Brain-wide quantification of c-Fos expression.

(A)Schematic representation of the habituation protocol typically used to acclimate mice. After being exposed to anesthetics for 90 minutes, the mice were euthanized. (B) Steps for data processing. Example of brain section registration to a corresponding coronal section from the Allen Brain Atlas. For Atlas rotation, the Allen reference atlas was rotated to mimic the slice angle of the experimental brain. Image registration maps the original coronal image (upper panel) to the corresponding Allen mouse brain atlas (lower panel). The registration module applies several geometric transformations (translation, rotation, and scaling) to optimize the matching of the original image to the anatomical structures. Fluorescence signals detected in the original image were projected onto the Allen Mouse Brain Atlas for quantification. Finally, the processed data undergo hierarchical clustering and network analysis to investigate the patterns of c-Fos expression and central network nodes. Figure 1—figure supplement 1. EEG and EMG power change after each treatment. The box represents the 25th-75th percentiles; the central line denotes the median; whiskers indicate maximum and minimum values. n = 6, 6, 8, 6 for the home cage, ISO, saline, and KET, respectively. (A) Normalized change in EEG power: ISO vs KET, P > 0.99; Saline vs KET, P = 0.01; Home cage vs ISO, P = 0.11. (B) Normalized change in EMG power: ISO vs KET, P = 0.36; Saline vs KET, P = 0.30; Home cage vs ISO, P = 0.02. Analyses were conducted using the Kruskal-Wallis test, followed by Dunn’s multiple comparisons tests.

Whole-brain distributions of c-Fos+ cells induced by ISO and KET.

(A) Hierarchical clustering was performed on the log relative c-Fos density data for ISO and KET using the complete linkage method based on the Euclidean distance matrix, with clusters identified by a dendrogram cut-off ratio of 0.5. Numerical labels correspond to distinct clusters within the dendrogram. (B) Silhouette values plotted against the ratio of tree height for ISO and KET, indicating relatively higher Silhouette values at 0.5 (dashed line), which is associated with optimal clustering. (C) The number of clusters identified in each treatment condition at different ratios of the dendrogram tree height, with a cut-off level of 0.5 corresponding to 4 clusters for both ISO and KET (indicated by the dashed line). (D) The bar graph depicts Z scores for clusters in ISO and KET conditions, represented with mean values and standard errors. One-way ANOVA with Tukey’s post hoc multiple comparisons. ns: no significance; ***P < 0.001. (E) Z-scored log relative density of c-Fos expression in the clustered brain regions. The order and abbreviations of the brain regions and the numerical labels correspond to those in Figure 2A. The red box denotes the cluster with the highest mean Z score in comparison to other clusters. CTX: cortex; TH: thalamus; HY: hypothalamus; MB: midbrain; HB: hindbrain. Figure 2—figure supplement 1. The c-Fos density in 53 brain areas for different conditions. (home cage, n = 6; ISO, n = 6 mice; saline, n = 8; KET, n = 6). Each point represents the c-Fos density in a specific brain region, denoted on the y-axis with both abbreviations and full names. Data are shown as mean ± SEM. Brain regions are categorized into 12 brain structures, as indicated on the right side of the graph.

Similarities and differences in ISO and KET activated c-Fos brain areas.

(A) Hierarchical clustering was performed on the log-transformed relative c-Fos density data for ISO and KET using the complete linkage method based on the Euclidean distance matrix, with clusters identified by a dendrogram cut-off ratio of 0.5. Brain region labels are provided in Supplementary Figures 4 and 5. (B) Silhouette values are plotted against the ratio of tree height from the hierarchical clustered dendrogram in Figure 3A. (C) The relationship between the number of clusters and the tree height ratio of the dendrogram for ISO and KET, with a cut-off ratio of 0.5 resulting in 3 clusters for ISO and 5 for KET (indicated by the dashed line). (D) The bar graph depicts Z scores for clusters in ISO and KET conditions, represented with mean values and standard errors. One-way ANOVA with Tukey’s post hoc multiple comparisons. ns: no significance; ***P < 0.001. (E) Z-scored log relative density of c-Fos expression within the identified brain region clusters. The arrangement, abbreviations of the brain regions, and the numerical labels are in accordance with Figure 3A. The red boxes highlight brain regions that rank within the top 10 percent of Z score values. The white boxes denote brain regions with an Z score less than −2. Figure 3—figure supplement 1. c-Fos density visualization across 201 distinct brain regions under various conditions. The graph depicts the c-Fos density levels for each condition, with data presented as mean and standard error. Brain regions with statistically significant differences are featured in Figures 4 and 5. Brain regions are organized into major anatomical subdivisions, as indicated on the left side of the graph. Figure 3—figure supplement 2. Region labels for the hierarchical clustering of the ISO group in Figure 3A.

c-Fos expression in distinct brain regions after exposure to KET.

(A) Representative immunohistochemical staining of MOB, AON, ORB, MPO, ACA, MO, TRS, PL, ILA, DP, LS, PVT, SO, PVH, RE, VISC, AI, CLA, EPd, PIR, COA, AUD, TEa, ECT, PERI, CeA, SS, DG, STN, RSP, APN, LAT, EW, DR, PAG, SLD, PB, TRN, NI, LC, NTS, and NI c-Fos+ cells from the indicated mice. Scale bar, 200 µm. (B) Representation of c-Fos density in brain regions exhibiting significant statistical differences. Data are shown as mean ± SEM. P values < 0.05. (C) Schematic cross-section of the mouse brain showing activated brain regions by KET. Different colors indicate distinct functional nuclei. The red nuclei are associated with the regulation of sleep-wakefulness, the blue-green nuclei are linked to analgesia, the yellow nuclei are associated with motor function, and the white nuclei are a composite of various functional nuclei. Figure 4—figure supplement 1. c-Fos expression in specific brain regions following normal saline administration. (A) Representative immunohistochemical staining of c-Fos+ cells in MOB, AON, ORB, MPO, ACA, MO, TRS, PL, ILA, DP, LS, PVT, SO, PVH, RE, VISC, AI, CLA, EPd, PIR, COA, AUD, TEa, ECT, PERI, CeA, SS, DG, STN, RSP, APN, LAT, EW, DR, PAG, SLD, PB, TRN, NI, LC, and NTS. Scale bar represents 200 µm.

c-Fos expression in distinct brain regions after exposure to ISO.

(A) Representative of brain regions with statistical differences c-Fos+ cells between the ISO group and home cage mice. Scale bar, 200 µm. (B) Representation of cell counts in brain regions exhibiting significant statistical differences between home cage and ISO. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. (C) Schematic cross-section of the mouse brain showing activated brain regions by ISO. Different colors indicate various functionally relevant nuclei. Red signifies nuclei involved in sleep-wake regulation, blue-green in pain management, blue in neuroendocrine function, pink in side-effect management, and white denotes nuclei exhibiting mixed functionalities. (D) The Venn diagram shows brain regions that are co-activated by ISO and KET and differentially activated brain regions. Figure 5—figure supplement 1. c-Fos expression in home cage group. (A) Representative immunohistochemical staining of c-Fos+ cells in PL, ILA, LSc, LSr, PIR, BST, VLPO, PVH, aPVT, SO, CeA, TU, PVi, ARH, EW, ENT, PB, LC, and NTS c-Fos+ cells from the indicated mice. Scale bar, 200 µm.

Generation of anesthetics-induced networks and identification of hub regions.

(A) Heatmaps display the correlations of log c-Fos densities within brain regions (CTX, CNU, TH, HY, MB, and HB) for various states (home cage, ISO, saline, KET). Correlations are color-coded according to Pearson’s coefficients. The brain regions within each anatomical category are organized by hierarchical clustering of their correlation coefficients. Full names and expression levels for each brain region are detailed in Supplementary Figure 3. (B) Network diagrams illustrate significant positive correlations (P < 0.05) between regions, with Pearson’s r exceeding 0.82. Edge thickness indicates correlation magnitude, and node size reflects the number of connections (degree). Node color denotes betweenness centrality, with a spectrum ranging from dark blue (lowest) to dark red (highest). The networks are organized into modules consistent with the clustering depicted in Figure 6—figure supplement 1A. Figure 6—figure supplement 1. Hierarchical clustering across different conditions. (A) Heatmaps depict the hierarchical clustering of brain regions within the home cage, ISO, saline, and KET groups, using Euclidean distance matrices calculated from correlation coefficients. Each brain region is represented by its abbreviation, with full names and expression levels detailed in Figure 3—figure supplement 1. Modules are demarcated within each dendrogram at a cut-off threshold of 0.7. (B) Silhouette scores are plotted against the dendrogram tree height ratio for each condition, with optimal cluster definition indicated by a dashed line at a 0.7 ratio. (C) The number of clusters formed at different cutoff levels. At a ratio of 0.7, ISO and saline treatments result in three clusters, whereas home cage and KET conditions yield two clusters. (D) The mean Pearson’s correlation coefficient (r) was computed from interregional correlations displayed in Figure 6A. Data were analyzed using one-way ANOVA with Tukey’s post hoc test, ***P < 0.001. Figure 6—figure supplement 2. Hub region characterization across different conditions: home cage (A), ISO (B), saline (C), and KET (D) treatments. Brain regions are sorted by degree, betweenness centrality, and eigenvector centrality, with each metric presented in separate bar graphs. Bars to the left of the dashed line indicate the top 20% of regions by rank, highlighting the most central nodes within the network. Red

The possible framework for KET and ISO-induced unconsciousness.

The distinct pathways of KET and ISO-induced unconsciousness can be explained by two contrasting mechanisms. The ‘top-down’ process attributes KET’s effect to widespread cortical activation (represented in yellow), with the temporal association areas (TEa) acting as the central node in the functional network (depicted in blue). Conversely, the ‘bottom-up’ approach suggests that isoflurane-induced unconsciousness stems from the activation of certain hypothalamic regions (highlighted in yellow), with the locus coeruleus (LC) acting as the hub node within the isoflurane-induced functional network. Nuclei activated by both anesthetics are shown in green. Adapted from (Mashour, 2014; Mashour and Hudetz, 2017; Reimann and Niendorf, 2020). PL, prelimbic area; ILA, infralimbic areas; SO, supraoptic nucleus; PVH, paraventricular hypothalamic nucleus; LC, locus coeruleus; SS, somatosensory cortex; CTX: cortex; TH: thalamus; HY, hypothalamus; MB; midbrain; HB, hindbrain.

Figure 4—source data 1. Summary of prior studies on ketamine activated brain regions identified via c-Fos immunostaining.

Figure 5—source data 1. Summary of prior studies on brain regions activated by isoflurane, as detected through c-Fos immunostaining.

EEG and EMG power change after each treatment. The box represents the 25th-75th percentiles; the central line denotes the median; whiskers indicate maximum and minimum values. n = 6, 6, 8, 6 for the home cage, ISO, saline, and KET, respectively. (A) Normalized change in EEG power: ISO vs KET, P > 0.99; Saline vs KET, P = 0.01; Home cage vs ISO, P = 0.11. (B) Normalized change in EMG power: ISO vs KET, P = 0.36; Saline vs KET, P = 0.30; Home cage vs ISO, P = 0.02. Analyses were conducted using the Kruskal-Wallis test, followed by Dunn’s multiple comparisons tests.

The c-Fos density in 53 brain areas for different conditions. (home cage, n = 6; ISO, n = 6 mice; saline, n = 8; KET, n = 6). Each point represents the c-Fos density in a specific brain region, denoted on the y-axis with both abbreviations and full names. Data are shown as mean ± SEM. Brain regions are categorized into 12 brain structures, as indicated on the right side of the graph.

c-Fos density visualization across 201 distinct brain regions under various conditions. The graph depicts the c-Fos density levels for each condition, with data presented as mean and standard error. Brain regions with statistically significant differences are featured in Figures 4 and 5. Brain regions are organized into major anatomical subdivisions, as indicated on the left side of the graph.

Region labels for the hierarchical clustering of the ISO group in Figure 3A.

Region labels for the hierarchical clustering of the KET group in Figure 3A.

c-Fos expression in specific brain regions following normal saline administration. (A) Representative immunohistochemical staining of c-Fos+ cells in MOB, AON, ORB, MPO, ACA, MO, TRS, PL, ILA, DP, LS, PVT, SO, PVH, RE, VISC, AI, CLA, EPd, PIR, COA, AUD, TEa, ECT, PERI, CeA, SS, DG, STN, RSP, APN, LAT, EW, DR, PAG, SLD, PB, TRN, NI, LC, and NTS. Scale bar represents 200 µm.

c-Fos expression in home cage group. (A) Representative immunohistochemical staining of c-Fos+ cells in PL, ILA, LSc, LSr, PIR, BST, VLPO, PVH, aPVT, SO, CeA, TU, PVi, ARH, EW, ENT, PB, LC, and NTS c-Fos+ cells from the indicated mice. Scale bar, 200 µm.

Hierarchical clustering across different conditions. (A) Heatmaps depict the hierarchical clustering of brain regions within the home cage, ISO, saline, and KET groups, using Euclidean distance matrices calculated from correlation coefficients. Each brain region is represented by its abbreviation, with full names and expression levels detailed in Figure 3—figure supplement 1. Modules are demarcated within each dendrogram at a cut-off threshold of 0.7. (B) Silhouette scores are plotted against the dendrogram tree height ratio for each condition, with optimal cluster definition indicated by a dashed line at a 0.7 ratio. (C) The number of clusters formed at different cutoff levels. At a ratio of 0.7, ISO and saline treatments result in three clusters, whereas home cage and KET conditions yield two clusters. (D) The mean Pearson’s correlation coefficient (r) was computed from interregional correlations displayed in Figure 6A. Data were analyzed using one-way ANOVA with Tukey’s post hoc test, ***P < 0.001.

Hub region characterization across different conditions: home cage (A), ISO (B), saline (C), and KET (D) treatments. Brain regions are sorted by degree, betweenness centrality, and eigenvector centrality, with each metric presented in separate bar graphs. Bars to the left of the dashed line indicate the top 20% of regions by rank, highlighting the most central nodes within the network. Red bars signify regions that consistently appear within the top rankings for both degree and betweenness centrality across the metrics.