Simultaneous whole-cell recording and calcium imaging to reveal electrically coupled neurons in Xenopus tadpoles

Neuronal populations connected by gap junctions can be revealed via dye coupling of small molecules like neurobiotin and lucifer yellow. However, the extent of dye diffusion between neurons varies with connexin subtype, loading method, and neuromodulation. Due to the increasing availability of GCaMP transgenic animals, we explore the possibility of revealing gap junctional coupling using Ca2+ imaging in the Xenopus laevis tadpole motor system. Reliable axo-axonal electrical coupling was previously found in excitatory descending interneurons (dINs) using paired recordings but not with neurobiotin dye coupling. Here, we made whole-cell patch-clamp recordings with Ca2+-supplemented intracellular solution to load Ca2+ into GCaMP6s-expressing neurons, followed by Ca2+ imaging to detect potential Ca2+ diffusion across coupled neurons. Successful membrane breakthroughs led to transient fluorescence increases in the patched neuron. However, increasing the Ca2+ concentration promoted membrane resealing and rapid loss of whole-cell recordings. Regardless of recording duration, loading-triggered fluorescence only lasted up to three minutes, suggesting rapid Ca2+ clearance. Pharmacologically blocking sarcoplasmic /endoplasmic reticulum Ca2+-ATPases and plasma membrane Na+/Ca2+ exchangers did not prolong fluorescence, although sustained fluorescence was achieved with positive current injections. Counter to our expectations, fluorescence increases in Ca2+-loaded dINs did not spread to neighboring dINs. Robust intracellular Ca2+ regulation mechanisms, membrane resealing, and long dIN axons likely hindered intercellular Ca2+ diffusion. Therefore, this approach is not appropriate for revealing electrical coupling within this system.