PACAP Modulation of Ca2+ Activity in Neonatal Mouse OB

项目摘要 腺苷酸环化酶激活多肽（PACAP）是一种多效性肽，在发育和整个生命过程中发挥作用[4]。PACAP敲除（KO）动物出生后不久就会死亡，原因是无法维持核心体温[5]。我们观察到，防止PACAP KO幼崽体温过低并不能消除存活率差，这表明其他功能，如嗅觉介导的进食可能受到干扰。PACAP及其G蛋白偶联受体（PAC 1）在嗅球中终生高表达，提示PACAP在嗅球神经元的发育、存活和更替中起重要作用。令人惊讶的是，目前还没有关于PACAP在调节发育中的OB神经元的Ca[2+]活性、迁移和成熟中的作用的研究。在其他CNS区域，自发振荡形式的Ca[2+]活性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在未成熟的神经元中是兴奋性的，并且与这种早期发育活动有关，但是对嗅觉中间神经元成熟的控制知之甚少。然而，我们的初步数据表明，PACAP诱导短暂的和持续的Ca[2+]振荡在多种类型的新生儿OB细胞。 我们的第一个目标将集中在发育中的GABA能颗粒细胞（GC）上，因为它们被认为表达PAC 1 R，并且可以使用GAD 2和Dlx 2- tdTomato转基因小鼠来鉴定。将评价发育中的GC的PACAP反应，并用GABA测试其成熟度，GABA是未成熟细胞的兴奋性神经递质，其维持高细胞内Cl-水平，并在GABA刺激下去极化。 第二个目标是确定PACAP在未成熟OB中间神经元迁移和成熟中的作用。这将使用对培养的GAD 2或Dlx 2 tdTomato小鼠OB细胞的迁移测定直接完成。还将检查与成熟相关的形态变化。 该建议将研究在出生后早期OB电路的发展过程中PACAP的作用，并基于OB和其他脑区的PACAP和PAC 1受体表达的研究。然而，我的研究是第一个动态成像研究PACAP在确定的新生儿OB细胞。由于很少有人知道的功能，PACAP在OB，我的建议的重点将是奠定基础，为未来的理解如何PACAP影响OB电路的发展。许多神经系统疾病，如自闭症、躁郁症和精神分裂症，被认为是源于神经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.