Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School, Rutgers University, 185 South Orange Avenue, Newark, NJ 07101-1709, United States; School of Graduate Studies, Rutgers Biomedical and Health Sciences Campus-Newark, Rutgers University, 185 South Orange Avenue, Newark, NJ 07101-1709, United States.
Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School, Rutgers University, 185 South Orange Avenue, Newark, NJ 07101-1709, United States.
Brain Res. 2022 Jul 15;1787:147919. doi: 10.1016/j.brainres.2022.147919. Epub 2022 Apr 15.
A technique combining fluorescence imaging with Ca indicators and single-cell laser scanning photostimulation of caged glutamate (LSPS) allowed identification of functional connections between individual neurons in mixed cultures of rat neocortical cells as well as observation of synchronous spontaneous activity among neurons. LSPS performed on large numbers of neurons yielded maps of functional connections between neurons and allowed calculation of neuronal network parameters. LSPS also provided an indirect measure of excitability of neurons targeted for photostimulation. By repeating LSPS sessions with the same neurons, stability of connections and change in the number and strength of connections were also determined. Experiments were conducted in the presence of bicuculline to study in detail the properties of excitatory neurotransmission. The AMPA receptor inhibitor, 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), abolished synchronous neuronal activity but had no effect on connections mapped by LSPS. In contrast, the NMDA receptor inhibitor, 2-Amino-5-phosphono-pentanoic acid (APV), dramatically decreased the number of functional connections between neurons while also affecting synchronous spontaneous activity. Functional connections were also decreased by increasing extracellular Mg concentration. These data demonstrated that LSPS mapping interrogates NMDA receptor-dependent connectivity between neurons in the network. In addition, a GluN2A-specific inhibitor, NVP-AAM077, decreased the number and strength of connections between neurons as well as neuron excitability. Conversely, the GluN2A-specific positive modulator, GNE-0723, increased these same properties. These data showed that LSPS can be used to directly study perturbations in the properties of NMDA receptor-dependent connectivity in neuronal networks. This approach should be applicable in a wide variety of in vitro and in vivo experimental preparations.
一种将荧光成像与 Ca 指示剂以及光解笼式谷氨酸（LSPS）的单细胞激光扫描光刺激相结合的技术，使得在大鼠新皮层细胞混合培养物中鉴定单个神经元之间的功能连接以及观察神经元之间的同步自发活动成为可能。对大量神经元进行 LSPS 可生成神经元之间功能连接的图谱，并允许计算神经元网络参数。LSPS 还提供了针对光刺激的目标神经元兴奋性的间接测量。通过对相同神经元重复进行 LSPS 会话，可以确定连接的稳定性以及连接数量和强度的变化。实验是在使用 Bicuculline 的情况下进行的，以详细研究兴奋性神经传递的特性。AMPA 受体抑制剂 6-氰基-7-硝基喹喔啉-2,3-二酮（CNQX）消除了同步神经元活动，但对 LSPS 映射的连接没有影响。相比之下，NMDA 受体抑制剂 2-氨基-5-磷戊酸（APV）显著减少了神经元之间的功能连接数量，同时也影响了同步自发活动。增加细胞外 Mg 浓度也会减少功能连接。这些数据表明 LSPS 映射询问了网络中神经元之间 NMDA 受体依赖性连接的特性。此外，GluN2A 特异性抑制剂 NVP-AAM077 降低了神经元之间的连接数量和强度以及神经元兴奋性。相反，GluN2A 特异性正调节剂 GNE-0723 增加了这些相同的特性。这些数据表明，LSPS 可用于直接研究 NMDA 受体依赖性连接特性的扰动在神经元网络中。这种方法应该适用于各种体外和体内实验制剂。
