Roles of CRF-PACAP systems in sleep in mice (Carlezon)

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SUMMARY: PROJECT 3 Sleep is affected in many psychiatric illnesses. Some with people with stress-related disorders (e.g., Post- Traumatic Stress Disorder [PTSD]) sleep less than normal, whereas others sleep more, or have fragmented sleep patterns that reflect more frequent bouts of sleep and wakefulness. There is considerable evidence that stress disrupts sleep, and we have shown that chronic stress in mice causes profound alterations in sleep and diurnal rhythms. In contrast, the effects of sleep dysregulation on brain systems that mediate stress effects are not thoroughly understood. Periods of poor or restricted sleep may produce molecular alterations that subsequently affect sleep. Work in rodents demonstrates important similarities and differences between CRF (corticotropin-releasing factor) and PACAP (pituitary adenylate cyclase-activating polypeptide) effects on behavior. Although both peptides increase acoustic startle, a measure of vigilance often used in preclinical and clinical studies of anxiety and fear, PACAP effects tend to be persistent (lasting >1 week), whereas CRF effects resolve quickly. These types of findings, together with known genetic alterations in CRF and PACAP systems in people vulnerable to stress-related illness, provide a rationale for comparing and contrasting the neurobiology of these peptides. The effects of CRF and PACAP on sleep have not been directly compared, particularly over extended periods of time. Our premise is that these peptide systems affect—and are affected by—sleep. Our hypothesis is that there is a reciprocal relationship between these peptides and sleep, such that activation of CRF and/or PACAP systems will affect sleep and, conversely, sleep restriction will affect CRF and/or PACAP systems, but that the persistence of these effects will differ. Tests will be conducted in male and female mice implanted with wireless transmitters that enable continuous collection of EEG, EMG, body temperature, and activity data for several weeks. Molecular analyses will focus on brain areas implicated in the emotional aspects of stress, including amygdala (AMG), bed nucleus of the stria terminalis (BNST), and prefrontal cortex (PFC), as well as areas more traditionally implicated in sleep and biological rhythms. We will also perform circuit mapping studies to provide insights on neural connections between regions that regulate emotional behavior and those that regulate sleep. In Aim 1, we will compare the effects of altering CRF and PACAP system function on sleep and biological (diurnal and circadian) rhythms. We will also examine the effects of PAC1R or CRFR1 ablation in AMG, following up on pilot data showing that stress increases PAC1R expression in this region. In Aim 2, we will directly compare the effects of acute and repeated sleep restriction on PACAP and CRF systems using RNAscope to quantify the magnitude of any changes and identify the cell-types in which changes occur. In Aim 3, we will use trans-synaptic viral labeling to map connections between circuits implicated in sleep and emotional behavior. Project 3 experiments are designed based on current knowledge, but as expected with Conte Centers, we will use new discoveries from all projects to refine and optimize our objectives via the Administrative Core.