3.1 S-Ketamine increased the sucrose preference percentage (SPP) in stressed rats
SPP can accurately assess the degree of anhedonia in rats (Luo et al. 2010), and we used it to validate the CUMS model and the antidepressant effect of s-ketamine. After completion of the CUMS procedure, the data of SPP meet normality (The P values of groups C, D, DK, DNK, and DN were 0.173, 0.275, 0.757, 0.275, 0.279) and homogeneity of variance (F(4, 55) = 0.855, P = 0.497). As shown in Fig. 1(A), significant intergroup differences were observed among five groups (F(4, 55) = 41.530, P < 0.001). The post hoc tests showed that the SPP in the CUMS-treated groups (group D, DK, DNK, and DN) was significantly lower than that of the group C (P < 0.001, respectively), but no statistical difference was observed between the four CUMS-treated groups (P = 1.000, respectively). After s-ketamine and NSC23766 treatment, the data of SPP meet normality (The P values of groups C, D, DK, DNK, and DN were 0.157, 0.894, 0.698, 0.416, 0.667) and homogeneity of variance (F(4, 55) = 0.118, P = 0.976). The intergroup statistical difference was observed among these five groups ( F(4, 55) = 74.030, P < 0.001). Post hoc tests showed that group DK exhibited increased levels in the SPP and significantly higher than that in the D group (P < 0.001). Compared to group DK, group DNK showed decreased levels in the SPP (P < 0.001). Figure 1(D).
3.2 S-Ketamine alleviated the open-field behavioral performance of stressed rats
Feeding frequency (RF) and number of crossed squares (NOCS) reflect the exploratory activities of rodents, and also used to evaluate the depression-like behavior in rats (Hallam et al. 2004). After completion of the CUMS procedure, the data of rearing frequency (RF) and number of crossed squares (NOCS) all meet normality (RF: the P values of groups C, D, DK, DNK, and DN were 0.522, 0.385, 0.376, 0.682, 0.304; NOCS: the P values of groups C, D, DK, DNK, and DN were 0.100, 0.251, 0.882, 0.292, 0.316) and homogeneity of variance (RF: F(4, 55) = 2.116, P = 0.091; NOCS: F(4, 55) = 1.530, P = 0.206). As shown in Fig. 1(B-C), significant intergroup differences were observed among five groups in terms of RF (F(4, 55) = 53.187, P < 0.001) and NOCS (F(4, 55) = 91.982, P < 0.001). The post hoc tests showed that the RF and NOCS in the CUMS-treated groups (group D, DK, DNK, and DN) was significantly lower than that of the group C (P < 0.001, respectively), but no statistical difference was observed for RF and NOCS between the four CUMS-treated groups (P = 1.000, respectively). After s-ketamine and NSC23766 treatment, the data of RF and NOCS meet normality (RF: the P values of groups C, D, DK, DNK, and DN were 0.075, 0.488, 0.523, 0.567, 0.431; NOCS: the P values of groups C, D, DK, DNK, and DN were 0.678, 0.243, 0.349, 0.459, 0.130) and homogeneity of variance (RF: F(4, 55) = 2.502, P = 0.053; NOCS: F(4, 55) = 1.439, P = 0.233). Significant intergroup differences were observed among five groups in terms of RF (F(4, 55) = 52.039, P < 0.001) and NOCS (F(4, 55) = 92.967, P < 0.001). The post hoc tests showed that group DK exhibited increased levels in RF and NOCS, and significantly higher than that in the D group (P < 0.001, respectively). Compared to group DK, group DNK showed decreased levels in RF and NOCS (both P < 0.001). Figure 1(E-F).
3.3 S-Ketamine up-regulated the level of Rac1's mRNA and protein expression in the hippocampus of stressed rats
To determine whether Rac1 plays an important role in the antidepressant effects of s-ketamine, we analyzed the level of Rac1's mRNA and protein expression in the hippocampus of stressed rats. The data of Rac1's mRNA and protein expression all meet normality (mRNA: the P values of groups C, D, DK, DNK, and DN were 0.588, 0.703, 0.812, 0.639, 0.687; protein expression: the P values of groups C, D, DK, DNK, and DN were 0.851, 0.692, 0.865, 0.360, 0.338) and homogeneity of variance (mRNA: F(4, 25) = 1.496, P = 0.233; protein expression: F(4, 25) = 0.653, P = 0.630). As shown in Fig. 2(A-C), the mRNA and protein level of Rac1 among all groups showed statistically significant differences (F(4, 25) = 52.234, P < 0.001 and F(4, 25) = 63.423, P < 0.001). Post hoc tests showed that CUMS induced expression decrease of Rac1 in hippocampal, and the mRNA, protein level of Rac1 in the group D were significantly lower than that of the group C (both P < 0.001). S-Ketamine up-regulated the expression level of Rac1, and the mRNA, protein expression of Rac1 in group DK exhibited higher readings than group D (both P < 0.001). However, NSC23766 failed to reverse the s-ketamine induced expression up-regulation of Rac1, and no statistical difference was observed for the mRNA and protein expression of Rac1 between the group DK and group DNK (P = 1.000 and P = 0.982).
3.4 S-Ketamine increased the activity of Rac1 in the hippocampus of stressed rats
Since the activated state GTP-Rac1 can reflect the function of Rac1, we further implemented the Rac1-GTP binding assay. The data of Rac1-GTP and Rac1-GTP/Total Rac1 ratio all meet normality (Rac1-GTP: the P values of groups C, D, DK, DNK, and DN were 0.762, 0.982, 0.760, 0.332, 0.369; Rac1-GTP/Total Rac1: the P values of groups C, D, DK, DNK, and DN were 0.753, 0.106, 0.797, 0.572, 0.463) and homogeneity of variance (F(4, 25) = 2.272, P = 0.090 and F(4, 25) = 0.881, P = 0.489). As shown in Fig. 2(D-E), the Rac1-GTP and Rac1-GTP/Total Rac1 ratio among all groups showed statistically significant differences (F(4, 25) = 340.523, P < 0.001 and F(4, 25) = 1083.035, P < 0.001). Post hoc tests showed that the levels of Rac1-GTP and Rac1-GTP/Total Rac1 ratio in group D were significantly lower than that of group C (both P < 0.001). S-Ketamine increased the activity of Rac1 in the hippocampus of stressed rats. Compared with group D, the levels of Rac1-GTP and Rac1-GTP/Total Rac1 ratio in group DK exhibited higher readings (both P < 0.001). However, NSC23766 reversed the s-ketamine induced up-regulation of Rac1activity, and the Rac1-GTP and the Rac1-GTP/Total Rac1 ratio showed a very low value in group DNK, which was significantly lower than those in the DK group (both P < 0.001).
3.5 S-Ketamine increased Tiam1 expression, and decreased α1-Chimaerin, Bcr expression in the hippocampus of stressed rats
To study the functional status of Rac1, we further detected the expression of potential upstream activity regulatory proteins Tiam1, α1-Chimaerin and Bcr. The data of these activity-regulating proteins all meet normality (Tiam1: the P values of groups C, D, DK, DNK, and DN were 0.457, 0.709, 0.439, 0.380, 0.689; α1-Chimaerin: the P values of groups C, D, DK, DNK, and DN were 0.653, 0.624, 0.617, 0.189, 0.427; Bcr: the P values of groups C, D, DK, DNK, and DN were 0.344, 0.501, 0.241, 0.919, 0.962) and homogeneity of variance (Tiam1: F(4, 25) = 0.472, P = 0.756; α1-Chimaerin: F(4, 25) = 1.174, P = 0.346; Bcr: F(4, 25) = 1.573, P = 0.212). As shown in Fig. 3(A-D), the expression levels of Tiam1, α1-Chimaerin and Bcr among all groups showed statistically significant differences (F(4, 25) = 164.463, P < 0.001; F(4, 25) = 67.307, P < 0.001; and F(4, 25) = 89.617, P < 0.001). Post hoc tests showed that the Tiam1 was significantly lower (P < 0.001), and α1-Chimaerin, Bcr were higher in group D than that of group C (P < 0.001 and P < 0.001). Compared with group D, the protein levels of Tiam1 exhibited increased expression (P < 0.001), and α1-Chimaerin, Bcr exhibited decreased expression in group DK (both P < 0.001). Compared with the DK group, the protein levels of Tiam1 and Bcr exhibited decreased expression (P < 0.001 and P = 0.002), and α1-Chimaerinin exhibited increased expression in groups DNK (P < 0.001).
3.6 S-Ketamine up-regulated the expression levels of synaptic protein Glur1, synapsin1 and PSD95 in the hippocampus of stressed rats
Synaptic proteins Glur1, synapsin1 and PSD95 were enriched in the postsynaptic membrane, and can regulate synaptic plasticity (Colledge et al. 2003; Huang et al. 2020). So we analyzed the effect of s-ketamine on the expression levels of these proteins. The data of synaptic proteins Glur1, synapsin1 and PSD95 all meet normality (Glur1: the P values of groups C, D, DK, DNK, and DN were 0.671, 0.442, 0.902, 0.108, 0.317; synapsin1: the P values of groups C, D, DK, DNK, and DN were 0.421, 0.488, 0.384, 0.788, 0.264; PSD95: the P values of groups C, D, DK, DNK, and DN were 0.591, 0.701, 0.610, 0.871, 0.331) and homogeneity of variance (Glur1: F(4, 25) = 1.460, P = 0.244; synapsin1: F(4, 25) = 0.723, P = 0.584; PSD95: F(4, 25) = 0.804, P = 0.534). As shown in Fig. 4(A-D), the expression levels of Glur1, synapsin1 and PSD95 among all groups showed statistically significant differences (F(4, 25) = 63.077, P < 0.001; F(4, 25) = 63.700, P < 0.001; and F(4, 25) = 82.779, P < 0.001). Post hoc tests showed that the expression levels of Glur1, synapsin1 and PSD95 were significantly lower in group D than that of group C (all P < 0.001). Compared with group D, their expression in group DK exhibited significantly increased levels (all P < 0.001). Compared with the DK group, the expression of Glur1, synapsin1 and PSD95 in groups DNK were significantly lower (P < 0.001, P < 0.001 and P = 0.002).
3.7 S-Ketamine increased the spines density in the hippocampal CA1 region of stressed rats
To determine whether the antidepressant effect of s-ketamine is related to changes in synaptic structure, we analyzed the dendritic spines density of hippocampal CA1 region in stressed rats. The data of spines density meet normality (The P values of groups C, D, DK, DNK, and DN were 0.446, 0.221, 0.888, 0.627, 0.245) and homogeneity of variance ( F(4, 25) = 0.177, P = 0.948). As shown in Fig. 5(A-B), the spine density among all groups showed statistically significant differences (F(4, 25) = 17.544, P < 0.001). Post hoc tests showed that the spine density was significantly lower in group D than that of group C (P < 0.001). Compared with group D, group DK exhibited increased levels of spines density (P = 0.010). Compared with the DK group, the spines density in groups DNK were lower (P = 0.004).
3.8 S-Ketamine alleviated the LTP impairments of Schaffer-CA1 in the hippocampus of stressed rats
Since LTP can intuitively reflect the process of synaptic plasticity, we implemented electrophysiological experiments through brain slices to study the internal mechanism of s-ketamine on the antidepressant effects in stressed rats. The data of fEPS slope meet normality (The P values of groups C, D, DK, DNK, and DN were 0.971, 0.693, 0.890, 0.693, 0.857) and homogeneity of variance (P = 0.985). As shown in Fig. 5(A-B), the fEPS slope among all groups showed statistically significant differences (F(4, 25) = 45.444, P < 0.001). Post hoc tests showed that CUMS induced LTP impairment in group D as compared to group C (fEPS slope: 145.9 ± 5.2% vs. 177.7 ± 6.6%) (P < 0.001). S-Ketamine effectively alleviated the LTP impairment caused by CUMS, as shown by the fEPS slope being higher in the DK group (164.47 ± 5.6%) than in the D group (P < 0.001). However, Rac1 inhibitor NSC23766 effectively reversed the protective effect of ketamine. Compared with the DK group, the fEPS slope in groups DNK (148.9 ± 5.2%) was lower (P = 0.001). Figure 6 (A, B).