Figures and data
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 were detected from the original image (upper panel), and once detected, they were projected onto the Allen Mouse Brain Atlas for quantification and network analysis by means of the detected signals labeled with yellow boxes.
Whole-brain distributions of c-Fos+ cells induced by ISO, KET, and control conditions.
(A) Scatter plot of four conditions in the space spanned by the first two principal components (PC1 versus PC2) of c-Fos density from 53 brain regions. (B) Line plot of coefficients of PC1 and PC2. Dots represent regions with absolute values larger than 75 percentiles. (C) The normalized c-Fos+ cells in 53 brain areas (Home cage, n = 6; ISO, n= 6 mice; Saline, n = 8; KET, n = 6). Brain areas are grouped into 12 generalized, color-coded brain structures. Abbreviations of the 53 brain areas and percentages of c-Fos+ cells are listed in supplementary table 1. Error bar, mean ± SEM. Mann-Whitney tests were corrected using the false discovery rate approach with a Q-value set at 0.05. Home cage vs ISO: *P<0.05, **P<0.01, ***P<0.001. Saline vs KET: #, P<0.05, ##, P<0.01, ###, P<0.001.
Similarities and differences in ISO and KET activated c-Fos brain areas.
(A) Scatter plot of four conditions in the space spanned by the first two principal components (PC1 versus PC2) of normalized c-Fos density from 201 brain regions. (B) Line plot of coefficients of PC1 and PC2. Dots represent regions with absolute values larger than 75 percentiles. (C) Activation patterns of ISO and KET in different parts of brain regions were summarized. ISO was predominantly characterized by increased activation of hypothalamic and cerebral nuclei while reducing activity in the cortex and midbrain, indicating that ISO primarily exerts its effects through a bottom-up mechanism. On the other hand, KET primarily activated the cortex and inhibited hypothalamus and midbrain activity, suggesting that KET regulates consciousness through a top-down mechanism.
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 cell counts 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.
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 values < 0.05. (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.
Generation of anesthetics-induced networks and identification of hub regions.
(A) Matrices showing interregional correlations for c-Fos expression at the home cage, ISO, saline, and KET. Colors reflect correlation strength based on Pearson’s r (color bar, right). Axes are numbered and correspond to brain regions listed in Supplementary Table 5. (B) Network graphs were generated by significant positive correlations (P < 0.05), as well as Pearson’s r > 0.82. The widths of the edges are proportional to the strength of the correlations and the size of the nodes is proportional to the degree of the node. Colors represent the major brain division (red, cerebral nuclei; purple, hypothalamus; orange, midbrain; brown, thalamus; blue, thalamus; green, the cerebral cortex. Network densities were noted in the left. (C) Mean r was calculated from interregional correlation coefficients. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. ***P < 0.001.
Identification of hub regions in the home cage (A), ISO (B), NS (C), and KET (D) groups.
Each brain region is ranked in descending order based on degree and betweenness centrality, respectively. The regions to the left of the dotted line represent the top 20% in our network. Black columns highlight brain regions that rank similarly in both degree and betweenness centrality. The within-module Z-scores and participation coefficients were calculated for each brain region in the networks. Brain regions characterized by a participation coefficient greater than 0.4 and a within-module degree Z-score greater than 1 are defined as hubs and are denoted by red dots.
The possible framework for ketamine and isoflurane induced unconsciousness.
The distinct pathways of ketamine and isoflurane 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 somatosensory cortex (SS) acting as the central node in the functional network (depicted in blue) and relative inhibition of hypothalamic sleep-promoting nuclei. Conversely, the “bottom-up” approach posits that isoflurane induced unconsciousness arises from the activation of hypothalamic sleep-promoting regions (indicated in yellow) and relative inhibition of cortical and thalamic nuclei, with the locus coeruleus (LC) serving as the hub node in the isoflurane induced functional network. Nuclei activated by both anesthetics are shown in green. Adapted from [3, 4, 52]. PL, prelimbic area; ILA, infralimbic areas; SON, supraoptic nucleus; PVH, paraventricular hypothalamic nucleus; TU, tuberal nucleus; LC, locus coeruleus; SS, somatosensory cortex; CTX: cortex; TH: thalamus; HY, hypothalamus; MB; midbrain; HB, hindbrain.
EEG and EMG power change after each treatment.
Box represents the 25th-75th percentiles; the central line denotes the median; whiskers indicate maximum and minimum values. n = 6, 6, 6, 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 test.
c-Fos expression in distinct brain regions after exposure to normal saline administration.
(A) Representative immunohistochemical staining of MOB, AON, ORB, MPO, ACA, MO, TRS, PL, ILA, DP, LS, PVT, SON, 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 c-Fos+ cells from the indicated mice. Scale bar, 200 µm.
c-Fos expression in home cage group.
(A) Representative immunohistochemical staining of PL, ILA, LSc, LSr, PIR, BST, VLPO, PVH, aPVT, SON, CeA, TU, PVi, ARH, EW, ENT, PB, LC, and NTS c-Fos+ cells from the indicated mice. Scale bar, 200 µm.
c-Fos-positive cell counts in each brain region normalized to the total cell count of their respective whole brains for the home Cage, ISO, saline, and KET groups. P1 denotes p-values for comparisons between the Home Cage and ISO groups, while P2 represents p-values for the contrasts between the Saline and KET groups. Statistical evaluations were undertaken using the Mann-Whitney test, followed by a false discovery rate (FDR) correction, with a threshold Q-value established at 0.05.
Sum of previous studies on ketamine activated brain regions detected by c-Fos immunostaining.
The sum of previous studies on isoflurane activated brain regions detected by c-Fos immunostaining.
Abbreviations of the relevant brain regions in Figure 3.
