No significant differences among the high, medium, and low jumpers were noted. Post hoc analyses for the dependent measure of velocity showed a significant difference between mice in the backward somersault ing and in the low stereotypy groups ( p =0.025) and in P2 ( p =0.043) and P3 ( p =0.016). When the velocity of each mouse was compared with their individual stereotypy frequency score, there was a tendency for mice with high frequency of stereotypy to swim slower ( r= -0.256, p =0.070). This was largely attributed to the swim speed among mice exhibiting vertical jumping (L, M, and H) ( r= -0.292, p =0.057). A main effect of time was f ound for all measures: latency ( F (2, 96)=38.1, p <0.001), distance traveled ( F (2, 96)=52.1, p <0.001), velocity ( F (2, 96)=32.3, p <0.001), the number of first correct arm entries ( F (2, 96)=20.0, p <0.001), the number of correct ( F (2, 96)=40.0, p <0.001) and error responses ( F (2, 96)=0.9, p= 0.452). The only measure for which a significant group by time interaction was found was distance traveled ( F (6, 96)=2.8, p =0.015). Mice in the low stereotypy gr oup increased the ve locity as the trials progressed, whereas mice in the backward somersaulting group stayed at relatively slow speed (Figure 3-5B). No group differences were found on the number of first correct arm entries ( F (3, 96)=1.6, p =0.205) (Figure 3-6B) and the number of correct ( F (3, 96)=2.5, p =0.067) (Figure 3-7B) and error responses ( F (3, 96)=0.9, p =0.452) (Figure 3-8B). In add ition, no significant difference was found in the total number of error responses ( F (3)=1.6, p =0.192) (Figure 3-9B) as well as a

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41 proportion of error responses across all sessions in the procedural phase ( F (3)=0.3, p =0.861) (Figure 3-10B). Next, each stereotypy score was regressed by the total number of correct or error responses made within P1-3. There were no relationship between rates of ster eotypy and correct ( r= -0.094, p =0.509) and error responses ( r= 0.045, p =0.750). Moreover, there wasnt any relationship between stereotypy scores and the total number of correct ( r= 0.050, p =0.724) and error responses ( r= 0.062, p =0.662) made in the procedural phase. Finally, one-way ANOVA for stereotypy groups showed that significant differences existed in days to reach the criterion ( F (3)=3.7, p =0.018). Post-hoc analyses revealed a significant difference between mice in the backward somersaulting group and in the low stereotypy group ( p =0.027) (Figure 3-14B). Reversal Learning During reversal training, no si gnificant differences among gr oups were found for latency ( F (3, 96)=1.2, p =0.335) (Figure 3-3B) or swim speed ( F (3, 96)=1.3, p =0.298) (Figure 3-5B). Conversely, main effects of stereotypy level we re found for the variables distance traveled ( F (3, 96)=3.1, p =0.036) (Figure 3-4B), the number of first correct arm entries ( F (3, 96)=8.8, p <0.001) (Figure 3-6B), and the number of correct ( F (3, 96)=8.9, p <0.001) (Figure 3-7B) and error responses ( F (3, 96)=5.3, p =0.003) (Figure 3-8B). Specifically, mice in the backward some rsaulting group traveled longer distances ( p =0.023), made fewer successful first correct arm entries ( p <0.001), and made fewer correct ( p <0.001) and more error responses ( p =0.002) than mice in the low stereotypy group. In addition, low stereotypy mice made significantly more first correct entry and correct responses, than mice in the medium stereotypy group ( p =0.001, p =0.001 respectively) and mice in the high

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42 stereotypy group made more first correct entry and correct respons es than mice in the backward somersaulting group ( p =0.042, p =0.047 respectively). A main effect of time was f ound for all measures: latency ( F (2, 96)=42.5, p <0.001), distance traveled ( F (2, 96)=76.8, p <0.001), velocity ( F (2, 96)=5.0, p =0.008), the number of first correct arm entries ( F (2, 96)=49.7, p <0.001), and the number of correct ( F (2, 96)=58.5, p <0.001) and error responses ( F (2, 96)=86.9, p <0.001). A group by time interaction was found for distance moved ( F (6, 96)=2.7, p =0.018) (Figure 3-4B), first correct arm entries ( F (6, 96)=4.3, p <0.001), and the number of correct ( F (6, 96)=6.3, p <0.001) (Figure 3-7B) and e rror responses (F(6, 96)=3.9, p =0.002) (Figure 3-8B). This effect seems to be due to the unexpected performance by mice in the high stereotypy group. In addition, the number of error responses ma de in R1 was positively correlated with the frequency of stereotypy ( r= 0.306, p =0.003) (Figure 3-13). This di fference was mainly due to mice showing vertical jumping ( r= 0.317, p =0.039). It is worth noting that number of error responses in the P1-3 and R1-3 was positively corre lated in mice exhibiti ng vertical jumping (L, M, and H groups) ( r= 0.310, p =0.043), suggesting that mice exhibi ting higher rates of stereotypy appear to perseverate more and mi ce with procedural learning defi cits exhibit reversal deficits. However, when the stereotypy score was regresse d against the total numbe r of error responses made by each mouse, no relationship between st ereotypy score and error responses was found ( r= 0.216, p =0.124). In addition, a significant differe nce was found in the total numb er of error responses across sessions in the reversal phase ( F (3)=8.1, p <0.001), where mice in the backward somersaulting group made significantly more error responses than any ot her groups (L, M, and H) ( p <0.001, p =0.012, p =0.002 respectively) (Figure 3-9B). There was also a significant difference in a

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43 proportion of error responses ( F (3)=3.9, p =0.014). Mice in the backward somersaulting group exhibited a significantly greater proportion of error responses th an mice in the low stereotypy group ( p =0.024) (Figure 3-10B). Figure 3-11B shows the proportion of mice making a correct res ponse in the first 24 trials of the reversal phase. In the firs t trial of the reversal phase (r1), almost a ll of the mice failed to make a correct response. Approximately 65% of mice in the low stereotypy group made a correct response in r8 and almost all found the plat form successfully by the middle of the second reversal session, 10% of mice in the backward somersaulting group made a correct response in r8 and the probability stayed below chance level thr ough r24. Similar reversal trends were seen for an error response in r1-24 (Figure 3-12B). Finally, significant differences were found in days required to reach a reversal criterion among different levels of stereotypy ( F (3)=8.9, p <0.001) (Figure 3-14B). Mice in the backward somersaulting group required significantly more days to complete the task than mice in the low ( p <0.001) and high ( p =0.003) stereotypy groups. In addi tion, mice in the middle stereotypy group required more days than mice in the low ( p =0.005) and high ( p =0.016) stereotypy groups. There was no correlation between days require d to meet criterion fo r procedural learning and days required to meet cr iterion for reversal learning ( r= 0.23, p =0.103). However, when the two topographies of stereotypy we re analyzed separately, the nu mber of days to reach both criteria was not correlated in jumpers ( r= -0.025, p =0.874), but were strongly correlated in backflippers ( r= 0.765, p =0.016). Therefore these results i ndicate that for mice exhibiting repetitive backward somersaulting deficits in procedural learning were associated with cognitive rigidity.

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44 HOUSING GROUPS SCEE AVERAGE COUNTS PER HOUR 0 200 400 600 800 1000 1200 Figure 3-1. Effects of environmen tal enrichment on stereotypy (vertical jumping and backward somersaulting): EE substantially attenuated the development and expression of stereotypy (p<0.001). Values expres sed are group means S.E.M. STEREOTYPY GROUPS HMLB AVERAGE STEREOTYPY SCORE 0 200 400 600 800 1000 1200 1400 1600 1800 Figure 3-2. Average stereotypy scores in four st ereotypy groups: Mice were categorized into H (n=15), M (n=14), L (n=14), or B (n=9 ) groups depending on their topography and frequency of stereotypy. Values e xpressed are group means S.E.M.

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45 A BLOCKS P1P2P3R1R2R3 LATENCY (SEC) 0 5 10 15 20 25 30 EE SC B BLOCKS P1P2P3R1R2R3 LATENCY (SEC) 0 5 10 15 20 25 30 35 H M L B Figure 3-3. Latency to reach the platform. A) Ef fects of housing conditions: EE mice reached the platform significantly faster than SC mi ce in the first three days of both the procedural ( p =0.001) and reversal ( p =0.046) phases. B) Effects of stereotypy levels: B mice took significantly longer to reach the platform than L mice in the first three days of the procedural phase ( p =0.029). There was no difference between stereotypy groups in the reversal phase ( p =0.335). Values expressed are group means S.E.M of eight daily trials.

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46 A BLOCKS P1P2P3R1R2R3 DISTANCE (PIXEL) 200 400 600 800 1000 1200 EE SC B BLOCKS P1P2P3R1R2R3 DISTANCE (PIXEL) 200 400 600 800 1000 1200 1400 1600 H M L B Figure 3-4. Distance traveled. A) Effects of housing conditions: EE mice swam significantly shorter distances than SC mice in the fi rst three days of both the procedural ( p =0.001) and reversal ( p =0.020) phases. B) Effects of st ereotypy levels: B mice traveled significantly longer distance than any other groups in the first three days of the procedural phase ( p <0.001). B mice traveled significan tly longer distance than L mice in the reversal phase ( p =0.023). Values expressed are group means S.E.M of eight daily trials.

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47 A BLOCKS P1P2P3R1R2R3 VELOCITY (PIXEL / SEC) 30 40 50 60 70 80 EE SC B BLOCKS P1P2P3R1R2R3 VELOCITY (PIXEL / SEC) 40 50 60 70 80 90 H M L B Figure 3-5. Velocity. A) Effects of housing conditions: EE mice sw am significantly faster than SC mice in the first three days of the procedural ( p <0.001) and reversal ( p =0.012) phases. B) Effects of stereotypy levels: B mice swam significantly slower than L mice in the first three days of the procedural phase ( p =0.029). No group difference was found in the reversal phase ( p =0.298). Values expressed are group means S.E.M of eight daily trials.

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48 A BLOCKS P1P2P3R1R2R3 # OF FIRST CORRECT ARM ENTRIES 0 2 4 6 8 10 EE SC B BLOCKS P1P2P3R1R2R3 # OF FIRST CORRECT ARM ENTRIES 0 2 4 6 8 10 H M L B Figure 3-6. The number of first correct arm entr ies. A) Effects of housing conditions: EE mice made significantly more correct entries than SC mice in the first three days of both the procedural ( p =0.020) and reversal ( p =0.007) phases. B) Effects of stereotypy levels: Although no group difference was found in the procedural phase ( p =0.205), L mice made significantly more correct entries than B ( p <0.001) and M mice ( p =0.001), and H mice made more correct entries than B mice ( p =0.042) in the first three days of the reversal phase. Values expressed are group means S.E.M of eight daily trials.

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49 A BLOCKS P1P2P3R1R2R3 # OF CORRECT RESPONSES 0 1 2 3 4 5 6 7 8 EE SC B BLOCKS P1P2P3R1R2R3 # OF CORRECT RESPONSES 0 2 4 6 8 10 H M L B Figure 3-7. The number of correct responses. A) Effects of housing conditions: EE mice made significantly more correct responses than SC mice in the first three days of both the procedural ( p =0.004) and reversal ( p =0.003) phases. B) Effects of stereotypy levels: Although no group difference was found in the procedural phase ( p =0.067), L mice made significantly more correct responses than B ( p <0.001) and M ( p =0.001) mice, and H mice made more correct responses than B mice ( p =0.047) in the first three days of the reversal phase. Values expressed are group means S.E.M of eight daily trials.

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50 A BLOCKS P1P2P3R1R2R3 # OF ERROR RESPONSES 0 1 2 3 4 5 6 7 8 EE SC B BLOCKS P1P2P3R1R2R3 # OF ERROR RESPONSES 0 1 2 3 4 5 6 7 8 H M L B Figure 3-8. The number of error responses. A) Effects of housing condi tions: SC mice made significantly more error res ponses than EE mice in the fi rst three days of both the procedural ( p =0.044) and reversal ( p <0.001) phases. B) Effects of stereotypy levels: There was no group difference in the procedural phase ( p =0.452), whereas B mice made significantly more error responses than L mice in the first three days of the reversal phase ( p =0.002). Values expressed are group means S.E.M of eight daily trials.

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51 A HOUSING GROUPS SCEE # OF ERROR RESPONSES 0 5 10 15 20 25 P R B STEREOTYPY GROUPS HMLB # OF ERROR RESPOSES 0 10 20 30 P R Figure 3-9. The total number of e rror responses made in the proce dural and reversal phases. A) Effect of housing conditions: SC mice made significantly more error responses than EE mice in both the procedural ( p =0.025) and reversal phases ( p =0.001). B) Effect of stereotypy levels: There was no significant difference among groups in the procedural phase ( p =0.192), whereas B mice made signific antly more error responses than L ( p <0.001), M ( p =0.012), and H ( p =0.002) mice in the reve rsal learning. Values expressed are group means S.E.M.

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52 A HOUSING GROUPS SCEE % OF ERROR RESPONSES 0.00 0.05 0.10 0.15 0.20 0.25 0.30 % P ERRORS % R ERRORS B STEREOTYPY GROUPS HMLB % OF ERROR RESPONSES 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 % P ERROR % R ERROR Figure 3-10. The proportion of erro r responses in the procedural and reversal phase. A) Effects of housing conditions: There was no difference between groups in the procedural phase (p=0.263), whereas SC mice exhibite d a significantly great er proportion of error responses than EE mice in the reversal phase (p<0.001). B) Effects of stereotypy levels: There was no significant differen ce among groups in the procedural phase (p=0.861), B mice exhibited a significantly grea ter proportion of e rror responses than L mice in the reversal phas e (p=0.024). Values expressed are group means S.E.M.

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53 A TRIALS r1r2r3r4r5r6r7r8r9r10r11r12r13r14r15r16r17r18r19r20r21r22r23r24 OCCURRENCE OF A CORRECT RESPONSE 0.0 0.2 0.4 0.6 0.8 1.0 EE SC B TRIALS r1r2r3r4r5r6r7r8r9r10r11r12r13r14r15r16r17r18r19r20r21r22r23r24 OCCURRENCE OF A CORRECT RESPONSE 0.0 0.2 0.4 0.6 0.8 1.0 H M L B Figure 3-11. Probability of making a correct response in the first 24 trials of the reversal phase. A) Effects of housing conditions: EE mice co mpared to SC mice quickly learned the new behavior. B). Effects of stereoty py levels: Although L mice learned the new behavior, B mice show a pattern of perseveration.

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54 A TRIALS r1r2r3r4r5r6r7r8r9r10r11r12r13r14r15r16r17r18r19r20r21r22r23r24 AVERAGE OCCURRENCE OF AN ERROR 0.0 0.2 0.4 0.6 0.8 1.0 EE SC B TRIALS r1r2r3r4r5r6r7r8r9r10r11r12r13r14r15r16r17r18r19r20r21r22r23r24 AVERAGE OCCURRENCE OF AN ERROR 0.0 0.2 0.4 0.6 0.8 1.0 H M L B Figure 3-12. Probability of making an error response in the first 24 trials of the reversal phase. A) Effects of housing conditions: EE mice co mpared to SC mice quickly learned to the previously relevant response. B) E ffects of stereotypy levels: Although L mice learned to inhibit the previously releva nt response, B mice show a pattern of perseveration.

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55 A STEREOTYPY SCORE (JUMPER) 050010001500200025003000 TOTAL # OF ERROR RESPONSES 0 5 10 15 20 25 30 B STEREOTYPY SCORE (JUMPERS) 020040060080010001200 TOTAL # OF ERROR RESPONSES 0 5 10 15 20 25 30 Figure 3-13. Regression of the total number of error responses in th e reversal phase by individual stereotypy score. A) Stereotypy score was pos itively correlated with the number of error responses (p=0.003). B) Stereotypy score was positively correlated with the number of error responses when six outliers were excluded (p=0.025).

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56 A HOUSING GROUPS SCEE DAYS TO THE CRITERION 0 2 4 6 8 10 P R B STEREOTYPY GROUPS HMLB DAYS TO THE CRITERION 0 2 4 6 8 10 12 14 P R Figure 3-14. Days required to reach the criter ion. A) Effects of housing conditions: SC mice required significantly more days to r each the criterion in the procedural ( p =0.012) and reversal ( p =0.033) phases. B) Effects of stereoty py levels: B mice spent significantly more days than L mice to reach the procedural criterion ( p =0.027). B mice spent significantly more days than L ( p <0.001) and H ( p =0.003) mice, and M mice spent more days than L ( p =0.005) and H ( p =0.016) to reach the reve rsal criterion. Values expressed are group means S.E.M.

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57 A B Figure 3-15. Effects of housing c onditions on trials to th e criterion. A) Trials to the procedural criterion: SC mice required significantly more trials to reach the procedural criterion ( p =0.021). B) Trials to the reve rsal criterion: SC mice re quired significantly more trials to reach the reversal criterion ( p =0.024).

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58 CHAPTER 4 DISCUSSION Restricted, repetitive behavi ors are commonly displayed in many neurodevelopmental disorders. Despite their prevalence in clinical populations, relatively little is known about the neurobiological mechanisms responsible for the de velopment and expression of these behaviors. A better understanding of the underlying alterations will aid in identifying etiology as well as developing a better treatment plan for individuals with repetiti ve behavior disorders. Valid animal models provide an important strategy in achieving these goals and are necessary for pursuing the neural mechanisms of this abnormal behavior. This study employed deer mice ( Peromyscus maniculatus ) as a model of repetitive motor behavior associated with ne urodevelopmental disorders. Th ese mice exhibit high rates of repetitive jumping or backward somersaulting wh en housed in standard rodent cages, and such behaviors can be attenuated or prevented by housing them in environmentally complex settings (Powell et al., 2000; Powell et al., 1999). In add ition, our work suggests th at deer mice exhibiting high rates of stereotypy have al tered cortico-basal ganglia circ uitry, specifically relatively decreased tone of the indirect pathway (Presti & Lewis, 2005). In this study, we attempted to examine hypothe sized impairments in st ereotypic deer mice in cognitive processes that are mediated in part by cortico-basa l ganglia circuitry. First, we tested striatally mediated procedural learning in mice displaying va rious rates of stereotypy, which were reared in either a standard rodent cage or an environmentally enriched condition. Since cortico-striatal circuitry involving the frontal cortex seems to play an important role in executive function (Dallery et al., 2004; Dias et al., 1996; Uylings et al ., 2003; van der Meulen et al., 2006), we further assessed one aspect of such higher-order function, cognitive flexibility, by

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59 switching of the stimulus-response (S-R) continge ncy (reversal learning) following acquisition of the procedural task. Effects of Housing Conditions on Stereotypy In agreement with our previous findings (P owell et al., 2000; Powell et al., 1999), deer mice reared in an environmentally enriched (EE) condition developed significantly lower rates of stereotypy compared to mice rear ed in standard cages (SC). On e novel feature of this study is that we categorized mice displaying backward so mersaulting as a separate group for statistical analysis. In past experiments, we have not sy stematically differentiated these two topographies (vertical jumps and backward somersaults), becaus e a majority of deer mice exhibited high rates of repetitive vertical jumps a nd only a minority engaged in more complex backward somersaults (Powell et al., 1999). As expected, most EE mice exhi bited low levels of jumping and most SC mice exhibited high rates of jumping or backward somers aulting. Although we wished to examine the interaction between housing conditio ns and levels of stereotypy on cognitive performance, we were not able to evaluate such effects due to the small number of mice in the EE-high stereotypy and SC-low stereotypy groups. Effects of Housing Conditions on Procedural Learning Studies have repeatedly demonstrated th at memory dependent on the hippocampal formation improves following exposure to EE in animals (Duffy et al., 2001; Lambert et al., 2005; Pham et al., 1999; Rampon et al., 2000; Winocur & Greenwood, 1999; Woodcock & Richardson, 2000). Since EE benefits a variety of other behaviors a nd relevant neurobiology, it is reasonable to hypothesize that the memory syst ems mediated by non-hippocampal regions such as the striatum (procedural learning) also improve followi ng EE. Although there have been

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60 several attempts to examine effects of EE on procedural learning (Frick & Fernandez, 2003; Frick et al., 2003; Schrijver et al., 2004), no significan t improvements were noted. For example, Frick and colleagues (Frick & Fernandez, 2003; Frick et al., 2003) exposed aged rats (18 and 28 months) to EE and assessed their performance in two ve rsions (spatial and cued) of Morris water maze tasks. Rats expos ed to EE conditions as adults performed significantly better than SC control rats in the spatial task, whereas EE did not reduce ageassociated cognitive impairments in the cued task. In contrast, our results showed that mice reared in EE condition performed significantly better with respect to all behavi oral measurements collected duri ng the procedural task (latency, distance traveled, velocity, the num ber of first correct arm entries, and the number of correct and error responses), and took fewer days to criterion compared to SC mice. This contradictory result from other groups could be explained by the age and species difference in sensitivity to surrounding environm ents. Whereas Frick and colleagues showed that age-related cognitive impairments in rats we re found specifically in the hippocampus and EE rescued associated deficits in rats, relatively younger deer mice were employed in this study. In addition, neurobiological alterations associated with repetitive beha vior in adult deer mice were primarily found in the motor cortex and dorsolatera l striatum (Turner et al., 2003; Turner et al., 2002). EE-associated improvements in hippocampally dependent memory are often associated with elevation of neurotrophic factors in the hippocampus (Pha m et al., 1999). We previously found that deer mice, which benefited from EE, ha d elevated levels of striatal brain derived neurotrophic factor (BDNF) (Turner & Lewis, 2 003). This neurotrophin plays a particularly important role in neuronal plasticity associ ated with learning and memory. In contrast, no

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61 increase in BDNF or nerve growth factor ( NGF) was noted within the hippocampus in these mice. The ameliorative effects of EE, therefore, seem ed to act preferentially upon the striatum in deer mice, resulting in improvements in perf ormance in the procedural-learning task. In summary, our current and previous work supports improvements of striatally mediated memory following EE in deer mice. Effects of Housing Condition s on Reversal Learning In the reversal experiment a ssessing cognitive flexibility, EE mice performed significantly better in all measurements during the first few da ys of the reversal task. Specifically, SC mice made significantly more perseverative errors befo re reaching the criteri on. Even after many days of training trials in the proce dural phase, they swam significan tly slower and longer distances during reversal learning, with no obvious motor abnormalities. Moreover, relatively more EE mice reached the cr iterion early in training according to the survival analysis. We found such significant di fferences for both the procedural and reversal phases, but the magnitude of diffe rence between the two groups was larger in the reversal phase. Although we hypothesized an effect of EE on execu tive function, not many studies have tested this effect (Jones et al., 1991; Schrijve r et al., 2004; Schrijver & Wurbel, 2001). The studies assessing the effect of EE on cognitive flexibi lity have focused primarily on the social component of EE. For example, Sc hrijver and Wurbel (2001) demonstrated that socially isolated rats had deficits in extradimen sional set-shifting from cu eto place-version of the radial arm maze tasks, and vice versa. Both th e social and isolated rats had intact cue and place memory as well as intradimensional se t-shifting within these tasks, however. In contrast, we exposed mice to an environm ent consisting of multiple EE factors (novelty, inanimate objects, social interaction, exercise increased space, etc. ), and found significant

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62 effects of EE on cognitive flexibility. Thus, thes e results suggest that a combination of multiple EE components benefits higher-order cognitive f unctions dependent in part on cortico-basal ganglia circuitry in deer mice, in addition to memory systems discussed above. Effects of Stereotypy Levels on Procedural Learning Among the four groups constituted by stereotypy level or topography (three levels of vertical jumping and backward somersaulting), imp airments in the procedural task were found in mice exhibiting backward somersaults. These mice traveled longer distance, swam slower, and required more days to criterion. There was no re lationship between rates of stereotypy and corrector error-response fre quency suggesting that mice of various levels of stereotypy explored the maze similarly. Thus contrary to our hypothesis, procedural-learning impairments in high-stereotypy mice were not found. Evidence for deficits in striatally mediated learning in autistic individuals is almost completely lacking, despite the si gnificance of these brain regions in the expression of repetitive behaviors. However, there has been a report of a deficit in the Serial Response Time Trial task (Mostofsky et al., 2000) in children with auti sm. Although performance in this task likely involves both cortico-striatal and cortico-cerebellar pathways, no attempt was made to relate reaction time to repetitive behaviors. We know relatively more about deficits in striatally mediated l earning and memory among individuals with Tourettes syndrome and obses sive-compulsive disorder (OCD). Marsh and colleagues (2004) recently reported an inverse correlation between tic and habit learning in children and adults with Tourette syndrome, wher eas declarative memory was not different from healthy controls. Patients with OCD performed better at the early course of proc edural learning but were impaired at the later phase, whereas declarative memory was intact (Roth et al., 2004).

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63 Thus, in these populations, restricted, repetitive be havior is associated wi th striatal dysfunctions, which, in turn, are associated with disrup tions in striatally mediated learning. Effects of Stereotypy Levels on Reversal Learning Of most interest in this e xperiment was the association be tween rates of stereotypy and cognitive rigidity. Our hypothesi s came from a series of stud ies done by Mason and colleagues (Garner & Mason, 2002; Garner et al., 2003a; Garner et al ., 2003b; Vickery & Mason, 2005), who reported in several species of animals that motor stereotypy associat ed with a restricted environment was positively correlated with perseverative behavior. The cognitive tasks employed included extinction, re versal learning, and a varia tion of the gambling task. Similarly, correlations between restricted, repe titive behavior and cognitive rigidity were recently demonstrated in autistic individuals (Lopez et al., 2005). Although it has been widely known that these individuals have deficits in executive function (Pennington & Ozonoff, 1996), its association with restricte d, repetitive behaviors had not previously been examined. Consistent with these findings, our results showed that stereotypy score was positively correlated with the number of e rror responses made during the be ginning of reversal training as well as before reaching the criter ion. Since error responses made especially during the first few days is indicative of how quickly, thus flexib ly, they switch the lear ning strategy, the results support our hypothesis that high rate s of stereotypy in deer mice is associated with cognitive rigidity. Perseverative behavior was promin ent in mice displaying backward somersaulting, which more frequently chose the inco rrect arm for their first response. However, mice in the middle-stereotypy group unexpectedly took as many days as mice in the backward somersaulting group to reach the re versal criterion, whereas mice in the highand low-stereotypy group performed equally. There were nine mice in total, wh ich did not reach the criterion, and four of them were in the mi ddle-stereotypy group (two mice from each housing

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64 condition). Relatively better perfor mance in mice displaying high ra tes of jumping was contrary to our hypothesis. Nonetheless, we did find an association between stereotypy and reversal learning. In this experiment, no interactions between housing conditions and st ereotypy levels could be examined and interpreted. Therefore, main effe cts of stereotypy levels on cognitive rigidity were largely skewed by housing conditions, since a large number of EE mice displayed low rates and most SC mice exhibited either high-ster eotypy jumping or backward somersaulting. However, of interest is that mice exhibiting back ward somersaults performed most poorly in both the procedural and the reversal tasks than mice displaying ver tical jumping. Considering that they all came from SC condition, we can speculate that cortico-ba sal ganglia circuitry in these mice was most perturbed. It is worth noting that we informally observe d that some of the backward somersaulters failed to swim straight but instead took either a clockwise or a countercloc kwise circular pattern during the visible platform Morris water maze task, which was used as a pretest. Thus we carefully observed their swim performance to exclude those with motor abnormalities. The remaining mice in this group, which appeared to be unimpaired in their swimming movements, swam significantly slower than mice in th e low-stereotypy group at the beginning of the procedural training, suggesting the possibility of slight motor abnormalities. This difference in swim speed, however, disappeared by the beginn ing of the reversal training. Thus the motor disadvantage in these mice seems relatively insignificant. There is accumulating evidence that the frontal co rtex subregions play an important role in cognitive flexibility, and activation of each area depends on the complexity of the task and types of set-shifting strategies required. Specificall y, the dorsolateral prefront al cortex or medial

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65 prefrontal cortex, in humans and non-human primates or rodents re spectively, appear to subserve higher-order extradimensional atte ntional changes, whereas the or bitofrontal cortex (OFC) is likely to mediate simple intradimensional changes (such as reversal lear ning) (Dias et al., 1996; Dias et al., 1997; Kim & Ragozzino, 2005; Ragozzino, 2002; Wallis et al., 2001). A double dissociation study of neuropsychologi cal task performance revealed that prefrontal dysfunctions of relatively localized areas are asso ciated with some neuropsychiatric disorders. Schizophrenic patients have deficits in Wisconsin Card Sorting test (WCST), which indexes dorsolateral prefrontal cortex impairment s, whereas OCD patients perform poorly in the Object Alternation Test and Gambling Task, which require normal OFC functions (Abbruzzese et al., 1995; Cavallaro et al., 2003). Yet, these disorders may not be exclusively associated with independent prefrontal dysfuncti ons, since both patient group pe rformed poorly in Tower of Hanoi task (Cavallaro et al., 2003). There are also contradictory repor ts on OCD patients who performed poorly in WCST (Head et al., 1989) or were as equally impaired as schizophrenic patients in the Object Alternati on Test (Spitznagel & Suhr, 2002). Neuroimaging studies support a ltered patterns of brain activ ation during executive function tasks in individuals with autism. These incl ude significantly increased activation of brain regions, which are not usually involved, in Go /No-Go task, spatial STROOP test, and SetShifting task (Schmitz et al., 2006). One speculati on of executive dysfuncti on in autistic patients is abnormal neural organization and activation of brain regions, which are not normally recruited for these tasks. Thus our results suggest that OFC-striatal circuitry is likel y impaired in stereotypic deer mice, since the cognitive task used involved simple reversal learning. Yet it is apparent that the OFC is not the only prefrontal region commonly perturbed in individuals with

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66 neurodevelopmental disorders associ ated with repetitive behaviors. For example, in addition to cognitive inflexibility, Lopez and colleagues (2005) also reported positive correlation of restricted, repetitive behaviors wi th deficits in another component of executive function, working memory, in autistic individuals. Other cognitive ta sks requiring different le vel of processes (e.g., extradimensional set-shifting) using varieties of executive function tasks will further elucidate the specific loci of impairments and its association with repetitive behaviors. In addition, as an extension of this study, we should be able to double dissociate impaired striatum-mediated procedural l earning and relatively intact hippocampally mediated spatial learning in deer mice. It is postulated that EE lowand high-stereotypy mice should not differ in the task testing spatial memory. Conversely, SC lowand high-stereot ypy mice should perform poorly on this task. Modeling a Wider Range of Rest ricted, Repetitive Behaviors Our findings in the reversal-learn ing task suggest that deer mice can be used as a model of not just stereotyped motor beha vior but also more complex repetitive behavior. Restricted, repetitive behaviors displayed in autistic individuals have been conceptually divided into two clusters: simple motor behaviors (e.g., body rock ing, hand flipping, and plat e spinning) and more complex higher-order behaviors (e.g., insisten ce on sameness and circumscribed interests) (Turner, 1999). Furthermore, two groups recently re ported that factor analyses of 12 items in the section of repetitive behaviors from Autism Dia gnostic Interview-Revised yielded two factors: repetitive sensorimotor actions and resistance to changes, supporting this categorization empirically (Cuccaro et al., 2003; Szatmari et al., 2005). Animal models of restricted, repetitive behavior generally focus on motor stereotypy, which is easier to measure than more complex behaviors. Cognitive rigidity shown in stereotypic deer mice in this experiment, especially those of backward somersaulting, resemble resistance to

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67 changes in autistic individuals. Although more systematic investigations are necessary, the results presented here will allow us to extend our animal model to include not only motor but also cognitive forms of restrict ed, repetitive behavior. These re sults will significantly enhance the applicability of this model to the wide range of restricted, repetiti ve behavior typical of autism and other neurodevelopmental disorders.

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80 BIOGRAPHICAL SKETCH The candidate was born in Tokyo, Japan to N obuo and Reiko Tanimura. She has one older sister, Chie. She completed high school at Meguro Seibi Gakuen in Tokyo. She moved to California in 1998 and received he r Bachelor of Arts in Psyc hology from California State University, Sacramento in May 2002 with honors. After working for a short period of time in Tokyo, she joined the laboratory of Dr. Mark H. Lewis and began her graduate education in August 2003. Currently she is pursuing her Ph.D. in the Behavioral Neuroscience program at the University of Florida.


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PROCEDURAL LEARNING AND COGNITIVE FLEXIBILITY
IN A MOUSE MODEL OF RESTRICTED, REPETITIVE BEHAVIOR
IN NEURODEVELOPMENTAL DISORDERS






















By

YOKO TANIMURA


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2006

































Copyright 2006

by

Yoko Tanimura



































To my parents, Nobuo and Reiko Tanimura,
and to my sister, Chie Sato, whose help and support made it possible for me
to pursue a career in science.









ACKNOWLEDGMENTS

I would like to express my sincere thanks to my adviser, Dr. Mark H. Lewis, for the

dedicated guidance and continuous help throughout my thesis work. I would also like to thank

my committee members, Dr. Darragh Devine and Dr. Timothy Vollmer, for their contributions. I

acknowledge and thank Ms. Bonnie I. McLaurin, our laboratory manager, for all the help

throughout the course of my work with animals, and Dr. Mark Yang for the help with analyzing

the data collected for the thesis.

Finally I would like to thank my friends in Gainesville, especially the ladies in the

Behavioral Neuroscience program, as well as those in Japan for encouraging me to finish this

work and to continue pursuing a career in science.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IST O F T A B L E S ...................................................................................................... . 7

LIST OF FIGURES ................................. .. ..... ..... ................. .8

A B S T R A C T ................................ .................. .......................... ................ .. 9

CHAPTER

1 INTRODUCTION ............... ................. ........... ......................... .... 11

Restricted, Repetitive Behaviors in Neurodevelopmental Disorders .................................. 11
Animal Models of Restricted, Repetitive Behaviors......... ..............................12
Stereotypy Associated with Central Nervous System Insults ......................................12
Pharmacologically Induced Stereotypy ................ ... ...............................14
Stereotypy Associated with Environmental Restriction......................................14
Deer M house M odel of Repetitive Behaviors ........... ....... ............................ ............. 16
Effects of Environmental Enrichm ent ..................... ...... ........................ ............... 17
Cortico-Basal Ganglia Circuitry and Repetitive Behaviors........................................18
Cortico-Basal G anglia Circuitry and Cognition ........................................ .....................21
Cortico-Basal Ganglia Circuitry and Cognitive Flexibility................ ...............23
Cortico-Basal Ganglia Circuitry and Environmental Enrichment............... ...................25
A im .................................................................................................. 2 7

2 M A TER IA L S A N D M ETH O D S ........................................ .............................................28

Subj ects ................ .... ................................. ............... 28
Housing Conditions ......................... ....... ... .. ... .. .. ............ .... 28
Stereotypy Assessment .............. ...... ............................. 29
A pparatu s ............... .... .. .......................... ..................................30
P retest A ssessm ent............................. ........................................................... ............... 30
Cognitive Assessment.......... ................. .. .. ... ....................3 1
Procedural Learning ....................... ......................... ...... ........... .... 31
R ev ersal L earn in g ......... ...... .................................. .................................. .... ...... .. 32
D ata A n a ly sis .................................................................................................................... 3 4

3 R E SU L T S .............. ... ................................................................35

Stereotypy A ssessm ent ........................................................................................... ............. 35
C o g n itiv e A sse ssm en t ..................................... .. .. ......................................... ............... 3 6
Effects of Housing Conditions: Enriched vs. Standard Caging ........................ ........36
Procedural Learning .................................. .. .. ......... .. .............36
Reversal Learning....................................................... 38









E effects of Stereotypy L evels.......................................................................... ................... 39
Procedural Learning ................................................ .. ........... .... ....... 39
R eversal Learning................................................... 41

4 D IS C U S S IO N ....... ............................................................................ 5 8

Effects of Housing Conditions on Stereotypy ............................................. ............... 59
Effects of Housing Conditions on Procedural Learning............... ....................................59
Effects of Housing Conditions on Reversal Learning ....................................... ...............61
Effects of Stereotypy Levels on Procedural Learning................................. ...... ...............62
Effects of Stereotypy Levels on Reversal Learning .................................... ............... 63
Modeling a Wider Range of Restricted, Repetitive Behaviors .................. ..................66

L IST O F R E FE R E N C E S ......... .. ................... ........................................................................68

B IO G R A PH IC A L SK E T C H .............................................................................. .....................80










LIST OF TABLES


Table


3-1 The frequency of repetitive vertical jumps and the number of deer mice in four
stereotype groups. ...........................................................................36


page









LIST OF FIGURES


Figure page

2-1 Representative swimming paths and responses................................. ............ 33

3-1 Effects of environmental enrichment on stereotypy (vertical jumping and backward
som ersaulting) ............................................................... ..... ..... ......... 44

3-2 Average stereotypy scores in four stereotypy groups.......................................................44

3-3 Latency to reach the platform. A) Effects of housing conditions. B) Effects of
stereotype levels ...................................................... .................. 45

3-4 Distance traveled. A) Effects of housing conditions. B) Effects of stereotypy levels.......46

3-5 Velocity. A) Effects of housing conditions. B) Effects of stereotypy levels.....................47

3-6 The number of first correct arm entries. A) Effects of housing conditions. B) Effects
of stereotype levels. ...................................................... ................. 48

3-7 The number of correct responses. A) Effects of housing conditions. B) Effects of
stereotype levels ...................................................... .................. 49

3-8 The number of error responses. A) Effects of housing conditions. B) Effects of
stereotype levels ...................................................... ................. 50

3-9 The total number of error responses made in the procedural and reversal phases. A)
Effect of housing conditions. B) Effect of stereotypy levels.............. .. ............. 51

3-10 The proportion of error responses made in the procedural and reversal phase. A)
Effects of housing conditions. B) Effects of stereotypy levels ............... ............... 52

3-11 Probability of making a correct response in the first 24 trials of the reversal phase. A)
Effects of housing conditions. B). Effects of stereotypy levels ...................................53

3-12 Probability of making an error response in the first 24 trials of the reversal phase. A)
Effects of housing conditions. B) Effects of stereotypy levels ............... ............... 54

3-13 Regression of the total number of error responses in the reversal phase by individual
stereotype score. ...........................................................................55

3-14 Days required to reach the criterion. A) Effects of housing conditions. B) Effects of
stereotype levels ...................................................... ................. 56

3-15 Effects of housing conditions on trials to the criterion. A) Trials to the procedural
criterion. B) Trials to the reversal criterion. ........................................... ............... 57









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

PROCEDURAL LEARNING AND COGNITIVE FLEXIBILITY
IN A MOUSE MODEL OF RESTRICTED, REPETITIVE BEHAVIOR
IN NEURODEVELOPMENTAL DISORDERS

By

Yoko Tanimura

December 2006

Chair: Mark H. Lewis
Major Department: Psychology

Restricted, repetitive behaviors (e.g., stereotypes, compulsions, rituals) in

neurodevelopmental disorders have been linked to alterations in cortical-basal ganglia circuitry.

Restricted, repetitive behavior has been shown to be associated specifically with deficits in

cognitive flexibility. Cognitive processes mediated by this circuitry (e.g., procedural learning,

executive function) are likely to be impaired in individuals exhibiting high rates of repetitive

behavior.

To test this hypothesis, we assessed both procedural learning and cognitive flexibility

(reversal learning) using a T-maze task in deer mice exhibiting various rates of repetitive

behavior (vertical jumping and backward somersaulting). These mice exhibited high rates of

stereotypy when reared in standard rodent cages, and such behavior was significantly prevented

by housing them in larger more complex environments.

The results showed that mice reared in complex environments exhibited significantly better

procedural and reversal learning than standard caged mice. Thus, early experience associated

with the prevention and attenuation of stereotypy was associated with better striatally mediated

learning and cognitive flexibility. In addition, mice exhibiting high rates of stereotypy (especially









backward somersaulting) performed most poorly in the reversal phase. The results indicate that

the expression of repetitive motor behavior is associated with perseverative behavior in a

learning and memory task. Our finding enhances the applicability of the deer mouse model of

spontaneous stereotypy to the wide range of restricted, repetitive behavior (e.g., rituals,

insistence on sameness) typical of neurodevelopmental disorders.









CHAPTER 1
INTRODUCTION

Restricted, Repetitive Behaviors in Neurodevelopmental Disorders

Restricted, repetitive behaviors encompass a range of abnormal behaviors, which are

common behavioral phenotypes of several neurodevelopmental disorders. In autism, repetitive

behaviors are expressed as motor stereotypes, repetitive manipulation of objects, and echolalia

as well as more complex behaviors such as compulsions, rituals, insistence on sameness, and

narrow and circumscribed interests (Bodfish et al., 2000; Lewis & Bodfish, 1998; Turner, 1999).

This wide range of repetitive behaviors is also typical of individuals with more severe forms of

mental retardation (Bodfish et al., 2000; Lewis & Baumeister, 1982).

Not surprisingly, motor stereotypes, or simple repetitive, often rhythmic motor

movements (e.g., hand flipping, body rocking), appear relatively early in development. More

complex repetitive behaviors (e.g., complex motor sequences and repetitive use of words)

emerge gradually with age (Militerni et al., 2002; Mooney et al., 2006). Complex repetitive

behaviors seem to involve cognitive as well as motor components, which are often accompanied

by some set of rules or a 'just right' criterion for completion (e.g., rituals and object

attachments). This cognitive rigidity may be a component of a broader profile of deficits in

executive function, which has been widely reported in individuals with autism (Pennington &

Ozonoff, 1996). The association between executive function, particularly cognitive flexibility,

and repetitive behaviors is now being investigated (Lopez et al., 2005).

Although a wide range of restricted, repetitive behavior is characteristic of

neurodevelopmental disorders, specific repetitive behaviors are the typical behavioral

phenotypes of certain genetic disorders. Two examples include repetitive self-biting of lips or

digits in Lesch-Nyhan disease (Cauwels & Martens, 2005) and skin picking in Prader-Willi









syndrome (Symons et al., 1999). Other psychiatric and neurological disorders may also be

associated with specific forms of repetitive behaviors. Two obvious examples include childhood

onset obsessive-compulsive disorder (OCD) (repetitive checking or washing) and Tourette

syndrome (vocal tics) (Cath et al., 2001; Muller et al., 1997).

In general, restricted, repetitive behaviors are considered abnormal and clinically

significant because they are stigmatizing, preclude or disrupt goal-directed actions, limit

interaction with the environment, and on occasion, are self-injurious. The pathophysiology of

these behaviors has received limited clinical study, although several neuroimaging and

pharmacological challenge studies have been published (Hollander et al., 2005; Malone et al.,

2005). A more complete understanding of neurobiological perturbations responsible for

repetitive behavior disorders would greatly facilitate the development of treatment options for

patients. Such an understanding will require use of valid animal models.

Animal Models of Restricted, Repetitive Behaviors

To study the underlying neurobiological basis of repetitive behaviors, several categories of

relevant animal models are available. These categories include stereotypy associated with central

nervous system (CNS) insults, pharmacologically induced stereotypy, and stereotypy induced by

rearing animals in restricted environments (Lewis et al., 2006). Although these models should

ideally reflect the wide range of repetitive behaviors displayed in individuals with

neurodevelopmental disorders, they tend to focus largely on motor stereotypy. This is because

stereotypy is observed relatively easily, whereas it is much more challenging to assess more

complex behavior that may be considered models of rituals or insistence on sameness in animals.

Stereotypy Associated with Central Nervous System Insults

Recent advances in genomics, particularly the ability to knock out specific genes, have

provided important information about the etiology of neuropsychiatric disorders. Of particular









interest is that some of these gene-manipulated animals demonstrate repetitive behaviors, which

resemble the repetitive behavior symptoms in clinical populations.

For example, a loss of function mutation in the methyl-CpG binding protein 2 (MeCP2)

gene is known as a major cause of Rett syndrome, and MeCP2 abnormalities are also reported in

individuals with mental retardation and autism (Carney et al., 2003; Meloni et al., 2000).

Compared to control animals, mice expressing truncated MeCP2 protein display more repetitive

forepaw movements, which resemble hand stereotypes (hand wringing and waving) commonly

engaged in by patients with Rett syndrome (Moretti et al., 2005; Shahbazian et al., 2002).

Excessive grooming resulting in hair removal and tissue damage has been reported in

Hoxb8 homozygous knockout mice (Greer & Capecchi, 2002). Expression of the Hoxb8 gene is

found in the orbitofrontal and anterior cingulate cortices, and caudate nucleus, which are the

brain regions comprising 'OCD circuitry' (Graybiel & Rauch, 2000). Excessive grooming in

Hoxb8 knockout mice has particular resemblance to trichotillomania, an OC spectrum disorder.

Similarly, a significantly increased number of brief head or body jerks is found in D1CT-7

transgenic mice expressing a neuropotentiating transgene in cortico-limbic glutamatergic

neurons, which seems to be overactivated in patients with OCD and Tourette's syndrome

(Nordstrom & Burton, 2002). Such behavior is suppressed by clonidine, an alpha-2 adrenergic

agonist that is commonly used to reduce tics in clinical population.

In addition to CNS insults via gene manipulations, prenatal exposure to environmental risk

factors has been shown to induce similar behavioral phenotypes to those seen in patients with

neurodevelopmental disorders. Prenatal exposure to valproic acid (VPA) has been advanced as a

potential etiological factor for autistic symptoms (Folstein & Rosen-Sheidley, 2001; Keller &

Persico, 2003). Exposure of this antiepileptic drug to rats on day 12.5 of gestation produced









neuropathological abnormalities similar to those reported in autistic individuals (e.g., altered

sensitivities to tactile stimuli, diminished prepulse inhibition, decreased social behaviors, and

hyperactivity including stereotypy-like behaviors) (Schneider & Przewlocki, 2005).

Pharmacologically Induced Stereotypy

Much of what we know about the neurobiological basis of motor stereotypy comes from

investigations using pharmacological models (Lewis & Baumeister, 1982; Lewis & Bodfish,

1998). These models have a direct human analogue, as clinically abnormal repetitive behaviors

were described in human amphetamine abusers as early as the late 1960s (Ellinwood, 1967;

Kramer et al., 1967). Such phenomena were also demonstrated in several species of animals

treated with amphetamine (Randrup & Munkvad, 1965). Subsequently, a wide range of other

pharmacological agents (e.g., phenmetrazine, L-dopa, morphine, methylphenidate, pemoline, and

monoamine oxidase inhibitors) that affect the nigro-striatal dopaminergic system was shown to

induce stereotypy in rats (Fog, 1972).

Systematic investigation of anatomical and neurochemical mechanisms underlying drug-

induced repetitive behavior implicated cortico-basal ganglia circuitry in the execution of these

movements. Intrastriatal and systemic administration of direct and indirect dopamine agonists

(e.g., amphetamine, apomorphine, and cocaine) as well as opiate agonists and NMDA receptor

antagonists (e.g., MK-801 and PCP) have consistently induced motor stereotypy (Ernst &

Smelik, 1966; Iwamoto & Way, 1977; Lewis et al., 1990; Segal et al., 1995; Vandebroek et al.,

1998; Vandebroek & Odberg, 1997). Cortico-basal ganglia circuitry will be discussed more in

detail in a later section.

Stereotypy Associated with Environmental Restriction

Repetitive motor behaviors not requiring pharmacological induction are frequently

reported in animals housed in zoo, farm, and laboratory environments (Mason, 1991). Examples









of these species-specific stereotypic behaviors include pacing, route-tracing, and feather-picking

in parrots and tits (Garner et al., 2003a; Garner et al., 2003b; Jenkins, 2001; Meehan et al.,

2004), bar-mouthing, vertical jumping, and backward somersaulting in rodents (Garner &

Mason, 2002; Powell et al., 1999; Vandebroek & Odberg, 1997), pacing, body-rocking, tail-

biting, and self-injurious behavior in rhesus monkeys (Lutz et al., 2003; Taylor et al., 2005),

pacing, somersaulting, and over-grooming in prosimians (Tarou et al., 2005), crib-biting,

boxwalking, and head-shaking in horses (Bachmann et al., 2003; McGreevy et al., 1995),

regurgitation and tongue flicking in pandas (Swaisgood et al., 2005), and head-twirling in minks

(Mason, 1993).

Since animals in the wild do not engage in such abnormal behaviors, stereotypy seems to

develop as a consequence of animals' responses to restricted environments, which limit

expression of species-typical behaviors. Conversely, alleviation of environmental deprivation

seems an effective means to reduce rates of spontaneous stereotypy (Garner et al., 2003a;

Meehan et al., 2004; Powell et al., 2000; Swaisgood et al., 2005; Turner et al., 2002).

Importantly, motor stereotypy associated with restricted environments has been

demonstrated to be qualitatively different from pharmacologically induced stereotypy in the

same species. For example, deer mice develop focused vertical jumping and backward

somersaulting as a consequence of restricted housing, whereas they exhibited excessive gnawing,

rearing, and locomotion following administration of apomorphine (Presti et al., 2004; Presti et

al., 2002). Similarly, although repetitive jumping is commonly displayed in captive bank voles,

NMDA receptor antagonist MK-801 and apomorphine elevated repetitive licking, sniffing, and

locomotion instead (Vandebroek et al., 1998; Vandebroek & Odberg, 1997). These findings









suggest the limitations of drug-induced stereotypy to model repetitive behavioral phenotypes of

neurodevelopmental disorders.

Motor abnormalities associated with environmental restriction are strikingly similar to the

phenomena (spontaneous, repetitive, topologically unvarying, functionless, and apparently

purposeless) in neurodevelopmental disorders. These individuals are likely to experience

environmental restriction early in life as a function of their handicap. Thus, it is possible that

similar neurodevelopmental perturbations spontaneously occur in stereotypic animals reared in

restricted environments and individuals with neurodevelopmental disorders. Studying the

neurobiological mechanisms of stereotypy using a model associated with environmental

restriction will advance our knowledge of the development of restricted, repetitive behavior

associated with neurodevelopmental disorders. To this end, we have adopted a mouse model of

stereotypy induced by environmental restriction. This model utilizes a species that can be studied

in the laboratory and whose stereotypy is spontaneous, persistent, and occurs early in

development.

Deer Mouse Model of Repetitive Behaviors

In our laboratory, we employ deer mice (Peromyscus maniculatus) as a model of repetitive

behavior disorders. These animals exhibit high rates of repetitive vertical jumping or backward

somersaulting when housed under standard laboratory conditions (Powell et al., 1999). These

behaviors emerge as early as weaning and persist for a prolonged period of time. Exposure to a

larger, more complex environment (environmental enrichment or EE) following weaning

significantly attenuates the development of these behaviors (Powell et al., 2000). After two

months of EE following weaning, approximately 80 percent of mice show low rates of

stereotyped behaviors, whereas close to 80 percent of mice reared in standard cages (SC) show









high rates of stereotyped behavior. Enrichment later in life is also beneficial, yet to a lesser

degree (Hadley et al., 2006; Powell et al., 2000).

Effects of Environmental Enrichment

The significance of environmental stimulation on behavior was first recognized through

the pioneering work of Hebb (1949). He reported that rats he brought home from his laboratory

to keep as pets were better at solving cognitive tasks than rats raised in the laboratory. Since this

early observation, EE has been used to demonstrate behavioral and brain plasticity in a large

number of studies using a variety of behavioral and biological measures or endpoints (Lewis,

2004; van Praag et al., 2000). It should be noted, however, that EE is a relative term and that

such experimental configurations cannot approximate the complexity and variability of the

animal's natural habitat.

EE may also have beneficial effects in preventing or attenuating the effects of CNS insult.

A higher educational background, cognitively challenging occupations, and high socioeconomic

status were found to lower the risk of Alzheimer's disease and cognitive impairment associated

with age (Moceri et al., 2001; Snowdon et al., 1996; Stern et al., 1994; White et al., 1994). In a

transgenic mouse model of Alzheimer's disease, EE prevented overproduction of amyloid-beta

protein and learning impairments associated with amyloid deposits (Jankowsky et al., 2005).

Furthermore, the neuroprotective role of EE was found in a 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP) mouse model of Parkinson's disease (Faherty et al., 2005), where EE

significantly attenuated the death of dopaminergic neurons in the substantial nigra pars compact

(SNpc). As another example, transgenic mice expressing human huntingtin transgene, a mouse

model of Huntington's disease, showed amelioration of motor abnormalities following EE. In

addition, EE rescued molecular alterations caused by transcriptional dysregulation in these mice,

such as reduction in brain derived neurotrophic factor (BDNF) in the hippocampus and striatum,









and dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP-32) deficits in the anterior

cortex (Spires et al., 2004).

Of particular interest are behavioral modifications in animal models of

neurodevelopmental disorders associated with repetitive behavior. A recent study showed that

mice with fragile X mental retardation 1 gene mutation, an animal model of Fragile X syndrome,

displayed hyperactivity, cognitive alterations, and immature dendritic and spine morphology

when housed in standard rodent cages, whereas enriched experiences normalized mutation-

associated cognitive and morphological impairments (Restivo et al., 2005). Similarly, VPA-

treated rats, modeling autism, exhibited stereotypy-like behaviors under standard laboratory

conditions, whereas VPA rats under EE conditions alleviated repetitive behaviors as well as

other parameters characteristic of autism (Schneider et al., 2006).

As suggested previously, our lab has demonstrated that EE markedly attenuates the

development of spontaneous stereotypy in deer mice. We have also examined the

neurobiological correlates of this experientially based attenuation. Specifically we found

elevated metabolic activity and dendritic spine density in the motor cortex and dorsolateral

striatum in EE mice exhibiting diminished levels of stereotypy (Turner et al., 2003; Turner et al.,

2002). Moreover, BDNF in the striatum was elevated in these enriched low-stereotypy mice

(Turner & Lewis, 2003). Thus, it is reasonable to hypothesize that altered activity within this

circuitry is responsible for the expression of repetitive behavior in deer mice.

Cortico-Basal Ganglia Circuitry and Repetitive Behaviors

The basal ganglia are a group of subcortical nuclei that regulate execution of motor and

cognitive programs. They receive inputs from all areas of the neocortex except the primary

visual and primary auditory cortices. The motor circuit that is hypothesized to be responsible for

the expression of stereotypy originates from the primary motor cortex and premotor area.









Glutamatergic neurons from these cortical regions enter the input nucleus of the basal ganglia

(dorsolateral striatum) and the output neurons from the basal ganglia regulate activation of

thalamocortical neurons. These neurons terminate at the somatosensory cortex, primary motor

cortex, and supplementary motor area, providing positive feedback to ongoing motor programs

in the primary motor cortex (Herrero et al., 2002; Parent & Hazrati, 1995).

There are two pathways that travel through the basal ganglia system: the direct pathway

and indirect pathway. The direct pathway consists of GABAergic medium spiny neurons directly

projecting from the dorsolateral striatum to the output nucleus of the basal ganglia, the globus

pallidus internal (GPi) and substantial nigra pars reticulata (SNpr) (striatonigral neurons). These

neurons selectively express D1 dopamine receptors that are co-localized with glutamate

receptors. They contain the neuropeptides dynorphin and substance P (Steiner & Gerfen, 1998).

The indirect pathway consists of GABAergic striatal medium spiny neurons (striatopallidal

neurons) projecting to the globus pallidus external (GPe), GABAergic neurons in the GPe

projecting to the subthalamic nucleus (STN), and finally glutamatergic neurons sending

excitatory projection to the GPi and SNpr. The striatopallidal medium spiny neurons express D2

receptors, and the neuropeptide enkephalin (Steiner & Gerfen, 1998).

Normal dopaminergic innervation in the striatum plays a crucial role in execution of

movements. D1 receptors in the striatonigral neurons are positively coupled to adenylyl cyclase

(Missale et al., 1998). Therefore, dopamine acts to amplify the glutamatergic corticostriatal

inputs, resulting in increased GABAergic inhibition of the GPi and SNpr. In contrast, D2

receptors in the striatopallidal neurons are negatively coupled to adenylyl cyclase (Missale et al.,

1998). Via D2 receptors, dopamine acts to diminish the corticostriatal inputs, resulting in

decreased inhibition of the GPe, hence causing this nucleus to exert even more inhibitory control









over the STN. This increased inhibition of the STN removes its excitatory influence on the GPi

and SNpr. Thus activation of D1 and D2 receptors both removes inhibitory tone of the basal

ganglia output neurons, ultimately resulting in disinhibition of the thalamocortical neurons to

provide positive feedback to motor programs.

Abnormal execution of movements is evident when the balance between the direct and

indirect pathway is disrupted. It is postulated that stereotypic behavior is expressed as a

consequence of a relative increase in striatonigral tone (Graybiel et al., 2000). This hypothesis is

supported by the finding that transgenic mice inducibly overexpressing the transcription factor

AFosB selectively in dynorphin-containing striatonigral neurons exhibit increased daily wheel

running, whereas such behavior was significantly reduced in mice overexpressing AFosB in

enkephalin-containing striatopallidal neurons (Werme et al., 2002).

To investigate alterations in cortico-basal ganglia circuitry in deer mice exhibiting

spontaneous stereotypy, we administered the selective D1 antagonist SCH23390 and the NMDA

antagonist MK-801 intrastriatally. These compounds both blocked spontaneous stereotypy

selectively (Presti et al., 2003). In contrast, the D2 receptor antagonist raclopride, unexpectedly

failed to reduce stereotypy (Presti et al., 2004). Finally, the mixed agonist apomorphine failed to

increase spontaneous stereotypes in deer mice when administered intrastriatally (Presti et al.,

2002).

In addition, we assessed neuronal activities of the direct and indirect pathways by

measuring neuropeptides dynorphin and enkephalin respectively. We found significantly

decreased leu-enkephalin content and significantly increased [dynorphin]/[enkephalin] content

ratios in high-stereotypy mice relative to low-stereotypy mice. Moreover, we saw a significant

negative correlation between striatal enkephalin content and frequency of stereotypy as well as a









significant positive correlation between the [dynorphin]/[enkephalin] content ratio and frequency

of stereotypy in these mice (Presti & Lewis, 2005).

Another line of evidence for potential neurobiological perturbations responsible for

repetitive behaviors comes from neuroimaging studies in clinical populations exhibiting

behaviors of interest. Increased right caudate volume was noted in individuals with autism

spectrum disorders, which was positively correlated with rates of repetitive behaviors, especially

scores of higher-order components (Hollander et al., 2005), such as compulsions, rituals, and

difficulties with minor change, and complex motor mannerisms (Sears et al., 1999). Patients with

trichotillomania displaying repetitive hair-pulling had significantly reduced volumes of the left

putamen (O'Sullivan et al., 1997). Moreover, tics in Tourette syndrome were inversely correlated

with changes of blood flow and oxygen concentration in the basal ganglia and thalamus

(Peterson et al., 1998).

These studies provide evidence for alterations of cortico-basal ganglia circuitry in animals

and individuals associated with repetitive behaviors. To further support alterations in this

circuitry in stereotypic animals, we wished to assess other non-stereotyped behaviors, which

require the intact basal ganglia system for normal function. We focused on cognitive behaviors

including striatally mediated procedural learning and cognitive flexibility, which require intact

fronto-basal ganglia circuitry.

Cortico-Basal Ganglia Circuitry and Cognition

The role of the striatum in mnemonic function, which is distinct from the hippocampally

dependent system, is increasingly appreciated. A number of studies demonstrated double

dissociation of two memory systems; the hippocampus mediates spatial, allocentric memory,

which relies on stimulus-stimulus relationships or Tolmanian cognitive mapping strategy,

whereas the striatum plays an important role in motor, egocentric memory, which in particular









emphasizes acquisition of Hullian stimulus-response (S-R) relationships (Packard & Knowlton,

2002; White & McDonald, 2002; Yin & Knowlton, 2006).

This dissociation of relatively independent memory systems is supported by the findings

that localized lesions of fimbria-fornix, a major input-output pathway of the hippocampus,

impaired spatial memory, whereas caudate lesions were associated with poor performance in

discrimination learning tasks, using spatial and cue versions of Morris water maze tasks

(McDonald & White, 1994; Packard & McGaugh, 1992) or win-shift and win-stay radial-arm

maze tasks (Kesner et al., 1993; Packard & White, 1991) respectively.

Different biochemical processing associated with these systems also provides evidence for

the mnemonic dissociation. For example, calcium-sensitive adenylyl cyclase activity was

increased to the greatest degree in the hippocampus after spatial learning, whereas calcium-

insensitive adenylyl cyclase was enhanced in several areas of the brain, especially the striatum,

following procedural learning (Guillou et al., 1999).

Furthermore, immediately after maze training that assessed either place or response

learning, both phosphorylated CREB (pCREB) and c-fos expression were increased in either

group of animals, which experienced place or response learning. After one hour, sustained

pCREB and c-fos immunoreactivity was observed in the hippocampus (the dentate gyms, CA1,

and CA3) following place learning, whereas pCREB activity was sustained in the striatum

(dorsolateral and dorsomedial) following response learning (Colombo et al., 2003). CREB is

widely known to contribute to memory consolidation and dynamic modulation of synaptic

strength. Thus, task acquisition seems to require initial activation of multiple brain regions, but

the region responsible for acquisition of the task later undergoes greater activation to consolidate

the learned strategy. Additionally, CREB mutation impaired performance in several tasks









associated with the dorsal striatum and altered corticostriatal long-term potentiation (LTP) and

depression (LTD) (Pittenger et al., 2006), supporting the involvement of this subcortical area in

learning, which is distinct from the hippocampally mediated learning.

This change in neural activation with learning is also supported electrophysiologically.

Graybiel and colleagues (Barnes et al., 2005; Jog et al., 1999) demonstrated that many of

dorsolateral striatal neurons were activated shortly after a discriminative auditory stimulus,

which indexed the location of a reward, in the simple discrimination T-maze task at the

beginning of training. However, as rats learned the S-R contingency, the neuronal firings at the

beginning (trial start) and end (reward) of the maze were enhanced and the firings at the tone and

turn were substantially attenuated. This shift in neuronal firings associated with procedural

learning was largely reversed during extinction learning.

In humans, functional magnetic resonance imaging (fMRI) studies provided evidence for

the involvement of the striatum in probabilistic classification task, which requires non-motor

procedural memory (Poldrack et al., 1999). Patients with the basal ganglia pathology, such as

Huntington's (Knopman & Nissen, 1991; Sprengelmeyer et al., 1995) and Parkinson's diseases

(Allain et al., 1995; Krebs et al., 2001), present a variety of cognitive impairments attributable to

the striatal memory system. Although some evidence exists (Mostofsky et al., 2000), not much is

known regarding S-R learning deficits in individuals with neurodevelopmental disorders who

exhibit restricted, repetitive behaviors.

Cortico-Basal Ganglia Circuitry and Cognitive Flexibility

Other domains of cognitive function, which require intact cortico-basal ganglia circuitry,

include executive function. Executive function is an umbrella term for cognitive processes such

as attention, planning, working memory, and cognitive flexibility, which require higher-order

mental acts. The disturbances of these processes are reported in many psychiatric disorders such









as schizophrenia and autism (Pennington & Ozonoff, 1996). According to recent human and

animal studies, these processes are largely mediated by the frontal cortex, which has strong

connections with the striatum in series of fronto-striatal loops (Dalley et al., 2004; Heyder et al.,

2004).

Of most interest to us among these subcomponents of executive function is cognitive

flexibility. Damage in subregions of the frontal cortex (dorsolateral prefrontal cortex and

orbitofrontal cortex) causes a difficulty in shifting a response or switching strategies when S-R

contingency changes. Thus, patients with frontal damage often repetitively respond to the

previously relevant, however no longer associated, stimuli. This perseverative behavior pattern

resembles simple motor stereotyped behaviors where both are inappropriately repeated and rigid

(Lewis & Bodfish, 1998). Hence, perturbations within the striatum could dysregulate not only

cortical-striatal loop inducing repetitive motor behavior, but also fronto-striatal loops impairing

cognitive flexibility.

To support this hypothesis, recent investigations presented evidence for the link between

stereotypy associated with restricted environment and cognitive rigidity. For example, blue and

marsh tits exhibiting repetitive route-tracing stereotypy showed a perseverative response pattern

in a gambling task (Garner et al., 2003a; Garner et al., 2003b), which has been used for cognitive

assessment in schizophrenia (Frith & Done, 1983) and OCD (Cavallaro et al., 2003). In this task,

subjects are instructed to search for the rule governing the reward presentation contingent on a

particular stimulus, when in fact there is no rule. Therefore their sequences of responses reveal

repetition of a specific response or repetitive pattern of responses.

A similar correlation was also found between cage stereotypes and reversal (where the

reinforcer was switched and associated to the opposing stimulus) or extinction (where the









reinforcer no longer existed) of S-R learning in bank voles (Garner & Mason, 2002; Garner et

al., 2003a) and bears (Vickery & Mason, 2005). Moreover, this relationship was recently

demonstrated by Lopez and colleagues (2005) in clinical populations. Scores on the executive

function task that indexed cognitive rigidity were positively correlated with severity of restricted,

repetitive behavior in autistic adults. This relationship between motor and perseverative

responses in the California Trail Making Test is of interest to many researchers, because

executive function deficits have been recognized in autistic individuals relatively independently

of repetitive motor behavior. Therefore, we wished to demonstrate such an association between

stereotypy and cognitive flexibility in our deer mice model.

Cortico-Basal Ganglia Circuitry and Environmental Enrichment

Evidence consistently supports that exposure to EE improves performance in various

cognitive tasks, which are at least partially dependent on the hippocampus (e.g., go/no go task,

object recognition task, contextual fear conditioning, spatial version of Morris water maze, and

open field test) (Duffy et al., 2001; Lambert et al., 2005; Leggio et al., 2005; Park et al., 1992;

Pham et al., 1999; Woodcock & Richardson, 2000).

EE has also been shown to attenuate impaired learning systems associated with

hippocampal (Will et al., 1983) and fimbria-fornix lesions (van Rijzingen et al., 1997),

immunolesioned basal forebrain cholinergic system (Paban et al., 2005), mutation in CA1-

specific NMDA receptor 1 subunit (Rampon et al., 2000), traumatic brain injury (Dahlqvist et

al., 2004; Gobbo & O'Mara, 2004; Rutten et al., 2002; Wagner et al., 2002), high fat diet

(Winocur & Greenwood, 1999), and age (Frick & Fernandez, 2003; Frick et al., 2003).

Biochemical indices of neural plasticity associated with learning improvement include increased

levels of neurotrophic factors (Pham et al., 1999), alteration of the cAMP-dependent protein









kinase dependence of LTP (Duffy et al., 2001), and increased phosphorylation of CREB

(Williams et al., 2001; Young et al., 1999) in the hippocampus.

Moreover, adult neurogenesis in the hippocampus is enhanced following EE. Bruel-

Jungerman and colleagues (2005) demonstrated that these new neurons actually contribute to

hippocampally mediated memory improvement associated with EE. Mice were housed either

under EE or standard laboratory conditions, and half of EE mice were treated with saline and the

other half were treated with antimitotic methylazoxymethanol acetate (MAM), which prevented

further cell division. There was significant elevation in 5-bromo-2'-deoxyuridine (BrdU)-

positive new born cells in EE naive mice compared to EE MAM mice and mice from standard

cages, implicating EE-dependent increase in neurogenesis in the dentate gyrus. Moreover, EE

naive mice showed statistically better long-term recognition memory than EE MAM-treated

mice, providing the first direct evidence for adult neurogenesis in the hippocampus following EE

and their functional participation in memory enhancement.

Thus, the role of EE on hippocampally mediated learning and memory has been

extensively studied behaviorally and neurobiologically. Fewer attempts to understand the

efficacy of EE on striatum-mediated learning have been made (Frick & Fernandez, 2003; Frick

et al., 2003; Schrijver & Wurbel, 2001). Frick and Fernandez (2003) demonstrated that age-

related memory impairment was rescued following EE in the spatial version of Morris water

maze, but not in the cued version of Morris water maze, which assesses procedural learning. An

increase in synaptophysin was evident in the hippocampus and frontoparietal cortex in EE mice,

but no such assessment was done for the striatum. Similarly, other studies failed to show

procedural learning enhancement following exposure to EE. Therefore, we wish to evaluate

carefully EE effect on striatum-mediated procedural learning in deer mice.









Conversely, studies support the effect of EE on cognitive flexibility. Isolation housing

impaired reversal learning (Jones et al., 1991; Schrijver et al., 2004) and extradimensional set-

shifting (Schrijver & Wurbel, 2001), isolated animals showing more perseverative responses

than their socially reared counterparts. Yet, effects of other EE components (e.g., cognitive

stimulation and exercise) and the neurobiological alterations associated with cognitive rigidity

have not been studied. Thus we wished to assess the effect of EE on cognitive flexibility in deer

mice.

Aim

Restricted, repetitive behaviors are clinically significant for several neurodevelopmental

disorders. Studies using pharmacologically induced animal models of stereotypy as well as our

deer mice model associated with restricted environment suggest that the relatively increased tone

of the direct pathway compared to the indirect pathway is responsible for the expression of

repetitive behaviors. To further confirm the alteration in the basal ganglia system in animals

displaying high rates of stereotypy, we hypothesized that other non-stereotypy behaviors

mediated by this brain region, such as procedural learning and cognitive flexibility, are also

impaired in these animals. To investigate these hypotheses, the present experiment examined the

relationship between spontaneously emitted stereotypy and these cognitive processes as well as

effects of EE on them using the water-filled T-maze.









CHAPTER 2
MATERIALS AND METHODS

Subjects

As we have previously demonstrated, both male and female Peromyscus maniculatus (deer

mice) develop high rates of persistent, spontaneously emitted stereotypy consisting of repetitive

vertical jumping and backward somersaulting when housed under standard laboratory conditions.

In most mice, these behaviors can be markedly attenuated by environmental enrichment. Fifty-

nine mice were originally designated for this experiment, but during the period from weaning to

the completion of the reversal phase, four mice died for unknown reasons and three mice were

excluded. Data from 52 mice (23 male and 29 female; 3-7 months old) were used for analysis.

Subjects were obtained from the breeding colony maintained in our laboratory, and at weaning

were randomly assigned to either standard cage (SC) or enriched environment (EE) housing

conditions.

Housing Conditions

Rodent chow and water were available ad libitum, and Cockatiel vita seed was provided

three times each week. The housing room was maintained at 20-250C and 50-70 % humidity.

Subjects were maintained on a 16:8-h light/dark cycle, with lights off at 10:00pm. All procedures

were performed in accordance with the guidelines set forth in the NIH Guide for the Care and

Use of Laboratory Animals and were approved by the University of Florida Institutional Animal

Care and Use Committee.

Mice assigned to EE were group-caged (6 same sex mice/cage) in large dog kennels (122 x

81 x 89 cm) consisting of two extra levels constructed of galvanized wire mesh and connected by

ramps of the same material. Bedding, a running wheel, shelters (similar opaque, concave object),

and various other objects (habitrail tubes, plastic toys, and mesh structures for climbing) were









placed in each kennel prior to introducing the mice. One oz. of Cockatiel vita seed was scattered

throughout the kennel three times each week to encourage foraging behavior. A running wheel

remained undisturbed in the kennel, but other objects (except those in which mice were hiding)

were removed and replaced with clean novel objects on a weekly basis.

For the SC condition, mice were caged (2-3 same sex mice/cage) in standard laboratory

rodent cages (48 x 27 x 15 cm). A half oz. of Cockatiel vita seed was placed at one corer of the

cage three times each week on the same schedule as the enriched cages in order to

counterbalance any nutrition effects with the EE mice.

Stereotypy Assessment

After being housed in their respective caging conditions for 60-180 days, the mice were

tested for rates of stereotyped behavior. Prior to cognitive assessment, mice from both SC and

EE conditions were tested for rates of stereotypy using a modified automated photocell detection

apparatus obtained from Columbus Instruments (Columbus, CO). This equipment uses

photocells located 13.5 cm above the floor, to quantify the number of interruptions made by

repetitive jumps or backward somersaults of a mouse during a given time period.

The session consisted of the eight hours of the dark cycle for two consecutive days. Any

single testing day involved three mice from the EE housing and two or three mice from the SC

housing sharing a common weaning date. The testing protocol involved removing mice from

their home cages and placing them singly in testing cages (22 x 15 x 28 cm) made of Plexiglas

prior to the beginning of the dark cycle. Food and water were provided, and the mice were left

undisturbed for two to three hours for habituation to the new cage and recovery from the stress of

handling.









All sessions were digitally video recorded for further identification of behavior phenotypes

and accuracy of the automated counters. Each animal received a stereotypy score that

represented the average stereotypy frequency per hour.

Apparatus

The testing apparatus consisted of a T-maze constructed from clear Plexiglas. Each arm

measured 7.5 x 32 x 18 cm. The platform (7.5 x 7.5 x 5 cm) made of the same Plexiglas material

was placed at the end of one arm. The maze was placed in a black plastic tank (85 cm in

diameter, 36 cm in height) and a white nylon curtain (85 cm in diameter, 60 cm in height)

surrounded the T-maze.

Prior to cognitive testing, the T-maze was filled with warm tap water (25 C) to a depth of

5.8 cm so that the clear platform was submerged 8 mm from the surface of the water. In order to

keep the temperature constant, the water was measured every eight trials and hot tap water was

added as needed. Non-toxic white paint was added to the water to ensure opacity.

A digital video camera was mounted 120 cm above the T-maze to make recordings of

swim trials. The camera was connected to a computer in the same room and used to measure

mice's escape latencies and track swimming paths by Ethovision" video tracking system (Noldus

Information Technology, the Netherlands).

Pretest Assessment

Mice were tested in the visible-platform version of Morris water maze task to detect any

motor or sensory abnormalities. The black tank was filled with warm tap water (25-280C) and the

white plastic platform (10 x 10 cm) was placed in the tank, 5 mm above the surface of water.

Mice were released from the edge of the tank and allowed to swim to the platform for up to 60

seconds. This was repeated four times and the location of the platform and the releasing point









were varied in every trial with an intertrial interval of approximately 15 minutes. During the

intertrial interval, the mice were returned to their cage in an adjoining room, towel dried and

placed underneath a heating lamp. Mice's swimming patterns were analyzed. Those mice

exhibiting apparent motor and sensory impairments were excluded from further cognitive

assessment (n=3).

On the following day, mice were exposed to the T-maze, which was placed in the tank and

filled with warm opaque water without the platform. Mice were released from the start arm

facing to the wall and allowed to explore the T-maze for 30 seconds. This was repeated six times

with intertrial interval of approximately 15 minutes. The swim paths were digitally recorded to

determine the existence of turning bias in each animal. Mice were determined to have a turning

bias, when they made more than five first entries to the same arm.

Cognitive Assessment

Procedural Learning

On the day following T-maze habituation, mice were tested for procedural memory in the

water-filled T-maze with the invisible platform. Each training session consisted of eight trials.

The platform was placed at the end of the east or west arm. The location of the platform was

pseudorandomly determined among mice except for those animals exhibiting a turning bias. For

those cases (n=6), the platform was positioned in the non-preferred arms. Four extra-maze cues

(different color, geometric shapes, approximately 15 x 15 cm in diameter) were attached to the

surrounding nylon curtain and their locations were randomly changed every trial.

A trial began by placing a mouse at the end of the start arm (facing the wall) and ended

when the mouse climbed onto the platform, or after 60 seconds had elapsed. If the mouse did not

escape after 60 seconds, it was gently guided to the platform, placed on the platform, and

removed from the maze. For each mouse there was a delay of approximately 15 minutes between









successive trials within a session. During the delay, the remaining mice were run on the same

trial. Thus, the intertrial interval varied slightly according to the mice's level of performance but

was approximately equal for all mice within each trial.

Completion of a trial was defined as spending two seconds on the platform. If a mouse

reached the platform but jumped out to the water again, the trial was continued until the mouse

reached the platform again and spent two seconds there. Each mouse was left on the platform for

15 seconds and then returned to the home cage, located in an adjoining room.

A correct response was defined as reaching the platform without entry into the opposite

arm (Figure 2-1). An incorrect response was scored when the mouse entered the arm not

containing the platform. When an animal swam all the way to the end (7.5 x 7.5 cm) of the arm

not containing the platform, the trial was recorded as an error response. The criterion for the

procedural phase was set as seven or eight correct responses per session for three consecutive

sessions.

Reversal Learning

A reversal-learning task was initiated the day after the animal met criterion for successful

completion of the procedural task. For this task, the same T-maze was employed except that the

platform was placed at the end of the opposite arm (East -* West, West -* East) so that mice

needed to inhibit the previously relevant response and learn the new stimulus-response

association. The criterion for this task was also set as seven or more correct responses for three

consecutive days.




















WetEs


Platform
Correct Arm


IStart Arm


East


Platform
Correct Arm







Start Arm


East


Platform
Correct Arm


Start Arm


Figure 2-1. Representative swimming paths and responses: A response in a single trial was

defined as either a correct, incorrect, or error response. The shaded area at the end of

east arm represents a submerged platform. The corresponding area of the opposite

arm represents the error zone. A) A correct response: completion of a trial without

entry to the incorrect arm. B) An incorrect response: completion of a trial with entry

to the incorrect arm. C) An error response: completion of a trial with entry to the error

zone at the end of the incorrect arm.


Error
Incorrect Arm


West


Error
Incorrect Arm


West


Error
Incorrect Arm


I


West


East









Data Analysis

A stereotypy score was calculated for each animal and a Student's t test was performed to

determine the effects of differential housing on stereotypy expression (Sigmastat, Systat

Software, CA). Based on the stereotypy score, groups of mice were constituted according to

different rates of stereotypy expression.

An analysis of variance (ANOVA) for repeated measures was performed to determine the

effect of housing condition and stereotypy levels on indices of cognitive performance including

latency, distance traveled, velocity, and the probability of making a correct or error response.

Further post hoc pairwise comparison tests (Tukey) were conducted to assess differences

between stereotypy groups where appropriate. Moreover, a Student's t test for housing condition

and a one-way ANOVA for stereotypy groups were performed for the days required to reach the

criterion. In addition, individual stereotypy scores were regressed against the number of correct

or error responses made. A p-value of .05 or less was adopted for assigning statistical

significance.









CHAPTER 3
RESULTS

Stereotypy Assessment

Stereotypy scores as measured by the automated photocell detection apparatus ranged from

38 to 2394 responses per hour for all animals. There was a significant housing effect on

stereotypy score (t(50)=4.1, p<0.001) with EE mice (n=25) exhibiting significantly lower rates of

stereotypy than SC mice (n=27) (Figure 3-1). When a small number of mice exhibiting backward

somersaulting, all of which were in the SC condition, were excluded from the analysis, a

significant difference in housing conditions was still found (t(41)=4.3, p=0.001).

To group mice according to rates of stereotypy, we first separated mice displaying

repetitive backward somersaulting (B) for the following reasons. First, this qualitatively distinct

behavior takes more time to complete a single response than does a vertical jump. Thus, the

frequency of backward somersaulting is typically less than that observed for vertical jumping.

Second, all of the mice exhibiting backward somersaulting (n=9) were in the SC condition.

Finally as shown in the Cognitive Assessment section below, this group of mice showed distinct

cognitive abnormalities.

The remaining mice showing vertical jumping were categorized according to whether their

frequency of jumping fell into the upper, middle, or lower third of the frequency range: high (H,

more than 761 counts per hour, n=15), middle (M, between 334 to 760, n=14), and low rate of

jumping (L, less than 334, n=14). The number of mice in each housing condition so categorized

is depicted in Table 3-1. The average stereotypy score per hour for each group is depicted in

Figure 3-2. We were not able to assess the interaction between stereotypy levels and housing

conditions due to the small number of mice in the enriched housing high stereotypy group and

the standard cage low and middle stereotypy group. According to photocell interruption









frequencies obtained for mice exhibiting backward somersaulting, five mice would have been

categorized as high stereotypy and two would have been categorized as middle and low each.

Table 3-1. The frequency of repetitive vertical jumps and the number of deer mice in four
stereotypy groups.
Stereotypy Group Stereotypy Score # of SC Mice # of EE Mice
High rates of jumping 761 < 12 3
Middle rates of jumping 334 760 4 10
Low rates of jumping < 333 2 12
Backward somersaulting (255-1268) 9 0



Cognitive Assessment

Since some mice reached criterion on the third day of training for both the procedural

learning and reversal learning experiments, we compared the average trial latencies, distance

traveled, and swim speed as well as the number of first correct arm entries, correct responses,

and error responses within a session for the first three days of the procedural (P1, P2, P3) and

reversal (R1, R2, R3) experiments. Thus, in order to examine the effects of housing condition,

we conducted 2 (housing condition) by 3 (days) ANOVAs with repeated measures on the second

factor. To assess the effects of level of stereotypy on cognitive function, we conducted 4

(stereotypy group) by 3 (days) ANOVAs with repeated measures on the second factor. For all

dependent variables assessed, there were no differences in sex or age, although there was a

tendency for females and for younger mice (3 months of age) to perform better than males and

older mice (7 months of age).

Effects of Housing Conditions: Enriched vs. Standard Caging

Procedural Learning

Significant main effects of housing condition were found on latency to reach the platform

(F(1, 100)=11.3, p=0.001) (Figure 3-3A), distance traveled (F(1, 100)=12.0, p=0.001) (Figure 3-

4A), velocity (F(1, 100)=20.5, p<0.001) (Figure 3-5A), the number of first correct entries (F(1,









100)=5.7, p=0.020) (Figure 3-6A), and the number of correct (F(1, 100)=9.0, p=0.004) (Figure

3-7A) and error responses (F(1, 100)=4.3, p=0.044) (Figure 3-8A). With respect to all of these

variables, EE mice performed better than SC mice.

There were also significant main effects of time for all the variables measured: latency

(F(2, 100)= 35.6,p<0.001), distance traveled (F(2, 100)=45.6,p<0.001), velocity (F(2,

100)=35.0, p<0.001), the number of first correct entries (F(2, 100)=22.2 p<0.001), and the

number of correct (F(2, 100)=42.2, p<0.001) and error responses (F(2, 100)=32.1, p<0.001).

No statistically significant interaction was found for latency (F(2, 100)=2.8, p=0.065),

distance traveled (F(2, 100)=1.9, p=0.155), velocity (F(2, 100)=0.3,p=0.739), the number of

first correct entries (F(2, 100)=1.3, p=0.272), and the number of correct responses (F(2,

100)=2.2, p=0.114). A statistically significant interaction was found, however, for error

responses (F(2, 100)=3.6, p=0.031). Specifically, EE and SC mice made a similar number of

error responses in P1, but significant differences between these groups were observed in P2

(p=0.008) and P3 (p=0.043).

Analysis of the first three days of procedural learning does not make use of all the data.

Thus, the number of error responses across all sessions was calculated. A significant difference

in housing condition was found for the number of error responses where SC mice made

significantly more error responses than EE mice (t(50)=-2.3, p=0.025) (Figure 3-9A). However,

the proportion of error responses (the total number of error responses divided by the total number

of responses in the procedural learning experiment) didn't differ between housing conditions

(t(50)=l. 1,p=0.263) (Figure 3-10A).

Finally a main effect of housing condition was found for days required to reach the

criterion, which ranged from 3 to 11 days (t(50)=2.6, p=0.012) (Figure 3-14A). In this, as in all









other measures, mice housed in an enriched environment performed better than mice housed in

standard cages in this procedural task.

In addition, the proportion of animals meeting criterion across totals was calculated by

survival analysis using the Cox proportional hazard model. There was a significant difference

(p=0.024), such that a greater proportion of EE mice met criterion in fewer trials than did SC

mice (Figure 3-15A).

Reversal Learning

A main effect of housing condition on latency to reach the platform was also observed in

the reversal phase (F(1, 100)=4.2, p=0.046) (Figure 3-3A). As seen in the procedural learning

phase, significant differences between EE and SC mice were also found for the other dependent

measures examined: distance traveled (F(1, 100)=5.8, p=0.020) (Figure 3-4A), velocity (F(1,

100)=6.9, p=0.012) (Figure 3-5A), the number of first correct arm entries (F(1, 100)=8.0,

p=0.007) (Figure 3-6A), and the number of correct (F(1, 100)=9.5, p=0.003) (Figure 3-7A) and

error responses (F(1, 100)=13.8, p<0.001) (Figure 3-8A).

A main effect of time was found on all measures employed: latency (F(2, 100)=42.3,

p<0.001), distance traveled (F(2, 100)=75.2, p<0.001), velocity (F(2, 100)=4.9,p=0.010), the

number of first correct arm entries (F(2, 100)=48.9, p<0.001), and the number of correct

(F(2)=53.8,p<0.001) and error responses(F(2, 100)=87.0,p<0.001). Specifically, EE mice swam

significantly faster on all R1-3 days (p=0.013, p=0.024, andp=0.027 respectively), and made

more correct and fewer error responses in R1 (p=0.013, p=0.010 respectively) and R2 (p<0.001,

p<0.001 respectively) than SC mice. There were no statistically significant interactions for any

of these measures.

In addition, EE mice made significantly fewer error responses compared to SC mice across

all sessions in the reversal phase (t(50)=-3.4, p=0.001) (Figure 3-9A). Similarly, SC mice









exhibited a significantly greater proportion of error responses compared to EE mice (t(50)=3.6,

p<0.001) (Figure 3-10A).

Figure 3-11A shows the proportion of correct responses in the first 24 trials of the reversal

phase. In the first trial of the reversal phase (rl), almost all the mice failed to make a correct

response. Yet, as the trials progressed, EE mice quickly learned the new S-R contingency. By the

end of the first session (r8), the proportion of correct response in SC mice was below chance

level (approximately 25%), whereas the proportion of correct responses for EE mice was

approximately 60%. Similarly, the proportion of error responses during reversal learning was

higher for SC mice compared to EE mice in rl-24 (Figure 3-12A). Finally, there was a main

effect of housing on days to reach criterion (t(50)=2.2, p=0.033) (Figure 3-14A), with mice

housed in the EE condition performing better than mice housed in the SC condition in the

reversal task.

In addition, the proportion of animals meeting criterion across totals was calculated for

survival analysis by the Cox proportional hazard model. There was a significant difference

(p=0.021), such that a greater proportion of EE mice met criterion in fewer trials than did SC

mice (Figure 3-15B).

Effects of Stereotypy Levels

Procedural Learning

A main effect of stereotypy level was found for latency (F(3, 96)=3.0, p=0.041) (Figure 3-

3B), distance traveled (F(3, 96)=6.9, p<0.001) (Figure 3-4B), and velocity (F(3, 96)=3.3,

p=0.029) (Figure 3-5B). Specifically mice in the backward somersaulting group spent a

significantly longer duration of time in the maze than the low stereotypy group (p=0.029). The

differences in latency between these groups were significant for the first (P1) (p=0.037) and

second (P2) (p=0.003) days. By day three (P3) all stereotypy groups took roughly equal amounts









of time for trial completion. Similarly, mice in the backward somersaulting group traveled the

longest distance to reach the platform compared to any other groups (L, M, and H) (p<0.001,

p=0.003, p=0.002 respectively) (Figure 3-4B). No significant differences among the high,

medium, and low jumpers were noted.

Post hoc analyses for the dependent measure of velocity showed a significant difference

between mice in the backward somersaulting and in the low stereotypy groups (p=0.025) and in

P2 (p=0.043) and P3 (p=0.016). When the velocity of each mouse was compared with their

individual stereotypy frequency score, there was a tendency for mice with high frequency of

stereotypy to swim slower (r=-0.256, p=0.070). This was largely attributed to the swim speed

among mice exhibiting vertical jumping (L, M, and H) (r=-0.292, p=0.057).

A main effect of time was found for all measures: latency (F(2, 96)=38.1, p<0.001),

distance traveled (F(2, 96)=52.1, p<0.001), velocity (F(2, 96)=32.3, p<0.001), the number of

first correct arm entries (F(2, 96)=20.0, p<0.001), the number of correct (F(2, 96)=40.0,

p<0.001) and error responses (F(2, 96)=0.9, p 0.452).

The only measure for which a significant group by time interaction was found was distance

traveled (F(6, 96)=2.8, p=0.015). Mice in the low stereotypy group increased the velocity as the

trials progressed, whereas mice in the backward somersaulting group stayed at relatively slow

speed (Figure 3-5B).

No group differences were found on the number of first correct arm entries (F(3, 96)=1.6,

p=0.205) (Figure 3-6B) and the number of correct (F(3, 96)=2.5,p=0.067) (Figure 3-7B) and

error responses (F(3, 96)=0.9, p=0.452) (Figure 3-8B). In addition, no significant difference was

found in the total number of error responses (F(3)=1.6, p=0.192) (Figure 3-9B) as well as a









proportion of error responses across all sessions in the procedural phase (F(3)=0.3, p=0.861)

(Figure 3-10B).

Next, each stereotypy score was regressed by the total number of correct or error responses

made within P1-3. There were no relationship between rates of stereotypy and correct (r=-0.094,

p=0.509) and error responses (r=0.045,p=0.750). Moreover, there wasn't any relationship

between stereotypy scores and the total number of correct (r=0.050, p=0.724) and error

responses (r=0.062, p=0.662) made in the procedural phase.

Finally, one-way ANOVA for stereotypy groups showed that significant differences

existed in days to reach the criterion (F(3)=3.7, p=0.018). Post-hoc analyses revealed a

significant difference between mice in the backward somersaulting group and in the low

stereotypy group (p=0.027) (Figure 3-14B).

Reversal Learning

During reversal training, no significant differences among groups were found for latency

(F(3, 96)=1.2,p=0.335) (Figure 3-3B) or swim speed (F(3, 96)=1.3, p=0.298) (Figure 3-5B).

Conversely, main effects of stereotypy level were found for the variables distance traveled (F(3,

96)=3.1, p=0.036) (Figure 3-4B), the number of first correct arm entries (F(3, 96)=8.8, p<0.001)

(Figure 3-6B), and the number of correct (F(3, 96)=8.9, p<0.001) (Figure 3-7B) and error

responses (F(3, 96)=5.3, p=0.003) (Figure 3-8B).

Specifically, mice in the backward somersaulting group traveled longer distances

(p=0.023), made fewer successful first correct arm entries (p<0.001), and made fewer correct

(p<0.001) and more error responses (p=0.002) than mice in the low stereotypy group. In

addition, low stereotypy mice made significantly more first correct entry and correct responses,

than mice in the medium stereotypy group (p=0.001, p=0.001 respectively) and mice in the high









stereotypy group made more first correct entry and correct responses than mice in the backward

somersaulting group (p=0.042, p=0.047 respectively).

A main effect of time was found for all measures: latency (F(2, 96)=42.5, p<0.001),

distance traveled (F(2, 96)=76.8, p<0.001), velocity (F(2, 96)=5.0, p=0.008), the number of first

correct arm entries (F(2, 96)=49.7, p<0.001), and the number of correct (F(2, 96)=58.5, p<0.001)

and error responses (F(2, 96)=86.9, p<0.001).

A group by time interaction was found for distance moved (F(6, 96)=2.7, p=0.018) (Figure

3-4B), first correct arm entries (F(6, 96)=4.3, p<0.001), and the number of correct (F(6, 96)=6.3,

p<0.001) (Figure 3-7B) and error responses (F(6, 96)=3.9, p=0.002) (Figure 3-8B). This effect

seems to be due to the unexpected performance by mice in the high stereotypy group.

In addition, the number of error responses made in R1-3 was positively correlated with the

frequency of stereotypy (r=0.306, p=0.003) (Figure 3-13). This difference was mainly due to

mice showing vertical jumping (r=0.317, p=0.039). It is worth noting that number of error

responses in the P1-3 and R1-3 was positively correlated in mice exhibiting vertical jumping (L,

M, and H groups) (r 0.310, p=0.043), suggesting that mice exhibiting higher rates of stereotypy

appear to perseverate more and mice with procedural learning deficits exhibit reversal deficits.

However, when the stereotypy score was regressed against the total number of error responses

made by each mouse, no relationship between stereotypy score and error responses was found

(r=0.216, p=0.124).

In addition, a significant difference was found in the total number of error responses across

sessions in the reversal phase (F(3)=8.1, p<0.001), where mice in the backward somersaulting

group made significantly more error responses than any other groups (L, M, and H) (p<0.001,

p=0.012, p=0.002 respectively) (Figure 3-9B). There was also a significant difference in a









proportion of error responses (F(3)=3.9, p=0.014). Mice in the backward somersaulting group

exhibited a significantly greater proportion of error responses than mice in the low stereotypy

group (p=0.024) (Figure 3-10B).

Figure 3-11B shows the proportion of mice making a correct response in the first 24 trials

of the reversal phase. In the first trial of the reversal phase (rl), almost all of the mice failed to

make a correct response. Approximately 65% of mice in the low stereotypy group made a correct

response in r8 and almost all found the platform successfully by the middle of the second

reversal session, 10% of mice in the backward somersaulting group made a correct response in r8

and the probability stayed below chance level through r24. Similar reversal trends were seen for

an error response in rl-24 (Figure 3-12B).

Finally, significant differences were found in days required to reach a reversal criterion

among different levels of stereotypy (F(3)=8.9, p<0.001) (Figure 3-14B). Mice in the backward

somersaulting group required significantly more days to complete the task than mice in the low

(p<0.001) and high (p=0.003) stereotypy groups. In addition, mice in the middle stereotypy

group required more days than mice in the low (p=0.005) and high (p=0.016) stereotypy groups.

There was no correlation between days required to meet criterion for procedural learning

and days required to meet criterion for reversal learning (r=0.23, p=0.103). However, when the

two topographies of stereotypy were analyzed separately, the number of days to reach both

criteria was not correlated in jumpers (r=-0.025, p=0.874), but were strongly correlated in

backflippers (r=0.765, p=0.016). Therefore these results indicate that for mice exhibiting

repetitive backward somersaulting deficits in procedural learning were associated with cognitive

rigidity.











1200


0 '


SC
HOUSING GROUPS


Figure 3-1. Effects of environmental enrichment on stereotypy (vertical jumping and backward
somersaulting): EE substantially attenuated the development and expression of
stereotypy (p<0.001). Values expressed are group means S.E.M.


H M L B
STEREOTYPY GROUPS


Figure 3-2. Average stereotypy scores in four stereotypy groups: Mice were categorized into H
(n=15), M (n=14), L (n=14), or B (n=9) groups depending on their topography and
frequency of stereotypy. Values expressed are group means S.E.M.

















25


20
UJ

o 15
> -

10.



5 -


P1 P2 P3 R1 R2 R3

BLOCKS


0
W 20-

O
Z
U. 15
-

10-


5-


0


P1 P2 P3


R1 R2 R3
R1 R2 R3


BLOCKS


Figure 3-3. Latency to reach the platform. A) Effects of housing conditions: EE mice reached the
platform significantly faster than SC mice in the first three days of both the
procedural (p=0.001) and reversal (p=0.046) phases. B) Effects of stereotypy levels:
B mice took significantly longer to reach the platform than L mice in the first three
days of the procedural phase (p=0.029). There was no difference between stereotypy
groups in the reversal phase (p=0.335). Values expressed are group means + S.E.M of
eight daily trials.


- EE
SC


I H
-0---- H
--y M
- --- L
------- B


u













1200

--- EE
SC
1000 -


LU
800-

Z
z
600
(,)


400



200
P1 P2 P3 R1 R2 R3

A BLOCKS


1600

H
1400 M
---- L

1200- ----"----- B
1200 -
-J
S1000


800 -

600


400 \


200
P1 P2 P3 R1 R2 R3

B BLOCKS


Figure 3-4. Distance traveled. A) Effects of housing conditions: EE mice swam significantly
shorter distances than SC mice in the first three days of both the procedural (p=0.001)
and reversal (p=0.020) phases. B) Effects of stereotypy levels: B mice traveled
significantly longer distance than any other groups in the first three days of the
procedural phase (p<0.001). B mice traveled significantly longer distance than L mice
in the reversal phase (p=0.023). Values expressed are group means + S.E.M of eight
daily trials.

















70
O
CS)
_J 60-
x
1-

50-
0
0
-j
LU
S40 -- EE
SC















30- --- i --- i --- i --- i ---- ---- -- -- -- --B
30
P1 P2 P3 R1 R2 R3

A BLOCKS



90



80-


s-




60--


LU-- H


------ B

40 "
P1 P2 P3 R1 R2 R3

g BLOCKS


Figure 3-5. Velocity. A) Effects of housing conditions: EE mice swam significantly faster than
SC mice in the first three days of the procedural (p<0.001) and reversal (p=0.012)
phases. B) Effects of stereotypy levels: B mice swam significantly slower than L mice
in the first three days of the procedural phase (p=0.029). No group difference was
found in the reversal phase (p=0.298). Values expressed are group means + S.E.M of
eight daily trials.
















LU EE
EE




I-
SC




LU
Q 6
0 4-

o
u- 2-
LU













1- 8 ----i--- L
A BLOCKS











S a-e /
0 /
1- 82- ->- --








u- 4 /
o

8 L

O


P1 P2 P3 R1 R2 R3

g BLOCKS


Figure 3-6. The number of first correct arm entries. A) Effects of housing conditions: EE mice
made significantly more correct entries than SC mice in the first three days of both
the procedural (p=0.020) and reversal (p=0.007) phases. B) Effects of stereotypy
levels: Although no group difference was found in the procedural phase (p=0.205), L
mice made significantly more correct entries than B (p<0.001) and M mice (p=0.001),
and H mice made more correct entries than B mice (p=0.042) in the first three days of
the reversal phase. Values expressed are group means + S.E.M of eight daily trials.















0 EE
7 SC
UI)
U) 6
Z

uo 5
Of


LU
0
0
w
1 -










P1 P2 P3 R1 R2 R3

A BLOCKS
10














H
L2
0




P1 P2 P3 R1 R2 R3

A BLOCKS





10
-0------ H

W a --y~- M




U) /6


-I_
O 4


0 2



0
P1 P2 P3 R1 R2 R3

g BLOCKS


Figure 3-7. The number of correct responses. A) Effects of housing conditions: EE mice made
significantly more correct responses than SC mice in the first three days of both the
procedural (p=0.004) and reversal (p=0.003) phases. B) Effects of stereotypy levels:
Although no group difference was found in the procedural phase (p=0.067), L mice
made significantly more correct responses than B (p<0.001) and M (p=0.001) mice,
and H mice made more correct responses than B mice (p=0.047) in the first three days
of the reversal phase. Values expressed are group means S.E.M of eight daily trials.













7 EE
SC
U)
LU 6
z
[ 5
LI.
w









P1 P2 P3 R1 R2 R3

A BLOCKS




7- I H

W 6 ------- B
O
02


















O
LLJ_
0 2























^1-
P1 P2 P3 R1 R2 R3

A BLOCKS













Figure 3-8. The number of error responses. A) Effects of housing conditions: SC mice made
significantly more error responses than EE mice in the first three days of both the
procedural (p=0.044) and reversal (p<0.001) phases. B) Effects of stereotypy levels:
There was no group difference in the procedural phase (p=0.452), whereas B mice
made significantly more error responses than L mice in the first three days of the
reversal phase (p=0.002). Values expressed are group means S.E.M of eight daily
trials.
-0--- -- H \
H
C) --U-- L
LU 6 ---4--- B
U)
z
0











P1 P2 P3 R1 R2 R3
B BLOCKS


Figure 3-8. The number of error responses. A) Effects of housing conditions: SC mice made
significantly more error responses than EE mice in the first three days of both the
procedural (p=0.044) and reversal (p<0.001) phases. B) Effects of stereotypy levels:
There was no group difference in the procedural phase (p=0.452), whereas B mice
made significantly more error responses than L mice in the first three days of the
reversal phase (p=0.002). Values expressed are group means + S.E.M of eight daily
trials.



































SC EE

HOUSING GROUPS


30

V)
LU
U)
O
0-
U)
L 20

0
O 1
D:
LU
LL
0 10
Ut


H M L B

STEREOTYPY GROUPS


Figure 3-9. The total number of error responses made in the procedural and reversal phases. A)
Effect of housing conditions: SC mice made significantly more error responses than
EE mice in both the procedural (p=0.025) and reversal phases (p=0.001). B) Effect of
stereotypy levels: There was no significant difference among groups in the procedural
phase (p=0.192), whereas B mice made significantly more error responses than L
(p<0.001), M (p=0.012), and H (p=0.002) mice in the reversal learning. Values
expressed are group means + S.E.M.
















0.25 E % R ERRORS
C)
u)
Z
O 0.20
u0

S 0.15
O

S0.10
LL
O

0.05-


0.00
SC EE

A HOUSING GROUPS



0.35

S% P ERROR
0.30 % R ERROR


) 0.25
O
U)
w 0.20


0 0.15


LL 0.10
0





H M L B

B STEREOTYPY GROUPS


Figure 3-10. The proportion of error responses in the procedural and reversal phase. A) Effects
of housing conditions: There was no difference between groups in the procedural
phase (p=0.263), whereas SC mice exhibited a significantly greater proportion of
error responses than EE mice in the reversal phase (p<0.001). B) Effects of stereotypy
levels: There was no significant difference among groups in the procedural phase
(p=0.861), B mice exhibited a significantly greater proportion of error responses than
L mice in the reversal phase (p=0.024). Values expressed are group means + S.E.M.













LU
U)
Z
O EE
0a SC
1-.
) 0.8






0.2 -
Of 0.6
of
0

LL 0.4
0

z
oi 0.2

C)
C-)
O

0.0 L












0 ____I_________________________
rl r2 r3 r4 r5 r6 r7 r8 r9 r10rl1r12r13r14r15r16r17r18r19r20r21r22r23r24

A TRIALS



LU 10

























Figure 3-11. Probability of making a correct response in the first 24 trials of the reversal phase.
z H0


w. o s l L




behavior, B mice show a pattern ofperseveration.

0 0

o o. .



000
rl r2 r3 r4 r5 r6 r7 r8 r9rlr11r12r13r14r15r16r17r18r19r20r21r22r23r24
B TRIALS


Figure 3-11. Probability of making a correct response in the first 24 trials of the reversal phase.
A) Effects of housing conditions: EE mice compared to SC mice quickly learned the
new behavior. B). Effects of stereotypy levels: Although L mice learned the new
behavior, B mice show a pattern of perseveration.















S---- EE
of \SC
LJ 0.8
Z
LL
O
LU 0.6
z
LU

D 0.4
O
0
LU
( 0.2
LU

0.0
r1 r2 r3 r4 r5 r6 r7 r8 r9 r10r11r12r13r14r15r16r17r18r19r20r21r22r23r24

A TRIALS



S1.0

O H
of *t--- M
U 0.8-- L
z 1 ---- --- B
LLZ
0\
LU 0.6 0.
z J

0.4 I1"r
P

0

(D 0.2 \

LU

0.0
rl r2 r3 r4 r5 r6 r7 r8 r9 r10r11r12r13r14r15r16r17r18r19r20r21r22r23r24

B TRIALS


Figure 3-12. Probability of making an error response in the first 24 trials of the reversal phase.
A) Effects of housing conditions: EE mice compared to SC mice quickly learned to
the previously relevant response. B) Effects of stereotypy levels: Although L mice
learned to inhibit the previously relevant response, B mice show a pattern of
perseveration.


















U)
LU 25-
,)
z
0


0-
U) 20 -
LU




LU
0 10 -



o 5

I-
0 -



A





30 -


U)
W 25-
U)
Z
0
0-
U) 20 -
LU


15-
LU
LL
U_
O 10-



1 5


0 200 400 600 800

STEREOTYPY SCORE (JUMPERS)


Figure 3-13. Regression of the total number of error responses in the reversal phase by individual

stereotypy score. A) Stereotypy score was positively correlated with the number of

error responses (p=0.003). B) Stereotypy score was positively correlated with the

number of error responses when six outliers were excluded (p=0.025).


S







S
S





* S


D 500 1000 1500 2000

STEREOTYPY SCORE (JUMPER)


0
00







S


3000
































200


1:


--

















z 8-
0

0-
O


6

I
I--
o 4


) 2-



0
SC EE

A HOUSING GROUPS



14


12
z
10
/ /

o 8
C-)

S6

U)
>4





H M L B

B STEREOTYPY GROUPS


Figure 3-14. Days required to reach the criterion. A) Effects of housing conditions: SC mice
required significantly more days to reach the criterion in the procedural (p=0.012) and
reversal (p=0.033) phases. B) Effects of stereotypy levels: B mice spent significantly
more days than L mice to reach the procedural criterion (p=0.027). B mice spent
significantly more days than L (p<0.001) and H (p=0.003) mice, and M mice spent
more days than L (p=0.005) and H (p=0.016) to reach the reversal criterion. Values
expressed are group means + S.E.M.





















S'I
-- I---,'-------





















0 20 60 80 1U

A






S- landard tt14
E-'iJ'ed ctge
E __






IL


-----------------













0ci 40 60 50 1 1




Figure 3-15. Effects of housing conditions on trials to the criterion. A) Trials to the procedural
criterion: SC mice required significantly more trials to reach the procedural criterion
2 --1-





I I I I I






criterion: SC mice required significantly more trials to reach the procedural criterion

(p=0.021). B) Trials to the reversal criterion: SC mice required significantly more
trials to reach the reversal criterion (p=0.024).









CHAPTER 4
DISCUSSION

Restricted, repetitive behaviors are commonly displayed in many neurodevelopmental

disorders. Despite their prevalence in clinical populations, relatively little is known about the

neurobiological mechanisms responsible for the development and expression of these behaviors.

A better understanding of the underlying alterations will aid in identifying etiology as well as

developing a better treatment plan for individuals with repetitive behavior disorders. Valid

animal models provide an important strategy in achieving these goals and are necessary for

pursuing the neural mechanisms of this abnormal behavior.

This study employed deer mice (Peromyscus maniculatus) as a model of repetitive motor

behavior associated with neurodevelopmental disorders. These mice exhibit high rates of

repetitive jumping or backward somersaulting when housed in standard rodent cages, and such

behaviors can be attenuated or prevented by housing them in environmentally complex settings

(Powell et al., 2000; Powell et al., 1999). In addition, our work suggests that deer mice exhibiting

high rates of stereotypy have altered cortico-basal ganglia circuitry, specifically relatively

decreased tone of the indirect pathway (Presti & Lewis, 2005).

In this study, we attempted to examine hypothesized impairments in stereotypic deer mice

in cognitive processes that are mediated in part by cortico-basal ganglia circuitry. First, we tested

striatally mediated procedural learning in mice displaying various rates of stereotypy, which

were reared in either a standard rodent cage or an environmentally enriched condition. Since

cortico-striatal circuitry involving the frontal cortex seems to play an important role in executive

function (Dallery et al., 2004; Dias et al., 1996; Uylings et al., 2003; van der Meulen et al.,

2006), we further assessed one aspect of such higher-order function, cognitive flexibility, by









switching of the stimulus-response (S-R) contingency (reversal learning) following acquisition of

the procedural task.

Effects of Housing Conditions on Stereotypy

In agreement with our previous findings (Powell et al., 2000; Powell et al., 1999), deer

mice reared in an environmentally enriched (EE) condition developed significantly lower rates of

stereotypy compared to mice reared in standard cages (SC). One novel feature of this study is

that we categorized mice displaying backward somersaulting as a separate group for statistical

analysis. In past experiments, we have not systematically differentiated these two topographies

(vertical jumps and backward somersaults), because a majority of deer mice exhibited high rates

of repetitive vertical jumps and only a minority engaged in more complex backward somersaults

(Powell et al., 1999).

As expected, most EE mice exhibited low levels of jumping and most SC mice exhibited

high rates of jumping or backward somersaulting. Although we wished to examine the

interaction between housing conditions and levels of stereotypy on cognitive performance, we

were not able to evaluate such effects due to the small number of mice in the EE-high stereotypy

and SC-low stereotypy groups.

Effects of Housing Conditions on Procedural Learning

Studies have repeatedly demonstrated that memory dependent on the hippocampal

formation improves following exposure to EE in animals (Duffy et al., 2001; Lambert et al.,

2005; Pham et al., 1999; Rampon et al., 2000; Winocur & Greenwood, 1999; Woodcock &

Richardson, 2000). Since EE benefits a variety of other behaviors and relevant neurobiology, it is

reasonable to hypothesize that the memory systems mediated by non-hippocampal regions such

as the striatum (procedural learning) also improve following EE. Although there have been









several attempts to examine effects of EE on procedural learning (Frick & Fernandez, 2003;

Frick et al., 2003; Schrijver et al., 2004), no significant improvements were noted.

For example, Frick and colleagues (Frick & Fernandez, 2003; Frick et al., 2003) exposed

aged rats (18 and 28 months) to EE and assessed their performance in two versions (spatial and

cued) of Morris water maze tasks. Rats exposed to EE conditions as adults performed

significantly better than SC control rats in the spatial task, whereas EE did not reduce age-

associated cognitive impairments in the cued task.

In contrast, our results showed that mice reared in EE condition performed significantly

better with respect to all behavioral measurements collected during the procedural task (latency,

distance traveled, velocity, the number of first correct arm entries, and the number of correct and

error responses), and took fewer days to criterion compared to SC mice.

This contradictory result from other groups could be explained by the age and species

difference in sensitivity to surrounding environments. Whereas Frick and colleagues showed that

age-related cognitive impairments in rats were found specifically in the hippocampus and EE

rescued associated deficits in rats, relatively younger deer mice were employed in this study. In

addition, neurobiological alterations associated with repetitive behavior in adult deer mice were

primarily found in the motor cortex and dorsolateral striatum (Turner et al., 2003; Turner et al.,

2002).

EE-associated improvements in hippocampally dependent memory are often associated

with elevation of neurotrophic factors in the hippocampus (Pham et al., 1999). We previously

found that deer mice, which benefited from EE, had elevated levels of striatal brain derived

neurotrophic factor (BDNF) (Turner & Lewis, 2003). This neurotrophin plays a particularly

important role in neuronal plasticity associated with learning and memory. In contrast, no









increase in BDNF or nerve growth factor (NGF) was noted within the hippocampus in these

mice.

The ameliorative effects of EE, therefore, seemed to act preferentially upon the striatum in

deer mice, resulting in improvements in performance in the procedural-learning task. In

summary, our current and previous work supports improvements of striatally mediated memory

following EE in deer mice.

Effects of Housing Conditions on Reversal Learning

In the reversal experiment assessing cognitive flexibility, EE mice performed significantly

better in all measurements during the first few days of the reversal task. Specifically, SC mice

made significantly more perseverative errors before reaching the criterion. Even after many days

of training trials in the procedural phase, they swam significantly slower and longer distances

during reversal learning, with no obvious motor abnormalities.

Moreover, relatively more EE mice reached the criterion early in training according to the

survival analysis. We found such significant differences for both the procedural and reversal

phases, but the magnitude of difference between the two groups was larger in the reversal phase.

Although we hypothesized an effect of EE on executive function, not many studies have tested

this effect (Jones et al., 1991; Schrijver et al., 2004; Schrijver & Wurbel, 2001).

The studies assessing the effect of EE on cognitive flexibility have focused primarily on

the social component of EE. For example, Schrijver and Wurbel (2001) demonstrated that

socially isolated rats had deficits in extradimensional set-shifting from cue- to place-version of

the radial arm maze tasks, and vice versa. Both the social and isolated rats had intact cue and

place memory as well as intradimensional set-shifting within these tasks, however.

In contrast, we exposed mice to an environment consisting of multiple EE factors (novelty,

inanimate objects, social interaction, exercise, increased space, etc.), and found significant









effects of EE on cognitive flexibility. Thus, these results suggest that a combination of multiple

EE components benefits higher-order cognitive functions dependent in part on cortico-basal

ganglia circuitry in deer mice, in addition to memory systems discussed above.

Effects of Stereotypy Levels on Procedural Learning

Among the four groups constituted by stereotypy level or topography (three levels of

vertical jumping and backward somersaulting), impairments in the procedural task were found in

mice exhibiting backward somersaults. These mice traveled longer distance, swam slower, and

required more days to criterion. There was no relationship between rates of stereotypy and

correct- or error-response frequency suggesting that mice of various levels of stereotypy

explored the maze similarly. Thus contrary to our hypothesis, procedural-learning impairments

in high-stereotypy mice were not found.

Evidence for deficits in striatally mediated learning in autistic individuals is almost

completely lacking, despite the significance of these brain regions in the expression of repetitive

behaviors. However, there has been a report of a deficit in the Serial Response Time Trial task

(Mostofsky et al., 2000) in children with autism. Although performance in this task likely

involves both cortico-striatal and cortico-cerebellar pathways, no attempt was made to relate

reaction time to repetitive behaviors.

We know relatively more about deficits in striatally mediated learning and memory among

individuals with Tourette's syndrome and obsessive-compulsive disorder (OCD). Marsh and

colleagues (2004) recently reported an inverse correlation between tic and habit learning in

children and adults with Tourette syndrome, whereas declarative memory was not different from

healthy controls. Patients with OCD performed better at the early course of procedural learning

but were impaired at the later phase, whereas declarative memory was intact (Roth et al., 2004).









Thus, in these populations, restricted, repetitive behavior is associated with striatal dysfunctions,

which, in turn, are associated with disruptions in striatally mediated learning.

Effects of Stereotypy Levels on Reversal Learning

Of most interest in this experiment was the association between rates of stereotypy and

cognitive rigidity. Our hypothesis came from a series of studies done by Mason and colleagues

(Garner & Mason, 2002; Garner et al., 2003a; Garner et al., 2003b; Vickery & Mason, 2005),

who reported in several species of animals that motor stereotypy associated with a restricted

environment was positively correlated with perseverative behavior. The cognitive tasks

employed included extinction, reversal learning, and a variation of the gambling task.

Similarly, correlations between restricted, repetitive behavior and cognitive rigidity were

recently demonstrated in autistic individuals (Lopez et al., 2005). Although it has been widely

known that these individuals have deficits in executive function (Pennington & Ozonoff, 1996),

its association with restricted, repetitive behaviors had not previously been examined.

Consistent with these findings, our results showed that stereotypy score was positively

correlated with the number of error responses made during the beginning of reversal training as

well as before reaching the criterion. Since error responses made especially during the first few

days is indicative of how quickly, thus flexibly, they switch the learning strategy, the results

support our hypothesis that high rates of stereotypy in deer mice is associated with cognitive

rigidity. Perseverative behavior was prominent in mice displaying backward somersaulting,

which more frequently chose the incorrect arm for their first response.

However, mice in the middle-stereotypy group unexpectedly took as many days as mice in

the backward somersaulting group to reach the reversal criterion, whereas mice in the high- and

low-stereotypy group performed equally. There were nine mice in total, which did not reach the

criterion, and four of them were in the middle-stereotypy group (two mice from each housing









condition). Relatively better performance in mice displaying high rates of jumping was contrary

to our hypothesis. Nonetheless, we did find an association between stereotypy and reversal

learning.

In this experiment, no interactions between housing conditions and stereotypy levels could

be examined and interpreted. Therefore, main effects of stereotypy levels on cognitive rigidity

were largely skewed by housing conditions, since a large number of EE mice displayed low rates

and most SC mice exhibited either high-stereotypy jumping or backward somersaulting.

However, of interest is that mice exhibiting backward somersaults performed most poorly in both

the procedural and the reversal tasks than mice displaying vertical jumping. Considering that

they all came from SC condition, we can speculate that cortico-basal ganglia circuitry in these

mice was most perturbed.

It is worth noting that we informally observed that some of the backward somersaulters

failed to swim straight but instead took either a clockwise or a counterclockwise circular pattern

during the visible platform Morris water maze task, which was used as a pretest. Thus we

carefully observed their swim performance to exclude those with motor abnormalities. The

remaining mice in this group, which appeared to be unimpaired in their swimming movements,

swam significantly slower than mice in the low-stereotypy group at the beginning of the

procedural training, suggesting the possibility of slight motor abnormalities. This difference in

swim speed, however, disappeared by the beginning of the reversal training. Thus the motor

disadvantage in these mice seems relatively insignificant.

There is accumulating evidence that the frontal cortex subregions play an important role in

cognitive flexibility, and activation of each area depends on the complexity of the task and types

of set-shifting strategies required. Specifically, the dorsolateral prefrontal cortex or medial









prefrontal cortex, in humans and non-human primates or rodents respectively, appear to subserve

higher-order extradimensional attentional changes, whereas the orbitofrontal cortex (OFC) is

likely to mediate simple intradimensional changes (such as reversal learning) (Dias et al., 1996;

Dias et al., 1997; Kim & Ragozzino, 2005; Ragozzino, 2002; Wallis et al., 2001).

A double dissociation study of neuropsychological task performance revealed that

prefrontal dysfunctions of relatively localized areas are associated with some neuropsychiatric

disorders. Schizophrenic patients have deficits in Wisconsin Card Sorting test (WCST), which

indexes dorsolateral prefrontal cortex impairments, whereas OCD patients perform poorly in the

Object Alternation Test and Gambling Task, which require normal OFC functions (Abbruzzese

et al., 1995; Cavallaro et al., 2003). Yet, these disorders may not be exclusively associated with

independent prefrontal dysfunctions, since both patient group performed poorly in Tower of

Hanoi task (Cavallaro et al., 2003). There are also contradictory reports on OCD patients who

performed poorly in WCST (Head et al., 1989) or were as equally impaired as schizophrenic

patients in the Object Alternation Test (Spitznagel & Suhr, 2002).

Neuroimaging studies support altered patterns of brain activation during executive function

tasks in individuals with autism. These include significantly increased activation of brain

regions, which are not usually involved, in Go/No-Go task, spatial STROOP test, and Set-

Shifting task (Schmitz et al., 2006). One speculation of executive dysfunction in autistic patients

is abnormal neural organization and activation of brain regions, which are not normally recruited

for these tasks.

Thus our results suggest that OFC-striatal circuitry is likely impaired in stereotypic deer

mice, since the cognitive task used involved simple reversal learning. Yet it is apparent that the

OFC is not the only prefrontal region commonly perturbed in individuals with









neurodevelopmental disorders associated with repetitive behaviors. For example, in addition to

cognitive inflexibility, Lopez and colleagues (2005) also reported positive correlation of

restricted, repetitive behaviors with deficits in another component of executive function, working

memory, in autistic individuals. Other cognitive tasks requiring different level of processes (e.g.,

extradimensional set-shifting) using varieties of executive function tasks will further elucidate

the specific loci of impairments and its association with repetitive behaviors.

In addition, as an extension of this study, we should be able to double dissociate impaired

striatum-mediated procedural learning and relatively intact hippocampally mediated spatial

learning in deer mice. It is postulated that EE low- and high-stereotypy mice should not differ in

the task testing spatial memory. Conversely, SC low- and high-stereotypy mice should perform

poorly on this task.

Modeling a Wider Range of Restricted, Repetitive Behaviors

Our findings in the reversal-learning task suggest that deer mice can be used as a model of

not just stereotyped motor behavior but also more complex repetitive behavior. Restricted,

repetitive behaviors displayed in autistic individuals have been conceptually divided into two

clusters: simple motor behaviors (e.g., body rocking, hand flipping, and plate spinning) and more

complex higher-order behaviors (e.g., insistence on sameness and circumscribed interests)

(Turner, 1999). Furthermore, two groups recently reported that factor analyses of 12 items in the

section of repetitive behaviors from Autism Diagnostic Interview-Revised yielded two factors:

repetitive sensorimotor actions and resistance to changes, supporting this categorization

empirically (Cuccaro et al., 2003; Szatmari et al., 2005).

Animal models of restricted, repetitive behavior generally focus on motor stereotypy,

which is easier to measure than more complex behaviors. Cognitive rigidity shown in stereotypic

deer mice in this experiment, especially those of backward somersaulting, resemble resistance to









changes in autistic individuals. Although more systematic investigations are necessary, the

results presented here will allow us to extend our animal model to include not only motor but

also cognitive forms of restricted, repetitive behavior. These results will significantly enhance

the applicability of this model to the wide range of restricted, repetitive behavior typical of

autism and other neurodevelopmental disorders.









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BIOGRAPHICAL SKETCH

The candidate was born in Tokyo, Japan to Nobuo and Reiko Tanimura. She has one older

sister, Chie. She completed high school at Meguro Seibi Gakuen in Tokyo. She moved to

California in 1998 and received her Bachelor of Arts in Psychology from California State

University, Sacramento in May 2002 with honors. After working for a short period of time in

Tokyo, she joined the laboratory of Dr. Mark H. Lewis and began her graduate education in

August 2003. Currently she is pursuing her Ph.D. in the Behavioral Neuroscience program at the

University of Florida.