University of Bergen, Department of Biomedicine, N-5009 Bergen, Norway, and
University of Bergen, Department of Biomedicine, N-5009 Bergen, Norway, and.
J Neurosci. 2019 Jan 23;39(4):627-650. doi: 10.1523/JNEUROSCI.2267-18.2018. Epub 2018 Nov 20.
In the rod pathway of the mammalian retina, axon terminals of glutamatergic rod bipolar cells are presynaptic to AII and A17 amacrine cells in the inner plexiform layer. Recent evidence suggests that both amacrines express NMDA receptors, raising questions concerning molecular composition, localization, activation, and function of these receptors. Using dual patch-clamp recording from synaptically connected rod bipolar and AII or A17 amacrine cells in retinal slices from female rats, we found no evidence that NMDA receptors contribute to postsynaptic currents evoked in either amacrine. Instead, NMDA receptors on both amacrine cells were activated by ambient glutamate, and blocking glutamate uptake increased their level of activation. NMDA receptor activation also increased the frequency of GABAergic postsynaptic currents in rod bipolar cells, suggesting that NMDA receptors can drive release of GABA from A17 amacrines. A striking dichotomy was revealed by pharmacological and immunolabeling experiments, which found GluN2B-containing NMDA receptors on AII amacrines and GluN2A-containing NMDA receptors on A17 amacrines. Immunolabeling also revealed a clustered organization of NMDA receptors on both amacrines and a close spatial association between GluN2B subunits and connexin 36 on AII amacrines, suggesting that NMDA receptor modulation of gap junction coupling between these cells involves the GluN2B subunit. Using multiphoton Ca imaging, we verified that activation of NMDA receptors evoked an increase of intracellular Ca in dendrites of both amacrines. Our results suggest that AII and A17 amacrines express clustered, extrasynaptic NMDA receptors, with different and complementary subunits that are likely to contribute differentially to signal processing and plasticity. Glutamate is the most important excitatory neurotransmitter in the CNS, but not all glutamate receptors transmit fast excitatory signals at synapses. NMDA-type glutamate receptors act as voltage- and ligand-gated ion channels, with functional properties determined by their specific subunit composition. These receptors can be found at both synaptic and extrasynaptic sites on neurons, but the role of extrasynaptic NMDA receptors is unclear. Here, we demonstrate that retinal AII and A17 amacrine cells, postsynaptic partners at rod bipolar dyad synapses, express extrasynaptic (but not synaptic) NMDA receptors, with different and complementary GluN2 subunits. The localization of GluN2A-containing receptors to A17s and GluN2B-containing receptors to AIIs suggests a mechanism for differential modulation of excitability and signaling in this retinal microcircuit.
在哺乳动物视网膜的杆状途径中，谷氨酸能的杆状双极细胞的轴突末梢与内丛状层中的 AII 和 A17 无长突细胞形成突触。最近的证据表明，这两种无长突细胞都表达 NMDA 受体，这引发了关于这些受体的分子组成、定位、激活和功能的问题。使用从雌性大鼠视网膜切片中突触连接的杆状双极细胞和 AII 或 A17 无长突细胞的双膜片钳记录，我们没有发现 NMDA 受体有助于在这两种无长突细胞中诱发的突触后电流的证据。相反，两种无长突细胞上的 NMDA 受体都被环境中的谷氨酸激活，而阻断谷氨酸摄取会增加它们的激活水平。NMDA 受体的激活也增加了杆状双极细胞中 GABA 能突触后电流的频率，这表明 NMDA 受体可以驱动 A17 无长突细胞释放 GABA。通过药理学和免疫标记实验揭示了一个显著的二分法，发现 AII 无长突细胞上含有 GluN2B 的 NMDA 受体和 A17 无长突细胞上含有 GluN2A 的 NMDA 受体。免疫标记还揭示了两种无长突细胞上 NMDA 受体的簇状组织以及 AII 无长突细胞上 GluN2B 亚基与连接蛋白 36 之间的紧密空间关联，这表明 NMDA 受体对这些细胞之间缝隙连接耦合的调制涉及 GluN2B 亚基。使用多光子 Ca 成像，我们验证了 NMDA 受体的激活会引起两种无长突细胞树突内细胞内 Ca 的增加。我们的结果表明，AII 和 A17 无长突细胞表达簇状的、 extrasynaptic NMDA 受体，具有不同的和互补的亚基，可能对信号处理和可塑性产生不同的贡献。谷氨酸是中枢神经系统中最重要的兴奋性神经递质，但并非所有谷氨酸受体都在突触处传递快速兴奋性信号。NMDA 型谷氨酸受体作为电压和配体门控离子通道，其功能特性取决于其特定的亚基组成。这些受体可以在神经元的突触和 extrasynaptic 部位找到，但 extrasynaptic NMDA 受体的作用尚不清楚。在这里，我们证明了视网膜 AII 和 A17 无长突细胞，在杆状双极细胞二联体突触的突触后伙伴，表达 extrasynaptic（而非突触）NMDA 受体，具有不同的和互补的 GluN2 亚基。GluN2A 包含的受体定位于 A17s，GluN2B 包含的受体定位于 AIIs，这表明在这个视网膜微电路中存在一种用于调节兴奋性和信号的机制。
