Combinative Protein Expression of Immediate Early Genes c-Fos, Arc, and Npas4 Along Aversive and Appetitive Experience-Related Neural Networks

Abstract

Expression of immediate early genes (IEGs) is critical for memory formation and has been widely used to identify the neural substrate of memory traces, termed memory engram cells. Functions of IEGs have been known to be different depending on their types. However, there is limited knowledge about the extent to which different types of IEGs are selectively or concurrently involved in the formation of memory engram. To address this question, we investigated the combinative expression of c-Fos, Arc, and Npas4 proteins using immunohistochemistry following aversive and rewarding experiences across subregions in the prefrontal cortex (PFC), basolateral amygdala (BLA), hippocampal dentate gyrus (DG), and retrosplenial cortex (RSC). Using an automated cell detection algorithm, we found that expression patterns of c-Fos, Npas4, and Arc varied across different brain areas, with a higher increase of IEG expressing cells in the PFC and posterior BLA than in the DG. The combinative expression patterns, along with their experience-induced changes, also differed across brain areas; the co-expression of IEGs increased in the PFC and BLA following experience, whereas the increase was less pronounced in the DG and RSC. Furthermore, we demonstrate that different area-to-area functional connectivity networks were extracted by different IEGs. These findings provide insights into how different IEGs and their combinations identify engram cells, which will contribute to a deeper understanding of the functional significance of IEG-tagged memory engram cells.

Keywords: context conditioning; co‐expression; immediate early genes; immunohistochemistry; memory engram cell.

FIGURE 1

Automated detection of IEG positive cells and the detection accuracy. (A) Top: Original images of c‐Fos, Npas4, Arc, and NeuN expressed cells in aBLA. Middle: Overlay of the manually detected positive cells (white “+” marks) on the original images. Bottom: Auto‐detected cells (colored cells), overlay of the manually detected positive cells and manually verified false‐positive detections (red “×” marks). Yellow “△” marks indicate cells overlooked in the manual detection but detected in the automated algorithm. Scale bars, 100 μm. (B) Evaluation of auto‐detection accuracy with Precision, Auto‐Manual match rate, and Sensitivity increase rate. Precision indicates false positive ratio in the automated cell detection per total detected cells, that is Precision = TP/(TP + FP), where true positive (TP) and false positive (FP) were identified manually after the automated detection (n = 7, 9, 7, and 9 sections for NeuN, c‐Fos, Npas4, and Arc). Auto‐Manual match rate indicates the ratio of cells which were both manually and automatically detected, such that Auto‐Manual match rate = Match/(Match + Eye_Only), where Match indicates the number of cells which were both manually and automatically detected, and Eye_Only indicates the number of cells identified only by manual detection (n = 5 sections for NeuN, n = 334 sections for c‐Fos, Npas4, and Arc). Sensitivity increase rate indicates ratio that cells were not detected manually but detected automatically, that Sensitivity‐increase rate = Auto_Only/(Match + Auto_Only), where Auto_Only indicates the number of cells which were not identified manually but detected automatically (n = 5 sections for NeuN, n = 334 sections for c‐Fos, Npas4, and Arc). (C) Correlation of automatically and manually detected cell number, with R indicating Pearson correlation coefficient (p < 0.001 for all). HC: N = 77, CFC: N = 129, RE: N = 128 sections. The sections used for accuracy analysis were randomly selected from the PFC, BLA, and DG. Total counted cells were, for manual‐ vs. auto‐detection, for c‐Fos positive cells, HC: 786 vs. 751 cells, CFC: 5914 vs. 5653 cells, and RE: 4604 vs. 4922 cells; for Npas4 positive cells, HC: 800 vs. 875 cells, CFC: 3548 vs. 3476 cells, and RE: 2713 vs. 2696 cells; for Arc positive cells, HC: 893 vs. 1965 cells, CFC: 4983 vs. 10,101 cells, and RE: 3415 vs. 9136 cells.

FIGURE 2

Expression of c‐Fos, Npas4, and Arc in multiple brain regions. Images of c‐Fos (red), Npas4 (green), Arc (blue), merged, and auto‐detected cells in the prelimbic (PL), infralimbic (IL), anterior basolateral amygdala (aBLA), posterior BLA (pBLA), dorsal dentate gyrus (dDG), and ventral DG (vDG), dorsal anterior RSC (aRSC), ventral aRSC, dorsal posterior RSC (pRSC), and ventral pRSC. Top: Home cage (HC); Middle: Contextual fear conditioning (CFC); Bottom: Reward experience (RE) groups. Scale bars, 40 μm. Larger field‐of‐view images are shown in Figures S2–S7.

FIGURE 3

Changes of cell densities and expression levels of c‐Fos, Npas4, and Arc positive cells. (A) Fold changes of c‐Fos, Npas4, and Arc positive cell densities by CFC and RE in each brain region, compared with HC group. (B) Fold changes of c‐Fos, Npas4, and Arc expression levels of cells by CFC and RE in each brain region, compared with HC group. Statistical tests were conducted between groups, as shown in Figures S8 and S9.

FIGURE 4

Co‐expression of IEGs in PL and IL. (A, E) Cell densities of each cell group with selective or combinative IEG expression in the PL (A) and IL (E). (B, F) Ratio of cell densities per all c‐Fos cells (left), Npas4 cells (middle), and Arc cells (right), in the PL (B) and IL (F). (C, G) Venn diagrams of c‐Fos, Npas4, and Arc positive cells in HC, CFC, and RE groups, in the PL (C) and IL (G). The size of the circles corresponds to cell densities, normalized by Arc cell density. (D, H) Average correlation of IEG expression in single cells, between c‐Fos versus Npas4 (left), c‐Fos versus Arc (middle), and Npas4 versus Arc (right), in the PL (D) and IL (H). For each group of bars, the left bar indicates HC, the middle indicates CFC, and the right indicates RE.

FIGURE 5

Co‐expression of IEGs in anterior and posterior BLA. (A, E) Cell densities of each cell group with selective or combinative IEG expression in aBLA (A) and pBLA (E). (B, F) Ratio of cell densities per all c‐Fos cells (left), Npas4 cells (middle), and Arc cells (right), in the aBLA (B) and pBLA (F). (C, G) Venn diagrams of c‐Fos, Npas4, and Arc positive cells in HC, CFC, and RE groups, in the aBLA (C) and pBLA (G). The size of the circles corresponds to cell densities, normalized by Arc cell density. (D, H) Average correlation of IEG expression in single cells, between c‐Fos versus Npas4 (left), c‐Fos versus Arc (middle), and Npas4 versus Arc (right), in the aBLA (D) and pBLA (H). For each group of bars, the left bar indicates HC, the middle indicates CFC, and the right indicates RE.

FIGURE 6

Co‐expression of IEGs in dorsal and ventral DG. (A, E) Cell densities of each cell group with selective or combinative IEG expression in the dDG (A) and vDG (E). (B, F) Ratio of cell densities per all c‐Fos cells (left), Npas4 cells (middle), and Arc cells (right), in the dDG (B) and vDG (F). (C, G) Venn diagrams of c‐Fos, Npas4, and Arc positive cells in HC, CFC, and RE groups, in the dDG (C) and vDG (G). The size of the circles corresponds to cell densities, normalized by Arc cell density. (D, H) Average correlation of IEG expression in single cells, between c‐Fos versus Npas4 (left), c‐Fos versus Arc (middle), and Npas4 versus Arc (right), in the dDG (D) and vDG (H). For each group of bars, the left bar indicates HC, the middle indicates CFC, and the right indicates RE.

FIGURE 7

Co‐expression of IEGs in RSC. (A, E, I, M) Cell densities of each cell group with selective or combinative IEG expression in the dorsal aRSC (A), ventral aRSC (E), dorsal pRSC (I), and ventral pRSC (M). (B, F, J, N) Ratio of cell densities per all c‐Fos cells (left), Npas4 cells (middle), and Arc cells (right), in the dorsal aRSC (B), ventral aRSC (F), dorsal pRSC (J), and ventral pRSC (N). (C, G, K, O) Venn diagrams of c‐Fos, Npas4, and Arc positive cells in HC, CFC, and RE groups, in the dorsal aRSC (C), ventral aRSC (G), dorsal pRSC (K), and ventral pRSC (O). The size of the circles corresponds to cell densities, normalized by Arc cell density. (D, H, L, P) Average correlation of IEG expression in single cells, between c‐Fos versus Npas4 (left), c‐Fos versus Arc (middle), and Npas4 versus Arc (right), in the dorsal aRSC (D), ventral aRSC (H), dorsal pRSC (L), and ventral pRSC (P). For each group of bars, the left bar indicates HC, the middle indicates CFC, and the right indicates RE.

FIGURE 8

Functional connectivity network of each IEG. (A, B, C) Left, inter‐regional correlation matrices for c‐Fos‐ (A), Npas4‐ (B), and Arc‐ (C) positive cell densities. Dendrograms above the correlation matrices are calculated using dissimilarity index 1 − |r|, with colors indicating |r| > 0.7 (dissimilarity index < 0.3). Right, connectivity network graphs of c‐Fos (A), Npas4 (B), and Arc (C) generated by connecting each brain region (node) based on the strong correlations (Pearson's |r| > 0.7) (right), for HC, CFC, and RE groups. The size of node circles in the network graphs corresponds to the number of connections (edges) the node has. (D, E) Quantification of network complexity. (D) Average number of edges per node across the 10 brain regions. (E) Average number of edges per effective node, the brain region which has at least one connection to another node, across brain regions. (F, G) Quantification of network dissimilarity. (F) Matrix of graph edit distance (GED) across the graphs of different IEG groups. Bar plot indicates average GED within HC, CFC, and RE groups. (G) Matrix of Sum of Differences in Edge‐Weight Values (SDEWV) across the graphs of different IEG groups. Bar plot indicates average SDEWV within HC, CFC, and RE groups (n = 7 graphs for each).