Graf R A, Cooke I M
Békésy Laboratory of Neurobiology, University of Hawaii at Manoa, Honolulu 96822.
J Neurobiol. 1994 Dec;25(12):1558-69. doi: 10.1002/neu.480251208.
Peptide-secreting neurons from crustacean X-organ regenerating in defined culture possess different ionic current profiles correlated with two distinct morphological types, veiling and branching; voltage-dependent Ca2+ current is prominent in neurons consistently extending large veils, but is small in neurons that repetitively branch. Intracellular free calcium levels ([Ca2+]i) have been implicated in the regulation of neurite outgrowth underlying the establishment of distinct morphologies. Here, basal [Ca2+]i was measured by fura-2 fluorescence ratio imaging from these morphologically distinct neurons and compared. Both morphological types can extend out processes over a [Ca2+]i range (approximately 50 to 300 nM) that is much greater than that reported for neurons of other phyla. Application of high K+ saline led to increases in [Ca2+]i in soma, neurite, and lamellipodium of veiling neurons. Increases were greater for veiling than branching neurons. These observations were consistent with the previous voltage clamp data for calcium currents. Media altered to perturb [Ca2+]i were used to assess the role of [Ca2+]i in veiling or branching outgrowth programs. Outgrowth of veiling cells was arrested by addition of 100 microM Cd2+, a calcium channel blocker. Outgrowth resumed following brief exposures to Cd2+. Branching neurons were unaffected by Cd2+. Cd2+ at lower levels (10 microM) had no effect on outgrowth of either neuronal type, whereas at higher levels (1 mM), outgrowth of both types was arrested. Reduction of extracellular sodium to 0.001 of normal concentration stopped veiling outgrowth, but branching outgrowth continued, although it was less robust. Addition of tetrodotoxin (1 microM) did not alter outgrowth of either neuronal type relative to controls. Thus, peptidergic neurons of differing intrinsic morphologies maintain similar basal [Ca2+]i levels under identical culture conditions, yet show differing sensitivities to manipulations influencing [Ca2+]i with respect to regenerative outgrowth, but not its form.
在特定培养条件下再生的甲壳类X器官中分泌肽的神经元具有不同的离子电流特征，这与两种不同的形态类型相关，即覆盖型和分支型；电压依赖性Ca2+电流在持续延伸大覆盖物的神经元中很突出，但在反复分支的神经元中很小。细胞内游离钙水平（[Ca2+]i）被认为参与了不同形态形成过程中神经突生长的调节。在此，通过fura-2荧光比率成像测量了这些形态不同的神经元的基础[Ca2+]i，并进行了比较。两种形态类型都能在一个[Ca2+]i范围内（约50至300 nM）伸出突起，这个范围比其他门类神经元的报道范围大得多。应用高钾盐水会导致覆盖型神经元的胞体、神经突和片状伪足中的[Ca2+]i增加。覆盖型神经元的增加幅度大于分支型神经元。这些观察结果与先前关于钙电流的电压钳数据一致。改变培养基以扰乱[Ca2+]i，用于评估[Ca2+]i在覆盖型或分支型生长程序中的作用。添加100 microM Cd2+（一种钙通道阻滞剂）会使覆盖型细胞的生长停止。短暂暴露于Cd2+后生长恢复。分支型神经元不受Cd2+影响。较低水平（10 microM）的Cd2+对两种神经元类型的生长均无影响，而在较高水平（1 mM）时，两种类型的生长均停止。将细胞外钠浓度降低至正常浓度的0.001会阻止覆盖型生长，但分支型生长仍会继续，尽管其强度较弱。添加河豚毒素（1 microM）相对于对照组而言，对两种神经元类型的生长均无改变。因此，不同内在形态的肽能神经元在相同培养条件下维持相似的基础[Ca2+]i水平，但在影响[Ca2+]i的操作对再生生长（而非其形式）方面表现出不同的敏感性。
