**Background:** Diet is a major contributor to health, but the effects of food hardness—and thus mastication—on brain function and behavior are poorly understood. Reduced chewing has been linked to cognitive decline, shortened lifespan, and increased oxidative stress in the brain. This study investigated whether long-term soft-food rearing in young mice alters cognitive function, motor skills, activity levels, and aggression, and explored underlying molecular changes in the hippocampus.
**Methods:** Three-week-old male C57BL/6N mice (n=65) were divided into three groups: Control (C, solid pellets, n=14), soft-fed (S, CE-2 powder with 60% water, n=31), and soft-fed then switched to solid diet after 3 months (SH, n=20). After 6 months of rearing, mice underwent behavioral testing: Y-maze for spatial memory, rotarod for motor coordination, aggressive biting behavior (ABB) measurement using an aggression response meter, and 24-hour locomotor activity monitoring. Blood was collected for serum biochemistry, corticosterone, and serotonin assays. Hippocampal gene expression was analyzed by real-time PCR for Glut1, c-Fos, Rbfox3 (NeuN), Bdnf, Maoa, Cdh13, 5-HT6-R, and AMPA receptor subunits (GluA1-4). Immunohistochemistry quantified c-Fos, NeuN, BDNF, MAOA, CDH13, serotonin, 5-HT2A, 5-HT2C, TPH2, and GluA1 protein expression in the hippocampus. Statistical analysis used one-way ANOVA with Tukey's post hoc test (p<0.05).
**Key Results:** Soft-fed mice showed significant weight gain from 8 weeks of age (p=0.008 vs. control). Food intake was higher in S group (7.42±0.83 g) vs. C (4.6±0.38 g, p<0.001), while water intake was lower (S: 1.73±0.22 g vs. C: 5.71±0.46 g, p<0.001). Serum albumin was similar across groups, but total cholesterol was elevated in S (119±6.75 mg/dL) vs. C (69±13.1 mg/dL, p=0.01) and SH (73.3±4.37 mg/dL, p=0.003). In the Y-maze, S group had significantly lower alternation rate vs. C (p=0.04), while SH group recovered vs. S (p=0.03). Rotarod latency was shorter in S vs. C (p=0.0007) and improved in SH vs. S (p<0.01). Hippocampal c-Fos mRNA and protein were reduced in S vs. C (p<0.01 and p<0.001, respectively) and increased in SH vs. S (p<0.001). NeuN-positive cells decreased in S vs. C (p<0.001) and increased in SH vs. S (p=0.009). Bdnf mRNA decreased in S vs. C (p=0.003) and BDNF immunostaining increased in SH vs. S (p=0.008). Nocturnal locomotor activity was significantly increased in S (p=0.04 vs. C) and SH (p=0.02 vs. C). Aggressive biting intensity was markedly higher in S (15.0±1.25 mNs) vs. C (0.78±0.26 mNs, p=0.001) and SH (3.84±0.73 mNs, p=0.005). Bite frequency was also higher in S (7.5±0.41 times) vs. C (1±0, p<0.001) and SH (2.33±0.88, p=0.023). Serum corticosterone was elevated in S (285±32.5 ng/mL) vs. C (152.3±13.3 ng/mL, p=0.02) and SH (152.67±17.42 ng/mL, p=0.023). Hippocampal Maoa mRNA and MAOA-positive cells were higher in S vs. C (p<0.001) and vs. SH (p=0.04 and p=0.02, respectively). Cdh13 mRNA was higher in S vs. SH (p=0.02). CDH13-positive cells were higher in S vs. C (p=0.001) and vs. SH (p=0.02). Serum serotonin was similar between C (97.6±13.19 ng/mL) and S (86.43±8.68 ng/mL, p=0.57) but lower in SH (27.8±5.61 ng/mL, p=0.003 vs. S, p=0.004 vs. C). Hippocampal 5-HT-positive cells were reduced in S and SH vs. C (p=0.03 and p=0.01, respectively). 5-HT2A-positive cells decreased in S and SH vs. C (both p<0.001). GluA1 protein expression increased in S vs. C (p=0.02). GluA1 mRNA decreased in S vs. C (p=0.008) and increased in SH vs. S (p<0.001). GluA3 mRNA decreased in S (p=0.002) and increased in SH (p=0.001). GluA4 mRNA increased in S vs. C (p=0.009) and decreased in SH vs. S (p=0.02). TPH2-positive cells increased in S vs. C (p=0.02) and decreased in SH vs. S (p=0.009).
**Clinical Implications:** This study provides the first evidence that long-term soft-food rearing in young mice increases aggressive behavior, alongside previously reported cognitive and motor deficits. The findings suggest that reduced mastication during development may alter hippocampal gene expression related to serotonin signaling, monoamine degradation (MAOA), cell adhesion (CDH13), and AMPA receptor subunit composition, potentially contributing to mood dysregulation and aggression. While these results are from a murine model, they underscore the potential importance of food hardness and chewing for healthy brain development. The partial reversibility of some changes upon switching to a solid diet suggests that dietary interventions may mitigate some negative effects. Further research in humans is needed to confirm whether soft-food diets during childhood could influence behavioral and cognitive outcomes.