Mechanisms underlying the effects of retinoic acid on neurite outgrowth and network plasticity

我的研究项目的主要目的是研究维生素A代谢物维甲酸对神经系统功能的影响。视黄酸对神经系统发育和再生以及成人大脑的学习和记忆都很重要。我们研究这些方面的维甲酸信号使用的中枢神经系统（CNS）的软体动物，滞流螺。与大多数脊椎动物不同，软体动物的成年中枢神经系统神经元可以再生。生长锥位于生长的神经纤维（神经突）的尖端，在发育和再生期间的轴突引导中起重要作用。在龙须草，生长锥培养再生中枢神经系统神经元是大而强大的，我们已经表明，他们响应并转向视黄酸，我们假设是作为一个重要的指导分子在发展和再生的焦点来源。众所周知，许多导向分子在脊椎动物和无脊椎动物物种中是非常保守的，因此，神经元是研究维甲酸诱导的生长锥转向的细胞和分子机制的理想选择，而这些机制目前在很大程度上是未知的。在下一个资助周期中，我们将研究维甲酸受体和潜在的下游第二信使在视黄酸引起的生长锥转向反应中的作用。 最近，我们还发现了一种非常新颖的“漂浮生长锥”现象，其中来自解剖的神经中枢神经系统的切断神经的再生神经突沿着空气-水界面生长。由于神经突生长和再生通常需要基质粘附，这是一种新的再生事件，将用于提供生长锥行为的新见解，这在细胞培养或完整动物中不容易解决。 使用神经元的另一个优点是，位于其中枢神经系统表面的许多神经元是单独可识别的，具有已知的递质，突触伴侣和功能。我们可以培养和/或电子记录参与特定行为的单个可识别神经元，例如进食或呼吸。此外，这些行为可以通过训练来改变，动物表现出学习和记忆。 我们已经证明，抑制维甲酸信号在拟南芥阻止长期记忆的形成，我们现在正在研究这种重要的信号分子影响的细胞和网络特性。为此，我们可以在细胞培养物中研究单个识别的神经元或突触，但也可以在完整的分离CNS或行为“半完整”制备物中研究嵌入其神经元网络中的这些相同识别的神经元，其中我们可以同时监测行为和神经元活动。这些研究为我们提供了一个独特的机会，利用相对简单的神经系统来研究维甲酸在记忆形成中的作用和潜在的网络变化。

The main aim of my research program is to study the effects of the essential Vitamin A metabolite, retinoic acid, on nervous system function. Retinoic acid is important for nervous system development and regeneration, as well as for learning and memory in the adult brain. We study these aspects of retinoid signaling using the central nervous system (CNS) of the mollusc, Lymnaea stagnalis. Unlike most vertebrate species, molluscan adult CNS neurons can regenerate. Growth cones, which are located at the tips of growing nerve fibres (neurites) play an important role in axon guidance during development and regeneration. In Lymnaea, growth cones of cultured regenerating CNS neurons are large and robust and we have shown that they respond to and turn toward a focal source of retinoic acid, which we hypothesize is acting as an important guidance molecule during development and regeneration. It is known that many guidance molecules are well conserved across vertebrate and invertebrate species, so Lymnaea neurons are ideal for investigating the cellular and molecular mechanisms underlying retinoic acid-induced growth cone turning, which are currently largely unknown. Over the next grant cycle, we will  investigate the role of retinoid receptors and potential downstream second messengers in the growth cone turning response to retinoic acid. Recently, we have also discovered an extremely novel "floating growth cone" phenomenon, where regenerating neurites from the cut nerves of a dissected Lymnaea CNS, grow along the air-water interface. As substrate-adhesion is normally required for neurite outgrowth and regeneration, this is a novel regenerating event which will be used to provide novel insights into growth cone behaviour that can not easily be addressed in cell culture or in the intact animal. Another advantage of using Lymnaea is that many neurons located on the surface of its CNS are individually identifiable, with known transmitters, synaptic partners and functions. We can culture and/or electrically record from single identifiable neurons involved in a specific behavior, such as feeding or respiration. Furthermore, these behaviours can be modified by training, and animals demonstrate both learning and memory.  We have shown that inhibiting retinoic acid signaling in Lymnaea prevents long-term memory formation, and we are now studying the cellular and network properties affected by this essential signaling molecule. To this end, we can study single identified neurons or synapses in cell culture, but also study these same identified neurons embedded in their neuronal networks in the intact isolated CNS, or a behaving "semi-intact" preparation, in which we can simultaneously monitor behavior and neuronal activity. These studies provide us with a unique opportunity to investigate retinoid functioning in memory formation and the underlying network changes, using a relatively simple nervous system.