Painful neonatal trauma alters subsequent fear and sensory function via changes in amygdalar CRF function

新生儿损伤和创伤，如发生在新生儿重症监护病房（NICU），诱导终身 认知、感觉和情感功能的变化。虽然机制尚不清楚，但我们 认为杏仁核CRF的发育改变可能是一种常见的促进机制。 杏仁核中央核CRF阳性细胞向腹外侧投射 中脑导水管周围灰质和外侧下丘脑，参与各种伤害反应行为的区域。在 此外，杏仁核CRF已被证明对恐惧和疼痛的获得或表达至关重要， 杏仁核CRF系统似乎对新生儿创伤特别敏感。为了调查此事，我们 实验室最近采用了“两次打击”的范例来产生创伤表型。第一，仿照 在NICU，新生大鼠幼仔在生命的第一周每天暴露于多次爪穿刺。二是 四个发育阶段（婴儿期、断奶期、青春期或成年期）之一的大鼠暴露于 创伤性恐惧条件反射我们的初步数据显示了两组行为变化， 在发育的时间进程上似乎有所不同。首先，断奶大鼠暴露于新生儿创伤和恐惧 条件反射在恐惧条件反射后表现出触觉超敏反应，这不能仅仅用年龄来解释， 新生儿创伤或恐惧条件反射。其次，新生儿疼痛的相当大的亚群（~25-33%） 受试者在以后的生活中表现出恐惧条件反射后的冻结受损。这种效应在断奶后出现 并随着年龄的增长而增强目前的建议进一步审查这些影响超过3个具体 目标。在目的1中，我们通过检查新生儿疼痛反应的行为异质性， 额外的疼痛刺激（炎性爪注射）、额外的激活应激源（约束）和额外的刺激（刺激）。 行为结果（非条件性焦虑测试;非反射性疼痛行为）。在目标2中，我们检查 杏仁核CRF的变化是否可以解释观察到的行为变化。例如，CRF 将使用PCR和无偏倚的PCR方法观察新生和活化应激后的表达水平。 体视学新产生的CRF-Cre大鼠与TD-番茄报告细胞系杂交将用于检查 回路解剖结构的变化，以及用于检查CRF+细胞活化变化的FOS的额外标记。 道追踪工具将用于确定该系统的不同投影是否存在差异 影响。在目标3中，我们操纵杏仁核CRF系统试图逆转新生儿创伤- 诱导表型在两次治疗期间，将使用CRF拮抗剂和激动剂的全身和局部注射。 新生儿和激活应激，以逆转过敏和情感表型。此外，本发明还提供了一种方法， 化学发生学方法可用于CRF-Cre大鼠，以破坏或增强CRF向特异性 目标的总之，这些实验肯定会验证新生儿创伤影响 通过杏仁核中CRF信号的改变来控制随后的行为。

Neonatal injury and trauma, such as occurs in the neonatal intensive care unit (NICU), induces life-long changes in cognitive, sensory and affective function. Although the mechanisms are as-of-yet unknown, we believe that developmental alteration of the Amygdalar CRF is likely to be a common contributing mechanism. Anatomically, CRF+ positive cells in the Central Nucleus of the Amygdala project to the ventrolateral Periaqueductal Grey and the Lateral Hypothalamus, regions involved in a variety of nocifensive behaviors. In addition, Amygdalar CRF has been shown to be critical for the acquisition or expression of both fear and pain, and the amygdala CRF system appears to be particularly sensitive to neonatal trauma. To investigate this, our lab has recently adapted a “two-hit” paradigm to produce a trauma phenotype. First, modeled after practices in the NICU, neonatal rat pups are exposed to multiple paw punctures daily for the first week of life. Second, at one of four developmental stages (infancy, weaning, adolescence or adulthood) rats are exposed to a traumatic fear conditioning procedure. Our preliminary data show two sets of behavioral changes, which appear to differ in developmental timecourse. First, weanling rats exposed to both neonatal trauma and fear conditioning show a tactile hypersensitivity following fear conditioning, that cannot be explained solely by age, neonatal trauma or fear conditioning alone. Second, a sizeable subpopulation (~25-33%) of neonatal pain subjects show impaired freezing following fear conditioning later in life. This effect is present following weaning and strengthens as a function of age. The current proposal further examines these effects over 3 specific aims. In aim 1, we further examine the behavioral heterogeneity in response to neonatal pain by examining additional painful stimuli (inflammatory paw injections), additional activating stressors (restraint) and additional behavioral outcomes (non-conditioned anxiety tests; non-reflexive pain behaviors). In aim 2, we examine whether changes in amygdalar CRF can explain the observed behavioral changes. For example, CRF expression levels following both neonatal and activating stress will be observed using PCR and unbiased stereology. Newly created CRF-Cre rats crossed with a TD-tomato reporter line will be used to examine changes in circuit anatomy, with additional labeling of FOS used to examine changes in CRF+ cell activation. Tract tracing tools will be used to determine whether distinct projections of this system are differentially affected. In aim 3, we manipulate the Amygdalar CRF system in an attempt to reverse the neonatal trauma- induced phenotype. Systemic and local injections of CRF antagonists and agonists will be used during both the neonatal and activating stress in order to reverse the hypersensitive and affective phenotypes. In addition, chemogenetic approaches can be used with CRF-Cre rats to disrupt or enhance CRF projections to specific targets. Together, these experiments will definitely test the hypothesis that neonatal trauma affects subsequent behavior via alternations in CRF signaling in the Amygdala.