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. 2023 Jul 13;18(7):e0286221.
doi: 10.1371/journal.pone.0286221. eCollection 2023.

Generalization of contextual fear is sex-specifically affected by high salt intake

Jasmin N Beaver  1   2 , Brady L Weber  1   2 , Matthew T Ford  1 , Anna E Anello  1   2 , Kaden M Ruffin  1 , Sarah K Kassis  1   2 , T Lee Gilman  1   2   3
Affiliations

Generalization of contextual fear is sex-specifically affected by high salt intake

Jasmin N Beaver et al. PLoS One. .

Abstract

A hallmark symptom of many anxiety disorders, and multiple neuropsychiatric disorders more broadly, is generalization of fearful responses to non-fearful stimuli. Anxiety disorders are often comorbid with cardiovascular diseases. One established, and modifiable, risk factor for cardiovascular diseases is salt intake. Yet, investigations into how excess salt consumption affects anxiety-relevant behaviors remains little explored. Moreover, no studies have yet assessed how high salt intake influences generalization of fear. Here, we used adult C57BL/6J mice of both sexes to evaluate the influence of two or six weeks of high salt consumption (4.0% NaCl), compared to controls (0.4% NaCl), on contextual fear acquisition, expression, and generalization. Further, we measured osmotic and physiological stress by quantifying serum osmolality and corticosterone levels, respectively. Consuming excess salt did not influence contextual fear acquisition nor discrimination between the context used for training and a novel, neutral context when training occurred 48 prior to testing. However, when a four week delay between training and testing was employed to induce natural fear generalization processes, we found that high salt intake selectively increases contextual fear generalization in females, but the same diet reduces contextual fear generalization in males. These sex-specific effects were independent of any changes in serum osmolality nor corticosterone levels, suggesting the behavioral shifts are a consequence of more subtle, neurophysiologic changes. This is the first evidence of salt consumption influencing contextual fear generalization, and adds information about sex-specific effects of salt that are largely missing from current literature.

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Conflict of interest statement

The authors declare that no competing interests exist.

Figures

Fig 1
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
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
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
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
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.
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Funding for this work was provided by Kent State University, and the Applied Psychology Center in the Department of Psychological Sciences at Kent State University.