Faculty of Health Sciences, Department of Physiology and Neurobiology, Ben-Gurion University of the Negev Beer-Sheva, Israel.
Front Neural Circuits. 2012 Feb 7;6:3. doi: 10.3389/fncir.2012.00003. eCollection 2012.
Deciphering the role of GABAergic neurons in large neuronal networks such as the neocortex forms a particularly complex task as they comprise a highly diverse population. The neuronal isoform of the enzyme nitric oxide synthase (nNOS) is expressed in the neocortex by specific subsets of GABAergic neurons. These neurons can be identified in live brain slices by the nitric oxide (NO) fluorescent indicator diaminofluorescein-2 diacetate (DAF-2DA). However, this indicator was found to be highly toxic to the stained neurons. We used this feature to induce acute phototoxic damage to NO-producing neurons in cortical slices, and measured subsequent alterations in parameters of cellular and network activity. Neocortical slices were briefly incubated in DAF-2DA and then illuminated through the 4× objective. Histochemistry for NADPH-diaphorase (NADPH-d), a marker for nNOS activity, revealed elimination of staining in the illuminated areas following treatment. Whole cell recordings from several neuronal types before, during, and after illumination confirmed the selective damage to non-fast-spiking (FS) interneurons. Treated slices displayed mild disinhibition. The reversal potential of compound synaptic events on pyramidal neurons became more positive, and their decay time constant was elongated, substantiating the removal of an inhibitory conductance. The horizontal decay of local field potentials (LFPs) was significantly reduced at distances of 300-400 μm from the stimulation, but not when inhibition was non-selectively weakened with the GABA(A) blocker picrotoxin. Finally, whereas the depression of LFPs along short trains of 40 Hz stimuli was linearly reduced with distance or initial amplitude in control slices, this ordered relationship was disrupted in DAF-treated slices. These results reveal that NO-producing interneurons in the neocortex convey lateral inhibition to neighboring columns, and shape the spatiotemporal dynamics of the network's activity.
解析 GABA 能神经元在新皮层等大型神经元网络中的作用是一项特别复杂的任务，因为它们构成了一个高度多样化的群体。酶一氧化氮合酶（nNOS）的神经元同工型由特定的 GABA 能神经元亚群在新皮层中表达。这些神经元可以通过一氧化氮（NO）荧光指示剂二氨基荧光素-2 二乙酸酯（DAF-2DA）在活体脑片中被识别。然而，这种指示剂被发现对染色神经元有很高的毒性。我们利用这一特性在皮质切片中诱导产生 NO 的神经元急性光毒性损伤，并测量随后细胞和网络活动参数的变化。新皮层切片在 DAF-2DA 中短暂孵育，然后通过 4×物镜进行照明。NADPH 黄递酶（NADPH-d）组织化学染色，nNOS 活性的标志物，显示处理后在照射区域消除了染色。在光照前后对几种神经元类型进行全细胞记录，证实了对非快速放电（FS）中间神经元的选择性损伤。处理过的切片显示轻度去抑制。复合突触事件在锥体神经元上的反转电位变得更加正，其衰减时间常数延长，证实了抑制性电导的去除。局部场电位（LFPs）的水平衰减在距刺激 300-400μm 的距离处显著降低，但当抑制作用不是通过 GABA(A) 阻滞剂胡椒碱非选择性减弱时，情况并非如此。最后，虽然在对照切片中，LFPs 沿 40Hz 刺激短串的抑制与距离或初始幅度呈线性关系，但在 DAF 处理的切片中，这种有序关系被打乱。这些结果表明，新皮层中产生 NO 的中间神经元向相邻柱传递侧向抑制，并形成网络活动的时空动力学。
