Leininger Eric, Belousov Andrei B
Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USA.
Brain Res. 2009 Jan 28;1251:87-102. doi: 10.1016/j.brainres.2008.11.044. Epub 2008 Nov 25.
Previous studies indicated that a long-term decrease in the activity of ionotropic glutamate receptors induces cholinergic activity in rat and mouse hypothalamic neuronal cultures. Here we studied whether a prolonged inactivation of ionotropic glutamate receptors also induces cholinergic activity in hippocampal neurons. Receptor activity was chronically suppressed in rat hippocampal primary neuronal cultures with two proportionally increasing sets of concentrations of NMDA plus non-NMDA receptor antagonists: 100 microM/10 microM AP5/CNQX (1X cultures) and 200 microM/20 microM AP5/CNQX (2X cultures). Using calcium imaging we demonstrate that cholinergic activity does not develop in these cultures. Instead, network-driven glutamate-dependent activity, that normally is detected in hyper-excitable conditions, reappears in each culture group in the presence of these antagonists and can be reversibly suppressed by higher concentrations of AP5/CNQX. This activity is mediated by non-NMDA receptors and is modulated by NMDA receptors. Further, non-NMDA receptors, the general level of glutamate receptor activity and CaMK-dependent signaling are critical for development of this network-driven glutamatergic activity in the presence of receptor antagonists. Using electrophysiology, western blotting and calcium imaging we show that some neuronal parameters are either reduced or not affected by chronic glutamate receptor blockade. However, other parameters (including neuronal excitability, mEPSC frequency, and expression of GluR1, NR1 and betaCaMKII) become up-regulated and, in some cases, proportionally between the non-treated, 1X and 2X cultures. Our data suggest recovery of the network-driven glutamatergic activity after chronic glutamate receptor blockade. This recovery may represent a form of neuronal plasticity that compensates for the prolonged suppression of the activity of glutamate receptors.
先前的研究表明，离子型谷氨酸受体活性的长期降低会在大鼠和小鼠下丘脑神经元培养物中诱导胆碱能活性。在此，我们研究了离子型谷氨酸受体的长期失活是否也会在海马神经元中诱导胆碱能活性。在大鼠海马原代神经元培养物中，通过两组浓度成比例增加的NMDA加非NMDA受体拮抗剂来长期抑制受体活性：100微摩尔/10微摩尔AP5/CNQX（1倍培养物）和200微摩尔/20微摩尔AP5/CNQX（2倍培养物）。使用钙成像技术，我们证明在这些培养物中不会产生胆碱能活性。相反，通常在过度兴奋条件下检测到的由网络驱动的谷氨酸依赖性活性，在存在这些拮抗剂的情况下，在每个培养组中重新出现，并且可以被更高浓度的AP5/CNQX可逆性抑制。这种活性由非NMDA受体介导，并受NMDA受体调节。此外，在存在受体拮抗剂的情况下，非NMDA受体、谷氨酸受体活性的总体水平以及CaMK依赖性信号传导对于这种由网络驱动的谷氨酸能活性的发展至关重要。使用电生理学、蛋白质印迹法和钙成像技术，我们表明一些神经元参数要么降低，要么不受慢性谷氨酸受体阻断的影响。然而，其他参数（包括神经元兴奋性、微小兴奋性突触后电流频率以及GluR1、NR1和βCaMKII的表达）会上调，并且在某些情况下，在未处理的、1倍和2倍培养物之间成比例上调。我们的数据表明，慢性谷氨酸受体阻断后，由网络驱动的谷氨酸能活性会恢复。这种恢复可能代表了一种神经元可塑性形式，可补偿谷氨酸受体活性的长期抑制。
