NPAS4 in the medial prefrontal cortex mediates chronic social defeat stress-induced anhedonia-like behavior and reductions in excitatory synapses

Abstract

Chronic stress can produce reward system deficits (i.e., anhedonia) and other common symptoms associated with depressive disorders, as well as neural circuit hypofunction in the medial prefrontal cortex (mPFC). However, the molecular mechanisms by which chronic stress promotes depressive-like behavior and hypofrontality remain unclear. We show here that the neuronal activity-regulated transcription factor, NPAS4, in the mPFC is regulated by chronic social defeat stress (CSDS), and it is required in this brain region for CSDS-induced changes in sucrose preference and natural reward motivation in the mice. Interestingly, NPAS4 is not required for CSDS-induced social avoidance or anxiety-like behavior. We also find that mPFC NPAS4 is required for CSDS-induced reductions in pyramidal neuron dendritic spine density, excitatory synaptic transmission, and presynaptic function, revealing a relationship between perturbation in excitatory synaptic transmission and the expression of anhedonia-like behavior in the mice. Finally, analysis of the mice mPFC tissues revealed that NPAS4 regulates the expression of numerous genes linked to glutamatergic synapses and ribosomal function, the expression of upregulated genes in CSDS-susceptible animals, and differentially expressed genes in postmortem human brains of patients with common neuropsychiatric disorders, including depression. Together, our findings position NPAS4 as a key mediator of chronic stress-induced hypofrontal states and anhedonia-like behavior.

Keywords: Anhedonia; NPAS4; RNA-sequencing; chronic social defeat stress; hypofrontality; mouse; neuroscience; prefrontal cortex.

Figure 1.. Social defeat stress induces NPAS4 expression in the medial prefrontal cortex (mPFC).

(A, B) Uniform manifold approximation and projection (UMAP) plot of the mPFC single cells colored by cell type (A) and Npsa4 mRNA expression (B). Cell types were defined by known markers and confirmed by predictive modeling using a single-cell mPFC atlas. (C) Donut chart represents the percentage of cell types that express Npas4 mRNA. (D) Dot plot represents the percentage of Npas4 mRNA expressing neurons in each cell type. (E) Schematic illustration of experimental timeline of gene expression analyses following acute social defeat stress and 10 days of chronic social defeat stress (CSDS). (F) Data plot represents the quantification of Npas4 mRNA expression following acute and chronic social defeat stress at 5 min, 15 min, 1 hr, and 24 hr (n = 5–10/condition). (G) Quantification of fold change in NPAS4-positive cell number following acute and chronic social defeat stress in subregions of the mPFC, including the anterior cingulate, prelimbic, and infralimbic cortices (n = 3–5/condition). (H) Quantification of mPFC NPAS4-positive cells relative to the number of CaMKIIα-positive cells in control/no-stress mice. (I, J) Data plot shows the percentage of CaMKIIα-, somatostatin (SST)-, and parvalbumin (PV)-positive cells in NPAS4-positive cells within the mPFC after acute stress and CSDS (n = 3–9/condition), as well as representative IHC images of NPAS4 colocalization in these respective cell type. Scale bar, 10 μm. Data shown are mean ± SEM; *p<0.05, ****p<0.0001. Also see Source data 1 for detailed statistical analyses.

Figure 1—figure supplement 1.. Social defeat stress induces NPAS4 and cFos expression in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC).

(A) Quantification of Npas4 mRNA expression in the NAc following acute and chronic social defeat stress at 15 min, 1 hr, and 24 hr (two-way ANOVA, Tukey’s post hoc analysis: control vs. acute stress at 15 min, p<0.0001, control vs. chronic stress at 15 min, p<0.0001, n = 5–10 per group). (B) Quantification of cFos mRNA expression in the mPFC following acute and chronic social defeat stress at 15 min, 1 hr, and 24 hr (two-way ANOVA, Tukey’s post hoc analysis: control vs. acute stress at 15 min, p<0.0001, control vs. chronic stress at 15 min, p<0.0001, acute vs. chronic stress at 15 min, p=0.0021, control vs. acute stress at 1 hr, p<0.0001, control vs. chronic stress at 1 hr, p<0.0001, acute vs. chronic stress at 1 hr, p=0.0055, n = 5–10 per group). (C) Data plot represents fold change of NPAS4 signal intensity in CaMKIIα-positive pyramidal excitatory neurons of the mPFC (one-way ANOVA, Tukey’s post hoc analysis: control vs. acute stress, p=0.0002, control vs. chronic stress, p=0.8222, acute vs. chronic stress, p=0.0001, n = 45–87 cells/3–5 animals/condition). Data shown are mean ± SEM; **p<0.01, ***p<0.001, ****p<0.0001. Also see Source data 1 for detailed for detailed statistical analyses.

Figure 2.. NPAS4 in the medial prefrontal cortex (mPFC) is required for chronic social defeat stress (CSDS)-induced anhedonia-like behavior.

(A) Schematic illustration of experimental timeline of behavioral test battery consisting of CSDS followed by social interaction (SI; C–F), sucrose preference (SP; G), elevated plus maze (EPM; H), sucrose self-administration, and progressive ratio testing (Suc-SA and PR; Figure 3A–D). (B) AAV2-Npas4 shRNA in the adult male mPFC decreases stress-induced NPAS4 protein expression. Left: representative image showing AAV2-shRNA expression viral vector-mediated eGFP expression in the adult mice mPFC. Right: quantification of NPAS4-positive cells/100 μm² (n = 4/condition). (C) and (D) CSDS decreases the time spent in the social interaction zone (C) and the social interaction ratio (D) in SC shRNA^PFC and Npas4 shRNA^PFC mice after CSDS (n = 18–25/condition). (E) and (F) CSDS increases the time spent in the avoidance corner zone and social avoidance ratio in SC shRNA^PFC and Npas4 shRNA^PFC mice (n = 16–24/condition). (G) CSDS-induced reduction of sucrose preference is blocked by Npas4 shRNA in the mPFC (F; n = 11–24). (H) CSDS reduces time spent in open arms (sec) in SC shRNA^PFC and Npas4 shRNA^PFC mice (n =14–18).

Figure 2—figure supplement 1.. NPAS4 in the medial prefrontal cortex (mPFC) is required for chronic social defeat stress (CSDS)-induced reduction of sucrose consumption.

Chart represents the amount of consumption of water and 1% sucrose solution in each day SC shRNA^PFC and Npas4 shRNA^PFC mice after CSDS (two-way ANOVA, Tukey’s post hoc analysis: control vs. CSDS in SC shRNA^PFC mice, p=0.0624, SC shRNA versus Npas4 shRNA animals with CSDS, p=0.0504, n = 11–24 per group). Data shown are mean ± SEM; Also see Source data 1 for detailed statistical analyses.

Figure 3.. NPAS4 in the medial prefrontal cortex (mPFC) regulates effort-based motivated behavior during sucrose SA following chronic social defeat stress (CSDS).

(A, B) Data plots showing the acquisition period of sucrose self-administration in SC shRNA^PFC and Npas4 shRNA^PFC mice after CSDS or no stress control condition, with no change in the number of sucrose delivery (A) and in the discrimination ratio between the active and inactive nosepokes (B; n = 14–18/group). (C) Data plot showing the maximum number of active nose pokes required to receive a sucrose reward (breakpoint) after CSDS in the PR test of both SC shRNA^PFC and Npas4 shRNA^PFC mice. Npas4 shRNA^PFC mice demonstrated a significantly higher PR breakpoint compared to control SC shRNA^PFC mice (n = 13–19/group). (D) Npas4 shRNA^PFC mice susceptible, but not resilience, to CSDS demonstrated a significantly higher breakpoint compared to SC shRNA^PFC mice after CSDS (n =3–14/group).

Figure 4.. NPAS4 regulates chronic social defeat stress (CSDS)-induced reductions in medial prefrontal cortex (mPFC) dendritic spine density and excitatory synaptic transmission.

(A, B) NPAS4 regulates CSDS-induced reduction of dendritic spine density in the mPFC. (A) Representative images showing AAV2-shRNA expression viral vector-mediated eGFP expression. Scale bar, 3 μm. (B) Quantification of dendritic spine density of deep layer mPFC pyramidal neurons from SC shRNAPFC and Npas4 shRNA^PFC mice after CSDS or in no stress controls (n = 34–55 branch/8 animals/condition). (C) Inter-event interval after Npas4 knockdown and CSDS. (D) Cumulative probability of inter-event interval after CSDS after SC shRNA^PFC and Npas4 shRNA^PFC. (E) Miniature excitatory postsynaptic current (mEPSC) amplitude after Npas4 knockdown and CSDS. (F) Cumulative probability of mEPSCC amplitude after CSDS after SC shRNA^PFC and Npas4 shRNA^PFC. (G) Representative mEPSC traces. (H) Paired-pulse ratio recordings after Npas4 knockdown and CSDS. Data shown are mean ± SEM; *p<0.05, ***p<0.001. Also see Source data 1 for detailed statistical analyses.

Figure 4—figure supplement 1.. Medial prefrontal cortex (mPFC) dendritic spine morphological analyses in the mPFC of SC shRNA^PFC and Npas4 shRNA^PFC mice after chronic social defeat stress (CSDS).

Data plots represent spine head diameter of AAV2-SC shRNA or Npas4 shRNA viral vector-mediated eGFP-positive mPFC pyramidal neurons after CSDS (two-way ANOVA, n =34–55 branch/8 animals/condition).

Figure 5.. NPAS4 regulates the expression of ribosomal and glutamatergic synapse genes.

(A, B) List of top differentially expressed genes in medial prefrontal cortex (mPFC) of Npas4 shRNAPFC mice (A) and corresponding volcano plot of all significant DEGs (FDR < 0.05, log2 (FC) > |0.3|, red) compared to those that were not significant (gray; B). (C) Npas4 DEG enrichment in gene modules that are deferentially regulated in Resilience and Susceptible animals in Bagot et al., 2016 and are dysregulated in neuropsychiatric disorders; Modules M1 and M15, as shown by PsychENCODE. (D) Gene ontology analysis of down- and upregulated DEGs in Npas4 shRNA^PFC mice. (E) Comparison of mPFC genes regulated by Npas4 shRNA^PFC compared to previously published Npas4 ChIP-seq data (Kim et al., 2010; Brigidi et al., 2019). (F) Overlap of significantly differential expression genes (p<0.05) in Npas4 shRNA^PFC mice (left; blue) and differential expression genes (p<0.05) in BA8/9 of human major depressive disorder (MDD) patients (right; pink). (G) ChIP-seq analysis of NPAS4 association with significant ribosome-related differential expression genes identified from this study.

Figure 5—figure supplement 1.. Differential expression genes in the medial prefrontal cortex (mPFC) of Npas4 shRNA mice.

Data plots represent the relative mRNA expression in the mPFC of SC shRNA and Npas4 shRNA mice (two-way ANOVA, Npas4, main effect of Npas4 shRNA, Tukey’s post hoc analysis, SC shRNA^PFC and Npas4 shRNA^PFC mice with acute social defeat stress [SDS], p=0.0005, Ache, main effect of Npas4 shRNA, p=0.0002, Arpp21, main effect of Npas4 shRNA, p=0.118, Dhcr7, main effect of Npas4 shRNA, p=0.0042, Hps4, main effect of Npas4 shRNA, p=0.0121, Nfix, main effect of Npas4 shRNA, p=0.0171, Sst, main effect of Npas4 shRNA, p=0.0281 [n = 7 per group]). Data shown are mean ± SEM; *p<0.05, **p<0.01. Also see Source data 1 for detailed statistical analyses.

Figure 6.. Summary for NPAS4 in the medial prefrontal cortex (mPFC) mediates chronic social defeat stress (CSDS)-induced anhedonia-like behavior and reductions in excitatory synapses.