GRABbing Serotonin During Fear

Serotonin plays a critical role in responding to potentially threatening stimuli and modulating the serotonin system can either exacerbate or ameliorate fear responses to subsequent encounters with fear-producing stimuli. Medications that inhibit serotonin reuptake are often used for stress disorders, but their effectiveness is variable and may dissipate with time. As part of a P50 Conte Center project, we found that repeated inescapable stress increased the expression of FKBP5 in mouse serotonin neurons using deep sequencing of the serotonergic translatome. This gene encodes FKBP51, a protein that acts as a co-chaperone for steroid receptors; its expression is dynamically regulated by complex physiological and epigenetic mechanisms, and it is poised to play a critical role in modulating glucocorticoid feedback mechanisms that impair stress resilience. We hypothesize that elevated FKBP5 expression in serotonin neurons interferes with hormonal feedback mechanisms that are necessary for successful adaptation of the serotonin system to stress. There has been scant research investigating the integration of steroid signaling with serotonergic function, and FKBP5 is positioned to act as a key integrator of systemic stress hormones and serotonergic function. In Aim 1 we will increase or decrease FKBP5 expression in serotonin neurons using intersectional strategies to investigate how this FKBP51 modulates fear learning and extinction. In Aim 2 we will use the novel 5-HT biosensor GRAB5-HT3.0 to measure serotonin release in amygdala in response to optogenetic stimulation or during fear conditioning. We have developed two novel Cre-dependent, conditional AAV vectors, one that expresses FKBP51 and GFP (for overexpression), and the other that contains floxed and inverted SaCas9 and a FKBP5-specific guide strand on separate transcriptional cassettes (for knockout). For Aim 1, we will inject these vectors into dorsal raphe nucleus of Pet1-Cre mice for serotonin neuron-specific expression. We will investigate fear learning, recall, extinction, and retrieval to investigate how high or low levels of FKBP5 in serotonin neurons affects passive coping in response to fearful stimuli. In Aim 2 will also inject a novel GRAB fluorescent biosensor to measure extracellular serotonin, which will be detected in response to graded optogenetic stimulation of the serotonin fibers before and after fear conditioning and in response to cues. This will reveal the effects of high or low levels of FKBP5 in serotonin neurons on serotonin release dynamics in amygdala. The results of these innovative experiments will frame a larger R01 proposal to investigate the impact of FKBP5 and steroid receptor signaling on serotonin neurons in a wider range of behaviors and brain regions and their potential role in future therapeutics. Project Number: 1R21MH137701-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Mental Health (NIMH) | Principal Investigator: John Neumaier | Institution: SEATTLE INST FOR BIOMEDICAL/CLINICAL RES, SEATTLE, WA | Award Amount: $359,433 | Activity Code: R21 | Study Section: Biobehavioral Regulation, Learning and Ethology Study Section[BRLE] View on NIH RePORTER: https://reporter.nih.gov/project-details/11137277