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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2010-08-31.

Permanent Link: http://ufdc.ufl.edu/UFE0021676/00001

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Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2010-08-31.
Physical Description: Book
Language: english
Creator: Lee, Linda
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Neuroscience (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Linda Lee.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Lewis, Mark H.
Electronic Access: INACCESSIBLE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021676:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021676/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2010-08-31.
Physical Description: Book
Language: english
Creator: Lee, Linda
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Neuroscience (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Linda Lee.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Lewis, Mark H.
Electronic Access: INACCESSIBLE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021676:00001


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1 NEUROPLASTICITY IN AN ANIMAL MODEL OF ABNORMAL STEREOTYPED MOTOR BEHAVIOR: EFFECTS OF ENVIRONMENTAL ENRICHMENT By LINDA WEN HUA LEE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FU LFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 200 8 Linda Wen Hua Lee

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3 T o my parents, Jau Ming and Chu Chen Lee.

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4 ACKNOWLEDGMENTS I am grateful to many people for their time and help throughout the course of my graduate studies. First and foremost, I would like to thank Drs. Mark H. Lewis, Michael A. King, Kevin K.W. Wang, Darragh P. Devine, and Edwin M. Meyer for ser ving on my supervisory committee. I would also like to thank my fellow labmates, both past and present. Finally, I would like to thank the faculty, graduate students, and administrative staff of the Department of Neuroscience and the Interdisciplinary Prog ram in Biomedical Sciences.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ......................... 9 ABSTRACT ................................ ................................ ................................ ................................ ... 11 CHAPTER 1 ABNORMAL STEREOTYPED MOTOR BEHAVIOR ................................ ....................... 13 Introduction ................................ ................................ ................................ ............................. 13 Prevalence and Significance ................................ ................................ ................................ ... 14 Human Stereotypies ................................ ................................ ................................ ......... 14 Animal Stereoty pies ................................ ................................ ................................ ........ 16 Neurobiological Basis of Stereotypy ................................ ................................ ...................... 17 Clinical Studies ................................ ................................ ................................ ................ 17 Animal Studies ................................ ................................ ................................ ................ 19 Animal Models of Stereotypy ................................ ................................ ................................ 21 Genetic Models of Stereotypy ................................ ................................ ......................... 21 Models of Stereotypy Associated with Insults to the Central Nervous System .............. 23 Models of Pharmacologically Induced Stereotypy ................................ .......................... 25 Models of Stereotypy Associated with Adverse Environmental Circumstances ............ 25 Deer Mice ( Peromyscus maniculatus ) ................................ ................................ ............. 27 2 ENVIRONMENTAL ENRICHMENT ................................ ................................ .................. 30 Historical Perspective ................................ ................................ ................................ ............. 30 Behavioral Consequences ................................ ................................ ................................ ....... 30 Structural and Morphological Modifications ................................ ................................ .......... 32 Regulation of Gene Expression ................................ ................................ .............................. 35 Effects on Neuronal Sign aling ................................ ................................ ................................ 37 3 SPECIFIC AIMS ................................ ................................ ................................ .................... 40 4 EFFECTS OF ENVIRONMENTAL ENRICHMENT ON THE REGULATION OF DARPP 32 IN AN ANIMAL MODEL OF ABNORMAL S TEREOTYPED MOTOR BEHAVIOR ................................ ................................ ................................ ............................ 42 Introduction ................................ ................................ ................................ ............................. 42 Materials and Methods ................................ ................................ ................................ ........... 45 Animals ................................ ................................ ................................ ............................ 45 Behavioral Testing ................................ ................................ ................................ ........... 45

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6 Western Blotting ................................ ................................ ................................ .............. 46 Stati stical Procedures ................................ ................................ ................................ ....... 48 Results ................................ ................................ ................................ ................................ ..... 48 Behavioral Data ................................ ................................ ................................ ............... 48 Levels of tot al DARPP 32, phospho Thr34 DARPP 32, and phospho Thr75 DARPP 32 ................................ ................................ ................................ ................... 48 Levels of phospho Thr34 DARPP 32/total DARPP 32 and phospho Thr75 DARPP 32/total DARPP 32 ................................ ................................ ........................ 49 Discussion ................................ ................................ ................................ ............................... 49 5 EFFECTS OF ENVIRONMENTAL ENRICHMENT ON PATTERNS OF FOSB EXPRESSION IN AN ANIMAL MODEL OF ABNORMAL STEREOTYPED MOTOR BEHAVIOR ................................ ................................ ................................ ............ 59 Introduction ................................ ................................ ................................ ............................. 59 Methods and Materials ................................ ................................ ................................ ........... 61 Animals ................................ ................................ ................................ ............................ 61 Behavioral Testing ................................ ................................ ................................ ........... 62 Western Blotting ................................ ................................ ................................ .............. 63 Double Label Immunohistochemistry ................................ ................................ ............. 64 Image Analysis ................................ ................................ ................................ ................ 65 Statistical Procedures ................................ ................................ ................................ ....... 65 Results ................................ ................................ ................................ ................................ ..... 66 Behavioral Data ................................ ................................ ................................ ............... 66 FosB Expression in Selected Brain Re gions ................................ ................................ 66 Regional Distribution of Striatal FosB/ FosB Expression ................................ ............. 67 Compartmental Distribution of Striatal FosB/ FosB Expression ................................ ... 68 Discussion ................................ ................................ ................................ ............................... 69 6 EFFECTS OF ENVIRONMENTAL ENRICHMENT AND ABNORMAL STEREOTYPED MOTOR BEHAVIOR ON SYNAPTIC PROTEIN EXPRESSION ......... 84 Introduction ................................ ................................ ................................ ............................. 84 Materials and Methods ................................ ................................ ................................ ........... 86 Animals ................................ ................................ ................................ ............................ 86 Behavioral Testing ................................ ................................ ................................ ........... 87 Western Blotting ................................ ................................ ................................ .............. 88 Statistical Analysis ................................ ................................ ................................ .......... 89 Results ................................ ................................ ................................ ................................ ..... 89 Behavioral Data ................................ ................................ ................................ ............... 89 Effects of Housing Condition and Stereotyp y on Synaptic Protein Expression ............. 90 Discussion ................................ ................................ ................................ ............................... 91

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7 7 EFFECTS OF ENVIRONMENTAL ENRICHMENT ON SPATIAL AND STIMULUS RESPONSE LEARN ING DEFICITS ASSOCIATED WITH ABNORMAL STEREOTYPED MOTOR BEHAVIOR ................................ ....................... 98 Introduction ................................ ................................ ................................ ............................. 98 Materials and Methods ................................ ................................ ................................ ......... 100 Animals ................................ ................................ ................................ .......................... 100 Quantification of Stereotypic Behavior ................................ ................................ ......... 101 Morris Water Maze (MWM) Tes ting ................................ ................................ ............ 102 Spatial MWM Task ................................ ................................ ................................ 103 Cued MWM Task ................................ ................................ ................................ ... 103 Western Blotting ................................ ................................ ................................ ............ 103 Data Analysis ................................ ................................ ................................ ................. 105 Results ................................ ................................ ................................ ................................ ... 105 Subjects ................................ ................................ ................................ .......................... 105 Morris Water Maze Testing ................................ ................................ ........................... 106 Spatial MWM Task ................................ ................................ ................................ 106 Cued MWM Task ................................ ................................ ................................ ... 108 Synaptic Proteins ................................ ................................ ................................ ........... 109 Learning induced changes in synaptic protein expression ................................ ..... 109 Relationships between MWM performance and synaptic protein levels ............... 110 Discussion ................................ ................................ ................................ ............................. 110 8 CONCLUSIONS ................................ ................................ ................................ .................. 127 Summary of Findings ................................ ................................ ................................ ........... 127 Future Directions ................................ ................................ ................................ .................. 129 LIST OF REFERENCES ................................ ................................ ................................ ............. 132 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 160

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8 LIST OF TABLES Table page 5 1 Stereotypy scores for experimental groups. ................................ ................................ ....... 74 5 2 brain regions. ................................ ..................... 75 5 3 ............... 76 7 1 Stereotypy scores of e xperimental groups. ................................ ................................ ...... 117 7 2 Immunoblotting analysis of synaptic marker proteins. ................................ .................... 118

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9 LIST OF FIGURES Figure page 2 1 An example of an enriched environment shown in comparison to a standard laboratory cage. ................................ ................................ ................................ .................. 39 4 1 Stereotypy scores for environmentally enriched, low stereotypy ; environment ally enriched, high stereotypy; standard caged, low stereotypy ; and standard caged, high stereotypy mice. ................................ ................................ ................................ ................. 55 4 2 Western blot analysis of total DARPP 32, phospho Thr34 DARPP 32, and phospho Thr75 DARPP 32 levels in the dorsolateral striatum. ................................ ....................... 56 4 3 Concentrations of phospho Thr34 DARPP 32 and phosp ho Thr75 DARPP 32 normalized relative to total DARPP 32 levels. ................................ ................................ .. 57 4 4 Relationship between the proportion of total DARPP 32 phosphorylated at Thr75 and stereotypy. ................................ ................................ ................................ ................... 58 5 1 Representative immunoblot for the detection of FosB isoforms in the striatum. .............. 77 5 2 Schematic illustrating striatal subregions. ................................ ................................ ......... 78 5 3 Photomicrographs of sections double stained for FosB/ FosB nuclei and MOR 1 rich striosomes ................................ ................................ ................................ .................. 79 5 4 Subregional distribution of Fo immunoreactive nuclei ................................ .... 80 5 5 Correlations between the estimated number of FosB/ FosB immunoreactive nuclei and stereotypy scores. ................................ ................................ ................................ ........ 81 5 6 Distribution of FosB/ FosB induction in the striosome and matrix compartments of the striatum. ................................ ................................ ................................ ........................ 82 5 7 Changes in ISMP value more closely parallel changes in stereotypy. .............................. 83 6 1 Diagrammatic representation of the dissection procedure. ................................ ................ 95 6 2 Effects of environmental enrichment and motor stereotypy on s ynaptophysin protein expression in the dorsolateral striatum. ................................ ................................ ............. 96 6 3 Effects of environmental enrichment and motor stereotypy on NCAM 140 protein expression in the cerebral cortex. ................................ ................................ ....................... 97 7 1 llustration depicting the timeline of experimental events. ................................ ............... 119 7 2 Performance duri ng spatial MWM task acquisition ................................ ....................... 120

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10 7 3 Performance during spatial MWM probe trials. ................................ .............................. 121 7 4 Performance during cued MWM task acquisition. ................................ .......................... 122 7 5 Performance during cued MWM probe trials. ................................ ................................ 123 7 6 Representative immunoblots of the synaptic proteins synaptophysin and neural cell adhesion molecule. ................................ ................................ ................................ ........... 124 7 7 Relationship between synaptophysin levels in the dorsolateral striatum and cued MWM performance. ................................ ................................ ................................ ........ 125 7 8 Relationship between NCAM 120 levels in the hippocampus and cued MWM performance. ................................ ................................ ................................ .................... 126

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11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy NEUROPLASTICITY IN AN ANIMAL MODEL OF ABNORMAL STEREOTYPED MOTOR BEHAVI OR: EFFECTS OF ENVIRONMENTAL ENRICHMENT By Linda Wen Hua Lee August 2008 Chair: Mark H. Lewis Major: Medical Sciences -Neuroscience Although abnormal, repetitive body movements (i.e., stereotypies) manifest in many neuropsychiatric illnesses, the neurobio logical basis of these behaviors remains poorly characterized. Deer mice ( Peromyscus maniculatus ) provide an animal model of stereotypy in which the expression of stereotyped behaviors varies inversely as a function of environmental complexity. The objecti ve of the present studies was to identify the molecular and cellular mechanisms that contribute to enrichment related behavioral plasticity. We first examined the impact of environmental manipulations on the functional status of DARPP 32, a key mediator of dopamine signaling. The data revealed an inverse relationship between stereotypy and the proportion of DARPP 32 phosphorylated at Thr75. Furthermore, enrichment related attenuation of stereotypy was also associated with a decrease in total DARPP 32 expres sion. Using FosB as an index of neuronal activation, our second study examined the effects of environmental enrichment on long term changes in gene expression. The results showed that stereotypy was associated with regionally and compartmentally specific patterns of FosB induction with the effects of enrichment localized primarily to the dorsolateral striatum.

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12 Our third study examined the effects of enrichment on synaptic protein expression. Stereotypy was associated with decreased levels of synaptophysin in the dors olateral striatum and neural cell adhesion molecule 140 (NCAM 140) in the cortex, whereas enrichment was associated with increased expression of synaptophysin and NCAM 140 in the dorsolateral striatum and cortex, respectively. Our final study examined whet her stereotypy would be associated with other behavioral deficits and, coincidentally, whether the beneficial effects of environmental enrichment would also extend to these deficits. The results showed that stereotypy was associated with deficits in both spatial and stimulus response learning and memory ability. In addi tion, we found that enrichment was able to counteract deficits in spatial but not stimulus response learning. Collectively, these findings suggest that decreases in stereotypy and associated deficits due to environmental enrichment are linked to regionally specific changes in neuronal communication, gene expression, and synaptic function. Hopefully, the novel mechanistic insights derived from this research will eventually reveal new therapeutic avenues for the treatment of human repetitive behavior disorder s.

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13 CHAPTER 1 ABNORMAL STEREOTYPED MOTOR BEHAVIOR Introduction Stereotypy refers to sequences of motor behavior which are characterized by excessive repetition, topographical invariance, and purposelessness (Lewis and Baumeister, 1982; Berkson, 1983; Mason, 1991; Ridley, 1994) These behaviors manifest in a wide variety of animal species, including man, and are striking in their frequency and intensity as well as in the rhythmical quality of their expression. Furthermore, because they occur absent a functional context and have, on occasion, been associated with maladaptive consequences, stereotypies are also generally regarded as being abnormal behaviors. Motor stereotypies represent one specific form of abnormal repetitive behavi or. Multiple categories of clinically relevant repetitive behaviors exist. These can include purely motoric behaviors, such as stereotyped body movements, self injurious behaviors, repetitive manipulation of objects, tics, dyskinesias, and akathisia, as we ll as more complex behaviors, such as repetitive verbal behaviors (e.g., echolalia), compulsions, rituals and routines, insistence on sameness, and preoccupation with narrow, circumscribed interests (Lewis and Bodfish, 1998) The studies comprising this dissertation employed an animal model of abnormal stereotyped motor behavior. However, animal behaviors can be interpreted in many different ways by many different researchers. Therefore, the following literature review will focus primarily on stereotypy, however, with the understanding that these behaviors may share considerable phenomenolo gical and conceptual overlap with a broad spectrum of abnormal repetitive behaviors.

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14 Prevalence and Significance Human Stereotypies Stereotypies are common in typically developing infants (Thelen, 1979) and often occur at subclinical levels within normal adult populations (Berkson et al., 1999; Rafaeli Mor et al., 1999) However, stereotypies can also become behaviorally dominant in many clini cal conditions. Indeed, the behavioral phenotypes of many neurodevelopmental (e.g., autism, mental retardation, Rett syndrome), psychiatric (e.g., schizophrenia, obsessive compulsive disorder, ongenital blindness or deafness, Nyhan syndrome, Prader Willi syndrome, Cri du Chat syndrome) disorders often include specific forms of stereotyped behaviors. For example, body rocking, head rolling, and hand flapping are among the most common topographies of stereotypic behavior observed in autistic and mentally retarded individuals. Repetitive hand movements (e.g., wringing, washing, clapping, and twirling) are characteristic of Rett syndrome (Temudo et al., 2007) while eye p ressing, eye rubbing, and eye poking are among the most prevalent stereotypies reported in congenitally blind children (Troster et al., 1991; Fazzi et al., 1999) Originally described in chronic abusers of amphetam ine (Ellinwood, 1967; Rylander, 1972; Ellinwood et al., 1973) stereotypy characterized by an intense fascination with the repetitive manipulation of objects (e.g., continual handling, assembl ing and disassembling, collecting, sorting, etc.) has also been therapy (Evans et al., 2004; Lawrence et al., 2007) Finally, repetitive self injurious behaviors, such as self biting and skin picking, feature prominently in the behavioral profiles of Lesch Nyhan (Robey et al., 2003) and Prader Willi patients (Symons et al., 1999) respectively.

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15 Although considered relatively innocuous by some clinicians, stereotypies can, nevertheless, have profound and multitudinous consequences on adaptive functioning (Matson et al., 1997) First, because they are often perceived as being inappropriate to context, stereotypies can be stigmatizing (Jones et al., 1990) As a result, this may limit participation in social activities. In fact, it has been shown that mentally retarded individuals with stereotypies display more negative nonverbal social skills and fewe r general positive social skills (Matson et al., 2006) and have greater difficulties in forming interpersonal relationships, communicating, and socializing than non stereotypic cont rols (Matson et al., 1997) Stereotypies are also considered problem behaviors because of their disruptive effects on learning. Stereotypies have been shown to impair the acquisition of new skills (Koegel and Covert, 1972; Dunlap et al., 1983) as well as the performance of established skills (Morrison and Rosales Ruiz, 1997) therefore, controlling stereotypies may be a necessary precondition for effective learning. Indeed, cor relational studies have shown that developmentally disabled individuals who exhibit more stereotypic behaviors also have lower IQs (Turner, 1999; Bishop et al., 2006) they can hinder sustained interaction with the environment. In extreme cases, severe and persistent stereotypies ca n even become crippling in their interference with intentional activities. This is the case in Rett syndrome where the gradual loss of purposeful hand movements is a tragic consequence seen with disease progression (Temudo et al., 2007) Perhaps the most clinically significant aspect of stereotypy is its r elationship to self injury. Stereotypies are frequently comorbid with and have been proposed as a potential precursor of self injurious behaviors (Kennedy et al., 2002; Richman and Lindauer, 2005; Richman, 2008)

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16 Well established self injurious behaviors are more difficult to treat than emerging self injurious behaviors. Therefore, it may be advantageous for clinicians to target stereotypies for therapeutic intervention antecedent to the development of more serious, self injurious behaviors. Animal Stereotypies Although mostly absent in wild populations, stereotypies are commonly observed in animals kept in captivity, such as those housed in zoo, farm, and laboratory environments. Similar to the distinct topographies observed in clinical stereotypies, animal stereotypies are species specific with widely varying morphologies. To illustrate, some examples of animal stereotypies include swaying and tr unk swinging in elephants (Elzanowski and Sergiel, 2006) pacing in bears (Vickery and Mason, 2004) and big cats (Jenny and Schmid, 2002; Bashaw et al., 2007) licking in giraffe and okapi (Bashaw et al., 2001) repetitive pawing in wombats (Hogan and Tribe, 2007) body rocking, self clasping, and self biting in nonhuman primates (Lutz et al., 2003; Mallapur and Choudhury, 2003; Tarou et al., 2005) sham chewing and bar mouthing in tethered sows (Cronin and Wiepkema, 1984) crib biting, box walking, and wind sucking in horses (Nicol, 1999) head twirling and nodding in farmed mink (Mason, 1993) route tracing in songbirds (Garner et al., 2003b) feather picking and per ch dancing in parrots (Garner et al., 2003a; Meehan et al., 2004; Garner et al., 2006) digging in gerbils (Wiedenmayer and Brunner, 1993) bar mouthing in bank voles (Garner and Mason, 2002) and wire gnawing and jumping in ICR mice (Wrbel et al., 1996) Indeed, stereotypies are the most common category of abnormal behavior reported in c aptive animals (Wrbel, 2001) with a recent estimate suggesting that stereotypies are displayed by over 85 million zoo, farm, and laboratory animals worldwide (Mason and Latham, 2004) The high occurrence of stereotypic behaviors in captive animals is a source of intense concern for animal caretakers. Among the deleterious consequences that have been attributed to stereotypy

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17 are impaired growth, self injury, decreased reproductive success, and increased mortality (Mason, 1991; Mason et al., 1995; Lutz et al., 2003) Because of their frequent association with adverse environmental circumstances and suboptimal health outcomes, stereotypies in captive populations have been construed as an indicator of poor animal welfare and a warning sign of potential suffering (Mason, 1991; Mason and Latham, 2004) Furthermore, with regard to the use of experimental animals, stereotypies have also been interpreted as symptomatic of abnormal brain functioning, thus, raising concerns about the validity, reliability, and replicability of scien tific research conducted using stereotypic laboratory animals (Garner, 2005) Neurobiological Basis of Stereotypy Despite its widespread prevalence and significance, the critical factors con tributing to the emergence and persistence of stereotypic behaviors remain poorly defined. However, while the specific mechanisms are unknown, substantial evidence from both clinical and animal studies indicates that behavioral stereotypy may originate as a consequence of basal ganglia dysfunction. Clinical Studies Many experimental approaches have been used to study stereotypic behaviors in clinical populations. For example, analysis of spontaneous eye blink rates can be employed as a non invasive probe of central dopamine activity (Karson, 1983) Several studies have shown that mentally retarded patients who exhibit frequent stereotyped behaviors have significantly lower blink rates than matched controls who do n ot engage in stereotypy, suggesting that hypodopaminergic function might be responsible for stereotypy in these subjects ( MacLean et al., 1985; Bodfish et al., 1995; Roebel and MacLean, 2007) Dopamine functioning in mentally retarded individuals has also been evaluated by measuring plasma concentrations of homovanillic acid (HVA), a dopamine metabolite (Lewis et al., 1996) In this study, individuals

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18 with high levels of stereotypy were found to have lower levels of HVA, providing further support for a hypothesis of dopamine deficiency in relation to stereotyp y. In addition to biobehavioral and biochemical markers of dopamine function, n europsychological tasks designed to detect basal ganglia pathology have also been used to study stereotypic subjects. As an example of one task, patients are presented with two stimuli over a series of trials and instructed to find the rule governing reward presentation. In actuality, the chosen stimulus is rewarded at random for each trial. Whereas normal controls will produce a random sequence of responses over time, subjects w ith damage to the basal ganglia will demonstrate a tendency to repeat a specific response, a state called recurrent perseveration. The degree of recurrent perseveration, as measured by this task, has been shown to be significantly correlated with t he sever ity of spontaneously occurring stereotypies i n both autistic (Turner and Russell, 1997) and schizophrenic (Frith and Done, 1983) patients. This relationship has also been demonstrated using other tasks to measure perseveration. For example, in a study of high functioning autistic individuals, a significant correlation was found between repetitive behavior scores and the number of perseverative responses in the Wisconsin Card Sorting Task (South et al., 2007) In another study of autistic adults (Lopez et al., 2005) subjects were given a battery of neuropsychological tests designed to measure executive functions (i.e., cognitive flexibility, working memory, response inhibition, planning, and flu ency). Of the processes examined, cognitive rigidity was the only executive deficit which predicted the restricted, repetitive symptoms of the autistic subjects, suggesting that the cognitive impairments associated with the repetitive behaviors were select ive for the basal ganglia rather than being global in nature. The presence of neuroanatomical abnormalities associated with stereotyped behaviors have also been localized to the basal ganglia using neuroimaging techniques. Reductions in the

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19 absolute volume of the caudate nucleus have been reported in autistic children with simple motor stereotypies involving repetitive movements of the arms and hands (Kates et al., 2005) Similarly, abnormalities in caudate volume have also been found in association with bot h lower order and higher order repetitive behaviors in autism (Sears et al., 1999) Interestingly, a positive corr elation was found between caudate volume and stereotyped motor mannerisms, a lower order behavior, whereas a negative correlation was found between caudate volume and higher order behaviors, such as compulsions, rituals, and resistance to change. This is i n contrast to another study of repetitive behaviors in autism wherein increases in right caudate and total putamen volumes were found to be driven primarily by the presence of higher order repetitive behaviors (Hollander et al., 2005) Although t he direction of change is different in these studies, these results collectively demonstrate that a relationship exists between structural abnormalities of the basal ganglia and repetitive behaviors in autism. On occasion, individual case reports within th e clinical literature have also documented the appearance of motor stereotypies following damage to basal ganglia structures. In one instance, a 17 year old boy began demonstrating circling behaviors and complex hand stereotypies after experiencing a right putaminal infarct (Maraganore et al., 1991) In another case, a previously healthy 27 year old woman with face, neck, and limb stereotypies was later found to have a lesion of the right caudate nucleus (Edwards et al., 2004) For the most part, these observations are congruent with hypothesis based experimental findings and also serve to provide further support for a role of compromised basal ganglia function in the pathogenesis of stereotyped behaviors. Animal Studies Experimental evidence from animal studies also lend credence to the theory that disruption s in the cortico basal gan glia neural circuits which control motor behavior culminate in

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20 stereotypy. In particular, pharmacological data have implicated the involvement of the nigrostriatal dopamine system in the production of stereotypies. It has been known since the late 1960s that motor stereotypies can be reliably induced in a wide variet y of animal species with dopaminergic stimulation of the striatum (Ernst and Smelik, 1966; Randrup and Munkvad, 1967; Fog, 1969) Furthermore, upon repeated stimulation, the behavioral response becomes progressiv ely augmented such that stereotypies are elicited more rapidly as well as demonstrate increased duration and intensity (Segal et al., 1980) In contrast, intrastriatal antagonism of dopamine receptors has b een shown to have an attenuating effect on stereotypic behaviors (Arnt, 1985; Presti et al., 2003) Obstruction of dopaminergic neurotransmission accomplished by interf ering with the biosynthetic production of dopamine (Odberg et al., 1987) or the destruction of dopamine neurons (Creese and Iversen, 1972; Fibiger et al., 1973) also produces a similar effect. In line with a critical role of dopamine i n the generation of stereotypies, genetic manipulations which increase extracellular levels of dopamine have also been found to evoke stereotyped patterns of behavior (Berridge et al., 2005) Furthermore, alterations in the density (Helmeste et al., 1981; Sharman et al., 1982; Fields et al., 1991; McBride and Hemmings, 2005) or sensitiv ity (Lewis et al., 1990) of dopamine receptors are also commonly associated with stereotypies. Similarly, changes in the concentration of the dopam ine metabolites, HVA and DOPAC, have been observed coincident with the development of stereotypic behaviors (Fry et al., 1981) Patterns of early gene activation in the neurochemically distinct, striosome and matrix, compartments of the striatum are highly accurate predictors of psychostimulant induced stereotypies (Canales and Graybiel, 2000; Capper Loup et al., 2002; Saka et al., 2004)

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21 Interestingly, stereotypies in rodent (Garner an d Mason, 2002) and avian (Garner et al., 2003b; Garner et al., 2003a) species are also correlated with impaired performance on neuropsychological tasks analogous to those used to detect basal ganglia pathology in clinical populations. Overall, a synthesis of the extant research tends to implicate the basal ganglia as the neural substrate of stereotypy. Nevertheless, it is clear that much still remains to be learned about the neurobiological basis of stereotypy. For this rea son, animal models of stereotypy may prove to be invaluable experimental tools. Animal Models of Stereotypy Animal models of stereotypy can generally be classified into four broad categories: (i) genetic models of stereotypy, (ii) models of stereotypy asso ciated with insults to the central nervous system, (iii) models of pharmacologically induced stereotypy, and (iv) models of stereotypy associated with adverse environmental circumstances. Genetic Models of Stereotypy Motor stereotypies have been observed i n conjunction with transgenic mouse models of various clinical disorders. Many neurodevelopmental disorders are characterized by patterns of behavior which may be described as restricted, repetitive, and stereotyped. Likewise, the behavioral aberrations ch aracteristic of their genetic models also include specific forms of stereotyped behavior. For example, mutations in the methyl CpG binding protein 2 ( MECP2 ) gene are responsible for the majority of Rett syndrome cases. Mice expressing truncated MeCP2 prote in exhibit repetitive forelimb movements resembling the distinctive hand stereotypies (e.g., hand wringing, waving, and clapping) observed in Rett syndrome patients (Shahbazian et al., 2002; Moretti e t al., 2005) The human genetic disorder Angelman syndrome (AS) results from a deletion or mutation in the 15q11 13 region of maternally derived chromosome 15. The

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22 GABRB3 gene, which codes for the 3 subunit of the GABA A receptor, lies within this region and is thought to contribute to the clinical manifestations of the disease. Paralleling the repetitive behaviors associated with AS, gabrb3 homozygous knockout mice show stereotyped behaviors r hours (Homanics et al., 1997; DeLorey et al., 1998) As another example, Ts65Dn mice are segmentally trisomic for the distal portion of mouse chromosome 16, the region containing murine orthologs to human chromosome 21, and provide a model for Down syndrome. The behavioral repertoire of Ts65Dn mice include s behaviors, such as repetitive jumping and cage top twirling, which recall the abnormal repetitive behaviors often seen in individuals with mental retardation (Turner et al., 2001) Because repetitive behaviors can be conceptualized as a neurobehavioral dimension which cuts across many neuropsychiatric disorders, mutant mouse models of other human repetitive behavior disorders, such as obsessive hold relevance to stereotypy. Hyperdopaminergic mice with a knockdown mutation of the dopamine transporter ( DAT (Berridge et al., 2005) Gene based knockdown of DAT reduces adult DAT expression to 10% of wild type levels and elevates extracellular dopamine concent rations in the striatum to 170% of wild type controls. Behaviorally, DAT knockdown mice perform syntactic grooming chains which become increasingly more fixed and less resistant to disruption. Similarly, compulsive grooming behaviors are also evident in mi ce with genetic deletion of SAP90/PSD95 associated protein 3 ( Sapap3 ), which codes for a postsynaptic scaffolding protein at excitatory synapses (Welch et al., 2007) SAPAP3 shows high levels of expression in the striatum; correspondingly, the cortico striatal synapses of Sapap3 null mice demonstrate multiple defects, including

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23 reduced glutamatergic transmission, altered NMDA receptor subunit composition, and changes in the structu re of the postsynaptic complex. In addition to transgenic mouse models, other genetic models of stereotypy have also been developed based on the observed heritability of the behavior. Selective breeding studies have shown that the offspring of stereotypic bank voles are six to seven times more likely to develop stereotypies than the offspring of non stereotypic animals (Schoenecker and Heller, 2000) This pattern of inheritance holds true when only one of the parents is stereotypic. Moreover, the specific topography of stereotypy which develops in the offspring appears to be strongly related to the maternal form of stereotypy. Similarly, a genetic basis for stereotypy has also been demonstrated in other species. Stereotypies are four times more common in the offspring of stereotypic female African striped mice ( Rhabdomys pumilio ) than in the offspring of non stereotypic females (Schwaibold and Pillay, 2001) Furthermore, cross fostering experiments indicate that this tendency to develop stereotypy is independent of non genetic factors (e.g., learned social behaviors) as the offspring of stereotypic mothers are still more likely to develop stereotypies than the offspring of non stereotypic mothers regardless of whether they are raised by their own biological mothe rs or a foster mother. Finally, genetic transmission of stereotypy has also been demonstrated in farm mink ( Mustela vison ), and the development of divergent lines of low stereotyping and high stereotyping mink has been used to conduct biological and welfa re related investigations into the significance of stereotypies (Jeppesen et al., 2004; Svendsen et al., 2007) M odels of Stereotypy Associated with Insults to the Central Nervous System The behavioral sequelae of targeted insults to the central nervous system often include the appearance of stereotypic behaviors. As mentioned previously, stereotypies are a defining feature of autism. Rather than targeting the major candidate genes or loci thought to be associated with

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24 autism, some animal models have examined the role of perinatal risk factors in the etiology of autism. These models have been generated based on the o bservation that prenatal exposure to teratogenic agents increases the risk of autism. For example, exposure to valproic acid (VPA), an anti epileptic drug, on embryonic day 12.5 in rats not only reproduces neuroanatomical abnormalities similar to those rep orted in autistic patients but also long term disturbances in postnatal behavior including increased time spent engaged in stereotypic activity (Ingram et al., 2000; Schneider and Pr zewlocki, 2005) Intriguingly, these behaviors appear to be sensitive to environmental manipulations as rearing VPA treated pups in an enriched environment ameliorates many of these behavioral deficits (Schneider et al., 2006) The pathogenesis of autism has also been linked to viral infection during early development. In support of this putative association, intracerebral inoculation of newborn rats with Borna disease virus induces neuroanatomical and neurochemical deficits correlated with those seen in autism and recapitulates many of the behavioral impair ments, including stereotypies, in common with clinical aspects of the human disease (Hornig et al., 1999) To demonstrate an autoimmune etiology for autism, studies have also been performed wherein rhesus monkeys were exposed prenatally to human IgG collected from the mothers of autistic children (Martin et al., 2008) Phenotypical analysis of these monkeys confirms that their behaviors are characterized by increases in whole body stereotypies. An autoimmune etiology has also been proposed levels of autoantibodies are infused into the ventrolateral striatum of rats, they begin to display high rates of oral stereotypies, such as wood chip eating, self gnawing, biting, licki ng, and taffy pulling (Taylor et al., 2002) As a final example of insult based stereotypy, administration of the

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25 dopaminergic neurotoxicant, 1 methyl 4 phenyl 1,2,3,6 tetrahydropyridine (MPTP), also results in acute increases in stereotyped behaviors, such as grooming or gnawing (Linder et al., 1987) Models of Pharmacologically Induced Stereotypy Insights into the molecular mechanisms which underlie stereotypy have been primarily derived from neuropharmacological investigations. Early experiments established the importance of central dopamine transmission in the generation of stereotypies by demonstrating that stereotypies can be induced in numerous mammalian species with intrastriatal or systemic administration of dopamine agonists (e.g., amphetamine, methamphetamine, apomorphine, cocaine, pemoline, methylphenidate, phenmetrazine) (Ernst and Smelik, 1966; Randrup and Munkvad 1967; Fog, 1969) Indeed, dopamine has received much of the focus of studies designed to elucidate the neurobiology of stereotypy; however, pharmacological agents which interact with other neurotransmitter systems have also been shown to consistently in duce stereotypies in laboratory animals. These compounds include NMDA antagonists (e.g., MK 801, AP 5, phencyclidine [PCP], dextromethorphan, dextrorphan, ketamine) (Schmidt, 1986; Tiedtke et al., 1990; Ishmael et al., 1998; Vandebroek et al., 1998) opiate agonists (e.g., morphine, endorphin, levorphanol, pentazocine, nalorphine) (Iwamo to and Way, 1977; Nistico et al., 1981) serotonin agonists (e.g., lysergic acid dietyhlamide [LSD] and 8 hydroxy 2 (di n propylamino) tetralin [8 OH DPAT] ) (Fog, 1969; Dourish et al., 1985) and GABA agonists (Scheel Kru ger et al., 1978) Models of Stereotypy Associated with Adverse Environmental Circumstances Stereotypies often develop in response to abnormal rearing conditions. Higher mammals, such as nonhuman primates, seem to be especially vulnerable to the effects o f partial or total social isolation during early development. In addition to profound deficits in social behavior, infant rhesus monkeys deprived of early social contact with their mother or peers exhibit more

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26 stereotypic behaviors (e.g., body rocking, sel f clasping, thumb sucking, and self wounding) than their group housed counterparts even after attempts are made to rehabilitate them by housing them with their normally reared peers (Cross and Harlow, 1965) (Harlow et al., 1965) Furthermore, when examined as adults, socially deprived monkeys show increased sensitivity to the effects of dopamine (Lewis et al., 1990) as well as substantial alterations in the neurochemical composition of the striatum (Martin et al., 1991) St ereotypies also arise in frustrative situations such as when animals are chronically deprived of the opportunity to perform highly motivated behaviors. For example, in the wild, Mongolian gerbils ( Meriones unguiculatus ) build elaborate networks of undergro und tunnels with numerous entrances to different chambers that serve as nest, food storage, and waste areas. Gerbils raised in standard laboratory cages are prevented from engaging in this natural behavioral pattern. As a result, they develop stereotypic d igging behaviors, which consist of long lasting digging bouts directed at the corners of their cages (Wiedenmayer and Brun ner, 1993) Neither variations in cage size (Wiedenmayer, 1996) nor digging substrate (Wiedenmayer, 1997b) alone are able to reduce these behaviors. In contrast, wh en raised in laboratory cages connected to artificial burrow systems, young gerbils do not develop stereotypic digging behaviors (Wiedenmayer, 1997a) However, these burrows can only prevent the development of stereotypy when the entrances appear tunnel like in shape (Wiedenmayer, 1997b; Waiblinger and nig, 2004) suggesting that the development of these behaviors is tied to the lack of highly specific environmental stimuli. Environmental res triction, in the form of standard laboratory caging, has also been shown to induce stereotypic behaviors in bank voles ( Clet h r ionomys glareolus ). The most common form of captivity induced stereotypy in bank voles is repetitive up and down jumping (Odberg,

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27 1986) Consistent with an etiological role for environmental factors, the augmentation of environmental complexity (e.g., the addition of shelter, nesting materia ls, climbing structures, and dietary enrichments) significantly reduces the development of these behaviors in young voles (dberg, 1987; Sorensen, 1987) Of note, stereotypies can also be pharmacologically induced in bank voles. Systemic injections of apomorphine elicit stereotyped licking (Vandebroek and dberg, 1997) whereas MK 801 administration induces intensive sniffing and unidirectional circling behaviors (Vandebroek et al., 1998) In both cases, not only are the topographies of drug induced stereotypy distinct from the stereotyped jumping normally seen in caged voles but the phar macological manipulations also have no effect on levels of captivity induced jumping stereotypy. These data suggest that restriction induced and drug induced stereotypies may not share the same underlying neural mechanisms and speak to the importance of id entifying valid animal models of clinical stereotypies. Deer Mice ( Peromyscus maniculatus ) Peromyscus maniculatus bairdii or prairie deer mice are wild type, muroid rodents among the most abundant of North American mammals. Extremely active animals, deer m ice require a relatively large home range in the wild of 242 to 3,000 m 2 (Wolff, 1989) When confined to sta ndard laboratory cages, these animals develop remarkably high rates of motor stereotypies. For this reason, our lab has adopted deer mice as an animal model of stereotypy. Despite their species specific nature, the stereotypic behavior s (i.e ., patterned r u nning, repetitive jumping, and backward somersaulting) exhibited by deer mice mimic the fundamental characteristics observed in clinical populations; i.e., these invariant and apparently functionless behaviors emerge early in development and persist throug hout the lifespan of the animal. Moreover, our lab has established that, while these stereotypies occur spontaneously under

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28 standard laboratory housing, their development may be delayed and their frequency reduced by rearing the mice in an enriched environ ment (Powell et al., 1999) The spontaneous nature of these behaviors is an important aspect of this model. By far, much of what is currently known about the neurobiology of stereotypy comes from studies of drug induced stereotypies. However, studies in our own (Presti et al., 2002; Presti et al., 2004a) and other labs (Vandebroek and dberg, 1997; Vandebroek et al., 1998) have demonstrated that the topographies of spontaneously emitted and pharmacologically induced stereotypies are often qualitatively different in the same species. Therefore, it is likely that they are subserved by different mechanisms. Clinically relevant stereotypies most often emerge spontaneously. As such, an animal model of spontaneous stereotypy would provide a more accurate representation of the pathophysiological mechanisms underlying clinical stereotypies. The fact that these behaviors are subject to environmental regulation is also of particular scientific utility. Environmental enrichment reduces stereotypy in de er mice. Environmental enrichment also recruits mechanisms similar to those active during neuroplasticity. By comparing the brains of environmentally enriched, low stereotypy mice to environmentally enriched, high stereotypy and standard caged mice, we can identify the specific mechanisms which underlie the ameliorative effects of environmental enrichment on stereotypy. This information is useful not only because it can provide clues into the biological basis of stereotypy but also because it can provide po tential targets for therapeutic applications. Finally and most importantly, this model also appears to demonstrate construct validity. As reviewed previously, clinical studies implicate basal ganglia dysfunction in the etiology of stereotypy. Similarly, ou r work with this model has linked the expression of stereotypic behaviors to alterations in the cortico basal ganglia loop circuits which control the selection and

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29 initiation of motor behavior (Presti et al., 2002; Turner et al., 20 02; Presti et al., 2003; Turner and Lewis, 2003; Turner et al., 2003; Presti et al., 2004a; Presti et al., 2004b; Presti and Lewis, 2005)

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30 CHAPTER 2 ENVIRONMENTAL ENRICH MENT Historical Perspective The concept of experience dependent plasticity was first proposed by Donald Hebb in the late 1940s (Hebb, 1949) In his revolutionary monograph The Organization of Behavior Hebb reported that rats he initially brought home as pets for his children later demonstrated superior problem solving ability when compared to their laboratory reared littermates. He concluded that yielded similar findings (Forgays and Forgays, 1952; Hymovitch, 1952) In the 1960s, R osenzweig and colleagues at the University of California Berkeley were able to expand on this specific neurochemical and neuroanatomical alterations in the brain (Rosenzweig and Bennett, 1996) More systematic investigations into the effects of environmental enrichment were to follow in later decades, most notably with the contributions of Greenough and co workers (Markham and Greenough, 2004) giving birth to what is now considered a classical line of research wherein the brains and behavior of animals reared in an enriched environment are compare d to those reared in a standard laboratory environment (Figure 2 1). Behavioral Consequences Environmental stimulation modifies a myriad of behavioral characteristics in normal, wild type animals. The beneficial effects of environmental enrichment on learn ing and memory ability are among the most well studied of these behavioral changes. Environmental enrichment has been shown to enhance performance in a wide variety of tasks designed to test spatial and non

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31 spatial learning (Krech et al., 1962; Nilsson et al., 1999; Duffy et al., 2001; Williams et al., 2001; Bruel Jungerman et al., 2005; Leggio et al., 2005; Barbelivien et al., 2006) as well as protect against memory decline due to normal aging (Winocur, 1998; Soffi et al., 1999; Kobayashi et al., 2002; Frick and Fernandez, 2003; Frick et al., 2003; Bennett et al., 2006; Harburger et al., 2007a; Harburger et al., 2007b) Environmental enrichment also confers behavioral advantages with respect to emotionality and stress responsiven ess. For example, enriched animals show decreased fear and anxiety not only when tested on artificial constructs, like the elevated plus maze (Chapillon et al., 1999; Benaroya Milshtein et al., 2004; Friske and Gammie, 2005; Pena et al., 2006) but also when confronted with natural stressors, like pred ators (Klein et al., 1994) Enriched animals also tend to be less neophobic and, as a result, exhibit greater novelty seeking and exploratory behaviors (Widman and Rosellini, 1990) Decreased reactivity to the deleterious effects of stress is also thought to contribute to the fewer depressive like behaviors observed in enriched animals (Brenes and Fornaguera, 2008; Brenes et al., 2008) The effects of environmental enrichment on social behaviors appear to be gender specific. When housed in an enriched envir onment, female mice generally display sociopositive behaviors (e.g., grooming cagemates), whereas male mice show increased aggression (Haemisch et al., 1994) Yet, in spite of the possible negative effects of aggressive behavior (e.g., increased stress, risks of injury, etc.), enriched male mice also show more play behaviors, an indicator of good welfare, implying that they are able to cope with the increased aggres sion brought about by enrichment (Marashi et al., 2003) Of direct relevance to the present studies, environmental enrichment also has a major impac t on the development of stereotypic behaviors. As reviewed previously (Chapter 1),

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32 environmental impoverishment is known to induce stereotypic behaviors in captive animals often with detrimental effects on the wellbeing of the animal. Consequently, environ mental enrichment has emerged as a means to eradicate these behaviors. In this context, enrichment is used as an animal husbandry tool; however, researchers have also utilized environmental stimulation as an experimental tool to improve functional outcome in animal models of human disease. Similar to the positive effects observed in studies of intact animals, these experiments have demonstrated that enrichment can ameliorate the cognitive deficits associated with fetal alcohol syndrome (Hannigan et al., 1993; Hannigan and Berman, 2000) developmental lead exposure (Schneider et al., 2001; Guilarte et al., 2003) fragile X syndrome (Restivo et al., 2005) Down syndrome (Martinez Cue et al., 2002) traumatic brain injury (Hamm et al., 1996; Hoffman et al., 2007; Kline et al., 2007) ischemic stroke (Dahlqvist et al., 2004) Hunti (Nithianantharajah et al., 2007) and (Arendash et al., 2004; Jankowsky et al., 2005; Berardi et al., 2007; Costa et al., 2007) In addition to its cognitive effects, exposure to an enriched environment can also accelerate the functional recovery of motor skills following tra umatic brain injury (Hoffman et al., 2007; Kline et al., 2007) and stroke (Ohlsson and Johansson, 1995; Johansson, 1996) (Jadavji et al., 2006; Steiner et al., 2006 ) and delay the onset and progression of motor symptoms in (van Dellen et al., 2000; Hockly et al., 2002; Schilling et al., 2004; Spires et al., 2004) Structural and Morphological Modifications The behavioral effects of e nvironmental enrichment are mediated through a host of structural alterations in the brain. First evidence for experience induced morphological plasticity was suggested by the observation that animals reared in an enriched environment possess heavier (Bennett et al., 1969) and thicker (Diamond et al., 1976) cerebral cortices than their standard

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33 caged counterparts. Subsequent studies were later able to pinpoint the underlying changes in neuronal, dendritic, and synaptic properties which contribute to these gross anatomical differences. Although there is a decrease in neuronal density following enrichment (Diamond et al., 1966) the neurons of enriched animals demonstrate larger cel l bodies and nuclei (Diamond et al., 1967) Enrichment related neuronal remodeling also involves changes in dendritic field dimensions: not only are there increases in the number and length of dendritic branches following enrichment (Wallace et al., 1992) but the dendritic arbor is also typified by the presence of more higher order branches (Volkmar and Greenough, 1972; Greenough and Volkmar, 1973; Greenough et al., 1973) In addition to synaptogenesis, as measured by increased dend ritic spine density (Turner and Greenough, 1985) or gr eater number of synapses per neuron (Sirevaag and Greenough, 1987; Jones et al., 1997) changes in existing synaptic morphology also occur in response to environmental stimulation. These can include inc reases in the size of pre and post synaptic components (Diamond et al., 1975; Sirevaag and Greenough, 1985) alterations in the shape of dendritic spine (Comery et al., 1996) th e appearance of perforations in the post synaptic density (Greenough et al., 1978) or the formation of multiple synaptic boutons (Jones et al., 1997) Many of these initial morphological studies focused on cortical regions; however, experience induced modifications in brain structure have since been demonstrated in several other regions, including the cerebellum (Floeter and Greenough, 1979; Greenough et al., 1986) superior colliculus (Fuchs et al., 1990) basal ganglia (Comery et al., 1995; Comery et al., 1996) and hippocampus (Faherty et al., 2003) The hippocampus, in particular, appears to be especially sensitive to the effects of environmental enrichment.

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34 Once regarded with a certain amount of scientific skepticism, adult neurogenesis in the mammalian brain is now a well accepted phenomenon. Exposure to an enriched environment, both during early adulthood (Kempermann et al., 1997) and old age (Kempermann et al., 2002) in duces hippocampal neurogenesis. Not only is this effect regionally specific to the hippocampus (Brown et al., 2003) but enrichment has also be en shown to reduce spontaneous apoptosis in the hippocampus in effect promoting the survival of the new neurons (Young et al., 1999) The newly generated cells develop into mature neurons with morphological characteristics and electrophysiological response properties similar to those found i n dentate granule cells (van Praag et al., 2002) Furthermore, when neurogenesis is blocked by treatment with an anti mitotic agent during the enrichment period enrichment related behavioral improvements in hippocampus dependent learning tasks are abolished (Bruel Jungerman et al., 2005) These data suggest that the new neurons are not only functionally integrated into the existing neural circuitry but are also critical for the behavioral improvements seen following enrichment. Although traditionally less well studied, environmental enrichment also impacts non neuronal elements in the brain. Astrocytic hypertrophy (Sirevaag and Greenough, 1987) and hyperplasia (Szeligo and Leblond, 1977; Sirevaag and Greenough, 1991) as well as increased ensheathment of synapses by astrocytic processes (Jones and Greenough, 1996) have been reported following enrichment. Similarly, increases in the volume fraction (Sirevaag and Greenough, 1987) and number (Szeligo and Leblond, 1977) of oligodendrocytes as well as increases in axon myelination (Juraska and Kopcik, 1988) have also been observed. In addition to glial proliferation, the cerebrovasculature of enriched animals are also characterized by larger, more elaborately branched capillaries (Black et al., 1987; Sirevaa g and Greenough, 1987; Sirevaag et al., 1988; Black et al., 1991) Because the spatiotemporal pattern of gliogenesis and

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35 angiogenesis often parallels the induction of experience dependent neuronal plasticity, it is hypothesized that these changes serve to support and optimize the changes in neuronal connectivity which occur as a result of enrichment. Regulation of Gene Expression Long lasting morphological plasticity in the brain requires changes in gene expression. Exposure to complex environmental stimul i enhances the activity of immediate early genes and transcription factors which regulate the expression of structural proteins involved in neuronal reorganization. Site specific induction of eg r 1 (also known as zif 268 NGFI A k rox 24 and zenk ) (Wallace et al., 1995; Pinaud et al., 2002; Toscano et al., 2006) arc (Pinaud et al., 2001) egr 2 (aka krox 20 ) (Ronnback et al., 2005) h omer 1a j unB (Koh et al., 2005) and CREB (Young et al., 1999; Williams et al., 2001) have all been reported foll owing enrichment. These increases are not elicited in response to other stimuli, such as handling, and only occur in regions known to experience enrichment related structural change. For the most part, levels of gene induction also correlate with improveme nts on learning tasks known to be affected by enrichment. In addition to experiments designed to elucidate the effects of environmental enrichment on individual genes, the advent of microarray technology has allowed researchers to characterize how the geno me changes as a whole in response to environmental stimulation. To this end, gene expression profiling has shown that enrichment upregulates genes involved in the synthesis or processing of DNA, RNA, and protein molecules, genes involved in the formation o f new synapses or the reorganization and strengthening of existing synapses, and genes involved in the regulation of neuronal excitability (Rampon et al., 2000) In contrast, genes encoding proteolytic and pro apoptotic proteins are downregula ted following enrichment. Many of these differences are apparent after only 3 hours of enrichment; however, with prolonged exposure to an enriched

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36 environment, a shift is observed towards the selective upregulation of genes involved in neuronal transmissio n and structural change. Microarray analysis has also been used to gain a better understanding of the molecular mechanisms which underlie the beneficial effects of environmental enrichment in animal models of disease. For instance, environmental enrichment produces a pronounced reduction in cerebral amyloid deposition in the APPswe PS1 (Lazarov et al., 2005) It also promotes the expression of immediate early genes as well as genes amyloid sequestration, endothelial function, and phospholipid metabolism. As many of these differentially expressed genes are known to regulate vasculogenesis, one theory amyloid clearance is related to modifications in the properties of the blood brain barrier. Despite the large scale analytical power afforded by microarrays, one potential l imitation of this approach is that it may only detect experience dependent changes in protein expression due to de novo protein synthesis and may not be able to identify changes in functional protein levels which arise due to posttranslational modification s, trafficking, or degradation. To circumvent this problem, a recent study used mass spectrometric methods to evaluate the global changes which occur in the hippocampal proteome following enrichment (McNair et al., 2007) In this experiment, the co alignment of principal cells, a feature unique to the anatomical organization of the hippocampus, was exploited to allow for independent analysis of the soma and dendritic fields. Functional classification of differentially express ed proteins revealed that structural and metabolic proteins were the most highly upregulated in response to enrichment. Signal transduction proteins also demonstrated differential expression; however, in contrast to the structural and metabolic proteins, w hich were upregulated in both the somatic and dendritic

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37 fractions, increased expression of signal transduction proteins were significantly more pronounced in the synaptosomal sample, suggesting a major contribution of neurotransmission related processes to the effects of environmental enrichment. Effects on Neuronal Signaling Intracellular signaling pathways lie at the nexus between environmental stimulation and experience dependent changes in gene expression. Environmental enrichment induces alterations in neuronal signaling via several mechanisms. For example, the functional properties of NMDA and AMPA receptors depend on their subunit composition. Enrichment has been shown to modify the expression of NMDA (i.e., NR1, NR2A, NR2B) and AMPA (i.e., GluR1 4) r eceptor subunits (Naka et al., 2005; Andin et al., 2007; Li et al., 2007) Furthermore, in many of these studies, these changes were observed concomitant with behavioral improvements known to involve glutamate mediated synaptic plasticity (Tang et al., 2001; Guilarte et al., 2003; Bredy et al., 2004) The effects of environmental enrichment on receptor expression also extend to other neurotransmitter systems as well: changes in dopamine (Keyvani et al., 2004; Del Arco et al., 2007) serotonin (Rasmuson et al., 1998) GABA (Keyvani et al., 2004) and adrenergic (Keyvani et al., 2004) receptor expression have all been reported following enrichment. In addition to neurotransmitter receptors, environmental enrichment also regu lates the expression of other proteins involved in neurotransmission. These can include neurotransmitter transporters (Zhu et al., 2005) (Andin et al., 2007) and ion channels ( Keyvani et al., 2004; Li et al., 2007) as well as various kinase/phosphatase proteins (Rampon et al., 2000; Keyvani et al., 2004; McNair et al., 2007) Augmentation of l ong term potentiation (LTP) is another mechanism by which enrichment can enhance synaptic transmission. One way enrichment accomplishes this is by altering the protein kinase A dependence of LTP (Duffy et al., 2001) Additionally, environmental

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38 manipulations a lso appear to be able to gate the efficacy of specific signaling pathways involved in the induction of LTP (Li et al., 2006) In young enriched mice, LTP induction can occur via cAMP dependent, NMDA receptor mediated activation of p38, a MAP kinase. Remarkably, this signaling cascade is completely non functional in s tandard caged mice. Furthermore, this pathway is also inactive in mature mice even if they are housed in an enriched environment later in life, implying a developmental time window. Overall, these studies show that environmental enrichment can exert multip le levels of control on neuronal signaling processes.

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39 Figure 2 1. An example of (a) an enriched environment is shown here in comparison to (b) a standard laboratory cage. inanimate and social stimul laboratory housing (Rosenzweig et al., 1978) In practice, experimental paradigms can vary widely from laboratory to laboratory although some common elements of enrichment include the addition of objects designed to provide cognitive or sensory stimulation, greater numbers of conspecific cagemates, increased spatial density, a running wheel for exercise, and novelty (i.e., routine replacement or reloc ation of enrichment items). Occasionally, foraging opportunities and dietary enrichments may also be provided.

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40 CHAPTER 3 SPECIFIC AIMS The overarching goal of this research was to identify the molecular and cellular mechanisms which contribute to environmentally mediated induction of functionally beneficial behavioral plasticity in an animal model of abnormal stere otyped motor behavior. We have observed frequent and intense stereotypies dominating the behavioral repertoire of deer mice ( Peromyscus maniculatus ). These motor stereotypies are extremely context dependent: phenotypically distinct groups of mice, i.e., hi gh stereotypy and low stereotypy, can be generated by differentially housing deer mice in either environmentally impoverished or environmentally enriched conditions, respectively. Previous work in our lab has demonstrated that enrichment related reduction of motor stereotypies in these animals correlates with region specific increases in measures of neuroplasticity. The objective of the present studies was to further explore and extend these findings. nd change over time in response to experience. Long term, experience dependent alterations in neural function can result from changes on multiple, interacting levels. The intracellular signaling pathways of the brain form the initial link between external stimuli and the translational machinery of the cell. In response to environmental stimulation and cell signaling, complex programs of gene expression are activated which modify the synaptic connections between neurons. Synaptic reorganization occurs as the brain remodels itself to absorb new information until, eventually, this neural plasticity gives rise If neuroplasticity allows an organism to adapt to its environment then the performance of stereotypy, a behavior marked by rigidity and invariance, implies an underlying impairment in neuroplasticity. Hence, the specific aims of this research were to determine the mechanisms by

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41 which environmental impoverishment restri cts and environmental enrichment restores neural and behavioral plasticity. 1. Converging lines of research link motor stereotypies to perturbations in dopaminergic neurotransmission. Dopamine and cAMP regulated phosphoprotein of 32kDa (DARPP 32) is a key p layer in dopamine signal transduction processes. Thus, the intertwining goals of our first specific aim were (i) to determine whether DARPP 32 would contribute functionally to stereotypy and (ii) to determine the impact of environmental manipulations on DA RPP 32 regulation. 2. Enduring changes in gene expression often occur following exposure to various forms of term, experience dependent neuroadaptations. Therefore, the go al of our second specific stereotypy and prolonged environmental enrichment. 3. On a molecular level, experience dependent neural plasticity can also manifest as lasting alterat ions in synaptic structure and function. For this reason, the goal of our third specific aim was to determine the sites of enrichment and stereotypy related changes in synaptic plasticity by analyzing levels of the synaptic proteins, synaptophysin and neu ral cell adhesion molecule (NCAM), in multiple brain regions. 4. The striatum does not mediate motor functions exclusively: Striatal neuropathology interferes with both striatal based motor activity and learning ability. As such, the goal of our final specific aim was to determine whether the striatal dysfunction associated with stereotypy would also affect the learning and memory functions of the striatum. As a corollary goal, we also sought to determine if the beneficial effects of environmental enrichment on stereotypy would also extend to improved behavioral performance on striatum dependent learning tasks.

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42 CHAPTER 4 EFFECTS OF ENVIRONME NTAL ENRICHMENT ON T HE REGULATION OF DAR PP 32 IN AN ANIMAL MODEL O F ABNORMAL STEREOTYP ED MOTOR BEHAVIOR Introduction Stereotypy refers to patterns of movements characterized by frequent and invariant repetition (Berkson, 1983; Mason, 1991) These behaviors occur absent a functional context and are a prominent feature of many psychiatric and neurological disorders, including autism, schizophrenia, and Rett syndrome. Persistent stereotypies are a source of distress for patients and concern for clinicians not only because they can be stigmatizing in social settings but also because they can severely interfere with more adaptive activities, essentially disabling some individuals. Under certain circumstances, stereotypies can also devolve into repetitive self injurious behaviors (e.g., head banging in autistic children, eye poking in the congenitally blind, and skin picking in Prader Willi patients), thereby assigning them a high priority for cli nical intervention (Kennedy et al., 2002) Although relatively little is known about the critical factor s contributing to the emergence and persistence of stereotypic behaviors, substantial evidence from both human and animal studies points to a relationship between stereotypy and disturbances of the nigrostriatal dopamine system. Human stereotypies appearin g in diverse clinical populations often share a common connection to dopamine. Several studies have linked alterations in dopamine functioning to the amount of stereotypy observed in subjects with mental retardation (Bodfish et al., 1995; Lewis et al., 1996; Roebel and MacLean, 2007) In chroni c abusers of amphetamine, behavioral stereotypy is a well documented side effect (Ellinwood et al., 1973) Similar ly, stereotyped disease patients (Evans et al., 2004; Lawrence et al., 2007) In studies using experimental animal models, genetic (Berridge et al., 2005) and pharmacological (Randrup and Munkvad, 1967;

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43 Segal et al., 1980) manipulations which result in increased levels of extracellular dopamine reliably induce stereotypies, while, conversely, stereotypies can be suppressed by blocking dopaminergic neurotransmission (Fog, 1972; Presti et al., 2003) Alterations in the density (Helmeste et al., 1981; Sharman et al., 1982; Fields et al., 1991; McBride and Hemmings, 2005) or sensitivity (Lewis et al., 1990) of dopa mine receptors are also commonly associated with stereotypy, lending further support for a role of dopamine in the expression of stereotyped behaviors. Dopamine and cAMP regulated phosphoprotein of 32kDa (DARPP 32) mediates many of the biological effects of dopamine (for a review see (Svenningsson et al., 2004) ). Highly enriched in the projection neurons of the striatum, DARPP 32 is a bifunctional signal transduction molecule whose biochemical properties are determined by its state of phosphorylation. When phosphorylated at Thr34 by protein kinase A (PKA), DARPP 32 is a potent inhibitor of protein phosphatase 1 (PP 1) (H emmings et al., 1984) On the other hand, when phosphorylated at Thr75 by cyclin dependent kinase 5 (Cdk5), DARPP 32 becomes an inhibitor of PKA activity (Bibb et al., 1999) Therefore depending on its site of p hosphorylation, DARPP 32 has the ability to modulate the activity of striatal output neurons by either amplifying or dampening the intracellular response to kinase/phosphatase signaling cascades. Because of the central role it plays in striatal signal tran sduction, DARPP 32 mediates many of the behavioral functions of dopamine, including the regulation of motor activity. N umerous studies have established that DARPP 32 participates in the motor response to various dopamine agonists, including c ocaine (Fienberg et al., 1998; Valjent et al., 2005; Zachariou et al., 200 6) amphetamine (Svenningsson et al., 2003) SKF 81297 (Scott et al., 2005) and apomorphine (Fienberg and Greengard, 2000) The motor side effects of pathological conditions

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44 affecting brain dopamine system s have also been linked to a bnormalities in DARPP 3 2 regulation. For example, dysregulation of DARPP 32 has been implicated in the pathogenesis of levodopa induced dyskinesias (Picconi et al., 2003; Hakansson et al., 2004; Aubert et al., 2005; Santini et al., 2007) Similarly, alterations in DARPP 3 2 expression hypothesized to contribute to its characteristic motor symptoms (Bibb et al., 2000; Spires et al., 2004) The goal of this study was t o determine whether DARPP 32 would also contribute functionally to motor stereotypies. Previous studies in mutant DARPP 32 mice have indicated that drug induced stereotypy requires DARPP 32 phosphorylation (Svenningsson et al., 2003) However, models o f stereotypic behavior which rely on administration of pharmacological agents may not offer the most appropriate paradigm with which to explore the underlying pathophysiology of clinical stereotypies. Indeed, it has been shown that the topography of sponta neously emitted and drug induced stereotypies are qualitatively different in the same species (Vandebroek et al., 1998; Presti et al., 2002; Presti et al., 2004a) suggesting that they also may not share the same neural substrates. The present study utilizes an animal model of spontaneous (i.e., non drug induced) stereotypy. When housed under standard laboratory conditions, deer mice ( Peromyscus maniculatus ) engage in high rates of motor stereotypies which occur absent any pharmacological challenge (Powell et al., 1999) Interestingly when reared in an enriched environment, the intensity of the stereotypies produced by these mi ce are substantially reduced (Powell et al., 1999) Environmental stimulati on has been shown to alter the expression of a number of genes involved in neuronal signaling (Rampon et al., 2000; Li et al., 2006; Li et al., 2007) including DARPP 32 (Spires et al., 2004) Therefore, a related goal of

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45 this study was to determine whether changes in the regulation of DARPP 32 might also be responsible for the decreases seen in stereotypic behaviors followi ng environmental enrichment Materials and Methods Animals Deer mice ( Peromyscus maniculatus bairdii ) were obtained from a breeding colony maintained at the University of Florida. The mice were housed in a room kept at 25C, maintained on a 16:8 light:dark cycle (lights off at 2200), and provided with access to food and water ad libitum At weaning (3 weeks of age), mice were randomly assigned to either standard cage (SC, n = 39) or environmentally enriched (EE, n = 40) housing conditions. Standard caged an imals were housed in standard laboratory rodent cages (29 33 15 cm) with two other same sex weanlings, while environmentally enriched animals were housed in groups of five or six in modified large dog kennels (122 81 89 cm). These enrichment kennel s were fitted with galvanized wire mesh to create multiple interconnected levels and equipped with bedding, a running wheel, and assorted ceramic and plastic objects varying in color, shape, and size. Throughout the 2 month experimental period, enrichment items were removed and replaced with novel objects on a weekly basis. To encourage foraging behaviors, approximately 50 grams of birdseed were also randomly distributed throughout the kennels three times a week. All animal procedures were approved by the U niversity of Florida Institutional Animal Care and Use Committee and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals Behavioral Testing Animals were tested for stereotypy after 2 months in their respective housin g conditions. Mice were removed from their home cages and placed singly into Plexiglas testing cages (22

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46 15 28 cm) two hours prior to the beginning of their dark cycle. Food, water, and bedding were provided, and the mice were left undisturbed for 2 ho urs to allow for habituation to the new environment and recovery from the stress of handling. Stereotypic behaviors were quantified using an automated testing apparatus (Columbus Instruments, Columbus, OH) designed to register stereotypic motor activity (e .g., repetitive jumping or backward somersaulting) as photobeam breaks. All testing sessions were recorded using a digital video surveillance system (GeoVision Inc., Taipei, Taiwan) and checked in order to verify the accuracy of the automated counts. Data were collected for two dark cycles, and the stereotypy score for each animal was calculated as the average number of photobeam interruptions per hour of testing. Following behavioral testing, the stereotypy scores for all animals tested were rank ordered, and animals belonging to the highest and lowest tercile of stereotypy scores were designated as high stereotypy (HS, greater than 600 photobeam breaks/hr) and low stereotypy (LS, less than 400 photobeam breaks/hr) animals, respectively. Animals with interm ediate stereotypy scores between 400 and 600 photobeam breaks/hr were not used for analysis of DARPP 32 protein levels. Western Blotting Levels of total (i.e., non phosphorylation specific) DARPP 32, phospho Thr34 DARPP 32, and phospho Thr75 DARPP 32 in th e dorsolateral striatum were determined using immunoblotting analysis. Animals (n = 10 18 per group) were sacrificed by cervical dislocation. The brains were rapidly removed, snap frozen in isopentane, and stored at 80C until processed for protein extrac tion. Briefly, 300 m thick coronal sections were collected through the striatum (AP: + 1.42 mm to +0.14 mm from bregma) (Franklin and Paxinos, 1997) in a cryostat held at 20C. Next, the d orsolateral striata were microdissected by punch method and sonicated in a

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47 homogenization buffer containing 20 mM Tris, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X ice and vortexed every 15 min for 1 hr before clarification via centrifugation at 16,000 g for 15 min. Subsequently, the supernatants were collected, and protein concentrations were determined using the DC Protein Assay kit (Bio Rad Laboratories, Hercules, CA), polyacrylamide gels and separated by SDS PAGE before transfer to polyvinylidene difluoride membranes. Membranes were then blocked with 5% n onfat milk in Tris buffered saline and 0.05% Tween (TBST) for 1 hr before incubation with the primary antibody (1:2,500 anti DARPP 32; 1:1,000 anti phospho Thr34 DARPP 32; and 1:1,000 anti phospho Thr75 DARPP 32; all antibodies from Cell Signaling Technolo gy, Beverly, MA) overnight at 4 C. The next day, blots were washed in TBST for 15 min before incubation with a horseradish peroxidase conjugated anti rabbit secondary antibody (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr at room temperature After washing with TBST, blots were developed using enhanced chemiluminescence and exposed to CL XPosure film (Pierce Biotechnology, Rockford, IL) for visualization of protein bands. To control for variations in sample loading, blots were then incubated in stripping buffer (62.5 mM Tris mercaptoethanol, 2% SDS) for 30 min at 50 C before being reprobed for tubulin (1:1,500, BioDesign, Saco, ME). Quantification of immunoreactive protein bands was done by densitometry using ImageJ software (v ersion 1.37, NIH, Bethesda, MD). Densitometric values for DARPP 32 bands were normalized relative to those of the loading control and are expressed as relative optical densities (ROD).

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48 Statistical Procedures All data are expressed as mean standard error of the mean (SEM). An independent samples t test (two tailed) was used to determine the effect of housing condition on stereotypy levels (SPSS, Chicago, IL). One way analyses of variance (ANOVAs) were used to determine group differences in stereotypy and p rotein expression with post hoc analysis of significant between protein concentrations and stereotypy were calculated according to the method of Pearson. The minim um level of significance was set at p < 0.05 for all analyses. Results Behavioral Data Consistent with our previous findings (Powell et al., 1999; Powell et al., 2000) environmental enrichment resulted in a marked attenuation of stereotypic behavior (t(77) = 2.36, p = 0.02). The interaction of housing condition with stereotypy yielded our four experimental groups of mice: environmentally enriched, low stereotypy (EE LS, n = 13), environmentally enriched, high stereotypy (EE HS, n = 13), standard caged, low stereotypy (SC LS, n = 10), and standard caged, high stereotypy (SC HS, n = 18). As shown in Figure 4 1, a one way ANOVA revealed significant differences (F(3,50) = 39.1, p < 0.001) in the frequency of s tereotypic behaviors between the high stereotypy groups (EE HS and SC HS) and the low stereotypy groups (EE LS and SC LS). Stereotypy scores for the low stereotypy groups were not significantly different from each other (p = 0.999). Likewise, stereotypy sc ores also did not significantly differ between the high stereotypy groups (p = 0.143). Levels of total DARPP 32, phospho Thr34 DARPP 32, and phospho Thr75 DARPP 32 We examined levels of total DARPP 32, phospho Thr34 DARPP 32, and phospho Thr75 DARPP 32 in the dorsolateral striatum based on previous studies in our lab (Turner et al., 2002;

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49 Turner and Lewis, 2003; Turner et al., 2003) implic ating this region as being the most sensitive to the effects of environmental enrichment and motor stereotypy. Our analysis revealed that levels of total DARPP 32 were significantly lower (F(3,50) = 4.15, p = 0.011) in EE LS mice when compared to EE HS, SC LS, and SC HS animals (Figure 4 2a). No group differences (F(3,50) = 0.823, p = 0.488) were found for phospho Thr34 DARPP 32 levels (Figure 4 2b), while significantly higher levels of phospho Thr75 DARPP 32 (F(3,50) = 4.56, p = 0.007) were observed in SC LS mice when compared to SC HS mice (Figure 4 2c). Levels of phospho Thr34 DARPP 32/total DARPP 32 and phospho Thr75 DARPP 32/total DARPP 32 Because there were significant differences in total DARPP 32 concentrations between groups, we normalized levels of the two phosphoproteins to total DARPP 32 levels to determine if differences in relative rather than overall phosphoprotein concentrations would more effectively differentiate the experimental groups. No group differences (F(3,50) = 1.21, p = 0.316) were found in phospho Thr34 DARPP 32/total DARPP 32 levels (Figure 4 3a). However, significantly higher (F(3,50) = 4.99, p = 0.004) levels of phospho Thr75 DARPP 32/total DARPP 32 were found in both EE LS and SC LS animals when compared to SC HS animals (Figure 4 3b). A significant main effect of stereotypy (t(52) = 3.12, p = 0.003) was also found for phospho Thr75 DARPP 32/total DARPP 32 with LS animals showing significantly greater levels of phospho Thr75 DARPP 32/total DARPP 32 than HS animals. Correlational analysis further confirmed the negative relationship (r(52) = 0.292, p = 0.032) between phospho Thr75 DARPP 32/total DARPP 32 and stereotypy (Figure 4 4). Discussion Previous studies from our lab have consistently demonstrated that early exposure to an en riched environment substantially reduces stereotypy in deer mice (Powell et al., 1999; Powell

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50 et al., 2000; Turner et al., 2002; Turner and Lewis, 2003; Turner et al., 2003) Moreover, we have shown that these decreases in stereotypy are accompanied by neurobiological changes exclusive to environmentally enriched, low stereotypy mice. Specifically, these studies demonstrated that enrichment related attenuation of stereotypy was associated with changes in various measures of synaptic plasticity, such as neuronal metabolic activity (Turner et al., 2002) dendritic morphology (Turner et al., 2003) and neurotrophin levels (Turner and Lewis, 2003) Here, we examined the effects of environmen tal enrichment on DARPP 32 regulation to determine if differences in the striatal signaling pathways which mediate dopamine function would also be associated with environmentally mediated decreases in stereotypy. The results show that environmentally enric hed, low stereotypy mice have lower levels of total DARPP 32 protein when compared to either environmentally enriched, high stereotypy or standard caged animals. Neurobiological differences which distinguish this group of mice from the other groups are sig nificant because they are specific for the beneficial effects of environmental enrichment. Therefore, this finding suggests that at least one additional mechanism by which enrichment functions to reduce stereotypy is by producing enduring changes in DARPP 32 protein expression. Changes in total DARPP 32 expression have important functional consequences for motor behavior. Genetic deletion of DARPP 32 results in decreased sensitivity to the motor stimulatory effects of a wide variety of substances, including cocaine (Fienberg et al., 1998) apomorphine (Fienberg and Greengard, 2000) SKF 81297 (Scott et al., 2005) para chloroamphetamine (Svenningsson et al., 2002) 5 HT (Svenningsson et al., 2002 ) and caffeine (Lindskog et al., 2002) In contrast, increases in total DARPP 32 levels have been associated with increases in abnormal motor behaviors, such as levodopa induced dyskinesias (Aubert et al., 2005) These

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51 observations are consistent with our finding of reduced stereotypy occurring concomitantly with decrea ses in total DARPP 32 expression in enriched, low stereotypy mice. However, given the disparate actions of the DARPP 32 phosphoproteins, any interpretations drawn from the analysis of total protein levels alone are incomplete because they omit consideratio n of DARPP 32 phosphorylation state. Indeed, while this decrease in total DARPP 32 levels was an important aspect of its regulatory control in environmentally enriched, low stereotypy animals, the data demonstrate that the relative proportion of DARPP 32 p hosphorylated at Thr75 (i.e., phospho Thr75 DARPP 32/total DARPP 32) was the best overall predictor of stereotypy level. Regardless of housing condition, stereotypy was inversely related to phospho Thr75 DARPP 32/total DARPP 32 levels; i.e., low stereotypy animals had higher levels of phospho Thr75 DARPP 32/total DARPP 32, whereas high stereotypy animals had lower levels of phospho Thr75 DARPP 32/total DARPP 32. Because phosphorylation at Thr75 converts DARPP 32 into an inhibitor of PKA, the physiological c onsequence of increased phospho Thr75 DARPP 32/total DARPP 32 in low stereotypy animals would be a relative decrease in PKA activity. As a result, downstream targets of PKA, which include neurotransmitter receptors, ion channels, and transcription factors, would be maintained at a predominantly dephosphorylated state. Presumably, the converse would be true in high stereotypy animals, and decreased phospho Thr75 DARPP 32/total DARPP 32 levels would result in a release of PKA inhibition and, therefore, an inc rease in neuronal excitability. Thus, the responsivity to endogenous dopamine would differ in high and low stereotypy animals. Previous findings in our lab support this hypothesis. Our research suggests that stereotypy might be expressed as a consequence o f imbalanced activity along the direct and indirect

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52 pathways of the basal ganglia (Presti et al., 2003; Presti and Lewis, 2005) Specifically, we have shown that hyperactivity of the indirect pathway is associated with low levels of stereotypy, whereas hypoactivity is associated with high levels of stereotypy (Presti and Lewis, 2005) The dopam ine D 2 receptors of the indirect pathway are negatively coupled to adenylyl cyclase activation. Therefore, enhanced activity of the indirect pathway in low stereotypy mice would result in a decrease in cAMP production and decreased activation of PKA. In co ntrast, decreases in the activity of the indirect pathway in high stereotypy mice would result in a relative increase in cAMP and increased levels of catalytically active PKA. As a consequence, dopamine signaling would be differentially potentiated in high and low stereotypy mice. No differences were found in either overall or normalized levels of phospho Thr34 DARPP 32. This is in contrast to studies of drug induced stereotypies which have indicated that this phosphorylation site may be crucial in the medi ation of abnormal repetitive behaviors. The motor stereotypies typically induced by D amphetamine, LSD, or PCP administration are all significantly reduced in DARPP 32 transgenic mice in which the Thr34 site has been replaced by a non phosphorylatable alan ine residue (Svenningsson et al., 2003) In other studies using Thr34Ala mutant mice, decreases in the motor stimulatory effect of acute cocaine challenge have also been observed (Valjent et al., 2005; Zachariou et al., 2006) The development of abnormal repetitive behaviors following L DOPA treatment also appears to i nvolve phosphorylation of DARPP 32 at Thr34. The striata of dyskinetic rats show abnormally high levels of phospho Thr34 DARPP 32 when compared to non dyskinetic rats (Picconi et al., 2003) Moreover, the severities of these repetitive movements are tightly correlated with levels of phospho Thr34 DARPP 32 (Santini et al., 2007) Nevertheless, these results notwithstanding, the lack of an effect of Thr34 phosphorylation in our study may be explained, in part, by the differences

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53 inherent between experimental paradigms of pharmacologically induced and spontaneously emitted s tereotypies. Drug induced stereotypies are typically elicited following acute administration of a pharmacological agent, whereas our model involves a behavior which is both chronic and persistent. Therefore, the results of the present study likely reflect the adaptive changes which occur following prolonged stereotypy rather than the immediate changes. In support of this, it has been shown that the site specific pattern of DARPP 32 phosphorylation differs following acute versus repeated psychostimulant admi nistration. For example, acute administration of cocaine increases phosphorylation at Thr34 and decreases phosphorylation at Thr75 (Nishi et al., 2000) whereas repeated administration induces behavioral sensitization and is associated with a decrease in phosphorylation at T hr34 and an increase in phosphorylation at Thr75 (Scheggi et al., 2004) This increase in phospho Thr75 DARPP 32 levels has been linked to enhanced Cdk5 activity (Bibb et al., 2001) and is thought to represent a homeostatic mechanism that develops in response to increased cAMP/PKA signaling during sensitization (Nestler, 2001) These data suggest that differences in the regulation of DARPP 32 can occur following short and long term exposure to the same stimulus. Another reason we might not have detected a difference in phospho Thr34 DARPP 32 concentrations may pertain to the contrasting actions of DARPP 32 in striatonigral and striatopallidal neurons. Activation of the cAMP/PKA pathway, and consequently phosphorylation of DARPP 32 at Thr34, in striatonigral neurons produces motor stimulation, whereas activation of the same pathway in striatopallidal neurons produces motor depression (Borgkvist and Fisone, 2007) Because the neuronal cell types of the s triatum are anatomically intermixed, it was not possible to differentiate the biochemical regulation of DARPP 32 in

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54 specific striatal cell subpopulations in this study using immunoblotting analysis. Therefore, additional studies may be necessary to more pr ecisely localize the changes in phosphorylation of DARPP 32 at Thr34 and Thr75. In summary, this study provides first evidence for a role of DARPP 32 in the mediation of spontaneously occurring repetitive behaviors. The data indicate that this is achieved primarily through the regulation of DARPP 32 phosphorylation at Thr75, although enrichment related attenuation of stereotypy also involved transcriptional control of overall DARPP 32 protein expression. Because DARPP 32 is an important modulator of the int racellular signaling events in dopaminoceptive neurons, these results offer further empirical support for a link between perturbed dopaminergic function and the expression of ster eotyped behaviors.

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55 Figure 4 1. Stereotypy scores for environmentally enriched, low stereotypy (EE LS, n = 13), environmentally enriched, high stereotypy (EE HS, n = 13), standard caged, low stereotypy (SC LS, n = 10), and standard caged, high stereotypy (SC HS, n = 18) mice. *** indicates p < 0.001 versus EE LS and SC LS mice as determined by

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56 Figure 4 2. Western blot analysis of (a) total DARPP 32, (b) phospho Thr34 DARPP 32, and (c) phospho Thr75 DARPP 32 levels in the dorsolateral striatum. Representat ive immunoblots for the detection of total DARPP 32 and each phosphoprotein are shown above their respective graphs. Densitometric values for DARPP 32 bands are tubulin, and are expressed as mean relat ive optical densities (ROD) SEM. indicates p < 0.05 versus EE LS, and ** indicates p < 0.01 versus SC

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57 Figure 4 3. Concentrations of (a) phospho Thr34 DARPP 32 and (b) phospho Thr75 DARPP 32 normalized relative to total DARPP 32 levels. indicates p < 0.05 and ** indicates p < 0.01 versus SC

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58 Figure 4 4. Relationship between the proportion of total DARPP 32 phosphorylated at Thr75 and stereotypy. Open symbols

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59 CHAPTER 5 EFFECTS OF ENVIRONME NTAL ENRICHMENT ON P ATTERNS OF FOSB EXPRESSION IN AN ANI MAL MODEL OF ABNORMA L STEREOTYPED MOTOR BEHAVIOR Introduction Abnormal repetitive behaviors have long been considered an important dimension of psychopathology. From it s earliest description (Kanner, 1943) to current diagnostic criteria (American Psychiatric Association, 1994) patterns of behaviors and interes ts which may be The phenomenological profile of these behaviors in autism is diverse and encompasses multiple categories of abnormal repetition, including s tereotyped body movements, self injurious behaviors, repetitive manipulation of objects, echolalia, compulsions, rituals and routines, insistence on sameness, and preoccupation with narrow, circumscribed interests. Although especially prominent in autistic symptomatology, repetitive behaviors are also characteristic of many other clinical disorders. Indeed, repetitive behaviors are associated with a wide variety of neurodevelopmental (e.g., mental retardation, Rett syndrome), psychiatric (e.g., schizophreni a, Despite their clinical significance, the pathophysiology of abnormal repetitive behaviors remains poorly defined. Nevertheless, ac cumulating evidence from both human and animal studies suggest a link between repetitive behaviors and basal ganglia dysfunction. For example, stereotyped body movements in individuals with mental retardation are associated with alterations in both biochem ical (Lewis et al., 1996) and biobehavioral (Bodfish et al., 1995; Bodfish et al., 2001; Roebel and MacLean, 2007) indices of dopamine functioning. Similarly, the frequency of repetitive motor behaviors in autistic (Turner and Russell, 1997) and schizophrenic

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60 (Frith and Done, 1983) patients is correlated with profound impairments on neuropsychological t asks designed to detect signs of basal ganglia pathology. Neuroimaging research has also type repetitive behaviors in autism (Sears et al., 1999; Hollander et al., 2005) In experimental animal models, motor stereotypies can be reliably induced with dopaminergic stimulation of the striatum (Randrup and Munkvad, 1967; Segal et al., 1980) With chronic administration of dopa mine agonists, behavioral sensitization, a state in which the behavioral response to subsequent drug exposure is potentiated, develops (Kalivas and Stewart, 1991) This state is marked by behavioral changes, including enhanced locomotor activity and the production of stereotypies, often accompanied by changes in gene expression. In fact, it has been demonstrat ed that patterns of early gene activation within the neurochemically distinct, striosome and matrix, compartments of the striatum can accurately predict the severity of psychostimulant induced stereotypies (Canales an d Graybiel, 2000; Capper Loup et al., 2002; Saka et al., 2004) By far, m uch of what has been learned about the neurobiological basis of abnormal repetitive behaviors has been derived from studies of drug induced stereotypies However, models of stereotyp ic behavior which rely on pharmacological induction may not offer the most appropriate paradigm with which to explore the underl ying pathophysiology of human repetitive behavior disorders Indeed, it has been shown that the topography of spontaneously emit ted and drug induced stereotypies are often qualitatively different in the same species (Vandebroek et al., 1998; Presti et al., 2002) suggesting that they also may not share the same neural substrates. Our lab has identified an anima l model of spontaneously occurring (i.e., non drug induced) stereotypy. When housed under standard laboratory conditions, deer mice ( Peromyscus

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61 maniculatus ) exhibit high rates of motor stereotypies which recapitulate key features of the abnormal repetitive behaviors observed in clinical phenomena; i.e., these stereotypies are spontaneous, emerge early in development, and persist throughout the lifespan of the animal. Remarkably, we have also discovered that the severity of these behaviors can be substantial ly reduced by rearing the mice in an enriched environment (Powell et al., 1999; Powell et al., 2000) T he goal s of this study were to determine whether specific patterns of FosB activation would serve to index the intensity of stereotypic behaviors in an animal model of spontaneous stereotypy and to determine whether the effects of environmental enrichment on stereotypy would correspond to changes in FosB induction. We chose to evaluate FosB because, unl ike other Fos family proteins, FosB levels accumulate rather than become desensitized with chronic stimulation (Chen et al., 1997) ; thus, it may provide a better marker for the long term, experience dependent neuroadaptations which occur following both persistent stereotypy and prolonged environmental enrichment. Methods and Materials Animals Deer mice ( Peromyscus maniculatus bairdii ) were obtained from a breeding colony maintained at the University of Florida. The mice were housed in a room kept at 25C, maintained on a 16:8 light:dark cycle (lights off at 2100), and provided with access to food and water ad libitum At weaning (3 weeks of age), mice were randomly assigned to either standard cage (SC, n = 49) or enriched (EE, n = 50) housing conditions. Standard caged animals were housed in standard l aboratory rodent cages (29 33 15 cm) with two other same sex weanlings, whereas environmentally enriched animals were housed in groups of five or six in

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62 modified large dog kennels (122 81 89 cm). These enrichment kennels were fitted with galvanized wire mesh to create multiple interconnected levels and equipped with bedding, a running wheel, and assorted ceramic and plastic objects varying in color, shape, and size. Throughout the 2 month experimental period, enrichment items were removed and replac ed with novel objects on a weekly basis. To encourage foraging behaviors, approximately 50 grams of birdseed were also randomly distributed throughout the kennels three times a week. All animal procedures were approved by the University of Florida Institut ional Animal Care and Use Committee and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals Behavioral Testing Animals were removed from their home cages and placed singly into Plexiglas testing cages (22 15 28 c m) two hours prior to the beginning of their dark cycle. Food, water, and bedding were provided, and the mice were left undisturbed for 2 hours to allow for habituation to the new environment and recovery from the stress of handling. Stereotypic behavior w as quantified using an automated testing apparatus (Columbus Instruments, Columbus, OH) designed to register stereotypic motor activity (e.g., repetitive jumping or backward somersaulting) as photobeam breaks. All testing sessions were recorded using a dig ital video surveillance system (GeoVision Inc., Taipei, Taiwan) and checked in order to verify the accuracy of the automated counts. Data was collected for two dark cycles, and the stereotypy score for each animal was calculated as the average number of ph otobeam interruptions per hour of testing. The stereotypy scores for all animals tested were rank ordered, and animals belonging to the highest and lowest tercile of stereotypy scores were designated as high (HS, greater than 600 photobeam breaks/hr) and l ow stereotypy (LS, less than 400 photobeam breaks/hr) animals,

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63 respectively. Animals with intermediate stereotypy scores between 400 and 600 photobeam breaks/hr were not used for analysis of FosB/ FosB expression. Western Blotting Levels of FosB (45 kDa) a nd FosB (35 37 kDa) in the striatum, motor cortex, sensory cortex, and hippocampus were determined using immunoblotting analysis. Animals (n = 6 7 per group) were sacrificed by cervical dislocation. The brains were rapidly removed, snap frozen in isopenta 20C. Tissue samples from the regions of interest were microdissected by punch method and sonicated in a homogenization buffer containing 20 mM Tris, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X 100, 10 every 15 min for 1 hr before clarification via centrifugation at 16,000 g for 15 min. The supernatants were collected, and protein concentrations were det ermined using the DC Protein Assay kit (Bio amounts of total protein from each sample were loaded onto 12% polyacrylamide gels and separated by SDS PAGE before transfer to polyvin ylidene difluoride membranes. Membranes were blocked with 5% nonfat milk in Tris buffered saline and 0.05% Tween (TBST) for 1 hr before incubation with the primary antibody (1:1,000 anti FosB, Santa Cruz Biotechnology) overnight at 4 C. The next day, blots were washed in TBST for 15 min before incubation with a horseradish peroxidase conjugated anti rabbit secondary antibody (1:5,000) for 1hr at room temperature. After washing with TBST, blots were developed using enhanced chemiluminescence and exposed to C L XPosure film (Pierce Biotechnology, Rockford, IL) for visualization of protein bands. To control for variations in sample loading, blots were then incubated in stripping buffer (62.5 mM Tris mercaptoethanol, 2% SDS) for 30

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64 min at 50 C before being reprobed for tubulin (1:1,500, BioDesign, Saco, ME). Quantification of immunoreactive protein bands was done by densitometry using ImageJ software (version 1.37, NIH, Bethesda, MD). The densitometric values for FosB bands were normalized relative to those of the loading control and are expressed as relative optical densities (ROD). Double Label Immunohistochemistry Following behavioral testing, animals (n = 5 per group) were deeply anesthetized via isoflurane inhalation and transcardially perfused with 0.9% saline followed by ice cold 4% paraformaldehyde. The brains were remo ved and post fixed in the same fixative overnight at 4C before equilibration in a cryoprotectant solution of 30% sucrose in 0.01 M phosphate buffered saline (PBS). Forty micron thick coronal sections were collected through the striatum, and s equential dua l antigen immunohistochemical staining was performed on free floating sections in PBS using antibodies raised against FosB (1:300, Santa Cruz Biotechnology) and opioid receptor 1 (MOR 1, 1:2,500, ImmunoStar, Hudson, WI). First, e ndogenous peroxidase acti vity was quenched by incubating the sections in 0.5% H 2 O 2 in 10% methanol for 15 min. Sections were then rinsed in PBS before blocking in 10% Normal Donkey Serum (NDS) in 0.3% Triton X 100 for 1 hr. S ubsequently, s ections were incubated with the primary an tibody in 1% NDS in 0.3% Triton X 100 overnight at 4C. The next day, tissues were rinsed in PBS before incubation with the appropriate biotinylated secondary antibody in 1% NDS in 0.3% Triton X 100 for 1 hr. Sections were then rinsed again before being in cubated in an avidin biotin peroxidase complex solution (Vectas tain Elite ABC kit, Vector Laboratories, Burlingame, CA) for 1 h r FosB immunoreactive nuclei were first visualized by reaction with diaminobenzidine (DAB) enhanced with 0.05% nickel ammo nium sulfate and 0.05% cobalt chloride which yield ed a dark

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65 blue black stain, before the sections were processed for MOR 1 rich striosomes using DAB which yield ed a light orange brown stain. Image Analysis For each animal, three equally spaced sections c orresponding to coronal planes ranging from approximately 1.2 mm to 0.48 mm anterior to bregma (Paxinos and Watson, 1998) were analyzed. The number of FosB/ FosB immunoreact ive nuclei in the striatum was estimated using the optical fractionator method (West et al., 1991) The contours of the striatum were outlined, and analysis was initiated from a random starting position within the striatum. The striatum wa s then systematically sampled by 250 m stepwise movements of the stage in the x and y directions. FosB/ FosB immunoreactive nuclei were counted when they came into focus, following the unbiased counting rules for the optical disector (Gundersen et al., 1988) The dimensions of the counting frame were set at 50 50 m, and the disector height was 15 m with the guar d volume offset 3 m from the tissue surface. This sampling scheme was designed to yield a coefficient of error (Glaser and Wilson, 1998) of less than 10% for each estimate. Point counting stereology was used to estimate the density of FosB/ FosB immunoreactive nuclei in the striosome and matrix compartments as well as the volume fraction of the striatum occupied by striosomes. For this analysis, a 10 10 m grid probe was superimposed over the counting frame, and FosB/ FosB immunoreactive n uclei were counted when they intersected the grid. Additionally, individual points were also identified as being striosome positive where they co localized with MOR 1 immunoreactive patches. Statistical Procedures All data are presented as mean standard error of the mean (SEM). An independent samples t test (two tailed) was used to determine the effect of housing condition on stereotypy

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66 levels (SPSS, Chicago, IL). One way analyses of variance (ANOVAs) were used to analyze behavioral data and FosB/ FosB de nsities with post hoc analysis of significant effects accomplished using Newman Keuls test. Correlations between FosB/ FosB activation and stereotypy scores were calculated according to the method of Pearson. The minimum level of significance was set at p < 0.05 for all analyses. Results Behavioral Data Consistent with our previous findings (Powell et al., 1999; Powell et al., 2000) environmental enrichment resulted in a marked attenuation of stereotypic b ehavior (t(97) = 4.089, p < 0.001). The interaction of housing condition with stereotypy yielded our four experimental groups: environmentally enriched, low stereotypy (EE LS, n = 12); environmentally enriched, high stereotypy (EE HS, n = 11); standard ca ged, low stereotypy (SC LS, n = 11); and standard caged, high stereotypy (SC HS, n = 12) mice. Stereotypy scores for the experimental groups are shown in Table 5 1. As expected, significant differences (F(3,42) = 75.4, p < 0.001 ) were found in the frequen cy of stereotypic behavior between the high stereotypy groups (EE HS and SC HS) and the low stereotypy groups (EE LS and SC LS). Stereotypy scores for the low stereotypy groups were not significantly different from each other. Likewise, stereotypy scores a lso did not significantly differ between the high stereotypy groups. FosB Expression in Selected Brain Regions In order to determine if the long term effects of environmental enrichment and motor stereotypy would localize to certain brain regions, we firs t examined FosB and FosB expression in the striatum, motor cortex, sensory cortex, and hippocampus using immunoblotting analysis (Table 5 2). These regions were selected based on their involvement in motor function (striatum and motor cortex) or their dem onstrated sensitivity (Rampon et al., 2000; van Praag et

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67 al., 2000) to the effects of environmental enrichment (hippocampus and cortical areas). Levels of FosB (45 kDa) in many samples were too low to allow for quantitative analysis. In contrast, the chronic FosB isoform (35 37 kDa) demonstrated measurable levels of expression throughout all regions examined and was also the predominant isoform expressed in the striatum (Figure 5 1). No differences were found in FosB levels between groups in the motor cortex (F(3,22) = 0.79, p = 0.512) or hippocampus (F(3,22) = 0.317, p = 0.813). However, FosB induction in the sensory cortex was significantly higher (F(3,22) = 3.93, p = 0.022) in EE LS mice than in EE HS, SC LS, or SC HS mice. A significant difference (F(3,2 2) = 4.54, p = 0.013) in FosB activation was also found in the striatum. Post hoc analysis revealed that this was due to significantly greater (p < 0.01) striatal concentrations of FosB in SC HS mice when compared to SC LS mice. Regional Distribution of Striatal FosB/ FosB Expression The Western blotting results indicated that, for standard caged mice, increased stereotypy was associated with increased FosB accumulation within the striatum. Given the functional and anatomical heterogeneity of the striatu m, we next used immunohistochemistry to more precisely characterize the anatomical distribution of FosB/ FosB expression. This was particularly important because our previous studies (Turner et al., 2002; Turner and Lewis, 2003; Turner et al., 2003) have indicated that the beneficial effects of environmental enrichment are specific to the dorsolateral striatum so there may have been a dilu tion effect in examining the entire striatum. To characterize the topographical distribution of FosB/ FosB activation, the striatum was subdivided along its dorsoventral and mediolateral axes into dorsolateral (DLS), dorsomedial (DMS), ventrolateral (VLS), and ventromedial (VMS) quadrants (Figure 5 2). EE LS mice

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68 showed significantly greater (F(3,16) = 28.5, p < 0.001) levels of FosB/ FosB in the dorsolateral striatum when compared to the other groups and significantly higher (F(3,16) = 4.28, p = 0.021) lev els of FosB/ FosB in the dorsomedial striatum when compared to the SC LS group (Figure 5 4). FosB/ FosB expression in both the ventrolateral (F(3,16) = 4.75, p = 0.015) and ventromedial (F(3,16) = 4.59, p = 0.017) regions of the striatum were significantly greater in the EE LS, EE HS, and SC HS groups than in the SC LS group. Correlations between the estimated number of FosB/ FosB immunoreactive nuclei and stereotypy scores in the four subregions were also calculated separately for enriched, standard caged, and all animals (Table 5 3). When the animals were analyzed altogether, no significant correlations were found between FosB/ FosB expression and stereotypy levels in any of the striatal subregions examined. However, when the data was segregated according to housing conditions, a distinct pattern emerged. For standard caged animals, increasing stereotypy was associated with increasing FosB/ FosB expression in all striatal subregions, whereas, for enriched animals, a significant correlation was found only in the dorsolateral striatum (Figure 5 5). Furthermore, in contrast to the standard caged animals, increasing stereotypy was associated with decreased FosB/ FosB induction in the enriched animals. Compartmental Distribution of Striatal FosB/ FosB Expression We next examined the distribution of FosB/ FosB expression in the striosome and matrix compartments of the striatum (Figure 5 3). For this analysis, we calculated an index of striosome to matrix predominance (ISMP) value, defined as the ratio of the densit y of FosB/ FosB immunoreactive nuclei in the striosomes to the density of FosB/ FosB immunoreactive nuclei in the matrix. The data revealed a significant difference in ISMP values (F(3,16) = 10.1, p = 0.001) between high (EE HS and SC HS) and low (EE LS an d SC LS) stereotypy animals with high

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69 stereotypy animals demonstrating significantly higher ISMP values (Figure 5 6) than low stereotypy animals. The relative distribution of compartmental FosB/ FosB activation, as measured by the ISMP value, also more clo sely paralleled changes in stereotypy than the density of FosB/ FosB expression in each compartment alone (Figure 5 7). These differences in compartmental FosB/ FosB activation did not appear to be result from differences in overall striosome volumes betwe en groups (data not shown). Discussion In this study, we mapped the regional distribution of FosB induction in order to identify the neuronal systems which are activated by persistent, spontaneous stereotypy and long term exposure to an enriched environme nt. The data demonstrate that, for standard caged mice, levels of FosB accumulate specifically in the striatum with prolonged stereotypic motor activity. As determined by immunoblotting analysis, levels of FosB were significantly higher in standard caged, high stereotypy mice when compared to standard caged, low stereo typy mice. Immunohistochemical analysis of FosB/ FosB expression further indicated that increasing stereotypy in standard caged animals was correlated with increasing FosB/ FosB induction in all striatal subregions. In contrast, the effects of enrichment o n stereotypy were localized only to the dorsolateral region of the striatum. Also, in contrast to the standard caged animals, increasing stereotypy in enriched animals was associated with decreasing levels of FosB/ FosB expression. Once established, stereo typies appear to become entrenched within the behavioral repertoire of deer mice and become increasingly intractable in response to further environmental manipulations (Hadley et al., 2006) Likewise, clinical stereotypies are also noted for being extremely refractory to treatment. Stria tal accumulation of FosB may mediate some of the neuroadaptations which lead to the continued persistence and maintenance of stereotypic

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70 behaviors. In support of this hypothesis, a role for FosB has also been implicated in other repetitive movement disor ders. movements (i.e., AIMs or dyskinesias) following chronic L DOPA treatment correlates temporally with a gradual increase in FosB levels (Andersson et al., 1999) Several lines of evidence have suggested that FosB mediates the development of these behaviors. First, increases in FosB levels are only observed in animals that develop dyskinesias. A subset of animals treated with L DOPA do not develop dyskinesias; these animals show levels of FosB comparable to those of control (i.e., saline treated) animals (Andersson et al., 1999) Second, experimental manipulations which decrease FosB expression also decrease dyskinetic symptoms. Intrastriatal injection of antisense FosB inhibits FosB expres sion and also attenuates dyskinetic symptoms (Chen et al., 2006) Likewise, pharmacological downregulation of FosB has also been shown to result in a reduction in dyskinetic symptoms (Westin et al., 2007) Third, the regional pattern of FosB induction has been shown to be somatotopically related to the types of abnormal involuntary movements observed. For example, locomotor AIMs are preferentially asso axial, limb, and orolingual AIMs are selectively the lateral s triatum (Andersson et al., 1999) The results of this study suggest that the beneficial effects of environmental enrichment on stereotypy are associated with long term alterations in the functioning of the dorsolate ral striatum. Environmentally enriched, low stereotypy mice showed significantly higher levels of FosB in the dorsolateral striatum than all other groups. Furthermore, a significant correlation

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71 between stereotypy and FosB activation was only observed in the dorsolateral striatum. These results are congruent with previous findings in our lab which hav e localized enrichment related differences in metabolic activity (Turner et al., 2002) dendritic morphology (Turner et al., 2003) and neurotrophin expression (Turner and Lewis, 2003) to the dorsolateral striatum of environmentally enriched, low stereotypy mice. These results are also functionally significant becaus e the dorsolateral striatum receives projections from the sensorimotor cortex which are responsible for mediating forelimb, trunk, and hindlimb movements (McGeorge and Faull, 1989; Ebrahimi et al., 1992) Thus, the striatal subregion which is the most sensitive to the eff ects of enrichment is also the subregion which is involved in producing the types of stereotypies (i.e., repetitive jumping, backflipping) which our mice express. In addition to the changes observed in the dorsolateral striatum, environmentally enriched, l ow stereotypy mice also showed higher levels of FosB in the sensory cortex than the other groups. Changes in the organizational features of somatosensory cortex have been shown to occur following environmental enrichment (Coq and Xerri, 1998) Indeed, active interaction with an enriched environment is required to produce the neurobiological effec ts of enrichment (Ferchmin and Bennett, 1975) Some of our animals do not benefit from the ameliorative effects of enrichment on stereotypy (i.e., the enriched, high stereotypy mice); these animals also do not show differences in FosB activation when compared to standard caged animals. This increase in FosB gene expression in the sensory cortex of enriched, low stereotypy mice might reflect their increased engagement with the environment and also contribute to some of the changes in FosB expression observed in the dorsolateral striatum. Another goa l of this study was to characterize the effects of environmental enrichment and stereotypy on the compartmental (striosome/matrix) distribution of FosB expression. Our data

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72 indicates that stereotypy is associated with enhanced FosB activation in the striosomes relative to activation in the matrix regardless of housing condition. Furthermore, we also found that the relative activation more closely p aralleled changes in stereotypy across housing conditions than activation in either of the compartments alone, suggesting coordinate regulation of the striatal compartments. These results are consistent with research performed using models of psychostimula nt induced stereotypy (Canales and Graybiel, 2000; Capper Loup et al., 2002; Saka et al., 2004) Predominant striosomal FosB expression has also been observed in conjunction with the development of AIMs following chronic L DOPA administration (Cenci et al., 1999; Sa ka et al., 1999) These findings suggest that behavioral stereotypy may originate as a consequence of a functional imbalance in the regulation of striosome and matrix compartments. Since the striosomes are thought to mediate reinforcement related behavior s (White and Hi roi, 1998) this change in the balance of gene activation with increasing stereotypy may indicate a shift towards more motivationally directed behaviors in high stereotyping animals. Altogether, these data demonstrate that chronic stereotypy and environme ntal enrichment induce regionally and compartmentally specific patterns of FosB activation and that FosB can mediate both adaptive and maladaptive behavioral plasticity depending on the form of chronic stimulation (stereotypic motor activity or environmental enrichment) experienced. This latter finding, in particular, suggests further investigation. The functional consequences of FosB induction on motor behavior can differ diametrically depending on the striatal cell population activated. The neuropeptides, dynorphin and enkephalin, are selectively expressed in the medium spiny neurons comprising the direct and indirect pathways, respectively. Activation of the direct pathway augments motor output, whereas activation of the indirect pathway attenuates motor output. Transgenic mice

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73 overexpressing FosB in dynorphin containing str iatal neurons show increased wheel running behavior relative to controls, whereas mice overexpressing FosB in enkephalin containing striatal neurons show less wheel running behavior (Werme et al., 2002) Similarly, activation in dynorphin positive neurons is associated with L DOPA induced dyskinesias (Andersson et al., 1999; Pavon et al., 2006) whereas a selective increase in enkephalin containing neurons following the c hronic administration of neuroleptic drugs is correlated with the appearance of extrapyramidal side effects. Our past research has indicated that stereotypy may be linked to imbalances in th e activity of the direct and indirect pathways (Presti et al., 2003; Presti et al., 2004a; Presti and Lewis, 2005) ; therefore, FosB may m ediate different forms of neural plasticity, in part, through cell specific expression. FosB could also exert its opposing effects through differential gene expression. In studies of chronic drug abuse, it has been shown that FosB activation following sh ort and long term exposure to cocaine results in the transcription of different target genes (McClung and Nestler, 2003) Furthermore, these differences in gene expression produced different behavioral effects: gene expression induced by short term FosB activation reduced the rewarding effects of cocaine, whereas long term FosB activation heightened drug reward. Th us, it is possible that, even though high levels of FosB were observed in both standard caged, high stereotypy and environmentally enriched, low stereotypy animals, different subsets of target genes were activated to produce differing behavioral effects. Future studies will be necessary to determine which of these mechanisms FosB utilizes to induce different forms of behavioral plasticity.

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74 Table 5 1. Stereotypy scores for experimental groups. Group n Stereotypy Scores EE LS 12 206 26 EE HS 11 815 40 SC LS 11 246 34 SC HS 12 989 69 Values represent mean stereotypy scores (photobeam breaks/hr) SEM.

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75 Table 5 2. Levels in selected brain regions Group Striatum Motor Cortex Sensory Cortex Hippocampus EE LS 0.94 0.0 7 0.89 0.07 0.75 0.05 b 0.78 0.05 EE HS 0.84 0.08 0.82 0.08 0.57 0.06 0.80 0.04 SC LS 0.71 0.07 0.76 0.08 0.51 0.05 0.74 0.05 SC HS 1.11 0.09 a 0.74 0.07 0.53 0.06 0.75 0.05 Values shown are expressed as mean relative optic al densities standard error of the mean. Significant values are shown in boldface. a indicates p < 0.01 vs SC LS, and b indicates p < 0.05 vs EE HS, SC LS, and SC HS.

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76 Table 5 3. Correlations between stereot ypy scores Subregion EE SC All DLS 0.87 b 0.789 b 0.326 DMS 0.506 0.897 a 0.064 VLS 0.091 0.893 a 0.417 VMS 0.29 0.7 c 0.258 Values represent correlation coefficients (r P ). Significant values are shown in boldface. a, b, and c indicate p < 0.001, p < 0.01, and p < 0.05, respectively.

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77 Figure 5 1. Representative immunoblot for the detection of FosB isoforms in the striatum (top panel). The same blot was re probed for tubulin (bottom panel) as a loading control. The blots show that the predominan t FosB isoform expressed in the striatum in all of the experimental groups is the chronic FosB (35 37 kDa) isoform ns indicates non specific bands.

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78 Figure 5 2. Schematic illustrating striatal subregions. The striatum was subdivided along its dorsoven tral and mediolateral axes into the dorsolateral (DLS), dorsomedial (DMS), ventrolateral (VLS), and ventromedial (VMS) quadrants.

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79 Figure 5 3. Photomicrographs of sections double stained for FosB/ FosB nuclei (black dots) and MOR 1 rich striosomes (gray patches) at (a) low and (b) high magnification Representative sections are from a SC HS animal.

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80 Figure 5 4. Subr egional distribution of immunoreactive nuclei in EE LS, EE HS, SC LS, and SC HS animals

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81 Figure 5 5. Correlations between t he estimated number of FosB/ FosB immunoreactive nuclei and stereotypy scores in the (a) dorsolateral, (b) dorsomedial, (c) ventrolateral, and (d) ventromedial quadrants of the striatum. Trendlines indicate significant correlations Open symbols ( ) indica te environmentally enriched animals. Closed symbols ( ) indicate standard caged animals.

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82 Figure 5 6. Distribution of FosB/ FosB induction in the striosome and matrix compartments of the striatum. The index of striosome to matrix predominance (ISMP) val ue corresponds to the ratio of FosB/ FosB density in the striosomes to FosB/ FosB density in the matrix. ** and *** indicate p < 0.01 and p < 0.001 versus EE LS and SC LS respectively.

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83 Figure 5 7. Changes in ISMP value more closely parallel changes in stereotypy than changes in the density of FosB/ FosB expression in the striosome or matrix alone. Values shown are for high stereotypy animals normalized relative to low stereotypy animals belonging to the same group.

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84 CHAPTER 6 EFFECTS OF ENVIRONME NTAL ENRICHMENT AND ABNORMAL STEREOTYPED MOTOR BEHAVIOR ON SY NAPTIC PROTEIN EXPRE SSION Introduction Stereotypies refer to abnormal repetitive motor behavior s which are characterized by topographical invariance and a lack of ob vious purpose or function (Lewis and Baumeister, 1982; Berkson, 1983; Mason, 1991) Although stereotypies normally occur in typically developing human infants a nd have, in fact, been hypothesized to enhance the early development of motor skills (Thelen, 1979) the persistence of these behaviors later in childhood and into adulthood is most often associated with neuropsychiatric and developmental disorders, such as autism and mental retardation In these cases, stereotypies (e.g., body rocking, head rolling, repetitive hand movements) are considered to be clinically relevant because they can become behaviorally dominant and interfere with more purposeful activities. Indeed, stereotypic individua ls often show deficits in social interaction (Matson et al., 2006) and are impaired in the acquisition of new skills (Koegel and Covert, 1972) as well as the performance of established skills (Morrison and Rosales Ruiz, 1997) including those necessary for normal adaptive functioning (Matson et al., 1997) Although absent in the wild, stereotypies are also freque ntly observed in animals kept in captivity, especially those housed in barren environments with limited opportunities to engage in species suboptimal housing conditions, the h igh occurrence of stereotypies in captive populations poses a major animal welfare concern. As environmental impoverishment has been demonstrated to induce stereotypic behaviors in a wide range of animal species (Cronin and Wiepkema, 1984; Mason, 1993; Wrbel et al., 1996; Bashaw et al., 2001; Garner et al., 2003b; Mallapur and Choudhury, 2003; Vickery and Mason, 2004; Elzanowski and Sergiel, 2006) attempts to provide

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85 ntial strategies to prevent or reduce these behaviors (Swaisgood and Shepherdson, 2005) In these experiments, it was discovered that even materials, etc.) yielded appreciable decreases in stereotypy. Alt hough these observations indicate a strong role for environmental regulation in the development and expression of stereotypic behaviors the specific pathophysiological mechanisms which give rise to motor stereotypies have yet to be clearly elucidated. In order to resolve the etiological factors more definitively, some researchers have utilized its contextual dependence to generate animal models of stereotypy based on environmental restriction. Our lab has adopted one such model. Despite its species specifi c nature, the abnormal repetitive motor behavior s (e.g., repetitive jumping, backwards somersaulting and patterned running) exhibited by deer mice ( Peromyscus maniculatus ) mimic the fundamental characteristics observed in clinical populations; i.e., these invariant and apparently functionless behaviors emerge early in development and persist throughout the lifespan of the animal. Moreover, our lab has established that, while these stereotypies occur spontaneously under standard laboratory housing, their de velopment may be delayed and their frequency reduced by rearing the m ice in an enriched environment (Powell et al., 1999; Powell et al., 2000) Based on the premise that it might ultimately lead to the der ivation of new therapeutic strategies for clinical stereotypies our previous line of research was aimed at deciphering the neural mechanisms underlying these beneficial effec ts of environmental enrichment. The effects of environmental enrichment on brain structure and function are well documented. Environmental enrichment increases brain weight (Bennett et al., 1969) cortical thickness (Diamond et al., 1976) dendritic branching (Greenough et al., 1973) and length

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86 (Wallace et al., 1992) dendritic spine density (Turner and Greenough, 1985) and synapse number and size (Sirevaag and Greenough, 1987) Exposure to an enriched environment also promotes neurogenesis (Kempermann et al., 2002) and reduces spontaneous apoptosis (Young et al., 1999) Additionally, increases in measures of non neuronal plasticity, such as angiogenesis and gliogenesis, have also been reported following enrichment (Dong and Greenough, 2004; Markham and Greenough, 2004) Previous work from our lab has linked enrichment rela ted attenuation of stereotypy to changes in neuronal metabolic activity (Turner et al., 2002) dendritic morphology (Turner et al., 2003) and neurotrophin expression (Turner and Lewis, 2003) These changes were not only specific to environmentally enriched, low stereotypy animals, but they were also regionally speci fic for areas involved in the control of motor behavior. The goal of this study was to determine whether region specific modifications in synaptic efficacy would represent an additional mechanism contributing to the effects of environmental enrichment on s tereotypy. To this end, we evaluated concentrations of the synaptic proteins, synaptophysin and neural cell adhesion molecule (NCAM), in various brain regions. Synaptophysin and NCAM are both considered reliable markers of synaptic density (Walaas et al., 1988; Jorgensen, 1995) Furthermore, increases in the expression of both proteins have been documented following enrichment (Koo et al., 2003; Nithianantharajah et al., 2004) Thus, we h ypothesized that analysis of these synaptic proteins would serve to index the molecular changes which occur as a consequence of enrichment related effects on stereotypy. Materials and Methods Animals Deer mice ( Peromyscus maniculatus bairdii ) were obtained from a breeding colony maintained at the University of Florida. The mice were housed in a room kept at 25C,

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87 maintained on a 16:8 light:dark cycle (lights off at 2200), and provided with access to food and water ad libitum At weaning (3 weeks of age), mi ce were randomly assigned to either standard cage (SC, n = 39) or environmentally enriched (EE, n = 40) housing conditions. Standard caged animals were housed in standard laboratory rodent cages (29 33 15 cm) with two other same sex weanlings, while en vironmentally enriched animals were housed in groups of five or six in modified large dog kennels (122 81 89 cm). These enrichment kennels were fitted with galvanized wire mesh to create multiple interconnected levels and equipped with bedding, a runni ng wheel, and assorted ceramic and plastic objects varying in color, shape, and size. Throughout the 2 month experimental period, enrichment items were removed and replaced with novel objects on a weekly basis. To encourage foraging behaviors, approximatel y 50 grams of birdseed were also randomly distributed throughout the kennels three times a week. All animal procedures were approved by the University of Florida Institutional Animal Care and Use Committee and were performed in accordance with the NIH Guid e for the Care and Use of Laboratory Animals Behavioral Testing Animals were tested for stereotypy after 2 months in their respective housing conditions. Mice were removed from their home cages and placed singly into Plexiglas testing cages (22 15 28 cm) two hours prior to the beginning of their dark cycle. Food, water, and bedding were provided, and the mice were left undisturbed for 2 hours to allow for habituation to the new environment and recovery from the stress of handling. Stereotypic behaviors were quantified using an automated testing apparatus (Columbus Instruments, Columbus, OH) designed to register stereotypic motor activity (e.g., repetitive jumping or backward somersaulting) as photobeam breaks. All testing sessions were recorded using a digital video surveillance system

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88 (GeoVision Inc., Taipei, Taiwan) and checked in order to verify the accuracy of the automated counts. Data were collected for two dark cycles, and the stereotypy score for each animal was calculated as the average number o f photobeam interruptions per hour of testing. Following behavioral testing, the stereotypy scores for all animals tested were rank ordered, and animals belonging to the highest and lowest third of stereotypy scores were designated as high stereotypy (HS, greater than 600 photobeam breaks/hr) and low stereotypy (LS, less than 400 photobeam breaks/hr) animals, respectively. Animals with intermediate stereotypy scores between 400 and 600 photobeam breaks/hr were not used for analysis of synaptic protein level s. Western Blotting Levels of synaptophysin and neural cell adhesion molecule (NCAM) in the cortex dorsolater al striatum, dorsomedial striatum, and hippocampus (Figure 6 1) were determined using Western blotting. A nimals were sacrificed by cervical disloc ation The b rains were rapidly removed, snap frozen in isopentane, and stored at 80 C until processed for protein extraction. Briefly, m thick coronal sections were collected in a cryostat held at 20C. Next, t issue samples from the regions of inter est were microdissected by punch method and sonicated in a homogenization buffer containing 20 mM Tris, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X Samples were then placed on ice and vortexed eve ry 15 min for 1 hr before clarification via centrifugation at 16,000 g for 15 min. Subsequently the supernatant was collected, and the protein concentrations were determined using the DC Protein Assay kit (Bio Rad Laboratories, Hercules, CA), following man loaded onto 12% polyacrylamide gels and separated by SDS PAGE before transfer to polyvi nylidene difluoride membranes. Afterwards, g els and blots were stained with Coomas sie blue and Ponceau S (Sigma), respectively, in order t o verify equal sample loading. The

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89 m embranes were then washed to remove excess stain and blocked with 5% nonfat milk in Tris buffered saline and 0.05% Tween (TBST) for 1 hr before incubation with the primary antibody (1:50,000 synaptophysin or 1:100 anti NCAM; both antibodies from Chemicon, Temecula, CA) overnight at 4 C. The next day, blots were washed in TBST for 15 min before incubation with the appropriate horseradish peroxidase conjugated secondar y antibody (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr at room temperature. Blots were then developed using enhanced chemiluminescence and exposed to CL XPosure film (Pierce Biotechnology, Rockford, IL) for visualization of protein bands Finally, q uantification of immunoreactive protein bands was done by densitometry using ImageJ software (version 1.37, NIH, Bethesda, MD). Statistical Analysis All data are expressed as mean standard error of the mean (SEM). Independent samples t tests (t wo tailed) were used to determine the effect of housing condition on levels of stereotypy (SPSS, Chicago, IL). Two way (2 2) factorial analyses of variance (ANOVAs) with housing condition (EE or SC) and stereotypy (LS or HS) as the two factors were used to evaluate differences in synaptic protein levels Correlations between protein concentrations and stereotypy scores were calculated according to the method of Pearson. The minimum level of significance was set at p < 0.05 for all analyses. Results Behavi oral D ata Consistent with our previous findings (Powell et al., 1999; Powell et al., 2000) environmental enrichment resulted in a marked attenuation of stereotypic behavior (t(77) = 2.36, p = 0.02). Beca use we hypothesized neurobiological changes would be more apparent in phenotypically distinct groups, only animals testing within the highest and lowest third of

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90 stereotypy scores were used for analysis of synaptic protein concentrations. The average stere otypy score for high stereotypy (HS, n = 31) animals was 906 52 photobeam breaks/hr, and the average stereotypy score for low stereotypy (LS, n = 23) animals was 258 17 photobeam breaks/hr. Of these animals, the average stereotypy score for EE animals (n = 26) was 534 65 photobeam breaks/hr, and the average stereotypy score for SC animals (n = 28) was 719 81 photobeam breaks/hr. Stereotypy scores were not significantly different between these two housing conditions (t(52) = 1.76, p = 0.08). Effects of Housing Condition and S tereotypy on S ynaptic Protein E xpression Levels of synaptophysin and neural cell adhesion molecule (NCAM) were evaluated in the cortex, dorsolateral striatum, dorsomedial striatum, and hippocampus (Figure 6 1). These areas were s elected based on their demonstrated sensitivity (Rampon et al., 2000; van Praag et al., 2000) to the effects of environmental enrichment (cortex and hippocampus) or their role in the control of motor behavior (striatum). Additionally, the dorsolateral and dorso medial subregions of the striatum were analyzed separately based on evidence suggesting anatomical and functional dissociation of the two regions (Voorn et al., 2004; Yin and Knowlton, 2006) Synaptophysin migrated as a single 38 kDa band, whereas NCAM was expressed as three isoforms, with molecular weights of 120, 140, and 180 kDa, in all regions examined. Optical densities w ere calculated separately for each NCAM isoform. Significant main effects of housing condition were found for synaptophysin levels in the dorsolateral striatum (F(1,50) = 4.86, p = 0.031, Figure 6 2a) and NCAM 140 levels in the cortex (F(1,50) = 5.14, p = 0.028, Figure 6 3a) with EE animals demonstrating higher levels of the synaptic proteins than SC animals. Although there were no significant main effects of stereotypy on synaptic protein levels, correlational analysis did reveal significant negative corre lations between stereotypy scores and synaptophysin concentrations in the dorsolateral striatum (r(52) = 0.381, p = 0.005, Figure 6 2b) and between

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91 stereotypy scores and NCAM 140 concentrations in the cortex (r(52) = 0.273, p = 0.046, Figure 6 3b). There were no differences in synaptic protein expression as a consequence of either housing condition or stereotypy in the dorsomedial striatum or hippocampus. No significant housing condition stereotypy interactions were found for any of the regions examined Discussion In this study, we examined the role of synaptic changes in the environmental regulation of stereotypy. Our results show that experience dependent plasticity was associated with region specific changes in synaptic protein content localized to t he dorsolateral striatum and cortex. Specifically, environmental enrichment increased the expression of synaptophysin in the dorsolateral striatum and NCAM 140 in the cortex. Increased levels of synaptophysin in the dorsolateral striatum and NCAM 140 in th e cortex were also associated with decreasing stereotypy. We also analyzed synaptic protein concentrations in the dorsomedial striatum and hippocampus; however, no differences in synaptophysin or NCAM expression were found as a result of either housing con dition or motor stereotypy in these areas. This regional pattern of effects is consistent with our previous research. We have demonstrated increases in neuronal metabolic activity (Turner et al., 2002) dendritic spine density (Turner et al., 2003) and brain derived neurotrophic factor (BDNF) expression (Turner and L ewis, 2003) in cortical and striatal regions following enrichment. This same regional distribution of increased neural plasticity was also evident when we compared low stereotypy animals to high stereotypy animals. Furthermore, these changes were regional ly selective as no effects of enrichment or stereotypy were found in any of the other areas examined, such as the hippocampus. Although we did not systematically differentiate between the dorsolateral and dorsomedial striatum in our previous studies, we an alyzed the striatal subregions separately in this study in

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92 order to more precisely delineate the regions involved in mediating the effects of environmental enrichment on stereotypy. Our results showed that synaptic modifications only occurred in the dorsol ateral subregion of the striatum. Whereas the dorsomedial striatum generally receives projections from association cortices, the dorsolateral striatum primarily receives afferent projections from the sensorimotor cortex, especially those involved in contro lling hindlimb, trunk, and forelimb movements (McGeorge and Faull, 1989; Ebrahimi et al., 1992) Thus, this finding suggests that the effects of environmental enrichment and stereotypy on synaptic plasticity were targeted specifically to the corticostriatal regions respon sible for the control of motor behavior Synaptic changes in the cortex and dorsolateral striatum were differentially indexed by the two synaptic proteins; i.e., synaptophysin was a better marker of enrichment and stereotypy related changes in the dorsola teral striatum, whereas NCAM 140 was better able to detect experience dependent synaptic modifications in the cortex. This suggests that different mechanisms may have been operative in inducing synaptic change in these two regions. Synaptophysin is an inte gral membrane component of presynaptic vesicles (Wiedenmann and Franke, 1985) As a specific marker of presynaptic terminals (Hiscock et al., 2000) synaptophysin is generally used to index synaptic density (Walaas et al., 1988) However, changes in synaptophysin expression have also been shown to reflect changes in neurot ransmitter release not related to changes in synapse number (Alder et al., 1992; Alder et al., 1995; Nakamura et al., 1999) Therefore, the changes in synaptophysin levels observed in the dorsolateral s triatum may indicate changes in synaptogenesis and/or presynaptic neurotransmission.

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93 NCAMs have also been validated as quantitative markers of synaptic density (Jorgensen, 1995) Of the three major NCAM isoforms expressed in the brain, only differences in cortical NCAM 140 levels were detected when comparing experimental groups. This isoform specificity is important because the different isoforms demonstrate different localizations and functions. For example, whereas NCAM 120 is found in glia and NCAM 180 is expressed primarily on postsynaptic membranes, NCAM 140 is expressed on both pre and post synaptic membranes (Noble et al., 1985; Persohn et al., 1989) As such, NCAM 140 can exert its effects on cell cell adhesion through either homophilic or heterophilic binding to stabilize synaptic connections (Schachner, 1997) In addition, NCAM 140 also plays a major role in altering neuronal morp hology by inducing neuritogenesis through its interactions with different signal transduction pathways (Niethammer et al., 2002) Regardless of the specific mechanisms involved, these changes in synaptic protein concentrations suggest increased synaptic reorganization. On a functional level, these structural alterations likely translate to changes in synaptic properties. Imp roved synaptic functioning, as mediated through greater synaptic efficacy, could contribute to the greater behavioral flexibility observed in low stereotypy mice. Although we did not find a significant interaction between housing condition and stereotypy o n synaptic protein levels, higher levels of synaptophysin and NCAM 140 were found in the dorsolateral striatum and cortex, respectively, of both enriched and low stereotypy mice. Thus, even though we cannot attribute enrichment related decreases in stereot ypy to the synaptic changes in these regions, the fact that both enrichment and decreasing stereotypy levels demonstrate identical patterns of region and protein specific effects is still suggestive of an

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94 association between the effects of environmental e nrichment on synaptic plasticity and the effects of environmental enrichment on stereotypy. Here, we employed immunoblotting techniques to assay synaptic protein concentrations due to the large sample size and multiple regions involved in our study. Althou gh this method is useful for high throughput analysis, changes in synaptic structure can only be inferred from changes in protein levels Therefore, future studies may be important to determine the exact morphological changes induced by environmental enric hment and motor stereotypy.

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95 Figure 6 1. Diagrammatic representation of the dissection procedure. Coronal sections shown are adapted from the atlas of Paxinos and Watson (1998). CTX, cortex; DLS, dorsolateral striatum; DMS, dorsomedial striatum; and HPC hippocampus.

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96 Figure 6 2. Effects of (a) environmental enrichment and (b) motor stereotypy on synaptophysin protein expression in the dorsolateral striatum. Representative blots of synaptophysin (38 kDa) are shown in the lower panel. indicates p < 0.05. Open symbols ( ) indicate environmentally enriched (EE) animals. Closed symbols ( ) indicate standard caged (SC) animals.

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97 Figure 6 3. Effects of (a) environmental enrichment and (b) motor stereotypy on NCAM 140 protein expression in the cerebral cortex. Representative blots of NCAM (expressed as three isoforms with apparent molecular weights of 120, 140, and 180 kDa) are shown in the lower panel. indicates p < 0.05. Open symbols ( ) indicate environmentally enriched (EE) animals. Closed symbols ( ) indicate standard caged (SC ) animals.

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98 CHAPTER 7 EFFECTS OF ENVIRONME NTAL ENRICHMENT ON S PATIAL AND STIMULUS RESPONSE LEARNING DE FICITS ASSOCIATED WI TH ABNORMAL STEREOTY PED MOTOR BEHAVIOR Introduction The multiple systems theory of memory organization is based on the hypothesis that different k inds of information are processed and stored in different parts of the brain. According to this hypothesis, the dorsal striatum mediates a form of learning and memory in which stimulus response associations are incrementally acquired (Packard and Knowlton, 2002) while the hippocampus processes information about the relationships among stimuli or events. I nitial evidence for this theory was based on observations that damage to a neuroanatomical structure selectively impaired the acquisition of some learning tasks while sparing others. In these experiments, it was noted that lesions to the hippocampal format ion selectively impaired the neostriatum impaired the acquisition of tasks requiring the use of stimulus procedural learning (Packard et al., 1989; Packard and McGaugh, 1992; McDonald and White, 1994) Since then, additional studies demonstrating the task dependent effects of intracerebral drug injections have provided further support that learning and memory abilities can be dissociated into independent neural systems (Packard and White, 1991; Packard et al., 1994; Packard and McGaugh, 1996; Packard and Teather, 1997a, b; Packard, 1999; Packard et al., 2001; Schroeder et al., 2002) More recently, at the molecular and cellular levels of analysis, the localization of learning induced changes in gene tra nscription (Colombo et al., 2003; Teather et al., 2005; Gill et al., 2007) and neurochemistry (Chang and Gold, 200 3; Pych et al., 2005) to specific brain regions continues to suggest that specialized regions of the brain subserve different mnemonic functions.

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99 Dissociation methodology has also been used to demonstrate the existence of multiple memory systems in humans Patients with damage to the medial temporal lobe (e.g., amnesiacs declarative memory system but perform normally on procedural learning tasks (Knowlton et al., 1994; Eldridge et al., 2002) In contrast, patients with diseases affecting the basal ganglia (e.g., requ ire the ability to probabilistically associate cues with specific task outcomes but are able to answer explicit, factual questions about the task (Knowlton et al., 1996a; Knowlton et al., 1996b; Marsh et al., 2004; Marsh et al., 2005) In the present study, we uti lize an analogous experimental approach to further investigate the behavioral functions of the striatum Our lab has characterized an animal model of striatal based neuropathology. We have observed frequent and intense motor stereotypies dominating the beh avioral repertoire of prairie deer mice ( Peromyscus maniculatus bairdii ). Neurobiological studies conducted using this model have implicated striatal dysfunction in the pathophysiology of these behaviors (Presti et al., 2002; Presti et al., 2003; Presti et al., 2004a; Presti et al., 2004b; Presti and Lewis, 2005) i ndicating that stereotypy may be a behavioral sign of striatal pathology. T he stereotypies expressed by these mice are notable in that they are extremely context dependent: phenotypically distinct groups of mice, i.e., low stereotypy and high stereotypy, c an be generated by differentially housing deer mice in either environmentally enriched or environmentally impoverished conditions, respectively (Powell et al., 1999; Powell et al., 2000) P revious findings from our lab have demonstrated that enrichment related attenuation of stereotypy in these animals is associated with increases in measures of synaptic plasticity localized to the striatum (Turner et al., 2002; Turner and Lewis, 2003; Turner et al., 2003) thereby suggesting

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100 that the beneficial effects of environmental enrichment on stereotypy are linked to improvements in striatal functio n This experiment was designed to accomplish three goals. First, because stereotypy is associated with striatal dysfunction and striatal dysfunction also interferes with stimulus response learning and memory abilities, we wanted to determine whether stere otypy would be selectively associated with impairments in stimulus response learning To this end we tested deer mice in a striatum dependent cued Morris water maze task. As a control, we also evaluated performance in a hippocampus dependent spatial Morri s water maze task. Our second goal was to determine whether the beneficial effects of environmental enrichment on stereotypy and therefore presumably striatal functioning, would also extend to behavioral performance in the water maze tasks Thus, the mice were reared in either standard cage or environmentally enriched housing conditions. Finally, our last goal wa s to determine whether differences in task performance would be associated with regionally specific changes in neuro plasticity Therefore, we anal yzed levels of the synaptic proteins synaptophysin and neural cell adhesion molecule ( NCAM ), following water maze training We selected these proteins as markers of synaptic plasticity based on previous research demonstrating experience dependent changes in their levels of expression following both enrichment (Frick and Fernandez, 2003; Koo et al., 2003; Gresack and Frick, 2004; Nithianantharajah et al., 2004; Lambert et al., 2005; Bennett et al., 2006; Nithianantharajah et al., 2007) and learning (Sandi et al., 2005; Venero et al., 2006; Derksen et al., 2007) Materials and Methods Anim als Deer mice ( Peromyscus maniculatus bairdii ) were obtained from a breeding colony maintained at the University of Florida. The mice were housed in a room kept at 25C,

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101 maintained on a 16:8 light:dark cycle (lights off at 2200), and provided with access t o food and water ad libitum At weaning (3 weeks of age), mice were randomly assigned to either standard cage (SC, n = 104) or environmentally enriched (EE, n = 112) housing conditions. Standard caged mice were housed in standard laboratory cages (29 33 15 cm) with two other same sex weanlings, while environmentally enriched animals were housed in groups of five or six in modified large dog kennels (122 81 89 cm). These enrichment kennels were fitted with galvanized wire mesh to create multiple inte rconnected levels and equipped with bedding, a running wheel, and assorted ceramic and plastic objects varying in color, shape, and size. Throughout the 10 wk differential housing period, enrichment items were removed and replaced with novel objects on a w eekly basis. To encourage foraging behaviors, approximately 50 grams of birdseed were also randomly distributed throughout the kennels three times a week. All animal procedures were approved by the University of Florida Institutional Animal Care and Use Co mmittee and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals Quantification of Stereotypic Behavior Animals were tested for stereotypy after 8 weeks in their respective housing conditions (see Figure 7 1 for a det ailed timeline of experimental events). Mice were removed from their home cages and placed singly into Plexiglas testing cages (22 15 28 cm) two hours prior to the beginning of their dark cycle. Food, water, and bedding were provided, and the mice were left undisturbed for 2 hours to allow for habituation to the new environment and recovery from the stress of handling. Stereotypic behaviors were quantified using an automated testing apparatus (Columbus Instruments, Columbus, OH) designed to register ste reotypic motor activity (e.g., repetitive jumping or backwards somersaulting) as photobeam breaks. All testing sessions

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102 were also recorded using a digital video surveillance system (GeoVision Inc., Taipei, Taiwan) and checked in order to verify the accurac y of automated counts. Data were collected for two dark cycles, and the stereotypy score for each animal was calculated as the average number of photobeam interruptions per hour of testing. Mice with stereotypy scores above 600 photobeam breaks/hr were des ignated as High Stereotypy (HS) animals, whereas mice with stereotypy scores below 400 photobeam breaks/hr were designated as Low Stereotypy (LS) animals. These values correspond to approximately the top and bottom third of stereotypy scores for all animal s tested. Animals with intermediate stereotypy scores between 400 and 600 photobeam breaks/hr were not used for Morris water maze testing or Western analysis of synaptic protein concentrations. Morris Water Maze (MWM) Testing The water maze was a black cir cular pool (94 cm in diameter and 37 cm high). Four starting positions (N, S, E, and W) were equally spaced around the perimeter of the pool, dividing it into four quadrants. The pool was filled with water maintained at 25C and made opaque with the additi on of non toxic white tempera paint. An escape platform (a 10 cm diameter Plexiglas disk covered with sheer nylon hose), which the mice can mount to escape the water, was submerged 2 cm below the surface of the water. Behavioral testing occurred during the light cycle between 0900 and 1100. For both the spatial and cued tasks, training consisted of 8 trials per day over the course of 7 consecutive days. In each trial, the animal was released, facing the wall of the maze, from one of the four starting positi ons and allowed to escape onto the hidden or cued platform. The position of the tester remained constant after placement of the animal into the maze, and each starting position was used twice during each 8 trial training session. After mounting the platfor m, the animal was allowed to remain on it for 10 sec before being placed into a holding cage for an intertrial interval of approximately 10 min. Three 60 sec

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103 probe trials were conducted: the first probe trial was conducted midway through training, the seco nd probe trial was conducted on the last day of training, and the final probe trial was conducted 3 days post training. Spatial MWM Task In the spatial version of the water maze task, the escape platform was always located in the center of the NW quadrant. The animals were required to navigate to the submerged platform using the extra maze spatial cues present in the room which included a computer, a video tracking system, a sink, and cabinets. The escape platform was removed during the probe trials. Animal s were released from three different starting positions for each of the three probe trials. Cued MWM Task In the cued version of the water maze task, a white foam cross (7 7 cm) was always suspended 10 cm above the submerged platform during training. The location of the escape platform was varied from trial to trial in order to prevent the use of a spatial strategy. Each of the four quadrants contained the platform on two of the 8 trials, and the platform was placed so that the distance (i.e., proximal or distal) and direction (i.e., left or right) of the platform relative to the starting point was counterbalanced across each daily block of 8 trials. During the probe trials, the escape platform was removed from the maze; however, the cue remained suspended in the center of a maze quadrant located distal to the starting position. The cue was placed in three different quadrants for each of the three probe trials. Western Blotting Levels of synaptophysin and neural cell adhesion molecule (NCAM) in the dorsolat eral striatum, dorsomedial striatum, hippocampus, and neocortex were determined using Western blotting. Tissue samples from control animals were obtained from a previous study. A nimals trained in MWM tasks were sacrificed by cervical dislocation f ollowing the final probe trial The

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104 b rains were rapidly removed, snap frozen in isopentane, and stored at 80 C until processed for protein extraction. Briefly, m thick coronal sections were collected on a cryostat held at 20C. Tissue samples from the regions of interest were microdissected by punch method and sonicated in a homogenization buffer containing 20 mM Tris, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X Samples were then placed on ice and vortexed every 15 min for 1 hr before clarification via centrifugation at 16,000 g for 15 min. Subsequently the supernatant was collected, and the protein concentrations were determined using the DC Protein Assay kit (Bio Rad Laboratories, from each sample were loaded onto 12% polyacrylamide gels and separated by SD S PAGE before transfer to polyvinylidene difluoride membranes. Loading of each gel was counterbalanced according to condition and group as much as possible. Gels and blots were also routinely stained with Coomassie blue and Ponceau S (Sigma), respectively, in order t o verify equal sample loading. The m embranes were then washed to remove excess stain and blocked with 5% nonfat milk in Tris buffered saline and 0.05% Tween (TBST) for 1 hr before incubation with the primary antibody (1:50,000 synaptophysin or 1 :100 anti NCAM; both antibodies from Chemicon, Temecula, CA) overnight at 4 C. The next day, blots were washed in TBST for 15 min before incubation with the appropriate horseradish peroxidase conjugated secondary antibody (1:5,000; Santa Cruz Biotechnology Santa Cruz, CA) for 1 hr at room temperature. Blots were then developed using enhanced chemiluminescence and exposed to CL XPosure film (Pierce Biotechnology, Rockford, IL) for visualization of protein bands Finally, q uantification of immunoreactive pro tein bands was done by densitometry using ImageJ software (version 1.37, NIH, Bethesda, MD).

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105 Data Analysis The Ethovision Pro (version 2.3, Noldus Information Technology, the Netherlands) video tracking system was used to acquire and calculate maze perform ance parameters (e.g., escape latencies, path length to platform, swim velocities, thigmotaxis, quadrant time, platform crossings, etc.). Wintrack (version 2.4) (Wolfer et al. 2001) was used to generate plots of group search preferences during the final probe trial (Figures 7 3 and 7 5). For this analysis, the water maze was subdivided into 20 20 square tiles, and the time spent in each tile, relative to the amount of time expected according to chance, was averaged for each group with the darker tiles indicating greater time spent in an area of the water maze. Statistical analyses were performed using SPSS software (Chicago, IL). All data are presented as mean standard err or of the mean (SEM). Morris water maze performance was analyzed using two way repeated measures analyses of variance (ANOVAs) with either Housing, Stereotypy, or Group as the independent variable and Training Day as the repeated measure. Separate one way ANOVAs without repeated measures were used to compare Group performance within the same Training Day. One way ANOVAs without repeated measures were also used to determine differences in probe trial and synaptic proteins data. Post hoc analysis of significa nt effects was accomplished using Newman Keuls test. Correlations between MWM other tests used are as indicated in the text. The minimum level of significance was se t at p < 0.05 for all tests. Results Subjects The interaction of housing condition and stereotypy yielded four experimental groups of mice: environmentally enriched, low stereotypy (EE LS); environmentally enriched, high

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106 stereotypy (EE HS); standard caged low stereotypy (SC LS); and standard caged, high stereotypy (SC HS). Because previous studies have suggested that behavioral flexibility may be impaired in stereotypic animals (Garner and Mason, 2002; Garner et al., 2003b; Garner et al., 2003a) separa te sets of the four groups were tested on spatial and cued versions of the MWM task. For analysis of synaptic marker proteins, a control condition, consisting of animals not exposed to water maze training, was also used. Stereotypy scores did not differ si gnificantly between the same groups assigned to different conditions. One mouse was unable to swim and was removed from MWM testing after the first trial. Two mice also failed to find the escape platform in any of the 56 training trials and were eliminated from behavioral analysis as non learners. The final sample sizes and stereotypy data for each condition (spatial MWM, cued MWM, and control) are shown in Table 7 1. Morris Water Maze Testing The mean escape latency, total distance moved, average swim velo city, thigmotaxis, and percent time spent in the platform quadrant were used as measures of water maze task acquisition. For the probe trials, several measures of performance (e.g., quadrant time, platform crossings, mean proximity to the platform, etc.) w ere calculated; however, the average number of platform crossings proved to be the most sensitive measure at detecting group differences for both tasks. Spatial MWM Task For the spatial MWM task, all groups learned to find the hidden platform as suggested by a significant (p < 0.001) main effect of Training Day for all parameters examined. However, significant differences in escape latency (F(1,42) = 9.21, p = 0.004), the total distance moved (F(1,42) = 8.79, p = 0.005), and NW (i.e., platform) quadrant ti me (F(1,42) = 6.18, p = 0.017) were found for the effect of Stereotypy with HS animals performing more poorly than LS

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107 animals. There were no significant Stereotypy Training Day interactions. A main effect of Housing was found for thigmotaxis (F(1,42) = 6 .12, p = 0.017) with SC mice showing significantly greater thigmotaxis than EE animals. Significant Housing Training Day interactions were also found for the total distance moved (F(6,252) = 2.28, p = 0.037) and time spent in the NW quadrant (F(6,252) = 3.19, p = 0.005). Group comparisons (Figure 7 2) revealed significant differences in escape latency (F(3,40) = 5.60, p = 0.003), distance (F(3,40) = 3.90, p = 0.016), and thigmotaxis (F(3,40) = 3.37, p = 0.028) between groups with the SC HS group performin g more poorly than the EE LS, EE HS, and SC LS groups which did not differ significantly from each other. One way ANOVAs, comparing group performance within individual training days, revealed that significant differences between the SC HS animals and the o ther groups occurred primarily during the last few days of training. No significant Group Training Day interactions were found. No differences in swim velocities were found as a function of Housing (F(1,42) = 0.766, p = 0.386), Stereotypy (F(1,42) = 1.69 p = 0.201), or Group (F(3,40) = 1.43, p = 0.249). No group differences (F(3,40) = 0.085, p = 0.968) in the number of platform crossings were observed during the first spatial probe trial which was conducted midway through training (Figure 7 3). However, by the last day of training (Probe Trial 2), all groups, except for the SC HS group, demonstrated significant improvements in performance. EE LS, EE HS, and SC LS mice performed significantly better (F(3,40) = 8.83, p < 0.001) than SC HS mice during the fi nal probe trial (Probe Trial 3) which was conducted 3 days post training. There was also a test: t(42) = 4.39, p < 0.001) on performance during the third probe trial with EE animals showing significantly more platform crossings than SC animals. The performance of the EE LS and EE HS groups did not differ between the second

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108 and third probe trials, whereas SC LS mice showed significantly fewer platform crossings (p < 0.05) during the final probe trial when compa red to the second probe trial. Cued MWM Task For the cued MWM task, all groups learned to find the cued platform as suggested by a significant (p < 0.001) main effect of Training Day for all parameters examined. There was a highly significant main effect of Stereotypy on escape latency (F(1,43) = 42.6, p < 0.001), distance moved (F(1,43) = 27.4, p < 0.001), thigmotaxis (F(1,43) = 26.8, p < 0.001), and cued quadrant time (F(1,43) = 28.4, p < 0.001) with HS animals performing more poorly than LS animals on a ll measures. Group comparisons (Figure 7 4) further confirmed this pattern of results: the EE HS and SC HS groups performed more poorly than the EE LS and SC LS groups on the measures of escape latency (F(3,41) = 17.2, p < 0.001), total distance moved (F(3 ,41) = 10.5, p < 0.001), thigmotaxis (F(3,41) = 10.1, p < 0.001), and cued quadrant time (F(3,41) = 12.7, p < 0.001). Significant Stereotypy Training Day (F(6,258) = 4.56 to 10.7, p < 0.001) and Group Training Day (F(18,246) = 2.11 to 4.66, p < 0.01) i nteractions were also found for all parameters examined except velocity. Although the EE groups appeared to perform consistently better than their SC counterparts, this trend failed to reach significance, and no significant main effects of Housing were fou nd for any of the measures examined. There were also no significant Housing Training Day interactions. As with the spatial task, there were no systematic differences in swim velocities due to Housing (F(1,43) = 0.001, p = 0.982), Stereotypy (F(1,43) = 1. 10, p = 0.300), or Group (F(3,41) = 0.638, p = 0.595). Analysis of cued probe trial performance (Figure 7 5) revealed that the EE LS group performed significantly better (F(3,41) = 9.13, p < 0.001) than all other groups in the first probe trial and signifi cantly better (F(3,41) = 6.49, p = 0.001) than the EE HS and SC HS groups in the final probe trial. All groups showed similar levels of performance on the last day of training

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109 (Probe Trial 2). When compared across probe trials, the EE LS group also showed significantly more platform crossings (F(2,33) = 4.71, p = 0.016) in the first probe trial than in the second probe trial. Synaptic Proteins Synaptophysin and neural cell adhesion molecule (NCAM) levels were evaluated in the dorsolateral striatum, dorsomed ial striatum, hippocampus, and neocortex using Western blotting analysis. The dorsal striatum and hippocampus were chosen based on their roles in stimulus response and spatial learning, respectively. The medial and lateral subregions of the dorsal striatum were analyzed separately based on evidence suggesting functional dissociation with regard to learning and memory (Yin and Knowlton, 2006) and the neocortex was selected based on previous studies suggesting its importance in mediating enrichment related effects on synaptic plasticity and learning and memory (Frick and Fernandez, 2003) Synaptophysin migrated as a single 38 kDa band, while NCAM was expressed as 3 isofo rms, with apparent molecular weights of 120, 140, and 180 kDa, in all regions examined (Figure 7 6). Learning induced changes in synaptic protein expression To determine learning induced changes in synaptic plasticity, the synaptic protein concentrations f or mice trained in the MWM were first normalized relative to the corresponding group of control animals not exposed to water maze training. One way ANOVAs were then used to make comparisons between different Groups (EE LS, EE HS, SC LS, SC HS) within the s ame Condition (spatial MWM, cued MWM, control) and between different Conditions within the same Group (Table 7 2). The results show that the EE LS group trained in the cued MWM had significantly higher levels of synaptophysin in the dorsolateral striatum t han all other groups trained in the cued MWM (F(3,41) = 9.35, p < 0.001), EE LS mice trained in the spatial MWM (F(2,34) = 5.52, p = 0.008), and control EE LS mice (F(2,34) = 5.52, p = 0.008). Levels of

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110 NCAM 180 were higher in the hippocampus of EE HS mice trained in the spatial MWM than in EE HS cued MWM or EE HS control mice (F(2,32) = 6.07, p = 0.006). SC LS mice trained in the spatial MWM also demonstrated higher hippocampal concentrations of NCAM 180 (F(2,28) = 3.53, p = 0.043) when compared to SC LS c ued MWM and SC LS control mice. EE LS mice trained in the spatial MWM showed significantly greater levels of NCAM 180 in the neocortex (F(2,34) = 6.32, p = 0.005) than either EE LS mice trained in the cued MWM or control EE LS mice. Similarly, EE HS mice t rained in the spatial MWM also showed significantly higher (F(2,32) = 4.74, p = 0.016) levels of NCAM 180 in the neocortex than EE HS control animals. Relationships between MWM performance and synaptic protein levels Correlational analyses were performed t o determine the relationship between overall synaptic protein levels and water maze performance. There were no significant correlations between synaptic protein levels and spatial MWM performance. In contrast, for the cued MWM, synaptophysin levels in the dorsolateral striatum were positively correlated with improved performance during both training and probe trials as indicated by a negative correlation (r(43) = 0.553, p < 0.001) between synaptophysin levels and the mean escape latency averaged over all 7 training days and a positive correlation (r(43) = 0.581, p < 0.001) between synaptophysin levels and the number of platform crossings during the final probe trial (Figure 7 7). The converse was true for NCAM 120 levels in the hippocampus which were invers ely correlated with cued MWM performance: hippocampal NCAM 120 levels were positively correlated (r(43) = 0.467, p = 0.001) with the average escape latency during training and negatively correlated (r(43) = 0.495, p = 0.001) with the number of platform cr ossings during the final probe trial (Figure 7 8). Discussion Our data demonstrate that stereotypy is associated with deficits in both spatial and stimulus response learning and memory. This was indicated by the performance of the high

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111 stereotypy groups re lative to the low stereotypy groups in the spatial and cued versions of the Morris water maze task, which required spatial and stimulus response learning, respectively. Standard caged, high stereotypy mice performed more poorly than all other groups in the spatial task, and both groups of high stereotypy mice performed more poorly than the low stereotypy mice in the cued task. Although these findings appear to be contrary to our initial hypothesis that stereotypy would be selectively associated with deficit s in only stimulus response learning, and not spatial learning, these results are not incongruent with a specific role for striatal dysfunction in the pathophysiology of stereotypy. Functional heterogeneity exists within the striatum. With respect to the r ole of the dorsal striatum in learning and memory, one theory suggests that the medial and lateral subregions support different mnemonic functions. According to this theory, the dorsolateral striatum selectively mediates stimulus response learning, whereas the dorsomedial striatum may also be involved in spatial learning. Previous studies have demonstrated that damage to the medial, but not lateral, portions of the dorsal striatum disrupts spatial learning in hidden platform Morris water maze tasks (Whishaw et al., 1987; Devan et al., 1999) and results in a preference for response strategies in tasks designed to simultaneously evaluate place versus cue guided behaviors (Devan et al., 1999; Devan and White, 1999; Yin and Knowlton, 2004) Given experimental evidence supporting a further dissociation of learning functions in the dorsal striatum, one interpretation of the present data is that the striatal pathology associated with stereotypy in our model encompasses both the dorsolateral and dorsom edial regions of the striatum with dysfunction of the dorsolateral striatum contributing to deficits in stimulus response learning and dysfunction of the dorsomedial striatum contributing to deficits in spatial learning. This interpretation is consistent w ith previous neurobiological studies from our lab. We

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112 have demonstrated differences in synaptic plasticity between high and low stereotypy mice localized to both the dorsolateral and dorsomedial regions of the striatum (Turner et al., 2002) In addition, our research has indicated that these differences are regionally specific for areas involved in motor control as no differences in synaptic structure or function were found in limbic areas, such as the hippocampus (Turner and Lewis, 2003; Turner et al., 2003) Another explanation for these results may pertain to possible interactions between the striatum and hippocampus during learning. Although once thought to be relatively independent, it is increasingly appreciated that complex interactions exist between memory systems. These interactions may be either cooperative or competitive. Thus, if the striatum and hippocampus normally cooperate during spatial learning, striatal pathology may also negatively impact spatial learning ability, thereby explaining the spatial learning deficit associated with stereotypy. Indeed, it has been shown that lesions to the st riatum can interfere with spatial MWM task performance in some cases (Block et al., 1993; Devan et al., 1999; Mura and Feldon, 2003) It should also be noted that this interpretation is not mutually exclusive with the aforementioned role for the dorsomedial striatum in spat ial learning. In fact, the presence of direct anatomical connections between the posterior dorsomedial striatum and entorhinal cortex (Totterdell and Meredith, 1997) sug gests that the dorsomedial striatum and hippocampus may influence each other to produce behavioral output, providing support for both interpretations. The spatial and stimulus response learning deficits associated with stereotypy did not appear to result f rom motor, sensory, or motivational components. There were no systematic differences in swim velocities between groups in either of the water maze tasks to indicate motor impairment or differences in swimming ability. For both tasks, all groups also showed increased time spent in the platform quadrant with training. Increases in this measure both require the

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113 ability to perceive extra maze and intra maze cues and indicate motivation to escape the water maze. Furthermore, we have also conducted other water ma ze studies using a fixed, visible escape platform. In these studies, standard caged, high stereotypy mice (i.e., the group that performed most poorly in both tasks) did not perform significantly differently from other groups (unpublished observations). Env ironmental enrichment alone was sufficient to counteract the observed deficits in spatial learning: environmentally enriched, high stereotypy mice performed significantly better than standard caged, high stereotypy mice in the spatial water maze task at le vels similar to the low stereotypy groups. Moreover, enrichment also promoted the retention of spatial memory 3 days post training; i.e., the number of platform crossings shown by the enriched, low stereotypy and enriched, high stereotypy groups did not de crease between the probe trials conducted during the last day of training and 3 days later. However, enrichment was unable to compensate for deficits in stimulus response learning since both groups of high stereotypy mice, enriched and standard caged, perf ormed more poorly than the low stereotypy groups in the cued task. Thus, there was a dissociation in the effect of environmental enrichment on performance in the two water maze tasks, suggesting that the central structures which mediate spatial and stimulu s response learning (i.e., the hippocampal formation and dorsal striatum, respectively) may have been differentially sensitive to the beneficial effects of environmental enrichment. Previous studies have demonstrated that the effects of environmental enric hment on learning and memory are highly dependent on the form of enrichment provided. For example, the effects of social and inanimate stimulation on spatial and reversal learning are dissociable, suggesting that they may act on different neural substrates (Schrijver et al., 2004) Certain individual elements of enrichment, such as voluntary exercise, have also been shown to

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114 contribute more greatly to the memory enhancing effect of enrichment than other p arameters (Lambert et al., 2005; Harburger et al., 2007b) Similarly, variations in the duration of enrichment also impact performance on l earning tasks (Bennett et al., 2006) Consistent with the results of this experiment, other studies have also demonstrated that enric hment improves performance specifically in spatial but not cued water maze tasks (Frick and Fernandez, 2003; Frick et al., 2003) Our enrichment paradigm incorporated many of the experimental parameters typically used in enrichment studies, including increased spatial density increased opportunities running wheels for exercise. Therefore, it is difficult to determine which of these parameters may have been responsible for the i mprovements seen in spatial task performance. However, environmental novelty was also a key aspect of our enrichment protocol: enriched mice were regularly exposed to different enrichment items arranged in different configurations. We speculate that this m ight have provided them with additional cognitive stimulation with respect to the formation of spatial maps, thereby enhancing their spatial learning abilities. The patterns of learning induced changes in synaptic protein expression were different followin g training in the two MWM tasks. However, the changes were generally localized to regions thought to mediate a specific learning task with the groups showing superior levels of task performance generally showing increased expression of the synaptic protein s. This suggests that lasting modifications in synaptic function occurred in the animals which performed well. For example, environmentally enriched, low stereotypy mice far outperformed the other groups in the cued water maze task. This group also had the highest levels of synaptophysin in the dorsolateral striatum when compared to the other groups trained in the cued task, environmentally enriched, low stereotypy mice trained in the spatial task, and environmentally

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115 enriched, low stereotypy control mice. Generally considered to be a reliable marker of synaptic density (Walaas et al., 1988; Eastwood et al., 2006) learning induced increases in levels of synaptophysin in the dorsolateral striatum of environmentally enriched, low stereoty py mice, therefore, suggest that improved performance in the cued MWM task was related to increased synaptogenesis within the dorsolateral striatum. Because synaptophysin is an integral membrane component of neurotransmitter containing presynaptic vesicles (Wiedenmann and Franke, 1985) these results further suggest that an increase in the efficacy of synaptic transmi ssion within the dorsolateral striatum may have contributed to better task performance. The overall positive correlation between cued MWM performance and synaptophysin levels in the dorsolateral striatum for all groups trained in the cued task also support s this conclusion. In the spatial water maze task, all groups, except for the standard caged, high stereotypy group, demonstrated improvements in spatial memory with training. Similarly, all groups, except for the standard caged, high stereotypy group, sho wed significant increases in NCAM 180 levels in either the hippocampus or neocortex when compared to their corresponding control group. Improved performance in the spatial MWM task requires the formation of spatial memory for the hidden platform. Experimen tal data support a role for neural cell adhesion molecules in long term memory storage (Welzl and Stork, 2003; San di, 2004) Several studies have demonstrated that interfering with NCAM function impairs performance in spatial learning tasks (Cremer et al., 1994; Arami et al. 1996; Cambon et al., 2003) whereas administration of synthetic peptides which mimic NCAM action improves spatial learning (Cambon et al., 2004; Klementiev et al., 2007) NCAM 180, in particular, has been shown to play a crucial role in synapse formation and sta bilization, especially during the synaptic remodeling processes which accompany long term potentiation (Schuster et al., 1998; Dityatev et al., 2000; Sandi et al., 2005) Therefore, these

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116 results suggest that enhanced synaptic connectivity, as mediated through increased NCAM 180 levels, in the hippocampus and neo cortex may have contributed to the improved performance of the enriched and standard caged, low stereotypy groups in the spatial MWM task. Although superior task performance generally corresponded to increases in synaptic protein expression, one notable ex ception was the inverse relationship between cued water maze performance and hippocampal NCAM 120 levels. As mentioned previously, within the conceptual framework of the multiple memory systems theory, interactions between memory systems can be competitive as well as cooperative. This finding indicates that increased synaptic plasticity in one region (i.e., the hippocampus) was detrimental to the performance mediated by another system (i.e., the dorsolateral striatum) and may represent a neural substrate of competitive interference. Altogether, these results demonstrate that the striatal dysfunction associated with stereotypy in deer mice is also linked to deficits in both spatial and stimulus response learning. Environmental enrichment alone was sufficient to counteract deficits in spatial learning and even enhanced the retention of spatial memory post training. However, enrichment was unable to compensate for deficits in stimulus response learning. Finally, the regional patterns of learning induced changes in synaptic protein expression were task specific and related to levels of performance.

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117 Table 7 1. Stereotypy scores of experimental groups. Spatial MWM Cued MWM Control Group n Stereotypy Score n Stereotypy Score n Stereotypy Score EE L S 12 209 35 12 212 31 13 264 20 EE HS 11 824 47 11 783 45 13 804 72 SC LS 10 166 35 11 247 36 10 250 29 SC HS 11 1087 82 11 1034 108 18 980 71 Values represent mean stereotypy scores (photobeam br eaks/hr) SEM.

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118 Table 7 2. Immunoblotting analysis of synaptic marker proteins. Dorsolateral Striatum Dorsomedial Striatum Hippocampus Neocortex Synaptic Protein Group Spatial MWM Cued MWM Spatial MWM Cued MWM Spatial MWM Cued MWM Spatial MWM Cued MWM Synaptophysin EE LS 98.3 5.4 120.5 4.5 a 101.8 5.6 103.7 5.3 97.5 6.8 107.7 6.1 99.3 4.1 101.9 4.6 EE HS 101.9 3.6 95.7 2.8 102.4 5.8 101.7 5.5 100.4 6.7 98.2 5.4 99.5 4.0 101.3 3.7 SC LS 10 3.4 3.1 100.0 5.0 99.2 7.6 102.3 4.8 97.9 8.8 98.0 7.5 101.0 3.1 101.2 3.8 SC HS 104.0 5.4 93.2 3.9 99.3 5.4 102.1 5.3 100.4 11.2 97.9 7.3 99.9 3.7 97.7 3.1 NCAM 120 EE LS 98.9 4.9 96.1 7.6 103.4 9.0 103. 6 6.0 101.5 8.9 89.8 8.1 101.2 7.4 103.9 6.9 EE HS 101.1 6.0 105.7 4.8 101.7 8.7 103.6 7.6 100.8 6.9 112.8 5.6 102.6 5.5 104.2 4.3 SC LS 98.2 7.4 98.2 7.6 103.7 6.6 104.6 7.6 103.8 9.4 98.1 7.6 104.0 5.0 104.4 4.4 SC HS 98.9 7.2 102.2 4.9 102.0 8.5 103.6 7.3 105.1 12.2 107.3 5.7 100.1 4.3 97.9 4.9 NCAM 140 EE LS 101.5 3.8 100.2 6.1 103.7 7.5 103.4 6.5 112.4 5.7 102.2 5.5 100.8 5.7 99.6 4.9 EE HS 98.9 4.3 100.1 3.8 103.0 7.4 103.3 6.4 123.9 8.4 111.3 6.7 100.7 6.6 100.4 5.6 SC LS 96.8 5.3 99.6 3.7 103.0 8.4 103.0 8.7 116.3 9.3 97.6 4.8 101.0 7.1 100.8 6.9 SC HS 99.4 6.7 98.5 6.3 101.9 7.1 102.7 6.8 111.8 11.2 104.2 5.6 99.3 6.1 100.2 8.1 NCAM 180 EE LS 103.0 7.8 102.6 6.2 114.0 7.4 103.2 6.6 119.6 8.5 100.5 11.4 138.1 8.4 d 102.7 7.7 EE HS 102.2 6.3 101.9 4.9 110.0 7.9 102.1 9.0 146.7 8.4 b 107.9 12.0 139.2 9.9 e 106.1 7.9 SC LS 102.3 8.0 103.0 3.9 110.0 7.3 102.9 9.5 142.3 10.3 c 107.6 11.0 122.3 9.5 105.3 7.9 SC HS 102.6 9.2 100.8 6.8 104.5 6.1 104.0 6.8 115.2 10.8 103.7 7.0 123.2 9.5 97.9 9.9 Densitometric values are normalized to the corresponding control group and are expressed as mean (% control) SEM. Significant values are shown in boldface. Symbols a, indicates p < 0.001 compared to EE HS, SC LS, and SC HS animals trained in the cued MWM a nd p < 0.05 compared to EE LS control and EE LS spatial MWM animals. b, indicates p < 0.01 compared to EE HS control and p < 0.05 compared to EE HS cued MWM animals. c, indicates p < 0.05 compared to SC LS control and SC LS cued MWM animals. d, indicates p < 0.01 compared to EE LS control and EE LS cued MWM animals. e, indicates p < 0.05 compared to EE HS control animals.

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119 Figure 7 1. Illustration depicting the timeline of experimental events. Animals were housed under either en riched or standard cage conditions starting from the time of weaning (3 wks) to the conclusion of Morris water maze (MWM) testing (13 wks). At 11 wks, animals were evaluated for stereotypy prior to training in either spatial or cued versions of the MWM tas k. The training schedule was identical for both tasks. Animals were trained for 7 days with probe trials conducted prior to the 4 th and 7 th days of training and 3 days following the final day of training. Numbers indicate the time in weeks (top line) and d ays (bottom line).

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120 Figure 7 2. Performance during spatial MWM task acquisition as measured by (a) mean escape latency, (b) total distance moved, (c) average velocity, (d) thigmotaxis, and (e) percent time spent in the NW (platform) quadrant.

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121 Figure 7 3. Performance during spatial MWM probe trials as measured by (a) the average number of platform crossings. Probe trials were conducted mid way through training (Probe Trial 1), on the last day of training (Probe Trial 2), and 3 days post training (Prob e Trial 3). For the final probe trial, (b) a representative swim path (left) and the time each group spent searching in different areas of the water maze (right) are also shown. Darker tiles indicate more time spent within an area of the water maze. ## and # indicate significant differences of p < 0.01 and p < 0.05, respectively. *** and indicate p < 0.001 and p < 0.05 vs SC HS, respectively.

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122 Figure 7 4. Performance during cued MWM task acquisition as measured by (a) mean escape latency, (b) total dis tance moved, (c) average velocity, (d) thigmotaxis, and (e) percent time spent in the cued (platform) quadrant.

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123 Figure 7 5. Performance during cued MWM probe trials measured by (a) the average number of platform crossings. Probe trials were conducted m id way through training (Probe Trial 1), on the last day of training (Probe Trial 2), and 3 days post training (Probe Trial 3). For the final probe trial, (b) a representative swim path (left) and the time each group spent searching in different areas of t he water maze (right) are also shown. Darker tiles indicate more time spent within an area of the water maze. # indicates a significant difference of p < 0.05. §§§ indicates p < 0.001 vs EE HS, SC LS, and SC HS. *** indicates p < 0.001 vs SC HS and p < 0.0 5 vs EE HS.

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124 Figure 7 6. Representative immunoblots of the synaptic proteins (a) synaptophysin and (b) neural cell adhesion molecule (NCAM). Synaptophysin migrated as a single 38 kDa band. NCAM was expressed as 3 isoforms with molecular weights of 120, 140, and 180 kDa.

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125 Figure 7 7. Relationship between synaptophysin levels in the dorsolateral striatum and cued MWM performance as measured by (a) the mean escape latency averaged over all training days and (b) the number of platform crossings during the final probe trial. Higher levels of synaptophysin in the dorsolateral striatum were associated with better performance in the cued MWM task. Open symbols ( ) indicate environmentally enriched animals. Closed symbols ( ) indicate standard caged animals.

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126 Figure 7 8. Relationship between NCAM 120 levels in the hippocampus and cued MWM performance as measured by (a) the mean escape latency averaged over all t raining days and (b) the number of platform crossings during the final probe trial. Higher levels of NCAM 120 in the hippocampus were associated with poorer performance in the cued MWM task. Open symbols ( ) indicate environmentally enriched animals. Close d symbols ( ) indicate standard caged animals.

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127 CHAPTER 8 CONCLUSIONS Summary of Findings Previous work in our lab has consistently demonstrated the ameliorative effects of environmental enrichment on stereotypy (Powell et al., 1999; Powell et al., 2000; Turner et al., 2002; Turner and Lewis, 2003; Turner et al., 2003; Hadley et al., 2006) The goal of this research was to identify the mechanisms which contribute to the impact of environmental manipulations on stereotypy. As reviewed previo usly (Chapter 1), the involvement of the nigrostriatal dopamine system in the production of motor stereotypies is well established. Given the importance of dopamine function in the mediation of stereotypic behaviors, our initial characterization of this mo del included neurobiological studies targeted at detecting differences in dopamine receptor density or endogenous dopamine content. Although these early studies failed to find differences in the density of striatal D 1 or D 2 dopamine receptors or in the con centration of striatal dopamine and its metabolites as a function of either housing condition or stereotypy level (Powell et al., 1999) subsequent studies from our lab have since demonstrated that differences in the response to dopamine do exist between high and low stereotypy animals (Presti et al., 2003; Presti and Lewis, 2005) The results of our first study (Chapter 4) reconciled these findi ngs by demonstrating that alterations in dopamine signaling represent one mechanism for the observed differences in dopamine sensitivity between high and low stereotypy animals. Our data demonstrate that stereotypy is inversely related to the proportion of DARPP 32 phosphorylated at Thr75 (phospho Thr75 DARPP 32/total DARPP 32). Because phosphorylation of DARPP 32 at Thr75 converts it into an inhibitor of PKA, these results suggest that decreased stereotypy is linked to lower tonic activation of the cAMP/PK A signaling cascade in striatal neurons, whereas

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128 increased stereotypy is associated with a release in PKA inhibition. We also found significantly lower levels of total DARPP 32 protein specifically in environmentally enriched, low stereotypy animals, there fore, suggesting that enrichment related decreases in stereotypy may involve synergistic regulation of DARPP 32 at both the transcriptional and post translational levels. Our second study (Chapter 5) examined the pattern of long term changes in neuronal ac tivation, as indexed by FosB induction, following chronic stereotypy and environmental specifically in the striatum with prolonged stereotypic activity. Further immunohis tochemical analysis of striatal subregions showed that, for standard caged animals, increasing stereotypy was effects of enrichment on stereotypy were localize d only to the dorsolateral region of the striatum, and, also in contrast to the standard cage controls, increasing stereotypy in enriched animals was (striosome/matrix) dist gene activation in the striosomes exceeded that in the matrix was a reliable indicator of m ediate both maladaptive and adaptive forms of long term, experience dependent neuroplasticity depending on the nature of the chronic stimulation (motor stereotypy or environmental enrichment) experienced. The goal of our third study (Chapter 6) was to dete rmine whether these differences in gene expression would translate to changes in synaptic structure and function. To this end, we evaluated levels of the synaptic proteins, synaptophysin and NCAM, in the cerebral cortex, dorsolateral striatum, dorsomedial striatum, and hippocampus. Our results show that enrichment

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129 increased concentrations of synaptophysin in the dorsolateral striatum and NCAM 140 in the cortex, whereas stereotypy decreased synaptophysin levels in the dorsolateral striatum and NCAM 140 level s in the cortex. No significant effects were found in the dorsomedial striatum or hippocampus. These results provide additional evidence that the effects of environmental enrichment and stereotypy on neuroplasticity are localized to the corticostriatal neu rocircuitry responsible for the control of motor behaviors. Finally, our last study (Chapter 7) investigated whether stereotypy would be associated with deficits in other behaviors mediated by the striatum and whether the beneficial effects of environmenta l enrichment would extend to these deficits. The results indicated that stereotypy was associated with deficits in both spatial and stimulus response learning and memory. Furthermore, we found that the effects of environmental enrichment on these impairmen ts in water maze performance were dissociable: enrichment alone was sufficient to counteract deficits in spatial learning and even enhanced spatial memory but did not have a significant effect on stimulus response learning. In order to determine whether th ese differences in behavior were mediated through experience dependent changes in synaptic plasticity, we again evaluated the synaptic proteins, synaptophysin and NCAM. The results of this analysis indicated that learning induced changes in synaptic protei n expression were task specific and generally corresponded to levels of performance. Future Directions Ultimately, the studies described herein strive to gain a more complete picture of how the brain is dynamically sculpted by complex environmental stimuli on a molecular level so as to exploit the therapeutic potential of this knowledge. In order for enrichment studies to guide the development of novel treatments, the mechanisms which underlie experience dependent plastic changes in the nervous system must first be identified so that approaches can be developed to

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130 enhance them when they play a beneficial role and downregulate them when they are maladaptive. We have sought to better elucidate the molecular substrates for the beneficial effects of environmenta l enrichment on stereotypy. However, much still remains to be learned. A recurring theme in many of these studies was the search for regional specificity. Based on our previous research, our focus has been on the dorsolateral striatum. Indeed, many of the findings in these studies recapitulate the results of our previous work. Nevertheless, the heterogeneous composition of the striatu m, both architectonically and functionally, deserves additional consideration. The striatum exhibits complex organization on several levels only some of which we have begun to systematically examine. For example, the topographical organization of connections to and from the striatum varies along all of its dimensional axes (i.e., rostro caudal, dorso ventral, and medio lateral). The striosome and matrix compartments of the striatum can also be further functionally segregated into their core/periphery and matrisome subdivisions. Striatal cell subpopulations, especially those comprising the direct and indirect pathways, are also op en to investigation. Future studies might also include other brain regions, especially those which interact with the striatum Despite the robust effects of early environmental enrichment on stereotypy, there also appears to be a critical period beyond whi ch the impact of environmental manipulations steadily begins to diminish. One approach that could be used to determine this sensitive period would be to incorporate a longitudinal design into our studies and examine deer mice at various developmental timep oints. Future studies could also continue to build upon the findings of these studies. For example, the effects of environmental enrichment and motor stereotypy on downstream effectors of DARPP 32 and

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131 with or indirectly influence each other, a clearer idea of the molecular and cellular processes which participate in the environmental regulation of stereotypic behaviors could gr adually emerge. Additionally, histological analysis could also be performed to confirm and clarify the synaptic changes implied by changing synaptophysin and NCAM 140 levels. Finally, as different forms of learning are functionally and anatomically dissoci able, other learning and memory tasks could also be evaluated to determine if they are also impacted by the neuropathology associated with stereotypy. By constructing a more comprehensive picture of the neurobiological processes which are involved in the p athogenesis of stereotypy as well as the beneficial effects of environmental enrichment on reducing stereotypy, the insights gained from these studies may eventually help to guide the development of therapeutic approaches for the treatment of human repetit ive behavior disorders.

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160 BIOGRAPHICAL SKETCH Linda Wen sciences (neurobiology), and psychology from Northwestern University and her doctoral degree in the medical sciences (neuroscience) from the Univers ity of Florida.