Anxiety and the Amygdala in the Prenatal Valproic Acid Exposure Model of Autism Open Access

Hennessey, Thomas (Fall 2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/mg74qm09p?locale=en
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Abstract

Confusion endures as to the exact role of the amygdala in relation to autism. The amygdala controls socioemotional behavior and has consistently been implicated in the etiology of Autism Spectrum Disorder (ASD). In addition, there is a strong association of anxiety disorders with both the amygdala and ASD. Precocious amygdala development is commonly reported in ASD youth with the degree of overgrowth positively correlated to the severity of ASD symptoms. Prenatal exposure to valproic acid (VPA) leads to an ASD phenotype in both humans and rats, and has become a commonly used tool to model the complexity of ASD symptoms in the laboratory. In this dissertation, we first examined abnormalities in gene expression in the amygdala and socioemotional behavior across development, then how VPA exposure predisposes rats to anxiety-like behavior after stress. Effects of VPA on time spent in social proximity and anxiety were sex dependent, with increased social abnormalities, as well as increased grooming, presenting in males after chronic stress. Adolescent VPA animals did not show a reduction in social behaviors. At postnatal day 10, gene pathways involved in nervous system and cellular development displayed predicted activations in prenatally exposed VPA amygdala samples. By juvenile age, however, transcriptomic and proteomic pathways displayed reductions in cellular growth and neural development. Alterations in immune pathways, calcium signaling, Rho GTPases, and protein kinase A signaling were also observed. As behavioral, developmental, and genomic alterations are similar to those reported in ASD, these results lend support to prenatal exposure to VPA as a useful tool for understanding how developmental insults to molecular pathways in the amygdala give rise to ASD-related syndromes. In addition, our results support the hypothesis that elevated rates of anxiety disorders in individuals with autism may be the result of a greater innate vulnerability to stress.

Table of Contents

Chapter 1: RDoC-based categorization of amygdala functions and its implications in autism .... 1

Abstract ........................................................................................................................................... 1

1. Introduction ................................................................................................................................ 2

2. The Amygdala: anatomy, history, development ..................................................................... 4

2.1 Anatomy ................................................................................................................................. 4

2.2 Historical context ................................................................................................................... 7

2.3 Development .......................................................................................................................... 8

3. Amygdala functions and RDoC ................................................................................................ 9

3.1 Negative Valence Systems (NVS) ......................................................................................... 9

3.1.1 Acute Threat .................................................................................................................... 9

3.1.2 Potential Threat and Sustained Threat .......................................................................... 11

3.1.3 Frustrative Non-reward ................................................................................................. 12

3.2 Positive Valence Systems (PVS) ......................................................................................... 13

3.2.1 Reward Learning ........................................................................................................... 13

3.2.2 Approach Motivation .................................................................................................... 14

3.3 Cognitive Systems ............................................................................................................... 15

3.3.1 Attention ....................................................................................................................... 15

3.4 The Social Processes ............................................................................................................ 16

3.4.1 Affiliation and Attachment ........................................................................................... 16

3.4.2 Social Communication .................................................................................................. 17

3.5 Arousal and Regulatory Systems ......................................................................................... 18

3.5.1 Arousal .......................................................................................................................... 18

3.5.2 Circadian Rhythms ........................................................................................................ 19

4. Amygdala dysfunction in Autism ........................................................................................... 19

4.1 Alterations in Development and Neurobiology ................................................................... 19

4.1.1 Volume differences ....................................................................................................... 20

4.1.2 Abnormal amygdala activity and social cognition ........................................................ 21

4.1.3 Damage to the amygdala ............................................................................................... 22

4.2 Valproic acid and autism ..................................................................................................... 23

4.3 Autism, anxiety, and oxytocin ............................................................................................. 25

5. Conclusion: synthesis of RDoC, the amygdala, and autism ................................................. 26

Chapter 2: Developmental disruption of amygdala transcriptome and socioemotional behavior in rats exposed to valproic acid prenatally …. 29

Abstract ......................................................................................................................................... 29

1. Background .............................................................................................................................. 31

2. Methods ..................................................................................................................................... 33

2.1 Animals ................................................................................................................................ 33

2.2 Drug administration ............................................................................................................. 34

2.3 Behavior ............................................................................................................................... 34

2.3.1 Maternal behavior ......................................................................................................... 34

2.3.2 Ultrasonic vocalizations (USVs) ................................................................................... 35

2.3.3 Nest-seeking response ................................................................................................... 35

2.3.4 Infant fear learning ........................................................................................................ 35

2.3.5 Startle to maternal odor ................................................................................................. 35

2.3.5 Open field and social behavior testing .......................................................................... 36

2.3.6 Acoustic startle .............................................................................................................. 37

2.4 Brain collection .................................................................................................................... 37

2.5 Next generation RNA sequencing ....................................................................................... 37

2.6 Proteomics ........................................................................................................................... 38

2.7 Statistics ............................................................................................................................... 39

3. Results ....................................................................................................................................... 40

3.1 Developmental disruption of social preference, fear expression, and anxiety-like behavior...... 40

3.2 RNA Sequencing ................................................................................................................. 43

3.3 Proteomics ........................................................................................................................... 48

4. Discussion ................................................................................................................................. 50

5. Conclusions ............................................................................................................................... 60

6. Supplemental Information ...................................................................................................... 62

Chapter 3: Increased Stress Vulnerability in Rats Exposed to Valproic Acid Prenatally ……… 87

Abstract ......................................................................................................................................... 87

1. Introduction .............................................................................................................................. 88

2. Methods ..................................................................................................................................... 89

2.1 Animals ................................................................................................................................ 89

2.2 Stress paradigm .................................................................................................................... 90

2.3 Behavioral testing ................................................................................................................ 90

2.4 Statistical analysis ................................................................................................................ 91

3. Results ....................................................................................................................................... 92

3.1 Juvenile Acute Stress ........................................................................................................... 92

3.1.1 Grooming ...................................................................................................................... 92

3.1.2 Open Field ..................................................................................................................... 94

3.1.3 Social Interaction .......................................................................................................... 94

3.2 Juvenile Chronic Stress ........................................................................................................ 96

3.2.1 Grooming ...................................................................................................................... 96

3.2.2 Open Field ..................................................................................................................... 97

3.2.3 Social Interaction .......................................................................................................... 98

3.3 Adolescent Chronic Stress ................................................................................................... 99

3.3.1 Open Field ................................................................................................................... 100

3.3.2 Social interaction ........................................................................................................ 100

4. Discussion ............................................................................................................................... 101

4.1 Anxiety ............................................................................................................................... 103

4.2 Social Behavior .................................................................................................................. 103

4.3 Human Implications ........................................................................................................... 105

Chapter 4: Conclusion and future directions………………………………………………...108

Literature Cited…………………………………………………………………………….…..115

List of Figures

Figure 1.1 The human amygdala…………………………………………………………………..4

Figure 1.2 Components, inputs, and outputs of the amygdala…………………………………….6

Figure 1.3 Developmental expansion of rat BLA principal neurons……………………………...10

Figure 2.1 Behavioral phenotype of prenatally-exposed VPA animals ………………………….41

Figure 2.2 VPA disrupts cellular growth, neural development, and immune function in amygdala gene pathways from P10-21 ... 45

Figure 2.3 Early enhancement but later deficit in cellular growth and neural development in amygdala from prenatal VPA exposed animals ……47

Figure 2.4 Pathways altered from prenatal VPA in both transcriptomic and proteomic samples............49

Figure 2.5 Alterations in amygdala genomic profile resulting from prenatal VPA exposure ......55

Figure 3.1. Grooming increased in the social novelty arena but not by acute stress.………...…93

Figure 3.2. Social preference decreased in male VPA rats after acute stress ….….……………94

Figure 3.3. Grooming enhanced in juvenile VPA males after chronic stress …………………...96

Figure 3.4. Social preference in juvenile male rats significantly reduced by chronic stress ……98

Figure 3.5. Social preference elevated in VPA exposed males after adolescent chronic stress..100

Figure 3.6. USS intensifies autism-like behavior in VPA compared to USS saline……………...102

List of Tables

Table 2.1 Outline of experiments………………………………………………………………...34

Table 2.2 Overlap between VPA model and ASD ……………………………………………….50

Supplementary Material

Figure S2.1. RNA sequencing pathways differentially altered across development between VPA and Saline amygdala. ...... 62

Table S2.1. Canonical RNA Sequencing Pathways differing from P10-21 in (A) both VPA and

Saline amygdala, (B) exclusively in Saline amygdala or (C) exclusively in VPA amygdala. ........ 63

Table S2.2. Canonical RNA Sequencing Pathways differing between Saline and VPA amygdala at (A) P10 and (B) P21. ........ 75

Table S2.3. Canonical Proteomic Pathways differing between Saline and VPA amygdala at P21. ......... 78

Table S2.4. Diseases and Functions RNA Sequencing Categories differing from P10-21 in (A) both VPA and Saline amygdala, (B) exclusively in Saline amygdala or (C) exclusively in VPA amygdala both VPA and Saline amygdala. .....80

Table S2.5. Diseases and Functions RNA Sequencing Categories differing between Saline and VPA amygdala at (A) P10 and (B) P21. ...... 83

Table S2.6. Diseases and Functions Proteomic Categories differing between Saline and VPA amygdala at P21. ....86

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