Dean J B, Huang R Q, Erlichman J S, Southard T L, Hellard D T
Department of Physiology and Biophysics, Wright State University, School of Medicine, Dayton, OH 45435, USA.
Neuroscience. 1997 Sep;80(1):21-40. doi: 10.1016/s0306-4522(97)00016-x.
Dye (Lucifer Yellow) and tracer (Biocytin) coupling, referred to collectively as anatomical coupling, were identified in 20% of the solitary complex neurons tested in medullary tissue slices (120-350 microm) prepared from rat, postnatal day 1-18, using a modified amphotericin B-perforated patch recording technique. Ten per cent of the neurons sampled in nuclei outside the solitary complex were anatomically coupled. Fifty-eight per cent of anatomically coupled neurons exhibited electrotonic postsynaptic potential-like activity, which had peak-to-peak amplitudes of < or = 7 mV, with the same polarity as action potentials; increased and decreased in frequency during depolarizing and hyperpolarizing current injection; was maintained during high Mg2+-low Ca2+ chemical synaptic blockade; and was measured only in anatomically coupled neurons. The high correlation between anatomical coupling and electrotonic postsynaptic potential-like activity suggests that Lucifer Yellow, Biocytin and ionic current used the same pathways of intercellular communication, which were presumed to be gap junctions. Anatomical coupling was attributed solely to the junctional transfer of Lucifer Yellow and Biocytin since potential sources of non-junctional staining were minimized. Specifically, combining 0.26 mM amphotericin B and 0.15-0.5% Lucifer Yellow produced a hydrophobic, viscous solution that did not leak from the pressurized pipette tip < or = 3 microm outer diameter) submerged in artificial cerebral spinal fluid. Moreover, unintentional contact of the pipette tip with adjacent neurons that resulted in accidental staining, another source of non-junctional staining, wits averted by continuously visualizing the tip prior to tight seal formation with infrared video microscopy, used here for the first time with Hoffman modulation contrast optics. During perforated patch recording which typically lasted for 1-3 h. Lucifer Yellow was confined to the pipette, indicating that the amphotericin B patch was intact. However, once the patch was intentionally ruptured at the end of recording, the viscous, lipophilic solution entered the neuron resulting in double labeling. Placing a mixture of amphotericin B, Biocytin and Lucifer Yellow directly into the pipette tip did not compromise tight seal formation with an exposed, cleaned soma, and resulted in immediate (<1 min) steady-state perforation at 22-25 degrees C. This adaptation of conventional perforated patch recording was termed "rapid perforated patch recording". The possible functional implication of cell-cell coupling in the dorsal medulla oblongata in central CO2/H+ chemoreception for the cardiorespiratory control systems is discussed in the second paper of this set [Huang et al. (1997) Neuroscience 80, 41-57].
使用改良的两性霉素B穿孔膜片钳记录技术，在出生后1 - 18天大鼠制备的延髓组织切片（120 - 350微米）中，对孤立复合体神经元进行测试，结果发现20%的神经元存在染料（路西法黄）与示踪剂（生物胞素）偶联，二者统称为解剖学偶联。在孤立复合体之外的核中采样的神经元，有10%存在解剖学偶联。58%的解剖学偶联神经元表现出电紧张性突触后电位样活动，其峰峰值幅度≤7毫伏，与动作电位极性相同；在去极化和超极化电流注入期间频率增加和降低；在高镁低钙化学性突触阻断期间持续存在；且仅在解剖学偶联的神经元中可测量到。解剖学偶联与电紧张性突触后电位样活动之间的高度相关性表明，路西法黄（Lucifer Yellow）、生物胞素（Biocytin）和离子电流利用了相同的细胞间通讯途径，推测这些途径为缝隙连接。解剖学偶联仅归因于路西法黄和生物胞素的连接性转移，因为非连接性染色的潜在来源已降至最低。具体而言，将0.26毫摩尔/升两性霉素B与0.15 - 0.5%路西法黄混合，可产生一种疏水、粘性溶液，该溶液不会从浸没在人工脑脊液中的外径≤3微米的加压移液器尖端漏出。此外，通过在使用霍夫曼调制对比光学首次在此处使用的红外视频显微镜形成紧密封接之前持续观察移液器尖端，避免了移液器尖端与相邻神经元的意外接触导致的意外染色，这是另一种非连接性染色来源。在通常持续1 - 3小时的穿孔膜片钳记录过程中，路西法黄局限于移液器中，表明两性霉素B膜片完整。然而，一旦在记录结束时故意破坏膜片，粘性的亲脂性溶液就会进入神经元，导致双重标记。将两性霉素B、生物胞素和路西法黄的混合物直接放入移液器尖端，不会影响与暴露、清洁的胞体形成紧密封接，并在22 - 25℃下导致立即（<1分钟）达到稳态穿孔。这种对传统穿孔膜片钳记录的改进被称为“快速穿孔膜片钳记录”。本系列第二篇论文[Huang等人（1997年），《神经科学》80卷，41 - 57页]讨论了延髓背侧细胞 - 细胞偶联在中枢二氧化碳/氢离子化学感受中对心肺控制系统可能的功能意义。
