psycho-social phenotype and brain n...

PSYCHO-SOCIAL PHENOTYPE AND BRAIN N-SULFATED HEPARAN

SULFATES IN MECP2 MUTANT MICE

A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

PSYCHOLOGY

MAY 2012

By

Brandon L. Pearson

Dissertation Committee:

Robert Blanchard, Chairperson D. Caroline Blanchard

Adrian Dunn Lorey Takahashi Timothy Tricas

ACKNOWLEDGEMENTS

Special thanks to my mother, Theresa Forshee for encouraging my curiosity and to my

wife, Daria Ebneter for her outstanding support and her incredible ability to keep me grounded.

Jaclyn Bettis, Erwin Defensor, Dr. Ksenia Meyza, Sam O’Hanlon, Larry Oasay, Dr.

Roger Pobbe, Amy Vasconcellos and Lace Yamamoto provided assistance with behavioral

studies. Ted Murphy constructed behavioral arenas. Michael Corley, Ashley Jensen, Dr.

Frederic Mercier and Dr. Roger Pobbe assisted with immunohistochemistry. Tina Carvalho of

the Biological Electron Microscope Facility (BEMF) at the University of Hawaii provided critical

confocal training and support. Funded by grant NIH MH081845-01 to Dr. Robert J. Blanchard.

i

ABSTRACT

A variety of inbred and mutant strains of mice exhibit impairments in social behaviors

and restricted, repetitive behaviors; these constitute face validity for autism models. However,

researchers rarely evaluate the psychological state eliciting the impaired social investigation and

there remain no reliable biomarkers for autism. A series of studies was conducted to assess

social behavior in methyl CpG binding protein 2 (MeCP2308/Y) mutant mice, which were

subsequently shown to be hypersocial. Analyses of psychostimulant-based behavioral

responses were assessed in the hypersocial MeCP2 mutant mice and in hypo-social BTBR

T+tf/J mice relative to their respective controls. This body of work suggests that dysregulated

reward processing in candidate mutant mice, and specifically, MeCP2 hypomorphs, may

underlie inflexible and abnormal social behaviors. N-sulfated heparan sulfate alterations in the

brain may underlie brain developmental and behavioral abnormalities in autism. To ascertain

whether MeCP2 mutation influences heparan sulfate proteoglycan systems, fluorescence

immunohistochemistry was performed on wild-type and mutant mouse brains throughout the

neurogenic lateral ventricle subventricular zone and it was shown that, counter to the prediction,

MeCP2308/Y mutants display normal levels of heparan sulfate and laminin extracellular matrix

markers in the neurogenic subventricular zone. Perhaps other brain regions or alternative

markers should be included in future analyses; yet, the overall influence of adult neurogenesis

and other brain plasticity processes in autism-relevant behavior deficits has not been fully

characterized. This body of work utilizes multiple levels of analyses in an attempt to elucidate

neurobiological and psychological contributions to abnormal and autism-relevant behavioral

disturbances.

ii

Table of Contents

CHAPTER 1: INTRODUCTION 1

1.1 MeCP2308/Y SOCIABILITY AND STEREOTYPY PHENOTYPE 2

1.2 INTRODUCTION 2

1.3 MATERIALS AND METHODS 4

1.3.1 Experimental Subjects 5

1.3.2 Experimental Design 5

1.3.3 Visible Burrow System 5

1.3.4 Three-Chamber Social Approach 6

1.3.5 Autogrooming 7

1.3.6 Repetitive Novel Object Contact Task/ Locomotion 8

1.3.7 Social Proximity 9

1.3.8 Urinary Scent Marking 9

1.3.9 Resident Intruder 10

1.3.10 Statistical Analyses 10

1.4 RESULTS 11

1.4.1 Body Weight 11

1.4.2 Visible Burrow System 11

1.4.3 Three-Chamber Social Approach 12


1.4.5 Repetitive Novel Object Contact Task/ Locomotion 13

1.4.6 Social Proximity 13

1.4.7 Urinary Scent Marking 13

1.4.8 Resident Intruder 13

1.5 DISCUSSION 14

iii

CHAPTER 2: EMOTIONAL CHARACTERISTICS OF MeCP2308/Y MUTANTS 25

2.1 INTRODUCTION 25



2.2.2 Experimental Design 26

2.2.3 Elevated Plus-Maze 27

2.2.4 Elevated Zero-Maze 27

2.2.5 Mouse Defense Test Battery 28

2.2.6 Female Urine Sniffing Test 29

2.3 RESULTS 29

2.3.1 Elevated Plus Maze 29

2.3.2 Elevated Zero Maze 30

2.3.3 Mouse Defense Test Battery 30

2.3.4 Female Urine Sniffing Test 30

2.4 DISCUSSION 30

CHAPTER 3: REWARD SYSTEMS IN MOUSE MODELS OF AUTISM 35

3.1 INTRODUCTION 36



3.2.2 Social Conditioned Place Preference 39

3.2.3 Social Interactions 39


3.2.5 Statistical Analysis 40

3.3 RESULTS 40

iv

3.3.1 Social Conditioned Place Preference 40

3.3.2 Social Interactions 41


3.4 DISCUSSION 42

3.5 PSYCOSTIMULANT REACTIVITY 48

3.5.1 Reward Tasks in MeCP2308/Y Mutants 50

3.5.2 Psychostimulant Response in Low-Social Mice 50

3.6 DISCUSSION 53

CHAPTER 4: N-SULFATED HEPARAN SULFATE ABUNDANCE AND DISTRIBUTION IN

MeCP2308/Y MICE 54

4.1 INTRODUCTION 54



4.2.2 Dual-Channel Indirect Fluorescent Immunohistochemistry 55

4.3 RESULTS 56

4.3.1 Laminin 56

4.3.2 Heparan Sulfate 56

4.3.3 Heparan/Laminin Ratio 56

4.4 DISCUSSION 57

CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS 61

5.1 NEUROBIOLOGY OF SOCIABILITY 61

5.2 THE ASSOCIATIONS OF NEURODEVELOPMENT AND NEUROPLASTICITY

WITH BEHAVIOR 62

v

REFERENCES 67

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LIST OF TABLES

Table 1. Review of MeCP2 Mutant Rodent Studies Assessing Social Behavior 64

Table 2. ANOVA Results for MeCP2308/Y in the Visible Burrow System 65

Table 3. Comparison of MeCP2308/Y Mutant and Wild-type Mice in the Mouse Defense

Test Battery 66

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LIST OF FIGURES

Figure 1. MeCP2308/Y Mutant Mice Show Increased Affiliation in the Visible Burrow

System 18

Figure 2. Three-Chamber Sociability in MeCP2308/Y Mutant and Wild-Type

Littermates 19

Figure 3. Subtle Alterations in Patterns of Autogrooming in MeCP2308/Y Mutant Mice 20

Figure 4. No Difference in MeCP2 Genotype-Related Performance in the Repetitive

Novel Object Contact Task 21

Figure 5. Patterns of Social Interaction in the Social Proximity Task 22

Figure 6. Elevated Scent Marking in MeCP2308/Y Mutant Mice 23

Figure 7. Comparison of Agonistic Behavior of MeCP2308/Y Mutant and Wild-Type Mice in

the Resident-Intruder Task 24

Figure 8. Elevated Plus-Maze (EPM) testing of MeCP2308/Y and Wild-Type

Littermates 33

Figure 9. Elevated Zero-Maze (EZM) Testing of MeCP2308/Y and Wild-Type

Littermates 35

Figure 10. Social Conditioned Place Preference Testing in B6 and BTBR Mice 45

Figure 11. Scan Sampling of Social Conditioning Sessions in B6 and BTBR Mice 46

Figure 12. Assessment of Autogrooming Behavior Before and After Social Conditioning

During Exploration 47

Figure 13. Cocaine-Induced Conditioned Place Preference Testing in B6 and BTBR

Mice 49

Figure 14. Locomotor Response to Repeated Cocaine Administration in MeCP2308/Y

Mutant and Wild-Type Mice 51

Figure 15. Acute Locomotor Response to IP Cocaine in B6 and BTBR Mice 52

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ix

Figure 16. Representative Confocal Fluorescent Immunohistochemistry Micrographs of

Mouse Brain Lateral Ventricle Immunolabeled for Laminin and Heparan Sulfate 59

Figure 17. Immunohistochemical Analyses of Brain Extracellular Matrix Markers Laminin

and Heparan Sulfate in MeCP2308/Y Wild-Type and Mutant Mice 60

1

CHAPTER 1: INTRODUCTION

Autism spectrum disorders (ASD) are a group of pervasive developmental

abnormalities characterized by three clusters of symptoms: deficits in reciprocal social

behaviors, impaired communication, and restricted interests and repetitive behaviors

(APA 2000). Though overall, ASD is highly heritable, the disorder is characterized by a

pronounced heterogeneity (Bill & Geschwind 2009). Less than 20% of ASD diagnoses

stem from known chromosomal or allelic disruptions (Benvenuto et al, 2009). Therefore,

most cases of ASD are idiopathic, with no specific endophenotypes or biomarkers to

aide in diagnosis or development of improved therapeutics. The diverse and complicated

pathophysiology of ASD potentially leads to an over-reliance on behavioral symptoms to

identify cases of non-syndromal neurodevelopmental disorders. A persistent goal of

clinicians and pre-clinical scientists will be to identify physiological abnormalities that are

present in ASD.

ASD disorders such as Rett syndrome and Fragile X mental retardation are

associated with known genetic mutations (MECP2 and FMR1, respectively), while others

such as Angelman syndrome, 15 q11-15 duplication syndrome and Williams syndrome

result from chromosome region deletions, duplications, or inversions across multiple

genes (i.e. Benvenuto et al, 2009). Since mice almost universally possess homologues

to these loci, knockout strategies have been performed and have yielded substantial

progress in the comprehension of these diseases. Much of the work presented here has

utilized such mutant mice.

In order for rodent models to be informative, they must exhibit reliable

approximations of the human condition. What’s more, the behavioral variables of the

rodent testing must be appropriate to the symptomatology of the disease. A variety of

rodent models have been developed which, on the surface, display many autism-like

characteristics (Moy & Nadler 2008). However, in order to make progress in revealing

consistent neurobiological markers for autism in rodent models, the systems underlying

the core deficits in ASD must be realized.

The following set of studies describes an innovative approach to assess explicit

components of social behavior in mice. These assessments encompass analyses of

emotional contributions to variation in affiliative social behavior with particular emphasis

on motivation. These approaches are based on the notion that social deficits are

maintained by alteration in social motivation, an impairment directly implicated in ASD

2

(Dawson et al, 1998; Dawson et al, 2005; Schultz 2005). Impaired social motivation can

encompass a lack of reinforcement for social interactions or a lack of reward for attention

to social stimuli (Scott-Van Zeeland et al, 2010). With regards to pre-clinical models and

experimental psychopharmacology, analyses of social reward mechanisms might be a

particularly suitable approach. In order to test for social motivation abnormalities, studies

were performed to compare mice with known social deficits to those with normal social

behavior, and to MeCP2308/Y mutant mice which were revealed to possess clear

enhancement of social behavior. Although this work could, and should be expanded in a

variety of directions, the final study assessed what is proving to be a potential biomarker

of altered brain function in animal models of ASD.

Despite vast advances in autism research, ASD remains a syndrome

characterized by behavioral impairments; no consistent diagnostic or prognostic

biomarkers have been identified. Identification is hindered by the fact that autism is not a

singular construct, but rather, a collection of neurodevelopmental abnormalities that

manifest as social impairments on cognitive and behavioral restrictions (Walsh et al,

2011). A potential improvement to pre-clinical approaches to the comprehension of ASD

will be to reveal the basic brain substrates of relevant symptoms in rodent models.

1.1 MeCP2308/Y Sociability and Stereotypy Phenotype

1.2 INTRODUCTION

Mutations of the MECP2 gene underlie the majority of cases of Rett syndrome

(Amir et al, 1999; Bienvenu et al, 2000). Investigations are revealing the complex

functions of this gene in intra- and inter-generational epigenetic regulation in non-

disease states, and in somatic and psychiatric disorders. The gene’s protein product,

methyl-CpG binding protein 2 (MeCP2), plays a prominent role in gene regulation due to

its recognition of promoter-specific methylated CpG sites, and its subsequent

interactions with co-repressors (Chahrour et al, 2008; Jørgensen & Bird, 2002; Lewis et

al, 1992; Samaco et al, 2009), as well as its influences on alternative splicing of mRNA

(Young et al, 2005) and microRNA (Wu et al, 2010). Females with MECP2 mutations

display a wide range of symptom severity, due to patterns of X-chromosome inactivation

(XCI), which in-turn leads to mosaicism (Braunschweig et al, 2004) and subsequent

complications in the clinical characterization as well as the generation of informative

mouse models. Mutations of MECP2 were previously considered embryonic lethal for

hemizygous males, but recently, MECP2 mutations have been noted in human males

3

and are associated with mental retardation, movement abnormalities, and other related

symptoms (Clayton-Smith et al, 2000; Dotti et al, 2002; Kleefstra et al, 2002; Orrico et al,

2000). Evidence is accumulating that partial loss-of function mutations of MECP2 may

contribute to neurodevelopmental and social disorders (Samaco et al, 2008).

Rett syndrome is characterized by normal early development, followed by a

pronounced regression at about 6-18 months in age with loss of verbal functions,

epilepsy, gastro-intestinal problems, stereotypies, and severe musculo-skeletal

deformations (Hagberg et al, 1983). Rett syndrome is classified as a pervasive

developmental disorder and is often considered an Autism Spectrum Disorder (ASD)

due to impairments in social communication and presence of restricted-repetitive

behavior (APA, 2000). Although Rett syndrome is comparatively rare (approximately

1:10,000-15,000, Hagberg et al, 1985) compared to ASD (1:110, CDC 2009) there has

been an upsurge of pre-clinical studies of single-gene and other ASD candidate mouse

models under the ultimate impetus to reveal the neural genetics and molecular signature

of developmental disorders affecting social behavior. These mouse models may play an

important role in elucidating some of the biology underlying ASD.

A variety of mutant MeCP2 mice have been engineered to study the

pathophysiology resulting from mutations in this gene (Ricceri et al, 2008). Newer cell

and brain region-specific null mice are proving valuable in the characterization of

pathways responsible for the phenotypic alterations associate with normal and perturbed

MeCP2 expression. Global, whole gene null MeCP2 male mice exhibit severe symptoms

such as seizures and reduced survivability (Chen et al, 2001; Guy et al, 2001; Pelka et

al, 2006). Truncated and conditional knockouts show a range of symptom severity, age

of onset, and mortality (see Calfa et al, 2011 for an exhaustive review).

Behavioral investigations have revealed an apparent decrease in social

interactions indexed by impaired nest building and social avoidance of an unfamiliar

stimulus mouse behind a partition within the home cage in MeCP2308/Y truncated protein

mutants (Moretti et al, 2005). Gemelli et al, (2006) demonstrated that conditional

postnatal knockout MeCP2 mice spent less time interacting with an unfamiliar mouse

located behind a wire mesh enclosure in an unfamiliar arena, relative to wild-type

littermates. Amygdala-specific siRNA knockdown of MeCP2 did not influence three-

chamber social approach behaviors but impaired juvenile play behaviors in male rats

(Kurian et al, 2008). With respect to social communication, MeCP2308/Y mutant mouse

4

pups show impaired ultrasonic vocalization calls upon separation from their mother (De

Filippis et al, 2010). These reductions in social behaviors in MeCP2 mutants generally

have not been attributable to impaired social recognition abilities (Ricceri et al, 2008).

In contrast with these findings, a number of recent studies suggest that MeCP2

mutations in rodent models may be associated with increased sociability. Three-

chamber social approach behavior was shown to be enhanced rather than decreased in

the Mecp21lox mutant (Schaevitz et al, 2010). Similarly, MeCP2 conditional knockout

mice (Sim1 –cre BAC transgenics) and their controls (Mecp2flox/+), who also show

reduced MeCP2 expression, both show increased sociability as indexed by more

proportional time in proximity to familiar and unfamiliar stimulus mice behind a wire-

mesh enclosure (Fyffe et al, 2008; Samaco et al, 2008, 2009). A recent report has

indicated that GABA-ergic neuron MeCP2 mutants show enhanced social interactions in

the partition test and in a modified three-chamber task (Chao et al, 2010). Mecp2llooxx//YY

hypomorphs have similar social approach compared to that of wild-types, but show an

apparent augmentation of social-novelty preference (Kerr et al, 2008). Table 1 provides

an overview of studies revealing alterations in social behavior in targeted mutations of

MeCP2 in rodents. Studies of MeCP2 mutant mice with no social behavior analyses

were not included. These studies indicate that, at a minimum, MeCP2 may be an

important gene for the investigation of mechanisms underlying social behaviors.

The purpose of the current study was to perform analyses of social behavior with

attempts to differentiate pro-social from aggressive or sexual motivations in social

interactions. A battery of behavioral tests in male MeCP2 wild-type and their hemizygous

mutant littermates was performed. The MeCP2308/Y mouse exhibits the same Rett

syndrome-like abnormalities as the whole gene knockouts, but with improved

background strain survivability and a later age of symptom onset (Shahbazian et al,

2002). This permitted the assessment of adult mice prior to the onset of severe sleep,

neurological, and musculo-skeletal deficits. The tests used were designed to reveal

subtle and overt distinctions in social interactions and repetitive behavior, which have

proven to be relevant variables for mouse models of neurodevelopmental disorders

(Arakawa et al, 2007; Defensor et al, 2011; Moy et al, 2004; Pearson et al, 2010, 2011;

Pobbe et al, 2010).

1.3 MATERIALS AND METHODS

5

1.3.1 Experimental Subjects

Experimental animals were bred from C57BL/6J-backcrossed stock obtained

from The Jackson Laboratory (B6.129S-Mecp2tm1Hzo/J, stock # 005439) and bred from

heterozygous mutant dams and hemizygous father sibling pairs. Stimulus mice used for

social behavior tests were adult CD-1 mice bred in-house from stock obtained from

Charles River Labs, and C57BL/6J (B6) mice bred from stock obtained from The

Jackson Laboratory. Mutant mouse genotype was determined according to the PCR

parameters obtained from The Jackson Laboratory with purified DNA collected from tail

biopsy after weaning at post-natal day 25. Since MeCP2 is an X-linked gene, only wild-

type (y/+) and hemizygous (y/-) males were obtained and compared in behavioral

testing. Mice were housed with up to five same-sex littermates under a 12-h light/dark

schedule with lights on at 0600h. Mice had ad libitum access to tap water and standard

laboratory rodent diet. Mice were 10-13 weeks old at the beginning of behavioral studies.

All procedures were performed according to protocols approved by the University of

Hawaii Laboratory Animal Service Institutional Animal Care and Use Committee.

1.3.2 Experimental Design

In total, nine mice per genotype were used as subjects. Mice were subjected to a

series of behavioral tests in a sequence intended to prioritize both the accumulating

effects of multiple testing and the influence of housing and experience on the measures.

All mice were weighed prior to the first behavioral test, and weighed again after testing

for urinary scent marking to determine baseline weight differences and weight gain

trajectories. One mutant animal showing degenerative features, severe weight loss and

seizures and was euthanized after the scent marking test; its data were excluded from

scent marking scores. No other animals showed complications during testing. One

mutant Visible Burrow System (VBS) colony (n=3) was not included in the analyses due

to a recorder error. Animals were moved to the behavioral testing room at least 30

minutes prior to testing and all arenas and equipment were cleaned with 70% ethanol

and dried completely between mice. Unless otherwise noted, testing was performed

under ambient fluorescent lighting between 0900 and 1800 hours. Temperature (22±1 ̊C)

and humidity (50-70%) were controlled in the experimental room.

1.3.3 Visible Burrow System (VBS)

6

Three days prior to behavioral testing, mice were anesthetized with an IP

injection of 12.5mg/ml of Avertin (2,2,2-Tribromoethanol, Sigma-Aldrich, St. Louis, MO)

at a dose of 336 mg/kg. Once mice were unresponsive, peroxide based hair bleach was

applied to the dorsal coat of each mouse so they were distinguishable in videotape

analysis. Mice were allowed to recover two days prior to being placed in the VBS

according to procedures outlined previously (Pobbe et al, 2010). Briefly, each colony

was housed in a rectangular, galvanized metal bin, 86 × 61 × 26 cm (H). The colony

arena consisted of three chambers, each 12 × 7 × 6 cm (H), which were positioned

behind a barrier wall extending across a short width (61 cm) of the bin, 30 cm from the

end wall. This wall separated an open surface area (30 × 61 × 26cm [H]) from the

chambers in the other compartment. These chambers were connected to the wall via

clear Plexiglas tubes 5 cm in diameter. Two of the three chambers, each connected to

the surface area via a Z shaped tube, were connected to each other via a straight clear

Plexiglas tube. The third chamber was connected only to the surface via a straight tube.

The animals could pass freely between each chamber and the surface area, or between

the two connected chambers, by these tubes. The experimental room was maintained

on a 12-h light/dark cycle (lights on at 0600h), being illuminated by fluorescent lamps

during the light period and by infrared light during the dark phase. An assistant who was

blind to genotype scored VBS videotapes by sub-sampling 30 second intervals every 10

minutes for the first 4 hours of the dark and light phases. The observer manually

recorded all occurrences of Approach Front, Approach Rear, Flight, Chase/Follow,

Vigorous behavior, Autogroom, Allogroom, and Huddle. Approach [to the] Front/Back of

another animal was defined in terms of a line bisecting the approached mouse,

perpendicular to the long axis of its body. Flight was defined as rapid locomotion away

from an approaching animal; Chase/Follow was rapid locomotion toward another animal,

or a slow approach toward an animal that was moving away. Vigorous behavior was

high-intensity chase, bite, or attack. Autogrooming was lick or rub self while

Allogrooming is lick or rub with paws of another animal. Huddle was characterized by a

mouse lying in contact with another animal for more than 10 seconds of the 30-second

time sample. Data were summarized separately for the dark and light phases, and the

mean frequency of each behavior for each genotype was compared across the three

days of testing.

1.3.4 Three-Chamber Social Approach

7

Twenty-four hours after removal from the VBS, mice were tested for social

approach behavior in the three chamber apparatus, which was constructed according to

published studies (Moy et al, 2004). Initially, mice are placed into the center of the

divided 41 x 70 x 28 cm (H) apparatus, which contained two empty, inverted wire cups

(Galaxy Pencil/Utility Cup, Spectrum Diversified Designs, Inc., Streetsboro, OH). Empty

glass jars of the same diameter were placed on top of the base of the wire cup to

prevent movement of the enclosures, or escape by stimulus mice. For the habituation

phase, the sliding doors were elevated and the mouse was permitted ten minutes to

explore the three chambers. At the end of the ten minute habituation session, mice were

placed back into the middle of the apparatus, the sliding doors were lowered, and an

unfamiliar adult (57-83 day old) male CD-1 mouse was placed into one of two outer wire

cups, and the doors were again lifted and the mouse was permitted to explore the entire

apparatus for ten minutes; this constituted the sociability phase. The time spent in each

compartment during both sessions was collected in real-time with two stopwatches by a

single observer who was blind to the genotype of the subject. During both the

habituation and sociability phases, cameras were mounted in front of both outer

compartments and connected to a DVD recorder. The frequency and duration of Rear,

Autogroom, Contact (with the stimulus cup), Sniff, Stretch-Attend, Quick-Withdraw, and

Nose-to-Nose were scored off-line using Noldus Observer software (Noldus Information

Technology, Wageningen, The Netherlands) for each of the two outer compartments

during the sociability phase for each subject.

1.3.5 Autogrooming

Detailed grooming analyses were performed as previously described (Pearson et

al, 2011). Mice were placed in a 14 x 7 x 30 (H) cm Plexiglas chamber for 20 minutes

under normal fluorescent lighting. An aluminum lid that permitted air circulation, but

prevented escape, was placed over the top. Two digital cameras were used to collect

videotapes from the front and side aspect so that the mouse’s grooming behavior was

always visible. Recorded DVDs were scored using Noldus software for the frequency

and duration of Paw Licking, Head Washing, Body Grooming, Leg Licking, and

Tail/Genital grooming. In addition to the collection of frequency and duration of body

directed grooming, the following variables were determined according to Kalueff et al,

(2007). A Bout was defined as at least one episode of any category of grooming, or an

uninterrupted sequence of grooming types. Bouts were divided by at least 6 seconds of

8

inactivity or by an activity other than grooming. An Interrupted Bout was defined as a

grooming bout that is interrupted by less than 6 seconds; the proportion of bouts that

were interrupted was calculated as (interrupted bouts/total bouts)*100. Transitions were

transfers between regional grooming subtypes. Incorrect transitions were transfers,

which did not follow the cephalo-caudal progression (0-No Grooming, 1-Paw Licking, 2-

Head Wash, 3-Body Groom, 4-Leg Licking, 5-Tail/Genital). Proportion of incorrect

transitions was calculated as (incorrect transitions/total transitions)*100.

1.3.6 Repetitive Novel Object Contact Task/Locomotion

On the day following the autogrooming analyses, mice were relocated to the

behavior room at least 30 minutes before habituation and testing for the repetitive novel

object contact task (Pearson et al, 2011). The habituation session consisted of placing a

mouse in a clean standard mouse cage containing 1 cm of bedding. No lid, water bottle,

or food hopper was present. A micro-isolator lid was modified by removing the filter

element and frame, and thick gauge wire bisecting both horizontal planes of the lid were

added which divided the overhead image into four equal sized compartments. The

number of transitions between quadrants and amount of time spent within each quadrant

was scored for a 10-minute habituation session under normal fluorescent illumination

using Noldus Observer software. This permitted assessment of any baseline differences

in motor activation or exploration tendencies of the genotypes.

On the following day, and at the same time of day as the prior habituation

session, mice were placed into the same apparatus which contained four small plastic

objects (a multicolored 3 cm long arrangement of Lego blocks, a green 4 cm long jacks

piece, a 1.5 cm3 multicolored die, and a 3.5 cm long white and red bowling pin) arranged

2 cm from the four corners. Video was collected for ten minutes and all instances of

investigation of the objects, and the order in which they were investigated, were scored

offline. Investigation was defined as clear facial or vibrissae contact with or burying of

the novel objects; merely passing or pausing by an object was insufficient for

investigation. The occurrence of repetitive contact with three and four toys and the

frequency of times that the mice buried each object were counted. Total frequency of

contact with each of the four toys, and the total number of burying episodes were also

calculated. In order to determine if there was a strain effect on the tendency to display

preferences for particular toys, the frequencies of contact with each object were ranked

9

in decreasing order from maximum to minimum preference (contact) values for each

subject, and the frequencies were averaged by genotype and compared.

1.3.7 Social Proximity

Social proximity testing was conducted in a clear rectangular chamber (7 ×14 ×

30 cm H) constructed of acrylic plastic according to previously reported parameters

(Defensor et al, 2011). The dimensions of this arena were identical to that used for

analyses of individual autogrooming behavior. For testing, the subject mouse and an

unfamiliar 5-7 month old male B6 mouse were placed into the chamber simultaneously

and an aluminum lid was placed over the top to prevent escape. Video from two

cameras providing front and side views was transferred to a video merge processor,

which combined both channels into a single side-by-side output. The availability of both

views aided in the discrimination of behaviors by reducing occlusion of one animal from

view by the other. The output from the video processor displaying both the front and side

view was transmitted to a DVD recorder for storage and subsequent analysis. The

frequencies of the following behaviors were manually quantified by an observer blind to

the subject’s genotype:

Nose-to-Nose- subject’s nose tip and/or vibrissae contact the nose tip

and/or vibrissae of the other mouse.

Nose-to-Anus- subject’s nose or vibrissae contacts the base of the tail or

anogenital region of the other mouse.

Crawl Over- subject’s forelimbs cross the midline of the dorsal surface of

the other mouse.

Crawl Under- subject’s head goes under the ventral surface of the other

mouse to a depth of at least the ears of the subject animal crossing the

midline of the other mouse’s body.

Upright- subject displays a reared posture oriented towards the other

mouse with head and/or vibrissae contact.

1.3.8 Urinary Scent Marking

Baseline scent marking in a divided arena was characterized on the first day of

testing, after seven days of single housing. Previous studies have demonstrated that

single housing is critical to establishing motivation to engage in detectable scent marking

10

(Arakawa et al, 2009). The scent-marking arena was an inverted rat cage with a steel

mesh divider wall installed to bisect the arena. This apparatus was placed on top of a 30

x 45 cm section of drawing paper, and the subject mouse was placed on one side for a

20-minute baseline session. Twenty-four hours later, the mouse was tested for urinary

scent marking to an unfamiliar CD-1 (91-103 days old) mouse stimulus placed

immediately before in the opposite half of the arena. At the end of the baseline and

social marking sessions the mice were removed and the number of fecal boli counted.

The placement of the compartments was marked and the paper was allowed to dry

overnight. The paper was then fixed and stained with a 6% solution of ninhydrin (Fisher)

in methanol and dried. To quantify the amount of urinary scent marking, a 1x1 cm

printed transparency grid was placed over the paper and the number of squares

containing a stained mark was counted manually by an assistant blind to the genotype of

the subject.

1.3.9 Resident Intruder

An unfamiliar, ten month old intruder B6 mouse was introduced into a resident

MeCP2 mutant or wild-type home cage, and the interactions between those animals

were recorded for five minutes. The latency to and duration of agonistic attack by the

resident was scored. Additionally, the frequency and duration of the following behaviors

was quantified: Face Sniff, Body Sniff, Anogenital Sniff, Tail Sniff, Vigorous Allogroom,

and Following. Mice analyzed in this test, having already been assessed in all of the

previous behavioral tasks were then euthanized by CO2 inhalation.

1.3.10 Statistical Analyses

Two-way repeated-measures analyses of variance (ANOVA) were performed to

compare body weights. Genotype was the between-subjects factor and the day of weight

measurement was the within-subjects repeated-measures factor. Two-way repeated-

measures ANOVAs were performed for data obtained from the VBS, with genotype as a

between-subjects factor, and day as the repeated-measures, within-subjects factor.

Separate analyses were performed across means from the dark and light phases.

Similarly, for the Three-Chamber Social Approach test, two-way repeated-measures

ANOVAs were performed for the duration of time spent within the two outer stimulus

chambers, and the frequency and duration values for each behavioral category except

that stimulus compartment (empty cup vs stimulus mouse) was the within-subjects

factor. Two-way repeated measures ANOVAs were applied to assess any differences in

11

the amount of time spent in each of the four compartments during the habituation phase

of the repetitive novel object investigation task and to compare the number of scent

marks during both scent marking conditions. For all ANOVA tests Bonferroni post-hoc

analyses were conducted to reveal any significant effects of genotype across the within-

subjects factor when significant main effects or interactions resulted. Independent t-tests

were performed to compare average frequencies and durations of each behavior

category in the autogrooming analysis, the social proximity task, the resident-intruder

test, and the number of lines crossed during the habituation phase of the repetitive novel

object contact task. Similarly, for the Three-Chamber Social Approach Task, the mean

frequency and duration of Stretch-Attend, Quick Withdraw, and Nose-to-Nose for each

genotype was compared with unpaired t-tests. When assumptions were violated for

these comparisons (non-normal distributions or unequal variances), nonparametric

Mann-Whitney U tests were performed instead. Statistica (v.6) and GraphPad PRISM

(v.4) software programs were used for statistical analyses and figures.

1.4 RESULTS

1.4.1 Body Weight

All mice were weighed before anesthetization for fur bleaching and on the

second day of scent marking. Mean ± 1 S.E.M. body weights before the VBS are as

follows: wild-type mice weighed 23.2 ± 0.6 g whereas mutant mice weighed 26.0 ± 0.7 g.

After the second scent marking session, wild-type mice weighed 22.7 ± 0.5 g and

hemizygous mice weighed 23.6 ± 1.0 g. A two-way repeated measures ANOVA revealed

no significant main effect of Genotype [F(1,16)=3.635, p=0.075], but a significant main

effect for Day [F(1,16)=16.21, p=0.001] and a significant Genotype x Day interaction

[F(1,16)=6.892, p=0.018, data not shown]. Post-hoc tests failed to indicate any

significant differences between genotypes within each day of weight measurement.

Hemizygous mice weighed slightly more at the beginning of experimentation, but no

significant weight differences were noted towards the end of behavioral assessments.

1.4.2 Visible Burrow System

Table 2 provides statistics for two-way repeated measures ANOVA for VBS

colonies for each behavior during light and dark phases. Figure 1 displays Mean ± 1

S.E.M frequencies of all behaviors in the VBS across the dark (upper panel) and light

(lower panel) phases for hemizygous and wild-type MeCP2 mice. Significant genotype

12

differences were of particular note. During the light phase, mutant mice engaged in

higher rates of frontal-oriented approaches and they also made fewer rear-oriented

approaches during the dark light cycle. Hemizygous mice had lower frequencies of

autogrooming during the light phase, with significantly more allogrooming in the dark

phase. Finally, hemizygous males showed increased rates of huddling in the dark phase.

1.4.3 Three-Chamber Social Approach

Mice displayed no significant side preferences in the habituation phase [WT,

t=1.529, p=0.146; KO, t=-0.910, p=0.376, data not shown]. Figure 2a displays the

amount of time spent in each compartment of the three-chamber social approach arena.

Duration of time spent in the center compartment is displayed for reference but was not

included in analyses. During the sociability phase, two-way ANOVA revealed a

significant main effect for Stimulus [F(1,32)=22.44, p<0.0001] indicating that both

genotypes spent a greater amount of time in the chamber associated with the stimulus

mouse compared to that containing the empty cup. The main effect for Genotype [F(1,32)=

0.003, p=0.959] and the Genotype x Stimulus interaction [F(1,32)=3.376, p=0.076] were

not statistically significant. Bonferroni post-hoc comparisons revealed no significant

difference between time in the social and non-social chambers for the wild-type mice

[t=2.051, p>0.05], but a significant increase in time spent in the social chamber for

hemizygous mice [t=4.649, p<0.001] (Figure 2a). Figures 2b&c present the frequency

and duration of behaviors scored during the sociability phase of the three-chamber task.

Significant main effects for Genotype for frequency and duration of contact [frequency:

F(1,16)=6.52, p=0.021; duration: F(1,16)=6.768, p=0.019] were found. Bonferroni post-hoc

tests indicated a significant genotype difference in the duration of contact with the empty

cup (t=2.626, p<0.05); mutant males displayed a reduced duration. There was also a

significant Stimulus main effect for the frequency and duration of sniffing [frequency:

F(1,16)=69.32, p<0.0001; duration: F(1,16)=60.05, p<0.0001]. No other statistically

significant main differences were found.

1.4.4 Autogrooming

Hemizygous mice showed a significantly increased frequency of tail/genital

grooming [t(16)=-2.7, p=0.016] and increased duration of paw licking [t(16)=-4.074,

p=0.0009] relative to wild-type littermates (Figures 3 a&b). No statistically significant

differences in the frequency or duration of other autogrooming sub-types were found.

Similarly, no significant differences in the number of bouts, interrupted bouts, proportion

13

of interrupted bouts, transitions between grooming stages, incorrect transitions, and

proportion of incorrect transitions were found (Figure 3c).

1.4.5 Repetitive Novel Object Contact Task/Locomotion

During the habituation phase, no significant differences were noted in the number

of lines crossed [t(16)=-0.598, p=0.558, data not shown] nor in the duration of time spent

within each quadrant [F(1,16)=0.827, p=0.377, data not shown]. Wild-type and hemizygous

mice investigated unfamiliar novel objects at a similar rate [F(1,64)=0, p=1] (Figure 4a).

Similarly, when proportional preferences for each toy were ranked and averaged for

each genotype, no significant difference in object preferences was found [F(1,32)=0.803,

p=0.377] (Figure 4b). Finally, when the number of identical three- and four-object

sequences was compared between genotypes, no differences were observed [F(1,64)=4E-

12, p=1] (Figure 4c). Taken together, MeCP2 mutants and wild-type mice are

indistinguishable in cognitive, object- investigation-based measures of stereotypy or

restricted interest.

1.4.6 Social Proximity

Figures 5 a&b display the frequency and duration of social behaviors of MeCP2

wild-type and mutant mice to unfamiliar B6 mice. Mann-Whitney U-tests indicated a

significant increase in the frequency of Crawl Under behavior in hemizygous mutants

[p=0.006]. They also showed significant elevations in the duration of Crawl-Over

[p=0.026] and Crawl-Under [p=0.010] behavior relative to wild-type littermates. Finally,

MeCP2 hemizygous males showed an increased duration of Nose-to-Face behavior

[p=0.040]. No other statistically-significant effects were noted in the social proximity

chamber between hemizygous and wild-type MeCP2 mice.

1.4.7 Urinary Scent Marking

Figure 6 presents the average number of 1 x 1cm squares containing scent

marks in the baseline and social scent marking conditions. Two-way repeated measures

ANOVA revealed a significant main effect of Genotype [F(1,32)=6.15, p=0.0195]. Neither

the main effect for scent marking Condition [F(1,32)=0.024, p=0.878] nor the Genotype x

Condition interaction were statistically significant [F(1,32)=0.280, p=0.601]. No significant

post-hoc differences were noted between Genotypes in either Condition. Therefore,

mutant MeCP2 mice show elevated scent marking across social and non-social

contexts.

1.4.8 Resident Intruder

14

No attack behavior was noted in any of the MeCP2 wild-type or mutant mice;

therefore, no attack variables are reported. Resident male MeCP2 hemizygous mutants

demonstrated lower average durations of following behavior than wild-type littermates

[t(15)=2.273, p=0.038]. No other significant differences were noted (Figure 7a,b).

1.5 DISCUSSION

Mutations of the MECP2 gene in humans and laboratory animals are associated

with a variety of neurological and behavioral alterations. However, the specific role of

this gene and the MeCP2 protein in normal variation in behavior and in disease states

remains to be elucidated. A major goal of this set of studies was to unravel the specific

types of abnormalities in social behaviors and stereotypies in male mice with targeted

mutations in Mecp2. Since existing reports are inconsistent in the directional influence of

mutations of this gene on social behavior, we performed a battery of tasks designed to

reveal subtle differences in social motivation under diverse social contexts. In the VBS, a

laboratory-based semi-natural environment in which subjects are free to establish their

own time budgets, MeCP2308/Y mutant males preferentially approach one another from

the front rather than the back, a preference which appears to be associated with pro-

social motivation (Arakawa et al, 2007). Mutant MeCP2308/Y mice also spent a greater

amount of time huddling during the dark phase, which indicates that they prefer contact

and spend less time alone compared to wild-type littermates. In the three-chamber

apparatus, whereas both genotypes spent more time in the side containing an unfamiliar

stimulus mouse, this effect was greater for the mutant mice. These results resemble

those noted in Mecp21lox and Mecp2flox mutants (Fyffe et al, 2008; Samaco et al, 2008,

2009; Schaevitz et al, 2010) albeit with some possible differences in interpretation in

light of other results in these studies (see below). Kerr et al, (2008) noted enhanced

social novelty preference for Mecp2lox/y mutants. However, a limitation of the Kerr et al,

(2008) study is that it did not counterbalance the social novelty phase, possibly

confounding social and object novelty (Pearson et al, 2010).

Mutant MeCP2308/Y mice have been reported to be hypoactive in the dark phase

and hyperactive in the light (De Filippis et al, 2010; Moretti et al, 2005). However, in the

present study, huddling behavior in mutants was comparable to that of wild-type mice in

the light phase, when mutants tend to be hyperactive, while allogrooming was elevated

in the dark phase, the period when mutants tend to be hypoactive. Moreover, we found

15

no genotype differences in locomotor activity in the habituation phase of the repetitive

novel object contact task which was assessed in the light phase. Similarly, an overall

decrease in contact with the stimulus cups in the three-chamber task was noted in

mutants. It is currently unclear why this might have occurred. However, confounding

distinctions in activity patterns seem an insufficient explanation, because mutants tend to

be hyperactive in the period when tested. Thus, the affiliative inclination of MeCP2

mutants does not appear to reflect hypoactivity in the dark phase and hyperactivity in the

light.

The social proximity task was designed to differentiate close-quarter differences

in social interactions (Defensor et al, 2011). In the current study, MeCP2308/Y mice

showed elevated frequencies and/or durations of crawl-under, crawl-over behaviors, and

nose-to-face investigation. Notably, low-social BTBR T+tf/J (BTBR) mice also displayed

increases in crawl-over and -under behaviors, which were interpreted as attempts to

avoid the other pair-member in a situation in which physical distance from that animal

could not be achieved; a view that was supported by findings that diazepam significantly

reduced this behavior in BTBRs (Defensor et al, 2011). In the present, highly social,

MeCP2308/Y mutants, these crawl-under/over results are consonant with their enhanced

social approach, investigation, and huddling in the VBS. An intriguing possibility is that

enhanced crawl under/over may nonetheless reflect a subtle social deficiency since

control mice (C57BL/6J) in the Defensor et al, (2011) study tended to orient to the pair-

partner mouse and move around it, rather than crawling under or over it, as might be

more appropriate for an inanimate object.

These data, including VBS indices, the three-chamber task, social proximity

findings, and urinary scent marking suggest that this specific partial loss of function

mutation of the MeCP2 gene in male mice produces an up-regulation of affiliative social

behavior. An additional goal of this set of studies was to understand further the

motivational components of these enhanced behaviors. Many commonly utilized social

behavior tasks (e.g. three chamber) do not inherently discriminate aggressive

motivations in approach and proximity variables. This is a particular issue for MeCP2

mutants because brain region and cell-specific MeCP2 mutants (Fyffe et al, 2008;

Samaco et al, 2009) were more aggressive in a resident-intruder situation, and also

spent more time in social proximity in the partition test, suggesting an aggressive

motivation in such approaches to the stimulus mouse. The present findings that the 308

16

mutant mice show higher levels of allogrooming are consonant with this interpretation, in

that intense bouts of allogrooming are often interpreted as agonistic behaviors reflective

of dominance motivations (“rough grooming”; Litvin et al, 2007; Long et al, 1972).

Similarly, increased scent marking for MeCP2308/Y in this study suggests an interpretation

in terms of enhanced aggressive or dominance motivations. This interpretation is not

supported by findings that MeCP2308/Y mice failed to show attack in the resident-intruder

task. However, overt attack may not be the most sensitive measure of dominance or

competitive motives, and an overall evaluation of the present data suggests that the

contribution of such motivation to social approach for the 308 mutant cannot currently be

dismissed.

Much of the early work with MeCP2308/Y mutant mice indicated that wild-type mice

seem to avoid contact with the mutant mice in the tube test and resident intruder task

(Shahbazian et al, 2002). An additional aspect of social interaction, relevant to

interpretation in terms of autism-like behaviors, is reciprocity. One possibility is that it is

the lack of reciprocation on the part of the recipient (stimulus) mouse that drives this

enhanced sociability in the mutants: Another is that the MeCP2308/Y mutants are deficient

in some behavioral, or perhaps physiological, characteristics that elicit sociality in WT

mice. In this context, the above-mentioned enhanced crawl under/over behaviors may

suggest such a social deficiency. These possibilities have yet to be tested, but suggest

some additional and potentially important aspects to the complex set of events that

constitute sociality in mammals. However, the present study revealed no differences in

the frequency of directed investigation towards mutant vs wild-type residents; and a

slight augmentation of duration of olfactory investigation including a significant increase

in duration of anogenital sniffing of mutants by intruder wild-types (data not shown) in the

resident intruder paradigm. The original 308/Y mutants in the Shahbazian et al, (2002)

study were on a mixed background, while those in the current study were on a C57BL/6J

background which may, in part, account of this difference.

Mouse models of ASD commonly display patterns of restricted repetitive

behaviors (Moy et al, 2008; Pearson et al, 2011). Published studies have characterized

hindlimb clasping when lifted by the tail, and forelimb stereotypies in MeCP2 mutants,

and it is thought that these might represent face validity in models of Rett syndrome

(Chen et al, 2001; Gemelli et al, 2006; Guy et al, 2001; Moretti et al, 2005; Shahbazian

et al, 2002). We found no disturbances in patterns of object investigation, and only

17

minor alterations in forepaw- and anogenital-directed grooming behaviors. The latter

results may extend previous discoveries of altered stereotypy in MeCP2 mutants by

noting that qualitative and region-specific differences in grooming may exist, but the

cephalo-caudal patterning appears to be normal.

Taken together, the hypersocial phenotype of MeCP2308/Y mutants demonstrates

one of the many conceptual issues arising from models of pervasive developmental

disorders- that autism-relevant features may occur without actual reductions in affiliative

social behavior. Mouse models of symptom- or domain-specific alterations in

neurodevelopmental disorders may be as informative as those that are generated in an

attempt to create an entire syndrome. To that end, a future goal might be to clarify the

distinct effects of the variety of region and allele targeted mutants of MeCP2 on specific

forms of social behavior, and the downstream targets of MeCP2 protein that influence

behavior, particularly throughout the developmental span. These results contribute to a

growing body of research that suggests MeCP2 is critical in the bi-directional fine-tuning

of behavioral responses, possibly up to, and including complex social interactions.

Figure 1. MeCP2308/Y Mutant Mice Show Increased Affiliation in the Visible Burrow System. Figures 1a and b display the frequencies of each of the eight behaviors scored during 24 scans across the first four hours of the dark (upper panel) and light phases (lower panel). Horizontal lines indicate a significant main effect of Day, and vertical lines indicate a significant Genotype main effect. y/+ denotes wild-type and y/- are hemizygous mutant mice. * p<0.05; ** p<0.01, *** p<0.0001. Symbols above individual means indicate a significant post-hoc effect: # p<0.05; $ p<0.001. Wild-type (y/+) N=9, mutant (y/-) N=6.

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Figure 2. Three-Chamber Sociability in MeCP2308/Y Mutant and Wild-Type Littermates. Both wild-type (y/+) and mutant mice (y/-) display increased duration of time in the chamber containing an unfamiliar mouse compared to an empty cup (Fig 2a). Post-hoc tests indicated a significant elevation in time spent in the stimulus mouse chamber compared to that with the empty cup for Mecp2 mutant mice. Frequency (Fig 2b) and duration (Fig 2c) of behaviors in each of the two outer stimulus compartments indicate that mutant mice have lower durations of contact with the cup in either chamber. Horizontal lines above columns indicate significant main effects for Genotype or Stimulus. Asterisks (*) indicate significant Genotype main effects while Stimulus main effects are indicated by an ampersand (&). * p<0.05, *** p<0.001, & p<0.001. Post hoc tests: # p<0.05. N=9/group.

19

Figure 3. Subtle Alterations in Patterns of Autogrooming in MeCP2308/Y Mutant Mice. Frequency and duration of body site-specific patterns of grooming (Fig 3a,b). Mecp2 mutant mice (y/-) show increased frequency of tail/genital grooming and an increased duration of paw licking compared to wild-type littermate controls (y/+). No significant differences were found in syntactical parameters of grooming (Fig 3c). *p<0.05; **p<0.01. N=9/group.

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Figure 4. No Difference in MeCP2 Genotype-Related Performance in the Repetitive Novel Object Contact Task. Wild-type and mutant MeCP2 mice show similar spontaneous investigation of novel toys (Fig. 4a,b). Similarly, wild-type (y/+) and mutant MeCP2 mice (y/-) show no differences in the number of sequenced visits (Fig. 4c). N=9/group.

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Figure 5. Patterns of Social Interaction in the Social Proximity Task. MeCP2 mutant mice (y/-) show increased frequency of crawl under (Fig 5a). Additionally, they show higher duration of nose-to-face interactions, as well as increased durations of crawl over and crawl under behavior (Fig 5b) compared to wild-type littermates (y/+). *p<0.05; **p<0.01. N=9/group.

22

Figure 6. Elevated Scent Marking in MeCP2308/Y Mutant Mice. Mutant mice (y/-) scent mark more to an empty chamber and when an unfamiliar adult CD-1 mouse is behind a divider compared to wild-type littermates (y/+). Wild-type (y/+) N=9, mutant (y/-) N=8. Genotype main effect * p<0.05.

23

Figure 7. Comparison of Agonistic Behavior of MeCP2308/Y Mutant and Wild-Type Mice in the Resident-Intruder Task. Mecp2 mutant mice (y/+) show similar frequencies of behavior to an unfamiliar intruder compared to their wild-type littermates (y/+)(Fig. 7a). Mutant mice show decrease duration of following behavior (Fig. 7b). *p<0.05. Wild-type (y/+) N=9, mutant (y/-) N=8.

24

25

CHAPTER 2: EMOTIONAL CHARACTERISTICS OF MECP2308/Y MUTANTS

2.1 INTRODUCTION

The protein methyl-CpG binding protein 2 (MeCP2) is transcribed from the X-

linked gene MECP2. MeCP2 is intricately involved in the regulation of gene expression

via its actions on methylated DNA and microRNA as well as influences on alternative

splicing (Chahrour et al, 2008; Jørgensen & Bird 2002; Lewis et al, 1992; Samaco et al,

2009; Wu et al, 2010; Young et al, 2005). Not only does MECP2 mutation underlie most

cases of Rett syndrome (Amir et al. 1999; Bienvenu et al. 2000), it has been implicated

in non-syndromal autism (Beyer et al, 2002; Carney et al, 2003; Nagarajan et al, 2006)

and other psychiatric disorders (Hammer et al, 2002; Van Esch et al, 2005). Emerging

literature also supports a central role of MECP2 in addiction processes because, in

rodents, psychostimulant drugs induce MeCP2 expression (Cassel et al, 2006), while

targeted mutation of MeCP2 alters amphetamine and cocaine sensitivity (Deng et al,

2010; Im et al, 2010). This has led to the hypothesis that fine tuning of MeCP2 protein

level in the brain is critical in normal brain functioning (Kaufmann et al, 2005) early in

brain development as well as in the adult (McGraw et al, 2011).

Shahbazian et al, (2002) created a transgenic mouse (MeCP2308/Y) that is

missing approximately one-third of the C-terminal region of the MeCP2 gene. This

partial-loss of function mutation is highly clinically relevant (e.g., Meloni et al, 2000; Wan

et al, 1999) and furthermore, permits the assessment of male mutants and mice that

reach adulthood prior to debilitating epilepsy and musculoskeletal abnormalities.

Constitutive MeCP2 knockout, as well as truncated and cell and brain-region specific

knockout mice show pronounced alterations in anxiety-related, cognitive and social

behaviors (see Calfa et al, 2011 for an exhaustive review). Interestingly, work from our

lab (Chapter 1 and Pearson et al, 2012) has noted a hypersocial phenotype in

MeCP2308/Y mice. More specifically, MeCP2308/Y mutants displayed an augmentation of

social approach and investigation with an absence of aggression. These results

complement findings from Schaevitz and colleagues (2010) noting increased three-

chamber sociability in Mecp21lox null mutants.

A variety of phenomena may contribute to the social characteristics of a given

mouse strain or engineered mutant. Cognitive or sensorimotor disturbances can

influence behaviors of interest and alterations in emotional reactivity influence the

26

behaviors expressed in a given paradigm (Crawley 2007a). It is entirely conceivable that

much of the social phenotype of a mouse (such as that of the MeCP2308/Y mutant mouse)

may reflect changes in emotional, defensive or cognitive disruptions. Previous reports

have indicated that MeCP2308/Y mice show several cognitive deficits such as spatial and

contextual fear memory (Moretti et al, 2006). Despite motor dexterity abnormalities

(Shahbazian et al, 2002; Pelka et al, 2006), sensory systems appear intact in MeCP2

mutants (Ricceri et al, 2008). Increases in anxiety and stress responsivity have been

found in MeCP2308/Y mice (McGill et al, 2006; Shabazian et al, 2002).

To replicate and extend previous reports, male MeCP2308/Y mutants and their

wild-type littermates were tested in the elevated plus-maze and the elevated zero-maze.

To determine whether the mutants show altered reactions to imminent threats, (fear) the

same animals were tested in the mouse defense test battery (MDTB) which is a series of

tests under which a threatening, predatory stimulus is presented under varying threat

distance and availability of escape. These tests were performed to assess potential

emotional phenotype characteristics of the MeCP2308/Y truncated mutant. We predicted

that the hyper-social phenotype of MeCP2 mutants would be accompanied by overt

decreases in anxiety and fear.



Experimental animals were bred from C57BL/6J-backcrossed stock obtained

from Jackson Laboratory (B6.129S-Mecp2tm1Hzo/J, stock # 005439) and bred from

heterozygous mutant dams and hemizygous father sibling pairs. Subject mouse

genotype was determined according to the PCR parameters from The Jackson

Laboratory with purified DNA collected from tail biopsy after weaning at post-natal day

25. Since MeCP2 is an X-linked gene, only wild-type (y/+: WT) and hemizygous (y/-: KO)

males were obtained and compared in behavioral testing. Stimulus mice of the CD-1

outbred strain were acquired from Charles River and bred in-house. Mice were housed

with up to five same-sex littermates under a 12-h light/dark schedule with lights on at

0600h. Mice had ad libitum access to tap water and lab rodent diet. All procedures were

performed according to protocols approved by the University of Hawaii Laboratory

Animal Service (LAS) Institutional Animal Care and Use Committee.

2.2.2 Experimental Design

27

Seventeen each of WT and MeCP2 KO mice were assigned to two experimental

cohorts. Eight of each were assessed in the elevated plus-maze, the female urine

sniffing test, and the cocaine-induced behavioral sensitization test. A second cohort of

nine of each genotype were tested in the elevated zero-maze and the mouse defense

test battery.

2.2.3 Elevated Plus-Maze

The test apparatus is based on that described by Lister (1987) and comprised of

two open arms (30 × 7 × 2 cm) and two closed arms (30 × 7 × 20) that extend from a

common central platform (5 × 5 cm). The apparatus was constructed from wood and

Plexiglas and was raised to a height of 40 cm above floor level. To prevent mice falling

off, a rim of Plexiglas (0.25 cm high) surrounded the perimeter of the open arms. One

ceiling-mounted video camera was used to record mouse behavior during the test and

the experimental room was illuminated with standard fluorescent lamps. The mean

intensity of luminosity on the open arms of the EPM was 55 lux. Testing was initiated by

placing the subject on the central platform of the maze, facing one of the open arms.

Test sessions lasted five minutes and, between subjects, the maze was thoroughly

cleaned with 70% ethanol and dry paper towels. The results were expressed as

frequency of entries into the closed and open arms, as well as percentage of time spent

in open arms with respect to total time in either closed or open arms. Ethological

measures comprised frequency scores for stretched attend postures (mouse stretches

forward and maintains a “flat-back” posture), head dips (mouse protrudes its head over

the edge of an open arm to a length equal to or exceeding the caudal aspect of the

pinnae).

2.2.4 Elevated Zero-Maze

The test apparatus consisted of an acrylic circular platform (6 cm in width)

composed of open and closed segments, with the latter being surrounded by 20 cm high

acrylic walls. The platform was raised to a height of 50 cm above floor level and the

diameter of the maze was 65 cm. To prevent mice falling off, a rim of Plexiglas (0.25 cm

high) surrounded the perimeter of the open segments. One ceiling-mounted video

camera was used to record the behavior during the test and the experimental room was

illuminated with standard fluorescent lamps. The mean intensity of luminosity on the

open segments of the zero-maze was 70 lux. Testing was initiated by placing the subject

in the center of the open arm. Test sessions lasted five minutes. The results were

28

expressed as mean entries into the closed and open sections as well as mean

percentage of time spent in open segments over total time spent in both open and

closed segments. Ethological measures comprised frequency scores for stretched

attend postures (mouse stretches forward and maintains a “flat-back” posture), head

dips (mouse protrudes its head over the edge of an open arm to a length greater than or

exceeding the caudal aspect of the pinnae). Videotapes collected for the elevated plus

and elevated zero mazes were scored by an observer blind to genotype using

commercially-available annotation software (Observer, Noldus Information Technology,

The Netherlands)

2.2.5 Mouse Defense Test Battery

Ten minutes before the test session began, an adult Sprague–Dawley rat

(average weight of 450 g) to be used as the predator stimulus was deeply anesthetized

with sodium pentobarbital (80 mg/kg, i.p.) in order to minimize its discomfort. The Mouse

Defense Test Battery (MDTB) consists of the following subtests:

1) Pre-test: subjects were placed into the MDTB apparatus for a 3-min

familiarization period during which total line crossings and wall rears were

recorded. Predator avoidance test: avoidance and escape distances were

measured when a predator stimulus (a hand-held rat) was brought up to the

subject at a speed of approximately 0.5 meters per second (m/s). Approach

was terminated when contact with the subject was made or if the subject ran

away from the approaching rat. This was repeated five times.

2) Chase/flight test: the anesthetized rat was brought up to the subject at a

speed of approximately 2.0 m/sec. Chase was initiated only when the subject

was standstill with its head oriented toward the rat, and completed when the

subject had traveled a distance of 14.4m (three laps in the runway). The time

spent by the mouse to travel this distance was recorded. Mean flight speed

(in m/s) was calculated based on the obtained latency to complete the three

laps. In addition, the number of stops (pauses in locomotion), orientations

(subject orients its head toward the oncoming rat) and reversals (subject

turned and ran in the rat direction) were also recorded.

3) Straight alley test: the runway was converted into a straight alley, 80 cm long,

by the closing of a door at one end and the placement of a removable barrier

at the other. The rat is placed at one end while the mouse begins the test at

29

the other. During a 30 sec period, the number of approach-withdrawals

(subject moves toward the rat more than 20 cm and then returns), as well as

voluntary contacts with the rat stimulus were recorded. Other measures

scored included immobility time (freezing) and the frequency of defensive

uprights.

4) Forced contact test: in this situation, the straight alley length was reduced to

40 cm. The rat was brought up by the experimenter in five sudden contacts

directed toward the subject. For each such contact, the number of

vocalizations, defensive uprights, jump attacks, jump escapes and bites were

recorded. This procedure was repeated three times.

5) Post-test: upon completion of the forced contact test, the alley doors were

opened allowing the subject free locomotion around the oval runway. The

subject exploratory activity was recorded for another 3 min period during

which the experimenter and the rat stimulus were out of sight. Line crossings

and wall rears were recorded.

2.2.6 Female Urine Sniffing Test

Reward sensitivity in MeCP2 mutants might underlie their behavioral phenotype.

To test this possibility, MeCP2 wild-type and knock out (KO) mice, previously analyzed

for social behavior and stereotypy, were tested in a female urine sniffing test of hedonia

according to procedures outlined in Malkesman et al, (2010). Fresh urine was collected

from adult female CD-1 mice and used as a point-source stimulus. Individually-housed

MeCP2 wild-type and mutant mice were initially exposed to a sterile foam-tipped surgical

swab containing 20 µL distilled water for a five minute session. Immediately following

this baseline session, they were permitted access to a new swab containing 20 μL fresh

female urine for an additional five minutes. Urine was collected for females primed to

soiled male mouse bedding for four consecutive days to ensure estrous and/or

proestrous status (Dalal et al, 2001), and pooled such that all samples were uniform.

The average duration of time sniffing the swab, which was defined as the subject

suspending all other activities while maintaining proximity of the nose to the tip of the

swab at a distance ≤ 2 cm, was analyzed for both sessions and compared by genotype.

2.3 RESULTS

2.3.1 Elevated Plus-Maze

30

No significant differences were noted in the frequency of entries into the areas of

the plus-maze arena (Figure 8a). Wild-type and knockout mice showed similar rates of

stretch-attend and head dip postures during the five minute test (Figure 8b). Finally,

there were no significant differences in the duration of time spent in the open, center and

closed arms of the elevated plus-maze as well in the proportion of time spent in the open

arm relative to the other arms (Figure 8c).

2.3.2 Elevated Zero-Maze

MeCP2 wild-type and mutant mice demonstrated similar frequencies of entries

into the open and closed portions of the elevated zero-maze (Figure 9a). Though the two

genotypes displayed similar rates of Stretch Attend postures, mutant mice showed a

significant elevation in the frequency of Head Dips (t(16)=-3.55, p=0.003, Figure 9b).

When the mean duration of time spent in the open and closed compartments was

compared for the wild-type and mutant mice, the results demonstrated no significant

differences. Furthermore, the proportion of time spent in the open arms was comparable

for the two genotypes (Figure 9c).

2.3.3 Mouse Defense Test Battery

Table 3 displays the mean and standard error for the MDTB parameters, and

independent t-test and Mann-Whitney U-test values. In brief, MeCP2 mutants showed an

enhanced avoidance distance of the predatory stimulus in the predator avoidance test.

Mutants showed enhanced flight speed, fewer stops, and more reversals in the

chase/flight component as well as fewer approaches and contacts; they also showed

increased freezing in the straight alley test. When the mice were exposed to forced

contact with the rat stimulus, mutants performed jump escape behaviors more than wild-

type mice. When mice were exposed to the post-test, when given free access to the

entire oval arena with an absence of the predatory stimulus, mutants performed more

rearing (exploratory) behaviors.

2.3.4 Female Urine Sniffing Test

Two-way ANOVA indicated no significant main effect of Genotype

(F(1,14)=0.004, p=0.952) or of the Stimulus (F(1,14)=1.522, p=0.238). The Genotype x

Stimulus interaction also failed to reach statistical significance (F(1,14)=0.291, p=0.598).

2.4 DISCUSSION

31

The purpose of these studies was to determine whether alterations in

fundamental affective and approach/withdrawal systems might underlie the unusual

social phenotype of MeCP2 mutant mice. More specifically, the prediction was tested

that enhanced affiliative sociability might be secondary to a reduction in anxiety or fear-

which would render novel stimuli or contexts less threatening. The overall hypothesis

that an augmentation of social affiliation might be the consequence of a general

enhancement of sensitivity to reinforcers was also tested. We noted normal anxiety in

the elevated plus- and zero-mazes but a complex enhancement of fear and defensive

responses in the mouse defense test battery. The latter finding was the opposite of the

predicted changes. Taken together these results suggest that the hyper-social

phenotype of MeCP2308/Y mutant mice cannot be attributed to a general effect of the

protein’s dysfunction on failures of the emotional systems showing appropriate risk

assessment and defensive responses to novel, potentially threatening social systems or

contexts. Likewise, the social phenotype cannot be understood solely in terms of overall

enhanced sensitivity to rewarding olfactory stimuli. Further testing will be necessary to

assess which sensory or emotional systems drive MeCP2308/Y hyper-sociability.

Previous studies of MeCP2 mutants have demonstrated increased anxiety

(Gemelli et al, 2006); however, others have demonstrated normal and decreased

anxiety, depending on the type of mutation (Guy et al, 2001; Pelka et al, 2006). Indeed,

our reports are contrary to others demonstrating elevated anxiety in the MeCP2308/Y

mutant (McGill et al, 2006; Shahbazian et al, 2002). A potential explanation of this

contradiction is that earlier reports utilize mutants on a hybrid background, while the

current study used mice fully backcrossed onto a C57BL/6J background (The Jackson

Laboratory Strain # 005439). Another consideration is that previous reports may

conclude that MeCP2 mutants possess an anxiety phenotype but this may be based on

open field test parameters, for instance, in the absence of light-dark box impairments

(e.g. Fyffe et al, 2008) or investigations of the plus or zero mazes. This conclusion would

be problematic given locomotor disturbances in many MeCP2 mutants. Altogether, the

influence of MeCP2 on anxiety may be complex, but our work does not support an

anxious phenotype in adult MeCP2308/Y mutants.

One limitation of our results is that the exploration parameters collected in the

elevated zero-maze might be affected by a ceiling effect. Subject mice were placed into

the open zone facing a closed portion and many of both genotypes inhibited exploration.

32

Therefore, proportion open area time approached one hundred percent; this could have,

to some degree masked the ability to demonstrate a difference. On the other hand, both

wild-type and mutant mice showed this lack of exploration around the perimeter of the

zero-maze and thus spent very little time in the protected areas. Although protocols for

elevated zero-maze often indicate placement of the subject into the closed area first,

others do not (e.g. Bader et al, 2011). In the future, researchers should consider placing

MeCP2 mutants and controls into the closed zone when using the elevated zero-maze to

reveal any abnormal anxiety-like behaviors in MeCP2 mutants. Alternatively, the light-

dark box may be a more appropriate measure of anxiety-like behavior in future analyses.

However, given the absence of anxiety-like behavior in the elevated plus maze, coupled

with the normal exploration noted in the mouse defense test battery prior to the

predatory threat stimulus presentation, it does not appear the MeCP2308/Y mice (on a

pure C57BL/6J background) display robust alterations in anxiety.

To explore the possibility that this mutation causes other affective disturbances,

MeCP2308/Y mice were tested for fear and panic-like parameters in the MDTB. Altogether,

mutant mice were substantially more defensive, as evidenced by augmented avoidance

and escape behaviors, as well as increases in active defensive responses to the

threatening predatory stimulus. These data suggest that not only are fear and defense

intact in the hypomorphs, it surpasses that of their normal littermates. Although fear

conditioning has been assessed in MeCP2308/Y mice, it has been shown to be normal, in

a context conditioning paradigm (but not in a cue-conditioning test) to footshock (Gemelli

et al, 2006; Shahbazian et al, 2002). Since fear conditioning and unconditioned fear

possess unique properties (Rosen et al, 2004), further research should analyze the

implications of altered unconditioned fear in MeCP2 mutants.

Figure 8. Elevated Plus-Maze (EPM) testing of MeCP2308/Y and Wild-Type Littermates. MeCP2 mutant (Y/-) and wild-type (Y/+) mice show similar frequencies of entries into the three areas of the EPM (a). They also display comparable durations and proportions of time in the open and closed arms (c). Rates of risk assessment and exploration behaviors were similar as well (b). N=8/group.

33

Figure 9. Elevated Zero-Maze (EZM) Testing of MeCP2308/Y and Wild-Type Littermates. MeCP2 mutant (Y/-) and wild-type (Y/+) mice show similar frequencies of entries into the open and closed areas of the EZM (a). They also display comparable durations and proportions of time in the open and closed areas (c). Rates of head dip exploration behaviors were increased in mutants (b). N=9/group.

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35

CHAPTER 3: REWARD SYSTEMS IN MOUSE MODELS OF AUTISM

Basic models of behavioral regulation (see Lang et al, 1998) tend to posit that

emotional systems regulate learning, experience and behavioral output which are, in turn

driven by internal motivational states. This simple model may be useful in attempts to

characterize central processes regulating psychiatric-disorder-relevant behavioral

displays in rodents. One potentially understudied system in murine models of autism is

motivation (Crawley 2007b). Since social motivation deficits have been implicated in

autism (Dawson 1998; Dawson et al, 2005; Waterhouse et al, 1996), pre-clinical

researchers should consider distinctions in sensitivity to rewarding stimuli in mice

showing variability in social approach and affiliation.

Much research in behavioral neuroscience has been concerned with the classical

and instrumental underpinnings of approach, avoidance and consummatory behaviors.

As such, many explanations of a given behavior tend to focus on reinforcement and

contingency. Although social interactions are a motivated behavior (Ikemoto & Panksepp

1999), very few murine models of autism have been assessed for aberrant brain reward

systems in conditions of disordered social behavior. In particular, neurodevelopmental

disorders with impaired sociability may result from an overall lack of motivation to

engage unfamiliar persons. This notion is supported by the fact that many individuals

with ASD show limited affiliation, communication and bonding with family members but

show a virtual absense of appropriate affiliation with others (Kanner 1943). A simple

approach to comprehend the mechanisms of these traits is to measure the hedonic

value of social reinforcers within rodent models. Any disturbances in brain reward

circuitry might contribute to, or reflect impairments in appropriate assignment of value to

exogenous social signals and potentially reveal a biomarker or target for therapeutics.

An aim complementary to analyses of emotional parameters is general assessment of

motivation and reward sensitivity in animal models of autism. Therefore the prediction

was made that since MeCP2308/Y mutants are hypersocial, they would show increased

sensitivity to a chemical reinforcer, and this altered corollary might underlie components

of their social phenotype.

To begin realizing the role of reward and hedonia in sociability, low-social BTBR

T+tf/J (BTBR) mice and normally-social C57Bl/6J (B6) mice were tested in a novel social

conditioned place preference task. More specifically, to verify that social interactions are

36

reinforcing in mice, a normal and socially-impaired strain were studied for positive

incentive evaluation-type behavior, to social interactions. Mice were tested in a hybrid

apparatus that was prepared by adding uniform black and white stripes to the outer two

compartments of two three chamber arenas- in doing so, allowing the distinct vertical

and horizontal stripes that are unique to the outer compartments to act as conditioned

stimuli in an a Pavolivan task. Rather than using a chemical reinforcer, one of the

unique contexts was paired with an unfamiliar conspecific during repeated days of

conditioning to assess the positive incentive value of the interaction. As outlined below-

while B6 mice showed a place preference for the side containing the CS associated with

social interactions, BTBR mice did not. This suggests that BTBR mice do not find the

interactions reinforcing, as predicted by the social motivation hypothesis.

3.1 INTRODUCTION

Autism spectrum disorder (ASD) is a highly heritable, but varied set of

neurodevelopmental abnormalities characterized by deficits in reciprocal social

interactions, communication, and restricted or repetitive behaviors (American

Psychological Association [APA], 2000). Increasingly, researchers are applying modern

molecular techniques and genetic association in order to determine the underlying

pathology of ASD (Betancur 2011; DiCicco-Bloom et al, 2006). Rodent models of ASD

have proven informative in the recapitulation of the syndrome, in revealing physiological

abnormalities and in pre-clinical drug screening (Silverman et al, 2010a).

Since ASD is conceptualized as a social disorder, a major research emphasis is

on revealing brain systems that regulate social behavior, and in particular, those that are

disturbed by environmental or genetic processes (Insel & Fernald 2004). Social behavior

encompasses a broad accumulation of constructs (emotion, and sexual and aggressive

motivation) and is modulated by a variety of stimuli and sensory modalities (olfactory,

visual and tactile); therefore, some specificity is required in the interpretation of

behavioral variables in social behavior tests (Insel & Fernald, 2004). One way to

approximate the positive incentive (pro-social or affiliative) motivation of social

interaction in rodents is through tests which measure value of social interactions via

conditioning. Relative motivation for social interaction has been tested with operant

lever-pressing for social contact (Evans et al, 1994), through conditioned place

preferences (CPP) to interactions with littermates (Panksepp et al, 2007; Panksepp &

37

Lahvis 2007) and via exposure to familiar and unfamiliar conspecifics (Calcagnetti &

Schechter 1992; Thiel et al, 2008; Trezza et al, 2009). In fact, the degree of social-

conditioned place preference in rodents corresponds directly to variability in social

motivation secondary to extent of previous social isolation (Trezza et al., 2009). In rats,

social interactions are sufficiently reinforcing in their ability to reverse cocaine CPP (Fritz

et al, 2011); however, restricting tactile contact with an unconditioned social stimulus

markedly attenuates or blocks social CPP (Kai et al, 2011). The degree of conditioned

place preference can therefore be considered an index of the positive incentive value

(Bardo & Bevins 2000) of social access and provides an outstanding measure of social

cognition in rodents.

Social motivation measures may provide complementary measures in animal

models of ASD (Crawley, 2007). In fact, attenuated conditioned place preference to

conspecifics has been demonstrated in fmr1 mutant mice, a model for Fragile X mental

retardation disorder (Pacey et al, 2011). In terms of translation, social motivation testing

may be critical in revealing brain substrates of impaired affiliative social behavior in ASD

(Dawson 2005; Waterhouse et al, 1996) and perhaps the development of such

impairments through altered assignment of reward to social stimuli (Dawson et al, 2005).

In the current study, a social reinforcement paradigm was developed to analyze

social conditioned place preference in BTBR T+tf/J (BTBR) mice. We present a

modification of the three-chamber social apparatus for the analysis of place preference

stemming from context-specific access to a novel juvenile conspecific. By applying this

social conditioning task to compare juvenile, inbred C57BL/6J (B6) mice to the socially-

impaired BTBR strain, we were able to demonstrate differential place preferences to a

context associated with social interactions.



C57BL/6J (B6) and BTBR T+tf/J (BTBR) mice were bred from stock obtained

from Jackson Laboratories (Bar Harbor, ME). Subject mice were weaned on postnatal

day 21-25 and housed with up to seven same-sex littermates. An equal number of male

and female mice from each strain were tested beginning at 25 - 30 days of age, at least

24 hours after being weaned. All mice were individually identified with numbered ear

tags.

38

3.2.2 Social Conditioned Place Preference

Three-chamber arenas (Nadler et al, 2004) were modified so that the two outer

compartments included alternating 3.8 cm black and white stripes on the walls; one

chamber contained vertical stripes while the other contained horizontal stripes (Figure

10a). The center compartment was comprised of black Plexiglas walls. Two separate

arenas were used simultaneously during testing- each of which had the opposite pattern

of stripes to eliminate confounding influences of baseline stripe orientation or side

preference. On the first day of testing (Day 0), all subjects were placed in the middle

compartment and the doors were lifted to permit full access to all three compartments.

Mice could freely explore the entire arena for twenty minutes during which time an

observer manually recorded the duration of time in each of the two striped outer

compartments with hand-held stopwatches. This session served as habituation and

permitted determination of baseline side preferences for each subject. Mice were then

randomly assigned to either 5 or 10 days of social conditioning, creating four groups: B6

5 day, B6 10 day, BTBR 5 day, and BTBR 10 day. In total, twenty mice from each strain

were used, representing an equal sex ratio, with ten each per condition. On the day

following baseline testing, and for 5 or 10 consecutive days, conditioning was performed.

Mice were randomly assigned to receive contact with a conspecific (unconditioned

stimulus) in either the horizontally- or vertically- striped (conditioned stimulus)

compartment. During conditioning, doors were left closed and the mice were placed into

the non-social chamber and permitted to explore alone for 10 minutes, then the mouse

was immediately removed and placed into the opposite compartment which contained

an unfamiliar age-matched male or female B6 mouse which had been placed in the

“social” chamber immediately prior to transferring the subject mouse. The subject mouse

and stimulus mouse were then permitted 10 additional minutes to explore or interact.

The side in which the subject mouse was initially placed was alternated daily; therefore,

the order in which they received the social and non-social sessions was

counterbalanced daily. An unfamiliar group of six naïve male and female mice (n=3/sex)

served as stimulus animals. As a result, mice conditioned for 10 days were exposed to

mice they were in contact with previously for the last four out of the ten days, while 5 day

conditioned mice were exposed to unfamiliar mice daily. Subjects of both sexes were

conditioned to stimuli of both sexes. On the three consecutive days following the last

conditioning session, mice were tested daily for place preference during a free

39

exploration session for 20 minutes, exactly as they were tested during the baseline

period.

3.2.3 Social Interactions

The degree of social conditioned place preference may be directly influenced by

the quality or quantity of interactions during conditioning. Mouse strains avoiding social

contact might show impaired social conditioning secondary to inadequate social contact

during conditioning. To account for this possibility, videotapes of conditioning sessions

were sub-sampled for the presence of social proximity. Proximity was defined as

maintaining a body distance (excluding tail) of less than or equal to 2 cm at any point in

a ten second scan collected every 60 seconds for each session per day. Therefore, the

presence/absence of proximity was sub-sampled for ten seconds at each of the nine

minutes (1-10, the first minute was excluded) for five or ten days, yielding at total of 45,

and 90 scans for the two conditions, respectively.

3.2.4 Autogrooming

BTBR mice show consistent enhancements of self-grooming rates compared to

B6 control mice (McFarlane et al, 2008). BTBR mice may avoid social contact in order to

perform high rates of such body care activities. This hypothesis is predicated on the

possibiliy that vigilance in a social context and self-grooming might be state behaviors at

odds with one another. Therefore, during baseline and the first testing session, the time

spent self-grooming was scored and calculated as a percentage time [grooming duration

(in seconds)/chamber duration x 100]; the calculated values on the first test day were

then subtracted from the baseline value to obtain a change in percent time spent

grooming in each of the three compartments during testing. I predicted that BTBR mice,

but not B6 mice would spend less proportional time grooming on the side they associate

with social isolation.

All sessions were videotaped from above with a digital camcorder connected to a

DVD recorder. Side preference testing was performed in real time by two observers;

social proximity and self-grooming were scored offline from DVDs by an assistant blind

to the strain and condition of the mouse. Testing arenas were sanitized with a 70%

ethanol solution and dried between subjects. Conditioning and testing was performed

between 0800 and 1300 hours under standard fluorescent lighting. Experiments and

husbandry were performed according to protocols approved by the University of Hawaii

Laboratory Animal Service (LAS) Animal Care and Use Committee.

40

3.2.5 Statistical Analysis

For three consecutive test days after conditioning, percent preference for the side

to which the subject mouse was socially conditioned was calculated [time in social

compartment/ (time in nonsocial compartment + time in social compartment) x 100] and

for graphical and statistical purposes, the baseline preference (calculated in the same

manner) for the social side was subtracted. Therefore, values represent the change in

percent from baseline, in the preference for the social conditioned chamber. A two-way

repeated measures analysis of variance (ANOVA) was performed with Strain and

Conditioning Days as the two factors and Test Day as the repeated measures variable.

To determine strain differences independent of testing day, a two-way ANOVA was

performed utilizing normalized social compartment preference scores collapsed across

all three days of testing. Similarly, two-way repeated measures ANOVAs were utilized to

compare mean percentages of scan observations during conditioning in which the

mouse was in proximity to the stimulus between the two strains within the five- and ten-

day conditioning groups. A series of one-way ANOVAs were performed to compare

mean durations of grooming during the habituation and first testing day as well as the

difference of the two, to compare proportion of time spent grooming in each of the three

chambers. In cases where ANOVA analyses reveal significant main effects or

interactions, Bonferroni post-hoc comparisons were performed to compare strain means.

3.3 RESULTS

3.3.1 Social Conditioned Place Preference

Two- way repeated measures ANOVA revealed a significant main effect of Strain

(F(1,36)=4.196, p=0.048), indicating that BTBR mice showed decreased percent

preference for the socially-conditioned side compared to B6 mice. The main effect for

the number of Conditioning Days (F(1,36)=1.765, p=0.192) failed to reach statistical

significance. Strain x Conditioning Days (F(1,36)=0.099, p=0.755), and Strain x

Conditioning Days x Test Day (F(2,72)=0.295, p=0.746) interactions were not statistically

significant. When mean chamber preferences were collapsed across the three days,

two-way ANOVA revealed a significant effect of strain (F(1,116)=9.914, p=0.003) but the

interaction between Strain and number of Conditioning Days failed to reach statistical

significance (F(1,116)=0.216, p=0.643). Interestingly, with the mean values collapsed

across all three days of testing, the Conditioning Days main effect bordered statistical

41

significant (F(1,116)=3.867, p=0.052) suggesting that the amount of conditioning might

influence the strength of place preference formation. Figure 10b presents percent

preference (-baseline) for the socially-conditioned side across three days of testing in B6

and BTBR 5- and 10-day conditioned mice. Figure 10c displays the average preference

for the social compartment across all three days. Positive values indicate preference for

the compartment associated with the social interactions while negative values indicate

an aversion or decrease in preference for the social compartment, relative to baseline.

3.3.2 Social Interactions

For mice conditioned for five days, there was no significant main effects of Strain

or Day, or a Strain x Day interaction (F(1,72)= 0.212, p=0.651; F(4,72)= 0.880, p=0.481;

F(4,72)=1.502, p=0.211, respectively). Similarly, there were no significant main effects of

Strain or Day, or Strain x Day interaction for mice conditioned for ten days to social

interactions (F(1,162)= 4.148, p=0.057; F(9,162)= 0.850, p=0.572; F(9,162)=, 0.391, p=0.938

respectively). These analyses indicate that although there was a slight decrease in the

percent time BTBR mice were in proximity during conditioning; this effect was not

statistically significant and perhaps also was not behaviorally relevant with regards to

opportunity to form social reward-related contingencies. BTBR mice showed similar

proportions of time in proximity during conditioning. Figure 11 presents mean

percentages of scan observations of social proximity for five (a) and ten (b) day

conditioned mice.

3.3.3 Autogrooming

Figure 12 presents the mean percent time self-grooming in each of the three

chambers of the conditioning apparatus during the baseline session (a) as well as at the

first day of testing (b) and the difference between the two (c), which represents the mean

change in percent time grooming after social conditioning. Although significant

differences in time spent grooming in each of the three chambers was noted, most of the

variance contributing the difference was between the outer two conditioning chambers

and the center. If BTBR mice were particularly avoidant of social interactions because of

their pre-occupation with self-grooming, we would expect decreased percent time

grooming in the social compartment after conditioning and this was not noted. In fact,

BTBR mice spent as much time self-grooming in the social chamber as they did the non-

social chamber (Figure 12b,c). This suggests that BTBR mice are not avoiding social

42

contact or failing to be reinforced to the presence of a conspecific in order to groom

themselves in a context associated with isolation.

3.4 DISCUSSION

Utilizing the commonly-utilized three-chamber apparatus for sociability testing,

we have validated a novel approach to analyze direct social motivation deficits by

demonstrating an absence of place-preference elicited by an animate social incentive in

BTBR mice- a strain known to exhibit face and construct validity for autism. Though our

results revealed that the number of conditioning days did not contribute a statistically

significant influence on the variance, the means (Figure 10b) strongly indicate that more

days of conditioning elicit a stronger place preference; an effect noted previously (Thiel

et al., 2008).

Subject mouse behavior throughout testing was assessed to determine if the

amount of voluntary social contact during conditioning was related to differences in

social conditioned place preference. We noted that both strains, independent of social

phenotype, showed similar rates of social proximity during conditioning. Therefore, the

absence of conditioned place preference to social interactions in BTBR mice cannot be

solely explained by avoidance of social interactions during conditioning sessions.

Since BTBR mice groom themselves at abnormally high levels and at highly

stereotyped patterns (McFarlane et al, 2008; Pearson et al, 2011), it was predicted that

BTBR mice might avoid social stimuli in order to perform bouts of self-grooming, which is

a time when mice cannot engage in high rates of vigilance. Our results demonstrate that

BTBR mice spent a similar proportion of time grooming in the area associated with social

interactions after conditioning when compared to the non-social conditioned chamber.

Furthermore, grooming patterns were somewhat incongruent across chambers during

baseline sessions, particularly for BTBR mice. To rule out baseline side and/or grooming

preferences, the difference between the baseline and first test session were calculated

and compared. Even when eliminating the baseline rates in grooming preferences,

BTBR mice were not preferentially grooming in the non-social compartment and avoiding

self-grooming while inside of the social compartment. These results suggest that social

deficits in BTBR mice are not secondary to avoidance of social stimuli because of an

urge to engage in self-grooming.

43

An inherent issue in learning-based assays of motivation is potential cognitive

disturbances in the candidate model. BTBR mice have been shown to have a deficit in

footshock-based fear conditioning (MacPherson et al, 2008). However, MacPherson et

al. (2008) demonstrated intact object recognition abilities in BTBR mice. Furthermore,

Silverman et al, (2010b) noted that BTBR mice have a higher nociception threshold,

suggesting that the aforementioned fear conditioning impairment (MacPherson et al,

2008) may have something to do with sensory, rather than cognitive disparities. Newer

work by Amodeo and colleagues (2012) indicates that BTBR mice have impairments in

reversal learning, an impairment that is highly relevant to restricted interests and

insistence on sameness deficits in ASD. Acquisition and maintenance of a rewarded

contingency are required for CPP, so the perseveration deficits noted by Amodeo et al,

(2012) in the BTBR mice would presumably have little or no effect. Ultimately, the

responses to non-social positive reinforcers (psychostimulants or sweetened solutions)

should be examined to determine whether learning and/or memory deficits, which are

independent of social incentive, influence conditioning performance in the BTBR strain.

An additional limitation is that one cannot rule out aggression and sex as

unconditioned stimulus in these tasks since rodents will operantly respond to contexts

associated with agonistic and sexual interactions (Bell et al, 2010; Martínez et al, 1995;

Mathews et al, 2005). Another consideration is whether subjects should be individually

housed of subjects prior to conditioning; this manipulation has been shown to increase

social play in juvenile rats (Trezza et al., 2009) and may increase the positive incentive

value of the unconditioned stimulus (Douglas et al, 2004). Single housing of male

subjects might cause increased drive for agonistic interactions. Alternatively, revealing

strain or genotype distinctions in social conditioned place preference without social

isolation in the home cage may be preferable. Other important parameters are whether

the stimulus animals are siblings or unfamiliar, and the age of both. The study presented

herein utilized juvenile mice; a design which may have effectively diminished aggressive

or sexual motivating factors in the conditioned response.

By utilizing place-preference conditioning testing, we have modified the standard

three-chamber sociability test for the use as a social reinforcement assay. Our results

strongly suggest that BTBR mice lack social motivation. We propose social conditioning

may be an alternative and/or complementary test, to social approach paradigms for

social impairments in rodent models of ASD. This technique may aide in determining

44

neurobiological system impairments and novel therapeutics in animal models of ASD

where social motivation is impaired.

Figure 10. Social Conditioned Place Preference Testing in B6 and BTBR Mice. (a) Social conditioned place preference arena. (b) While B6 mice increased the proportion of time spent on the side associated with social interactions, BTBR mice showed a significantly decreased conditioned place preference to repeated social interactions over three consecutive days of testing. (c) When preference was collapsed across three days of testing, B6 mice showed a clear percent preference for the social conditioned side, relative to baseline (subtracted) and to BTBR mice. N=10/group.

45

Figure 11. Scan Sampling of Social Conditioning Sessions in B6 and BTBR Mice. No significant differences were noted in the percent of scan observations wherein the B6 or BTBR mice were in proximity to the stimulus mouse for those conditioned for five days (a) or for ten days (b). N=10/group.

46

Figure 12. Assessment of Autogrooming Behavior Before and After Social Conditioning During Exploration. Mean percentages of time spent self-grooming during baseline (a), test day one (b), and the difference between baseline and test day as a measure in change in self-grooming preferences for mice before and after social conditioning. Values represent Mean ± 1 S.E.M. Horizontal lines denote significant main effects: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Bonferroni pairwise post-hoc comparisons: # p ≤ 0.05, λ p ≤ 0.01, p ≤ 0.001, Ψ p ≤ 0.0001. N=10/group.

47

48

3.5 PSYCHOSTIMULANT REACTIVITY

A potential confound to the social conditioning results would be the presence of

any cognitive impairments in BTBR mice, particularly in conditioning; this would

influence the ability of the mice to generate contingency, independent of social

motivation. Previous reports have demonstrated fear conditioning impairments (to

footshock) in the strain, relative to B6 mice (MacPherson et al, 2008). An obvious

challenge to the interpretation that BTBR mice are impaired in learning or memory is that

BTBR mice possess a higher nociception threshold (Silverman et al, 2010b). As such,

conditioning deficits should be assessed to non-painful reinforcers as well.

To ascertain whether SCPP impairments in BTBR mice stem from lack of social

reinforcement or a cognitive deficit, a cocaine-induced conditioned place preference was

performed in the same arenas. Figure 13 illustrates the change, relative to baseline, in

preference for the side associated with cocaine administration (4 days each of

alternating saline and cocaine conditioning). Two-way repeated measures analysis of

variance (ANOVA) revealed a significant strain effect. However, neither strain showed a

reliable increase in preference for the side associated with the chemical reinforcer. In

fact, BTBR mice showed an apparent aversion to the conditioning side, which is virtually

meaningless since it was independent of treatment. If forced to speculate, one could

argue that BTBR mice are more avoidant of the side the mouse was last placed into

after an injection, relative to B6 mice. Given the small sample size, and the degree of

variance, it would be difficult to assert that this is demonstrative of intact active

avoidance conditioning. Differential psychostimulant conditioning should be investigated

in these both the BTBR and MeCP2 strains in the future to fully realize their

characteristics specific to non-social positive associations.

Figure 13. Cocaine-Induced Conditioned Place Preference Testing in B6 and BTBR Mice. Percent preference for the CS-US paired side, relative to pre-conditioning baseline for three consecutive days in B6 and BTBR mice treated with saline or cocaine. Positive numbers represent increased preference for the US-paired side. Both strains failed to show a reliable conditioned place preference to cocaine administration. N=5/group.

49

50

3.5.1 Reward Tasks MeCP2308/Y Mutants

MeCP2 mutant and wild-type mice were tested for acute and chronic locomotor

responses to cocaine, a potent, dopamine (DA) indirect agonist, in a cocaine-induced

behavioral sensitization experiment. Mutant mice showed a decreased acute response

to cocaine, with neither genotype differing in behavioral sensitization to the drug (Figure

14). These data complement emerging work showing the pervasive and bi-directional

influence of MeCP2 on psychostimulant response and the psychostimulant influence on

MeCP2 expression. This test was performed to test the hypothesis that the hypersocial

phenotype of the mutant mice might result from enhanced reward sensitivity. The

obtained results are opposite to the prediction, and may indicate that the lack of MeCP2

protein may result in decreased reward sensitivity, driving an enhanced appetitive drive

to social engagement. A multitude of tests will need to be performed to assess this

possibility.

3.5.2 Psychostimulant Response in Low-Social Mice

A logical extension of the psychostimulant results in MeCP2 KO mice is to

determine whether the low-social BTBR mouse strain would show the opposite -

enhancement of acute locomotor response to cocaine. Within the CIPP experiment

mentioned previously, locomotor responses during the 20 minute conditioning sessions

to saline and cocaine, were analyzed from videotapes. BTBR mice showed a slight, but

consistent attenuation of locomotor response to cocaine, relative to saline, than did B6

mice (Figure 15). These data (reduced acute psychostimulant responses) are similar to

aforementioned data in the cocaine-induced behavioral sensitization task for MeCP2

mutants. Attenuated acute response to a psychostimulant drug may reflect DA

abnormalities in both MeCP2 mutant and BTBR mice despite opposing social

phenotypes.

* *

Figure 14. Locomotor Response to Repeated Cocaine Administration in MeCP2308/Y

Mutant and Wild-Type Mice. Upon daily, repeated injection of cocaine (10mg/kg) in an open field arena, MeCP2 mutant mice showed significantly decreased distance on the first day, relative to wild-type mice (a). Similarly, they showed significantly reduced total distance traveled (b) and velocity (c) on day 1. N=9/group.

51

Figure 15. Acute Locomotor Response to IP Cocaine in B6 and BTBR Mice. BTBR mice show an attenuated locomotor response to cocaine (10mg/kg) relative to saline injection, compared to B6 mice when accounting for the locomotor behavior in the previous day of saline injection. N=5/group.

52

53

3.6 DISCUSSION

Despite widespread analysis of the role of the mesolimbic reward neurocircuitry

in specific forms of social behaviors such as pair bonding and maternal behavior (e.g.

Champagne et al, 2004; Liu & Wang 2003), there has been very little consideration of

the role of this system in disturbances in other forms of social affiliation. The specific role

or function of reward processing in the brain of animal models of autism as well as in

human neurodevelopmental syndromes has not been sufficiently studied.

The neurobiology of behavior, but more specifically complex social behavioral

phenotypes are being considered in light of emerging evidence of dynamic changes in

genome regulation and neural plasticity (Hofmann 2003). These processes have vast

implications for normal and abnormal functioning, particularly with regards to

development, cognition and coping in humans and model organisms alike. Furthermore,

neural and behavioral plasticity phenomena, themselves, may prove to be critical and

effective targets for novel therapeutics (e.g. Manji et al, 2003).

The candidate mouse strains assessed here (BTBR inbred line and MeCP2

hypomorphs vs. wild-type inbred controls) show opposite, but nonetheless aberrant

behavioral displays relevant to autism. In light of emerging work demonstrating

pronounced disruptions of normal genetic and cellular plasticity cascades in these

candidates (Asaka et al, 2006; Deng et al, 2010; Martinowich et al, 2003; Stephenson et

al, 2011), it seems relatively evident that neural plasticity might be directly involved in

their syndrome-related behaviors. Pre-clinical research should aim to characterize the

specific mechanisms (ie. neurogenesis, synaptogenesis, epigenetics etc.) and their

interactions with known or predicted risk factors that culminate in pathology and

neurodevelopmental syndromes.

54

CHAPTER 4: N-SULFATED HEPARAN SULFATE ABUNDANCE AND DISTRIBUTION

IN MECP2308/Y MICE

4.1 INTRODUCTION

A major goal in research into autism and other related neurodevelopmental

disorders is to identify biomarkers that may aide in detection and treatment of these

troubling abnormalities (Walsh et al, 2011). Since MeCP2 protein regulates transcription

of a variety of genes, and MeCP2308/Y mutants have alterations in brain development and

neuroplasticity, a logical hypothesis would be that MeCP2308/Y mutants show alterations

in brain systems which regulate dynamic cyto-architectural and neurotrophic processes.

Emerging research has demonstrated that the subependymal layer of the lateral

ventricle (LV) contains small fractal structures (fractones) immunoreactive for laminin

(LAM) and n-sulfated heparan sulfate (HS) proteoglycans (Mercier et al, 2002).

Research suggests these constituents are crucial for growth factor binding and cognate

receptor signaling- processes which directly regulate cell proliferation and neurite growth

(Kerever et al, 2007). Their abundant presence in the sub-ependymal layer of the lateral

ventricle subventricular zone suggests a direct role in neurogenesis since this region and

the dentate gyrus are the two main adult neurogenic zones (Ming & Song 2005). More

specifically, n-sulfated HS sequesters a variety of growth and guidance factors that are

critical to normal stochastic processes in the adult brain (Yamaguchi 2001). Any

disturbances in the distribution or function of these HS proteoglycans, at any point in the

life-span, but particularly early in development, might severely disrupt brain circuit

formation and function.

Laminins are glycoproteins that are important constituents of the basement

membrane extracellular matrix (Timpl et al, 1979) and are expressed by endothelial and

epithelial cells (Foidart et al, 1980). As the most abundant glycoprotein constituent of

the basement membrane, laminin has central roles in cell-to-cell interactions as well as

is diverse signaling functions (Martin & Timpl 1987). Laminin is the first detectable

glycoprotein present in the basement membrane during embryogenesis, and binds

directly to heparan sulfate proteoglycans (Martin & Timpl 1987).

BTBR mice, an outstanding behavioral model of all three symptom clusters of

autism, show vast reductions in the amount of HS in the subventricular zone of the LVs

compared to B6 mice (Blanchard et al, 2012, Meyza et al, 2012). Furthermore, BTBR

55

mice show corpus callosum agenesis and a reduced hippocampal commissure

(Wahlsten et al, 2003). Although this work is ongoing, it suggests a potential mechanism

which might underlie aberrant brain development and the display of abnormalities in

social and repetitive behaviors reminiscent of autism. A major goal is to determine the

extent to which proteoglycan disturbances might affect normal functioning in models of

autism and other pervasive neurodevelopmental disorders. The following experiment

was designed to determine if n-sulfated heparan sulfate disturbances are characteristic

of MeCP2 mutant mice.



Adult (pnd 56-88) male MeCP2 WT and KO mice (n=4/genotype) were

euthanized and whole brains were removed and flash frozen in isopentane cooled to -

80ºC. Brain sections (10 µM) were collected and mounted on poly l-lysine (Sigma 1/5

dilution) coated glass slides and stored at -20ºC.

4.2.2 Dual-Channel Indirect Fluorescent Immunohistochemistry

Prior to immunolabeling, sections were fixed in acetone (-20ºC) for two minutes.

Sections were hybridized in wells created on the slide with a hydrophobic barrier pen

(Vector Labs). Primary antibodies directed at laminin (Sigma: rabbit IgG 1/1000) and HS

(Seikagaku Corp: 10E4: mouse IgM 1/400) were incubated for 120 minutes. Secondary

antibodies (Invitrogen: AlexaFluor 546 goat anti-rabbit IgG and AlexaFluor 488 goat anti-

mouse IgM) recognized the laminin and HS primaries, respectively and were incubated

for 40 minutes. After several rinses, slides were mounted and coverslipped with a

mounting medium containing DAPI nuclear marker (Vector Labs).

Microscope images (20X) were collected on an Olympus Fluoview 1000 laser

scanning confocal microscope utilizing a multi-argon laser for DAPI quantification as well

as red and green HeNe lasers for the laminin and heparan sulfate immunoreactivity (ir),

respectively, at the University of Hawaii Biological Electron Microscope Facility. High

resolution (1024 x 1024 pixel) images of the lateral wall of the right lateral ventricle were

combined as a montage in Photoshop and imported into ImageJ software (NIH) as 8 bit

RGB images. The images were divided into stacks which yielded grayscale images of

each of the three channels (Figure 16). The polygon selection tool was used to trace the

entire dorsal-ventral extent of the subventricular zone of the lateral aspect of the lateral

56

ventricle (caudate side) at each of five sections per subject (Bregma +1.0; +0.5; 0.0; -

0.5; and -1.0. Area, integrated density and mean gray value were collected for the

experimental zone selected as well as for three background areas which were used to

calculate corrected total fluorescence [Integrated density – (area of selected zone x

mean fluorescence of background images)] according to procedures previously

published (e.g. Burgess et al, 2010). Mean corrected total fluorescence for each

genotype at each region for the laminin- and heparan sulfate-immunoreactivity (LAM-ir,

HS-ir, respectively) as well as the heparan sulfate/laminin ratio (HS/LAM-ir) was

compared with Two-way analyses of variance (ANOVA) with Strain and AP Point as the

between- and within-subjects factors, respectively.

4.3 RESULTS

4.3.1 Laminin

Figure 17a displays the mean level of laminin fluorescence (mean signal intensity

in arbitrary units) across the five anterio-posterior sub-ventricular walls. No significant

difference was noted in the mean LAM-ir across AP Points (F(4,24)=1.150, p=0.357).

Likewise, there was no significant main effect of Genotype across the AP extent

(F(1,24)=0.165, p=0.699). Finally, the Genotype x AP Point interaction failed to reach

statistical significance (F(4,24)=1.120, p=0.370).

4.3.2 Heparan Sulfate

Figure 17b displays the mean level of heparan sulfate fluorescence across the

five anterio-posterior sub-ventricular zones. No significant difference was noted in the

mean HS-ir across AP Points (F(4,24)=1.976, p=0.130). Likewise, there was no significant

main effect of Genotype across the AP extent (F(1,24)=0.162, p=0.702). Finally, the

Genotype x AP Point interaction failed to reach statistical significance (F(4,24)=1.800,

p=0.162).

4.3.3 Heparan/Laminin Ratio

Figure 17c displays the mean level of heparan sulfate:laminin fluorescence

across the five anterio-posterior sub-ventricular walls. No significant difference was

noted in the mean HS/LAM-ir across AP Points (F(4,24)=425, p=0.789). Likewise, there

was no significant main effect of Genotype across the AP extent (F(1,24)=0.212, p=0.661).

Finally, the Genotype x AP Point interaction failed to reach statistical significance

(F(4,24)=1.138, p=0.363).

57

4.4 DISCUSSION

Despite widespread behavioral anomalies in male mice with a truncated form of

the MeCP2 gene, the subventricular zone heparan sulfate/ laminin system appears to be

relatively intact in MeCP2308/Y mutant mice. These data are in stark contrast to those

obtained when comparing normal B6 mice to BTBR mice. BTBR mice show pronounced

reductions of both heparan and laminin in the lateral ventricle subventricular zone

(Meyza et al, 2012). It is thought that this distruption might be secondary to or causal to

the brain anatomical abnormalities such as callosal agenesis and collapsed anterior

lateral ventricles of this autism candidate strain (Meyza et al, 2012). In the current study,

the same coordinates were assessed for similar disturbances in wild-type and

MeCP2308/Y mutants, but no differences emerged.

Since heparan sulfates are large multi-unit and varying proteoglycan molecules,

they are not under transcriptional control, per se. Rather, a variety of enzymes modify

the structure of the polysaccharide side chains of the complex molecules largely through

sulfation patterns, which in turn affects its affinity for growth factors (Kerever et al, 2007;

Turnbull et al, 2001). On the contrary, these regulatory enzymes are under

transcriptional/translational regulation. MeCP2 mutation directly affects the abundance of

their transcripts in microarray analyses (Chahrour et al, 2008). Furthermore, though not

to any extent like that of BTBR mice, MeCP2 mutant mice show alterations in brain

growth factors (Nag et al, 2008) as well as brain morphological alterations suggesting

perturbed cellular development (Stearns et al, 2007). Ultimately, no differences in the

LAM-ir were expected, as the basement membrane signal should be relatively stable.

The main rationale for including it in these assays is to provide a unit of the measured

amount of basement membrane to determine how much of it, and to what extent, it is

immune-positive for heparan sulfate; thus the HS/LAM variable.

Since MeCP2 protein regulates expression of methyl-transferases and other

such heparan sulfate proteoglycan enzymes, this strongly suggests that MeCP2308/Y

truncated mutants might have altered abundance and distribution of n-sulfate heparan

sulfate in the brain subventricular zones. We did not note such an effect. The case that

N-sulfated regions on heparan sulfate proteoglycans are not disrupted with this particular

mutation, but other enriched domains are abnormal (ie. O-sulfated zones), is a distinct

possibility that has not been assessed here. Furthermore, alterations in N-sulfated

58

heparan sulfates may occur in the brain of MeCP2308/Y mutants but in areas outside of

the anterio-posterior boundaries (Bregma +1.0 to -1.0) and/or in areas outside of the

subventricular zone. Alterations might also exist in other developmental times

(embryonically, and early postnatal periods) and these were not noted with the analyses

performed in adult animals. Altogether, the absence of significant findings here does not

preclude a role of heparan sulfate proteoglycans in the abnormal behavioral and

physiological phenotype of this candidate mutant, but it does exemplify the possibility

that the HS system does not underlie altered neurodevelopmental trajectories in animal

models of autism or in those clinically affected with ASD.

DAPI HS MERGELAM

DAPI LAM HS MERGE

a

hgfe

b c d

Figure 16. Representative Confocal Fluorescent Immunohistochemistry Micrographs of Mouse Brain Lateral Ventricle Immunolabeled for Laminin and Heparan Sulfate. Ventral lateral ventricle revealed by DAPI nuclear marker (a) as well as laminin (b)and heparan sulfate (c) and the three channels merged (d, e). The three channels were separated into grayscale images (e-g) and the subventricular zone was manually traced (dotted yellow lines). ImageJ intensity analyses were performed in the selected regions to quantify the fluorescence immunoreactivity in the laminin (f) and heparan sulfate (g) channels.

59

a

b

c

Figure 17. Immunohistochemical Analyses of Brain Extracellular Matrix Markers Laminin and Heparan Sulfate in MeCP2308/Y Wild-Type and Mutant Mice. Laminin immunoreactivity (LAM-ir) throughout the antero-posterior extent of analysis was comparable (a). Likewise, no difference in the heparan sulfate immunoreactivity (HS-ir) was evident (b). The ratio of HS to LAM was also similar across the coordinates for wild-type (WT) and mutant (KO) mice (c). N=4/group.

60

61

CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS

Utilizing an engineered mouse mutant with alterations in social behavior, and

comparing its emotional and motivation characteristics in reference to normal siblings

and an inbred strain with known alterations in social behaviors, strides have been made

in characterizing the role of emotion, motivation and brain subventricular development

systems in mouse models of autism. Although the MeCP2308/Y mutant showed an

accentuation of social behavior rather than a deficit, this specific phenotype might prove

exceptionally useful in determining the brain mechanisms of altered social behavior. This

possibility is supported by the fact that the various types of engineered MeCP2

mutations can cause opposing and varied influences on social behaviors (Table 1).

Other findings of note in this mutant candidate were subtle changes in autogrooming

patterns that might show face validity to the motor stereotypies characteristic of Rett

syndrome (Hagberg et al, 1983). Furthermore, the influences of the MeCP2308/Y mutation

on fear-related anxiety responses and reactions to cocaine are notable. Despite these

advances, these results raise more questions than are answered. Altogether, the search

for novel comprehension of the pathobiology and treatment of autism-spectrum

disorders will require continued advances in basic and translational research into the

fundamental mechanisms of social and repetitive behavior symptoms.

5.1 NEUROBIOLOGY OF SOCIABILITY

Three major theoretical constructs are essential to our comprehension of the role

of the nervous system in social behaviors, but more specifically in the understanding of

the brain in social cognition (Adolphs 2001). More specifically, when assessing the

mechanisms of autism-relevant social disturbances, key questions must be regarded

with respect to what the deficits might reflect; the first is how the organism - more

specifically, the nervous system receives and processes exogenous social information

(Young et al, 2002). Disturbances in social information processing might contribute

greatly to the development of social impairments in neurodevelopmental disorders and

this influence must be considered in rodent models of ASD (Crawley 2004).

A second and perhaps equally important set of processes are the influences of

the gathered social information on emotional processes and motivation (Adolphs 2001).

Take, for example, the immense body of literature examining the differential activation of

62

the amygdala, a set of limbic nuclei principally involved in emotional processing, in

response to facial expressions and other social signals in ASD (Baron-Cohen 2000). The

notion that emotion- principally, the evolutionarily-preserved basic emotion systems,

might be directly involved in decisions about when and how to engage social stimuli

(which are potentially threatening or rewarding) are quite comprehensible. Similarly, the

motivational and emotional systems can thus integrate and differentially regulate the

motoric output systems which underlie behavior (Adolphs et al, 2001). This regulatory

model suggests how the organism is afforded additional opportunities for social learning

and reward (through feed-forward experience) which shape the cognitive and emotional

experience of a social entity.

The work illustrated here has been concerned mainly with assessing the

emotional and motivational contributions to pro-social or affiliative forms of social

behavior in male mice - forms of affiliation that are not secondary to reproduction. The

theoretical framework should apply also to other forms of social behavior (parenting,

mating and aggression) but the emphasis here is to analyze behaviors in a manner that

informs the neurobiology of social avoidance in ASD.

5.2 THE ASSOCIATIONS OF NEURODEVELOPMENT AND

NEUROPLASTICITY WITH BEHAVIOR

Discoveries surrounding the dynamic and experience-driven changes in the brain

and genome are fast revolutionizing biological and psychological sciences. Yet,

researchers have only scratched the surface in the revelation of relationships between

genes, experience and cellular plasticity (neuron/neurite/synapse proliferation and

development) and behavioral phenotypes. Crucial studies will be those that reveal direct

relationships between dynamic changes within the brain and forms of behavior that are

disturbed in autism. What’s more, the developmental conditions and exogenous

perturbations that regulate these brain changes will need to be understood in greater

detail. Based upon the data presented here and the expanding literature of the role of

MeCP2, there is a substantial amount of support that engineered mutations of the

MeCP2 gene can reveal mechanisms of the relationships between neural plasticity, DNA

methylation, histone modification and behaviors relevant to psychological and behavioral

impairments throughout development and within adulthood. This notion is best illustrated

by studies demonstrating MeCP2 is critical for normal neuroplastic responses to

63

psychostimulant drugs of abuse (Deng et al, 2010) and those showing that post-mortem

tissue collected from those affected with Rett syndrome have decrements in

hippocampal (CA1 pyramidal cell) spine density and structure, suggesting synaptic

plasticity abnormalities (Chapleau et al, 2009). Altogether, impairments in neural activity-

dependent phosphorylation of MeCP2 have been shown to regulate function and

development of the nervous system through its gene-regulatory influences stemming

from chromatin influences (Cohen et al, 2011). How MeCP2 is involved in social

cognition will require intensive assessment, but this suggests that the impairments in

response to drugs of abuse might also underlie abnormalities in the socio-emotional

phenotype of MeCP2 hypomorphs.

64

Table 1. Review of MeCP2 Mutant Rodent Studies Assessing Social Behavior

Mutant Region/Cell Background Reduced/Impaired Reference MeCP2308/Y Constitutive

Truncation 129SvEv or 129SvEv x C57BL/6J

Tube Test Avoidance Shahbazian et al. 2002

MeCP2308/Y Constitutive Truncation

129/SvEv Tube Test Avoidance; Nest Building; Divided Homecage Proximity; Interaction with Unfamiliar Juvenile

Moretti et al. 2005

MeCP2CKO Forebrain Conditional

B6 Divided Homecage Proximity

Gemelli et al. 2006

Rat siRNA Amygdala Sprague Dawley Juvenile Play Kurian et al. 2008

Mecp2Flox/Y CNS-Conditional Flanked Hypomorph

129S6/B6 Nest Building Samaco et al. 2008

MeCP2308/Y Constitutive Truncation

B6 USVs Maternal Separation

De Filippis et al. 2010

MeCP2CKO GABA Neurons

129;FVB Nest Building Chao et al. 2010

Mutant Region/Cell Background Increased/Enhanced Reference MeCP2lox/Y Constitutive

Flanked Hypomorph

129S1/SvImJ;B6/CBA Three Chamber Social Preference and Novelty Preference

Kerr et al. 2008

Mecp2Flox/Y CNS-Conditional Flanked Hypomorph

129S6/B6 Proximity in Partition Test

Samaco et al. 2008

MeCP2flox/+, and MeCP2CKO

Flanked WT, and Hypothalamus

129SvEV/B6 Proximity in Partition Test#

Fyffe et al. 2008

MeCP2flox/Y, and MeCP2CKO

Flanked WT, DA/NE- and 5-HT-Neurons

129S6SvEV (flox), 129;FVB (DA/NE), 129;B6 (5-HT)

Proximity in Partition Test#

Samaco et al. 2009

MeCP2flox, and MeCP2CKO

Flanked WT, GABA-, and FB GABA Neurons

129S6SvEV (flox), 129;FVB (GABA)

Proximity in Partition Test; Three-Chamber Social Preference

Chao et al. 2010

MeCP21lox CNS-Conditional

B6 Three-Chamber Social Preference and Social Novelty Preference

Schaevitz et al. 2010

# Signifies accompanying aggression in mutant

Table 2. ANOVA Results for MeCP2308/Y in the Visible Burrow System

Main Effects Interaction Behavior Phase Genotype Day Genotype x Day Approach

Front Dark F(1,26)=0.84, p=0.376 n.s. F(2,26)=11.58, p=0.003** F(2,26)=0.52, p=0.603

n.s.

Light F(1,32)=13.88, p=0.0018**

F(2,32)=1.43, p=0.254 n.s.

F(2,32)=1.43, p=0.254 n.s.

Approach Rear Dark F(1,26)=5.47, p=0.036* F(2,26)= 0.41, p=0.669

n.s. F(2,26)=0.76, p=0.478 n.s.

Light F(1,32)=2.84, p=0.112 n.s. F(2,32)=3.58, p=0.040* F(2,32)=2.07, p=0.143

n.s.

Flight Dark F(1,26)=1.30, p=0.276 n.s.

F(2,26)=1.49, p=0.243 n.s.

F(2,26)=2.05, p=0.149 n.s.

Light F(1,32)=0.55, p=0.468 n.s.

F(2,32)=1.14, p=0.332 n.s.

F(2,32)=1.14, p=0.332 n.s.

Chase/Follow Dark F(1,26)=1.50, p=0.243 n.s.

F(2,26)=1.68, p=0.205 n.s.

F(2,26)=0.57, p=0.570 n.s.

Light F(1,32)=0.08, p=0.780 n.s.

F(2,32)=3.22, p=0.053 n.s.

F(2,32)=1.50, p=0.239 n.s.

Autogroom Dark F(1,26)=0.17, p=0.687 n.s. F(2,26)= 3.94, p=0.032* F(2,26)=0.02, p=0.976

n.s.

Light F(1,32)=6.34, p=0.023* F(2,32)=4.31, p=0.022* F(2,32)=0.72, p=0.496 n.s.

Allogroom Dark F(1,26)=9.74, p=0.008**

F(2,26)= 2.20, p=0.131 n.s.

F(2,26)=1.59, p=0.224 n.s.

Light F(1,32)=2.42, p=0.140 n.s.

F(2,32)=1.99, p=0.154 n.s.

F(2,32)= 0.97, p=0.389 n.s.

Huddle Dark F(1,26)=36.77, p<0.0001***

F(2,26)= 45.20, p<0.0001*** F(2,26)=4.21, p=0.026*

Light F(1,32)=0.47, p=0.505 n.s. F(2,32)=1.92, 0.163 n.s. F(2,32)=1.24, p=0.303

n.s.

Vigorous Dark F(1,26)=0.65, p=0.435 n.s.

F(2,26)=0.65, p=0.530 n.s.

F(2,26)=0.65, p=0.530 n.s.

Light - - -

65

66

Table 3. Comparison of MeCP2308/Y Mutant and Wild-type mice in the Mouse Defense

Test Battery

Y/+ Y/-

Pre-test activity Mean ± 1 S.E.M. Result

Line crossings 108.56 ± 9.48 99.11 ± 9.31 t(16)= 0.71, p=0.487

Rears 6.33 ± 1.34 6.00 ± 1.40 t(16)= 0.17, p=0.866

Predator avoidance test

Avoidance distance (cm) 36.67 ± 14.14 141.11 ± 34.90 U=14.5, p=0.021 Escape distance (cm) 431.11 ± 39.03 400.00 ± 44.72 t(16)=0.52, p=0.607

Chase/flight test

Flight speed (m/sec) 0.315 ± 0.03 0.512 ± 0.04 t(16)= -4.37, p<0.001 Stops 11.90 ± 1.24 7.67 ± 1.03 t(16)= 2.62, p=0.018 Jump Escapes 0.00 ± 0.00 0.33 ± 0.17 t(16)= -2.00, p=0.063

Reversals 1.22 ± 0.52 0.00 ± 0.00 t(16)= 2.35, p=0.032 Straight alley test

Approaches/withdrawals 1.33 ± 0.29 0.22 ± 0.15 t(16)= 3.43, p=0.003 Contacts 0.89 ± 0.26 0.00 ± 0.00 t(16)= 3.41, p=0.004 Freezing (sec) 0.00 ± 0.00 1.78 ± 0.83 t(16)= -2.14, p=0.048 Uprights 3.89 ± 0.61 2.56 ± 0.82 t(16)= 1.31, p=0.210

Forced contact test

Vocalizations 1.11 ± 0.75 0.11 ± 0.11 U=26.5, p=0.115

Uprights 5.56 ± 1.44 2.33 ± 0.62 U=19.5, p=0.061

Jump attacks 0.56 ± 0.24 0.33 ± 0.30 t(16)= -1.08, p=0.297

Jump escapes 2.33 ± 0.60 6.56 ± 0.94 t(16)= -3.77, p=0.002 Bites 0.00 ± 0.00 0.22 ± 0.22 t(16)= -1.00, p=0.332

Post-test

Line crossings 108.22 ± 6.41 117.56 ± 10.04 t(16)= -0.78, p=0.445

Rears 14.44 ± 0.90 20.22 ± 1.67 t(16)= -3.05, p=0.008

n=9/ group.

67

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