Fv1000 bx61 confocal laser scanning microscope

Olympus FV1000 BX61 confocal laser scanning microscope (RRID:SCR_020337) was used to acquire the images and optical sections were collected in z-stacks. Fluoview software (Olympus) was used to analyze the images. From each mouse, 20 dI3 INs and 20 MNs were selected, and the number of boutons was quantified manually using orthogonal views to confirm apposition onto the cell body. For quantification of sensory synapses on dI3 INs and MNs, VGLUT1⁺ boutons were quantified on the entire soma and proximal dendrites (up to 100 μm from the soma) on confocal images. Similarly, VGLUT2⁺ boutons were counted to estimate the number of central excitatory synapses onto dI3 INs and MNs. In addition, GAD65⁺ boutons contacting VGLUT1⁺ terminals were counted, and the fraction of VGLUT1⁺ boutons with GAD65⁺ boutons was calculated to estimate presynaptic inhibition of sensory inputs to dI3 INs and MNs. To estimate the surface area of the soma, thresholding of the image was applied using ImageJ software. Specifically, a rectangle was drawn around the cell of interest and the threshold was adjusted until the surface of the cell had become white while the background remained black. The area of the soma was then measured. To measure the size of the boutons, the optical section containing the largest area of each button was selected and the maximum diameter of the bouton was measured using ImageJ.

Cells after 4 days of culture were dropped onto an ITO electrode and covered with a cover glass so that the number of the cells per surface area is approximately the same as that in the case of electrochemical measurements performed with ITO electrode. The imaging of autofluorescence from the cells was performed with FV1000/BX61 confocal laser scanning microscope (Olympus, Japan) by utilizing Cy5-filter sets. Data collection was performed with FV10-ASW Version 04.02; data analysis was performed with FV10-ASW Version 04.02 and OriginPro 2021b (64-bit) SR2 Version 9.8.5.212.