GABA-active neurosteroids in contextual fear responses

描述(申请人提供)：神经类固醇涉及焦虑症、谱系障碍和抑郁症的病因。我们最近报道，在创伤后应激障碍患者中，脑脊液中别孕酮(Allo)水平的降低与创伤后应激障碍再发和伴发抑郁的增加相关(Biol。精神科60,704-713。2006)。Allo是GABA作用于GABAA受体的一种有效的正变构调节剂，由51-还原酶I型(51-RI)和31-羟基类固醇脱氢酶(31-HSD)顺序作用于皮质边缘谷氨酸能神经元。因此，PTSD患者大脑中Allo的缺陷可能导致GABA能神经传递的缺陷，并导致PTSD重新体验和抑郁症状的增加。在社交隔离(4周)的小鼠中，在内侧额叶皮质、海马体和杏仁核表达神经类固醇生物合成下调的小鼠，过度的情景恐惧反应可以通过给予神经类固醇生成药物，包括氟西汀(Floxetine)来正常化(PNAS 105,5567-5572)。2008年)，剂量太低，不能抑制5-羟色胺的再摄取(Curr Opin Pharmacol 9，24-30。2009年)。这些观察结果支持这样的假设：Flx通过上调选定皮质边缘区域的Allo水平来减少应激诱导的过度情景恐惧反应。该R21建议的目的是确定Allo是否在应激诱导的过度背景恐惧反应和受损的恐惧消退的小鼠模型中介导了Flx的影响。此外，还将阐明氟尿嘧啶诱导的行为/生化作用的电路和分子机制。目标1将研究Flx是否通过增加皮质边缘Allo水平来扰乱恐惧记忆的获得，促进消退，并防止恐惧记忆在消亡后恢复。目的2研究氟尿嘧啶刺激皮质边缘结构中Allo生物合成的机制。Allo的含量将使用气相色谱-质谱仪(GC-MS)在离散的皮质边缘区域进行量化，这使得可以测量激光显微解剖的小鼠大脑结构中神经类固醇的毫微摩尔量。神经类固醇生成酶的表达将通过定量竞争套式RT-PCR和Western印迹进行检测。酶分析将确定Flx是否增加了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来刺激脑片中Allo的生物合成。了解Flx增加局部皮质边缘回路中Allo的生物利用度的机制(S)，可能有助于解释GABAA受体活性神经类固醇在调节应激诱导的过度背景恐惧反应中的作用，从而定义一个生物标记物，可以作为药物的靶点，改善包括广泛性焦虑、恐慌和创伤后应激障碍在内的衰弱神经精神障碍。 公共卫生相关性：GABAA受体活性神经类固醇别孕酮与几种精神疾病有关，包括焦虑、创伤后应激障碍和抑郁症，也参与了这些疾病的小鼠模型。这项建议的目的是在几个小鼠模型中确定：a)皮质边缘别孕酮水平在调节上下文恐惧反应和受损的恐惧消退中的作用；b)参与神经类固醇生成剂(如氟西汀)药理作用的大脑结构；以及c)氟西汀上调大脑别孕酮水平的机制。

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.