Simultaneous Whole-Cell Recording and Calcium Imaging Does Not Reveal Electrically Coupled Neurons in Xenopus...

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 motor system of Xenopus laevis tadpole of either sex. 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 GCaMP6-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 3 min, 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.