Kulik A, Nishimaru H, Ballanyi K
Physiologisches Institut, Universität Göttingen, D-37073 Göttingen, Germany.
J Neurosci. 2000 Nov 1;20(21):7905-13. doi: 10.1523/JNEUROSCI.20-21-07905.2000.
Ca(2+) imaging and (perforated) patch recording were used to analyze the mechanism of GABA- and glycine-induced depolarizations in lumbar motoneurons of spinal cord slices from fetal rats. In fura-2 ester-loaded cells, the agonist-induced depolarizations increased Ca(2+) by up to 100 nm. The GABA- and glycine-evoked Ca(2+) transients were suppressed by bicuculline and strychnine, respectively. Their magnitude decreased by approximately 50% between embryonic days 15.5 and 19.5. The Ca(2+) increases were abolished by Ca(2+)-free superfusate and attenuated by approximately 65% by nifedipine, showing that the responses were mediated by voltage-activated Ca(2+) channels. The Ca(2+) rises were potentiated by >300% immediately after removal of Cl(-) from the superfusate but recovered to values of 50-200% of control during repeated agonist administration in Cl(-)-free saline. Bumetanide gradually suppressed the Ca(2+) increases by >75%. Subsequent removal of Cl(-) reconstituted the responses and increased, upon repeated agonist application, the peak Ca(2+) rises to values above control. Removal of HCO(3)(-) from the Cl(-)-free (bumetanide-containing) superfusate reversibly abolished both the agonist-induced Ca(2+) rises and depolarizations that were reestablished by formate anions. In Cl(-)-containing superfusate, removal of HCO(3)(-) decreased both the peak and duration of the agonist-evoked membrane depolarization and Ca(2+) response. Our findings show that HCO(3)(-) efflux has a major contribution to depolarizations mediated by GABA(A) and glycine receptor-coupled anion channels in prenatal neurons. We hypothesize that the HCO(3)(-)-dependent depolarizing component, which is likely to produce an intracellular acidosis, might play an important role during the early postnatal period when the Cl(-)-dependent component gradually shifts to hyperpolarization.
采用钙离子成像和（穿孔）膜片钳记录技术，分析γ-氨基丁酸（GABA）和甘氨酸诱导的胎鼠脊髓切片腰段运动神经元去极化的机制。在装载了fura-2酯的细胞中，激动剂诱导的去极化使细胞内钙离子浓度（Ca(2+)）升高达100 nM。GABA和甘氨酸诱发的Ca(2+)瞬变分别被荷包牡丹碱和士的宁抑制。在胚胎第15.5天至19.5天之间，它们的幅度下降了约50%。无钙灌流液可消除Ca(2+)的升高，硝苯地平使其减弱约65%，表明这些反应是由电压激活的钙离子通道介导的。从灌流液中去除氯离子后，Ca(2+)的升高立即增强>300%，但在无氯盐水中重复给予激动剂期间恢复到对照值的50 - 200%。布美他尼逐渐抑制Ca(2+)的升高>75%。随后去除氯离子可恢复反应，并在重复给予激动剂时使Ca(2+)的峰值升高超过对照值。从无氯（含布美他尼）灌流液中去除碳酸氢根离子可逆地消除了激动剂诱导的Ca(2+)升高和由甲酸根离子重建的去极化。在含氯离子的灌流液中，去除碳酸氢根离子可降低激动剂诱发的膜去极化峰值和持续时间以及Ca(2+)反应。我们的数据表明，碳酸氢根离子外流对产前神经元中由GABA(A)和甘氨酸受体偶联阴离子通道介导的去极化有主要贡献。我们推测，依赖碳酸氢根离子的去极化成分可能在出生后早期发挥重要作用，此时依赖氯离子的成分逐渐转变为超极化，这种成分可能会导致细胞内酸中毒。
