Identification of subunits contributing to synaptic and extrasynaptic NMDA receptors in Golgi cells of the rat cerebellum
Identification of subunits contributing to synaptic and extrasynaptic NMDA receptors in Golgi cells of the rat cerebellum
复制标题
DOI：
10.1111/j.1469-7793.2000.00147.x
复制
发表时间：
2000-04-01
影响因子：
5.5
通讯作者：
Cull-Candy, SG
中科院分区：
文献类型：
--
作者：
Misra, C;Brickley, SG;Cull-Candy, SG
1.为探讨小脑高尔基体细胞N-甲基-D-天冬氨酸受体(NMDAR)的特性，对出生后14天(P14)大鼠小脑片进行膜片钳记录。方法：1.为验证细胞特性，用神经生物素填充细胞，共聚焦显微镜观察。NR2B亚基选择性NMDAR拮抗剂伊芬地尔(10 MU M)使全细胞NMDA诱发电流减少约80%。锌离子螯合剂N，N，N‘，N’-四-(2-吡啶甲基)-乙二胺(TPEN；1mU M)不影响NMDA诱发电流，提示不存在含有NR2A亚单位的NMDARs。高尔基体细胞的由外向外的斑块显示出一群“高电导”的50ps NMDAR开口。这些都被伊芬地尔抑制，其IC(50)为19 nM。这些细胞的斑块也含有“低电导”的NMDAR通道，具有NR2D亚单位受体的特征。它们的主电导为39ps，次电导水平为19ps，两个能级之间的跃迁具有明显的不对称性。与包含NR2D的受体的预期一样，这些事件不受异丙酚的影响。刺激平行纤维或自发产生的NMDAR介导的EPSCs成分不受1 mU M TPEN的影响。然而，在10 mU的米芬地尔存在下，它被减少了(类似于60%)，留下了残留的NMDAR介导的电流，呈现出快速衰减动力学。因此，这不太可能是由NR1/NR2D亚单位组成的受体引起的。我们的结论是，在小脑高尔基体细胞中，高电导和低电导的NMDAR通道分别来自NR2B和NR2D受体。我们在这些细胞中没有发现含有NR2A受体的证据。虽然包含NR2B的受体存在于突触和突触外的膜上，但我们的结果表明，NR1/NR2D受体不参与EPSC，似乎局限于突触外的膜。
1. To investigate the properties of N-methyl-D-aspartate receptors (NMDARs) in cerebellar Golgi cells, patch-clamp recordings were made in cerebellar slices from postnatal day 14 (P14) rats. To verify cell identity, cells were filled with Neurobiotin and examined using confocal microscopy.2. The NR2B subunit-selective NMDAR antagonist ifenprodil (10 mu M) reduced whole-cell NMDA-evoked currents by similar to 80 %. The NMDA-evoked currents were unaffected by the Zn(2+) chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN; 1 mu M) suggesting the absence of NMDARs containing NR2A subunits.3. Outside-out patches from Golgi cells exhibited a population of 'high-conductance' 50 pS NMDAR openings. These were inhibited by ifenprodil, with an IC(50) of 19 nM.4. Patches from these cells also contained 'low-conductance' NMDAR channels, with features characteristic of NR2D subunit-containing receptors. These exhibited a main conductance of 39 pS, with a sub-conductance level of 19 pS, with clear asymmetry of transitions between the two levels. As expected of NR2D-containing receptors, these events were not affected by ifenprodil.5. The NMDAR-mediated component of EPSCs, evoked by parallel fibre stimulation or occurring spontaneously, was not affected by 1 mu M TPEN. However, it was reduced (by similar to 60 %) in the presence of 10 mu M ifenprodil, to leave a residual NMDAR-mediated current that exhibited fast decay kinetics. This is, therefore, unlikely to have arisen from receptors composed of NR1/NR2D subunits.6. We conclude that in cerebellar Golgi cells, the high- and low-conductance NMDAR channels arise from NR2B- and NR2D-containing receptors, respectively. We found no evidence for NR2A-containing receptors in these cells. While NR2B-containing receptors are present in both the synaptic and extrasynaptic membrane, our results indicate that NR1/NR2D receptors do not contribute to the EPSC and appear to be restricted to the extrasynaptic membrane.