Chiacchiaretta Martina, Latifi Shahrzad, Bramini Mattia, Fadda Manuela, Fassio Anna, Benfenati Fabio, Cesca Fabrizia
Center for Synaptic Neuroscience and Technology, Fondazione Istituto Italiano di Tecnologia, Largo Rosanna Benzi 10, Genova 16132, Italy.
Department of Experimental Medicine, University of Genova, Viale Benedetto XV 3, Genova 16132, Italy.
J Cell Sci. 2017 Apr 15;130(8):1435-1449. doi: 10.1242/jcs.198564. Epub 2017 Mar 2.
Extracellular pH impacts on neuronal activity, which is in turn an important determinant of extracellular H concentration. The aim of this study was to describe the spatio-temporal dynamics of extracellular pH at synaptic sites during neuronal hyperexcitability. To address this issue we created ex.EGFP, a membrane-targeted extracellular ratiometric pH indicator that is exquisitely sensitive to acidic shifts. By monitoring ex.EGFP fluorescence in real time in primary cortical neurons, we were able to quantify pH fluctuations during network hyperexcitability induced by convulsant drugs or high-frequency electrical stimulation. Sustained hyperactivity caused a pH decrease that was reversible upon silencing of neuronal activity and located at active synapses. This acidic shift was not attributable to the outflow of synaptic vesicle H into the cleft nor to the activity of membrane-exposed H V-ATPase, but rather to the activity of the Na/H-exchanger. Our data demonstrate that extracellular synaptic pH shifts take place during epileptic-like activity of neural cultures, emphasizing the strict links existing between synaptic activity and synaptic pH. This evidence may contribute to the understanding of the physio-pathological mechanisms associated with hyperexcitability in the epileptic brain.
细胞外pH值会影响神经元活动，而神经元活动反过来又是细胞外H⁺浓度的一个重要决定因素。本研究的目的是描述神经元兴奋性过高期间突触部位细胞外pH值的时空动态变化。为了解决这个问题，我们创建了ex.EGFP，一种靶向细胞膜的细胞外比率型pH指示剂，它对酸性变化极为敏感。通过实时监测原代皮层神经元中的ex.EGFP荧光，我们能够量化惊厥药物或高频电刺激诱导的网络兴奋性过高期间的pH值波动。持续的过度活动导致pH值下降，在神经元活动沉默后这种下降是可逆的，且发生在活跃突触处。这种酸性变化并非归因于突触囊泡H⁺流入突触间隙，也不是由于膜表面H⁺-V-ATP酶的活性，而是由于Na⁺/H⁺交换体的活性。我们的数据表明，在神经培养物的癫痫样活动期间会发生细胞外突触pH值变化，强调了突触活动与突触pH值之间存在的紧密联系。这一证据可能有助于理解与癫痫大脑中兴奋性过高相关的生理病理机制。
