Université Paris Descartes, 75006 Paris, France.
Neuropharmacology. 2012 Sep;63(4):624-34. doi: 10.1016/j.neuropharm.2012.05.010. Epub 2012 May 17.
Photolysis is widely used in experimental neuroscience to isolate post-synaptic receptor activation from presynaptic processes, to determine receptor mechanisms in situ, for pharmacological dissection of signaling pathways, or for photostimulation/inhibition in neural networks. We have evaluated new caged neuroactive amino acids that use 4-methoxy-7-nitroindolinyl- (MNI) or 1-(2-nitrophenyl)ethoxycarbonyl (NPEC) photoprotecting groups to make caged ligands specific for glutamate receptor sub-types. Each was tested for interference with synaptic transmission and excitability and for receptor-specific actions in slice preparations. No adverse effects were found at glutamate receptors. At high concentration, MNI-caged, but not NPEC-caged ligands, interfered with GABA-ergic transmission. MNI-caged amino acids have sub-microsecond release times suitable for investigating mechanisms at fast synaptic receptors in situ. MNI-NMDA and MNI-kainate were synthesized and tested. MNI-NMDA showed stoichiometric release of chirally pure NMDA. Wide-field photolysis in cerebellar interneurons produced a fast-rising sustained activation of NMDA receptors, and localized laser photolysis gave a fast, transient response. Photolysis of MNI-kainate to release up to 4 μM kainate generated large inward currents at resting membrane potential in Purkinje neurons. Application of GYKI 53655 indicated that 40% of the current was due to AMPA receptor activation by kainate. Signaling via metabotropic glutamate receptors (mGluR) does not require fast release rates. NPEC cages are simpler to prepare but have slower photorelease. Photolysis of NPEC-ACPD or NPEC-DHPG in Purkinje neurons generated slow inward currents blocked by the mGluR type 1 antagonist CPCCOEt similar to the slow sEPSC seen with parallel fiber burst stimulation. NPEC-AMPA was also tested in Purkinje neurons and showed large sustained inward currents selective for AMPA receptors with little activation of kainate receptors. MNI-caged l-glutamate, NMDA and kainate inhibit GABA-A receptors with IC₅₀ concentrations close to the maximum concentrations useful in receptor signaling experiments.
光解广泛应用于实验神经科学，用于将突触后受体激活与突触前过程分离，以确定原位受体机制，用于信号通路的药理学剖析，或用于神经网络的光刺激/抑制。我们评估了新的笼形神经活性氨基酸，它们使用 4-甲氧基-7-硝基吲哚基（MNI）或 1-（2-硝基苯基）乙氧基羰基（NPEC）光保护基团来制作针对谷氨酸受体亚型的笼形配体。每种配体都经过测试，以确定其对突触传递和兴奋性的干扰以及在切片制剂中的受体特异性作用。在谷氨酸受体上未发现不良反应。在高浓度下，MNI-笼形，但不是 NPEC-笼形配体，干扰 GABA 能传递。MNI-笼形氨基酸具有亚微秒的释放时间，适合原位研究快速突触受体的机制。合成并测试了 MNI-NMDA 和 MNI- kainate。MNI-NMDA 显示出手性纯 NMDA 的化学计量释放。小脑中间神经元的宽场光解产生快速上升的 NMDA 受体持续激活，而局部激光光解则产生快速瞬态响应。用 MNI-kainate 光解释放高达 4 μM kainate，在浦肯野神经元的静息膜电位下产生大的内向电流。应用 GYKI 53655 表明，电流的 40%是由 kainate 激活 AMPA 受体引起的。代谢型谷氨酸受体（mGluR）的信号传递不需要快速释放速率。NPEC 笼形物更易于制备，但光释放速度较慢。在浦肯野神经元中用光解 NPEC-ACPD 或 NPEC-DHPG 产生的内向电流被 mGluR 类型 1 拮抗剂 CPCCOEt 阻断，类似于平行纤维爆发刺激时观察到的缓慢 sEPSC。还在浦肯野神经元中测试了 NPEC-AMPA，发现它对 AMPA 受体具有大的持续内向电流，对 kainate 受体的激活作用很小。MNI-笼形 l-谷氨酸、NMDA 和 kainate 以接近用于受体信号转导实验的最大浓度抑制 GABA-A 受体。
