Imperatorin Influences Depressive-like Behaviors: A Preclinical Study on Behavioral and Neurochemical Sex Differences

Imperatorin, a naturally derived furanocoumarin, exerts promising neuropharmacological properties. Therefore, it might be applicable in the treatment of brain diseases such as depression. In the present project, we aimed to investigate the sex-dependent effects of imperatorin (1, 5, and 10 mg/kg) on behavior and neurochemistry associated with antidepressant effects. The depressive-like behaviors of male and female Swiss mice were investigated in a forced swim test (FST). Subsequently, High-Performance Liquid Chromatography (HPLC) was used to evaluate the level of serotonin, its metabolite, 5-HIAA, and noradrenaline, in mouse brains. The study revealed that only males responded to imperatorin (1 and 5 mg/kg) treatment and caused an antidepressant effect, such as with respect to depressive-like behaviors, lowering immobility time and increasing immobility latency. The HPLC analysis demonstrated that serotonin levels in the prefrontal cortex of females decreased with the middle dose of imperatorin (5 mg/kg), while in the male prefrontal cortex, the lower dose (1 mg/kg) boosted serotonin levels. There were no evident changes observed with respect to noradrenaline and serotonin metabolite levels in the male hippocampus. To conclude, we propose that imperatorin has antidepressant potential, seemingly only in males, influencing brain serotonin level, but the direct mechanism of action requires further investigation.


Introduction
Depression is a common psychiatric disorder characterized by a persistent feeling of despair along with an inability to experience pleasure [1]. Nowadays, depression is a leading cause of disability, and despite being a major contributing factor to the global burden of disease, current treatments face significant limitations, while elements of the etiopathology of the disease remain complex and elusive. Therefore, there is a need for personalized therapies and targeted medicines with higher and faster efficacy as well as fewer side effects.

Discussion
The current project investigated the potential influence of imperatorin on depressivelike behaviors in the FST and associated neurochemical changes in both sexes. There were perceivable alterations in the animals' behavioral response following imperatorin treatment, supported by variations in 5-HT and NA levels. In addition, the decrease in immobility duration following imperatorin application (1 and 5 mg/kg) was evident in males but not in females, as shown for the first time in this study ( Figure 1).
Indeed, the present results showed that imperatorin (1 mg/kg and 5 mg/kg) and imipramine (30 mg/kg) influence depressive-like behaviors only in male not in female Swiss mice. Regarding imipramine, our findings are in line with a previous study conducted by Carrier and Kabbaj, where the social isolation of rats caused anxiolytic and antidepressant effects of imipramine only in males [24]. Collectively, our findings suggest that males responded more effectively to imperatorin treatment than females as depressivelike behavioral indices were lower (i.e., immobility time) and appeared later (i.e., immobility latency) than the saline male control group.
Moreover, imperatorin (1, 5, and 10 mg/kg) exerted a perceivable dose-dependent effect on depressive-like behaviors in male mice, whereas, as mentioned, no such effect was observed in female mice. The weaker response to the highest dose of imperatorin might be caused by the liver enzymes' saturation that turns imperatorin into two active metabolites, xanthotoxol and heraclenin [25]. The pharmacological effects of the active metabolites were evaluated in several previous studies. Xanthotoxol showed neuroprotective anti-inflammatory properties and a blockage of the calcium channels [26,27]. On the other hand, heraclenin, the second active metabolite of imperatorin, was also found to possess anti-inflammatory properties [28]. Thus, we may hypothesize that the activity of imperatorin is linked to its metabolites, xanthotoxol and heraclenin, a decreased immobility duration, and elongated immobility latency, although further studies are required to explain this mechanism.
It is also of interest that imperatorin did not decrease the immobility time in the FST of females. A similar pattern, with males but not females responding to the treatment, was recently observed by our group after treatment with another furanocoumarin, xanthotoxin [29]. In the current study, as in the previous study with xanthotoxin [29], at baseline, male vehicle-treated animals had a slightly, but not statistically significant, longer immobility duration than females. Several conflicting results have been reported regarding baseline sex differences in the FST [30]. However, the behavioral response to the treatment with coumarins and the typical decrease in immobility time in the FST appears to be more prominent in males than in females. Such findings are in agreement with clinical evidence revealing that men respond faster and more favorably to imipramine than women [31]. Thus, our study corroborates that imipramine and, importantly, imperatorin-derived antidepressants may not be optimal for treating depression in women.
Our results showed that the acute administration of imperatorin influenced tissue levels of 5-HT only in the female hippocampus. Additionally, only the 1 mg/kg dose of imperatorin increased 5-HT tissue levels in the female prefrontal cortex, although no behavioral effects were observed. Concerning the 5-HT turnover ratio (5-HIAA/5-HT), it is evident that males displayed a more extensive release and/or metabolism of 5-HT than females in both the prefrontal cortex and hippocampus. Imipramine (30 mg/kg) reduced the sex difference in 5-HT turnover in both brain structures. All doses of imperatorin in the prefrontal cortex decreased 5-HT turnover in males. However, it was still higher than in females, where 5-HT release and/or metabolism stayed unchanged, regardless of the applied dose compared to the vehicle-treated control group. On the contrary, in the hippocampus, doses of 5 and 10 mg/kg of imperatorin increased the speed of 5-HT release and/or metabolism. The different tendencies of changes in 5-HT levels after imperatorin and xanthotoxin injections may result from the disparity in mechanisms of action, drug metabolism, and/or the different pharmacological activity of active metabolites. Indeed, substantial evidence shows significant sex differences in the pharmacokinetics of antidepressants [21][22][23][30][31][32]. This is a limitation of the present study that requires further experiments to examine the level of active metabolites and their activity in the brain. Based on current data, we can speculate that the pharmacokinetics of imperatorin in female and male mice are also differentiated and cause discrepancies in the observed behavioral and neurochemical effects.
Interestingly, a correlation exists between the behavior of animals in the FST and the level of the 5-HT turnover ratio in the prefrontal cortex [32]. The lower 5-HT turnover ratio in the prefrontal cortex but not in the hippocampus correlates well with the lower immobility time in the FST. Clinical studies revealed that brain 5-HT turnover was significantly elevated in unmedicated patients with major depressive disorder, compared with healthy subjects. Following effective therapy with an antidepressant drug, brain 5-HT turnover was reduced [33]. Thus, we may conclude that a reduced 5-HT turnover ratio is a marker of the antidepressant efficacy of imipramine in male mice tested in our study.
Regarding NA, the only doses of imperatorin that increased the NA tissue levels were 1 and 5 mg/kg in the female, but not in the male prefrontal cortex. This finding does not correlate well with the behavioral response and perhaps it further corroborates that female neurotransmitter systems are more sensitive to change following pharmacological manipulations. There was no influence on the NA tissue levels in the hippocampus, so the hippocampus might not be engaged in the imperatorin's mechanism of action.
A recent in vitro study showed that imperatorin could have an affinity to the 5-HT 7 receptor, which is involved in the neurobiology of depression and its pharmacological antagonism decreased immobility in the FST [34], Therefore, it is possible that the influence on depressive-like behaviors observed in males could be related to the effect of imperatorin on 5-HT receptors without necessarily affecting the neurotransmitter levels. The monoamine theory of depression states that alongside 5-HT and NA, dopamine might also be involved in disease development and treatment response [35]. Based on this theory, another explanation could be that imperatorin targets the adenosine and dopamine systems. Indeed, the structure-based optimization study on synthetic coumarin 3-(4-Bromophenyl)-8-hydroxycoumarin revealed high antagonistic potency and selectivity to the A3 receptor [36]. There are also findings demonstrating that adenosine receptors are colocalized with dopamine receptors, thus influencing their activity [37]. Since dopamine D2 receptor activity is usually inhibited by adenosine A2A receptors co-localized on the same population of striatal neurons adenosine may play a crucial role in dopamine signaling modulation. Additionally, adenosine A2A receptors and dopamine D4 receptors are expressed in the striatal and cortical areas, respectively, which are known to be involved in depression [38]. An investigation of the dopaminergic system represents an interesting avenue for future research into imperatorin's mechanism of action.

Animals
Two-month-old Swiss mice of both sexes, males g, were group-housed under the conditions determin Center in Lublin. Rights and permission of the Local Et (permission number 2/2019; 51/2020) were obtained. temperature (22 °C), and humidity conditions (55%) w tation. Mice were divided into 10 treatment groups, 5 and 5 of which were female (n = 10/group). All fema estrous cycle because of the randomization rule in o addition, mice were randomly assigned to different menter was blinded to the allocation when conducting tor Activity test and for the outcome assessment, accor

Spontaneous Locomotor Activity
Previous studies with imperatorin conducted by o ratorin (1-10 mg/kg) does not alter the locomotor act has not been studied in females, we performed a pilot e on female locomotor activity. Female mice were rand line, imperatorin (1, 5, and 10 mg/kg), or imipramine ( immediately following injections in the Columbus In

Animals
Two-month-old Swiss mice of both sexes, males (n = 50) and females (n = 50), 20-25 g, were group-housed under the conditions determined by the Experimental Medicine Center in Lublin. Rights and permission of the Local Ethical Committee on Animal Testing (permission number 2/2019; 51/2020) were obtained. Light cycle (12 h light/12 h dark), temperature (22 • C), and humidity conditions (55%) were stable throughout experimentation. Mice were divided into 10 treatment groups, 5 of which were male (n = 10/group) and 5 of which were female (n = 10/group). All females were in different phases of the estrous cycle because of the randomization rule in order to avoid bias in the study. In addition, mice were randomly assigned to different treatment groups, and the experimenter was blinded to the allocation when conducting the FST and Spontaneous Locomotor Activity test and for the outcome assessment, according to ARRIVE guidelines.

Spontaneous Locomotor Activity
Previous studies with imperatorin conducted by our group [4][5][6] revealed that imperatorin (1-10 mg/kg) does not alter the locomotor activity of male mice. As imperatorin has not been studied in females, we performed a pilot experiment on imperatorin's effects on female locomotor activity. Female mice were randomly allocated to receive either saline, imperatorin (1, 5, and 10 mg/kg), or imipramine (30 mg/kg). The test was performed immediately following injections in the Columbus Instruments Opto-Varimex-4 Auto-Track (transparent cages: 43 × 43 × 32 cm 3 , two rows of photocells), as previously described [29]. The examination lasted 30 min and the distance traveled was measured.

Forced Swim Test (FST)
The FST was performed 30 min after acute drug administration, and the duration of immobility behavior, as well as the immobility latency, were measured. The procedure was performed and recorded as previously described [29]. The recordings were analyzed using the open-source software Kinoscope [39].

Neurochemical Assays
To estimate neurotransmitters concentration in the brain, mice were sacrificed immediately after the FST by decapitation. The hippocampi and prefrontal cortices were dissected on ice, rinsed with saline, weighed, and snap-frozen on dry ice. Samples were stored at −80 • C until analysis. The homogenization of dissected tissues was performed as previously described [40]. Reverse phase ion-pair chromatography on an isocratic pump (YL9112 Instrument Co., Ltd., Gyeonggi-do, Korea) was used together with an electrochemical detector (BASi LC-EC, Bioanalytical Systems, Inc., Indianapolis, IN, USA) to determine tissue monoamine levels, as previously described, [40]. In addition to the assay of 5-HT and 5-HIAA tissue levels, the 5-HT turnover ratio was also calculated (5-HIAA/5-HT ratio). This index corresponds to the serotonergic activity being better than 5-HT or 5-HIAA tissue levels alone because it reflects 5-HT release and/or metabolic activity as described extensively elsewhere [41][42][43]. The results are expressed as ng/mg of wet tissue weight.

Statistical Analysis
Results were analyzed using a two-way ANOVA comparing sex (female vs. male) and drug treatment (control vs. imperatorin/imipramine), followed by post hoc multiple pairwise comparisons with Bonferroni's correction using GraphPad Prism version 8. Statistical significance was indicated when p < 0.05 and results are expressed as mean ± SD.

Conclusions
To conclude, in our study, despite the observed antidepressant effect in the FST following imperatorin administration in males, the mobilization of the serotoninergic system was not consistent with the behavioral response. Several changes were observed in the female brain neurochemistry in the absence of a related behavioral response. Thus, it may be suggested that the serotoninergic and noradrenergic mechanisms are not crucial for the observed decrease in depressive-like behaviors caused by imperatorin, and it is necessary to look for other possible pathways. Additionally, we are aware of the study's limitations regarding the duration of the drug administration. Typical therapies of depression last at least several weeks. Thus, another step of our research will be a long-term administration of imperatorin to establish its antidepressant effects.