Jensen J B, Schousboe A, Pickering D S
PharmaBiotec Research Center, Department of Pharmacology, The Royal Danish School of Pharmacy, Copenhagen.
J Neurosci Res. 1998 Oct 15;54(2):273-81. doi: 10.1002/(SICI)1097-4547(19981015)54:2<273::AID-JNR15>3.0.CO;2-5.
The developmental expression of calcium (Ca2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in cultured neocortical neurons was evaluated by using cobalt uptake, a histochemical method that identifies cells expressing Ca2+-permeable, non-N-methyl-D-aspartate (non-NMDA) receptors. At a concentration of 500 microM, AMPA was found to stimulate cobalt uptake only late in development, resulting in staining of 2.7%+/-0.3% of the neurons maintained in culture for 12 days in vitro (DIV). When AMPA receptor desensitization was blocked with 50 microM cyclothiazide, the developmental profile of cobalt uptake mediated by 25 microM AMPA changed dramatically. The cobalt staining now appeared in young cultures (5 DIV), and the percentage of stained cells increased from 3.4%+/-0.2% at 5 DIV to 21.7%+/-1.6% at 12 DIV. The effect of 200 microM kainate was similar to that seen with 25 microM AMPA plus 50 microM cyclothiazide, resulting in 17.7%+/-0.3% stained neurons at 12 DIV. The cobalt uptake was specific to AMPA and kainate receptors because NMDA receptors and voltage-gated calcium channels were found not to mediate any cobalt staining. In addition, 10 microM 6-nitro-7-sulphamoylbenzo-[f]-quinoxaline-2,3-dione (NBQX) was able to prevent all staining at 5 and 8 DIV and most of the staining at 12 DIV, indicating that the non-NMDA ionotropic glutamate receptors are involved in cobalt uptake into the neurons. The AMPA receptor-selective antagonist GYKI 53655 was used to differentiate between cobalt influx through AMPA- or kainate-preferring receptors. After pretreatment with concanavalin A (con A), an inhibitor of kainate receptor desensitization, cobalt uptake was assessed after stimulation by 200 microM kainate in the presence of 25 microM GYKI 53655. No cobalt staining was observed under these conditions, indicating that most if not all of the cobalt influx induced by kainate was mediated through AMPA receptor channels.
通过使用钴摄取法，一种能识别表达钙离子通透、非N-甲基-D-天冬氨酸（非NMDA）受体细胞的组织化学方法，评估了培养的新皮质神经元中钙离子（Ca2+）通透的α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）和海人藻酸受体的发育表达情况。在500微摩尔浓度下，发现AMPA仅在发育后期刺激钴摄取，导致在体外培养12天（DIV）的神经元中有2.7%±0.3%被染色。当用50微摩尔环噻嗪阻断AMPA受体脱敏时，由25微摩尔AMPA介导的钴摄取的发育情况发生了显著变化。钴染色现在出现在年轻培养物（5 DIV）中，染色细胞的百分比从5 DIV时的3.4%±0.2%增加到12 DIV时的21.7%±1.6%。200微摩尔海人藻酸的作用与25微摩尔AMPA加50微摩尔环噻嗪的作用相似，在12 DIV时导致17.7%±0.3%的神经元被染色。钴摄取对AMPA和海人藻酸受体具有特异性，因为发现NMDA受体和电压门控钙通道不介导任何钴染色。此外，10微摩尔6-硝基-7-氨磺酰基苯并-[f]-喹喔啉-2,3-二酮（NBQX）能够在5和8 DIV时阻止所有染色，并在12 DIV时阻止大部分染色，表明非NMDA离子型谷氨酸受体参与了钴进入神经元的过程。使用AMPA受体选择性拮抗剂GYKI 53655来区分通过AMPA或海人藻酸偏好受体的钴内流。在用刀豆球蛋白A（con A）预处理，一种海人藻酸受体脱敏抑制剂后，在25微摩尔GYKI 53655存在下，用200微摩尔海人藻酸刺激后评估钴摄取。在这些条件下未观察到钴染色，表明海人藻酸诱导的大部分（如果不是全部）钴内流是通过AMPA受体通道介导的。
