Adult male mice (C57BL/6) aged 8 - 9 weeks were purchased from Hubei Provincial Laboratory Animal Public Service Center (Wuhan, China) and housed under standard conditions (12-h light/dark cycle, room temperature 18˚C - 25˚C, 40% - 50% humidity) with access to food and water ad libitum. The protocols were approved by the committee on the Ethics of Animal Experiments of South-Central Minzu University.

Since depressed patients, especially patients with double-phase emotional disorders, there is a certain seasonal law [13]. This experiment was conducted in the alternate season of winter and spring, from October 27, 2019 to February 1, 2020, for a total of 96 days. Sixty mice were randomly divided into four groups (control group, mild, moderate and severe salt deficiency groups) with 15 mice in each group. Each mouse from the treated and control groups was raised in a separate cage. The mice in control group were fed with standard diet (salt content 0.26%) purchased from Commercial animal feed company and the salt content of foodstuff was decreased by 10% (salt content 0.234%), 30% (salt content 0.182%) and 50% (salt content 0.13%), respectively, in the mild, moderate and severe salt deficiency groups compared with the control group. Before the beginning of the experiment, all mice were tested for the sucrose preference, and the data were used as the benchmark value of depressive-like behavior state of mice. After the beginning of the experiment, the food intake of mice within 24 hours was measured and recorded every four days. An appropriate amount of foodstuff was weighed and put on the feed rack of cage, and after 24 hours, the remaining foodstuff was weighed and the data were recorded for a total of 20 times during the experiment. The mice were weighed with an electronic scale at an interval of about 10 days and the data were recorded for a total of 9 times during the experiment. Before the end of the experiment, the mice were tested for sucrose preference again.

Sucrose preference test (SPT) was performed to observe anhedonia-like behavior. 1% sucrose solution was prepared and filtrated with 0.22 μ membrane filter. All mice were habituated to 1% sucrose solution and water for 24 hours. On the testing day, mice were deprived of water for 24 hours and then presented with two bottles, one containing 1% sucrose and the other with drinking water. Twenty-four hours later, the bottles were removed and weighed. The sucrose preference was expressed as mean sucrose consumption relative to total fluid consumption [14].

Data were analyzed using Graphpad prism v 8.0. For multiple group comparisons and to determine treatment effects, one-way ANOVA post hoc Tukey’s multiple comparison tests were used to determine statistical significance. For comparisons between two groups, either unpaired or paired Student’s t test (two tailed) was performed to determine statistical significance. P < 0.05 was considered as statistically significant.

During this experiment, the observation on 15 mice in each group was completed except that 2 and 3 mice died in the mild and severe salt deficiency group, respectively. Overall, the food intake of mice in each group fluctuated up and down to a certain extent in 20 tests (Figure 1(a)). After the analysis of variance on the food intake data of mice in each group, it was found that there was no significant difference between each group before the day 17. The food intake of mice in the four groups had similar change trends since the day 21, and was significantly higher in the moderate and severe salt deficiency groups than that of the control group. Such differences disappeared on the day 29, but reappeared in the next two tests. Interestingly, the differences disappeared again on day 45, and then the differences between the moderate and severe salt deficiency group and the control group reached the maximum from the day 49 to day 63. After that, the food intake of mice showed a little or no difference between the groups until the end of experiment with the exception of that it was significantly higher in the severe salt deficiency group than that of the control group (P < 0.001) on the day 96 (Figure 1(b)). It should be noted that there was no significant difference in food intake between the mild salt deficiency group and the control group from the beginning to the end of the experiment. Based on these results, it could be concluded that the food intake of mice in each salt deficiency group has three stages including an adaptation period (13 - 45 days), the large intake period (49 - 67 days) and the maintenance period (71 - 96 days). In addition, by analyzing the data in each group on day 96, it was found that the food intake of mice was negatively correlated with the salt content of feed (Table 1), that is, the lower the salt content, the higher the food intake.

Figure 2 shows the change trend of the average body weight of mice in each group in 9 tests, and overall, the body weight of mice in each group presents an upward trend. However, according to the statistical analysis, the significant differences in the body weight of the mild and severe salt deficiency group were only found on day 54, 65 or 73 as compared to the control group (P < 0.05). In the last test on day 96, the body weight of mice in the mild salt deficiency group was significantly lower than that of the other three groups, but there were no significant differences between the moderate and severe salt deficiency group, and the moderate salt deficiency group and the control (Figure 2(b)). It was also found that the salt content of feed was negatively correlated with the body weight of mice (Table 1). Interestingly, the body weight of mice in the mild salt deficiency group began to deviate from that in the severe salt deficiency group from the second test and remained until the end of the experiment, suggesting that the long-term intake of mild salt deficiency diet has a greater impact on body weight as compared with the severe salt deficiency diet.

Con: control group; I, II and III: mild, moderate and severe salt deficiency groups.

The sucrose preference values were decreased in the low, medium and severe salt deficiency group, but the significant differences were found in the medium and severe group, and unexpectedly, there was also significant difference in the control group (Figure 3).