The Role of Corticotropin-Releasing Hormone-Binding Protein in Binge Drinking and Alcohol Dependence

Alcoholism is a chronic and progressive disorder characterized by periods of excessive drinking, withdrawal and abstinence, and eventual relapse. Both genetic and environmental factors contribute to the development of alcohol addiction, with stress being a critical environmental factor. Corticotropin-releasing hormone (CRH) is the key regulator of the mammalian stress response, and dysregulation of the CRH system is observed in binge drinking and alcohol dependence. CRH-binding protein (CRH-BP) is a secreted glycoprotein that binds CRH with a very high affinity, thereby regulating CRH receptor activation. Numerous studies have identified SNPs in the CRHBP gene that are associated with alcoholism, suggesting a role for CRH-BP in vulnerability to alcohol abuse. In this thesis, I investigated the role and regulation of CRH-BP in mouse models of binge drinking and alcohol dependence. Using in situ hybridization, I determined that repeated cycles of drinking in the dark, a mouse model of binge drinking, decrease CRH-BP mRNA expression in the medial prefrontal cortex, a region involved in executive function and regulation of emotion and behavior, including responses to stress. In a mouse model of alcohol dependence, the chronic intermittent ethanol (CIE) vapor paradigm, I observed a decrease in CRH-BP mRNA at peak alcohol withdrawal in the anterior paraventricular nucleus of the thalamus, a novel participant in the stress/reward circuitry. In a CIE paradigm that included periods of voluntary ethanol drinking, I detected a decrease in CRH-BP mRNA in the ventral tegmental area and an increase in CRH-BP mRNA in the bed nucleus of the stria terminalis, two key brain regions in the CRH and reward systems that have been implicated in control of excessive ethanol consumption. Interestingly, studies using CRH-BP KO mice suggest that the complete absence of CRH-BP may prevent increases in dependence-induced alcohol consumption. Together, these studies demonstrate changes in CRH-BP levels that may result in altered CRH receptor signaling within the stress and reward pathways in both binge drinking and dependence. I also examined the cell type-specific expression of CRH-BP in the PFC to begin to define the neural circuits in which CRH-BP is expressed. Using dual in situ hybridization, I detected CRH-BP mRNA predominantly in inhibitory, somatostatin-expressing interneurons of the PFC, suggesting that CRH-BP may be acting locally within the PFC to mediate its effects on CRH receptors on pyramidal neurons. These colocalization studies provide the basis for future studies to manipulate CRH-BP in a cell-type specific manner to further elucidate its role in mouse models of excessive alcohol consumption. Finally, I conducted signaling experiments that begin to address the mechanisms by which CRH-BP modulates CRH activity at the two CRH receptors. I demonstrated that CRH-BP inhibits CRH-mediated activation of CRH receptors and the resulting increases in cAMP in LβT2 cells, an effect that is partially reversed by the CRH-BP ligand inhibitor, CRH6-33. Lastly, I optimized a calcium assay for future experiments to assess CRH-BP modulation of CRH receptor signaling through the Gαq/PLC/PKC/calcium signaling pathway. Overall, the results from this thesis expand our knowledge on the role of the CRH-BP and the CRH system in alcohol use and addiction and begin to define the potential roles of CRH-BP within circuits of the stress and reward system and its mechanism of action.PHDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138762/1/kketches_1.pd

Cell Type-Specific Expression of Corticotropin-Releasing Hormone-Binding Protein in GABAergic Interneurons in the Prefrontal Cortex

Corticotropin-releasing hormone-binding protein (CRH-BP) is a secreted glycoprotein that binds CRH with very high affinity to modulate CRH receptor activity. CRH-BP is widely expressed throughout the brain, with particularly high expression in regions such as the amygdala, hippocampus, ventral tegmental area and prefrontal cortex (PFC). Recent studies suggest a role for CRH-BP in stress-related psychiatric disorders and addiction, with the PFC being a potential site of interest. However, the molecular phenotype of CRH-BP-expressing cells in this region has not been well-characterized. In the current study, we sought to determine the cell type-specific expression of CRH-BP in the PFC to begin to define the neural circuits in which this key regulator is acting. To characterize the expression of CRH-BP in excitatory and/or inhibitory neurons, we utilized dual in situ hybridization to examine the cellular colocalization of CRH-BP mRNA with vesicular glutamate transporter (VGLUT) or glutamic acid decarboxylase (GAD) mRNA in different subregions of the PFC. We show that CRH-BP is expressed predominantly in GABAergic interneurons of the PFC, as revealed by the high degree of colocalization (>85%) between CRH-BP and GAD. To further characterize the expression of CRH-BP in this heterogenous group of inhibitory neurons, we examined the colocalization of CRH-BP with various molecular markers of GABAergic interneurons, including parvalbumin (PV), somatostatin (SST), vasoactive intestinal peptide (VIP) and cholecystokinin (CCK). We demonstrate that CRH-BP is colocalized predominantly with SST in the PFC, with lower levels of colocalization in PV- and CCK-expressing neurons. Our results provide a more comprehensive characterization of the cell type-specific expression of CRH-BP and begin to define its potential role within circuits of the PFC. These results will serve as the basis for future in vivo studies to manipulate CRH-BP in a cell type-specific manner to better understand its role in stress-related psychiatric disorders, including anxiety, depression and addiction

Novel Roles for CRF‐Binding Protein and CRF Receptor 2 in Binge Drinking

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134133/1/acer12897.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134133/2/acer12897_am.pd

Binge Drinking Decreases Corticotropin‐Releasing Factor‐Binding Protein Expression in the Medial Prefrontal Cortex of Mice

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/133535/1/acer13119.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/133535/2/acer13119_am.pd

Twelve-hour rhythms in transcript expression within the human dorsolateral prefrontal cortex are altered in schizophrenia

Twelve-hour (12 h) ultradian rhythms are a well-known phenomenon in coastal marine organisms. While 12 h cycles are observed in human behavior and physiology, no study has measured 12 h rhythms in the human brain. Here, we identify 12 h rhythms in transcripts that either peak at sleep/wake transitions (approximately 9 AM/PM) or static times (approximately 3 PM/AM) in the dorsolateral prefrontal cortex, a region involved in cognition. Subjects with schizophrenia (SZ) lose 12 h rhythms in genes associated with the unfolded protein response and neuronal structural maintenance. Moreover, genes involved in mitochondrial function and protein translation, which normally peak at sleep/wake transitions, peak instead at static times in SZ, suggesting suboptimal timing of these essential processes

Twelve-hour rhythms in transcript expression within the human dorsolateral prefrontal cortex are altered in schizophrenia.

Twelve-hour (12 h) ultradian rhythms are a well-known phenomenon in coastal marine organisms. While 12 h cycles are observed in human behavior and physiology, no study has measured 12 h rhythms in the human brain. Here, we identify 12 h rhythms in transcripts that either peak at sleep/wake transitions (approximately 9 AM/PM) or static times (approximately 3 PM/AM) in the dorsolateral prefrontal cortex, a region involved in cognition. Subjects with schizophrenia (SZ) lose 12 h rhythms in genes associated with the unfolded protein response and neuronal structural maintenance. Moreover, genes involved in mitochondrial function and protein translation, which normally peak at sleep/wake transitions, peak instead at static times in SZ, suggesting suboptimal timing of these essential processes