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

Bowers, Mallory Elva (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/nk322f09x?locale=en
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Abstract

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

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

3.3.2.1 Elevated Plus Maze ______________________________60

3.3.2.2 Open Field Test ________________________________60

3.3.2.3 Shock Reactivity ________________________________61

3.3.2.4 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

4.3.2.1 Elevated plus maze ______________________________95

4.3.2.2 Open field test _________________________________95

4.3.2.3 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

Figure Index

Figure. 2.2-1 Schematic Diagram of Mammalian Fear Circuitry _____________________52

Table. 2.3-1 The effect of opioid manipulation on fear/anxiety models ________________53

Table. 2.4-1 Modulation of the cholecystokinin system in fear/anxiety models ___________54

Table. 2.5-1 The effect of NPY manipulation on fear/anxiety models _________________55

Figure. 3.4-1 Manipulation of the cannabinoid system acutely alters cued fear expression ___76

Figure. 3.4-2 Manipulation of the cannabinoid system acutely alters cued fear expression ___77

Figure. 3.4-3 Baseline measures in cholecystokinin B receptor (CCKBR) knockout mice ____78

Figure. 3.4-4 CCKBR knockout mice exhibit normal locomotion ____________________79

Figure. 3.4-5 Wild-type and CCKBR knockout littermates do not differ significantly on elevated plus maze measures of time spent on closed arms, entries on open arms, entries on closed arms, and time in center ___________________________________________________80

Figure. 3.4-6 CCKBR knockout mice exhibit normal cued fear learning ________________81

Figure. 3.4-7 Knockout of CCKBR blunts Cnr1 antagonist-mediated increases in freezing across cued fear expression and extinction retention test days __________________________82

Figure. 3.4-8 Cnr1-positive fibers form perisomatic baskets around CCKBR-positive cell bodies in the BLA ________________________________________________________84

Figure. 3.4-9 CCKBR colocalizes with markers of excitatory and inhibitory neurons in the BLA

________________________________________________________________86

Figure. 3.4-10 At a dose of 0.1, 0.3, and 1 mg/kg, URB597 does not affect locomotion ______88

Figure3.4-11 Schematic of putative Cnr1-CCKBR interaction during cued fear expression: future directions _________________________________________________________89

Table. 3.4-12 Antibodies used in immunohistochemistry experiments ________________90

Figure 4.4-1 Cnr1 knockout subjects exhibit increased anxiety-like behavior ___________108

Figure 4.4-2 At a dose of 1 mg/kg, the CCKBR antagonist L-365,260 decreases freezing across multiple days of cued fear extinction in Cnr1 knockout mice, not in wild-type littermates ___109

Figure 4.4-3 At a dose of 0.3 and 3 mg/kg, the CCKBR antagonist L-365, 260 does not affect freezing across multiple days of extinction in Cnr1 wild-type and knockout littermates ____110

Figure 4.4-4 Administration of a Cnr1 antagonist, SR141716A, blocks within-session extinction of cued freezing; expression of CCBR mRNA may underlie differences in within-session extinction between C57BL/6J versus Cnr1 transgenic mice _______________________111

Figure 4.4-5 Cnr1 activation inhibits release of CCK from amygdala punch _____________112

Figure 5.4-1 Schematic of behavioral protocol, genotype validation, and measure of motor behavior _________________________________________________________124

Figure 5.4-2 Unlike males, female Cnr1 knockout subjects do not exhibit increased anxiety-like behavior compared to Cnr1 knockout littermates ______________________________125

Figure 5.4-3 Ovariectomy does not increase anxiety-like behavior in Cnr1 knockout females 126

Figure 5.4-4 A Cnr1 antagonist, SR141716A, increases anxiety-like behavior in adult male and female C57BL/6J mice _______________________________________________127

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