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

由于急性治疗后发现其亲社会，抗焦虑和抗应激作用，神经肽催产素（OXT）被认为是与社会情绪缺陷相关的精神疾病的治疗选择。然而，一些动物和人类研究报告了慢性氧化疗法的不良行为影响。在第一个资助期，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CRFR2α的选择性剪接而增加焦虑。而CRFR2α介导焦虑作用，可溶性sCRFR2α变体发挥焦虑作用。然而，sCRFR2α在其他行为或神经内分泌环境中的作用尚不清楚。为了延续我们的研究结果，我们的目标是通过监测下丘脑基因表达模式，以及进入血液和不同大脑区域的分泌，研究sCRFR2α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和抗利尿激素系统）的（re）活性。我们将进一步研究scfr2α是否参与基础条件下、社会创伤和慢性社会心理应激条件下的社会和情感行为的调节，分别使用小鼠社会恐惧条件（SFC）和慢性从属群体住房（CSC）模型。这些小鼠范式在sCRFR2α背景下的相关性是，暴露于SFC和CSC分别导致社交恐惧和焦虑相关行为的增加，以及OXT系统的改变。在这里，我们将使用特定的反义寡核苷酸（GapmeRs）、靶点阻滞剂和重组sCRFR2α肽注入确定的感兴趣区域来操纵选择性剪接和sCRFR2α的区域可用性，并评估对情绪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scfr2 α表达的影响，并研究特定区域scfr2 α操纵对SFC和CSC诱导的行为改变的影响。为了揭示sCRFR2α信号传导的机制，我们将首先确定神经元和星形胶质细胞培养中表达和剪接sCRFR2α的主要细胞类型，然后我们将通过共免疫沉淀和随后的质谱分析来表征原代小鼠神经元和过表达sCRFR2α的星形胶质细胞中选定的sCRFR2α信号级联和细胞内蛋白相互作用伙伴。该项目将有助于了解OXT慢性治疗的不良后果，包括刺激CRFR2α的选择性剪接，这与慢性OXT诱导的焦虑水平增加有因果关系。

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.