Striatal melanocortin-4 receptor control of action flexibility Open Access
Heaton, Elizabeth (Fall 2023)
Abstract
More than half of all individuals in treatment for substance use disorder (SUD) will relapse. Inflexibility in selecting between familiar, habitual behaviors that have been rewarded in the past (drug seeking) and novel strategies that might be more advantageous (rehabilitation) may be a factor that preserves SUD. The dorsomedial striatum (DMS) is a brain region that receives and integrates glutamatergic input from cortical and subcortical regions required for goal-directed action selection. However, the factors in the DMS responsible for coordinating this incoming information remain incompletely understood. This dissertation begins by describing the extra-hypothalamic functions of the melanocortin-4 receptor (MC4R), a receptor that is well-positioned in the DMS to control flexible, goal-directed action. Next, I report that MC4R in the DMS appears to propel familiar reward-seeking behavior (habit), even when it is not fruitful, and moderating MC4R presence improves the capacity for goal-directed behavior. I then demonstrate that this process requires inputs from the orbitofrontal cortex, a brain region canonically associated with response strategy switching. Then, I further investigate how striatal melanocortin systems propel familiar behaviors, particularly via interaction with the central nucleus of the amygdala (CeA). I demonstrate that MC4R-expressing cells in the DMS are 1) predominantly expressed on dopamine D1-type receptor-expressing medium spiny neurons and 2) are necessary and sufficient for controlling the capacity of mice to arbitrate between actions and habits. I next use site-selective gene silencing and pharmacological techniques to reveal that MC4R presence suppresses goal seeking. I also find that MC4R-expressing neurons are functionally integrated into an amygdalo-striatal circuit that suppresses action flexibility in favor of routinized behaviors. Additionally, I use publicly available spatial transcriptomics datasets to reveal differences in the gene transcript correlates of Mc4r across the striatum, with considerable co-variation in dorsal structures. Guided by these results, I lastly discovered that MC4R function in the dorsolateral striatum complements that in the DMS, here suppressing habitual behavior. Together, these findings provide insight into the molecular and circuit-level mechanisms by which MC4R in the DMS propels habitual behavior. This dissertation thus illuminates mechanistic factors that support the development of automatized routines when flexible decision making is no longer adaptive, which may provide insight into therapeutic targets for neuropsychiatric disorders in which decision making is impaired.
Table of Contents
CHAPTER 1: Extra-hypothalamic functions of the melanocortin-4 receptor...... 1
1.1 Context and author's contribution 2
1.2 Abstract 2
1.3 Introduction 2
1.3.1 Overview of the central melanocortin system 3
1.4 Functions of MC4R outside of the hypothalamus 5
1.4.1 Cortex 5
1.4.2 Hippocampus 7
1.4.3 Amygdala 11
1.4.4 Striatum 12
1.5 Discussion 14
1.6 Framework and overview of the dissertation 15
CHAPTER 2: Selective breeding reveals control of reward-related action selection by the melanocortin-4 receptor................................................................. 21
2.1 Context, author's contribution, and acknowledgement of reproduction 22
2.2 Abstract 22
2.3 Introduction 23
2.4 Results 25
2.4.1 Individual differences in reward-related response strategies in mice 25
2.4.2 Individual differences in instrumental response strategies are associated with striatal protein composition 27
2.4.3 MC4R control of action strategies 28
2.4.4 MC4R control of action strategies via the OFC 31
2.5 Discussion 33
2.6 Methods 38
2.6.1 Subjects 38
2.6.2 Ages of mice at testing 39
2.6.3 Test of action strategies 39
2.6.4 Breeding strategy 40
2.6.5 Reinforcer devaluation 40
2.6.6 Intracranial surgery and viral vectors 42
2.6.7 CNO administration and timing in DREADDs experiments 43
2.6.8 Assessments of food intake 43
2.6.9 Histology 43
2.6.10 Immunoblotting 44
2.6.11 Dendritic spine imaging and reconstruction 45
2.6.12 Statistics and reproducibility 46
2.7 Funding 48
2.8 Acknowledgements 48
CHAPTER 3: Striatal melanocortin systems propel familiar actions via interaction with the central nucleus of the amygdala............................................. 57
3.1 Context and author's contribution 58
3.2 Abstract 58
3.3 Introduction 59
3.4 Results 60
3.4.1 MC4R-expressing cells in the DMS bidirectionally control action flexibility 60
3.4.2 Reducing Mc4r in the DMS prompts flexible behavior 62
3.4.3 MC4R in amygdalo-DMS circuits controls action selection 64
3.4.4 Spatial transcriptomics reveals a diversity of Mc4r expression co-variates across the striatum 66
3.4.5 MC4R acts as a molecular brake on DLS function in prompting habit-like behavior 68
3.5 Discussion 69
3.5.1 Striatal MC4R controls action flexibility 70
3.5.2 MC4R and amygdalo-striatal interactions suppress action flexibility 72
3.5.3 Conclusions 74
3.6 Methods 75
3.6.1 Animals 75
3.6.2 RNAScope 76
3.6.3 Surgery and viral vectors 76
3.6.4 Instrumental response training 79
3.6.5 Test for response flexibility 79
3.6.6 CNO administration 80
3.6.7 Cocaine administration 81
3.6.8 Setmelanotide administration 81
3.6.9 Locomotion and ad libitum feeding 81
3.6.10 Histology 82
3.6.11 Immunofluorescence imaging 82
3.6.12 Synaptoneurosome preparation 82
3.6.13 Western blotting 83
3.6.14 Trans-synaptic retrograde tracing 84
3.6.15 Statistical analysis 84
3.7 Funding 85
3.8 Acknowledgements 86
CHAPTER 4: Concluding remarks....................................................................... 113
4.1 Abstract 114
4.2 Etiological motivations of decision-making behavior 114
4.3 Physiological motivation: Hunger 115
4.3.1 The cycle of hunger 115
4.3.2 Hunger and behavioral flexibility 117
4.4 Security motivation: Stress 118
4.4.1 The hypothalamic-pituitary-adrenal axis 119
4.4.2 Stress and behavioral flexibility 119
4.5 Social motivation: Pair bonding 121
4.5.1 The neurobiology of pair bonding 121
4.5.2 Pair bonding and behavioral flexibility 122
4.6 Conclusions 123
APPENDIX A: Publications to which the author has contributed..................... 126
REFERENCES......................................................................................................... 127
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