Adult neurogenesis in the ventral hippocampus decreased among animal models of neurodevelopmental disorders

Abstract

Introduction: Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by deficits in social interaction and communication, along with restricted and repetitive behaviors. Both genetic and environmental factors contribute to ASD, with prenatal exposure to valproic acid (VPA) and nicotine being linked to increased risk. Impaired adult hippocampal neurogenesis, particularly in the ventral region, is thought to play a role in the social deficits observed in ASD.

Methods: In this study, we investigated social behavior and adult hippocampal neurogenesis in C57BL/6J mice prenatally exposed to VPA or nicotine, as well as in genetically modified ASD models, including IQSEC2 knockout (KO) and NLGN3-R451C knock-in (KI) mice. Sociability and social novelty preference were evaluated using a three-chamber social interaction test. Adult hippocampal neurogenesis was assessed by BrdU and DCX immunofluorescence to identify newborn and immature neurons.

Results: VPA-exposed mice displayed significant deficits in social interaction, while nicotine-exposed mice exhibited mild impairment in social novelty preference. Both IQSEC2 KO and NLGN3-R451C KI mice demonstrated reduced adult neurogenesis, particularly in the ventral hippocampus, a region associated with social behavior and emotion. Across all ASD mouse models, a significant reduction in BrdU+/NeuN+ cells in the ventral hippocampus was observed, while dorsal hippocampal neurogenesis remained relatively unaffected. Similar reductions in DCX-positive cells were identified in VPA, nicotine, and NLGN3-R451C KI mice, indicating impaired proliferation or differentiation of neuronal progenitors.

Discussion: These findings suggest that impaired adult neurogenesis in the ventral hippocampus is a common hallmark across ASD mouse models and may underlie social behavior deficits. This study provides insight into region-specific neurogenic alterations linked to ASD pathophysiology and highlights potential targets for therapeutic interventions.

Keywords: IQSEC2; adult hippocampal neurogenesis; autism spectrum disorder; neuroligin 3; prenatal nicotine exposure; valproic acid.

Figure 1

Experimental design and characterization of VPA mouse model. (A) Timeline of the experimental procedures, illustrating the administration of valproic acid (VPA) or saline at embryonic day (E) 13.5, followed by behavioral testing, BrdU administration, and immunohistological analysis at specific postnatal days. (B) Survival rate of VPA-exposed mice compared to controls. Prenatal exposure to 450 mg/kg VPA significantly decreased survival, whereas 150 mg/kg VPA showed similar survival rates to controls. (C) Representative DAPI-stained coronal sections of dorsal and ventral hippocampus, showing no significant morphological differences between control and VPA-exposed mice. (D) Body weights of VPA-exposed and control mice measured at several time points indicate no significant differences across groups. Scale bars = 1 mm. Results are presented as mean ± SEM.

Figure 2

Social interaction deficits are observed in VPA-exposed mice. (A) Diagram of the three-chamber social preference test. S1 represents a stranger mouse, and E represents an empty cage. (B) Representative heatmap images depicting exploratory behavior in control and VPA-exposed mice during the social preference test. (C) Time spent in each area during the social preference phase indicates reduced interaction with S1 in VPA-exposed mice compared to controls. (D) Social preference index (S1–E) showing significantly diminished social preference in VPA mice. (E–H) Social novelty preference phase: diagram of the test (E), heatmap images (F), and quantification (G,H) showing reduced interaction with a novel stranger (S2) in VPA-exposed mice, suggesting impaired social novelty recognition. Results are presented as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired student’s t-test was applied to compare Con vs. VPA; paired student’s t-test was applied to compare S1 vs. E.

Figure 3

Reduction of adult hippocampal neurogenesis is observed in VPA-exposed mice. (A) Representative immunofluorescence images showing BrdU (red) and NeuN (green) co-labeling in dorsal and ventral hippocampus of control and VPA mice. BrdU/NeuN double-positive cells indicate newborn neurons. Inset represents zooming in of the location indicated by a square on the main image. Arrows indicate BrdU/NeuN double-positive cells. Scale bars = 100 μm or 20 μm (insets). (B) Quantification reveals a significant reduction in the number of BrdU+/NeuN+ cells in both dorsal and ventral hippocampus of VPA mice compared to controls. (C) Immunofluorescence images of doublecortin (DCX, red)-positive cells in dorsal and ventral hippocampus. The brain structure was visualized with DAPI (blue). Inset represents zooming in of the location indicated by a square on the main image. An arrow indicates DAPI/DCX double-positive cells. Scale bars = 100 μm or 20 μm (inset). (D) The cell density of DCX-positive immature neurons shows a significant decrease in both dorsal and ventral hippocampus of VPA mice compared to controls, indicating impaired neurogenesis. Results are presented as mean ± SEM, *p < 0.05, **p < 0.01, Student’s t-test.

Figure 4

Social behavior deficits are observed in prenatal nicotine-exposed (PNE) mice. (A) Diagram of the social preference test setup. (B) Heatmap images of exploratory behavior in PNE and control mice during the social preference phase. (C,D) Quantification of time spent in each area (C) and contact preference (D) during the social preference phase indicates no significant difference between PNE and control mice. (E–H) Social novelty preference test: diagram (E), heatmap images (F), and quantification (G–H) show reduced preference for a novel stranger (S2) in PNE mice, indicating impaired social novelty recognition. Results are presented as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired student’s t-test was applied to compare Con vs. VPA; paired student’s t-test was applied to compare S1 vs. E.

Figure 5

Reduction of adult hippocampal neurogenesis is selectively observed in the ventral hippocampus of PNE mice. (A) Representative immunofluorescence images showing BrdU (red) and NeuN (green) labeling in the dorsal and ventral hippocampus of control and PNE mice. Inset represents zooming in of the location indicated by a square on the main image. Arrows indicate BrdU/NeuN double-positive cells. Scale bars = 100 μm or 20 μm (insets). (B) Quantification reveals a reduction in BrdU+/NeuN+ cells in the ventral hippocampus of PNE mice compared to controls. (C,D) DCX staining (red) (C) and quantification (D) show reduced cell density of DCX-positive immature neurons (per mm) in the ventral hippocampus of PNE mice. In Figure 5C, the brain structure was visualized with DAPI (blue). Inset represents zooming in of the location indicated by a square on the main image. An arrow indicates DAPI/DCX double-positive cells. Scale bars = 100 μm or 20 μm (inset). Results are presented as mean ± SEM, *p < 0.05, **p < 0.01, Student’s t-test.

Figure 6

Reduction of adult hippocampal neurogenesis is selectively observed in the ventral hippocampus of IQSEC2 KO and NLGN3-R451C KI mice. (A,C) Quantification of BrdU+/NeuN+ double-positive cells in the hippocampus of IQSEC2-KO (A) and NLGN3-R451C KI (C) mice. Reduction in BrdU+/NeuN+ cells is observed in the ventral hippocampus of both mouse models. (B,D) Quantification of DCX-positive cells in the hippocampus of IQSEC2-KO (B) and NLGN3-R451C KI (D) mice. DCX-positive cell density was reduced in the ventral hippocampus of NLGN3-R451C KI mice compared to WT controls. Results are presented as mean ± SEM, *p < 0.05, **p < 0.01, Student’s t-test.