Schmidt John T, Fleming Matthew R, Leu Byunghee
Department of Biological Sciences and Center for Neuroscience Research, University at Albany-SUNY, 1400 Washington Avenue, Albany, New York 12222, USA.
J Neurobiol. 2004 Feb 15;58(3):328-40. doi: 10.1002/neu.10286.
Visual activity refines developing retinotectal maps and shapes individual retinal arbors via an NMDA receptor-dependent mechanism. As retinal axons grow into tectum, they slow markedly and emit many transient side branches behind the tip, assuming a "bottlebrush" morphology. Some branches are stabilized and branch further, giving rise to a compact arbor. The dynamic rate of branch addition and deletion is increased twofold when MK801 is used to block NMDA receptors, as if this prevents release of a stabilizing signal such as arachidonic acid (AA) from the postsynaptic neuron. In optic tract, AA mediates NCAM and L1 stimulation of axon growth by activating presynaptic protein kinase C (PKC) to phosphorylate GAP-43 and stabilize F-actin, and, if present in tectum, this growth control pathway could be modulated by postsynaptic activation. To test for the effects on arbor morphology of blocking PKC or AA release, we examined DiO-labeled retinal axons of larval zebrafish with time-lapse videomicroscopy. Bath application of the selective PKC inhibitor bisindolylmaleimide from 2 or 3 days onward doubled the rate at which side branches were added and deleted, as seen with MK801, and also prevented maturation of the arbor so that it retained a "bottlebrush" morphology. In order to selectively block the PKC being transported to retinal terminals, we injected the irreversible inhibitor calphostin C into the eye from which the ganglion cells were labeled, and this produced both effects seen with bath application. In contrast, there were no effects of control injections, which included Ringers into the same eye and the same dose into the opposite eye (actually much closer to the tectum of interest), to rule out the possibility that the inhibitor leaked from the eye to act on tectal cells. For comparison, we examined arbors treated with the NMDA blocker MK801 at half-hour time-lapse intervals, and detected the twofold rise in rates of branch addition and deletion previously reported in Xenopus larvae, but not the structural effect seen with the PKC inhibitors. In addition, we could produce both effects seen with PKC inhibitors by using RHC80267 to block AA release from DAG lipase, indicating that AA is the main drive for PKC activation. Thus, the results show a distinct role of AA and presynaptic PKC in both maturation of arbor structure and in the dynamic control of branching. The effects on branch dynamics were present regardless of the level of maturity of arbor structure. The fact that they mimicked those of MK801 suggests that presynaptic PKC may be involved in the NMDA receptor-driven stabilization of developing retinal arbors.
视觉活动通过一种依赖NMDA受体的机制来优化正在发育的视网膜-脑顶盖图谱并塑造单个视网膜树突分支。当视网膜轴突生长进入脑顶盖时，它们会显著减慢，并在轴突尖端后方发出许多短暂的侧支，呈现出“刷状”形态。一些分支会稳定下来并进一步分支，形成一个紧密的树突分支。当使用MK801阻断NMDA受体时，分支添加和删除的动态速率会增加两倍，这似乎是因为它阻止了诸如花生四烯酸（AA）等稳定信号从突触后神经元释放。在视束中，AA通过激活突触前蛋白激酶C（PKC）使GAP-43磷酸化并稳定F-肌动蛋白，从而介导NCAM和L1对轴突生长的刺激，并且，如果脑顶盖中存在这种生长控制途径，它可能会受到突触后激活的调节。为了测试阻断PKC或AA释放对树突分支形态的影响，我们使用延时视频显微镜检查了斑马鱼幼体中用DiO标记的视网膜轴突。从2或3天开始浴用选择性PKC抑制剂双吲哚马来酰胺，会使侧支添加和删除的速率增加一倍，这与使用MK801时的情况相同，并且还会阻止树突分支的成熟，使其保持“刷状”形态。为了选择性地阻断转运到视网膜终末的PKC，我们将不可逆抑制剂钙泊三醇C注射到标记了神经节细胞的眼中，这产生了与浴用药物相同的两种效果。相比之下，对照注射没有效果，对照注射包括向同一只眼睛注射林格氏液以及向对侧眼睛注射相同剂量（实际上更接近感兴趣的脑顶盖），以排除抑制剂从眼睛泄漏并作用于脑顶盖细胞的可能性。为了进行比较，我们以半小时的时间间隔检查了用NMDA阻断剂MK801处理的树突分支，检测到了之前在非洲爪蟾幼体中报道的分支添加和删除速率两倍的增加，但没有检测到PKC抑制剂所产生的结构效应。此外，我们可以通过使用RHC80267阻断二酰甘油脂肪酶释放AA来产生与PKC抑制剂相同的两种效果，这表明AA是PKC激活的主要驱动力。因此，结果表明AA和突触前PKC在树突分支结构成熟以及分支的动态控制中具有独特的作用。无论树突分支结构的成熟程度如何，对分支动态的影响都存在。它们模拟了MK801的效果这一事实表明，突触前PKC可能参与了NMDA受体驱动的发育中视网膜树突分支的稳定过程。
