Fig. 6: In vivo optical recording of neuronal activity in cold-sensing neurons.

Optical voltage recordings of cold (0 °C)-sensitive primary sensory neurons from naïve (a), CFA (b), or SN-CCI (c) animals. Insets, images of DRG cell bodies (white dotted lines) expressing ASAP4.4-Kv. Yellow lines indicate 1.1 kHz line scan regions where ASAP4.4-Kv optical recording signals were acquired. Each trace is the response of a single DRG neuron; (right) expanded view of the boxed region. Purple bars indicate the timing of the stimulus application. Representative line scan images in b are shown under the trace. The red arrow marks one of the action potentials, and the red box indicates action potential bursts. Scale bar: 5 μm. d Mean area under the curve (AUC) of ASAP4.4-Kv z-score signals (each dot represents an individual recorded neuron; number of cells = 7, 6 and 5 for naïve, CFA, and SN-CCI groups, respectively, from three biologically independent mice per group; Kruskal-Wallis test with Dunn’s post-hoc test) and GCaMP3 Ca²⁺ transients (violin plot with median (solid line) and quartiles (dashed lines); number of cells = 58 and 34 for naïve and CFA, respectively, from three biologically independent mice per group; two-tailed Mann-Whitney U-test) in L5 DRG neurons in response to noxious cold stimulus, and the number of total activated neurons (each dot represents one animal; number of biologically independent mice = 5 per group; two-tailed Mann-Whitney U-test). e In vivo intact DRG Ca²⁺ imaging from naïve or CFA-treated Pirt-GCaMP3 mice. (left) Averaged images before application of cold stimulus (0°C). (right) Averaged images after application of cold stimulus (0°C). White arrowheads indicate spontaneously activated neurons without application of stimulus. Yellow arrowheads indicate DRG neurons activated by cold (0°C). Scale bar: 100 μm. Ca²⁺ transients from DRG neurons in response to application of cold stimulus (0°C) to the hindpaw of Pirt-GCaMP3 mice (grey), from one (f) or three (g) animals for each treatment. Traces of averaged Ca²⁺ transients from each group are shown in black or orange. All data are presented as mean values ± SEM.