A rapid and sensitive assay of intercellular coupling by voltage imaging of gap junction networks

Cell Communication and Signaling

Methodology

Federico Ceriani¹  Fabio Mammano² 

[1] Dipartimento di Fisica e Astronomia “G. Galilei”, Università di Padova, 35131, Padova, Italy;Istituto Veneto di Medicina Molecolare, Fondazione per la Ricerca Biomedica Avanzata, Via G. Orus, 2, 35129, Padova, Italy;Dipartimento di Fisica e Astronomia “G. Galilei”, Università di Padova, 35131, Padova, Italy;Istituto Veneto di Medicina Molecolare, Fondazione per la Ricerca Biomedica Avanzata, Via G. Orus, 2, 35129, Padova, Italy;Istituto di Neuroscienze, Consiglio Nazionale delle Ricerche, 35131, Padova, Italy;

关键词: Connexins;    Electrical coupling;    Dye coupling;    Genetic deafness;    Voltage sensitive dye;    Digital phase−sensitive detector;   

DOI  :  10.1186/1478-811X-11-78

 received in 2013-08-30, accepted in 2013-10-01,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundA variety of mechanisms that govern connexin channel gating and permeability regulate coupling in gap junction networks. Mutations in connexin genes have been linked to several pathologies, including cardiovascular anomalies, peripheral neuropathy, skin disorders, cataracts and deafness. Gap junction coupling and its patho–physiological alterations are commonly assayed by microinjection experiments with fluorescent tracers, which typically require several minutes to allow dye transfer to a limited number of cells. Comparable or longer time intervals are required by fluorescence recovery after photobleaching experiments. Paired electrophysiological recordings have excellent time resolution but provide extremely limited spatial information regarding network connectivity.ResultsHere, we developed a rapid and sensitive method to assay gap junction communication using a combination of single cell electrophysiology, large–scale optical recordings and a digital phase–sensitive detector to extract signals with a known frequency from Vf2.1.Cl, a novel fluorescent sensor of plasma membrane potential. Tests performed in HeLa cell cultures confirmed that suitably encoded Vf2.1.Cl signals remained confined within the network of cells visibly interconnected by fluorescently tagged gap junction channels. We used this method to visualize instantly intercellular connectivity over the whole field of view (hundreds of cells) in cochlear organotypic cultures from postnatal mice. A simple resistive network model reproduced accurately the spatial dependence of the electrical signals throughout the cellular network. Our data suggest that each pair of cochlear non−sensory cells of the lesser epithelial ridge is coupled by ~1500 gap junction channels, on average. Junctional conductance was reduced by 14% in cochlear cultures harboring the T5M mutation of connexin30, which induces a moderate hearing loss in connexin30T5M/T5M knock–in mice, and by 91% in cultures from connexin30−/− mice, which are profoundly deaf.ConclusionsOur methodology allows greater sensitivity (defined as the minimum magnitude of input signal required to produce a specified output signal having a specified signal−to−noise ratio) and better time resolution compared to classical tracer–based techniques. It permitted us to dynamically visualize intercellular connectivity down to the 10th order in non−sensory cell networks of the developing cochlea. We believe that our approach is of general interest and can be seamlessly extended to a variety of biological systems, as well as to other connexin−related disease conditions.

【 授权许可】

CC BY   
© Ceriani and Mammano; licensee BioMed Central Ltd. 2013