Fig 2: Patch-seq of tdTomato-positive cells of retinal sections. (A) Schematic of the Patch-seq procedure in this paper (created with BioRender.com). (B) Expression patterns of a subset of marker genes for retinal cell classes and types. Pax6 and Slc32a1 are marker genes for ACs; Lhx1 for horizontal cells; Vsx2 for bipolar cells; Slc17a6 for RGCs; Arr3 for cones; Slc1a3 for Müller cells; Gfap for Astrocytes; Pecam1 for endothelium; Pdlim1 for pericytes and Ptprc for microglia. INL: cell bodies in the INL (n = 135 cells); GCL: cell bodies in the GCL (n = 17 cells). (C) Uniform manifold approximation and projection (UMAP)s showing the distribution of Patch-seq ACs (n = 152 cells) with cell bodies located in GCL (n = 17 cells) and INL (n = 135 cells). (D) UMAPs showing the expression of marker genes of known AC types. Gad1, Gad2, and Slc6a1 are the marker genes for GABAergic ACs; Slc6a9 for glycinergic ACs; Ppp1r17 and Ebf3 for non-GABAergic non-glycinergic ACs; Chat for SACs; Vip for VIP-expressing ACs; Slc17a8 for VGluT3-expressing ACs; Gjd2 for A-II ACs; and Calb2 for calretinin. It can be seen that tdTomato-positive cells highly express markers of ACs and GABAergic neurons, as well as gene encoding calretinin.

Fig 3: Immunofluorescence cFos, VIP, and AVP signals.a A schematic depicting the timeline of the experiment: For half of the animals (n = 3 wild-type and three GluA1 KO mice), no light pulse was given prior to perfusion and they were perfused at ~ZT15.5 (day 1, LP-); the remaining animals perfused on day 2 (n = 3 wild-type and three GluA1 KO mice) received a nocturnal light pulse (LP+) which started at ZT14. Star symbols indicate the Zeitgeber time of perfusion, which was ~90 min after the onset of the nocturnal light (~ZT15.5). Thus, the ZTs at which perfusion was performed were matched between LP- and LP+ conditions. b Representative images of immunofluorescence cFos, VIP, and AVP staining in the SCN (scale bar = 100 µm). In the middle multi-panels, brighter colours indicate higher immunofluorescence (F) intensity, whereas darker colours indicate lower F intensity. A schematic delineating the core (ventromedial) vs. shell (dorsolateral) subregions of the SCN is shown on the right (D dorsal, V ventral, M medial, and L lateral). c Mean F intensities (±standard errors of the mean) from all three channels across the entire mediolateral extent of the SCN. Nocturnal-light-induced cFos signals were attenuated in GluA1 knockouts (*two-way ANOVA for LP+ conditions, Genotype × Mediolateral Extent interaction p = 0.019). VIP and AVP data were pooled across LP- and + LP (n = 6 per genotype). The vertical dashed lines represent the location of the midline, and the 60-µm bins adjacent to the dashed lines (i.e. the middle two bins with the lowest F intensities) are the location of the 3rd ventricle. d Example images of immunofluorescence cFos staining in the motor cortex, area M1/M2 (scale bar = 100 µm). The image of the Nissl-stained coronal section on the left was retrieved from the Allen Mouse Brain Atlas, plate reference number 100048576_241 [125]. The number of cFos+ cells in M1/M2 of wild-type animals, but not GluA1 knockouts, was reduced in response to nocturnal light (**p < 0.01). Variability in cFos+ cell count in KO LP- and KO LP+ conditions (pink and red bars) was higher than that in the WT LP- condition (black bar), as indicated by Levene’s tests for equality of variances (†p < 0.05; ‡0.05 < p < 0.06).

Fig 4: Expression of ARHGAP15 across the major CIN subtypes. (A) In the left column, maximum intensity projections of z-stack images (10 serial image planes; z step size = 2 µm) of the somatosensory cortex of P45 Arhgap15 LacZ/+ ;GAD67-eGFP brains immunostained for ß-GAL and PVALB, CALB2, SST, or VIP. In the central column, maximum intensity projections of z-stack images (10 serial image planes; z step size = 0.5 µm) of the regions inside the dashed boxes on the left. In the right column, orthogonal projections of the regions inside the dashed boxes in the middle showing the co-localization of the PVALB, CALB2, SST, or VIP and the ß-GAL signals. Scale bars: 200 µm (left), 20 µm (middle). (B–E) Percentage of ß-GAL/PVALB (B), ß-GAL/CALB2 (C), ß-GAL/SST (D), and ß-GAL/VIP (E) double-positive cells over the total of PVALB, CALB2, SST, and VIP-positive cells, respectively. n = 3 mice; about 120 cells were evaluated for ß-GAL expression for each bin in each brain. Data are presented as mean ± SEM.

Fig 5: CIN distribution across layers in the adult cortex. (A) Maximum intensity projections of z-stack images (10 serial image planes; z step size = 2 µm) of coronal sections of WT and Arhgap15 LacZ/LacZ P45 somatosensory cortices immunostained for PVALB, CALB2, SST, and VIP. (B–E) Average density of PVALB- (B), CALB2- (C), SST- (D), and VIP- (E) positive neurons in each bin of P45 WT and Arhgap15 LacZ/LacZ somatosensory cortices. At least 4 sections from 5 different mice were analyzed for each genotype. p (PVALB) > 0.99 for all bins; p(CALB2, from bin 1 to 1-10) = 0.99, 0.71, 0.99, 0.47, 0.003, 0.004, 0.005, 0.52, 0.99, 0.99, 0.65; p (SST, from bin 1 to 1-10) = 0.78, 0.32, 0.78, 0.78, 0.78, 0.44, 0.60, 0.72, 0.18, 0.02, 0.78; p(VIP, from bin 1 to 1-10) = 0.07, 0.51, 0.99, 0.76, 0.59, 0.03, 0.77, 0.99, 0.33, 0.72, 0.98. Data are presented as mean ± SEM. p-values were calculated using unpaired multiple t-test corrected for multiple comparisons using the Holm-Sidak method. * = p< 0.05; ** = p< 0.01.