Heredia Dante J, Schubert Douglas, Maligireddy Siddhardha, Hennig Grant W, Gould Thomas W
Department of Physiology and Cell Biology, University of Nevada School of Medicine Reno, NV, USA.
Front Cell Neurosci. 2016 Dec 1;10:276. doi: 10.3389/fncel.2016.00276. eCollection 2016.
The failure to transmit neural action potentials (APs) into muscle APs is referred to as neuromuscular transmission failure (NTF). Although synaptic dysfunction occurs in a variety of neuromuscular diseases and impaired neurotransmission contributes to muscle fatigue, direct evaluation of neurotransmission by measurement of successfully transduced muscle APs is difficult due to the subsequent movements produced by muscle. Moreover, the voltage-gated sodium channel inhibitor used to study neurotransmitter release at the adult neuromuscular junction is ineffective in embryonic tissue, making it nearly impossible to precisely measure any aspect of neurotransmission in embryonic lethal mouse mutants. In this study we utilized 3-(N-butylethanimidoyl)-4-hydroxy-2H-chromen-2-one (BHC), previously identified in a small-molecule screen of skeletal muscle myosin inhibitors, to suppress movements without affecting membrane currents. In contrast to previously characterized drugs from this screen such as N-benzyl-p-toluene sulphonamide (BTS), which inhibit skeletal muscle myosin ATPase activity but also block neurotransmission, BHC selectively blocked nerve-evoked muscle contraction without affecting neurotransmitter release. This feature allowed a detailed characterization of neurotransmission in both embryonic and adult mice. In the presence of BHC, neural APs produced by tonic stimulation of the phrenic nerve at rates up to 20 Hz were successfully transmitted into muscle APs. At higher rates of phrenic nerve stimulation, NTF was observed. NTF was intermittent and characterized by successful muscle APs following failed ones, with the percentage of successfully transmitted muscle APs diminishing over time. Nerve stimulation rates that failed to produce NTF in the presence of BHC similarly failed to produce a loss of peak muscle fiber shortening, which was examined using a novel optical method of muscle fatigue, or a loss of peak cytosolic calcium transient intensity, examined in whole populations of muscle cells expressing the genetically-encoded calcium indicator GCaMP3. Most importantly, BHC allowed for the first time a detailed analysis of synaptic transmission, calcium signaling and fatigue in embryonic mice, such as in mutants reported here, that die before or at birth. Together, these studies illustrate the wide utility of BHC in allowing stable measurements of neuromuscular function.
神经动作电位（APs）无法转化为肌肉动作电位被称为神经肌肉传递失败（NTF）。尽管突触功能障碍在多种神经肌肉疾病中都会出现，且神经传递受损会导致肌肉疲劳，但由于肌肉产生的后续运动，通过测量成功转导的肌肉动作电位来直接评估神经传递是困难的。此外，用于研究成年神经肌肉接头处神经递质释放的电压门控钠通道抑制剂在胚胎组织中无效，这使得精确测量胚胎致死小鼠突变体中神经传递的任何方面几乎变得不可能。在本研究中，我们使用了3 -（N - 丁基乙亚胺基）- 4 - 羟基 - 2H - 色烯 - 2 - 酮（BHC），它先前在骨骼肌肌球蛋白抑制剂的小分子筛选中被鉴定出来，可抑制运动而不影响膜电流。与该筛选中先前表征的药物如N - 苄基 - 对甲苯磺酰胺（BTS）不同，BTS抑制骨骼肌肌球蛋白ATP酶活性但也阻断神经传递，而BHC选择性地阻断神经诱发的肌肉收缩而不影响神经递质释放。这一特性使得对胚胎和成年小鼠的神经传递进行详细表征成为可能。在BHC存在的情况下，以高达20 Hz的频率对膈神经进行强直刺激产生的神经动作电位成功转化为肌肉动作电位。在更高的膈神经刺激频率下，观察到了NTF。NTF是间歇性的，其特征是失败的肌肉动作电位之后跟着成功的肌肉动作电位，成功转导的肌肉动作电位的百分比随时间减少。在BHC存在下未能产生NTF的神经刺激频率同样未能导致使用新型肌肉疲劳光学方法检测到的肌肉纤维峰值缩短的损失，或在表达基因编码钙指示剂GCaMP3的整个肌肉细胞群体中检测到的细胞质钙瞬变峰值强度的损失。最重要的是，BHC首次使得对胚胎小鼠的突触传递、钙信号传导和疲劳进行详细分析成为可能，例如在此处报道的在出生前或出生时死亡的突变体中。总之，这些研究说明了BHC在实现神经肌肉功能稳定测量方面的广泛用途。
