Fig. 5: In vivo visual stimulation with drifting gratings induces transient depolarizing events selectively on microglial processes.

a Schematic of the experimental procedure for generation of TMEM-Cre;ASAP3 knock-in mice. b Immunostaining images confirming ASAP3 expression in MGs. Scale bars, 10 μm and 5 μm. c Schematic of the experimental procedure for ex vivo two-photon imaging of ASAP3 signals in MGs of acute brain slices. d, e Representative images of ASAP3 signals on the somata and processes of MGs in response to different voltages resulting from perfusion of ACSF with different KCl concentrations. Scale bar, 5 μm. f Negative correlation between ASAP3 fluorescence intensity and MP. g Schematic of the experimental procedure for in vivo two-photon ASAP3 imaging. h Example of ASAP3 fluorescence changes in the soma and process of an MG from a WT mouse induced by visual stimulation. Scale bar, 10 μm. i Left: amplitude changes of ASAP3 signals in the soma and process (Soma: 0.10 ± 0.10 vs Process: 2.93 ± 0.19, n = 14 cells from 5 mice; two-sided unpaired t-test) of MGs in WT mice. Right: MP changes in the soma and process (Soma: 0.64 mV ± 0.65 mV vs Process: 17.33 mV ± 0.96 mV, n = 14 cells from 5 mice; two-sided unpaired t-test) of MGs in WT mice. j Example of ASAP3 fluorescence changes in the process of an MG in a THIK-1 cKO mouse induced by visual stimulation. Scale bar, 10 μm. k Quantification of the amplitude change of ASAP3 signals in the processes of MGs in WT and THIK-1 cKO mice during and after visual stimulation (WT: Stimulus: 2.93 ± 0.19 vs After: 0.09 ± 0.13, n = 14 cells from 5 mice; two-sided paired t-test. cKO: Stimulus: 3.78 ± 0.21 vs After: 3.52 ± 0.17, n = 14 cells from 5 mice; two-sided paired t-test. After: WT vs cKO, two-sided unpaired t-test). Note that the dataset of WT stimulus group is shared with that of the Process group in the left panel in i. l Representative images of ASAP3 signals on the processes of MGs responding to visual stimulation, before and after injection of the muscarinic receptor specific blocker scopolamine. Scale bar, 10 μm. m Amplitude changes in ASAP3 signals before and after scopolamine application (Ctrl: 2.91 ± 0.22 vs Scopolamine: 0.11 ± 0.10; two-sided paired t-test; n = 13 cells from 5 mice). n mRNA copy number of the NALCN coding gene Nalcn in AISa-MGs vs AISn-MGs (AISa-MG: 182.60 ± 88.83, n = 8 mice vs AISn-MG: 18.14 ± 13.85, n = 7 mice; two-sided Mann–Whitney test). o Confocal images showing NALCN protein expression on the microglial primary process. Scale bar, 2 μm. p Quantification of NALCN protein expression on the processes of AISa-MGs vs AISn-MGs (AISa-MG: 0.23 ± 0.01 vs AISn-MG: 0.12 ± 0.01, n = 4 mice; two-sided unpaired t-test). q Representative images of ASAP3 signals on the processes of MGs responding to visual stimulation, before and after injection of the NALCN specific blocker CP96345. Scale bar, 10 μm. r Amplitude changes of ASAP3 signals in MGs before and after CP96345 application (Ctrl: 2.85 ± 0.26 vs CP96345: 0.58 ± 0.20; two-sided paired t-test. n = 7 cells from 4 mice). Whiskers of box plots extend to the 2.5th and 97.5th percentiles of the data. Error bars indicate SEM.