GABA-active neurosteroids in contextual fear responses

描述（由申请人提供）：神经类固醇与焦虑谱系障碍和抑郁的病因有关。我们最近报道，在创伤后应激障碍中，脑脊液异孕酮（Allo）水平的降低与PTSD再经历和共病抑郁症的增加相关。精神病学60,704-713。2006)。Allo是GABA受体作用的一种有效的正变构调节剂，在皮质边缘谷氨酸能神经元中通过51-还原酶I型（51-RI）和31-羟基类固醇脱氢酶（31- hsd）的顺序作用产生。因此，PTSD患者大脑中Allo的缺失可能导致gaba能神经传递的缺失，以及PTSD再体验和抑郁症状的增加。在社会隔离（4周）的小鼠中，内侧额叶皮质、海马体和杏仁核表达神经类固醇生物合成下调，过度的情境恐惧反应可以通过给予神经类固醇药物，包括氟西汀（FLX）来正常化（PNAS 105,5567 -5572）。2008年)，剂量过低而不能抑制血清素再摄取（Curr Opin Pharmacol 9,24 - 30）。2009)。这些观察结果支持以下假设：FLX通过上调特定皮质边缘区域的Allo水平来减少压力引起的过度情境恐惧反应。本R21提案的目的是确定Allo是否介导FLX在应激诱导的过度情境恐惧反应和恐惧消退受损小鼠模型中的作用。flx诱导的行为/生化作用的电路和分子机制也将被阐明。AIM 1将研究是否通过增加皮质边缘Allo水平，FLX破坏恐惧记忆的获得，促进消退，并阻止消退后恐惧记忆的恢复。AIM 2将研究FLX刺激皮质边缘结构中Allo生物合成的机制。将使用气相色谱-质谱（GC-MS）在离散皮质边缘区域量化Allo含量，该方法可以测量激光显微解剖小鼠脑结构中神经类固醇的飞摩尔量。采用定量竞争性巢式RT-PCR和Western blot检测神经甾体生成酶的表达。酶促试验将确定FLX是否增加51-RI和/或31-HSD对底物或辅因子的亲和力。最后，通过使用几种放射性底物，我们将研究FLX是否通过激活51-RI或31-HSD来刺激脑切片中Allo的生物合成。了解FLX增加局部皮质边缘回路中Allo生物利用度的机制可以帮助解释GABAA受体活性神经类固醇在调节应激诱导的过度情境恐惧反应中的作用，从而定义一种生物标志物，可以通过药物来改善衰弱的神经精神疾病，包括广泛性焦虑、恐慌和创伤后应激障碍。

DESCRIPTION (provided by applicant): 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.