Fig. 1: In vivo optical detection of electrically synchronous, spontaneous neuronal activity using ASAP4.4-Kv voltage imaging in intact DRG neurons.

Paired optical recordings of two DRG somata (white dotted lines) expressing ASAP4.4-Kv in naïve (a), CFA (b), or SN-CCI (c) animals. Yellow lines indicate 1.1 kHz line scan regions where ASAP4.4-Kv fluorescent signals were acquired. In b, representative traces show electrical de-synchronization between a pair of adjacent DRG neurons approximately 1 hr after systemic injection of gap junction blocker, CBX (100 mg/kg, i.p.). The subthreshold voltage dynamics from recording neurons were gradually diminished following de-synchronization by gap junction blocker. The red arrow marks one of action potentials and red box indicates action potential burst. d Mean area under the curve (AUC) of ASAP4.4-Kv z-score signals in L5 DRG neurons from naïve, CFA, or SN-CCI animals (each dot represents an individual recorded neuron; number of cells = 10 from three biologically independent mice per group; Kruskal-Wallis test with Dunn’s post-hoc test). e Percentage of synchronous pairs in total DRG neuron pairs recorded from CFA mice without (control) and with systemic CBX injection (each dot represents an individual CFA-treated animal; number of biologically independent CFA mice = 7 and 4 for control (without CBX treatment) and CBX-treated groups, respectively; two-tailed Mann-Whitney U-test). f Correlation coefficients were calculated from DRG neuron pairs recorded from CFA mice without (control) and with systemic CBX injection (each dot represents one pair of DRG neurons; number of DRG pairs = 5 and 10 for control (without CBX treatment) and CBX-treated groups, respectively, from three biologically independent mice per group; two-tailed Mann-Whitney U-test). Neuron pairs exhibit electrical correlation, whereas the correlation is dissociated by gap junction blocker. Fluorescence traces showing respiratory motion or heartbeat-like rhythmic events, or digital artifacts were not considered as electrical synchronization in the analysis. g Optical ASAP4.4-Kv signals from DRG neurons of different size reveal various action potential shapes, in agreement with Aβ, Αδ and C-type nociceptor properties. Grey box on the line scan image indicates regions of interest (ROI). Red arrow marks one of action potentials recorded from ROIs, with expanded view on the right. Scale bar in inset image: a–c: 10 μm. g: 10 μm. All data are presented as mean values ± SEM.