Article Figures & Data
Figures
- Figure 1.
Timeline of experimental procedures, behavioral tests, and outcome measures. Abbreviations: PND, postnatal day; CDU, control diet unexposed; CDE, control diet exposed; WDU, Western high-fat diet unexposed; WDE, Western high-fat diet exposed; ASR, acoustic startle reflex; OFT, open field test; EPM, elevated plus maze; FST, forced swim test; MRI, magnetic resonance imaging; IMH, immunohistochemistry; WB, Western blot; PBG, postprandial blood glucose; ELISA, enzyme-linked immunosorbent assay.
- Figure 2.
Exposure to PS evokes short-term avoidance behaviors. A, Heat maps depicting the time spent in the threat zone (area of the cage with cat urine odor) and safe zone during PS exposure at P88. B, Analyses showed marked differences in approach–avoidance behaviors. Rats exhibited a strong preference for the safe zone (F(1,32) = 78.87, p < 0.0001). The diet type did not alter this behavior (F(1,32) = 1.43 × 10−10, p = 1.00). No interactions were found between the time spent in the odor threat zone and diet type consumed (F(1,32) = 0.68, p = 0.42). All data are presented as the mean ± SEM; n = 8-10 rats/group.
- Figure 3.
WD reduces traumatic stress-induced startle responses. A, We found that the maximum ASR amplitude increased at 1 week post-PS (PS: F(1,25) = 5.43, p = 0.028; diet: F(1,25) = 1.41, p = 0.25; interactions: F(1,25) = 1.00, p = 0.33). Post hoc testing revealed a significant increase in ASR amplitude in CDE rats when compared with controls (p < 0.05). B, WDE rats exhibited reduced ASR responses during the last recording block when compared with CDE rats (diet: F(1,23) = 7.28, p = 0.013; Tukey’s adjusted for WDE vs CDE, p = 0.028). PS exposure (F(1,23) = 2.73, p = 0.11) or diet–PS exposure interactions (F(1,23) = 1.43, p = 0.24) did not affect the short-term habituation of the ASR. C, TW-ANOVA revealed no significant effect of diet (F(1,26) = 0.35, p = 0.56), PS (F(1,26) = 0.39, p = 0.54), or interaction between these factors (F(1,26) = 0.87, p = 0.36) on the PPI of the ASR. Data presented are presented as the mean ± SEM. Post hoc test, *p < 0.05, **p < 0.01, n = 10–23 rats.
- Figure 4.
WD confers vulnerability to OFT anxiety-like behaviors following traumatic stress exposure. A, PS exposure reduced the number of entries to the center of the OFT (PS: F(1,23) = 14.48, p = 0.0009; diet: F(1,23) = 0.11, p = 0.75; diet × PS interactions: F(1,23) = 2.04, p = 0.17). Post hoc comparisons indicated that the effect of PS was particularly robust in WDE rats when compared with WDU controls (Tukey’s test adjusted, p = 0.0036). B, ANOVA revealed that PS decreased the duration in the center (F(1,23) = 5.02, p = 0.035), while diet (F(1,23) = 0.83, p = 0.37) and diet–PS exposure interactions (F(1,23) = 0.97, p = 0.34) showed no significant main effects. C, PS exposure reduced the total distance traveled in the open field arena (F(1,23) = 32.85, p = 0.0001). Analyses revealed no significant main effect of the diet (F(1,23) = 0.37, p = 0.55) or diet–PS exposure interactions (F(1,23) = 0.049, p = 0.83) on locomotor activity in the open field arena. D, PS exposure increased indices of anxiety on the OFT (PS: F(1,23) = 25.97, p < 0.0001). The main effects of the diet (F(1,23) = 0.37, p = 0.55) and diet–PS exposure interactions (F(1,23) = 1.35, p = 0.26) showed no statistical significance on the OFT anxiety index. E, OFT heat map analyses show that thigmotaxis was particularly robust in the WDE group. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, n = 6–10 rats/group.
- Figure 5.
WD increases anxiety-like behaviors in the EPM following PS. Each open arm was subdivided into three zones (zone 1, proximal; zone 2, medial; zone 3, distal) to eliminate false-positive results in arm entry statistics. A, The diet type significantly affected the number of entries to the medial section of the open arms (diet: F(1,24) = 4.68, p = 0.041). WDE rats showed a reduced number of entries to zone 2 and zone 3 of the open arms when compared with CDE rats (p < 0.05). PS exposure (F(1,24) = 3.23, p = 0.085) or diet–PS exposure interactions (F(1,23) = 1.93, p = 0.18) did not affect the number of entries to the medial zone of the open arms. B, Notably, we found a significant main diet effect on the time the rats spent in the proximal zones of the EPM open arms (for zone 1: F(1,23) = 4.81, p = 0.039). Post hoc testing revealed that WDE rats spent less time in the proximal and medial parts of the open arms when compared with CDE rats (p < 0.05). PS exposure main effects (F(1,23) = 0.25, p = 0.62) or diet–PS exposure interactions (F(1,23) = 2.30, p = 0.14) showed no statistical significance. PS exposure decreased the time spent in the distal zones of the EPM open arms (for zone 3: F(1,23) = 6.59, p = 0.017). Analyses revealed no significant diet (F(1,23) = 0.80, p = 0.38) or diet–PS exposure interactions effects (F(1,23) = 0.55, p = 0.47). C, PS exposure had a significant effect on the EPM index of anxiety (F(1,24) = 5.65, p = 0.026). Post hoc analyses revealed that the PS-exposed rats exhibited an increased anxiety index when compared with controls (WDE vs WDU, p = 0.022). Analyses revealed no significant main effects of the diet (F(1,24) = 2.03, p = 0.17) or diet–PS exposure interactions (F(1,24) = 2.94, p = 0.099) on the anxiety index. D, We found that the diet type significantly affected the frequency of when the nose point of the rats was in the head-dipping areas (F(1,24) = 4.61, p = 0.042). Post hoc comparisons indicated a decreased nose in head-dipping zone frequency in WDE rats when compared with the CDE rats (p < 0.05). PS exposure (F(1,24) = 3.60, p = 0.070) and diet–PS exposure interactions (F(1,24) = 3.96, p = 0.058) did not have a significant effect on head-dipping zone frequency. E, PS exposure increased SAP frequency (PS: F(1,24) = 7.17, p = 0.013). Post hoc testing showed increased SAPs in the WDE rats when compared with WDU rats (p < 0.05). The diet type (F(1,24) = 3.37, p = 0.079) and diet–PS exposure interactions (F(1,24) = 0.98, p = 0.33) did not show significant main effects on SAP frequency. F, We found no significant main effect of diet (F(1,24) = 0.94, p = 0.16), PS exposure main effect (F(1,24) = 1.54, p = 0.23), or interaction between these factors (F(1,24) = 2.76, p = 0.11) on the total distance traveled during EPM testing. G, Heat maps show that WDE rats spent more time in the closed arms when compared with CDE rats. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, n = 6–10 rats/group. For A and B: *p < 0.05 or **p = 0.01 for CDE vs WDE; #p < 0.05 for WDU vs WDE.
- Figure 6.
WD increases FKBP51 protein levels in the hippocampus. A, Representative Western blot shows increased FKBP51 levels in the brains of the rats that consumed the WD. B, Analyses showed a significant main effect of the diet on brain FKBP51 levels (diet effect: F(1,19) = 15.45, p = 0.0009), PS exposure (F(1,19) = 0.51, p = 0.49), or interaction between diet and PS exposure (F(1,19) = 0.42, p = 0.52) did not show significant main effects on FKBP51 levels. C, Representative blot from micropunched anxiety-related brain regions showed distinctive FKBP51 levels. D, Analyses revealed a significant increase in FKBP51 levels in the hippocampus of the WDE rats when compared with CDE rats (F(1,16) = 9.88; p = 0.0063, p = 0.021). Diet did not alter FKBP51 levels in the amygdala or medial prefrontal cortex. Although it did not reach statistical significance, we found a trend for more prefrontal cortex FKBP51 levels when compared with the amygdala and the hippocampus (F(2,16) = 3.58, p = 0.052). No significant interactions between diet and the studied brain regions were revealed (F(2,16) = 2.44, p = 0.12). Data are expressed as the mean ± SEM. *p < 0.05. Amygdala (AMY), n = 3–4 rats; medial prefrontal cortex (mPFC), n = 3–4; HPC, n = 4–5.
- Figure 7.
WD and traumatic stress exposure alter hippocampal volume in a region-specific manner. A, We found a significant main effect of PS on total brain volume (F(1,11) = 11.31, p = 0.0063). Post hoc comparisons indicated that PS exposure increased the brain volume in WDE rats when compared with CDU rats (p = 0.036). TW-ANOVA revealed no significant main diet effects (F(1,11) = 0.91, p = 0.36) or interactions (F(1,11) = 1.13, p = 0.31) on total brain volume. B, WDU rats showed reduced HPC volumes when compared with CDU rats (diet effect: F(1,11) = 9.59, p = 0.010; post hoc test, p = 0.0080). Although analyses revealed no significant main PS effects (F(1,11) = 0.13, p = 0.73), we found a significant interaction between diet and PS exposure (F(1,11) = 10.62, p = 0.0076) on total brain volume. C, The left HPC volume was significantly affected by the diet (F(1,11) = 7.34, p = 0.020) and PS exposure (F(1,11) = 5.88, p = 0.034). Post hoc comparisons revealed reduced HPC volumes in WDU rats when compared with CDU rats (WDU vs CDU, p = 0.022) and WDE rats (WDU vs WDE, p = 0.015). We also found significant interactions between the diet and PS exposure (F(1,11) = 7.38, p = 0.020). D, The diet type (F(1,11) = 3.74, p = 0.079) and PS exposure (F(1,11) = 0.00091, p = 0.98) did not have a significant effect on right HPC volumes (interaction: F(1,11) = 4.83, p = 0.050). E, We found reduced ventral HPC volumes in the rats that consumed the WD (F(1,11) = 6.93; p = 0.023). We found no significant main effects of PS exposure (F(1,11) = 0.39, p = 0.54) or interactions (F(1,11) = 3.27, p = 0.098) on ventral HPC volumes. F, Interestingly, PS exposure led to increased dorsal HPC volumes (F(1,11) = 7.45; p = 0.020). We found no significant main effects of the diet type (F(1,11) = 0.19, p = 0.67) or significant interactions between diet and PS exposure (F(1,11) = 1.23, p = 0.29). G, The WD had a significant effect on increasing the left lateral ventricle volume (F(1,11) = 5.95; p = 0.033). We found no significant main effects of PS exposure (F(1,11) = 1.33, p = 0.27) or interactions (F(1,11) = 2.59, p = 0.14). H, We found no significant main effects of diet (F(1,11) = 1.23, p = 0.29), PS exposure (F(1,11) = 0.39, p = 0.54), or interactions (F(1,11) = 2.13, p = 0.17) on the right lateral ventricular volume. *p < 0.05, **p < 0.01; n = 3–5 brains/group. BV, Brain volume; LV, lateral ventricle.
- Figure 8.
WD consumption led to hippocampal atrophy and ventricular enlargement. A, Representative MR images showing reductions in the anteroposterior hippocampal volume in the rats that consumed the WD. The asterisk indicates larger lateral ventricles in the rats that consumed the WD. B, C, 3D coronal and ventral oblique MRI reconstructions show the impact of the WD on the hippocampal (B; red, arrowheads) and ventricular (C; yellow, arrowheads) volumes.
- Figure 9.
A WD alters neurovascular integrity in the hippocampus. A, Representative photomicrograph showing FKBP51 expression in hippocampal Glut-1+ endothelial cells in a rat that consumed the WD. B, Immunoreactivity specificity was confirmed by omitting primary antibodies. C, D, Fluorescence photomicrographs show that the hippocampus of rats that consumed the CD (C) had more tomato-lectin+ (TL) staining compared with the hippocampus of animals consuming the WD (D). E, F, Representative photomicrographs depict smaller blood vessel diameter in the rats that consumed the CD (E) when compared with rats that consumed the WD (F). G, Analyses revealed reduced TL+ staining area in the animals consuming the WD (t(6) = 2.69, p = 0.036). H, No significant differences were observed in the blood vessel diameter when diet groups were compared. BV, Blood vessel; M, microglia. n = 4 brains per group. Student’s t test, *p < 0.05. Scale bars, 25 μm.
- Figure 10.
Pearson’s correlations show significant associations between obesity and stress biomarkers. A–C, Scatter plots show a significant relationship between plasma triglyceride and leptin levels (A), leptin levels and the EPM anxiety index (B), and leptin levels and ventral hippocampal volume (C). Pearson’s correlation was significant at p < 0.05. Solid line, Linear regression; outer dashed line, 95% confidence intervals; CDU, clear circles; CDE, gray circles; WDU, black circles; WDE rats, red circles.
Tables
CD WD Carbohydrates (% kcal) 64.7 43.1 Protein (% kcal) 18.8 15.5 Fat (% kcal) 16.5 41.4 Total kcal 3.77 4.57 Fatty acids (g/100 g) C4:0 ND 0.68 C6:0 ND 0.44 C8:0 ND 0.29 C10:0 ND 0.43 C12:0 ND 0.59 C14:0 ND 2.10 C16:0 0.81 5.59 C18:0 0.32 2.71 Total saturated 1.13 12.83 C16:1 0.04 0.42 C18:1 1.57 5.50 Total monosaturated 1.61 5.92 C18:2n6 3.55 1.04 C18:3n3 0.48 0.36 C20:4n6 ND 0.10 C20:5n3 ND ND C22:5n3 ND ND C22:6n3 ND ND Total polyunsaturated 4.09 1.50 The level of dietary fat was ∼7% of dry weight in the CD or 20% in the WD. Gas chromatography coupled with mass spectrometry revealed high levels of saturated fatty acids (11-fold difference) and monosaturated fatty acids (4-fold difference) in the WD when compared with the CD. The CD contained higher levels of omega-6 polyunsaturated fatty acids. ND, Not determined.
Biometric parameter Group p Value CDU CDE WDU WDE Diet PS Interaction Body weight (g) 429 ± 7.6 461 ± 3.6 462 ± 13.6 506 ± 4.3*,*** <0.0001 <0.0001 0.409 Food consumption (kcal/d) 72 ± 5.4 84 ± 3.8 84 ± 1.3 98 ± 1.4*,*** 0.002 0.001 0.749 Corticosterone (ng/0.5 g feces) 8.2 ± 1.7 7.6 ± 1.5 6.8 ± 0.7 7.1 ± 0.5 0.206 0.827 0.505 FBG (mg/dl) 137 ± 4.7 134 ± 7.5 165 ± 9.2 137 ± 7.8 0.087 0.086 0.170 PBG (mg/dl) 127 ± 9.4 167 ± 8.8**** 129 ± 5.1 109 ± 4.7*** 0.002 0.199 0.001 Plasma leptin (pg/ml) 612 ± 82.8 603 ± 163.7 1211 ± 257.6 1553 ± 282.2*** 0.006 0.481 0.459 Plasma triglycerides (mg/dl) 626 ± 18.1 581 ± 37.7 679 ± 35.7 643 ± 38.9 0.116 0.252 0.900 The rats that consumed the WD showed significant alterations in body weight and food consumption when compared with animals consuming the CD (n = 12–16 rats/group). Exposure to traumatic stress (F(1,24) = 25.12; p < 0.0001) and WD consumption (F(1,24) = 25.81, p < 0.0001) increased body weights. No interactions were observed between traumatic stress and diet (F(1,24) = 0.71, p = 0.40). Notably, we found that the rats that consumed the WD showed a significant increase in caloric intake (PS: F(1,10) = 18.96, p = 0.0014; diet: F(1,10) = 13.99, p = 0.0038; PS × diet interaction: F(1,10) = 0.086, p = 0.78; n = at least 6 cages/group; 2 rats per cage). We found no significant differences in FBG levels between the diet and stress groups (PS: F(1,24) = 3.20, p = 0.086; diet: F(1,24) = 3.18, p = 0.087; interaction: F(1,24) = 2.01, p = 0.17). However, we found significant main effect interactions in PBG levels (PS: F(1,24) = 0.0016, p = 0.97; diet: F(1,24) = 2.03, p = 0.17; interaction: F(1,24) = 11.66, p = 0.0023; n = 12–16 rats/group). The WD and PS did not alter corticosterone (CORT) levels (diet effect: F(1,24) = 1.69, p = 0.21; PS: F(1,24) = 0.049, p = 0.0.83); interaction: F(1,24) = 0.46, p = 0.51; n = 12-13 rats/group). Values are presented as the mean ± SEM. The diet type had a significant effect on plasma leptin levels (diet: F(1,11) = 11.53, p = 0.0060; PS: F(1,11) = 0.53, p = 0.48; interaction F(1,11) = 0.59, p = 0.46). Post hoc testing revealed that WDE rats had significantly higher plasma leptin levels when compared with those of CDE rats (p = 0.0456). Interestingly, plasma triglyceride levels were not affected by the diet and stress exposure (diet: F(1,12) = 2.87, p = 0.12; PS: F(1,12) = 1.45, p = 0.25; interaction F(1,12) = 0.016, p = 0.90). Bold denotes statistically significant effects.
*p < 0.05.
***p < 0.001.
****p < 0.0001.
Behavior CD WD p Value ASR (vMax, mV) 346.90 ± 66.0 267.60 ± 40.0 n.s. PPI (%) 67.98 ± 3.4 72.89 ± 5.0 n.s. EPM-entries (frequency) 5.17 ± 1.8 7.63 ± 1.8 n.s. EPM-anxiety index (index from 0 to 1) 0.47 ± 0.0 0.56 ± 0.1 n.s. FST-high mobility (time duration, %) 10.88 ± 1.6 12.34 ± 1.3 n.s. Values are presented as the mean ± SEM. Baseline ASR, PPI of the ASR, and EPM and FST indices of anxiety were evaluated at P58 (4 weeks after WD consumption started and 4 weeks before traumatic stress). No significant differences were observed between diet groups (p > 0.05, n = 8–23 rats per group/behavioral outcome). n.s., Not significant.
WD effect PS effect WD × PS interactions Biometrics Body weight ↑ ↑ n.s. Caloric consumption ↑ ↑ n.s. Fasting blood glucose n.s. n.s. n.s. Postprandial blood glucose n.s. n.s. s. Behavioral ASR n.s. ↑ n.s. ASR habituation ↑ n.s. n.s. Prepulse inhibition n.s. n.s. n.s. OF center entries and duration n.s. ↑ n.s. OF anxiety index n.s. ↑ n.s. OF distance traveled n.s. ↓ n.s. EPM open arm entries and duration ↓ ↓ n.s. EPM anxiety index ↑ ↑ n.s. EPM head dipping zone ↓ n.s. n.s. EPM stretched postures n.s. ↑ n.s. FST mobility n.s. n.s. n.s. Endocrine Corticosterone levels n.s. n.s. n.s. FKBP51 protein levels ↑ n.s. n.s. Plasma leptin levels ↑ n.s. n.s. Plasma triglycerides n.s. n.s. n.s. Anatomical Brain volume n.s. ↑ n.s. HPCV ↓ n.s. s. Ventral HPCV ↓ n.s. n.s. Dorsal HPCV n.s. ↑ n.s. Left HPCV ↓ n.s. s. Right HPCV n.s. n.s. n.s. Left lateral ventricle volume ↑ n.s. n.s. Right lateral ventricle volume n.s. n.s. n.s. Histological HPC microglia density/area n.s. n.d. n.d. HPC blood vessel density/area ↓ n.d. n.d. HPCV, HPC volume; OF, open field; s., significant main effect; n.s., nonsignificant main effect; n.d., not determined. Our cross-scale study design allowed for the identification of fundamental relationships among neuroanatomical and neurovascular structure, neuroendocrine function, and behavior. Table shows statistically significant main effects of the WD, PS, or interactions as determined by two-way ANOVA. Arrows depict whether the WD or PS significantly altered the outcome measures and the direction of change.
In this issue
