0 Tatiana Guevara April 24, 2018 Brain, Cognition and Development Lab; Department of Psychology Mentor: Lisa Scott, Ph.D.
1 Index Abstract 2 Introduction 3 Methods 5 Study specific behavioral schemes (Table 1 and 2) 7 Results 8 Behaviors rated on a scale Placebo Group Results (Figure 2 Table 3) 9 Intervention Group Results (Figure 3 Table 4 ) 9 Timed Behaviors Placebo Group Results (Figure 4 Table 5 ) 10 Intervention Group Results (Figure 5 Table 6 ) 10 Discussion 11 Conclusion 14 List of Abbreviations 15 Acknowledgements and References 16
2 Abstract Prader Willi Syndrome (PWS) is a genetic disorder typically brought about by a deletion in the paternal chromosome 15q11 q13 region. The symptoms of PWS range based on age and other individual differences. In infancy, the clinical manifestations of PWS inc lude hypotonia, failure to thrive, and low levels of oxytocin. The diverse effect of oxytocin on the brain makes it a great candidate for an intervention. The purpose of this thesis is to evaluate the effect of intranasal oxytocin intervention on infants w ith PWS. A double blind, randomized controlled trial was conducted where infants were recorded while they looked at pictures on a computer screen. Participants (N=15, average age: 4 months) were recorded before and after a short course (5 days) of interven tion. The videos collected were scored for attentiveness based on a study specific behavioral scheme. Once all the videos were coded, the study was unblinded to analyze the data and the results of intervention and placebo group were compared. For behaviors that were rated on a scale, the placebo group received lower scores from pre to post while the intervention group maintained their scores. The intervention group also spent more of their time during that task looking at the computer screen than the plac ebo group in the post test. These results supported the idea that the oxytocin intervention improved attentiveness, a behavior that has yet to be investigated in other studies.
3 Introduction Prader Willi Syndrome (PWS) is a genetic disorder whose origin is in a defect on the chromosome 15q11 q13 region 1 PWS is diagnosed most efficiently through DNA methylation. Typical individuals have both a methylated and unmethylated allele while individuals with PWS o nly have a methylated allele 1 Research has outlined three principle genetic mechanisms which lead to PWS: deletion of the parental chromosome 15q11 q13 region, maternal uniparental disomy 15, and imprinting defects. In 65 75% of PWS cases, the disorder was caused by the deletion of t he parental chromosome 15q11 q13 region 1 This region is present near the centromere on the long arm of chromosome 15. When this deletion occurs, many important genes are lost including the genes coding for GABA receptions which can lead to down regulation of certain activities. The phenotype for PWS range from nutritional defects to cognitive impairments, and change Current studies focus on specific clinical manifestations during certain ages. In infancy, mos t research focuses on the feeding difficulties that infants with PWS experience. This is primarily because these feeding difficulties may lead to failure to thrive and have other negative consequences. Developmental delays have not been shown to be manifes ted until ear ly childhood (2 6 years old) 1 When developmental delays have been studied, they are often compared to those in autism spectrum disorder 2 A study by Descheemaeker et al., investigated the relationship between pervasive developmental disorder (synonymous with autism spectrum disorder) and PWS. The study showed that participants with PWS (average age: 22 years) showed developmental delays that were s ignificantly similar to some symptoms of pervasive developmental disorder. While these results were help characterize some of the developmental delays experienced by individuals with PWS, they are also evidence of the
4 lack of representation of infants in PWS research. The focus of this thesis is to provide further insight into th e development and behaviors of infants with PWS. Another clinical manifestation of PWS in infancy is a deficiency in oxytocin containing neurons in the hypothalamic p araventricular nucleus (PVN) 3 Oxytocin is a hormone with the amino acid sequence: cysteine tyrosine isoleucine glutamine aspargine cyteine proline leucine glycine (Figure 1). As a neuropeptide, little is known about the pathway of oxytocin in the brain but advances in brain imagining have allowed several hypotheses to be made regarding the effect of oxytocin on the brain and behavior Oxytocin is one of principle hormones which influence social behaviors, especially parent infant bonding and pair preferences. It can be found in peripheral locations (such as in blood or saliva) or in the cerebr al spinal fluid (CSF) 4 While there are only theories describing the pathway of oxytocin, it is hypothesized that oxytocin reaches the peripheral locations and/ or the CSF by dendritic release 5 In the central nervous system, oxytocin can exert an effect on a variety of brain areas because the hormone has a long half life and lacks specificity 6 fMRI studies have shown that oxytocin activates neural areas including the anterior cingulate cortex and the m edial prefrontal cortex 7 Oxytocin has been investigated as an intervention for infants with PWS possibly because it has been connected to mechanisms controlling hunger. The main form of administration in studies using oxytocin is intranasal, because it is less invasive than intravenous and can cross the blood brain barrier. The extent to which intranasal oxytocin intervention influences an individual depends on factors like: nasal struct ure and dosage specification 8 The topography of the infant nasal cavity is not well known so there is variability in the form of administration of this intervention. There is also Figure 1 Molecular structure of oxytocin
5 inconsistency in the literature regarding the formulat ion of oxytocin intervention 8 Einfeld, et al., investigated the effects of internasal oxytocin on social behavior in individuals with PWS (average age: 18 years old). The dosages tested were 18 and 32 IU (international units) for participants 13 15 years old and 24 and 40 IU for participants 16 years and older. After an 8 week administratio n period, oxytocin showed very little impact on almost all the social behaviors meas ured 3 Another study, looked at the effects of this intervention on 4 month old infants with PWS Only one dose was studied, 4 IU, and it was administered over a seven day period. It was reported that infants showed significant improvements in eye contact and general level of activity as measured by the Alarm Distress Baby Scale 9 Both studies differ in methodology so all that can be determined is that more research needs to be done. One important thing to note is that both studies focus only on social behaviors because those behaviors are predicted to be most affected by oxytocin. There is little to no research looking at the effects of intranasal oxytocin intervention on ot her behaviors of infants with PWS. Thus, the aim of this thesis was to investigate the impact of internasal oxytocin intervention on the attentiveness of infants with PWS while presented a task. Attentiveness was measured indirectly by scoring infants for behaviors including: affect, head control, attention to screen, and arm/leg movements. Methods Study Overview This thesis was conducted as part of the behavioral portion of a double blind randomized controlled trial investigation the clinical and behavioral effects of intranasal oxytocin intervention on infants with PWS. A pre determined dose of oxytocin was administe red to participants every day during a five day period. Randomization was based on 2:1 trail as to maximize the number of participants who received the intervention. Clinical and behavioral measures were taken before and after the intervention period. The behavioral measures were
6 scored by analyzing videos of the participants while engaged in a task. These measures were then analyzed and qualitative trends were assessed. Study Participants Participants for this study were recruited from a national PWS list serv. Fifteen infants with PWS resulted from this recruitment effort. The average age of the infants who participated in the pre test was 4 months 7 days and 3 months and 7 days for the post test. Consent P rocedures Participants were positioned in front of a computer monitor where images of female A video camera was positioned near the computer monitor to reco rd the participant as they watched the computer screen. This was done both before and after the intervention period. The video recordings were then analyzed using the Datavyu software. A total of 28 videos were collected during the experiment. In both th e pre test and post test there was one participant that was not recorded during the task. The average video length was 15 minutes and the median video length was 7 minutes. It was decided that the first three minutes of each video would be analyzed to maxi mize inclusion. From this criterion, the data pool for this thesis comprised of 13 pre test videos and 10 post test videos. Two experimenters with expertise in development constructed a study specific scheme of behaviors which would be used to analyze the recordings. All the behaviors in the scheme dealt with attentiveness. T wo types of behaviors were analyzed : behaviors rated on a scale (Table 1) and timed behaviors (Table 2 ).
7 T able 1 Scheme for behaviors rated on a scale. A score of 1 is describes th e least desirable behavior, while a score of 4 describes a the most desirable behavior. Affect Head Control 1 = Crying 2 = Irritable 3 = Content 4 = Smiling 1 = Support always needed 2 = A lot of support needed 3 = Little support needed 4 = No support needed Eye Control General Arousal 1 = Unable to control 2 = Difficult to control 3 = Some difficulty controlling 4 = Easily moves eyes 1 = Asleep 2 = Falling asleep 3 = Awake with little to no movement 4 = Awake with arm/ foot movement Timed Behavior Description Attention to face Looking at a person present in the testing room Attention to screen Eyes focused around the center of the computer monitor Successful redirections the computer monitor Arm/leg movements Burst of movements, grouped together Back arches Back arched, head titled back, and limbs outstretched
8 esults Behaviors rated on a scale The average scores for affect, head control, eye control, and general arousal for the placebo group (Figure 2) and the intervention group (Figure 3) are shown. The average score for all but one behavior (affect) decreased from pre test to post test in the placebo group. Participants in the placebo group were generally irritable, had poor head and eye control, and were often falling asleep during the task. There was no general trend for the behaviors in the intervention group. Participants in this group wer e generally content, had poor head control, had okay eye control, and were awake for most of the task. Timed Behaviors The percent duration for the timed behaviors in the placebo group (Figure 4 ) and the intervention group (Figure 5 ) are shown Participants in the placebo group spent most of th eir time looking at the screen in the pre test but shifted to looking at faces in the post test. The number of successful redirections from pre test to post test decreased in the placebo group. Participants in the intervention group spent most of their time in both the pre test and post test looking at the computer screen. In this group there were increases in attention to screen and successful redirections, and decreases in arm/leg movements, and attention to faces. Overall, participants in
9 both groups spent about 50% of the task period engaging in the behaviors outline by the study specific scheme Fi gure 2. Average score for behaviors rated on a scale for the placebo group. N=5 for pre test and N=3 for post test. Error bars reflect standard deviation of the average of each b ehavior. Figure 3 Average score for behaviors rated on a scale for the intervention group. N=8 for the pre test and N=7 for post test. Error bar s reflect standard deviation of the average of each behavior. 0 1 2 3 4 5 Affect Head Control Eye Control General Arousal Average Score Behavior Pre-test Post-Test 0 1 2 3 4 Affect Head Control Eye Control General Arousal Average Score Behavior Pre-test Post-Test Table 3. Average score for behaviors rated on a scale during pre test and post test for the placebo group. Behavior Pre test mean Post test mean Affect Head Control Eye Control General Arousal 2. 7 0.2 1. 3 0.5 2.5 0.5 2.7 0.6 2. 7 0.6 1 0 2 2 2 1 Table 4 Average score for behaviors rated on a scale during pre test and post test for the intervention group. Behavior Pre test mean Post test mean Affect Head Control Eye Control General Arousal 2.9 0.2 1.3 0.5 0.7 3.4 0.4 3.0 0.1 1 .5 0 .9 3.3 0.5 3.1 0.5
10 Figure 4. Average percent duration for timed behaviors in the placebo group. N=5 for the pre test and N=3 for post test. Error bars reflect standard deviation of the average of each behavior. Figure 5. Average percent duration for timed behaviors in the intervention group. N=8 for the pre test and N=7 for post test. Error bars reflect standard deviation of the average of each behavior. 0% 10% 20% 30% 40% Attention to Face Attention to Screen Sucessful Redirections Arm/Leg Movement Back Arching Percent Duration Behaviors Pre-Test Post-Test 0% 10% 20% 30% 40% 50% 60% Attention to Face Attention to Screen Sucessful Redirections Arm/Leg Movement Back Arching Percent Duration Behaviors Pre-Test Post-Test Table 5 Average percent duration for timed behaviors during pre test and post test for the intervention group. Behavior Pre test % dur. Post test % dur. Attention to face Attention to screen Successful Redirections Arm/Leg Movement Back Arching 0.2 0.4 19 15 6 10 15 12 1 1 14 25 7 7 0.3 0.4 15 15 1 2 Note: All values are percentages Table 6 Average percent duration for timed behaviors during pre test and post test for the intervention group. Behavior Pre test % dur. Post test % dur. Attention to face Attention to screen Successful Redirections Arm/Leg Movement Back Arching 12 12 27 8 3 5 29 26 1 2 7 5 34 6 5 3 15 8 1 1 Note: All values are percentages
11 Discussion The results of this thesis showed that oxytocin may influence several behaviors associated with attentiveness in infants with PWS. An analysis of the behaviors rated on a scale showed that the scores for the placebo group worsened from pre test to post test in almost of the behaviors. This is a meaningful result since the intended purpose of the placebo group is to show that no changes occur from pre test to post test. This maintenance effect was, however, seen in the intervention group. These two trends suggest that t he oxytocin may work to prevent the impairment of the attention related behaviors studied rather than improving the behaviors. The intranasal oxytocin intervention may function to compensate for the hypothalamic hyposecretion of oxytocin 3 that occurs in in fants with PWS. It is also important to note that head control improved in the intervention group. This result is of clinical significance because one of the symptoms of PWS in infancy is hypotonia and the participants in the intervention group showed impr ovement head control. Head control is also indicative of attentiveness; once the study was unblinded it was noted that during the post test participants in the intervention group moved their head less and appeared to be more focused on the images they were presented. Two of the behaviors analyzed, eye control and general arousal, were also measured in a randomized controlled trial by Tauber et al. a Both behaviors increased significantly in that study 5 while eye control stayed constant and general arousal w orsened in this thesis. These behaviors were scored using different behavioral schemes so a direct comparison cannot be done, however this observation serves to further emphasize the need for more research in this field. of oxytocin on the attentiveness of infants with PWS. The first meaningful compari son between groups is the difference in the percent of time participants spent looking at the faces of individuals a = The average age of participants in the Tauber study was 4 months old.
12 in the testing room. There were typically around five individuals in the testing room with the participant including a t least one parent/guar dian, two experimenters, and a doctor. In the placebo group, participants spent more of the task period looking at these individuals than the images presented to them; in other words, they became more distract from pre test to post test (0.4% to 14%). On t he other hand, participants in the intervention group decreased the percent of time they looked at individuals in the room from 12% in the pre test to 7% in the post test. These results were also reflected in the percent of time participants looked at the computer monitor. The increase in attention to face experienced by participants in the placebo group was paired with a decrease in attention to screen. The same is true for participants in the intervention group, their decrease in attention to face was acc ompanied by an increase in attention to screen. It is possible that oxytocin that was administered activated areas such as the prefrontal cortex, which is implicated in moderating social behavior, or the anterior cingulate cortex, which is involved in atte ntion allocation 7 The idea that participants in the intervention group were more attentive to the task was also supported by the decrease in arm and leg movements. When typically developing infants were presented with the experimental task, they remained reasonably still. Thus, participants in the intervention group were able to replicate the behaviors of typically developing infants in the post test (arm and leg movements decreased from 29% to 15%) Another example of participant attentiveness was in the measures of successful redirections. Whenever a participant shifted focus to something other than the computer monitor, the experimenters attempted refocus them by directing their attention to the screen with a noise producing toy. If the participant began to look at the computer monitor again, then that event was classified as a successful redirection. Successful redirections decreased in the placebo group from 6% to 0.3%, but increased in the intervention group from 3% to 5%. The re is an elaborate, neural mechanism required to recognize
13 a noise and shift focus; thus, the increase in successful redirections in the intervention group highlights the increased attentiveness of the participants as well as a potential increase in cognitive ability. One of the strength of t his thesis is that it is a first of its kind study which to investigate the effects of intranasal oxytocin intervention on the attentiveness of infants with PWS. It is important to study how a wide variety of behaviors are affected by a n intervention such as intranasal oxytocin because oxytocin lacks specificity and can affect many different areas of the brain 6 This thesis showed that the intranasal oxytocin intervention positively influenced behaviors related to attention, and supporte d the idea that overall attentiveness of infants with PWS can improve with this intervention. One potential limitation of this thesis is the scheme used to assess attention related behaviors. The scheme used by the video coder was created after a few pre t est videos of participants were viewed and two experimenters noted behavior that was different than typically developing infants. This may have introduced some bias into the analysis process because the behaviors analyzed were specific to the participants in the experimental pool. The use of a study specific behavioral scheme also impedes the results of this thesis from being compared to other studies. It would be useful to repeat the methodology of this thesis but analyze the videos collected with a discip line accepted behavioral scheme, like the Codi ng Interactive Behavior Scale 9 Another limitation to this study was that only one person analyzed the videos collected and an inter rater reliability measure was not possible to report. Whenever possible, two or more video coders should be used to verify both the validity of the scores given and the scheme used. Finally, the results of this study remain clinically significant because improvements in attention related behaviors were produced after a relative sh ort intervention period (5 days). This is an improvement from the Tauber, et al. study which showed improvements is social behaviors after
14 seven days of intervention. Intranasal oxytocin is a clinic specific intervention which will required a family to be displaced while the intervention takes place, thus it becomes important to maximize the use of the time available. The fact that this intervention showed positive results in just five days is an advantage. Conclusion The purpose of this thesis was to ex plore the impact of internasal oxytocin intervention on the attentiveness of infants with Prader Willi Syndrome The results revealed that a short administration period of intranasal oxytocin positively influenced attention related behaviors. The first ca tegory of behaviors analyzed were behaviors rated on a scale. The oxytocin intervention produced a sort of ceiling effect where the score for the intervention group was the same from pre test to post test, while the placebo group received lower scores from pre test to post test. The second category of behaviors was timed behaviors. In the intervention group, the percent of time participants focused on the computer monitor increased from pre test to post test and the percent of time focused on faces decrease d. The opposite trend was characteristic of the placebo group. This reflects the idea that, on average, participants in the intervention attention improved their attention to the task while participants in the placebo group become more distracted. This the sis is one of the first studies to look at the effects of oxytocin on the attentiveness of infants with PWS. It would be beneficial to replicate this experiment, addin g in an ERP component, to see what areas of the brain are activated throughout the course of the task. This would allow for additional information to the current understanding that oxytocin effects many different areas of the brain. Future research should be directed toward formulation and determining the minimum effective dose and administrat ion time of intranasal oxytocin administration.
15 List of Abbreviations CSF Cerebral S pinal Fluid fMRI Functiona l Magnetic Resonance Imaging GABA Aminobutyric Acid IU International Units PVN Paraventricular Nucleus PWS Prader Willi Syndrome
16 A cknowledgements I thank Dr. Lisa Scott and Dr. Ryan Barry Anwar for their help running both the pre test and post test, as well as interpreting the behavioral data acquired. I also thank Dr. Jennifer Mil ler and her Prader Willi clinic which was responsible for the administration of the intervention. Finally, I would like to thank the all the parents of the infants who participated in this study. References 1. Angulo, M. A.; Butler, M. G.; Cataletto, M. E. J Endocrinol Invest 2015 38 (12), 1249 1263. 2. Descheemaeker, M. J.; Govers, V.; Vermeulen, P.; Fryns, J. P. Am J Med Genet Part A 2006 140A (11), 1136 1142. 3. Einfeld, S. L.; Smith, E.; Mcgregor, I. S.; Steinbeck, K.; Taffe, J.; Rice, L. J.; Horstead, S. K.; Rogers, N.; Hodge, M. A.; Guastella, A. J. Am J Med Genet Part A 2014 164A (9), 2232 2239. 4. Stevens, F. L.; Wiesman, O.; Feldman, R.; Hurley, R. A.; Taber, K. H. J Neuropsychiatry Clin Neurosci 2013 25 (2), 96 100. 5. Ross, H. E.; Young, L. J. Frontiers in Neuroendocrinology 2009 30 (4), 534 547. 6. Bethlehem, R. A.; Honk, J. V.; Auyeung, B.; Baron Cohen, S. J Psyneuen 2012 38 (7), 962 974. 7. Wade, M.; Hoffmann, T. J.; Wigg, K.; Jenkins, J. M. Genes, Brain and Behavior 2014 13 (7), 603 610. 8. Demayo, M. M.; Song, Y. J. C.; Hickie, I. B.; Guastella, A. J. Pediatr Drugs 2017 19 (5), 391 410. 9. Tauber, M.; Boulanouar, K.; Diene, G.; et al. Pediatrics 2017 139 (2).