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The Role of Anxiety in a Rat Model of Self-Injury

Permanent Link: http://ufdc.ufl.edu/UFE0045465/00001

Material Information

Title: The Role of Anxiety in a Rat Model of Self-Injury
Physical Description: 1 online resource (62 p.)
Language: english
Creator: Yuan, Xiaomeng
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: anxiety -- sib
Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Self-injurious behavior (SIB) is a debilitating characteristic that is highly prevalent in autism, Lesch-Nyhan disease and fragile X syndrome. Although pathological anxiety is also prevalent among these neurodevelopmental disorders, the relationship between anxiety and self-injury has not been adequately characterized. 1) Experiment 1: the open field test(OFT) and elevated plus maze (EPM) were used to assess innate expression of anxiety-related behaviors (ARB) in rats before they were assigned to vehicle-treated(peanut oil, n=6) or pemoline-treated (75 mg/kg/day or 100mg/kg/day, s.c., n=10per dose) groups. The expression of self-injury was assessed by multiple measures. The low dose of pemoline produced self-injury in 6/10 rats; whereas the high dose did so in all the rats. Correlations between the ARB in the OFT and self-injury were detected, which were absent for the EPM. 2) Experiment 2:independent groups of rats were injected with FG7142 (0, 1, 3, or 10mg/kg,i.p., n=9 per dose) for 5 days, twice daily. Immediately following that,pemoline (75mg/kg, s.c.) was administered to all groups. The expression of self-injury was elevated in the FG7142-treated groups compared to the vehicle.These results suggest that anxiety contributes to vulnerability for pemoline-induced self-injury. Additional studies of the neurobiological basis of vulnerability for self-injury are currently under investigation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Xiaomeng Yuan.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Devine, Darragh P.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045465:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045465/00001

Material Information

Title: The Role of Anxiety in a Rat Model of Self-Injury
Physical Description: 1 online resource (62 p.)
Language: english
Creator: Yuan, Xiaomeng
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: anxiety -- sib
Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Self-injurious behavior (SIB) is a debilitating characteristic that is highly prevalent in autism, Lesch-Nyhan disease and fragile X syndrome. Although pathological anxiety is also prevalent among these neurodevelopmental disorders, the relationship between anxiety and self-injury has not been adequately characterized. 1) Experiment 1: the open field test(OFT) and elevated plus maze (EPM) were used to assess innate expression of anxiety-related behaviors (ARB) in rats before they were assigned to vehicle-treated(peanut oil, n=6) or pemoline-treated (75 mg/kg/day or 100mg/kg/day, s.c., n=10per dose) groups. The expression of self-injury was assessed by multiple measures. The low dose of pemoline produced self-injury in 6/10 rats; whereas the high dose did so in all the rats. Correlations between the ARB in the OFT and self-injury were detected, which were absent for the EPM. 2) Experiment 2:independent groups of rats were injected with FG7142 (0, 1, 3, or 10mg/kg,i.p., n=9 per dose) for 5 days, twice daily. Immediately following that,pemoline (75mg/kg, s.c.) was administered to all groups. The expression of self-injury was elevated in the FG7142-treated groups compared to the vehicle.These results suggest that anxiety contributes to vulnerability for pemoline-induced self-injury. Additional studies of the neurobiological basis of vulnerability for self-injury are currently under investigation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Xiaomeng Yuan.
Thesis: Thesis (M.S.)--University of Florida, 2013.
Local: Adviser: Devine, Darragh P.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045465:00001


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1 T HE ROLE OF ANXIETY IN A RAT MODEL OF SELF INJURY By XIAOMENG YUAN A MASTER THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCI ENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Xiaomeng Yuan

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3 To my parents Dan Ding & Daotian Yuan and my beloved husband Yu Wang

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4 ACKNOWLEDGMENTS Im grateful to my advisor Dr. Darragh P. Devine. Without the encouragement he gave to me whenever I encountered difficulties and the tremendous effort he put in helping me design and conduct the experiments, this project could not be finished successfully. Im also grateful to my committee members Dr. Mark H. Lewis and Dr. Serge i Zolotukhin, for the time and effort they made to contribute in helping me think and rethink the meanings behind the project and culturing my ability to think critically and scientifically.

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5 TABLE OF CONTENTS p age ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ..................................................................................................................... 9 CHAPTER 1 INTRODUCTION .................................................................................................... 11 2 METHODS .............................................................................................................. 14 Animals and D rugs ................................................................................................. 14 Animals ............................................................................................................. 14 Drugs ................................................................................................................ 14 Experimental Procedures ........................................................................................ 14 Experiment 1 .................................................................................................... 14 Behavioral prescreening ........................................................................... 14 Pemoline treatment .................................................................................... 17 Assays of SIB and ancillary behaviors ....................................................... 17 Termination ................................................................................................ 18 Statistical analyses .................................................................................... 19 Experiment 2 .................................................................................................... 20 Open field t est (OFT) ................................................................................. 20 Drug treatments ......................................................................................... 20 Assays of SIB and other ancillary behaviors .............................................. 20 Termination ................................................................................................ 21 Statistical analyses .................................................................................... 21 3 R ESULTS ............................................................................................................... 23 Experiment 1 ........................................................................................................... 23 Evaluation of B alanced G roup A ssignments .................................................... 23 Dose E ffects of the P emoline T reatment on S elf Injury .................................... 23 B etweenG roup Differences in Ancillary B ehaviors .......................................... 24 Corre lations between the A verage M easures of S elf Injury and the M easures of I nnate A nxietyRelated B ehaviors ............................................ 24 Correlations between the average measures of self injury and the OFT measures ................................................................................................ 24 Correlations between the average measures of self injury and the EPM measures ................................................................................................ 25

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6 Correlations between the average measures of self injury and the locomotion response to mild stress in the CCT ....................................... 25 Correlations between the A verage D uration of A ncillary B ehaviors and the M easures of I nnate A nxietyRelated B ehaviors ............................................ 25 Experiment 2 ........................................................................................................... 26 Evaluation of B alanced G roup A ssignments .................................................... 26 FG7142Indu ced Elevation in Expression of S elf Injury ................................... 26 BetweenGroup Differences in Ancillary Behaviors .......................................... 27 4 DISCUSSION ......................................................................................................... 49 Dose E ffects of P emoline on S elf Injury .................................................................. 49 Relations between I nnate A nxiety Related B ehaviors and S elf Injury .................... 49 Relations between L ocomotor R esponse to M ild S tress and S elf Injury ................. 51 FG7142Induced E levation in E xpression and S everity of S elf Injury ..................... 52 Implications for N eurodevelopmental D isorders ..................................................... 56 Conclusion .............................................................................................................. 56 LIST OF REFERENCE S ............................................................................................... 58 BIOGRAPHICAL SKETCH ............................................................................................ 62

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7 LIST OF TABLES Table page 1 1 Rating scale of tissue in jury ................................................................................ 22 3 1 The correlations between the average measures of self injury and the measures of innate anxiety related behaviors.. .................................................. 28 3 2 The correlations between the average duration of the ancillary behaviors and the measures of innate anxiety related behaviors. ............................................. 29 3 3 Euthanized rats in experiment 2 ......................................................................... 30

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8 LIST OF FIGURES Figure page 3 1 Evaluation of balanced group assignments for Experiment 1 ............................. 31 3 2 T he dose effects of the pemoline treatment ...................................................... 32 3 3 Betweengroup differences in duration of ancillary behaviors in E xperiment 1. ....................................................................................................................... 33 3 4 The correlations between self injury and innate anxiety related behaviors in the OFT a t the pemoline dose of 75 mg/kg ........................................................ 34 3 5 The correlations between self injury and innate anxiety rel ated behaviors in the OFT at the pemoline dose of 100 mg/kg ...................................................... 36 3 6 The correlations between self injury and innate anxiety related behaviors in the EPM at the pemoline dose of 75 mg/kg ........................................................ 38 3 7 The correlations between self injury and innate anxiety related behaviors in the EPM at the pemoline dose of 100 mg/kg ...................................................... 40 3 8 Th e correlations between self injury and locomotor response to mild stress in CCT at the pemoline dose of 75 mg/kg .............................................................. 42 3 9 The correlations between self injury and locomotor response to mild stress in CCT at the pemoline dose of 100 mg/kg ........................................................... 44 3 10 Evaluation of balanced group assignments for Experiment 2 ............................. 46 3 11 FG7142i n duced e levation in e xpression of self i njury ...................................... 47 3 12 Betweengroup differences in duration of ancillary behaviors in experiment 2. ....................................................................................................................... 48

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9 Abstract of Master T hesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE ROLE OF ANXIETY IN A RAT MODEL OF SELF INJURY By Xiaomeng Yuan May 2013 Chair: Darra gh P. Devine Major: Psychology Self injurious behavior (SIB) is a debilitating characteristic that is highly prevalent in autism, LeschNyhan disease and fragile X syndrome. Although pathological anxiety is also prevalent among these neurodevelopmental disorders, the relationship between anxiety and self injury has not been adequately characterized. 1) Experiment 1: the open field test (OFT) and elevated plus maze (EPM) were used to assess innate expression of anxiety related behaviours (ARB) in rats before they were assigned to vehicle treated (peanut oil, n=6) or pemolinetreated (75 mg/kg/day or 100mg/kg/day, s.c., n=10 per dose) groups. The expression of self injury was assessed by multiple measures. The low dose of pemoline produced self injury in 6/10 rats; whereas the high dose did so in all the rats. Correlations between the ARB in the OFT and self injury were detected, which were absent for the EPM. 2) Experiment 2: independent groups of rats were injected with FG7142 (0, 1, 3, or 10mg/kg, i.p., n= 9 per dose) for 5 days, twice daily. Immediately following that, pemoline (75mg/kg, s.c.) was administered to all groups. The expression of self injury was elevated in the FG7142treated groups compared to the vehicle. These results suggest that anxiety contributes to vulnerability

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10 for pemolineinduced self injury. Additional studies of the neurobiological basis of vulnerability for self injury are currently under investigation.

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11 CHAPTER 1 INTRODUCTION Self injury is a devastating characteristic which is prevalent among people with a variety of neurodevelopmental disorders including autism (Richards et al., 2012), fragile X syndrome (Symons et al., 2010), and LeschNyhan syndrome (Schretlen et al., 2007). Intriguingly, anxiety disorders are also found in th ese and other neurodevelopmental disorders (Hallett et al., 2013; Schretlen et al., 2007; Symons et al., 2010). In addition, the fragile X patients with self injurious behaviors (SIB) were more likely to have anxiety disorders compared to the fragile X pat ients who did not self injure (Symons et al., 2010). However, the potential relationship between anxiety and self injury has not been clearly established in any of these neurodevelopmental disorders. Some recent studies in animal models lend support to the notion that anxiety and self injury might be related. For example, high levels of innate anxiety related behaviors have been found in SAPAP3 (Welch et al., 2007), Slitrk5 (Shmelcov et al., 2010), and Shank3 (Pea et al., 2011) mutant mice, all of which ex hibit stereotypic over grooming that results in tissue injury. In a rodent model of self injury, moderate doses of pemoline (an indirect monoamine reuptake inhibitor) can induce SIB in about 50% of rats, demonstrating individual differences in vulnerabilit y for SIB (Kies and Devine, 2004). In addition, dopamine depletion was found in rats repeatedly treated with pemoline (Muehlmann and Devine, 2008). This resembles the dopamine loss in LeschNyhan syndrome (Ernst et al., 1996), although the degree of deplet ion is far less in the pemoline model, than it is in the genetic syndrome. In another study using the pemoline model (Muehlmann et al., 2011), rats with higher innate locomotor response to the mild stress of a novel environment were more self injurious tha n rats with lower locomotor

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12 response, suggesting a possible relation between innate emotional responsiveness and SIB vulnerability. However, the potential contribution of the innate level of anxiety related behaviors to individual differences in vulnerabil ity for SIB has not been directly tested. In the first of two studies contained herein, both the elevated plus maze test and the open field test were used to prescreen rats to rats in order to obtain a stable measure of their innate anxiety level, before t he SIB inducing pemoline treatment was applied to the same group of rats. Thus, we were able to explore the correlation between innate anxiety related behaviors and individual differences in vulnerability for pemolineinduced SIB in this study. Pharmacological research with both humans and nonhuman primates provides evidence for the potential role of anxiety in SIB, by demonstrating beneficial effects of drugs with anxiolytic properties. One openlabel case report (Brahm et al., 2008) indicated that the anxiolytic drug buspirone diminished self injurious behaviors in an autistic woman, although this should be interpreted with caution because of coadministration of antiepileptic and antipsychotic drugs and staff changes during the treatment. Both buspirone and fluoxetine (an antidepressant drug with anxiolytic actions) attenuated SIB in nonhuman primates (Fontenot et al., 2005; Fontenot et al., 2009), whereas diazepam produced inconsistent effects on pemolineinduced SIB in rats (Mueller and Nyhan, 1982). However, the impact of anxiogenic agent on SIB has only been tested in one study, using nonhuman primates, which produced mixed results. (Major et al., 2009). FG7142 (a partial inverse agonist of GABAA receptors) increases the expression of anxiety related behaviors in the Vogel conflict test (Stephens et al., 1987), elevated plus maze test (Atack et al., 2005; Dawson et al.,

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13 2006), elevated T maze test (Bueno et al., 2005; Sena et al., 2003) and social interaction test (Hackler et al., 2007). Also, it inc reases expression of both c fos protein and mRNA in brain regions involved in anxiety relatedbehaviors, which include amygdala, hypothalamus, bed nucleus of stria terminalis, etc. (for review, see Evans and Lowery, 2007). In the second study contained her ein, we tested the effects of FG7142 on frequency and severity of self injury induced by pemoline in rats.

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14 CHAPTER 2 METHODS Animals and D rugs Animals Sixty two male Wistar rats (Charles River Laboratories, Raleigh, NC) weighing 176~200g were used in thi s study, with 26 of them used in experiment 1 and 36 rats used in experiment 2. All the rats were paired housed in standard polycarbonate cages (43 cm21.5 cm25.5 cm) under a 12 hr:12 hr light/dark schedule (lights on at 7:00 am) with controlled temperate and humidity. Standard rat chow (Lab Diet 5001) and tap water were available ad libitum. Each rat was acclimated to the housing conditions for a week prior to the start of the experiment. All the experimental procedures were preapproved by the Institutional Animal Care and Use Committee at the University of Florida, and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Drugs Pemoline (Spectrum Chemicals, New Brunswick, New Jersey) was suspended in peanut oil at 50 mg /m L FG7142 (SigmaAldrich Inc., St. Louis, MO) was suspended in 1.0% (w/v) carboxymethylcellulose (CMC) at concentrations of 0, 1, 3, or 10 mg/m L Experimental Procedures Experiment 1 Behavioral p re screening Three behavioral tests: elevated plus maze ( EPM), open field test (OFT) and circular corridor test (CCT) were conducted on experimental days 1, 3, and 5, respectively, between 0800 and 1300.

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15 Elevated plus maze (EPM). The EPM apparatus was constructed with black acrylic, with four 45cm 12cm arms ext ended from one 12cm 12cm central platform Two opposing closed arms were surrounded by 45cm high walls, and the two open arms had 0.6cm high ledges. The height of the maze was 90cm above the floor. Illumination (provided by a light bulb above the central platform of the maze) was 4660 lux in the open arms, 1930 lux in the closed arms and 4345 in the central platform. A video camera suspended above the maze was used to record the rats activities during tests. At the beginning of the test, one individual rat was placed on the central platform facing one of the closed arms. Then it was allowed to explore the maze during the 5 minute test session All activit ies during the period were digitally recorded and assessed using Noldus Observer software ( Noldus I nformation Technology ) by the experimenter Latency to enter the open arm s was defined as the interval between the beginning of the session and the first time when the rat placed all four paws into any one of the two open arms. T he time spent in the closed arms was defined as the total duration of all visit s to the closed arms during the test session, which beg a n when the rat s four paws were into one of the closed arms, and ended when the rat s four paws were into one of the open arms. The maze was cleaned with 4% bleach and dried between each individual test session. Open field test (OFT). The OFT apparatus was a 90cm 90cm black acrylic arena (open area) connect ed to a 20cm 30cm start box (closed area) The open area and start box were separated by a 20cm 20cm acrylic door. The door was connected to a rope that allowed it to be opened from outside the test room. The walls of the

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16 apparatus were 60cm high, and there was an open top. The illumination was 1215 lux in the closed area and 2829 lux in the open area. A digital camera mounted above the apparatus was used to record the rats behaviors during the test. Each rat was individually placed into the start box and the door separating the open area and the start box was kept closed for 1 min. The door was then opened remotely and a 5 minute test session started. B ehaviors during the test session w ere assessed using Noldus O bserver software by the experimenter Latency to enter the open area wa s measured as the interval from the moment when the door was opened t o the first time when the rat placed all four paws i n to the open are a. Time spent in the closed area was measured as the total time of all visits to the closed area during the test session, which started/ended when the rat placed all four paws into the closed/open area. The apparatus was cleaned with 4% bleach and dried between each individual test session Circular corridor test (CCT). T he CCT apparatus was a plastic bucket with another smaller bucket set within it. The center of the smaller bucket was aligned with that of the larger one, forming a 7 cm wide circular corridor between the walls of the two buckets. The outer diameter of the circular corridor was 44cm. The illumination during tests was 8 12 lux within the circular corridor provided by a lamp placed on the floor outside the larger bucket. At the beginning of the test, each rat was individually placed into the circular corridor and then the video camera above the apparatus was used to record the rat s activities during the 1hour test ses sion. B ehavioral stress responsiveness to the mild stress of this novel environment was scored as the counts of line crossing s within the circular corridor. A transparent sheet of acetate with two lines

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17 perpendicular to each other was placed over the vi deo image on the computer monitor to divide the image of the circular corridor into 4 quadrants. A line crossing was counted each time the rat s snout crossed a line, any individual line was not count ed again until the rat crossed another different li ne. The circular corridor was cleaned with 4% bleach, dried and then filled with fresh standard bedding between each individual test session The rats were transferred to single housing in identical polycarbonate cages upon completion of the CCT. Pemolin e treatment Pemoline treatment was applied between Day 13 to Day 22. Independent groups of rats were assigned to drug treatments in a balanced manner based on their counts of linecrossing s in the CCT The r ats were injected once daily with 0, 75, or 100 mg/kg, s.c, at 09 001000 during the treatment. The pemoline injections were administered either at the nape of the neck, or the dorsal flank near the right hindlimb. Each group ha d 10 rats except the control group that had 6 rats Assays of SIB and ancilla ry behavior s At 0 900 and 17 00 of each day during the pemoline treatment, each rat was inspected to examine physical evidence of injury. Each rat was held before a digital camera to record any injur y sites (denuded skin, erythema, edema and open lesions). T issue injury scores were assigned to each rat according to a 04 rating scale (See table 1) Injury sites were digitally recorded and still images were captured. The size of each injury site was measured using MCID image analysis software ( Imaging Researc h Inc., St. Catharines, ON, Canada) Average tissue injury scores and average injury size were calculated across days for each rat.

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18 Digital recordings of the rats behaviors were recorded using digital cameras for 4 time samples each day between 06000615, 12001215, 18001815 and 24000015 for 5 minutes each time, recording the rats behaviors from their home cages throughout the pemoline treatment (total 200 minutes for each rat). Self injurious oral contact and other ancillary behaviors (stereotypy, grooming, and rearing) were assessed by the experimenter using "Noldus Observer software. Self injurious oral contact was defined as all oral contact that stayed fixed on any particular body site for 2 seconds or longer. For each of the 5minute time samples, the total duration summed from all bouts of self injurious oral contact was quantified. Average duration of oral contact was calculated across all 40 time samples for each rat. Grooming was defined as whenever a rat made oral contact along its torso or li mbs, staying fixed at any particular site for less than 2 seconds. Stereotypy was defined as whenever a rat continuously engaged in any of the following behaviors for over 3 seconds: cage licking, head bobbing and/or digging/burrowing/sniffing through the bedding. Since the expression of the specific forms of stereotypy was different for each individual rat, the stereotypy scores are reported as aggregate scores that compile the total duration of all these stereotyped behaviors. Rearing was defined as whenever a rat raised both of its forepaws off the bedding for at least 3 seconds. The daily average duration of each of the ancillary behaviors was calculated across the four time samples from the same day for each rat. For each of the ancillary behaviors, average duration was calculated across all 40 time samples for each rat. Termination The rats were terminated 24 hrs after the final pemoline injection on the morning of the 23th experimental day, except those that were euthanized early due to severe

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19 tissue damage (i.e. reached score 4 according to the 04 rating scale: Table. 1 (Kies and Devine, 2004) ). Statistical analyses One way analyses of variance (ANOVAs) were used to compare each measure of the behavioral tests (EPM, OFT and CCT) between doses to ass ess balanced group assignment. Differences in each of the measures of self injury and ancillary behaviors (grooming, stereotypy and rearing) between pemoline dose groups were analyzed using repeated measures analyses of variance (RM ANOVAs). Significant ef fects (p<0.05) were further analyzed with the Bonferroni post tests. The relationships between two measures of self injury (daily average duration of oral contact and injury size and tissue injury score) and the measures of the anxiety related behaviors i n the EPM and OFT (latency to enter the open arms, time spent in the closed arms, latency to enter the open area, time spent in the closed area) were each evaluated with Pearsons correlation analyses. Also, the relationships between the tissue injury scor e and the measures of the anxiety related behaviors in the EPM and OFT were each evaluated with Spearmans correlation analyses. Results were treated as significant if the P value (1tailed) was less than 0.05. Similarly, the relationships between measures of stress responsiveness in the CCT (novelty induced locomotor counts) and any measure of self injury were also evaluated by Pearsons (or Spearmans for tissue injury score) correlation analyses. Again, the relationships between daily average duration of the ancillary behaviors (grooming, stereotypy and rearing) and the behavioral measures in the EPM, OFT and CCT (latency to enter the open arms, time spent in the closed arms, latency to enter the open area, time spent in the closed area, and novelty induc ed

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20 locomotor counts) were each evaluated with Pearsons correlation analyses. Results were treated as significant if the P value (1tailed) was less than 0.05. Experiment 2 Open field t est (OFT) O n d ay 1 of Experiment 2 (i.e. after 7 days acclimation) t he OFT was conducted according to the procedure described for Experiment 1. Drug treatments The drug treatments were administered during experimental days 37. Upon initiation of the drug treatments, the rats were singly housed in identical polycarbonate cages in order to ascertain that all injuries were self inflicted. The rats were assigned to different doses of the FG7142 treatment in a balanced manner based on their performance in the OFT (the time spent in the closed area). These independent groups o f rats were injected with FG7142 (0, 1, 3, or 10mg/kg, i.p., n=9 per group) twice daily at 08000845 and 17001745 throughout the treatment. Immediately following the FG7142 injection each morning, pemoline (75mg/kg, s.c) was administered to all groups. The pemoline injections were administered either at the nape of the neck, or the dorsal flank near the right hindlimb. Assays of SIB and other ancillary behaviors Rats were inspected in the afternoon the day before the start of the drug treatments. Throughout the treatments, daily inspections were conducted at 1700 1745 for each rat before FG7142 was administered, according to the procedures described for Experiment 1 The t issue injury scores were assigned using the previously described tiss ue injury rating scale (Table. 1 1 ). Time sample recording s of the rats behaviors were collected by digital cameras 4 times a day for 5 minutes each time between 0115-

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21 0200, 08450930, 13151400, and 17451830, recording the rats behaviors from their home cages through ou t the treatments S elf injurious oral contact and ancillary behaviors (stereotypy grooming, and rearing) were assessed using "Noldus O bserver software by the experimenter according to the procedures described for Experiment 1; except that the self injur ious oral contact was counted only when the oral contact stayed fixed at any particular body site for 5 seconds or longer, and grooming was defined as whenever a rat made oral contact along its torso or limbs while staying fixed at any particular site less than 2 seconds. Termination The r ats were terminated on the morning of day 8 except those that were euthanized early due to severe tissue damage (i.e. reached score 4 according to the self injury rating scale: Table. 11 ). Statistical analyse s One way analyses of variance ( ANOVA s) were used to compare OFT measures ( l atency to enter the open area and time spent in the closed area ) between doses to assess balanced group assignment Differences in each of the measures of self injury and ancillary behaviors (grooming, stereotypy and rearing) between FG7142 dose groups were analyzed using repeated measures analyses of variance (RM ANOVAs ). Significant effects (p<0.05) were further analyzed with the Bonferroni post tests.

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22 Table 1 1. Rating scale of tissu e injury Score Severity Description 0 No SIB No tissue damage 1 Very Mild SIB Slight edema, pink moist skin, involves small area 2 Mild SIB Moderate edema, slight erythema, slightly denuded skin, involves medium area, and/or involves multiple sites 3 Moderate SIB Substantial edema and erythema, large area, substantially denuded skin, and/or involves multiple sites 4 Severe SIB Clear/open lesions, and/or amputation of digit, requires immediate euthanasia

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23 CHAPTER 3 R ESULTS Experiment 1 Evaluation of B alanced G roup A ssignments Since the rats were assigned to the groups in a counterbalanced manner based upon the locomotor scores in the CCT, oneway ANOVAs were used to evaluate how evenly the groups were distributed in scores on CCT and the other two pre screening tests F or the counts of line crossings in the CCT, the oneway ANOVA revealed no significant between groups differences (F2, 23= 0.2379, P>0. 1; Fig 3 1 A ) among different pemoline doses. For the measures in the EPM, the o ne way ANOVAs revealed no significant betweengroups differences for the time spent in the closed arms (F2 23=1.856, P>0.1; Fig 3 1 B ) or the latency to enter the open arms (F 2 23= 1.081, P> 0. 1; Fig 3 1 C ). For the measures in the OFT, the oneway ANOVAs rev ealed no significant betweengroups differences for the time in the closed ar ea (F2, 23= 2.644, P>0. 05: Fig. 1 D ) or the latency to enter the open ar ea (F 2, 23= 1.209 P>0.1 ; Fig. 3 1 E ). Dose E ffects of the P emoline T reatment on S elf I njury The measures of pemolineinduced s elf injury (the duration of oral contact, the injury size and the tissue injury score) were higher in the two pemolinetreated groups than in the vehicletreated group and were higher at the high dose (pemoline 100mg/kg) compared with the low dose ( pemoli ne 75 mg/kg). For the duration of oral contact (Fig 3 2A), the 2 way RM ANOVAs revealed a significant main effect of dose (F2, 198=1 0 31 p<0.001), a significant main eff ect of time (F9, 198= 4 55 p <0.0001) and a significant dose x time interaction (F1 8, 198= 2.04 p< 0. 01) For the injury size (Fig 3 2B), the 2way RM ANOVAs revealed a significant main effect of dose (F2, 230= 1 5 41,

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24 p<0.0001), a significant main effect of time (F10, 230=14.97, p<0.0001), and a significant dose x time interaction (F20, 230=8.80, p<0.0001). For the t issue injury score (Fig 3 2C) 2 way RM ANOVAs revealed a significant main effect of dose (F 2, 230=24.58, p<0.0001), a significant main effect of time (F 10, 230=26.92, p<0.0001), and a significant dose x time interaction (F 20, 230=13.80, p<0.0001). The percentage of rats with tissue injury across each day of the treatment is shown in Fig 3 2D. B etween G roup Differences in Ancillary B ehaviors For the duration of gro oming (Fig 3 3 A), the 2way RM ANOVAs revealed a significant main effect of dose (F2, 198= 7.53 p<0.01) whereas the effect of time (F9, 198= 0.87, p>0.1), and the dose x time interaction (F1 8, 198= 0.77 p> 0. 1 ) were not significant. For the duration of stere otypy (Fig 3 3 B), the 2 way RM ANOVAs revealed a significan t main effect of dose (F2, 198=1 2 72 p<0.001), a significant main eff ect of time (F9, 198= 12.71, p <0.0001) and a significant dose x time interaction (F1 8, 198= 3.60 p< 0. 0001) For the duration of rearing (Fig 3 3 C), the 2 way RM ANOVAs revealed a signif icant main effect of dose (F2, 198= 3.84 p<0.05), a significant main eff ect of time (F9, 198= 8.84, p <0.0001) and a significant dose x time interaction (F 1 8, 198= 2.27, p< 0. 005 ). Correlations between the A verage M easures of S elf I njury and the M easures of I nnate A n xiety R elated B ehaviors Correlations between the average measures of self injury and the OFT measures At the low dose (pemoline 75 mg/kg) group, each pair of t he Pearson correlations between self injury measures (average oral contact duration an d average injury size) and the OFT measures (time spent in the closed area and latency to enter the open area) was significant. The S pearman correlation between the average tissue injury score and the OFT measures was also significant for the low dose group (see Fig. 3 4

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25 and Table 3 2) At the high dose (pemoline 100mg/kg) group, the correlation was significant between the average injury size and the time spent in the closed area (see Fig 3 5 and Table 3 2 ) Correlations between the average measures of self injury and the EPM measures T here were no significant correlations between measures of anxiety related behavior on the EPM and measures of self injury for the low dose (see Fig 3 6 and Table 3 2). For the high dose (pemoline 100mg/kg) groups, the Pearson correlations between the average duration of oral contact and the time spent in the closed arms were significant. Also at the high dose, the Pearson correlation between the average injury size and time spent in the closed arms was significant (see Fig 3 7 and Table 3 2 ) Correlations between the average measures of self injury and the locomotion response to mild stress in the CCT The Pearson correlation between the average injury size and the counts of line crossings in the CCT was significant at the low dose (pemoline 75mg/kg ; see Fig 3 8 Table 3 2 ) And there was no significant correlation detected for the high dose (see Fig 3 9 Table 3 2). Correlations between the A verage D uration of A ncillary B ehaviors and the M easures of I nnate A nxiety R elated B eh aviors The Pearson correlations between the average duration of ancillary behaviors (grooming, stereotypy and rearing) and the measures in the EPM, the OFT and the CCT (time spent in the closed arms, latency to enter the open arms, time spent in the closed area, latency to enter the open area, and the counts of line crossing) are illustrated in Table 3. The Pearson correlation between the average duration of grooming and the time spent in the closed arms in the EPM was significant for the vehicletreated gr oup

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26 (see Table 3 3). Also, the correlation between the average duration of stereotypy and the time spent in the closed arms in the EPM was significant at the pemoli ne dose of 100 mg/kg (see Table 3 3). Experiment 2 Evaluation of B alanced G roup A ssignments Since the group assignments were counterbalanced on the basis of time spent in the closed area of the OFT in this experiment, t he 1 way ANOVAs confirmed that there were no significan t betweengroups differences in the time spent in the closed ar ea (F3 32= 0.0071, P>0. 1 ) Likewise, there were no significant betweengroups differences in the latency to enter the open ar ea (F 3 32 = 0.3240, P>0.1). (See Fig 3 10.) FG7142I nduced Elevation in Expression of S elf I njury Nine rats were euthanized before complet ion of the treatments due to severe tissue damage (i.e. reached score 4 according to the 0 4 rating scale ) (See T able 3 4). Thus, the missing data of the self injury measures for the 9 rats were replaced by the corresponding data of the last day before the euthanization from the euthanization day to the end of the experiment. The FG7142 treated groups had higher scores on the duration of oral contact compared with the vehicletreated group (Fig 3 11A ) The 2way RM ANOVAs revealed a significant main effec t of dose (F3, 128= 3.26 p< 0.0 5 ) and a significant main eff ect of time (F4 128=2 0 42 p <0.0001) on the duration of oral contact. The dose x time interaction was not significant (F12, 128=1. 17 p> 0. 05). For the t issue injury score (Fig 3 11B ) t wo way RM ANOVAs revealed no significant eff ect of dose (F3 160=2. 06 p> 0. 1) on the tissue injury score, but there were significant effect s of time (F5, 160= 97.44, p< 0.0 001 ) and of the dose x time interaction (F15, 160=1. 96 p< 0. 05) Also, the

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27 percentage of rats w ith tissue injury across each day of the treatments was demonstrated in Fig 3 11C Between G roup Differences in Ancillary Behaviors The 2 way RM ANOVAs revealed a significant eff ect of time (F4 128= 4.01, p<0.01) on the duration of grooming (Fig. 3 12A ), but there were no significant effect s of dose (F3, 128= 0.84 p>0.1 ) or the dose x time interaction (F12, 128= 0.32 p>0.1) The 2 way RM ANOVAs revealed a significant eff ect of dose (F3 128= 3.27, p<0.05) and time (F4 128= 19.10, p<0.0001) and on the durat ion of stereotypy (Fig 3 12B ), but there were no significant effect s of the dose x time interaction (F12, 128= 0.93, p>0.1 ) T he 2 way RM ANOVAs revealed a significant eff ect of time (F4 128= 30.10, p<0.0001 ) on the duration of rearing (Fig 3 12C ), but th ere was no significant effect of dose (F 3, 128= 0.69, p>0.1) or the dose x time interaction (F12, 128= 1.44, p>0.1)

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28 Table 3 1. The correlations between the average measures of self injury and the measures of innate anxiety related behaviors. The Pearson s r was calculated for each pair of the correlations between the measures in OFT, EPM or CCT and the average duration of oral contact or the average injury size. The Spearson s rho was calculated for each pair of the correlations between the measures in OF T, EPM or CCT and the average tissue injury score. All significant correlations are depicted in bold font. The significance of each correlation is depicted as *p<0.05 and **p<0.01 (1tailed). One rat in the low dose group (pemoline 75mg/kg) was excluded fr om analyses with the duration of oral contact due to data loss, which left 9 rats in the group. OFT EPM CCT time closed area latency open area time closed arm latency open arm line crossing p value r or rho p value r or rho p value r or rho p value r or rho p value r or rho 75 mg/kg (n=9 or 10) oral contact 0.0066 ** 0.7796 0.0028 ** 0.8309 0.4624 0.03696 0.43 0.06897 0.104 0.4643 injury size 0.0242 0.6355 0.0163 ** 0.6739 0.4749 0.02299 0.305 0.1845 0.0235 0.6382 tissue injury score 0 .0195 0.6667 0.0441 0.5749 0.4191 0.07927 0.5 0.006098 0.2801 0.2141 100 mg/kg (n=10) oral contact 0.1913 0.3105 0.4697 0.02768 0.0429 0.5695 0.2865 0.2034 0.0839 0.4725 injury size 0.0245 0.634 0.3476 0.1421 0.006 ** 0.7522 0.1678 0. 3406 0.4822 0.01626 tissue injury score 0.2801 0.2067 0.4191 0.07295 0.0893 0.462 0.2135 0.2857 0.2801 0.2128

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29 Table 3 2 The correlations between the average duration of the ancillary behaviors and the measures of innate anxiety related behaviors The Pearson s r was calculated for each pair of correlations between the measures in OFT, EPM or CCT and the average duration the ancillary behaviors. The significance of each correlation was depicted as *p<0.05 (1 tailed). One rat in the low dose group (pemoline 75mg/kg) was excluded from analyses with the duration of ancillary behaviors due to data loss caused by a computer error, which left 9 rats in the group. OFT EPM CCT time closed area latency open area time closed arms latency open arms line cr ossing p value r p value r p value r p value r p value r 0 mg/kg (n=6) grooming 0.4674 0.04344 0.0718 0.6722 0.0172 0.8447 0.4255 0.09961 0.2213 0.3915 stereotypy 0.0945 0.6202 0.3025 0.2698 0.409 0.122 0.4143 0.1148 0.2051 0.4174 rearing 0.2389 0.3643 0.292 0.2851 0.0764 0.6612 0.2426 0.3586 0.0624 0.6957 75 mg/kg (n=9) grooming 0.2382 0.2735 0.3229 0.1786 0.4494 0.04976 0.2222 0.2928 0.4886 0.01115 stereotypy 0.3474 0.1527 0.477 0.02254 0.4871 0.01263 0.2178 0.2983 0.1104 0. 4529 rearing 0.2054 0.3139 0.2437 0.267 0.1951 0.3271 0.2699 0.2367 0.1028 0.4664 100 mg/kg (n=10) grooming 0.4345 0.06012 0.4136 0.07949 0.2221 0.2737 0.2927 0.197 0.3664 0.124 stereotypy 0.2232 0.2724 0.4602 0.0364 0.0127 0.6958 0.1301 0.3937 0.1046 0.4348 rearing 0.4651 0.03196 0.382 0.1092 0.302 0.1875 0.1129 0.4208 0.1734 0.3333

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30 Table 33. Euthanized rats in experiment 2 total # of rats euthanized euthanization time ( # of rats e u thanized at the time) 0 mg/kg FG7142 1 day 3 (1) 1 mg/ kg FG7142 3 day 3 (1), day 4 (1), day 5 (1) 3 mg/kg FG7142 4 day 3 (2), day 4 (1), day 5 (1) 10 mg/kg FG7142 1 day 4 (1)

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31 Fig ure 3 1. Evaluation of b alanced g roup a ssignments for Experiment 1. The balance of the group assignment w as confirmed as the means of A) the counts of line crossings in the CCT This balancing of assignment based on the CCT scores also produced groups that were balanced on the B) time spent in the closed arms in the EPM, C) latency to enter the open arms in t he EPM, D) time spent in the closed area in the OFT, E) latency to enter the open area in the OFT (n=10 for the pemolinetreated groups, n=6 for the vehicle group). Values expressed are group means SEM. A E D C B

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32 Fig ure 3 2 The dose effects of the pemoline treatme nt A) D uration of oral contact, B) i njury size and C) t issue injury score D) percentage of rats with tissue injury Significant differences between the high dose (pemoline 100mg/kg), the low dose (pemoline 75 mg/kg) and the vehicle groups are pr esented as follow s: *P<0.05 **P<0.01, ***P<0.001 for comparisons between the pemoline dose at 100 mg/kg and the vehicle ; # P<0.05, ## P<0.01, ### P<0.001 for comparisons between the pemoline dose at 100 mg/kg and 75mg/kg As four rats reached the tissue i njury score 4 in the high dose group were euthanized at the morning of day 4 the missing data points of oral contact injury size and tissue injury scores from day 4 to the end of the experiment w ere replaced by the corresponding data of day 3 for those r ats n=10, for the pemolinetreated groups; n=6, for the vehicle group. Values expressed are group means SEM. A B C D

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33 Fig ure 3 3. Betweengroup differences in duration of ancillary behaviors in experiment 1. A) Duration of grooming, B) d uration of steretypy; C) d uration of rearing. Significant differences among the high dose (pemoline 100mg/kg), the low dose (pemoline 75 mg/kg) and the vehicle groups are presented as follow s: +P<0.05 ++P<0.01, +++P<0.001 for comparisons between the pemoline dose a t 75 mg/kg and vehicle; *P<0.05, ***P<0.001 for comparisons between the pemoline dose at 100 mg/kg and vehicle; # P<0.05 for comparisons between the pemoline dose at 100 mg/kg and 75mg/kg As four rats reached the tissue injury score 4 in the high dose group were euthanized at the morning of day 4 the missing data points of the duration of the ancillary behaviors from day 4 to the end of the experiment w ere replaced by the corresponding data of day 3 for those rats n=10 for the pemolinetreated groups; n= 6 for the vehicle group. A B C

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34 Fig ure 3 4 The correlations between self injury and innate anxiety related behaviors in the OFT at the pemoline dose of 75 mg/kg A) T he significant positive correlation (p< 0.0 1, r = 0.7796) between the average duration of oral c ontact and the time spent in the closed area. B ) The significant positive correlation (p< 0.0 1, r = 0.8309) between the average duration of oral contact and the latency to enter the open area. C ) The significant positive correlation (p< 0.0 5 r = 0.6355) between the average injury size and the time spent in the closed area. D ) The significant positive correlation (p< 0.0 5 r = 0.6739) between the average injury size and the latency to enter the open area. E ) The significant positive correlation (p< 0.0 5, r = 0.6667) b etween the average tissue injury score and the time spent in the closed area F) The significant positive correlation (p< 0.0 5 r = 0.5749) between the average tissue injury score and the latency to enter the open area. One rat in the low dose group (pemoline 75mg/kg) was excluded from analyses with the duration of oral contact due to data loss, which left 9 rats in the group.

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35 A B C D E F

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36 Fig ure 3 5. The correlations between self injury and innate anxiety related behaviors in the OFT at the pemoline dose of 100 mg/kg. A) T he re was no significant correlation (p> 0.0 5 r = 0.3105) between the average duration of oral contact and the time spent in the closed area. B ) The re was no significant correlation (p > 0.0 5 r = 0.02768) between the average duration of oral contac t and the latency to enter the open area. C ) The re was a significant neg tive correlation (p < 0.0 5 r = 0.634) between the average injury size and the time spent in the closed area. D ) The re was no significant correlation (p> 0.0 5 r = 0.1421) between the average injury size and the latency to enter the open area. E ) The re was no significant correlation (p> 0.0 5, r = 0.2067) between the average tissue injury score and the time spent in the closed area. F) The was no significant correlation (p> 0.0 5 r = 0.07295) bet ween the average tissue injury score and the latency to enter the open area.

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37 A B C D E F

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38 Fig ure 3 6 The correlations between self injury and innate anxiety related behaviors in the EPM at the pemoline dose of 75 mg/kg A) T he re is no significant correlation (p > 0.0 5 r = 0.03696) between the average duration of oral contact and the time spent in the closed ar ms B ) The re is no significant correlation (p > 0.0 5 r = 0.06897) between the average duration of oral contact and the latency to enter the open ar ms C ) The re is no significant correlation (p > 0.0 5 r = 0.02299) between the average injury size and the time spent in the closed ar ms D ) The was no significant correlation (p > 0.0 5 r = 0.1845) between the average injury size and the latency to enter the open ar ms E ) The re is no significant correlation (p> 0.0 5, r = 0.07927) between the average tissue injury score and the time spent in the closed ar ms. F) The re was no significant correlation (p> 0.0 5 r = 0.006098) between the average tissue injury score and the latency to enter the open ar ms One rat in the low dose group (pemoline 75mg/kg) was excluded from analyses with the duration of oral contact due to data loss, which left 9 rats in the group.

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39 D B F E C A

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40 Fig ure 3 7 The correlations between self injury and inn ate anxiety related behaviors in the EPM at the pemoline dose of 100 mg/kg. A) T he re is a significant negative correlation (p < 0.0 5 r = 0.5695) between the average duration of oral contact and the time spent in the closed ar ms B ) The re is no significant co rrelation (p> 0.0 5 r = 0.2034) between the average duration of oral contact and the latency to enter the open ar ms C ) The re is a significant negative correlation (p< 0.0 1 r = 0.7522) between the average injury size and the time spent in the closed ar ms D ) The was no significant correlation (p> 0.0 5 r = 0.3406) between the average injury size and the latency to enter the open ar ms E ) The re is no significant correlation (p > 0.0 5, r = 0.462) between the average tissue injury score and the time spent in the clos ed ar ms. F) The re was no significant correlation (p> 0.0 5 r = 0.2857 ) between the average tissue injury score and the latency to enter the open ar ms

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41 C E A B D F

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42 Fig ure 3 8 The c orrelations between self injury and locomotor response to mild stress in CCT at t he pemoline dose of 75 mg/kg. A) The re was no significant correlation (p> 0.0 5 r = 0.4643) between the average oral contact and the counts of line crossing. B) The re was a significant neg ative correlation (p< 0.0 5 r = 0.6382 ) between the average injury size and the counts of line crossing C) There was no significant correlation (p> 0.0 5 r = 0.2141) between the average tissue injury score and the counts of line crossing

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43 B A C

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44 Fig ure 3 9 The c orrelations between self injury and locomotor response to mild stress in CCT at the pemoline dose of 100 mg/kg. A) The re was no significant correlation (p> 0.0 5 r = 0.4725) between the average oral contact and the counts of line crossing. B) The re was no significant correlation (p> 0.05 r = 0.01626) between the average inju ry size and the counts of line crossing. C) The re was no significant correlation (p> 0.0 5 r = 0.2128) between the average tissue injury score and the counts of line crossing

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45 B C A

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46 Fig ure 3 10. Evaluation of b alanced g roup a ssignments for Experiment 2. The balance of the group assignment s was confirmed as the means of A) time spent in the closed area in the OFT and B) latency to enter the closed area in the OFT were not significantly different across all groups with different FG7142 doses (n=9, for each dose and the vehicle). A B

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47 Fig ure 3 11. FG7142i nduced e levation in e xpression of self i njury A) Duration of oral contact; B) tissue injury score, C) percentage of rats with tissue injury. Significant differences among the different FG7142 doses and the vehicle are presented as follow: +p<0.05 and ++p<0.01 for comparisons between FG7142 3mg/kg and vehicle; *p<0.05 for comparisons between FG7142 1 mg/kg and vehicle; #p<0.05 for comparisons between FG7142 10mg/kg and 1 mg/kg. The data on day 5 of two rats in the vehicle groups were replaced by the corresponding data of day 4, because one rat was injected at the dose of FG7142 1mg/kg and the other rat was injected at the dose of FG7142 3mg/kg by mistake both at the morning of day 5. A B C

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48 Fig ure 3 12. Between group differences in duration of ancillary behaviors in experiment 2. A) Duration of grooming, B) duration of stereo typy; C) duration of rearing. The significant difference between the group of FG7142 10 mg/kg and the group of FG7142 1mg/kg is presented as #P<0.05. The data on day 5 of two rats in the vehicle groups were replaced by the corresponding data of day 4, because one rat was injected at the dose of FG7142 1mg/kg and the other rat was injected at the dose of FG7142 3mg/kg by mistake on the morning of day 5. A B C

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49 CHAPTER 4 DISCUSSION Dose E ffects of P emoline on S elf I njury T he expression of self injury among different pemoline doses was doseorderly for all four dependent measures (percentage of rats that self injured, self injurious oral contact injury size and tissue injury scores), which was in concordance with a previous finding ( Kies and Devine, 2004) It is not clear why the magnitude of the expression of self injury induced by the 100 mg/kg/day dose was larger in the current study than it was in the previous study although the strain differences and the improved method for getting pemoline into suspension might contribute to this discrepancy The 100 mg/kg/day dose of pemoline caused moderate to severe self injury in all the rats (except 1 rat only exhibit ed very mild self injury) of the group with near ly half of the rats euthanized before completion of the treatment, whereas the 75 mg/kg/day dose produced mild to moderate self injury in 6/10 rats of the group. It appears that the low dose (75 mg/kg pemoline) is a more appropriate dose for study ing individual differences in SIB vulnerability as the high dose might be too powerful to allow the individual differences in SIB vulnerability to be express ed. Thus, the low dose (75 mg/kg pemoline) was used in the experiment 2 studying the effects of FG7142. Relations between I nnate A nxiety R elated B ehaviors and S elf I njury Since p ositive correlations were consistently detected between the measures of anxiety related behavior in the OFT, and the self injury measures in the rats treated with the low dose of pemoline, it appears that higher level s of innate anxiety related behaviors were correlated with increased vulnerability for pemolineinduced SIB Although one small negative correlation between average injury size and time spent in

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50 the closed area was detected at the high dose, there were no consistent outcomes found between measures of self injury and indices of anxiety related behaviors in OFT at this dose. One possible explanation for the inc onsistency in results of the high dose group could be that, the reliability in terms of revealing individual differences in SIB etiology was impaired by the sever ity of the self injury produced by the high dose of pemoline. In order to obtain a stable measure of the innate anxiety related behaviors in rats, both the EPM and OFT were conducted in E xperiment 1. However, there were discrepancies between the two tests of anxiety related behavior (EPM and OFT) in terms of the pattern of correlations between anx iety related behavior s and vulnerability for pemolineinduced self injury One possible explanation is that there are intrinsic differences in the procedures and the design of the apparatuses. As the open arms of the EPM were 90 cm high above the floor, an d the open area of the OFT was at floor level, exploration of the open arms of the EPM may have constituted a greater challenge than exploration of the open area of the OFT did. In addition, as the EPM test session started right away when each individual rat was placed o nto the maze, the stress of being handled by the experimenter might have a substantial impact on how the rat behaves at the beginning of the test. In contrast, the OFT started after 1minute habituation in the start box (which is also diffe rent from OFT procedures that have been reported in other publications ) T his potentially decreased the handling effects on the results of the OFT. Furthermore, there was no prior handling before the EPM whereas the OFT was conducted after the rats experienced handling during the EPM This again might make the latter test less stressful than the former one. It has been reported that

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51 Wistar rats exhibit elevated expression of anxiety related behaviors compared to Long Evans rats (Shaw et al., 2009). Although there is no direct evidence, the Wistar rats that were used in this study could be particularly sensitive to the stress of the EPM, obscuring the individual differences in the individual rats behaviors Moreover, it has been reported that the EPM and OFT may measure different aspects of emotionality. I n one study the anxiolytic drug NKP608 (a NK1 receptor antagonist) exerted anxiolytic like effects on the OFT but not on the EPM in male spontaneously hypertensive rats (SHR) Conversely it had a partial anxiolytic like effect on the EPM but not OFT in male Lewis (LEW) rats (Vendruscolo et al., 2003). However, it should be noted that the version of the OFT used in the current study was different from that used in these other experiments. Relations between L ocomot or R esponse to M ild S tress and S elf I njury A p revious study using the CCT reported positive correlations between the locomotor counts and self injury measures ( Muehlmann et al., 2011) These correlations were not found in the current study and ther e wa s one negative correlation between the average injury size and the locomotors counts. However, there i s evidence that the positive correlations detected in the previous study were driven by highly responsive rats with high locomotor counts. The locomot or counts in the previous study ranged from 156 to 448 (mean score = 2 3 1. 50), whereas the locomotor counts in the present study ranged from 70 to 290 (mean score = 160.03). This probably contributed to discrepancies between the two studies. In addition, st rain differences (Wistar vs. Long Evans) of rats used in the two studies might also contribute to the discrepancies in results.

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52 FG7142I nduced E levation in E xpression and S everity of S elf I njury Since the FG7142treated rats exhibited greater duration of oral contact, larger tissue injury scores, and a greater percentage of these rats engaged in self injury (current results) and since FG7142 increases the expression of anxiety related behaviors in a variety of behavioral tests ( Atack et al. 2005; Bueno et al. 2005; Dawson et al. 2006; Hackler et al. 2007 ; Sena et al. 2003; Stephens et al. 1987), the outcomes of Experiment 2 further support the hypothesis that anxiety related behaviors and self injury a re positively correlated, as suggested by the results from E xperiment 1. It is likely that the elevated expression of SIB in the FG7142treated rats in this study is due to elevated anxiety level s. Because beside its effects on anxiety related behaviors, the ability of FG7142 ( a partial inverse agonist of the GABAA receptor ) to increase anxiety states is supported by several lines of evidence regarding neurochemical and neurophysiological changes induced by FG7142 administration. Many studies found that FG7142 increased expression of cfos protein and m RNA in brain regions involved in anxiety relatedbehaviors including prefrontal cortex (PFC), anterior cingulate cortex (ACC), amygdala, paraventricular thalamic nucleus of hypothalamus (PVN), bed nucleus of stria terminalis (BNST), nucleus accumbens (NAc), and striatum ( Kurumaji et al., 2003 ; Lyss et al., 1999) In terms of potential neuro physiological effects, FG7142 augmented the elevation of plasma corti costerone induced by the OFT ( Fernandez et al ., 2004). Also, FG7142 (20 mg/kg, i.p.) increased extracel lular corticosterone in the basolateral amygdala complex of male Wistar rats ( Bouchez et al., 2012), which is a brain region highly involve d in anxiety. However, changes in mRNA of corticotropinreleasing factor ( CRF a neuropeptide regulating corticostero ne level and increasing anxiety related behaviors) w ere not detected after

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53 administration of FG7142 (10mg/kg) in male Wistar rats (Funk et al., 2006) and C57 BL/6J mice (Funk et al., 2009) among many brain regions related to anxiety. Future work a ssessing changes in corticosterone, CRF and their receptors in relation to self injury mechanisms might be helpful to provide further explanations on the FG7142induced elevation of self injury. For more than 20 years the foremost neurobiological explanation for S IB was that it is driven by D1 dopamine receptor mediated supersensitivity of striatal medium spiny neurons (MSNs) (Breese et al., 1985) suggesting the involvement of dopamine neurotransmission. However, contr ibutions of dopaminergic and non dopaminergic neurotransmission in other parts of the brain have not been studied as closely. Indeed FG7142induced changes in dopamine neurotransmission were found within many brain regions involved in anxiety. Increased e xtracellular dopamine release in medial prefrontal cortex (mPFC) but not in striatum was observed after administration of FG7142 (15 mg/kg i.p.), as assessed by in vivo microdialysis (Moghaddam et al., 1990). Increased concentration of 3, 4 d ihydroxyphenyl acetic acid ( DOPAC a dopamine metabolite) was also found in the medial prefrontal cortex in rats after FG7142 administration (15 mg/kg i.p.) (Attack et al., 2005). In addition, increased extracellular concentration of homovanillic acid (HVA, another metabolite of dopamine) in NAc was detected start ing from day 3 of a subchronic FG7142 treatment and this measure peaked at day 5 ( Brose et al., 1987) whereas these effects were not found in striatum. FG7142 also increased tyrosine hydroxylase activity in PFC and ventral tegmental area in vivo but not in caudate putamen or substantia nigra, whereas it decreased tyrosine hydroxylase activity in striatum in vitro (Knorr et al., 1989). Thus it is possible that

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54 interactions between FG7142 and dopamine neurotransmis sion within brain regions related to anxiety contributed to the observed adverse effects of FG7142 on self injury in the current study. Intriguingly, FG7142induced increase in dopamine turnover was blocked by the glycine/NMDA antagonist (+) HA966 (Murphy et al., 1996), suggesting the involvement of glutamate neurotransmission. Glutamate neurotransmission is also implicated in pemolineinduced self injury (King, Au, and Poland, 1995; Muehlmann and Devine, 2008), as the NMDA antagonist MK801 decreased self i njury in the pemoline model. This line of evidence suggests a potential overlap between FG7142 induced anxiety and pemoline induced self injury, which might explain the augmentation of pemolineinduced self injury during repeated FG7142 treatment. A s revi ewed by Etkin et al. ( 2011) the prefrontal cortex is likely to exert a modulatory function on anxiety related behaviors by regulating neuronal activities within amygdala. mPFC inputs decreased the possibility of firing in neurons of basolateral complex of amygdala (BLA), possibly by activating inhibitory interneurons within this brain region. This t op down suppression from mPFC to amygdala was disrupted by excessive stimulation on PFC dopamine receptors (Rosenkranz et al., 2001). It is possible that FG7142 induced changes in dopamine neurotransmission would also impair the PFC control over amygdala. T his needs to be tested with further experiments Despite the prominent role of the amygdala in anxiety, the role of this region in self injury is not clear. H owever, a case study reported that deep brain stimulation in basolateral amygdala improved SIB in an autistic boy (Sturm et al., 201 2 ) Nevertheless, this result should be interpreted cautiously because it is a single case,

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55 uncontrolled design, and the res ults were based on subjective scores documented by his parents who didn t received psychiatric psychological or medical training. Beside the associations between FG7142 and dopamine neurotransmission, there is also evidence suggesting involvement of sero tonin neurotransmission in FG7142induced elevation of anxiety. For example, pretreatment with the selective 5 HT2C receptor antagonist SB 242084 blocked FG7142 induced increases in anxiety related behaviors and the increase in bloodoxygen level dependent (BOLD) signal in amygdala, hypothalamus and hippocampus ( Hackler et al., 2007). However, the role of serotonin in self injury remains controversial. F luoxetine, a selective serotonin reuptake inhibitor ( SSRI) attenuated SIB (Fontenot et al., 2005; Fontenot et al., 2009 ) in non human primates Pretreatment with another SSRI, paroxetine significantly increased the severity of SIB (Turner et al., 1999) in the pemoline model in rats Based on current knowledge, it is possible that serotonin neurotransmission is involved in anxiety related behaviors augmented by FG7142 whereas it is not clear if it has a role in FG7142induced elevation in self injury. Regarding all evidence discussed above about changes in neurotransmission within brain regions involved in anxiety, one possibility is that FG7142 induced increases in self injury might be due to certain overlaps between the neurochemical mechanisms underlying anxiety and self injury. However, more effort needs to be expended on elucidating the underlying neurochemical mechanisms of SIB, especially for the role of dopamine serotonin and glutamate, as well as the n euroanatomical structures that are involved, before we can further confirm (or reject) this possibility.

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56 Implications for N eurodevelopmental D isor d ers Although the comorbidity between anxiety and self injury i s not well established, anxiety disorders have been reported among self injurious patients with neuro developmental disorders According to a metaanalysis study, about 40% of young people with autis tic spectrum disorders (ASD) meet diagnostic criteri a for at least one DSM IV anxiety disorder (V an Steensel et al., 2011). Since approximately 50% of people with ASD exhibit SIB ( Richards et al., 2012) there is a potential for significant overlap between these subsets of autistic individuals In the case of fragile X syndrome (FXS), the proportion of SIB patients has been estimated at about 55% ( Richards et al., 2012). A nd there is a significantly higher ratio of cooccurring anxiety found in those individuals with FXS who self injured compared with those who did not self injure ( Symons et al. 2010) As for Lesch Nyhan disease (LND) for which self injury is its hallmark, higher anxious/depressed level was observed in LND patients compared to both healthy controls and patients with Lesch Nyhan variants (LNV) who did not self injure ( Schretlen et al., 2005). These findings converge to suggest a potential overlap between pathological mechanisms underlying anxiety and self injury, yet no conclusion can be reac hed without further information regarding the specific comorbid ity between anxiety and self injury among the above mentioned neurodevelopmental disorders. If there actually are correlations between anxiety and self injury in defined clinical populations causal relations still cannot be easily determined unless we can explain more fully the underlying neurobiological/ ne u ropathological mechanisms. Conclusion This study provided evidence for the role of anxiety in self injury induced by pemoline in a rat model, by demonstrating the positive correlations between innate

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57 anxiety related behaviors and self injury, and the adverse effect of an anxiogenic agent FG7142 on self injury. Future studies assessing anxiety related behavioral /hormonal changes before, duri ng and after the pemoline treatment and investigating neurochemical changes underlying FG7142induced augmentation of self injury will be helpful in order to facilitate a greater understanding of the role of anxiety in self injury.

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62 BIOGRAPHICAL SKETCH Xiaomeng Yuan was born in the Changsha City of Hunan Province, China She received her bachelor degree at Beijing Forestry University with concentration of biotechnology. At May 201 3 she received her master degree of science from B ehavioral and C ognitive N euroscience Program in the Department of Psychology at University of Florida.