Synaptic Mechanisms in the Mammalian Retina

我们的工作重点是研究视网膜内部的特殊突触和电路。今年是实验室的重大转变，我们正在努力将各种类型的技术放在适当的位置，这些技术将在未来几年推动我们的实验方法。 我们扩大了对视网膜内无长突细胞所建立的抑制性突触连接的研究，以了解前馈和反馈抑制如何在这个网络中对信号处理做出贡献。我们先前发现，A17无长突细胞通过一种独立于膜去极化或电压门控钙通道的释放过程向杆状双极细胞带突触提供快速的GABA能反馈(Chavez等人，2006年)。这种快速反馈可能对于防止杆状双极细胞突触终末易释放的小泡迅速耗尽至关重要(Singer和Diamond，2006)。我们最近的工作表明，这种反馈扩大了这些突触编码亮度和计算对比度的范围(手稿正在准备中)。此外，另一项研究表明，反馈抑制增强了杆状通路中对单光子吸收的突触反应的增益(提交的手稿)。 此外，我们将电生理和解剖学(EM)数据与数学模拟相结合，以探讨突触带在调节突触前膜小泡的传递和释放中的作用。我们的发现表明，只需将囊泡沿着二维条带被动扩散，条带就可以实现类似于传送带的功能。这些模拟还对囊泡沿着丝带扩散的分子机制做出了可检验的预测。一份手稿已经完全准备好，即将提交。 我们正在结合电生理学、成像方法和细胞/网络建模来探索定向选择性神经节细胞中的树突整合。我们还利用大量转基因小鼠系来成像特定的细胞类型，这将使我们能够记录突触耦合的锥体双极神经节细胞对。这使我们能够研究在电路的不同阶段方向选择性是如何编码的。这项工作还表明，NMDA受体在神经节细胞树突中发挥着独特的作用，以放大定向选择信号。一份手稿正在准备中。 我们还在努力了解NMDA受体如何有助于神经节细胞树突的发育完善，这些树突精确地分层在内丛状层，即视网膜内部的突触神经纤维层。我们已经成功地结合多种遗传工具获得了一只小鼠，在发育过程中，NMDA受体可以在单一类型的神经节细胞中的任何时候被敲除。这个项目花了很长时间来准备，但在小鼠身上进行的初步实验非常有希望，并表明我们将对NMDA受体在视网膜发育中的作用获得令人兴奋的新见解。 我们在反馈抑制方面的工作使我们进行了一项长期的努力，以系统地了解无长突细胞如何对视网膜内部的视觉信号做出贡献。由于有40多种不同的无长突细胞，这一前景可能有点压倒性。首先，我们已经确定了可以通过遗传手段识别和操纵的窄视野和宽视野的无长突细胞。我们已经获得了Cre在某些无长突细胞中特异表达的小鼠系，然后将它们与小花系杂交，使表达Cre的神经元在化学上被沉默(通过依赖Cre的设计者受体的表达，而设计者受体仅由特制药物激活)。这种沉默对神经节细胞信号的影响将用微电极阵列来检测。此外，钙和电压指示物的细胞特异性表达将使我们能够检查这些细胞中的树突信号，这些信号可能是这些细胞中视觉处理的基础(参见Grimes等人，2010)，这是通过体细胞电生理记录无法获得的。 我们还在研究突触和神经元的生物物理性质如何随着昼夜周期或暗适应的不同阶段而变化。最初我们关注的是AII无长突细胞中BK通道功能的变化，无长突细胞在夜间和日间视觉中扮演着不同的角色(见Oesch和Diamond，2010)。一份手稿正在准备最初的生理工作，我们已经开始与Kevin Briggman博士合作挖掘密集重建的视网膜组织，以识别相关视网膜回路的变化。 最后，我们正在扩展我们的电子显微镜研究，与Tom Reese和Richard Leapman合作，探索光感受器和视杆双极细胞中突触带的详细超微结构。到目前为止，EM断层扫描使我们能够检测到将突触小泡拴在突触带和突触前膜上的蛋白质细丝。这种方法可能使我们第一次从形态上辨别停靠和预置的突触小泡。我们对听觉系统中的毛细胞带状突触进行了类似的分析，发现突触后的专门化可以优化这些突触的信号传递(提交的手稿)。

Our work focuses on specialized synapses and circuitry in the inner retina. This year represents a major transition in the lab, and we are working hard to put various types of technology in place that will drive our experimental approach for years to come. We have expanded our study of inhibitory synaptic connections made by amacrine cells within the inner retina, to understand how feedforward and feedback inhibition contributes to signal processing in this network. We previously discovered that A17 amacrine cells provide rapid GABAergic feedback to rod bipolar cell ribbon synapses via a release process that is independent of membrane depolarization or voltage-gated calcium channels (Chavez, et al., 2006). This rapid feedback may be essential to prevent the rapid depletion of readily-releasable vesicles from the rod bipolar cell synaptic terminal (Singer and Diamond, 2006). Our recent work indicates that this feedback extends the range over which these synapses encode luminance and compute contrast (manuscript in preparation). In addition, another study indicates that feedback inhibition enhances the gain of synaptic responses in the rod pathway to the absorption of single photons (manuscript submitted). In addition, we have combined electrophysiological and anatomical (EM) data with mathematical simulations to explore the role of the ribbon in regulating the delivery and release of vesicles at the presynaptic membrane. Our findings suggest that the ribbon may achieve a "conveyor belt" like function simply with passive diffusion of vesicles along the two-dimensional ribbon. These simulations also make testable predictions regarding the molecular mechanism by which the vesicles diffuse along the ribbon. A manuscript is fully developed and is nearing submission. We are combining electrophysiology, imaging approaches and cellular/network modeling to explore dendritic integration in directionally-selective ganglion cells. We are also taking advantage of numerous transgenic mouse lines to image specific cell types, which will enable us to record from synaptically coupled cone bipolar - ganglion cell pairs. This has enabled us to examine how directional selectivity is encoded at various stages of the circuit. This work also suggests that NMDA receptors play a unique role in ganglion cell dendrites to amplify the directionally selective signal. A manuscript is in preparation. We are also working to understand how NMDA receptors contribute to the developmental refinement of ganglion cell dendrites, with are precisely stratified within the inner plexiform layer, the synaptic neuropil of the inner retina. We have successfully combined multiple genetic tools to acquire a mouse in which the NMDA receptors can be knocked out in a single type of ganglion cell at any time during development. This project has taken a long time to prepare, but initial experiments with the mice are very promising and suggest that we will gain exciting new insights in the role of NMDA receptors in retinal development. Our work on feedback inhibition has led us to undertake a long-term effort to understand, in a systematic way, how amacrine cells contribute to visual signaling in the inner retina. With over 40 different kinds of amacrine cell, this prospect can be a bit overwhelming. To start, we have identified narrow- and wide-field amacrine cells that can be identified and manipulated by genetic means. We have acquired mouse lines in which CRE is expressed specifically in certain amacrine cells and then cross them with floxed lines enabling the CRE-expressing neurons to be silenced chemically (through CRE-dependent expression of designer receptors exclusively activated by designer drugs). The impact of this silencing on ganglion cell signaling will be examined with a microelectrode array. In addition, the cell-specific expression of calcium and voltage indicators will enable us to examine dendritic signaling in these cells that likely underlies visual processing in these cells (see Grimes, et al., 2010) that is not accessible with somatic electrophysiological recordings. We are also studying how the biophysical properties of synapses and neurons change with different phases of the circadian cycle or dark adaptation. Initially we have focused on changes in BK channel function in AII amacrine cells, which play distinct roles in night and daytime vision (see Oesch and Diamond, 2010). A manuscript is in preparation with the initial physiological work, and we have begun to collaborate with Dr. Kevin Briggman to mine densely reconstructed retinal tissue to identify changes in the relevant retinal circuitry. Finally, we are extending our electron microscopy studies, in collaboration with Tom Reese and Richard Leapman, to explore the detailed ultrastructure of synaptic ribbons in photoreceptors and rod bipolar cells. So far, EM tomography enables us to detect protein filaments that tether synaptic vesicles to the ribbon and the presynaptic membrane. This approach may enable us to discern morphologically, for the first time, docked and primed synaptic vesicles. We have applied a similar analysis to hair cell ribbon synapses in the auditory system and found postsynaptic specializations that optimize signal transfer at these synapses (manuscript submitted).