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

我的研究计划的主要目的是研究必需的维生素A代谢物维甲酸对神经系统功能的影响。视黄酸对神经系统的发育和再生，以及成人大脑的学习和记忆都很重要。我们利用软体动物（lynaea stagnation）的中枢神经系统（CNS）来研究类视黄醇信号的这些方面。与大多数脊椎动物不同，软体动物成年中枢神经系统神经元可以再生。生长锥位于生长中的神经纤维（神经突）的尖端，在发育和再生过程中对轴突的引导起着重要的作用。在lynaea中，培养的再生中枢神经系统神经元的生长锥大而结实，我们已经证明它们对维甲酸有反应并转向一个焦点来源，我们假设维甲酸在发育和再生过程中起着重要的指导分子作用。众所周知，许多引导分子在脊椎动物和无脊椎动物物种中都是保守的，因此淋巴神经元是研究视黄酸诱导生长锥转向的细胞和分子机制的理想选择，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，我们将研究类视黄酮受体和潜在的下游第二信使在视黄酸对生长锥转向反应中的作用。***最近，我们还发现了一种极其新颖的“漂浮生长锥”现象，即淋巴中枢神经系统的切割神经的再生神经突沿着空气-水界面生长。由于底物粘附通常是神经突生长和再生所必需的，这是一个新的再生事件，将用于对生长锥行为提供新的见解，这些行为在细胞培养或完整动物中不易解决。****使用lynaea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可单独识别的，具有已知的递质，突触伙伴和功能。我们可以培养和/或电记录参与特定行为的单个可识别神经元，如进食或呼吸。此外，这些行为可以通过训练来改变，动物既能学习也能记忆。我们已经证明，抑制lynaea中的视黄酸信号传导可以阻止长期记忆的形成，我们现在正在研究受这一重要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中研究单个已识别的神经元或突触，也可以在完整的分离中枢神经系统或行为“半完整”的准备中研究嵌入其神经元网络中的相同已识别的神经元，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来研究类维生素a在记忆形成中的功能和潜在的网络变化，使用一个相对简单的神经系统。********

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.********