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1 ANXIETY RELATED BEHAVIOR IS POSITIVELY CORRELATED WITH SEVERITY OF PEMOLINE INDUCED SELF INJURY By WILLIAM LIN A THE S IS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013
2 2013 William Lin
3 To my mom
4 ACKNOWLEDGMENTS I thank my mother for supporting my graduate career I thank Dr. Darragh Devine for being my mentor. I appreciate the support I received from my brother and sister. I also am grateful that my dad encouraged me to study behavioral neuroscience.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 6 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREV IATIONS ................................ ................................ ............................. 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 10 2 MATE RIALS AND METHODS ................................ ................................ ................ 13 Animals ................................ ................................ ................................ ................... 13 Anxiety Testing ................................ ................................ ................................ ....... 13 Elevated Plus Maze ................................ ................................ .......................... 13 Open Field Test ................................ ................................ ................................ 14 Stress Responsive Test ................................ ................................ .......................... 15 Drug Treatment ................................ ................................ ................................ ....... 16 Analyses of Self Injury ................................ ................................ ............................ 16 Stat istical Analysis ................................ ................................ ................................ .. 17 3 RESULTS ................................ ................................ ................................ ............... 20 4 DISCUSSION ................................ ................................ ................................ ......... 32 LIST OF REFERENCES ................................ ................................ ............................... 36 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 40
6 LIST OF TABLES Table page 2 1 Tissue injury rating scale for rats treated with pemoline ................................ ..... 19 3 1 Percentages of rats that exhib ited oral contact injury across ten days ............... 24 3 2 Spearman correlation between injury ranking score vs anxiety related variables and number of injury sites vs anxiety related behavior variables ........ 24
7 LIST OF FIGURES Figure page 3 1 Ten day pattern of self injury indices.. ................................ ................................ 25 3 2 Percentage of rats that self injure across ten days. ................................ ............ 26 3 3 Elevated plus maze durations.. ................................ ................................ ................................ ........... 27 3 4 Elevated plus maze variables and injury area ....... 28 3 5 Open field measurements and their correlation with area of self injury. ............. 29 3 6 Open field variables and their correlation with duration of oral contact. .............. 30 3 7 Hesitation to enter open arena had no signif icant correlation with any measure of self injury. ................................ ................................ ........................ 31 3 8 Circular corridor scores and their correlation with indices of self inju ry.. ............ 31
8 LIST OF ABBREVIATIONS BLA Basolateral nucleus of amygdala CeA Central nucleus of amygdala CCT Circular corridor CRF Corticotropin releasing factor EPM Elevated plus maze OFT Open field test SIB Self injurious behavior
9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science ANXIETY RELATED BEHAVIOR IS POSITIVELY CORRELATED WITH SEVERITY OF PEMOLINE INDUCED SELF INJURY By William Lin May 2013 Chair: Darragh Devine Major: Psychology Self injurious behavior ( SIB ) is devastating characteristic that is commonplace in many neurodevelopmental disorders. There are very few studies that examined the role of anxiety related behavior in predicting severity of SIB. In thi s study, pemoline model of SIB wa s employed to investigate anxiety relate d behavior as a predictor o f vulnerability for SIB in Long Evans rats. The pemol ine dose used in the present study wa s 125mg/kg. The anxiet y tests used in this project were the elevated plus maze ( EPM) and the open field test ( OFT) The self injury i ndices examined in this study were oral contact duration, tissue injury area, injury scores, a nd number of injury sites. It wa s found that anxiety related behavior measurements from the EPM correlated with these indices of self injur y. However, the open field test yielded no correlat ion with any of the self injury indices. Also, the present study found no correlation between locomotor response to novelty, tested using the circular corridor apparatus, and any indices of self injury. In summary, the finding of this current project ind icate d that anxiety related behavior can be correlated with SIB and is worthy of future investigations.
10 CHAPTER 1 INTRODUCTION Self injurious behavior ( SIB) is a destructive phenotype that is commonly expressed in intellectually handicapped populations with autism ( Baghdadli et al., 2003), Lesch Nyhan syndrome ( Anderson and Ernst, 1994), Prader Willi syndrome ( Hellings and Warnock 1994), and ot her neurodevelopmental disorders. There have been studies that indicate SIB is a learned behavior in which the afflicted individual responds to positive and negative social reinforcement. However, in a significant proportion of the intellectually challen ged self injurers, their SIB is not influenced by social environmental contingencies ( Iwata et al., 1994). In addition, individuals suffering from paternal deletion diagnosis of Prader Willi syndrome demonstrate a greater severity of self injury when comp ared to individuals afflicted with the uniparental disomy diagnosis ( Hartley et al., 2005). These findings suggest that there are biological bases to SIB. There is evidence from clinical finding s and animal models that this biological basis of SIB involves dopaminergic neurotransmission. All animal models of self injury involve dopamine dysfunctions ( Devine and Muehlmann 2009), and this patholog y is also found in children suffering from developm ental disorders such as Lesch Nyhan syndrome ( Saito et al., 1999; Lloyd et al., 1981) and autism ( Ernst et al., 1997; Xiao et al., 2005)which involve SIB. For example, in Lesch Nyhan syndrome ( LNS), there is a decrease in dopamine transporter binding in t he striatum ( Wong et al., 1996). Self injurious behavior has been studied in various animal models, including repeated pemoline administration ( Meuhlmann et al., 2007). Pemoline model has a good predictive validity. Evidence for this validity includes th e fact that rats with pemoline induced SIB respond to medications such as risperidone, valproate, and
11 topiramate which show ed promise in human clinical studies ( Muehlmann et al., 2007). Pemoline is a good model for SIB also because it targets the dopamine rgic system ( Devine and Muehlmann, 2009). Pemoline i s an indirect monoamine agonist; therefore its action s also include blockade of reuptake of serotonin and norepinephrine. Pemoline induced self injury is dose orderly and at doses of pemoline, 100mg/k g, not all rats exhibit SIB ( Kies and Devine, 2004). This allows one to examine individual vulnerability for SIB. We are using the pemoline model to investigate the biological bases for SIB, specifically the roles of anxiety and stress responsiveness in predicting vulnerabilities for SIB. Individual vulnerability for SIB is also observed in human populations. For example, in certain studies, 50% of the sample autistic children exhibit ed SIB ( Baghdadli et al., 2003). Another example is the finding that 80 90% of children with Prader Willi syndrome exhibit ed skin picking SIB ( Clark et al., 1989; Symons et al., 1999). The four dependent measures of self injury SIB indices used in the current project measure different qualitative aspects of self injury. Oral contact duration describes the motor aspect of SIB and injury area alone does not take into consideration the severity of the injury areas involved. For example when valproate was tested in the pemoline model of SIB, there was a dissociation between oral contact duration and tissue injury area ( i.e. no reduction in oral contact duration whereas tissue injury area was reduced) ( Muehlmann et al., 2007), this indicate d that these two variables measured different aspects of self injury. The injury ranki ng scor es although subjective, measure aspects of self injury that are not addressed by oral contact duration and tissue injury area. The number of injury sites measures an aspect of self
12 injury that provides additional information to the tissue injury ar ea. In summary, interpretation of SIB in the pemoline model is most informed if all four indices of self injury are considered. We study anxiety as a predictor of vulnerability for SIB because emotional distress often precedes SIB in human self injurers, and relief of such emotional distress is observed following SIB. Although most neurobiological examinations of SIB focus ed on striatal dopamine function, there appear to be important interactions between dopaminergic neurotransmission and amygdaloid funct ion. The amygdala extend glutamatergic neural projections to the nucleus accumbens ( Robinson and Beart, 1988 ), and there are dopaminergic projection s from ventral tegmental area ( VTA), medial prefrontal cortex ( mPFC), and substantia nigra ( SN) to the amyg dala. There are interactions between dopaminergic neurotransmission with GABA function in the amygdala ( Mora et al., 2010). Other researchers have identified basal ganglia functions that are correlated with anxiety. For example, Corticotropin releasing factor ( CRF ) administration int o the nucleus accumbens le a d to greater anxiety related behavior in the elevated plus maze ( EPM) ( Chen et al., 2012). Finally, deep bra in stimulation in the NAcS ameliorate d anxiety symptoms ( Sturm et al., 2003; Bewernick et al., 2010). Since basal ganglia is implicated in SIB ( Cromwell and King, 2004), these shared neural substrate for anxiety and SIB implicate that anxiety related behavior might be correlated with SIB.
13 CHAPTER 2 MATERIALS AND METHODS Animals Thirty six male Long Evans rats ( 150 175g) were pair housed in a room with a 12h/12h light/dark schedule ( lights on at 7:00am) for two weeks. Standard rat chow and tap water were ava ilable ad libitum. The rats acclimated to housing condition during the first week. During the morning of the Monday of the second week, the rats were tested for expression of anxiety related behavior using the elevated plus maze ( EPM ) During the morning of the 3 rd day of the second week, the rats were tested for expression of anxiet y related behavior using the open field test ( OFT). On the morning of the 5 th day of the second week, the circular corridor test ( CCT) which constitute a novel mild stressor, was administered to elicit and evaluate individual locomotor behavior of the ra ts. The rats were single housed at the start of pemoline tre atment ( to ascertain that the injury was self inflicted) which took place on the first morning of the third week, and throughout the pemoline treatment schedule ( which lasted nine days) ending on the tenth day. Anxiety Testing Elevated Plus Maze The elevated plus maze ( EPM) was constructed out of two open and two closed arms that create d a cross with a 12cmX12cm central platform The horizontal dimensions of both the open arm and the closed arm were 45cm X 12cm. The walls of the closed arm were 45cm tall. The distance between the EPM platform and the floor was 90 cm. The illumination of the open arm was 50 lux and the illumination of the closed arm was 21 lux. This elevated plus maze test was conducted between 9:30am
14 and 11:00am. A camera was located at the ceiling above the center of the EPM apparatus to rec apparatus was cleaned with 4% bleach pri or to the testing of each rat. The camera was t urned on and then the behavior was recorded for a total of five minutes starting immediately after the placement of the rat onto the center platform. At the end of the fiv tail was marked with an identification stripe. The variables of interest measured in this test were latency to enter the open arm, hesitation to enter the open arm, total time spent in the open arm. For all three of these measures the avoidance of the open arms was interpreted as anxiety related behavior. During the scoring, an open arm entry was considered only when the rat placed all four paws into this region. The time in open arm concluded when the rat placed all four paws into the closed arm. If the rat only placed two forepaws onto the open arm and then withdrew the two forepaws, this time duration was recorded as hesitation to enter the open arm. Similarly, a closed arm entry was considered only if the rat placed all fo ur paws into the closed arm. Open Field Test The open field test ( OFT) apparatus consisted of a start box ( 20cm X 30cm) and an open arena ( 90cm X 90cm). The height of each open field wall was 60cm. The start box had a door ( 20cm X 20cm) which led into the open arena. The illumination of the start box was 11 lux and the illumination of the center of the open arena region was 31 lux. This test was done between 9:30 am till 11:00 am. A camera was located at the top of the ceiling above the center of th to the testing of each rat, the apparatus was cleaned with 4% bleach. The open field test began by placing the rat into the start box. After one minute, the door connecting
15 the start box and th e open are na was opened remotely allowing the rat to choose between the start box and the open arena. The start box door remained open throughout the duration of the test. After the door of the start box was opened, the behavior was recorded for five minutes During scoring, a transparent grid consisting of 16 equal sized squares, where the 4 squares along the wall where the start box was located were zone 1, and the 8 squares along the side and back walls were designated zone 2. Finally, the central 4 squ ares were designated zone 3. The variables recorded in this OFT were latency to enter the open arena ( zone 1), latency to enter zone 3, total time in the open arena ( zone 1 3), total time in zone 3, and hesitation to enter the open arena ( zone 1). An ent rance into a region was considered only if the rat placed all four paws into the specified region. If the rat placed two forepaws into the open arena and retrieved the two forepaws, this duration was recorded as hesitation to enter the open arena. The la tency to enter the open arena measurement consisted of the time elapsed before the rat entered into the open arena. Similarly, latency to zone three consisted of time elapsed before the rat placed all four paws into zone three. Stress Responsive Test Circ ular Corridor Test. The circular corridor test ( CCT ) apparatus was composed of a small plastic bin put into a large plastic bin forming a circular corridor ( 7 cm wide, 44 cm outer diameter). Small lamps were placed on the floor, six inches from the outer wall of the circular corridors ( providing less than 10 lux of illumination in the corridor). This test was done between 9:30am and 11:00am. A camera was located on to the testing of each rat, the circular corridor was cleaned with 4% bleach and then the floor of the corridor was covered with beddings. Each rat was tested individually in the
16 circular corridor for 60min. The variable of interest in this study was the number of line crossings. For scoring the video, a transparent sheet was placed over the image of the circular corridor with crossed lines that divided the apparatus into four equal quadrants. Each time a rat crossed a line entering a quadrant, a single locomotor count was scored. Another crossing was not scored until the rat crossed a different line. The circular corridor scores were used to balance rats receiving pemoline and peanut oil ( ( toward CCT)rats self injure more th an low responsive ( toward CCT) rats. Drug Treatment Pemoline was suspended in peanut oil at 50mg/ml. It was stirred constantly starting on the afternoon before the day that the injections took place. Rats wer e weighed before each injection The rats w ere injected once daily with pemoline ( 125 mg/kg, s.c.) or peanut oil ( times 125mg/kg then divided by 50mg/ml, s.c.) between 9:30 and 10:30 a.m. for nine consecutive days. Analyses of Self Inju ry Each rat was inspected for self injury and placed before a camera showing the and tail once right before the first injection and twice daily throughout the nine days of the pemoline treatment regimen, and finall y once on the morning of the tenth day. A subjective injury score was recorded during the inspection based on a scale of zero to four ( see table 2 1 ). A n one centimeter mark sta ndardized tissue injury area evaluation using the MCID image analysis program, using still images of tissue injury captured from the video. The total numbers of tissue
17 injuries were also recorded. In addition, video cameras with night vision were aimed c ontinuously at the rats starting on the first day of pemoline treatment until the tenth day. The camera recorded 15min of data every three hours for a total of eight sets of video information including behaviors such as oral contact duration ( orally fixed site for more than 2 seconds), stereotypy ( cage licking, head bobbing, sniffing that e ndured for 3 seconds or more), rearing ( when the rat lifted both of its forepaws for more than 3 seconds), and grooming ( oral contact that change sites with no more than 2 seconds at a fixed site). Out of each 15 min sample, the first five minutes were scored unless the image was not optimal ( i.e. the rat facing the rear of the cage). Each video was scored using Noldus Observer software. For data analysis, the total tim e of oral contact duration was divided by total time of obs ervation to create a percentage of time of oral contact duration ( self injury). Similar overall percentages for the ten days of pemoline treatment were calculated for grooming, stereotypy, and rea ring. Statistical A nalysis The percentage of rats that self injured across the ten days was analyzed using oral contact durations. The trajectory of the four indices of SIB ( oral contact duration, tissue injury area, injury scores, and number of injury si tes) was obtained for the ten days of pemoline regimen for the 18 rats. For rats that received an injury ranking score of 4, euthanasia was performed and the missing data for the remaining days were filled with the final values of the injury indices for the rat. Repeated measure ANOVA were conducted on all four indices of self injury between pemoline and vehicle group with time ( 10 days) and pemoline ( drug/vehicle) as independent variables. Bonferroni post test comparisons were conducted for each of the ten days between pemoline and
18 oral contact duration, injury area, injury score, and number of injury sites. Outcomes were categorized as significant if the p value is less than 0.05. Several linear regression ( Pearson correlation) were c onducted to determine the relationship between an xiety related behavior measures obtained using elevated plus maze ( EPM ) and open field test ( OF T ) and indices of self injury specifically duration of oral contact and tissue injury area since thes e are con tinuous variables. Elevated plus anxiety related behaviors included total time in the open arm, latency to enter the open arm, and hesitation to enter the open arm. The open field anxiety related behavior included total time in the open arena, lat ency to enter the open arena, total time in zone 3 and latency to enter zone 3, and hesitation to enter the open arena. Locomotor response to circular corridor measurements ( stress responsiveness) was also analyzed using Pearson correlation to determine its relationship with two indices of self injury which are continuous variables: Oral contact duration and tissue injury area. A Pearson correlation analysis was further used to compare anxiety related behaviors obtained using EPM and OF T specifically tot al time in the open arm vs. total time in the open arena, latency to enter the open arm vs. latency to enter the open arena, and hesitation to enter the open arm vs. hesitation to enter the open arena. A Spearman correlation analysis was conducted to find the strength of the relationship between injury scores/ number of tissue injuries ( since these are discrete variables), and anxiety related behavior ( from both EPM and OFT)/ stress responsi veness.
19 Table 2 1 Tissue injury rating scale for rats treated with pemoline Injure score Description Detailed description 0 No injury No presentation of tissue damage 1 Very mild Very minor edema, pink moist skin, involves minute area 2 Mild Medium edema, very minor er ythema/denuding, involves moderate area or multiple sites 3 Moderate Considerable edema/e rythema/denuding, involves great size of area, and can include multiple sites 4 Open lesion Any open rupture in the skin, requires prompt euthanasia
20 CHAPTER 3 RESULTS All 18 rats s elf injured by day three. T his pattern persisted until day 7, whence the percentage slightly decreased and then plateaued until day 10 ( see table 3 1 and Fig 3 2). The pattern across the ten days for injury area, oral contact duration ( % of time the rat spent on self injuring), injury score and number of injury sites are shown in Fig 3 1. RM ANOVA analysis conducted on SIB values ( oral contact duration, injury area, injury score, number of injury si tes) for pemoline and vehicle group across ten days reached significant effect of time, pemoline, and time/pemoline interaction for all four indices of self injury: Oral contact duration ( time: F=8.303, p<0.0001, pemoline: F=33.76, p<0.0001, interaction:F= 17.145, p<0.0001), Injury score ( time: F=12.41, p<0.0001, pemoline: F=39.29, p<0.0001, interaction: F=12.41, p<0.0001), injury area ( time: F=5. 617, p<0.0001, pemoline: F=10.96 p<0.0025, interaction: F=5.617, p<0.0001) and number of injury sites ( time: F=1 3.46, p<0.0001, pemoline: F=35.94, p<0.0001, interaction: F=13.46, p<0.0001). All four indices of self injury exhibit ed an inverted U shape across the ten days of pemoline regimen. The results of this project indicated that anxiety relate d behavior obta ined using elevated plus maze ( EPM ) wer e linearly correlated with indices of self injury but the open field test ( OFT ) did not yield significant linear correlation with any indices of self injury. Therefore, a linear regression analysis was conducted with variables of EPM and variables of OF T M easurements obtained from EPM did not correlate with measurements obtained fr om OF T Specifically, Pearson correlation analysis of the latency to enter the open arm and latency to enter the open arena yielded non significant results ( p= 0.6078, R^2= 0.01684). Also, there was no correlation between
21 measurements total time in the open arm and total time in the open arena ( p=.6112, R^2=.01652). Finally, there was no linear correlation between hesitation to enter the open arm and hesitation to enter the open arena ( p=0.4251, R^2=.04019) In this project, anxiety related behavior mea sured using EPM demonstrated significant linear correlation with indices of self injury. Specifically, rats that spend less time in the open arm exhibit ed greater indices of oral contact duration, tissue injury area, injury ranking scores, and number of t issue injury. In the elevated plus maze total time in the open arm value negatively correlated with oral contact duration ( p= 0.001 and R^2=0 0.4991) ( see fig 3 3b). The latency to enter the open arm variable was positively correlated with oral contact duration ( p= 0.0051, R^2= 0.3970 ( see fig 3 3a). However, the hesitation to enter the open arm variable was not linearly correlated with oral contact duration ( p=0.2164, R^2=0.09382) ( see Fig 3 3 c). Other self injury indice s such as tissue injury area ne gatively correlated with the total time in the open arm measures ( p=0.0077, R^2=0.3668, see Fig 3 4b)but not with latency to enter the open arm ( p=0.0531, R^2=0.2142, see Fig 3 4a) or hesitation to enter the open arm ( p=0.1182, R^2=0.1456 see Fig 3 4c). T he Spearman correlation analysis on injury ranking score and number of injury sites, with anxiety related variables yielded results in table 3 2. The injury ranking score correlated with all three measures of EPM variables while its correlations with open field variables were all non significant. The number of injury sites demonstrated a positive correlation with latency to enter the open arm, and negative correlation with total time in the open arm variable In summary, anxiety related behavior measured using EPM indicated the greater the anxiety related behavior the greater the self injury induced by pemoline.
22 The Pearson correlation analysis of OFT and self injury returned non significant results for all comparisons of OF T variables with oral contact d uration/tissue injury area. Specifically, the two variables latency to enter the open arena and total time in the open arena showed no linear correlation with tissue injury area ( p=0.2776, R^2=.07320; p=0.3921, R^2=.04613) ( see fig 3 5a, 3 5 c). The latenc y to enter the open arena and oral con tact duration were not linearly correlated ( p = 0.1314, R^2 = 0.1365) ( 3 6a). The total time in the open arena and oral contact durati on regression analysis yielded no n significant results ( p = 0.8276, R^2 = 0.003053) ( see fig 3 6c). The zone 3 variables ( latency to enter zone 3 and total time in zone 3) yielded non significant linear regression ( Pearson correlation) results with indices of self injury specifically tissue injury area and oral c ontact duration: latency to enter zone 3 vs. injury area ( p=0.4069, R^2=0.04337) ( see fig 3 5b); Latency to enter zone 3 vs. oral contact duration ( p=0.2315, R^2=0.08816) ( see fig 3 6b); total time in zone 3 vs. injury area ( p=0.9273, R^2=.0005363, fig 3 5d ); Total time in zone 3 vs. oral contact duration ( p=0.3014, R^2=0.06654) ( see fig 3 6d). All measures of hesitation to enter the open arena did not linearly correlate with injury area and oral contact duration ( see fig 3 7): hesitation to enter the open arena vs. injury area ( p=0.4062, R^2=0.04351) ( see fig 3 7b ); Hesitation to enter the open arena vs. oral contact duration ( p=.3177, R^2=.06234) ( see fig 3 7a ). Spearman correlation conducted on the open field variables with injury ranking score and number of injury sites yielded non significant p values for all comparisons ( see table 3 2). The Pearson correlation analysis concerning circular corridor scores and indices of self injury, the oral contact duration and tissue injury area, yielded non signifi cant
23 results ( p=0.5185, R^2=0.03277; p=0.6285, R^2=0.01498) ( see fig 3 8). The Spearman correlation analysis indicated that both injury ranking score and number of tissue injury area did not correlate with circular corridor counts ( see table 3 2). In summ ary, all four indices of self injury did not correlate with circular corridor results.
24 Table 3 1 P ercentages of rats that exhibited oral contact injury across ten days Day 1 2 3 4 5 6 7 8 9 10 Oral contact 61% 94% 100% 100% 100% 100% 100% 94% 94% 94% Table 3 2 Spearman corr elation between injury ranking score vs anxiety related variables and number of injury sites vs anxiety related behavior variables Injury rank score Number of injury sites Injury rank score Number of injury sites Circular corridor P=0.1791 Sr= 0.3314 P=0.1767 Sr= 0.3332 Total time in z3 P=0.7197 Sr= 0.09093 P=0.8774 Sr= 0.03916 Hesitation to open arm P=0.014 Sr=0.5677 P=0.1309 Sr=0.3698 Latency to enter z3 P=0.7344 Sr=0.08598 P=0.3712 Sr=0.2242 Hesitation to enter open arena P=0.2694 Sr=0.2750 P=0.1404 Sr=0.3615 Total time in open arena P=0.2872 Sr=0.2654 P=0.8498 Sr=0.04806 Latency to enter open arm P=0.0042 Sr=0.6401 P=0.0055 Sr=0.6254 Latency to enter open arena P=0.3699 Sr=0.2248 P=0.0843 Sr=0.4180 Total time in open arm P=0.0258 Sr= 0.5234 P=0.0193 Sr= 0.5452
25 A B C D Fig ure 3 1. Ten day pattern of self injury indices A) I njury area B ) Injury score C ) Oral contact duration and D ) N umber of injury sites. *=p value< 0.05, **=p value<0.01 for comparison of pemoline and vehicle group.
26 Fig ure 3 2. P ercentage of rats that self injure across ten days.
27 Fig ure 3 3. Elevated plus maze sis with oral contact durations. A) Total time in the open arm and B) l atency to enter the open arm variable had significant correlation with oral contact duration variable whereas C) h esitation to enter the open arm did not correla te with oral contact d uration A B C
28 Fig ure 3 4. Only B) total time in the open arm variable was linear l y correlated with injury area A) Latency to enter the open arm and C) h esitation to enter the open arm did not have significant correlation with injury area A B C
29 A B C D Fig ure 3 5. Open field measurements and their correlation with area of self injury. A)Latency to enter the open arena and injury area. B) Latency to enter zone 3 and injury area. C) Total time in the open arena and injury area. D) Total time in zone 3 and injury area. None of the open field variables had significant correlation with injury area
30 A B C D Fig ure 3 6 Open field variables and their correlation with duration of oral contact. A) Latency to enter the open arena and oral contact duration. B) Latency to enter zone 3 and oral contact duration. C) Total time in the open arena and oral contact duration. D) Total time in zone 3 and oral contact duration. All four open field variables yielded non significant correlation with oral contact duration measurements
31 A B Fig ure 3 7. Hesitation to en ter open arena had no significant correlation with any measur e of self injury A ) O ral contact duration B ) Injury area A B Fig ure 3 8. Circular corridor scores and their correlation with i ndices of self injury. A ) Injury area, B ) O ral contact duration All correlations are non significant.
32 CHAPTER 4 DISCUSSION In this project, anxiety related variables obtained from elevated plus maze ( EPM) are correlated with all four indices of self injury. Visual inspection of the correlatio n between total time in the open arm and oral contact duration indicate that these two variables have a broad range of individual differences in their values. Same broad range of values can be observed for the correlation between total time in the open ar m and tissue injury area. Elevated plus maze measurements fall on three factors: anxiety, locomotion, and risk assessment. The latency to enter the open arm and total time in the open arm are variables that loa d heavily on the anxiety factor while the h esitation to enter the open arm loads also on risk assessment ( Ramos and Mormede, 1998). However, there were no correlations between anxiety related behavior obtained from open field test ( OFT ) and any indices of SIB. There are several explanation s for t his discrepancy. It was found that repeated OFT enhanced the thigmotaxis behavior of the mi ce ( Leppanen et al., 2006). However, it is questionable whether such behavior in mice will generalize to rats. It is al so possible that the EPM and OFT measure dif ferent types of anxiety related behavior. Evidence for this notion included the following fact: Lewis rats tested with chlordiazepoxide demonstrat ed anxiolytic effect on EPM, but not in OF T Chlordiazepoxide with NKP608, a tachykinin NK 1 receptor antagonist lead to anxiolytic effect in OF T but not in EPM for spontaneously hypertensive rats ( SHR) ( Vendruscolo et al., 2003). In summary, elevated plus maze and open field test might measure different aspects of the anxiety related behavior. One of the main differences between EPM and OF T in the current project was that in the OFT the rats had the opportunity to habituate to the start box for one minute, thus
33 ena. The locomotor response scores in the presen t project ranged from 79 to 283 while suggested that all th e rats in the current project wer e low responders, indicating that the r oward circular corridor test were different ( possibly due to shipping to the correlation with all four indices of self injury. A research area where anxie t y related behavior had a role predicting vulnerability for SIB involved that of primates. One such finding is that diazepam decreased SIB in 50% of monkeys that already exhibited SIB. However, diazepam increased SIB in the remaining 50% of treated monke ys. This discrepancy might be explained by any of the f ollowing four hypotheses: ( 1) T he monkeys that had reduced SIB have a different form of anxiety related disorder than those that developed greater SIB in response to diazepam. ( 2) T he two groups are afflicted with the same anxiety disorder but were at a different stage of the anxiety dysfunction ( 3) These two groups of monkeys differed in their sensitivity to anxiolytic medications through different GABA receptor subunit composition ( 4) Finally, th e group that had increased SIB in response to diazepam might not have anxiety as a contributing factor for their SIB ( Tiefenbacher et al., 2005). the expression of SIB is another study where FG7142, an anxiogeni c drug, increase SIB in 50% of monkeys that already have SIB ( Major et al., 2009). There are human clinical findings that suggested anxiety as a predictor of vulnerability for SIB, specifically deep brain stimulation of basolateral nucleus of
34 amygdala ( BL A) which in one case study ameliorated SIB significantly and reduced anxiety ( Sturm et al., 2013). However, co stimulation of the central nucleus of the amygdala and supra amygdaloid projection system yielded no beneficial ef fect. Basolateral nucleus of amygdala is one of the nuclei of the amygdala and is involved in the processing of anxiety response. However, it should be noted that this is observed i n one single subject and the self injurious behavior measurement relied on a SIB scale devised and used by the parents, who were not blind to the deep brain stimulation experimental protocol. Furthermore, there were no controls in this study. Other clinical mutilation ( Price et al. 2001). In this study, the anterior cingulate gyrus and media orbitofrontal regions were lesioned using bilateral radiofrequency thermocoagulation. The anterior cingulate gyrus is an interface linking the medial prefrontal cortex and the amygdala with important functions involving the cognitive and emotional association of anxiety related response ( Kim and Gorman, 2005). However, this leucotomy study involved only five subjects ( one subject did not display sustained improvement) and had no controls. Despite the substantial problems in these clinical studies, the data indicate some support for the concept that anxiety is related to the expression of SIB. Investigation of the role of anxiety related behavior as a predictor of vulnerability for SIB can benefit intellectual challen ged population such as autism, Prader W illi syndrome, and Lesch Nyhan syndrome. The importance of these investigation s is also due to other psychiatric illness that exhibit SIB such as major depression, anxiety disorders, schiz ophrenia, eating disorders, substance abuse, and posttraumatic st ress disorder ( Haw et al., 2001; Herpertz et al ., 1997; Klonsky et al., 2003; Zlotnick et al.,
35 1999). The urgent need of treatment for SIB is also reflected in statistics such as the fact th at 4% of the entire US population have a past history of SIB ( Briere and Gil, 1998) and 14% of the entire college students also commit self injur y ( Favazza et al., 1989; Gratz, 2001; Whitlock et al., 2006). Future direction may include investigation of ot her These medication studies are important because these two medications do not cause dependency and they are a different class of medications apart from benzodiazepines. Other future direction may be to test pemoline induced SIB in CRF1 receptor knockout mice. Corticotropin releasing factor 1 receptor is suggested to be involved in anxiety related behavior. It is found in neural structure ranging from the amygdala to nu cleus accumbens shell. Injection of CRF into amygdala enhance s anxiety related behavior in the EPM ( Davis et al., 2010 ). It has also been found that forebrain CRF 1 receptor knockout mice demonstrated less anxiety related behavior in open field, compared to control mice when both group are subjected to early life stressor ( Wang et al., 2012).
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40 BIOGRAPHICAL SKETCH William Lin came to US from Taiwan when he was t welve yea rs old. William went to Hume Fogg high school in Nashville, TN. William went to Columbia University in the city of New York studying biochemistry and biophysics from 1993 to 1995. When William entered Columbia, he received 34 AP credits. In 2008, He received Bachelor of S cience in psychology from Stony Brook University. He also received Master of S cience in psychology for Spring of 2013.