Neurosteroids are involved in the etiopathology of anxiety spectrum disorders and depression. We recently reported that in PTSD, a decrease of CSF allopregnanolone (Allo) levels was correlated with increased PTSD re-experiencing and comorbid depression (Biol. Psychiatry 60, 704-713. 2006). Allo, a potent positive allosteric modulator of GABA action at GABAA receptors, is produced in corticolimbic glutamatergic neurons by the sequential action of 51-reductase type I (51-RI) and 31- hydroxysteroid dehydrogenase (31-HSD). Thus, a deficit of Allo in the brain of PTSD patients could result in a deficit of GABAergic neurotransmission and in increased PTSD re-experiencing and depressive symptoms. In socially isolated (4 weeks) mice that express a neurosteroid biosynthesis downregulation in the medial frontal cortex, hippocampus, and amygdala, excessive contextual fear responses can be normalized by administering neurosteroidogenic drugs, including fluoxetine (FLX) (PNAS 105, 5567-5572. 2008) at doses too low to inhibit serotonin reuptake (Curr Opin Pharmacol 9, 24- 30. 2009). These observations support the hypothesis that: FLX reduces stress-induced excessive contextual fear responses by upregulating Allo levels in selected corticolimbic areas. The purpose of this R21 proposal is to determine whether Allo mediates the effects of FLX in mouse models of stress-induced excessive contextual fear responses and impaired fear extinction. The circuitry and molecular mechanisms underlying FLX-induced behavioral/biochemical actions will also be elucidated. AIM 1 will investigate whether by increasing corticolimbic Allo levels, FLX disrupts acquisition of fear memory, facilitates extinction, and prevents the reinstatement of fear memory following extinction. AIM 2 will study the mechanism by which FLX stimulates Allo biosynthesis in corticolimbic structures. Allo content will be quantified in discrete corticolimbic areas using gas chromatography-mass spectrometry (GC-MS), which allows measurement of femtomolar amounts of neurosteroids in laser microdissected mouse brain structures. Neurosteroidogenic enzyme expression will be investigated by quantitative competitive nested RT-PCR and Western blot. Enzymatic assays will establish whether FLX increases the affinity of 51-RI and/or 31-HSD for the substrate or cofactor. Finally, by using several radioactive substrates, we will study whether FLX stimulates Allo biosynthesis in brain slices by activating 51-RI or 31-HSD. An understanding of the mechanism(s) whereby FLX increases Allo bioavailability in local corticolimbic circuits could help explain the role of GABAA receptor-active neurosteroids in the modulation of stress-induced excessive contextual fear responses and thereby define a biomarker that could be targeted by drugs that improve debilitating neuropsychiatric disorders, including generalized anxiety, panic, and PTSD. PUBLIC HEALTH RELEVANCE: The GABAA receptor-active neurosteroid allopregnanolone is involved in several psychiatric disorders, including anxiety, PTSD, and depression and also in mouse models of these disorders. The goal of this proposal is to identify in several mouse models: a) the role of corticolimbic allopregnanolone levels in the modulation of contextual fear responses and impaired fear extinction;b) the brain structures involved in the pharmacological action of neurosteroidogenic agents, such as fluoxetine;and c) the mechanism whereby fluoxetine upregulates brain allopregnanolone levels.