A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae
A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae
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DOI：
10.1080/01677060490894522
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发表时间：
2004-04-01
影响因子：
1.9
通讯作者：
Wu, CF
中科院分区：
文献类型：
--
作者：
Feng, YF;Ueda, A;Wu, CF
血淋巴样HL 3盐水（Stewart等人，1994）和标准盐水（Jan & Jan，1976）是两种广泛用于果蝇幼虫神经肌肉接头生理记录的沐浴溶液。已经确定，在HL 3盐水中更好地维持幼虫制剂的寿命。然而，HL 3可以产生与先前在标准盐水中的发现不一致的结果，特别是在温度敏感性和膜兴奋性表型上。在野生型幼虫中，在标准盐水（含有4 mM Mg 2+和1.8mM Ca 2+）中的兴奋性连接电位（EJPs）不被温度升高至39- 40 ℃所阻断，这与低于这些温度的未受损幼虫运动一致。在30 ℃时，在HL 3生理盐水（含20 mM Mg ~（2+）和1.5mM Ca ~（2+））中，EJPs被阻断。对于温度敏感突变体nap（ts）和帕拉（ts），EJP阻断温度分别从标准盐水中的约29和33 ℃降低到HL 3中的约23和26 ℃。复合动作电位记录证实，节段性神经动作电位更容易被阻断的温度升高，在HL 3比在标准盐水。轴突兴奋性抑制HL 3，即使在室温下，证明了在野生型幼虫的不应期延长。类似的抑制发生在超兴奋的双突变体Eag Sh，其在标准盐水中保持高频率的自发EJP，但在HL 3中显示出快速下降的EJP频率。HL 3生理盐水的应用也强烈地抑制了在通过K+通道阻断剂或通过先前在标准生理盐水中描述的eag Sh突变去除复极化机制后的延长的递质释放。这些差异表明，高的二价阳离子含量在HL 3可能赋予表面电荷屏蔽效应，抑制神经膜兴奋性。我们发现，对HL 3盐水进行最小调整，将Mg 2+离子浓度从20 mM降低至4 mM，足以解决差异。在保留幼虫神经肌肉制备物的寿命的同时，改良的HL 3盐水（HL3.1）恢复了已建立的野生型EJP特性以及先前在标准盐水中记录的几种广泛使用的温度敏感和超兴奋突变体的表型。
The hemolymph-like HL3 saline (Stewart et al., 1994) and standard saline (Jan & Jan, 1976) are two widely used bathing solutions for physiological recordings at the Drosophila larval neuromuscular junction. It has been established that longevity of larval preparations is better maintained in HL3 saline. However, HL3 can produce results that are inconsistent with previous findings in standard saline, particularly on temperature sensitivity and membrane excitability phenotypes. In wild-type larvae, the excitatory junctional potentials (EJPs) in standard saline (containing 4mM Mg2+ and 1.8mM Ca2+) were not blocked by a temperature increase up to 39-40degreesC, consistent with unimpaired larval locomotion below these temperatures. However, in HL3 saline (containing 20mM Mg2+ and 1.5mM Ca2+), EJPs were blocked at 30degreesC. As for temperature-sensitive mutants nap(ts) and para(ts), the EJP-blocking temperatures were decreased from about 29 and 33degreesC in standard saline to about 23 and 26degreesC in HL3, respectively. Compound action potential recordings confirmed that segmental nerve action potentials were more readily blocked by a temperature increase in HL3 than in standard saline. Axonal excitability was suppressed in HL3 even at room temperatures, as evidenced by a lengthened refractory period in wild-type larvae. Similar suppression occurred for the hyper-excitable double mutant eag Sh, which maintained high-frequency spontaneous EJPs in standard saline but showed a rapidly declining EJP frequency in HL3. Application of HL3 saline also strongly suppressed the prolonged transmitter release following removal of repolarization mechanisms by K+ channel blockers or by the eag Sh mutation previously described in standard saline. These discrepancies suggest that the high divalent cation content in HL3 may confer a surface charge screening effect to suppress nerve membrane excitability. We found that a minimal adjustment of the HL3 saline, decreasing the Mg2+ ion concentration from 20 to 4mM, was sufficient to resolve the discrepancies. While retaining the longevity of the larval neuromuscular preparation, the modified HL3 saline (HL3.1) restored the established wild-type EJP properties as well as phenotypes of several widely used temperature-sensitive and hyper-excitable mutants previously documented in standard saline.