Interaction between the cholecystokinin and endogenous cannabinoid systems in cued fear expression and extinction retention Open Access

Bowers, Mallory Elva (2015)

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Posttraumatic stress disorder (PTSD) is thought to develop, in part, from improper inhibition of fear. Accordingly, one of the most effective treatment strategies for PTSD is exposure-based psychotherapy. Pavlovian fear conditioning and extinction using rodent models is a valid analog of trauma consolidation and exposure therapy. Pavlovian fear conditioning involves repeated co-presentation of a neutral stimulus, often an auditory tone, with an aversive, unconditioned stimulus (US) so that the test subject learns that the neutral, now conditioned, stimulus (CS) predicts an incoming US. As the subject learns that the CS is predictive of the US the subject will exhibit fear behavior in response to the CS. Conversely, extinction involves repeated presentations of the CS so that the test subject learns that the CS no longer signals an incoming US and inhibits fear behaviors. Separate studies have implicated the cholecystokinin (CCK) and endocannabinoid systems in fear; however, there is a high degree of anatomical colocalization between the cannabinoid 1 receptor (Cnr1) and CCK in the basolateral amygdala (BLA), which is critical for Pavlovian fear conditioning and extinction and emotion regulation. Although most research has focused on GABA and GABAergic plasticity as the mechanism by which Cnr1 mediates fear inhibition, we hypothesize that an interaction between Cnr1 and CCKBR is critical for fear extinction processes. This dissertation reports on a behavioral interaction between the CCK and endocannabinoid systems in cued fear expression and extinction retention that is likely mediated by functional CCKBR/Cnr1 cross-talk in the amygdala. First, the behavioral effect of Cnr1 antagonist administration was measured in C57BL/6J and CCKBR transgenic mice. Additionally, BLA Cnr1 and CCKBR immunoreactivity was examined. Second, the behavioral effect of CCKBR antagonist administration in Cnr1 transgenic mice was measured. In the same set of experiments, functional and genetic interactions between Cnr1 and CCKBR were assessed. Finally, sex differences in anxiety-like behavior of Cnr1 transgenic mice were assessed. These results provide much needed, novel evidence that Cnr1 contributes to cued fear expression via an interaction with the CCK system. Dysfunctional Cnr1-CCKBR interactions might contribute to the etiology of, or result from, fear-related psychiatric disease.

Table of Contents

Chapter 1: A brief overview and framework. 17

1.1 An overall framework and perspective on the dissertation. 18

Chapter 2: Neuropeptide regulation of fear and anxiety: implications of cholecystokinin, endogenous opioids, and neuropeptide Y. 21

2.1 Context, Author's Contribution, and Acknowledgement of Reproduction. 22

2.2 Introduction. 22

2.3 Opioids. 25

2.4 Cholecystokinin (CCK). 34

2.5 Neuropeptide Y. 42

2.6 Discussion. 50

Chapter 3: Interaction between the cholecystokinin and endogenous cannabinoid system in cued fear expression. 56

3.1 Context, Author's Contribution, and Acknowledgement of Reproduction. 57

3.2 Introduction. 57

3.3 Methods. 59

3.3.1 Animals. 59

3.3.2 Behavior. 60 Elevated Plus Maze. 60 Open Field Test. 60 Shock Reactivity. 61 Associative Fear Conditioning and Extinction. 61

3.3.3 Drugs. 62

3.3.4 Immunohistochemistry. 63

3.3.5 Statistics. 63

3.4 Results. 64

3.4.1 The cannabinoid system is critical for cued fear expression. 64

3.4.2 Global CCKB receptor knockout has no effect on baseline measures of weight, shock reactivity, or anxiety-like behavior. 65

3.4.3 CCKBR knockout mice exhibit normal cued fear acquisition, expression, and extinction. 65

3.4.4 Knockout of CCKBR blunts Cnr1 antagonist-mediated increases in freezing during cued fear expression and extinction retention. 66

3.4.5 Cnr1-positive fibers form perisomatic baskets around CCKBR-positive cell bodies in the BLA. 67

3.4.6 CCKBR colocalizes with markers for excitatory and inhibitory neurons in the BLA. 68

3.5 Discussion. 68

Chapter 4: Functional and genetic interaction between the endogenous cannabinoid and cholecystokinin systems may underlie expression of conditioned fear. 91

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

4.2 Introduction. 92

4.3 Methods. 94

4.3.1 Animals. 94

4.3.2 Behavior. 95 Elevated plus maze. 95 Open field test. 95 Associative fear conditioning and extinction. 96

4.3.3 Ex vivo CCK release in amygdala slice. 97

4.3.4 CCK octapeptide (non-sulfated) enzyme immunoassay. 98

4.3.5 Drugs. 98

4.3.6 RNA extraction, cDNA synthesis, and quantitative PCR (qPCR). 98

4.3.7 Statistics. 99

4.4 Results. 99

4.4.1 Cnr1 knockout increases anxiety-like behavior. 99

4.4.2 Administration of the CCKBR antagonist, L-365,260, decreases freezing across multiple days of cued fear extinction in Cnr1 knockout mice, not in wild-type littermates. 100

4.4.3 Administration of a Cnr1 antagonist, SR141716A, blocks within-session extinction of cued freezing; differential expression of CCBR mRNA may underlie cued fear extinction behavior in C57BL/6J versus Cnr1 transgenic mice. 101

4.4.4 Cnr1 activation inhibits release of CCK from amygdala punch. 102

4.5 Discussion. 103

Chapter 5: Anxiety-like behavior in cannabinoid 1 receptor (Cnr1) knockout mice is sex-dependent. 113

5.1 Context, Author's Contribution, and Acknowledgement of Reproduction. 114

5.2 Introduction. 114

5.3 Methods. 115

5.3.1 Animals. 115

5.3.2 Surgery. 116

5.3.3 Elevated plus maze. 116

5.3.4 Drugs. 116

5.3.5 Statistics. 117

5.4 Results. 117

5.4.1 Genetic validation of Cnr1 knockout; Wild-type and Cnr1 knockout male and female littermates exhibit normal locomotor behavior. 117

5.4.2 Female Cnr1 knockout subjects do not exhibit increased anxiety-like behavior compared to male Cnr1 knockout littermates. 117

5.4.3 Ovariectomy does not increase anxiety-like behavior in Cnr1 knockout females. 118

5.4.4 A Cnr1 antagonist, SR141716A, increases anxiety-like behavior in male and female C57BL/6J mice. 119

5.5 Discussion. 120

Chapter 6: Translationally informed treatments for PTSD. 128

6.1 Context, Author's Contribution, and Acknowledgement of Reproduction. 129

6.2 Introduction. 129

6.3 Pharmacotherapies. 130

6.3.1 D-cycloserine (DCS). 130

6.3.2 Cannabinoids. 132

6.3.3 Glucocorticoids. 134

6.3.4 Opioids/Morphine. 135

6.3.5 SSRIs/Antidepressants. 137

6.3.6 Norepinephrine/Propranolol. 139

6.4 Device-based treatments. 141

6.5 Discussion. 142

Chapter 7: Discussion. 144

7.1 Summary of results. 145

7.2 Integration of findings. 146

7.3 Implications and future directions. 147

References. 150

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