Berdan R C, Bulloch A G
Department of Physiology, University of Alberta, Edmonton, Canada.
Brain Res. 1990 Dec 24;537(1-2):241-50. doi: 10.1016/0006-8993(90)90364-h.
Our aim was to determine whether neural activity in the form of sodium-dependent action potentials play a role in the formation, maintenance and specificity of electrical synapses between regenerating neurons. We axotomized buccal neurons of the mollusc, Helisoma trivolvis, and placed ganglia into organ culture in the absence or presence of tetrodotoxin (TTX), a specific sodium channel blocker. Electrical coupling was measured using intracellular microelectrodes positioned within the soma of identified neurons. Neurite outgrowth was assessed by epifluorescence microscopy after filling neurons by iontophoresis with Lucifer yellow. Previous studies found that two days after axotomy transient electrical synapses form between heterologous neurons (e.g. buccal neurons 4 and 5). Five days after axotomy these transient connections disappeared and a new electrical synapse was stabilized between the paired buccal neurons 5. To determine whether blocking neural activity with TTX affected the specificity and formation of new electrical synapses, we examined electrical coupling between the heterologous neurons 4 and 5 two days after axotomy, and the paired buccal neurons 5 five days after axotomy. Our electrophysiological recordings indicated that different neurons in the buccal ganglion varied in their sensitivity to TTX (i.e. sensitivity of buccal neurons 19 greater than 5 greater than 4), but spontaneous activity was abolished in all 3 neurons by 2 x 10(-5) M TTX. Furthermore, the inhibitory effects of TTX occurred within seconds of superfusion and persisted for at least 6 days. Inhibition of activity by TTX could be reversed after superfusion with normal saline. Neurite outgrowth from axotomized neurons was not appreciably altered in the presence of TTX. Furthermore, no differences in the incidence of electrical coupling or the coupling resistance were detected between neurons 4 and 5 two days after axotomy and organ culture in the presence of TTX. However, electrical coupling between the symmetrically paired neurons 5 was elevated in the presence of TTX after 5 days. We conclude from these results that neural activity in the form of sodium-dependent action potentials does not play an important role in the formation or breaking of transient electrical synapses during neuronal regeneration in the mollusc Helisoma trivolvis.
我们的目的是确定以钠依赖性动作电位形式存在的神经活动是否在再生神经元之间电突触的形成、维持和特异性方面发挥作用。我们切断了软体动物三角帆蚌颊神经元的轴突，并将神经节置于有无河豚毒素（TTX，一种特异性钠通道阻滞剂）的器官培养中。使用位于已识别神经元胞体中的细胞内微电极测量电耦合。在用离子电泳法用荧光黄填充神经元后，通过落射荧光显微镜评估神经突生长。先前的研究发现，轴突切断后两天，异源神经元（如颊神经元4和5）之间形成短暂的电突触。轴突切断后五天，这些短暂连接消失，成对的颊神经元5之间形成新的稳定电突触。为了确定用TTX阻断神经活动是否会影响新电突触的特异性和形成，我们在轴突切断后两天检查了异源神经元4和5之间的电耦合，以及轴突切断后五天成对的颊神经元5之间的电耦合。我们的电生理记录表明，颊神经节中的不同神经元对TTX的敏感性不同（即颊神经元19的敏感性大于5大于4），但2×10⁻⁵ M TTX使所有3个神经元的自发活动均消失。此外，TTX的抑制作用在灌注后数秒内出现，并持续至少6天。用生理盐水灌注后，TTX对活动的抑制作用可逆转。在TTX存在的情况下，轴突切断神经元的神经突生长没有明显改变。此外，在轴突切断后两天以及在TTX存在的器官培养中，神经元4和5之间的电耦合发生率或耦合电阻没有差异。然而，五天后在TTX存在的情况下，对称成对的神经元5之间电耦合增强。从这些结果我们得出结论，在三角帆蚌神经元再生过程中，以钠依赖性动作电位形式存在的神经活动在短暂电突触的形成或破坏中不发挥重要作用。
