The functional role of the chronic oxytocin-induced soluble CRF receptor 2α splice variant

由于其亲社会，抗焦虑和抗应激作用后发现急性治疗，神经肽催产素（OXT）被认为是一种治疗选择与社会情绪缺陷相关的精神疾病。然而，一些动物和人类研究报告了慢性OXT治疗的不良行为影响。在第一个资助期间，我们发现慢性OXT治疗通过激活下丘脑室旁核（PVN）中促肾上腺皮质激素释放因子（CRF）受体CRFR 2 α的选择性剪接增加焦虑。CRFR 2 α介导抗焦虑作用，而可溶性sCRFR 2 α变体发挥致焦虑作用。然而，sCRFR 2 α在其他行为或神经内分泌环境中的作用尚不清楚。为了继续我们的研究结果，我们的目标是通过监测下丘脑基因表达模式以及分泌到血液和不同脑区中来研究sCRFR 2 α是否调节应激相关神经内分泌系统（下丘脑-垂体-肾上腺（HPA）轴、OXT和加压素系统）的（再）活性。我们将进一步研究sCRFR 2 α在基础条件下、在社会创伤和慢性心理社会应激条件下是否参与社会和情绪行为的调节。这些小鼠范例在sCRFR 2 α背景下的相关性是由于暴露于SFC以及CSC分别导致社交恐惧和焦虑相关行为增加以及OXT系统改变。 在此，我们将使用特异性反义寡核苷酸（GapmeR）、靶点阻断剂和重组sCRFR 2 α肽注入已确定的感兴趣区域，操纵sCRFR 2 α的选择性剪接和区域可用性，并评估对情绪性和自然发生的社会偏好行为的影响。我们将进一步监测暴露于SFC或CSC对sCRFR 2 α表达的影响，并研究SFC和CSC诱导的行为改变的特定区域sCRFR 2 α操纵的影响。为了揭示sCRFR 2 α信号传导的机制，我们将从鉴定神经元和星形胶质细胞培养物中sCRFR 2 α表达和剪接的主要细胞类型开始，在此之前，我们将通过免疫共沉淀和随后的质谱分析来表征过表达sCRFR 2 α的原代小鼠神经元和星形胶质细胞中选定的sCRFR 2 α信号级联和sCRFR 2 α的细胞内蛋白相互作用伴侣。该项目将有助于对OXT长期治疗的不良后果（包括刺激CRFR 2 α的选择性剪接）的认识仍然有限，CRFR 2 α的选择性剪接与慢性OXT诱导的焦虑水平升高有因果关系。

Due to its pro-social, anxiolytic and anti-stress effects found after acute treatment, the neuropeptide oxytocin (OXT) is considered a therapeutic option for psychiatric disorders associated with socio-emotional deficits. However, several animal and human studies reported adverse behavioral effects of chronic OXT treatment. During the first funding period we revealed that chronic OXT treatment increases anxiety via activating alternative splicing of the corticotropin releasing factor (CRF) receptor CRFR2α in the hypothalamic paraventricular nucleus (PVN). Whereas CRFR2α mediates anxiolytic effects, the soluble sCRFR2α variant exerts an anxiogenic effect. However, the role of sCRFR2α in other behavioral or neuroendocrine contexts is unknown. In continuation of our results we aim to study, whether sCRFR2α modulates the (re)activity of stress-related neuroendocrine systems (hypothalamo-pituitary-adrenal (HPA) axis, OXT and vasopressin systems) by monitoring hypothalamic gene expression patterns, and secretion into blood and within distinct brain regions. We will further study, whether sCRFR2α is involved in the regulation of social and emotional behaviors under basal conditions, and under conditions of social trauma and chronic psychosocial stress using mouse models of social fear conditioning (SFC) and of chronic subordinate colony housing (CSC), respectively. The relevance of these mouse paradigms in the context of sCRFR2α is given as exposure to SFC as well as CSC results in increased social fear and anxiety-related behavior, respectively, and alterations in the OXT system. Here, we will manipulate alternative splicing and the regional availability of sCRFR2α using specific antisense oligonucleotides (GapmeRs), target site blockers and recombinant sCRFR2α peptide infused into the identified region(s) of interest and assess consequences on emotionality and naturally occurring social preference behavior. We will further monitor the consequences of exposure to either SFC or CSC on sCRFR2α expression, and study the consequences of specific regional sCRFR2α manipulation of SFC- and CSC-induced behavioral alterations.To reveal mechanisms of sCRFR2α signaling we will start with identifying the major cell type(s) of expression and splicing of sCRFR2α in neuronal and astrocytic cell cultures, before we will characterize selected sCRFR2α-signaling cascades and intracellular protein interaction partners of sCRFR2α in primary mouse neurons and astrocytes overexpressing sCRFR2α by co-immunoprecipitation and subsequent analysis using mass spectrometry. The project will contribute to the still limited knowledge regarding the adverse consequences of chronic treatment with OXT including the stimulation of alternative splicing of the CRFR2α, which is causally involved in the chronic OXT-induced increase in anxiety level.