ARHGEF10 expression in different brain regions
To examine ARHGEF10 expression in the central nervous system, we measured the protein level by Western blot analysis. As shown in Fig. 1a, ARHGEF10 protein was widely expressed in the wild-type (WT) mouse brain, especially in the frontal cortex and amygdala. Notably, the absence of ARHGEF10 protein in the knockout mouse brain was confirmed by Western blotting (Fig. 1b). Nissl staining of serial coronal brain sections revealed a similar brain structure between WT and Arhgef10 knockout mice (Fig. 1c).
Arhgef10 knockout mice display social deficits in the three-chamber test
To explore whether Arhgef10 knockout affects social interaction, we employed the three-chamber paradigm to test sociability and social recognition. After habituation, a novel same-sex mouse was placed within the plastic cylinder (stranger mouse 1) in one chamber, and one empty cylinder was placed in another chamber. We found that WT mice spent a significantly longer time in the chamber with a stranger mouse than in the empty chamber (Fig. 2a; F1,22 = 36.517, p < 0.001 for genotype × chamber by two-way ANOVA; F1,22 = 24.831, p < 0.001 for chamber; Tukey’s post hoc comparisons were used to examine the differences between the empty cylinder and stranger mouse 1, WT: p < 0.05; KO: p > 0.05; n = 12 for each). However, Arhgef10 knockout mice did not spend a longer time in the chamber with the stranger mouse (Fig. 2a). Upon further evaluation of the social interactions, we found that WT mice spent significantly and markedly more time in close interaction with the stranger mouse by sniffing the holes of the cylinder, indicating normal social ability (Fig. 2b; F1,22 = 52.971, p < 0.001 for genotype × chamber by two-way ANOVA; p < 0.05 for post hoc comparisons between the empty cylinder and the stranger mouse, n = 12). Arhgef10 knockout mice showed no significant preference between these two cylinders, indicating that they did not exhibit interest in the stranger mouse (Fig. 2b; p > 0.05 for post hoc comparisons between the empty cylinder and the stranger mouse, n = 12). Since enhanced locomotor activity may increase the possibility of contacts between mice, we further examined the number of entries into each chamber. We found that the number of entries into these two chambers were similar in WT mice and Arhgef10 knockout mice (Fig. 2c; F1,22 = 1.607, p = 0.218 for genotype × chamber, by two-way ANOVA).
In the test for social novelty preference, we further examined social recognition in WT and Arhgef10 knockout mice. Mice naturally exhibit a preference for social novelty, spending more time with a new mouse than with a familiar mouse as a social stimulus. In addition to stranger 1 in the original cylinder, another stranger mouse (stranger 2) was placed in the second cylinder. WT mice showed a preference for exploring the compartment with the novel mouse, stranger 2, compared with the chamber containing stranger 1 (Fig. 2d and e; F1,22 = 5.843, p = 0.024 for genotype × chamber, by two-way ANOVA; post hoc for comparisons between stranger 1 and stranger 2 revealed p < 0.05, n = 12 for WT, and p < 0.05, n = 12 for KO). Although Arhgef10 knockout mice spent more time in the chamber with the first stranger mouse than in the chamber with the second stranger mouse (Fig. 2d), the time spent in social interactions, such as sniffing or tail rattling or time spent near the cylinder containing the stranger mice was similar between the first and the second stranger mouse (Fig. 2e). Keeping up with the decreased social interaction in sociability test, it was found that the social novelty preference was affected in Arhgef10 knockout mice. However, the longer time spent in the chamber with the first stranger mouse without more social interaction may imply that Arhgef10 knockout mice need more time to become familiar with the stranger mouse during the experiment. Both WT and Arhgef10 knockout mice showed a comparable number of entries into the compartment with stranger 1 and the one with the novel mouse, stranger 2 (Fig. 2f; F1,22 = 0.983, p = 0.602 for genotype × chamber, by two-way ANOVA).
Increased locomotor activity in Arhgef10 knockout mice
The open field test was used to evaluate the general locomotor and exploratory activity of the mice. Arhgef10 knockout mice showed a higher level of locomotor activity in open field (F1, 30 = 12.296, p = 0.001, n = 16) (Fig. 3a). In a 60-min open field test, Arhgef10 knockout mice exhibited significantly enhanced locomotor activity compared with WT mice at 0–5, 30–35, and 45–50 min (Fig. 3b, Tukey’s test for multiple comparisons, p < 0.05). There was no significant difference in rearing activity between WT and knockout mice (Fig. 3c). In addition, there was no significant difference between WT and Arhgef10 knockout mice in the amount of activity occurring in the center of the field (Fig. 3d).
Reduction of anxiety-like behavior in Arhgef10 knockout mice
The EPM was used to measure the level of anxiety-like behavior in the mice. The time spent in the open arms or closed arms is used as an index to define the level of anxiety-like behavior. It was found that Arhgef10 knockout mice displayed less anxiety-like behavior. The time spent in the open arms was significantly increased in Arhgef10 knockout mice (open arms: F1,23 = 11.04, p = 0.003; closed arms: F1,23 = 14.481, p = 0.001, n = 13 for WT vs Arhgef10 KO) (Fig. 4a), indicating that anxiety-like behavior was reduced in knockout mice. Upon analyzing the number of entries into each arm, we found that the number of entries into the open arms was significantly increased in Arhgef10 knockout mice (open arms: F1,23 = 6.301, p = 0.02; closed arms: F1,23 = 2.431, p = 0.134 for WT vs Arhgef10 KO) (Fig. 4b). The results demonstrated that there was a reduction of anxiety-like behavior in Arhgef10 KO mice.
Reduction of depression-like behavior in Arhgef10 knockout mice
To further examine whether other mood-related behaviors were also affected in Arhgef10 knockout mice, animals were subjected to the tail suspension test (TST) and the forced swim test (FST). Increased immobility and floating time are indicative of depression-related behavior. Interestingly, Arhgef10 knockout mice showed a significant reduction of immobility in both the FST (151.1 ± 9.47 s, and 62.43 ± 1.5 s for WT and KO mice, respectively) and TST (207.0 ± 6.013 s and 131.2 ± 13.47 for WT and KO, respectively) (Fig. 4c, d). The duration of immobility time in both the FST and the TST was significantly shorter in Arhgef10 knockout mice than in WT mice (F1,17 = 39.175, p < 0.0001 in FST; F1,17 = 24.109, p = 0.0013 in TST; n = 9 and 10 for WT and KO, respectively). These results indicated that there was reduction of depression-like behavior in Arhgef10 KO mice.
Pre-pulse inhibition is unaffected in Arhgef10 knockout mice
The pre-pulse inhibition (PPI) test is used to evaluate sensory gating in mice. Acoustic startle responses provide information on the sensorimotor processes of the animal in response to acoustic stimuli. Arhgef10 knockout mice had a normal startle amplitude in response to 120 dB acoustic stimuli, indicating that the reflexive contraction of the muscles in response to acoustic stimuli was normal (F1.9 = 1. 171, p = 0.337, n = 5–6) (Fig. 5a). Moreover, Arhgef10 knockout mice also exhibited a reduced acoustic startle response when the acoustic startle stimulus was preceded by a weaker acoustic stimulus, indicating a normal PPI response in comparison with WT mice (two-way ANOVA for genotype × pre-pulse dB, main effect of genotype: F1.9 = 1. 0366, p = 0.8524; main effect of pre-pulse dB: F2,18 = 25.27, p < 0.001.) (Fig. 5b).
Arhgef10 knockout mice exhibit normal spatial learning in the Morris water maze test
Spatial learning behavior was measured using the Morris water maze test. For four training days, Arhgef10 knockout mice displayed normal learning ability with typical decreases in escape latency (F = 0.260, p = 0.618, n = 6–7) (Fig. 5c). In the probe test, both WT and Arhgef10 knockout mice spent much more time in the target quadrant than in opposite or adjacent quadrants (Fig. 5d), indicating that Arhgef10 knockout mice exhibited normal spatial learning.
Increased norepinephrine (NE) and serotonin (5-HT) levels in the frontal cortex and amygdala of Arhgef10 knockout mice
To further explore the possible underlying mechanisms leading to the behavioral changes caused by functional loss of ARHGEF10, we investigated the neurochemical composition of different brain regions of Arhgef10 knockout mice. The frontal cortex, striatum, hippocampus, and amygdala from WT and KO mice were analyzed by HPLC with electrochemical detection (ECD) to determine the content of dopamine, 5-HT, NE, and their metabolites. The content of NE in the frontal cortex and amygdala was significantly elevated in Arhgef10 knockout mice (two-way ANOVA for genotype × brain regions, main effect of genotype: F1, 10 = 6.776, p = 0.0264 and brain regions F3, 30 = 3.250, p = 0.0354; interaction: F3, 30 = 2.764, p = 0.0591). Post hoc comparisons between WT and KO revealed significant differences in the frontal cortex and amygdala (Fig. 6a). Serotonin content in the amygdala and hippocampus was also increased in Arhgef10 knockout mice compared with WT mice (two-way ANOVA for genotype × brain regions, main effect of genotype: F1, 10 = 11.57, p = 0.0068 and brain regions (F3, 30 = 24.650, p < 0.0001; interaction: F3, 30 = 1.814, p = 0.1659). Post hoc comparisons between WT and KO revealed significant differences in the frontal cortex and amygdala. Dopamine in the striatum was also increased in Arhgef10 knockout mice compared with WT mice (two-way ANOVA for genotype × brain regions, main effect of genotype: F1, 10 = 23.47, p = 0.0007 and brain regions F (3, 30) = 731.5, p < 0.0001; interaction: F3, 30 = 20.36, p < 0.0001). Post hoc comparisons between WT and KO revealed a significant difference in the striatum (Fig. 6c). However, there were no differences in the metabolites of these monoamines between WT and Arhgef10 knockout mice (Fig. 6d, e).
Reduction of MAO-A expression in Arhgef10 knockout mice
Since NE and 5-HT levels were significantly elevated in Arhgef10 knockout mice, we measured the expression of MAO-A and MAO-B, the key enzymes that degrade NE and 5-HT, in the corresponding brain areas using Western blotting. MAO-A levels between WT and Arhgef10 knockout mice were evaluated by two-way ANOVA (two-way ANOVA for genotype × brain regions, main effect of genotype: F1, 12 = 4.305, p = 0.0602 and brain regions F3, 36 = 2.286, p = 0.0953; interaction: F3, 36 = 2.324, p = 0.0913). Post hoc comparisons between WT and KO revealed significant differences in frontal cortex and amygdala. Further comparison of these differences found significantly reduced MAO-A levels in the frontal cortex and amygdala (Fig. 7a). In contrast with MAO-A levels, no statistically significant difference was found in MAO-B levels between WT and Arhgef10 knockout mice (two-way ANOVA for genotype × brain regions, main effect of genotype: F1, 4 = 3.163, P = 0.1499 and brain regions F3, 12 = 2.688, p = 0.0934; interaction: F3, 12 = 0.3990, p = 0.7563) (Fig. 7b). Furthermore, we also examined the enzymes dopamine β-hydroxylase (DBH) and tryptophan hydroxylase (TPH), which are involved in the synthesis of NE and 5-HT, respectively. WT and Arhgef10 knockout mice had similar protein levels of both DBH (two-way ANOVA for genotype × brain regions, main effect of genotype: F1, 4 = 0.03559, p = 0.8595 and brain regions F3, 12 = 7.614, p = 0.0041; interaction: F3, 12 = 0.2854, p = 0.8350) and TPH (two-way ANOVA for genotype × brain regions, main effect of genotype: F1, 4 = 0.5692, p = 0.4926 and brain regions F3, 12 = 6.591, p = 0.0070; interaction: F3, 12 = 1.190, P = 0.3549) in the tested brain regions (Fig. 7c, d).