Deconstructing the Role of Zona Incerta in Fear Generalization Pubblico

Abstract

The ability of an animal to assess threat and express appropriate fear is crucial for survival. Generalization of learned fear allows information from a previous experience to be used flexibly in a dynamic environment and is adaptive in nature. In contrast, persistent generalization of fear towards neutral, non-aversive cues is maladaptive and a core symptom of trauma- and anxiety-related disorders. Knowledge about the neural circuitry underlying fear generalization is primarily concentrated around the canonical tripartite neural circuit comprising of the amygdala, hippocampus and prefrontal cortex. Very few studies have attempted to understand the contribution of thalamic and sub-thalamic brain regions to fear generalization. This dissertation utilizes a rodent model of discriminative auditory fear learning to examine the role of subthalamic zona incerta (ZI) in mediating fear generalization. First, using C-FOS immunohistochemistry, we report an inverse relationship between ZI activation and fear generalization such that the animals that generalized fear had lower number of C-FOS expressing cells in the ZI. Subsequently, we demonstrate that chemogenetic activation of the ZI reduces fear generalization and chemogenetic inhibition of the ZI results in fear generalization. Given the considerable presence of GABAergic neurons in the ZI, we probed the role of these cells in mediating fear generalization. Using cell-specific chemogenetic manipulations, we demonstrate that the GABAergic neurons in the ZI bidirectionally modulate fear generalization. Further, our anterograde tracing studies reveal dense efferent GABAergic projections from the ZI to the thalamic nucleus reuniens (RE), dorsolateral periaqueducatal gray, ventral periaqueducatal gray, and posterior hypothalamus. With the RE implicated in maintaining specificity of fear memories, we chose to examine whether the ZI à RE GABAergic projections modulate fear generalization. In vitro electrophysiological recordings reveal that GABAergic inputs from the ZI evoke inhibitory post-synaptic currents (IPSCs) in the RE. Using cell-specific and projection-specific optogenetics, we show that activation of GABAergic projections from ZI to the RE in vivo prevented fear generalization. The experimental results contained in this dissertation establishes a central role for ZI in fear generalization and provides novel evidence for the influence of an inhibitory incerto-thalamic circuit in controlling fear memory specificity. This work contributes to a growing body of research on the role of thalamic and sub-thalamic influences in fear expression and inhibition and underscores the complex and dynamic nature of fear memories involving multiple, parallel neural pathways.

Table of Contents

LIST OF FIGURES. xi

CHAPTER 1: INTRODUCTION.. 1

1.1 Context for the dissertation. 1

1.2 The neurobiology of fear inhibition. 2

1.3 Behavioral protocols to study fear extinction and fear generalization. 4

1.4 Canonical view of the neurobiology of fear inhibition. 6

1.4.1 Amygdala. 6

1.4.3 Prefrontal Cortex. 8

1.4.4 Hippocampus. 9

1.4.5 The tripartite synaptic circuit 11

1.5 Non-canonical circuits for fear inhibition. 13

1.5.1 Anterior Cingulate Cortex. 13

1.5.2 Insular Cortex. 15

1.5.3 Thalamic and sub-thalamic influence on fear inhibition. 16

1.5.4 Nucleus Reuniens. 17

1.5.5 Paraventricular nucleus of thalamus. 18

1.5.6 Zona Incerta. 20

1.6 A framework for the dissertation. 24

CHAPTER 2: Bidirectional regulation of fear generalization by the zona incerta. 30

2.1 Context, Author’s Contribution, and Acknowledgement of Reproduction. 30

2.2 ABSTRACT.. 31

2.3 INTRODUCTION.. 32

2.4 MATERIALS AND METHODS. 35

2.4.1 Animals. 35

2.4.2 Auditory fear conditioning to test fear generalization. 35

2.4.3 Stereotaxic surgeries. 36

2.4.4 C-FOS immunohistochemistry & Cell Counting. 37

2.4.6 Histology. 37

2.4.7 Open field test 38

2.4.8 Electrophysiology. 38

2.4.9 Statistical Analysis. 39

2.5 RESULTS. 40

2.5.1 High intensity foot-shock training leads to fear generalization. 40

2.5.2 Decreased neuronal activity in the ZI accompanies increased fear generalization. 40

2.5.3 Increasing cellular activity in the ZI reduces fear generalization that manifests after conditioning with high intensity foot-shocks. 41

2.5.4 Decreasing cellular activity in the ZI results in fear generalization after conditioning with low intensity foot-shocks. 42

2.6 DISCUSSION.. 44

CHAPTER 3: GABAergic cells in the zona incerta mediate fear generalization. 61

3.1 Context, Author’s Contribution, and Acknowledgement of Reproduction. 61

3.2 ABSTRACT.. 62

3.3 INTRODUCTION.. 63

3.4 MATERIALS AND METHODS. 66

3.4.1 Animals. 66

3.4.2 Discriminative auditory fear conditioning to test fear generalization. 66

3.4.3 Stereotaxic viral injections. 67

3.4.4 Histology. 67

3.4.5 Open field test 68

3.4.6 Slice preparation and recording. 68

3.4.7 Statistical Analysis. 69

3.5 RESULTS. 70

3.5.1 Selective targeting of GABAergic neurons in the ZI 70

3.5.2 Increasing activity of GABAergic cells in the ZI reduces fear generalization. 70

3.5.3 Decreasing activity of GABAergic cells in the ZI induces fear generalization. 71

3.5.4 GABAergic projections from the ZI target the thalamus, hypothalamus and midbrain. 72

3.6 DISCUSSION.. 74

CHAPTER 4: GABAergic projections from zona incerta to thalamic reuniens regulate fear generalization 92

4.1 Context, Author’s Contribution, and Acknowledgement of Reproduction. 92

4.2 ABSTRACT.. 93

4.3 INTRODUCTION.. 94

4.4 MATERIALS AND METHODS. 96

4.4.1 Animals. 96

4.4.2 Virus injection and fiber optic implantation. 96

4.4.3 Behavioral procedures 97

4.4.4 Optogenetic stimulation. 98

4.4.5 Slice preparation and electrophysiological recording. 98

4.4.6 Histology and immunohistochemistry. 99

4.4.7 Statistical Analysis. 100

4.5 RESULTS. 101

4.5.1 Selective optogenetic targeting of GABAergic projections from the ZI to RE. 101

4.5.2 Functional validation of GABAergic projections from the ZI to RE. 101

4.5.3 Optical activation of ZI à RE GABAergic projections reduces fear generalization. 102

4.5.4 Optical activation of ZI à RE GABAergic projections does not produce non-specific changes in fear responses. 103

4.6 DISCUSSION.. 104

CHAPTER 5: DISCUSSION.. 120

5.1 Summary of results. 121

5.2 Integration of key findings 125

5.3 Implications. 128

5.4 Future directions. 130

5.5 Conclusions 131

REFERENCES. 132

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Psychology, Behavioral Sciences Biology, Neuroscience Health Sciences, Mental Health
Parola chiave	PTSD Zona Incerta Fear Inhibition Optogenetics Chemogenetics
Committee Chair / Thesis Advisor	Brian George Dias, Emory University
Committee Members	Robert Liu, Emory University David Weinshenker, Emory University Nancy Forger, Georgia State University Larry Young, Emory University

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