Tempia F, Alojado M E, Strata P, Knöpfel T
Laboratory for Neuronal Circuit Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
J Neurophysiol. 2001 Sep;86(3):1389-97. doi: 10.1152/jn.2001.86.3.1389.
The metabotropic glutamate receptor 1 (mGluR(1)) plays a fundamental role in postnatal development and plasticity of ionotropic glutamate receptor-mediated synaptic excitation of cerebellar Purkinje cells. Synaptic activation of mGluR(1) by brief tetanic stimulation of parallel fibers evokes a slow excitatory postsynaptic current and an elevation of intracellular calcium concentration (Ca2+) in Purkinje cells. The mechanism underlying these responses has not been identified yet. Here we investigated the responses to synaptic and direct activation of mGluR(1) using whole cell patch-clamp recordings in combination with microfluorometric measurements of Ca2+ in mouse Purkinje cells. Following pharmacological block of ionotropic glutamate receptors, two to six stimuli applied to parallel fibers at 100 Hz evoked a slow inward current that was associated with an elevation of Ca2+. Both the inward current and the rise in Ca2+ increased in size with increasing number of pulses albeit with no clear difference between the minimal number of pulses required to evoke these responses. Application of the mGluR(1) agonist (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) by means of short-lasting (5-100 ms) pressure pulses delivered through an agonist-containing pipette positioned over the Purkinje cell dendrite, evoked responses resembling the synaptically induced inward current and elevation of Ca2+. No increase in Ca2+ was observed with inward currents of comparable amplitudes induced by the ionotropic glutamate receptor agonist AMPA. The 3,5-DHPG-induced inward current but not the associated increase in Ca2+ was depressed when extracellular Na+ was replaced by choline, but, surprisingly, both responses were also depressed when bathing the tissue in a low calcium (0.125 mM) or calcium-free/EGTA solution. Thapsigargin (10 microM) and cyclopiazonic acid (30 microM), inhibitors of sarco-endoplasmic reticulum Ca2+-ATPase, had little effect on either the inward current or the elevation in Ca2+ induced by 3,5-DHPG. Furthermore, the inward current induced by 3,5-DHPG was neither blocked by 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy] ethyl-1H-imidazole, an inhibitor of store operated calcium influx, nor by nimodipine or omega-agatoxin, blockers of voltage-gated calcium channels. These electrophysiological and Ca2+-imaging experiments suggest that the mGluR(1)-mediated inward current, although mainly carried by Na+, involves influx of Ca2+ from the extracellular space.
代谢型谷氨酸受体1（mGluR(1)）在出生后发育以及离子型谷氨酸受体介导的小脑浦肯野细胞突触兴奋的可塑性中发挥着重要作用。通过对平行纤维进行短暂强直刺激来使mGluR(1)发生突触激活，可在浦肯野细胞中诱发缓慢的兴奋性突触后电流以及细胞内钙浓度（Ca2+）升高。这些反应背后的机制尚未明确。在此，我们使用全细胞膜片钳记录结合对小鼠浦肯野细胞中Ca2+的显微荧光测量，研究了对mGluR(1)突触激活和直接激活的反应。在用药物阻断离子型谷氨酸受体后，以100Hz对平行纤维施加两到六个刺激会诱发一个缓慢的内向电流，该电流与Ca2+升高相关。内向电流和Ca2+的升高幅度均随着脉冲数量增加而增大，尽管诱发这些反应所需的最小脉冲数量之间没有明显差异。通过置于浦肯野细胞树突上方的含有激动剂的移液管施加持续时间较短（5 - 100毫秒）的压力脉冲来应用mGluR(1)激动剂（S）-3,5 - 二羟基苯甘氨酸（3,5 - DHPG），诱发的反应类似于突触诱导的内向电流和Ca2+升高。用离子型谷氨酸受体激动剂AMPA诱导出的幅度相当的内向电流未观察到Ca2+增加。当细胞外Na+被胆碱替代时，3,5 - DHPG诱导的内向电流受到抑制，但与之相关的Ca2+增加却未受影响，不过令人惊讶的是，当将组织置于低钙（0.125mM）或无钙/EGTA溶液中时，这两种反应也都受到抑制。毒胡萝卜素（10 microM）和环匹阿尼酸（30 microM），即肌浆内质网Ca2+ - ATP酶的抑制剂，对3,5 - DHPG诱导的内向电流或Ca2+升高几乎没有影响。此外，3,5 - DHPG诱导的内向电流既不被1 - [2 - （4 - 甲氧基苯基） - 2 - [3 - （4 - 甲氧基苯基）丙氧基]乙基 - 1H - 咪唑（一种储存操纵性钙内流抑制剂）阻断，也不被电压门控钙通道阻滞剂尼莫地平或ω - 芋螺毒素阻断。这些电生理和Ca2+成像实验表明，mGluR(1)介导的内向电流虽然主要由Na+携带，但涉及细胞外空间Ca2+ 的内流。
