Barmack N H, Qian Z
Neurological Sciences Institute, Oregon Health Sciences University, Beaverton, OR 97006, USA.
Neuroscience. 2002;113(1):235-50. doi: 10.1016/s0306-4522(02)00008-8.
Optokinetic stimulation activates visual climbing fiber pathways that synapse upon contralateral floccular Purkinje cells. Long-term horizontal optokinetic stimulation causes a progressive decrease in gain of the optokinetic reflex and leads to the subsequent genesis of a prolonged negative optokinetic afternystagmus. Since the flocculus is involved in adaptation to optokinetic stimulation, we used the technique of differential display reverse transcription-polymerase chain reaction to explore transcriptional changes in the flocculus evoked by long-term optokinetically evoked climbing fiber discharge. Several differentially transcribed gene products were isolated and sequenced. One of these, calbindin mRNA, was expressed in relatively decreased abundance in the flocculus that received increased climbing fiber input. Decreased transcription of calbindin mRNA was confirmed by northern blots. Hybridization histochemistry was used to localize calbindin mRNA to Purkinje cells and confirmed decreased transcription of calbindin mRNA in Purkinje cells located in folium 1 of the flocculus. Western blots and immunohistochemistry localized the climbing fiber-evoked decreased expression of calbindin to Purkinje cells in folia 1 of the flocculus. The expression of four other calcium-binding proteins in the flocculus was not influenced by optokinetic stimulation. Changes in expression of calbindin could be evoked by decreases in intracellular calcium associated with climbing fiber-evoked decreases in Purkinje cell simple spike activity.The application of differential display reverse transcription-polymerase chain reaction has provided a positive screen for several molecules in addition to calbindin whose expression is affected by naturally evoked activity in a major synaptic pathway to the cerebellum. Further experiments will be required to specify the functional role of each of these molecules.
视动刺激激活视觉攀缘纤维通路，该通路与对侧绒球浦肯野细胞形成突触。长期水平视动刺激会导致视动反射增益逐渐降低，并随后引发长时间的负性视动后眼震。由于绒球参与对视动刺激的适应，我们使用差异显示逆转录-聚合酶链反应技术来探索长期视动诱发的攀缘纤维放电所引起的绒球转录变化。分离并测序了几种差异转录的基因产物。其中之一，钙结合蛋白mRNA，在接受增加的攀缘纤维输入的绒球中表达丰度相对降低。通过Northern印迹证实了钙结合蛋白mRNA转录的减少。杂交组织化学用于将钙结合蛋白mRNA定位到浦肯野细胞，并证实位于绒球小叶1的浦肯野细胞中钙结合蛋白mRNA转录减少。蛋白质免疫印迹和免疫组织化学将攀缘纤维诱发的钙结合蛋白表达减少定位到绒球小叶1的浦肯野细胞。视动刺激不影响绒球中其他四种钙结合蛋白的表达。与攀缘纤维诱发的浦肯野细胞简单锋电位活动减少相关的细胞内钙减少可引起钙结合蛋白表达的变化。差异显示逆转录-聚合酶链反应的应用除了钙结合蛋白外，还为几种受小脑主要突触通路中自然诱发活动影响其表达的分子提供了阳性筛选。还需要进一步的实验来确定这些分子各自的功能作用。
