Fig 2: Layer-specific and cell type-specific induction of IEG expression in neocortex after SD. A, Proportion of time spent in ad libitum sleep between ZT0 and ZT6 for mice used for fluorescence in situ hybridization experiments. B, Cumulative frequency distribution of A/P coordinates (relative to bregma) for brain sections used in analysis. C, A representative image of neocortical in situ hybridization for Arc (magenta), Cfos (yellow), Homer1a (red), and Pvalb (green). DAPI staining shown in blue. D, Anatomical regions for quantification were demarcated manually (shown in orange). Within these anatomic regions, Pvalb (green) fluorescence delineated Pvalb+ and non- Pvalb+ ROIs. An automated protocol was used to calculate the total fluorescence intensity and area of each ROI (and background). Scale bars: 100 µm. E, Example of IEG and Pvalb fluorescence. F, Pvalb+ ROI demarcation. Scale bar: 5 µm. G, Representative images showing neocortical riboprobe labeling for ubiquitously expressed (+ control) housekeeping genes UbC (magenta), Hprt1 (green), PpiB (red), and Polr2a (yellow). Negative control probes targeting DapB mRNA, a gene expressed in Bacillus subtilis, shown in the same regions. Inset regions are shown at higher magnification; scale bars: 100 and 10 µm, respectively. H, Representative images of neocortical IEG expression after 6 h of ad libitum sleep (n = 5 mice) or SD (n = 6 mice). Inset regions are shown at higher magnification on right. Scale bars: 100 µm (images) and 10 µm (insets). I, Six-hour SD significantly increased Arc expression among non-Pvalb+ cells (blue) across neocortex as a whole, and within layers 2/3, 4, and 5, and increased Cfos and Homer1a expression in layers 4 and 5. J, Six-hour SD significantly increased Cfos expression among Pvalb+ cells (magenta) in layer 2/3; no other significant changes were observed. K, When analysis was restricted to IEG+ Pvalb+ cells (magenta, box pattern), SD significantly increased Homer1a levels among Homer1a+ Pvalb+ cells in whole cortex; no other significant changes were observed. Violin plots show distribution of values for individual mice; *p < 0.05, **p < 0.01, Holm–Sidak post hoc test versus sleep.

Fig 3: Validation and experimental design for TRAP. A, Antibody staining for Rpl22-HA (green) and PV (red) is shown for dorsal hippocampal subregions DG (top), CA3 (middle), and CA1 (bottom) of a Camk2a::RiboTag mouse. Scale bars: 100 µm. B, Example of automated protocol used for Camk2a::RiboTag mice to quantify non-specific expression (i.e., overlap of PV+ with HA+ expression regions; quantified in D within the pyramidal cell layer of CA3). Areas of overlapping fluorescence were determined using automated detection of HA+ (green fluorescence, labeled in yellow) and PV+ (red fluorescence, labeled in magenta) areas. Scale bars: 50 µm. C, HA expression in PV+ interneurons was validated with immunohistochemistry in PV::RiboTag mice, using the method described in B. D, Areas of PV+ and HA+ overlap (as a proportion of total HA+ area) in Camk2a::RiboTag sections were minimal. E, F, HA+ and PV+ overlapping expression presented as a proportion of total PV+ or HA+ cell counts (red and green, respectively; E) and total PV+ or HA+ area (red and green, respectively, F) in PV::RiboTag sections. G, Experimental design for cell type-specific ribosomal profiling. Camk2a::RiboTag (blue) and PV::RiboTag (violet) transgenic mice were killed after a 3- or 6-h period of ad libitum sleep (sleep) or SD starting at lights on (ZT0). Ribosome-associated mRNAs were affinity purified from hippocampus and neocortex separately. H, Enrichment of markers for glia (Mbp, Gfap), non-PV+ inhibitory neurons (NPY, SOM), PV+ neurons (Griar4, Gad67, Pvalb), and excitatory neurons (Vglut1, Vglut2, Camk2a) calculated as ??CT between affinity purified (RiboTag) mRNA and input mRNA from neocortex or hippocampus. Data presented as log(2) transformed fold changes and are shown separately for SD and sleep conditions. Gene expression was normalized to housekeeping gene pairs according to their respective condition (see Table 1). Values indicate mean ± SEM with propagated error; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one sample t test against a hypothetical value of 0.

Fig 4: Triple fluorescence labeling of the net markers WFA and aggrecan (ACAN) combined with the immunodetection of parvalbumin (PARV) exemplarily shown for the retrosplenial cortex (A–A'') and the hippocampal CA3 region (B–B'') in non-affected control tissue from young adult mice. In (A), the lectin-histochemical Cy2 staining of WFA reveals numerous PNs in the retrosplenial cortex, which also displays considerable ACAN-ir in (A'). The overlay in (A'') elucidates several net-like structures with lectin-binding sites but devoid of visible ACAN-ir, frequently around PARV-containing neurons. In parallel and as expected, many PNs are positive for both markers. WFA-positive structures in the CA3 region appear sparsely scattered (B), whereas more ECM structures exhibit ACAN-ir (B'). The overlay elucidates that net components with ACAN-ir outweigh those with WFA binding and are associated with PARV-immunoreactive structures. Scale bars (A'') (also valid for A,A'), 75 µm, (B'') (also valid for B,B'), 75 µm.

Fig 5: Cell type-specific and region-specific changes in hippocampal IEG expression after SD. A, Representative in situ images showing DG, CA1, and CA3 riboprobe labeling for + control and – control genes, as in Figure 3G. Inset regions are shown at higher magnification; scale bars: 100 and 10 µm, respectively. B, Representative images of IEG expression after 6 h of ad libitum sleep (n = 5 mice) or SD (n = 6 mice). In situ hybridization is shown for Arc (magenta), Cfos (yellow), Homer1a (red), and Pvalb (green). Inset regions are shown at higher magnification on right. Scale bars: 100 µm (images) and 10 µm (insets). C, Six-hour SD significantly increased Cfos expression among non-Pvalb+ (blue) cells in CA3; no other significant changes observed. D, E, No significant changes were observed within DG, CA3, or CA1 in Pvalb+ cells (magenta; D) or IEG+ Pvalb+ cells (magenta, box pattern; E). Violin plots show distribution of individual subjects; **p < 0.01, Holm–Sidak post hoc test versus sleep.