Regulation of Behavioral Flexibility by the Orbitofrontal Cortex and Amygdala Open Access

Zimmermann, Kelsey Sage (2015)

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In order to survive in a constantly changing environment, organisms must be able to learn that certain actions or stimuli are predictive of specific outcomes. Equally important is the ability to recognize when a formerly predictive relationship changes and the learned association is no longer relevant or viable. This flexibility in learning facilitates the suppression of previously meaningful behaviors in favor of new, more appropriate responses. The formation of both reward-related and aversion-based associations is known to rely in part on the basolateral amygdala (BLA), which shares rich reciprocal connections with the prefrontal cortex (PFC). The orbitofrontal cortex (OFC) is a highly conserved subregion of the PFC that is necessary for encoding changes to learned associations and facilitating behavioral flexibility. For instance, when an expected outcome is not delivered upon the presentation of a formerly predictive stimulus or the completion of a learned response, the OFC encodes this violation and modifies the previously acquired association accordingly, in part through interactions with the BLA. This process of recognizing changes in contingencies and appropriately changing behavioral responses is an important aspect of goal-directed decision-making. We hypothesize that plasticity within the BLA and the ventrolateral subregion of the OFC (VLO) is necessary for the formation and modification of associative memories, and that functional connectivity between these two regions is critical for goal-directed action selection. This dissertation first reports that activity of Brain-Derived Neurotrophic Factor (BDNF) within the BLA is necessary for both reward-related and fear-based associative conditioning. Next, anatomical and functional connectivity between the VLO and the BLA in mice is described within the context of appetitive instrumental conditioning; here we show that plasticity within the VLO, as well as connectivity between the VLO and the BLA, are necessary for flexible, goal-directed decision-making. Finally, we demonstrate that long-term potentiation in the VLO is necessary for behavioral flexibility in both reward-based action-outcome conditioning and fear-based stimulus-outcome conditioning. Together, these results provide novel insight into how the OFC and the amygdala process information about emotionally salient stimuli in order to mediate associative learning and behavioral flexibility.

Table of Contents

Table of Contents

Chapter 1: Introduction 12

1.1 A Framework and Context for the Dissertation 13

Chapter 2: Bdnf Deletion or TrkB Impairment in the Amygdala Inhibits both Appetitive and Aversive Conditioning 18

2.1 Context, Author's Contribution, and Acknowledgement of Reproduction 19

2.2 Abstract 19

2.3 Introduction 20

2.4 Materials and Methods 21

2.4.1 Subjects 22

2.4.2 Lentiviral Vectors 22

2.4.3 Surgery 23

2.4.4 Histology 23

2.4.5 Behavioral Testing 24

2.5 Results 26

2.5.1 Site-specific Bdnf deletion and TrkB inhibition in the BLA 26

2.5.2 Knockdown of amygdala Bdnf does not affect baseline anxiety 26

2.5.3 Bdnf knockdown in the BLA impairs FPS. 27

2.5.4 Bdnf knockdown in the BLA and dominant-negative impairment of TrkB in the amygdala delay

CPP and impair extinction. 27

2.6 Discussion 29

Chapter 3: Connections of the Mouse Orbitofrontal Cortex and Regulation of Action Selection by BDNF-TrkB 37

3.1 Context, Author's Contribution, and Acknowledgement of Reproduction 38

3.2 Abstract 38

3.3 Introduction 39

3.4 Materials and Methods 41

3.4.1 Subjects 41

3.4.2 Surgery 41

3.4.3 Instrumental Conditioning 42

3.4.4 Extinction Conditioning 43

3.4.5 Drug Treatment 44

3.4.6 Dendritic Spine Imaging and Enumeration 44

3.4.7 Histology 45

3.4.8 BDNF Quantification 46

3.4.9 Statistical Analyses 46

3.5 Results 46

3.5.1 OFC projections to the dorsal striatum, BLA, and perirhinal cortex are topographically organized

in the mouse. 47

3.5.2 VLO-amygdala interactions coordinate outcome-based decision-making. 49

3.5.3 Augmenting TrkB activity increases sensitivity to action-outcome associations. 51

3.5.4 7,8-DHF and Rho-kinase inhibition correct response strategies following Bdnf

silencing. 53

3.5.5 Gi-DREADD-mediated VLO silencing impairs goal-directed action selection. 54

3.6 Discussion 55

Chapter 4: The orbitofrontal cortex regulates behavioral flexibility in both appetitive and aversive domains 74

4.1 Context and Author's Contribution 75

4.2 Abstract 75

4.3 Introduction 76

4.4 Materials and Methods 78

4.4.1 Subjects 78

4.4.2 Surgery 78

4.4.3 Drugs 79

4.4.4 Instrumental Conditioning 79

4.4.5 Fear Conditioning and Extinction 80

4.4.6 Histology 81

4.4.7 Electrophysiology 81

4.4.8 Statistical Analyses 83

4.5 Results 84

4.5.1 VLO inhibition obstructs goal-directed decision-making. 84

4.5.2 VLO inhibition obstructs retention of fear extinction. 85

4.5.3 Goal-directed response strategies correlate with fear extinction retention. 86

4.5.4 Gi-DREADD activation increases the threshold for LTP. 87

4.6 Discussion 88

Chapter 5: Discussion 100

5.1 Summary of Results 101

5.2 Integration of Findings with the Current Literature 103

5.2.1 Anatomical Connectivity 104

5.2.2 Contribution to Fear Conditioning and its Extinction 109

5.2.3 Translational Implications 112

5.3 Implications and Future Directions 113

5.4 Conclusions 116

Appendix A: Publications 121

References 122

Figure Index

Figure 1-1 BDNF-Dependent Model of Associative Learning and Behavioral Flexibility 17

Figure 2-1: Site-specific Bdnf knockdown and TrkB dominant negative inhibition with lentiviral vector approaches. 32

Figure 2-2: Knockdown of Bdnf is specific to the infused region and is not associated with general anxiety-like effects. 33

Figure 2-3: Bdnf knockdown in the BLA disrupts FPS. 35

Figure 2-4: Bdnf knockdown in the BLA and dominant-negative impairment of TrkB in the amygdala delay CPP

and impair extinction. 36

Figure 3-1: The VLO innervates the dorsal and central striatum and projects to the BLA and ITCs of the amygdala. 61

Figure 3-2: Representative photomicrographs of the striatum and amygdala show innervation of the striatum and

retrograde labeling of cell bodies in the BLA. 63

Figure 3-3: The DLO/AI innervates the lateral and ventral striatum, and sends topographically organized

projections to the posterior AI, PRh, and BLA. 65

Figure 3-4: BDA infusions into the AI/DLO reveal bilateral rostrocaudal innervation of the striatum and

topographically organized innervation of the PRh and BLA. 67

Figure 3-5: VLO-selective Bdnf knockdown interferes with goal-directed action selection, resulting in reflexive habits. 68

Figure 3-6: Functional disconnection of the VLO and amygdala results in reflexive habits. 70

Figure 3-7: 7,8-DHF rescues goal-directed decision-making and regulates VLO dendritic spines. 71

Figure 3-8: Gi-DREADD-mediated silencing of the VLO results in stimulus-response habits. 73

Figure 4-1: DREADD-mediated inhibition of the VLO prevents stable consolidation of response-outcome

contingency degradation. 94

Figure 4-2: DREADD-mediated inhibition of the VLO impairs between-session retention of fear extinction. 96

Figure 4-3: The expression of goal-directed decision-making strategies correlates with the retention of fear extinction.97

Figure 4-4: Activation of Gi-DREADDs in the VLO increases the threshold for LTP induction. 98

Figure 5-1: Subregions of the Rodent PFC 117

Figure 5-2: Posterior Insula Projections to the Amygdala 118

Figure 5-3: Indirect Connectivity Between the VLO and the BLA 119

Figure 5-4: Anatomical and Functional PFC-Amygdala Connectivity 120

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