Fig 1.
Study timeline and fear conditioning procedures.
A) Mice were assigned to either a control (0.4% NaCl) or high salt (4.0% NaCl) diet for two or six weeks. Experiment 1 involved diet manipulation for two weeks, at which time mice underwent context fear training, followed 48 h later by context fear testing. Experiment 2 (grey shading) involved diet manipulation for six weeks, at which time mice underwent context fear training, followed 48 h later by context fear testing. Experiment 3 involved diet manipulation for two weeks, at which time mice underwent context fear training, followed four weeks later (while continuing the same diet manipulation) by context fear testing. B) Mice trained with a mild foot shock in the Training Context were tested 48 h (Experiments 1, 2) or four weeks (Experiment 3) later in either the Training Context or the Neutral Context. The Training Context included a metal grid floor, visible illumination, patterned background, and 70% ethanol scent. The Neutral Context included a smooth acrylic floor, infrared illumination, no background, and Windex scent. C) Control no shock mice were exposed to the Training Context, but were never administered a foot shock. These mice were tested at the same timelines for Experiments 1–3 in the Training Context, to assess for any baseline influences of diet consumption on fear behavior.
Fig 2.
Context fear training across Experiments.
Mice assigned to 0.4% NaCl represented by blue symbols, mice assigned to 4.0% NaCl represented by red symbols; mice to be tested in Training Context represented by squares and solid lines, mice to be tested in Neutral Context represented by circles and dotted lines. All mice were trained in the Training Context with five, pseudorandomized mild (1 s, 0.8 mA) foot shocks during a 6 min training session. Percent freezing during training of mice in A) Experiment 1, B) Experiment 2 (grey shading), and C) Experiment 3. Baseline percent freezing averaged across the first two minutes of the training session and plotted along x-axis as 0 time point. Average percent freezing for each 30 sec period following each of the five mild foot shocks are plotted along x-axis as Post-shock Periods 1–5. Experiment 1: 0.4% NaCl females Training Context, n = 7; 0.4% NaCl females Neutral Context, n = 9; 4.0% NaCl females Training Context, n = 8; 4.0% NaCl females Neutral Context, n = 9; 0.4% NaCl males Training Context, n = 8; 0.4% NaCl males Neutral Context, n = 9; 4.0% NaCl males Training Context, n = 7; 4.0% NaCl males Neutral Context, n = 8. Experiment 2: 0.4% NaCl females Training Context, n = 9; 0.4% NaCl females Neutral Context, n = 8; 4.0% NaCl females Training Context, n = 8; 4.0% NaCl females Neutral Context, n = 8; 0.4% NaCl males Training Context, n = 9; 0.4% NaCl males Neutral Context, n = 8; 4.0% NaCl males Training Context, n = 8; 4.0% NaCl males Neutral Context, n = 10. Experiment 3: 0.4% NaCl females Training Context, n = 8; 0.4% NaCl females Neutral Context, n = 9; 4.0% NaCl females Training Context, n = 9; 4.0% NaCl females Neutral Context, n = 8; 0.4% NaCl males Training Context, n = 7; 0.4% NaCl males Neutral Context, n = 8; 4.0% NaCl males Training Context, n = 8; 4.0% NaCl males Neutral Context, n = 8. Data are graphed as mean ± 95% confidence interval. ✣indicates p<0.05 difference between males to be tested in Neutral Context and consuming 0.4% and 4.0% NaCl diets at indicated time points.
Fig 3.
Context fear expression across contexts and Experiments.
Mice assigned to 0.4% NaCl represented by blue symbols, mice assigned to 4.0% NaCl represented by red symbols; mice tested in Training Context represented by squares and solid lines, mice tested in Neutral Context represented by circles and dotted lines. Percent freezing during minutes two through six of the 10 min testing session are graphed for all mice. Testing occurred A) 48 h after training in Experiment 1, during which mice underwent two weeks of diet manipulation; B) 48 h after training in Experiment 2 (grey shading), during which mice underwent six weeks of diet manipulation; and C) four weeks after training in Experiment 3, during which mice underwent six total weeks of diet manipulation (training occurred after two weeks of diet manipulation). Experiment 1: 0.4% NaCl females Training Context, n = 7; 0.4% NaCl females Neutral Context, n = 9; 4.0% NaCl females Training Context, n = 8; 4.0% NaCl females Neutral Context, n = 9; 0.4% NaCl males Training Context, n = 8; 0.4% NaCl males Neutral Context, n = 9; 4.0% NaCl males Training Context, n = 7; 4.0% NaCl males Neutral Context, n = 8. Experiment 2: 0.4% NaCl females Training Context, n = 9; 0.4% NaCl females Neutral Context, n = 8; 4.0% NaCl females Training Context, n = 8; 4.0% NaCl females Neutral Context, n = 8; 0.4% NaCl males Training Context, n = 9; 0.4% NaCl males Neutral Context, n = 8; 4.0% NaCl males Training Context, n = 8; 4.0% NaCl males Neutral Context, n = 10. Experiment 3: 0.4% NaCl females Training Context, n = 8; 0.4% NaCl females Neutral Context, n = 9; 4.0% NaCl females Training Context, n = 9; 4.0% NaCl females Neutral Context, n = 8; 0.4% NaCl males Training Context, n = 7; 0.4% NaCl males Neutral Context, n = 8; 4.0% NaCl males Training Context, n = 8; 4.0% NaCl males Neutral Context, n = 8. Data are graphed as mean ± 95% confidence interval. *p<0.05, **p<0.01, ***p<0.001 indicate difference between Training Context and Testing Context within same sex and diet. ✣✣✣indicates p<0.001 difference between females and males on same diet and tested in same context.
Fig 4.
Serum osmolality in context fear conditioned mice across Experiments.
Mice assigned to 0.4% NaCl represented by blue symbols, mice assigned to 4.0% NaCl represented by red symbols; mice tested in Training Context represented by squares and solid lines, mice tested in Neutral Context represented by circles and dotted lines. Serum osmolality in context fear conditioned mice from A) Experiment 1, B) Experiment 2 (grey shading), and C) Experiment 3. Experiment 1: 0.4% NaCl females Training Context, n = 8; 0.4% NaCl females Neutral Context, n = 9; 4.0% NaCl females Training Context, n = 8; 4.0% NaCl females Neutral Context, n = 9; 0.4% NaCl males Training Context, n = 8; 0.4% NaCl males Neutral Context, n = 9; 4.0% NaCl males Training Context, n = 8; 4.0% NaCl males Neutral Context, n = 8. Experiment 2: 0.4% NaCl females Training Context, n = 9; 0.4% NaCl females Neutral Context, n = 9; 4.0% NaCl females Training Context, n = 8; 4.0% NaCl females Neutral Context, n = 8; 0.4% NaCl males Training Context, n = 9; 0.4% NaCl males Neutral Context, n = 9; 4.0% NaCl males Training Context, n = 9; 4.0% NaCl males Neutral Context, n = 9. Experiment 3: 0.4% NaCl females Training Context, n = 8; 0.4% NaCl females Neutral Context, n = 9; 4.0% NaCl females Training Context, n = 9; 4.0% NaCl females Neutral Context, n = 8; 0.4% NaCl males Training Context, n = 7; 0.4% NaCl males Neutral Context, n = 8; 4.0% NaCl males Training Context, n = 7; 4.0% NaCl males Neutral Context, n = 7. Data are graphed as mean ± 95% confidence interval.
Fig 5.
Log-transformed serum corticosterone levels in fear conditioned mice across Experiments.
Mice assigned to 0.4% NaCl represented by blue symbols, mice assigned to 4.0% NaCl represented by red symbols; mice tested in Training Context represented by squares and solid lines, mice tested in Neutral Context represented by circles and dotted lines. Log-transformed serum corticosterone levels in context fear conditioned mice in A) Experiment 1, B) Experiment 2 (grey shading), and C) Experiment 3. Experiment 1: 0.4% NaCl females Training Context, n = 6; 0.4% NaCl females Neutral Context, n = 7; 4.0% NaCl females Training Context, n = 7; 4.0% NaCl females Neutral Context, n = 7; 0.4% NaCl males Training Context, n = 8; 0.4% NaCl males Neutral Context, n = 9; 4.0% NaCl males Training Context, n = 8; 4.0% NaCl males Neutral Context, n = 8. Experiment 2: 0.4% NaCl females Training Context, n = 8; 0.4% NaCl females Neutral Context, n = 9; 4.0% NaCl females Training Context, n = 7; 4.0% NaCl females Neutral Context, n = 7; 0.4% NaCl males Training Context, n = 9; 0.4% NaCl males Neutral Context, n = 9; 4.0% NaCl males Training Context, n = 8; 4.0% NaCl males Neutral Context, n = 10. Experiment 3: 0.4% NaCl females Training Context, n = 7; 0.4% NaCl females Neutral Context, n = 8; 4.0% NaCl females Training Context, n = 8; 4.0% NaCl females Neutral Context, n = 7; 0.4% NaCl males Training Context, n = 8; 0.4% NaCl males Neutral Context, n = 8; 4.0% NaCl males Training Context, n = 8; 4.0% NaCl males Neutral Context, n = 7. Data are graphed as mean ± 95% confidence interval. ✣indicates p<0.05 difference between females and males on same diet and tested in same context.