PACAP Modulation of Ca2+ Activity in Neonatal Mouse OB

项目摘要 垂体腺苷环化酶激活多肽(PACAP)是一种多效肽，在发育和生命过程中具有多种功能[4]。PACAP基因敲除(KO)动物将在出生后不久死亡，因为无法保持核心体温[5]。我们的观察表明，防止PACAP KO幼崽体温过低并不能消除低存活率，这表明其他功能，如嗅觉调节的喂养可能会受到干扰。PACAP及其G蛋白偶联受体(PAC1)在嗅球(OB)的整个生命过程中都有高表达，提示PACAP在嗅球内神经元的发育、存活和替换中起重要作用。令人惊讶的是，还没有关于PACAP在调节发育中的OB神经元的Ca[2]活性、迁移和成熟方面的作用的研究。在其他中枢神经系统区域，自发振荡形式的钙[2]活动对于发育中的神经元回路的建立和存活至关重要，特别是在感觉输入之前[6]。GABA在未成熟神经元中是兴奋性的，并与这种早期发育活动有关，但对嗅觉中间神经元成熟的控制知之甚少。然而，我们的初步数据显示，PACAP在新生儿OB的多种细胞类型中诱导瞬时和持续的Ca[2]振荡。 我们在第一个目标中的重点将放在发育中的GABA能颗粒细胞(GC)上，因为它们被认为表达PAC1Rs，并且可以通过Gad2-和Dlx2-tdTomato转基因小鼠进行鉴定。发育中的GC将被评估PACAP反应，并用GABA测试它们的成熟度，GABA将是未成熟细胞的兴奋性神经递质，这些未成熟细胞维持细胞内高氯离子水平，并通过GABA刺激去极化。 第二个目标将是确定PACAP在未成熟的OB中间神经元迁移和成熟中的作用。这将通过在培养的GAD2或Dlx2 td番茄小鼠OB细胞上进行迁移分析直接完成。与成熟相关的形态变化也将被检查。 这项建议将研究PACAP在OB回路早期发育中的作用，并基于对OB和其他脑区PACAP和PAC1受体表达的研究。然而，我的研究是第一次在识别的新生儿OB细胞中进行PACAP的动态成像研究。由于对PACAP在OB中的作用知之甚少，我的建议的重点将是为未来了解PACAP如何影响OB电路的发展奠定基础。许多神经疾病，如自闭症、双相情感障碍和精神分裂症，被认为起源于神经元回路早期发育的问题。了解PACAP在早期回路发育中的作用可能为这些毁灭性疾病提供新的治疗靶点。此外，了解PACAP如何影响发育中的中间神经元可能有助于设计损伤或疾病后成人神经发生的策略。

Project Summary Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a pleiotropic peptide with functions in development and throughout life [4]. PACAP knockout (KO) animals will die shortly after birth due to an inability to maintain core body temperature [5]. Our observation that preventing hypothermia in PACAP KO pups does not eliminate poor survival suggests that other functions such as olfactory mediated feeding may be disturbed. Both PACAP and its G-protein coupled receptor (PAC1) are highly expressed in the olfactory bulb (OB) throughout life suggesting PACAP plays an important role in the development, survival and replacement of neurons within the bulb. Surprisingly, there have been no studies on the role of PACAP in modulating the Ca[2+] activity, migration, and maturation of developing OB neurons. In other CNS regions, Ca[2+] activity in the form of spontaneous oscillations is critical for the establishment and survival of developing neuronal circuitry, especially prior to sensory input [6]. GABA is excitatory in immature neurons and has been implicated in this early developmental activity but the control of maturation in olfactory interneurons is poorly understood. However, our preliminary data show that PACAP induces transient and sustained Ca[2+] oscillations in multiple cell types of the neonatal OB. Our focus in the first aim will be on the developing GABAergic granule cells (GCs) because they are thought to express PAC1Rs and can be identified using GAD2- and Dlx2- tdTomato transgenic mice. The developing GCs will be evaluated for PACAP responses and their maturity tested with GABA, which will be an excitatory neurotransmitter for the immature cells which maintain high intracellular Cl- levels and depolarize with GABA stimulation. The second aim will be determining the role of PACAP in the migration and maturation of immature OB interneurons. It will be directly accomplished using migration assays on cultured GAD2 or Dlx2 tdTomato mice OB cells. Morphological changes associated with maturation will also be examined. This proposal will examine the role of PACAP during early postnatal development of OB circuitry and is based on studies of PACAP and PAC1 receptor expression in the OB and other brain regions. However, my studies are the first dynamic imaging studies of PACAP in identified cells of neonatal OB. Because little is known about the function of PACAP in the OB, the emphasis of my proposal will be to lay the foundation for future understanding of how PACAP affects the development of OB circuitry. A number of neurological diseases such as autism, bipolar disease, and schizophrenia are thought to originate from problems in early development of neuronal circuits. Understanding the role of PACAP in early circuitry development may provide a novel therapeutic target for these devastating diseases. Furthermore, understanding how PACAP affects developing interneurons may aid in designing strategies for adult neurogenesis following injury or disease.