Introduction
Major depression affects millions of people worldwide and contributes to their disability. Besides the well-defined depressive symptoms, patients often report cognitive disturbances, which significantly deteriorate their functioning. Therefore, the scientists still search for the new compounds with increased efficacy and positive influence on cognition.
Current antidepressants worsen (e.g. tricyclic antidepressants) or have no influence (e.g. selective serotonin reuptake inhibitors) on cognitive function (for review see: Biringer et al.
2009). Some Authors suggested that reboxetine, bupropion, duloxetine or venlafaxine might have more beneficial effect on cognitive function than other antidepressants. However, recent meta-analysis of clinical trials showed that only vortioxetine significantly improved cognition in depressed patients (McIntyre et al.
2016). Positive influence on cognitive function was most likely due to the drug’s broad receptor profile. Studies on animals showed that both 5-HT
1A and 5-HT
7 receptor ligands might enhance cognitive function in rodents (reviewed in Glikmann-Johnston et al.
2015 and Meneses
2014). Interestingly, 5-HT
1A antagonism facilitated memory retention possibly via 5-HT
7 activation, and 5-HT
7 receptor could improve emotional memory upon reduced 5-HT
1A receptor transmission (Stiedl et al.
2015).
We previously reported significant antidepressant- and anxiolytic-like effects of two dual 5-HT
1A and 5-HT
7 receptor antagonists in various behavioral tests in rodents (Waszkielewicz et al.
2015; Pytka et al.
2015a). As a continuation of our previous experiments, in this study we aimed to investigate whether chronic administration of 1-[(2,6-dimethylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-14) and 1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-15) caused antidepressant-like effects and elevated serotonin levels in the murine hippocampus. We also evaluated cholinolytic properties and the influence of acute administration of both compounds on cognitive function in mice.
Discussion
In the present study we found that dual 5-HT1A and 5-HT7 antagonists i.e. HBK-14 and HBK-15 possessed antidepressant-like activity and increased serotonin levels in the hippocampus after chronic treatment. HBK-15 possessed very weak cholinolytic properties, whereas HBK-14 did not block muscarinic receptors. Moreover, acute administration of HBK-15 enhanced memory of mice and significantly ameliorated memory impairment caused by scopolamine.
Although the data are inconsistent some studies show that 5-HT
1A and 5-HT
7 receptor blockade might have potential in the treatment of major depressive disorder (reviewed in Pytka et al.
2016c). Researchers proved that 5-HT
1A receptor deficient mice were less immobile in the forced swim test than wild-type control (Parks et al.
1998; Heisler et al.
1998). This suggests that the lack of functional 5-HT
1A receptors favors a less depressed phenotype. Similarly, deletion of 5-HT
7 gene (Guscott et al.
2005) as well as 5-HT
7 receptor antagonists (Wesołowska et al.
2006) resulted in antidepressant-like effect in various animal tests. Altogether, these findings indicate that 5-HT
1A and 5-HT
7 receptor antagonists may have potential as antidepressants.
Our previous studies revealed that dual 5-HT
1A and 5-HT
7 receptor antagonists i.e. HBK-14 and HBK-15 possessed significant antidepressant-like properties after acute treatment (Waszkielewicz et al.
2015; Pytka et al.
2015a). Both compounds showed α
1-adrenolytic properties, and only HBK-15 did not lower blood pressure at antidepressant-like doses after single administration (Pytka et al.
2016b). Interestingly, none of the compounds affected blood pressure after chronic administration (Pytka et al.
2016d). Here, we evaluated antidepressant-like activity of HBK-14 and HBK-15 after chronic administration in mice. Both compounds injected for 21 days showed antidepressant-like activities in the mouse forced swim test. None of them affected locomotor activity of mice; therefore, the observed effects were specific. Both compounds increased swimming, whereas had no influence on climbing behaviors. Since serotonin-targeting antidepressants increase swimming behavior in rodents (Nakatomi et al.
2008), we concluded that the serotonergic system was involved in the antidepressant-like effect of the studied compounds. HBK-15 showed stronger antidepressant-like properties than HBK-14, which is in agreement with our previous findings (Pytka et al.
2015a). Interestingly, HBK-15 administered chronically showed antidepressant-like properties at lower dose (0.625 mg/kg) than the dose active after acute treatment (1.25 mg/kg) (Pytka et al.
2015a). This is a common phenomenon for antidepressants, since antidepressant effect requires adaptive changes at the neuronal receptor level. Similarly, fluoxetine (Contreras et al.
2001) or
Hypericum perforatum (Lozanondash and Rodriguez-L
2010) administered chronically showed antidepressant-like effects at lower doses than those active after acute injection.
According to the serotonin hypothesis a deficit in brain serotonergic activity might be a cause of depression or an important vulnerability factor in this disease. The findings are divergent, but some studies reported a decrease in serotonin or its metabolite levels in the brains of suicide victims or suicide attempters (for review, see Mann et al.
1989). The reduced levels of serotonin were most frequently found in brainstem. Although there are many other theories, the serotonin hypothesis is still to date, as most antidepressants in clinical use enhance the serotonergic neurotransmission.
Since many antidepressants elevate serotonin levels, we investigated the influence of studied compounds on the level of serotonin in the hippocampus after acute and chronic treatment. Scientists proved that hippocampus plays a central role in major depression (for review see: Campbell and Macqueen
2004). Our experiments showed that chronic (but not acute) treatment with the studied compounds caused a significant increase in the level of hippocampal serotonin. In both cases the increase in serotonin levels was in parallel with a decrease in the immobility of mice in the forced swim test. Since hippocampus plays crucial role in mood disorders and reduced serotonin levels might occur in depression, we believe that the fact that the studied compounds elevate hippocampal serotonin levels might be beneficial in depressed individuals.
Cognitive dysfunction is very common among patients with major depressive disorder and significantly affects their capacity to function (Darcet et al.
2016). Carvalho et al. (
2015) suggested that several factors might contribute to cognitive dysfunction in major depressive disorder i.e. hyperactive hypothalamic-pituitary-adrenal axis, an increase in oxidative and nitrosative stress, increased apoptosis or diminished neurotrophic support. The cognitive impairments mostly occur during depressive episodes and include deficits in executive functions (attention, processing speed, cognitive flexibility) or learning and memory.
Bearing that in mind, in the second part of our studies, we examined the influence of dual 5-HT
1A and 5-HT
7 antagonists on learning and memory in mice after acute administration. Since the blockade of muscarinic receptors may cause various unfavorable effects such as memory impairment, we first investigated potential cholinolityc properties of the studied compounds. Our experiments revealed that HBK-14 possessed no, and HBK-15 very weak and negligible cholinolytic activity. Both compounds reduced carbachol maxima at the concentration of 10 μM, which suggested a non-specific or additional site of interaction (most likely not related to muscarinic receptors). In comparison, previous experiments performed in our laboratory showed that pA
2 value for atropine (cholinolytic drug) was 8.985 (Mogilski et al.
2015), which was a much higher value than pK
B (5.99) obtained for HBK-15.
The data on the effects of 5-HT
1A and 5-HT
7 receptor ligands on learning and memory in rodents are ambiguous. Galeotti et al. (
2000) as well as Tsuji et al. (
2002) proved that stimulating 5-HT
1A receptor in mice promoted learning and memory. Opposite results presented Madjid et al. (
2006), who reported that 5-HT
1A antagonists facilitated aversive learning in mice. Interestingly, the Authors also showed that 8-OH-DPAT (5-HT
1A agonist) displayed biphasic effect on retention times. Similarly, studies on the role of 5-HT
7 receptor in cognitive function are also conflicting (reviewed in Meneses
2014). Nevertheless, 5-HT
7
−/− mice showed impaired contextual hippocampal-dependent learning and decreased long-term synaptic plasticity in the hippocampus (Roberts et al.
2004). Moreover, both genetic and pharmacological inactivation of 5-HT
7 receptor in mice resulted in deficits in hippocampus-associated spatial memory in the location recognition test (Sarkisyan and Hedlund
2009).
In the present study, we determined the influence of both dual 5HT
1A and 5-HT
7 antagonists on learning and memory using the step-through passive avoidance test, which is a hippocampus-dependent memory task. In this test animals need to inhibit their natural tendency to enter the dark chamber. We showed that only HBK-15 administered alone possessed memory-enhancing properties in passive avoidance task. Moreover, the compound ameliorated memory deficits induced by scopolamine in this test. HBK-14 and fluoxetine were inactive in the step-through passive avoidance task. Interestingly, in both experiments HBK-15 displayed an inverted U-shaped dose-effect curve (0.625 mg/kg). This nonlinear relationship was frequently reported in pharmacological studies on cognitive functions and memory (Baldi and Bucherelli
2005). Although an inverted U-shaped dose-effect is widely described, it is very poorly understood. Scientists proposed several theories (e.g. arousal hypothesis), but the effect is most likely multifactorial and therefore difficult to explain.
Studies suggested that 5-HT
7 receptor blockade resulted in the cognitive deficits in mice (Sarkisyan and Hedlund
2009; Freret et al.
2014). Eriksson et al. (
2008) showed that even though SB-269970 (administered alone before the training session) had no effect on retention latency, it enhanced amnestic effects of 8-OH-DPAT in the passive avoidance test in mice. Since 8-OH-DPAT is also 5-HT
7 receptor agonist, the Authors concluded that 5-HT
7 receptor stimulation by 8-OH-DPAT counteracts 5-HT
1A receptor-mediated impairments in hippocampal-dependent contextual learning. Our findings are in agreement with the above studies, since our previous experiments revealed that HBK-15 compared with HBK-14 showed around three-fold stronger antagonistic properties at 5-HT
1A and weaker at 5-HT
7 receptor (Pytka et al.
2015a). We believe that these slight differences in HBK-14 and HBK-15 receptor profiles might be responsible for the observed differences in the activity in passive avoidance task.
The limitation of our study was the fact that antidepressant-like activity of the compounds was not tested using animal model of depression (e.g. chronic unpredictable mild stress). Since depression is closely associated with stress, this would provide more insight into the therapeutic potential of studied compounds. Furthermore, we should also confirm memory-enhancing activity of HBK-15 using other tests, such as Morris water maze or Y-maze, as well as after chronic treatment.
Therefore, in future studies we plan to examine antidepressant-like properties of both compounds utilizing animal models of depression. We also intend to evaluate the influence of acute and repeated administration of HBK-15 on learning and memory using other behavioral paradigms in rodents.