*3.4. The 5-HT2A Receptors*

The 5-HT2A receptors, like the others of the 5-HT<sup>2</sup> family, are preferentially coupled to the G protein of the Gq/11 type, so their activation increases the cellular level of inositol phosphate and, consequently, the cytosolic concentration of calcium ions. The 5-HT2A receptors are distributed postsynaptically and presynaptically throughout the brain at serotonergic terminals, with the greatest concentration in the neocortex [140–142]. Recent anatomical and functional studies suggest that 5-HT2A receptors are also present presynaptically as heteroreceptors, where they may enhance glutamatergic neurotransmission and participate in memory processes [143]. It has also been demonstrated that the 5-HT2A receptors of the cerebral cortex are located on GABAergic interneurons as well as glutamatergic projection neurons in the brains of humans and rodents [42,144].

Many antidepressants and antipsychotic drugs possess a relatively high binding to 5-HT2A receptors [145]. Although there is no direct correlation between the affinity of these drugs for 5-HT2A receptors and clinically effective doses, there is ample evidence that the 5-HT2A receptor plays a role in the pathomechanism of depression [20,146]. Some antidepressants mediate their action partly via the antagonism of 5-HT2A receptors [147]. In addition, chronic treatment with antidepressants, such as tricyclic antidepressants, monoamine oxidase inhibitors, mianserin, mirtazapine, or sertraline, decreased the number of 5-HT2A receptors in rodents [148]. Chronic electroconvulsive shock treatment resulted in the upregulation of cortical 5-HT2A receptors in rodents [149].

Several clinical trials have shown that atypical antipsychotics [150] and the antidepressant mirtazapine with an affinity for α2-adrenoceptors and 5-HT2A receptors [151] augment the clinical response to SSRIs in treatment-resistant patients [76]. A common feature of these substances is their ability, at clinical doses, to block responses to signals mediated by 5-HT2A receptors [152]. Such downregulation could, inter alia, explain why the side effects of SSRIs diminish after 2 or 3 weeks. The high co-expression of 5-HT1A and 5-HT2A receptors in the neocortex [153] may indicate that the blockade of 5-HT2A receptors enhances 5-HT1A receptor-mediated neurotransmission in the cortical and limbic regions, an activity associated with antidepressant efficacy. The chronic administration of 5-HT2A receptor antagonists has been shown to result in a paradoxically negative regulation of 5-HT2A receptors [154,155], which may be beneficial in the treatment of depression. Moreover, preclinical studies indicate that 5-HT2A antagonists have anxiolytic properties, as demonstrated by ritanserin, a 5-HT2A antagonist with anxiolytic effects in humans [156].

Another issue is the relationship between the 5-HT2A receptor and the noradrenergic system in relation to depression [157]. Studies have shown that the activation of 5-HT2A receptors as a result of treatment with SSRIs causes an increase in serotonin levels in GABA neurons. This inhibits the neuronal activity of norepinephrine through the prolonged release of GABA [158–160]. In turn, citalopram, in addition to reducing norepinephrine firing, also has the effect of lowering basal and evoked extracellular norepinephrine levels in the amygdala [161]. This may underlie SSRI ineffectiveness in resistant depression. The co-administration of an SSRI and a 5-HT2A receptor antagonist trazodone (as well as atypical antipsychotics, such as quetiapine, risperidone, olanzapine, and aripiprazole) reversed this inhibitory effect in noradrenergic neurons in rats and might be beneficial in the treatment of resistant depression [160,162–164]. Increasing evidence shows that 5-HT2A receptor antagonists display antidepressant effects. EMD 281014 (Figure 5), a 5-HT2A receptor antagonist, showed significant activity in the FST in congenital learned helpless rats [165]. A similar effect was shown by another 5-HT2A receptor antagonist, FG5893, which significantly shortened the immobility time in the FST [166]. The selective 5- HT2A receptor antagonist, M100907, enhanced the antidepressant-like behavioral effects of fluoxetine [167], suggesting that a selective 5-HT2A receptor blockade may complement the behavioral effects of serotonin transporter inhibition. In contrast, recent studies in rats have shown that the functional disturbance of the 5-HT2A receptor in the medial prefrontal cortex may contribute to postpartum mental disorders, including depression and psychosis [168]. In addition, prefrontal 5-HT2A receptors may both have beneficial and negative effects on cognition, which might explain the aggravation of cognitive deficits after the onset of SSRI treatment in depressed patients, as well as the limited efficacy of second-generation antipsychotics that act as 5-HT2A receptor antagonists against the strongly debilitating cognitive symptoms of schizophrenia and other psychiatric disorders [169]. A deficiency in 5-HT2A receptors has also been shown to alter the metabolic and transcriptional, but not behavioral, consequences of chronic unpredictable stress in mice [170]. The 5-HT2A blockade or SSRI-induced downregulation of 5-HT2A may lead to emotional blunting in patients. It is, therefore, very likely that 5-HT2A receptors may have different functions depending on the region of the brain: Another issue is the relationship between the 5-HT2A receptor and the noradrenergic system in relation to depression [157]. Studies have shown that the activation of 5-HT2A receptors as a result of treatment with SSRIs causes an increase in serotonin levels in GABA neurons. This inhibits the neuronal activity of norepinephrine through the prolonged release of GABA [158–160]. In turn, citalopram, in addition to reducing norepinephrine firing, also has the effect of lowering basal and evoked extracellular norepinephrine levels in the amygdala [161]. This may underlie SSRI ineffectiveness in resistant depression. The co-administration of an SSRI and a 5-HT2A receptor antagonist trazodone (as well as atypical antipsychotics, such as quetiapine, risperidone, olanzapine, and aripiprazole) reversed this inhibitory effect in noradrenergic neurons in rats and might be beneficial in the treatment of resistant depression [160,162–164]. Increasing evidence shows that 5-HT2A receptor antagonists display antidepressant effects. EMD 281014 (Figure 5), a 5-HT2A receptor antagonist, showed significant activity in the FST in congenital learned helpless rats [165]. A similar effect was shown by another 5-HT2A receptor antagonist, FG5893, which significantly shortened the immobility time in the FST [166]. The selective 5-HT2A receptor antagonist, M100907, enhanced the antidepressant-like behavioral effects of fluoxetine [167], suggesting that a selective 5-HT2A receptor blockade may complement the behavioral effects of serotonin transporter inhibition. In contrast, recent studies in rats have shown that the functional disturbance of the 5-HT2A receptor in the medial prefrontal cortex may contribute to postpartum mental disorders, including depression and psychosis [168]. In addition, prefrontal 5-HT2A receptors may both have beneficial and negative effects on cognition, which might explain the aggravation of cognitive deficits after the onset of SSRI treatment in depressed patients, as well as the limited efficacy of second-generation antipsychotics that act as 5-HT2A receptor antagonists against the strongly debilitating cognitive symptoms of schizophrenia and other psychiatric disorders [169]. A deficiency in 5-HT2A receptors has also been shown to alter the metabolic and transcriptional, but not behavioral, consequences of chronic unpredictable stress in mice [170]. The 5-HT2A blockade or SSRI-induced downregulation of 5-HT2A may lead to emotional blunting in patients. It is, therefore, very likely that 5-HT2A receptors may have different functions depending on the region of the brain:

Moreover, preclinical studies indicate that 5-HT2A antagonists have anxiolytic properties, as demonstrated by ritanserin, a 5-HT2A antagonist with anxiolytic effects in humans [156].

*Int. J. Mol. Sci.* **2021**, *22*, 9015 11 of 32

**Figure 5.** 5-HT2A receptor antagonists: EMD 281014, FG5893 and M100907. **Figure 5.** 5-HT2A receptor antagonists: EMD 281014, FG5893 and M100907.


### *3.5. The 5-HT2B Receptors*

The 5-HT2B receptor is expressed mainly in peripheral tissues, especially in the liver, kidneys, and heart, and its distribution in the brain is low [171]. In the central nervous system, the 5-HT2B receptor is present in septal nuclei, the dorsal hypothalamus, and the medial amygdala at levels similar to those found in the stomach [171]. The 5-HT2B receptor, mRNA, is found in the dorsal raphe nucleus, suggesting a potential autoreceptor role [172]. The 5-HT2B receptors are coupled to the Gq protein, which activates PLC (phospholipase C)/PKC (protein kinase C) and increases the concentration of calcium ions in the cytosol.

The knowledge about the function of the 5-HT2B receptor in the CNS is limited; however, there are reports of the antidepressant properties of selective 5-HT2B receptor agonists [173]. The presence of 5-HT2B receptors in the dorsal raphe and their stimulatory role in 5-HT release has been demonstrated [173]. The pharmacological or genetic inactivation of the 5-HT2B receptor abolished the effects of chronic treatment with SSRIs, and the stimulation of 5-HT2B receptors induced an SSRI-like response in behavioral and neurogenic tests. In turn, the genetic inactivation of 5-HT2B receptors in serotonergic neurons eliminated the neurogenic effects of fluoxetine [173]. It has recently been confirmed that 5-HT2B receptors directly and positively regulated the activity of serotonin neurons [174]. In addition, the stimulation of the 5-HT2B receptor via fluoxetine in astrocyte cell cultures resulted in the phosphorylation of extracellular signal-regulated kinases and the transactivation of the EGF (epidermal growth factor) receptor [175]. A reduced level of astroglial (but not neuronal) 5-HT2B receptors in a mouse model of Parkinson's disease was also reported, which paralleled the development of the depression-like phenotype [176]. The stimulation of astroglial 5-HT2B receptors may, therefore, be beneficial in treating depressive disorders [177].

Considering the role of peripherally located 5-HT2B receptors, potential new antidepressants acting on 5-HT2B receptors may adversely affect the function of the respiratory and circulatory systems [17,20]:


### *3.6. The 5-HT2C Receptors*

The 5-HT2C receptors are mainly located in the choroid plexuses, cerebral cortex, hippocampus, substantia nigra, and cerebellum. They bind preferentially with Gq/11 and increase the concentrations of inositol phosphates and cytosolic Ca2+. Like 5-HT2A receptors, they are involved in the regulation of mood, motor behavior, and appetite [178].

Several classes of antidepressants have an affinity for 5-HT2C receptors. Although these receptors are usually somatodendritic, in some regions they are also present on axon terminals [179]. The location of 5-HT2C receptors in relation to serotonergic and GABAergic neurons in the anterior raphe nuclei demonstrates complex systemic relationships in the brain. It has been shown that 5-HT2C receptors are preferentially located on GABAergic interneurons (and not on serotonergic neurons). This suggests that the stimulation of GABAergic interneurons by 5-HT2C receptors plays an important role in the suppression of serotonergic cell firing in the dorsal raphe and surrounding areas [180]. The immunoreactivity of the 5-HT2C receptor has also been described in GABAergic cells in the PFC [181] and in the dopaminergic and GABAergic neurons of the mesolimbic pathway [182].

A potent 5-HT2C receptor antagonist, S32006 (Figure 6), showed antidepressant activity in rodent behavioral tests and increased dopamine and norepinephrine levels in the frontal cortex [183]. This compound reduced immobility in the FST in mice, suppressed anhedonia in a chronic mild stress model, and increased cell proliferation and BDNF expression in the dentate gyri of rats [183]. In contrast, the inverse agonist of the 5-HT2C receptor,

*Int. J. Mol. Sci.* **2021**, *22*, 9015 13 of 32

chronic treatment [184].

S32212, showed an antidepressant effect in the FST in rats after both acute and chronic treatment [184]. of postsynaptic serotonin receptors [188]. Other selective 5-HT2C receptor agonists have also been effective in animal models of depression and obsessive–compulsive disorder [189].

expression in the dentate gyri of rats [183]. In contrast, the inverse agonist of the 5-HT2C receptor, S32212, showed an antidepressant effect in the FST in rats after both acute and

On the other hand, some studies report that 5-HT2C agonists have been shown to be active in animal models of depression, suggesting an antidepressant-like effect [185,186]. WAY-163909, a selective 5-HT2C receptor agonist, elicited a rapid antidepressant effect in a rat FST that was blocked by the 5-HT2C/2B receptor antagonist, SB206553 [186]. Moreover, after chronic treatment, WAY163909 reduced the hyperactivity associated with olfactory bulbectomy in rats [186,187]. It is possible that the mediated antidepressant effects of these compounds were due to the stimulation of 5-HT2C receptors and the resulting activation

**Figure 6.** 5-HT2C receptor ligands: S32006, S32212, WAY-163909 and SB206553. **Figure 6.** 5-HT2C receptor ligands: S32006, S32212, WAY-163909 and SB206553.

Preclinical data show that the antagonism of 5-HT2C receptors increases the neurochemical and behavioral effects of SSRIs. Examples include: the increase in the effect of SSRIs on extracellular 5-HT concentrations in the hippocampus and cortex [190,191], or a significant increase in the effect of SSRIs in behavioral models of depression by selective and non-selective 5-HT2C antagonists [190]. Additionally, 5-HT2C receptors have been shown to be involved in the anti-immobility effect of antidepressants in the FST, increasing the serotonin level in the synapse [192]. Few studies suggest that 5-HT2C receptor antagonists alone may also exhibit antidepressant-like properties. The inactivation of 5-HT2C receptors has been shown to potentiate On the other hand, some studies report that 5-HT2C agonists have been shown to be active in animal models of depression, suggesting an antidepressant-like effect [185,186]. WAY-163909, a selective 5-HT2C receptor agonist, elicited a rapid antidepressant effect in a rat FST that was blocked by the 5-HT2C/2B receptor antagonist, SB206553 [186]. Moreover, after chronic treatment, WAY163909 reduced the hyperactivity associated with olfactory bulbectomy in rats [186,187]. It is possible that the mediated antidepressant effects of these compounds were due to the stimulation of 5-HT2C receptors and the resulting activation of postsynaptic serotonin receptors [188]. Other selective 5-HT2C receptor agonists have also been effective in animal models of depression and obsessive–compulsive disorder [189].

SSRI-induced serotonin release in rodents [190]. However, 5-HT2C receptor antagonists administered separately had no effect on serotonin levels [191]. An altered editing of the mRNA-encoding 5-HT2C receptors has been reported in the PFC of depressed suicide victims [193]. The desensitization of these receptors has been observed in patients after chronic treatment with SSRIs [194]. Preclinical data show that the antagonism of 5-HT2C receptors increases the neurochemical and behavioral effects of SSRIs. Examples include: the increase in the effect of SSRIs on extracellular 5-HT concentrations in the hippocampus and cortex [190,191], or a significant increase in the effect of SSRIs in behavioral models of depression by selective and non-selective 5-HT2C antagonists [190].

The 5-HT2C receptors are also involved in the tonic modulation of dopaminergic activity [195]. The role of the dopaminergic system in schizophrenia, along with the antagonism of atypical antipsychotics towards the 5-HT2C receptors, has aroused interest in this receptor for the treatment of schizophrenia [196]. Conversely, the ineffectiveness of SSRIs in some patients may be due to the serotonin-related inhibition of the neuronal activity of Additionally, 5-HT2C receptors have been shown to be involved in the anti-immobility effect of antidepressants in the FST, increasing the serotonin level in the synapse [192]. Few studies suggest that 5-HT2C receptor antagonists alone may also exhibit antidepressantlike properties. The inactivation of 5-HT2C receptors has been shown to potentiate SSRIinduced serotonin release in rodents [190]. However, 5-HT2C receptor antagonists administered separately had no effect on serotonin levels [191].

dopamine in the ventral capping region via 5-HT2C receptors [157]. Escitalopram has been shown to reduce the stimulation of dopamine neurons by activating 5-HT2C receptors located on GABA neurons. Some studies indicate that the co-administration of SSRIs with An altered editing of the mRNA-encoding 5-HT2C receptors has been reported in the PFC of depressed suicide victims [193]. The desensitization of these receptors has been observed in patients after chronic treatment with SSRIs [194].

5-HT2C receptor antagonists (including atypical antipsychotics, such as aripiprazole) may eliminate the inhibitory effects on dopaminergic neurons in rat brains and restore the effect of the SSRI [163]. The aforementioned 5-HT2C receptor antagonist S32006, with a potential antidepressant- and anxiolytic-like effect, increased dopamine levels in the frontal cortex of rats and enhanced dopaminergic neuron firing [183]. The modulation of dopaminergic activity may, therefore, be beneficial in the development of antidepressants due to the above-mentioned activity of 5-HT2C receptor ligands. Recent studies demonstrate the contradictory effect of 5-HT2C receptors on the effects of SSRIs on motor function and affective behavior, highlighting the potential benefits of 5-HT2C receptor antagonists both The 5-HT2C receptors are also involved in the tonic modulation of dopaminergic activity [195]. The role of the dopaminergic system in schizophrenia, along with the antagonism of atypical antipsychotics towards the 5-HT2C receptors, has aroused interest in this receptor for the treatment of schizophrenia [196]. Conversely, the ineffectiveness of SSRIs in some patients may be due to the serotonin-related inhibition of the neuronal activity of dopamine in the ventral capping region via 5-HT2C receptors [157]. Escitalopram has been shown to reduce the stimulation of dopamine neurons by activating 5-HT2C receptors located on GABA neurons. Some studies indicate that the co-administration of SSRIs with 5-HT2C receptor antagonists (including atypical antipsychotics, such as aripiprazole) may eliminate the inhibitory effects on dopaminergic neurons in rat brains and restore the effect of the SSRI [163]. The aforementioned 5-HT2C receptor antagonist S32006, with a potential antidepressant- and anxiolytic-like effect, increased dopamine levels in the frontal cortex of rats and enhanced dopaminergic neuron firing [183]. The modulation of dopaminergic activity may, therefore, be beneficial in the development of antidepressants due to the above-mentioned activity of 5-HT2C receptor ligands. Recent studies demonstrate the contradictory effect of 5-HT2C receptors on the effects of SSRIs on motor function and affective behavior, highlighting the potential benefits of 5-HT2C receptor antagonists both for reducing SSRI motor side effects and enhancing the therapeutic antidepressant and anxiolytic effects [197].

Both 5-HT2C receptor agonists and antagonists exhibit antidepressant-like activity, and there is still a need to further define the role of this receptor subtype in depression: and there is still a need to further define the role of this receptor subtype in depression: • Several classes of antidepressants have an affinity for 5-HT2C receptors. Alterations

for reducing SSRI motor side effects and enhancing the therapeutic antidepressant and

Both 5-HT2C receptor agonists and antagonists exhibit antidepressant-like activity,

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#### *3.7. The 5-HT<sup>3</sup> Receptors 3.7. The 5-HT3 Receptors*

anxiolytic effects [197].

The activation of the 5-HT<sup>3</sup> receptor leads to a rapid opening of the transmembrane channel, resulting in an increase in the conductivity of Na+/K<sup>+</sup> ions and an immediate influx of extracellular Ca2+ ions. This, in turn, triggers the release of neurotransmitters and/or peptides. The 5-HT<sup>3</sup> receptors are found throughout the brain and CNS and the highest density of 5-HT<sup>3</sup> receptors was found in the spinal cord and brainstem. The 5-HT<sup>3</sup> receptors present in the dorsal vagal complex are involved in the control of the emetic mechanism [142]. Many 5-HT<sup>3</sup> receptor antagonists have been developed as antiemetics for use in cancer chemotherapy. In the 1990s, lithoxetine, an antidepressant combining serotonin reuptake and 5-HT<sup>3</sup> receptor antagonism, was developed to prevent SSRI-induced gastrointestinal side effects [198]. In the forebrain, on the other hand, 5-HT<sup>3</sup> receptors were present mainly in structures of the limbic system, such as the hippocampus, amygdala, and entorhinal cortex [199]. The activation of the 5-HT3 receptor leads to a rapid opening of the transmembrane channel, resulting in an increase in the conductivity of Na+/K+ ions and an immediate influx of extracellular Ca2+ ions. This, in turn, triggers the release of neurotransmitters and/or peptides. The 5-HT3 receptors are found throughout the brain and CNS and the highest density of 5-HT3 receptors was found in the spinal cord and brainstem. The 5-HT3 receptors present in the dorsal vagal complex are involved in the control of the emetic mechanism [142]. Many 5-HT3 receptor antagonists have been developed as antiemetics for use in cancer chemotherapy. In the 1990s, lithoxetine, an antidepressant combining serotonin reuptake and 5-HT3 receptor antagonism, was developed to prevent SSRI-induced gastrointestinal side effects [198]. In the forebrain, on the other hand, 5-HT3 receptors were present mainly in structures of the limbic system, such as the hippocampus, amygdala, and entorhinal cortex [199].

The 5-HT<sup>3</sup> receptors are involved in the control of dopamine and acetylcholine release. They also control the functioning of the GABAergic system. Activity towards other neurotransmission systems is the main mechanism of action for 5-HT<sup>3</sup> receptor ligands. The 5-HT<sup>3</sup> receptors are expressed on different types of GABAergic interneurons in the forebrain [200,201]. The physiological stimulation of serotonergic neurons stimulates cortical (and possibly hippocampal) GABAergic neurons. This likely results in the inhibition of neighboring excitatory neurons by GABA<sup>A</sup> and GABA<sup>B</sup> receptors [201]. The 5-HT3 receptors are involved in the control of dopamine and acetylcholine release. They also control the functioning of the GABAergic system. Activity towards other neurotransmission systems is the main mechanism of action for 5-HT3 receptor ligands. The 5-HT3 receptors are expressed on different types of GABAergic interneurons in the forebrain [200,201]. The physiological stimulation of serotonergic neurons stimulates cortical (and possibly hippocampal) GABAergic neurons. This likely results in the inhibition of neighboring excitatory neurons by GABAA and GABAB receptors [201].

Preclinical studies suggest that the 5-HT<sup>3</sup> receptor plays a role in mental disorders [17]. The 5-HT<sup>3</sup> antagonists show antidepressant-like activity in various animal models [202]. The systemic administration of tropisetron (a 5-HT<sup>3</sup> receptor antagonist) prevented restraint stress-induced dopamine release in the nucleus accumbens and prefrontal cortex in rats. This suggested that 5-HT<sup>3</sup> receptors mediated the stress-dependent activation of dopaminergic neurotransmission [203]. Tropisetron additionally exerted an antidepressantlike effect in FST in rats. This effect was abolished after a pretreatment with mCPGB (1-(m-chlorophenyl)-biguanide), a potent 5-HT<sup>3</sup> receptor agonist (Figure 7) [204]. Preclinical studies suggest that the 5-HT3 receptor plays a role in mental disorders [17]. The 5-HT3 antagonists show antidepressant-like activity in various animal models [202]. The systemic administration of tropisetron (a 5-HT3 receptor antagonist) prevented restraint stress-induced dopamine release in the nucleus accumbens and prefrontal cortex in rats. This suggested that 5-HT3 receptors mediated the stress-dependent activation of dopaminergic neurotransmission [203]. Tropisetron additionally exerted an antidepressant-like effect in FST in rats. This effect was abolished after a pretreatment with mCPGB (1-(m-chlorophenyl)-biguanide), a potent 5-HT3 receptor agonist (Figure 7) [204].

**Figure 7.** 5-HT3 receptor ligands: tropisetron and mCPGB. **Figure 7.** 5-HT<sup>3</sup> receptor ligands: tropisetron and mCPGB.

Some antidepressants with different mechanisms of action exhibit functional 5-HT3 receptor antagonism [205]. Chronic treatment with fluoxetine desensitizes 5-HT3 receptors [206], and SERT knockout mice show increased 5-HT3 receptor density compared to wild-Some antidepressants with different mechanisms of action exhibit functional 5-HT<sup>3</sup> receptor antagonism [205]. Chronic treatment with fluoxetine desensitizes 5-HT<sup>3</sup> receptors [206], and SERT knockout mice show increased 5-HT<sup>3</sup> receptor density compared to wild-type mice [207]. It has been suggested that the antidepressant effect of SSRIs is partially dependent on the blockade of 5-HT<sup>3</sup> receptors [208]. The relatively new multimodal antidepressant drug vortioxetine [209,210] displays nanomolar binding affinities to the SERT (K<sup>i</sup> = 1.6 nM) and other serotonin receptors, including 5-HT3, 5-HT1A, 5-HT7,

5-HT1B and 5-HT1D, with K<sup>i</sup> values of 3.7 nM, 15 nM, 19 nM, 33 nM and 54 nM, respectively [102]. Vortioxetine antagonism at the 5-HT<sup>3</sup> receptor [211] may underlie its faster onset of action [212]. Rodent experiments show that the antidepressant-like effect should be attributed to postsynaptic, rather than presynaptic, 5-HT<sup>3</sup> antagonism, since the presynaptic and somatodendritic 5-HT<sup>3</sup> receptor blockade reduces serotonin levels [203]. The antidepressant and/or anxiolytic effects recently demonstrated by some 5-HT<sup>3</sup> receptor antagonists in animal models of depression may result from the modulation of the hypothalamic–pituitary-adrenal axis, interaction with the serotonergic system, or antioxidant properties [213–218].

The agonism of the 5-HT<sup>3</sup> receptor reduces the antidepressant effect in the FST in rats [219], while the antagonism of the 5-HT<sup>3</sup> receptor reduces the immobility time in the FST [220]. Ondansetron, a 5-HT<sup>3</sup> receptor antagonist, confirms these observations; it exhibits antidepressant properties in the TST (tail suspension test) and FST, also enhancing the effect of fluoxetine [220]. In a model of chronic unpredictable stress in mice, the administration of ondansetron reversed depressive behavior affecting the hypothalamicpituitary-adrenal axis [221]. Moreover, in mice with streptozotocin-induced diabetes, the drug displayed antidepressant and anxiolytic properties, possibly through the antagonism of the 5-HT<sup>3</sup> receptor [222]. Behavioral studies with ondansetron (and tropisetron) also suggested an interaction of 5-HT<sup>3</sup> and NMDA receptors, as well as an involvement of the nitric oxide-cyclic guanosine monophosphate pathway inhibition in the observed antidepressant-like effects [223,224]. Studies on genetically modified animals confirm the role of 5-HT<sup>3</sup> receptors in the antidepressant effect. The 5-HT<sup>3</sup> receptor knockout mice were reported to display an antidepressant-like phenotype [225].

In vitro electrophysiology studies showed that low-dose citalopram treatment desensitized the 5-HT1A receptor only in the dorsal raphe nucleus of 5-HT<sup>3</sup> knockout mice, while high dose treatment caused similar 5-HT1A autoreceptor desensitization in 5-HT<sup>3</sup> knockout and wild types [225]. Hence, lower doses of citalopram may be effective when 5-HT<sup>3</sup> receptors are deactivated. It has also been shown that the blockade of the 5-HT<sup>3</sup> receptor by ondansetron enhances the effect of citalopram on extracellular serotonin levels in the rat forebrain [102]. The use of combined SSRIs and 5HT<sup>3</sup> receptor antagonists is proposed as an improvement strategy to be tested in the treatment of depressive disorders [226]. The neurochemical, electrophysiological, and behavioral consequences of the repeated administration of this drug combination will need to be assessed.

The properties of 5-HT<sup>3</sup> receptor antagonists have also been used to alleviate substance abuse, which is often associated with most psychiatric disorders, including MDD [227]. The 5-HT<sup>3</sup> receptor antagonists have been reported to be effective in reducing ethanol and morphine intake [228]. It is worth noting that various antipsychotics are non-competitive 5-HT<sup>3</sup> receptor antagonists, and this may contribute to their efficacy [229]. There is likely to be an association between 5-HT<sup>3</sup> receptors and anxiety behavior [230]. The 5-HT<sup>3</sup> antagonists reverse helpless behavior in rats [231] and abolish the emotion-potentiated startle effect in humans [232]:


### *3.8. The 5-HT<sup>4</sup> Receptors*

The 5-HT<sup>4</sup> receptors in the CNS are mainly located in the putamen, caudate nucleus, hippocampus, nucleus accumbens, globus pallidus, and substantia nigra. To a lesser extent, these receptors are present in the neocortex, raphe and pontine nuclei, and thalamus [233].

Studies using positron emission tomography show a slightly more limited regional distribution of 5-HT<sup>4</sup> receptors in the human brain, showing a high density of this receptor in the caudate–putamen and much lower densities in the frontal cortex and hippocampus [234]. The 5-HT<sup>4</sup> receptors are coupled to the Gs protein, which activates adenylate cyclase/PKC and increases the intracellular level of cAMP. Regarding peripheral tissues, these receptors play an important role in the heart, gastrointestinal tract, adrenal glands, and urinary bladder [235].

There are reports linking the 5-HT<sup>4</sup> receptor with depressive disorders [236]. Preclinical models of depression, such as the olfactory bulbectomized and glucocorticoid heterozygous receptor mice, show that the expression of 5-HT<sup>4</sup> receptors increased in the ventral hippocampus or striatum, respectively [237], while in the Flinders-sensitive line rat model of depression, the downregulation of 5-HT<sup>4</sup> receptors was observed in the ventral and dorsal hippocampus [238].

The 5-HT<sup>4</sup> receptor subtype is involved in the modulation of synaptic plasticity [239], which is influenced by antidepressants [240]. The signaling of the 5-HT<sup>4</sup> receptor may modulate the function of the dentate gyrus of the hippocampus by increasing the neurogenesis and expression of neurotrophic factors, which may contribute to the antidepressant effects of drugs that enhance serotonergic transmission [241]. The 5-HT<sup>4</sup> receptor interacts with the p11 protein, which determines the antidepressant activity mediated by 5-HT1B and 5-HT<sup>4</sup> receptors [242].

In addition, 5-HT<sup>4</sup> knockout mice show an enhanced response of serotonergic neurons to citalopram [243]. Thus, 5-HT<sup>4</sup> receptors are possibly involved in the activation of 5-HT neurons during SERT inhibition. As observed for 5-HT1A and 5-HT2A receptors [37,40], 5-HT<sup>4</sup> receptors in the PFC control the firing rate of midbrain serotonergic neurons via descending inputs [244]. In addition, 5-HT<sup>4</sup> receptors mediate synaptic transmission between the dentate gyrus and the CA3 field of the hippocampus. Fluoxetine was observed to normalize the mossy fiber pathway by activating 5-HT<sup>4</sup> receptors [245]. Chronic treatment with fluoxetine and venlafaxine (but not reboxetine) decreased the 5-HT<sup>4</sup> receptor density in rat brain [246]. Although the 5-HT<sup>4</sup> receptor antagonist, SB 204070A, showed no independent effect and did not reduce the immobility time in the FST in naive rats [247], another receptor antagonist, GR 125487, blocked fluoxetine activity in a mouse corticosteroneinduced depression model [248]. Therefore, this study suggests that the activation of 5-HT<sup>4</sup> receptors mediates the antidepressant-like effects of fluoxetine. It has been shown that a knockout of the 5-HT<sup>4</sup> receptor can induce some adaptive changes in mice, leading to depression and anxiety-like behavior. Moreover, 5-HT<sup>4</sup> receptor knockout mice do not respond to fluoxetine in the olfactory bulbectomized model of depression and anxiety [249]. On the other hand, some studies suggest that the behavioral effects of fluoxetine in the corticosterone-induced model of depression and anxiety do not appear to be dependent on 5-HT<sup>4</sup> receptors [250].

Preclinical studies show that the administration of the 5-HT<sup>4</sup> agonists, RS67333 and prucalopride (Figure 8), reduces the immobility time in the FST, thus demonstrating the potential of the 5-HT<sup>4</sup> receptor as a molecular target of a potential new generation of antidepressants [251]. The agonism of the 5-HT<sup>4</sup> receptor may also play a role in the cognitive deficits associated with MDD. The use of RS67333 in chronic neuroendocrine animal models of depression/anxiety resulted in the restoration of induced learning and memory disorders [252]. Moreover, the studies show that administration of RS67333 and prucalopride causes 5-HT1A autoreceptor desensitization, increased the tonus on hippocampal postsynaptic 5-HT1A receptors, and increased CREB phosphorylation and neurogenesis in the hippocampus [251]. These parameters, which characterize the functioning of the brain, are used in antidepressant therapies. Importantly, these effects are noticeable after 3 days of treatment [251], while they are usually only seen after 2–3 weeks of treatment with SSRIs due to the latency phenomenon. The faster response to 5-HT<sup>4</sup> agonism has been suggested to be a result of the parallel rapid and sustained activation of 5-HT neuronal firing in the dorsal raphe nucleus [253]. Increased serotonergic neuronal firing may also

underlie the apparently superior efficacy of 5-HT<sup>4</sup> agonists over SSRIs because the reuptake inhibitory effect depends on the basal rates of 5-HT cell firing. Since the 5-HT<sup>4</sup> receptor is not expressed in the raphe nuclei, the ability of 5-HT<sup>4</sup> receptors to stimulate the firing of 5-HT neurons appears to involve the activation of receptors located on neurons in the PFC [244]. The identity of the cells expressing 5-HT<sup>4</sup> receptors and their connections to the serotonergic neurons of the dorsal raphe nucleus are not yet well understood. It is possible that they project to other regions, contributing to the antidepressant effect of 5-HT<sup>4</sup> agonists [253]. apparently superior efficacy of 5-HT4 agonists over SSRIs because the reuptake inhibitory effect depends on the basal rates of 5-HT cell firing. Since the 5-HT4 receptor is not expressed in the raphe nuclei, the ability of 5-HT4 receptors to stimulate the firing of 5-HT neurons appears to involve the activation of receptors located on neurons in the PFC [244]. The identity of the cells expressing 5-HT4 receptors and their connections to the serotonergic neurons of the dorsal raphe nucleus are not yet well understood. It is possible that they project to other regions, contributing to the antidepressant effect of 5-HT4 agonists [253].

treatment [251], while they are usually only seen after 2–3 weeks of treatment with SSRIs due to the latency phenomenon. The faster response to 5-HT4 agonism has been suggested to be a result of the parallel rapid and sustained activation of 5-HT neuronal firing in the dorsal raphe nucleus [253]. Increased serotonergic neuronal firing may also underlie the

*Int. J. Mol. Sci.* **2021**, *22*, 9015 17 of 32

**Figure 8.** 5-HT4 receptor agonists: RS67333 and prucalopride. **Figure 8.** 5-HT<sup>4</sup> receptor agonists: RS67333 and prucalopride.

The activation of the 5-HT4 receptor may be a useful adjunct to antidepressant therapy, both to accelerate the onset of clinical antidepressant effects and to target cognitive symptoms that are not effectively treated with current therapies [254]: The activation of the 5-HT<sup>4</sup> receptor may be a useful adjunct to antidepressant therapy, both to accelerate the onset of clinical antidepressant effects and to target cognitive symptoms that are not effectively treated with current therapies [254]:


#### *3.9. The 5-HT6 Receptors 3.9. The 5-HT<sup>6</sup> Receptors*

The 5-HT6 receptors are the postsynaptic receptors most expressed in the striatum, nucleus accumbens, olfactory tubercle, and cortex. They are also moderately dense in the The 5-HT<sup>6</sup> receptors are the postsynaptic receptors most expressed in the striatum, nucleus accumbens, olfactory tubercle, and cortex. They are also moderately dense in the amygdala, hippocampus, hypothalamus, thalamus, and cerebellum [255].

amygdala, hippocampus, hypothalamus, thalamus, and cerebellum [255]. This serotonin receptor subtype has been found to play a role in learning and memory [256] as well as in the central regulation of hunger and satiety behavior [257]. The 5-HT6 receptors may, therefore, serve as a novel molecular target for the improvement of cognitive functions [258]. Several of the tricyclic antidepressants (e.g., amitriptyline) and atypical antidepressants (e.g., mianserin) exhibit nanomolar 5-HT6 binding and antagonistic activity [259]. This fact, as well as the distribution of the 5-HT6 receptor in the limbic and cortical regions of the brain, may suggest that 5-HT6 receptors play an important role in the pathogenesis and/or treatment of depression [259]. The 5-HT6 antagonists (SB-399885, Figure 9) show antidepressant activity in the FST and in the TST in rodents (rats and mice) [260]. In addition, the combination of an ineffective dose of SB-399885 with ineffective doses of imipramine, desipramine, bupropion, or moclobemide has been shown to exert antidepressant effects in the rat FST [261]. This suggests that the inhibition of the This serotonin receptor subtype has been found to play a role in learning and memory [256] as well as in the central regulation of hunger and satiety behavior [257]. The 5-HT<sup>6</sup> receptors may, therefore, serve as a novel molecular target for the improvement of cognitive functions [258]. Several of the tricyclic antidepressants (e.g., amitriptyline) and atypical antidepressants (e.g., mianserin) exhibit nanomolar 5-HT<sup>6</sup> binding and antagonistic activity [259]. This fact, as well as the distribution of the 5-HT<sup>6</sup> receptor in the limbic and cortical regions of the brain, may suggest that 5-HT<sup>6</sup> receptors play an important role in the pathogenesis and/or treatment of depression [259]. The 5-HT<sup>6</sup> antagonists (SB-399885, Figure 9) show antidepressant activity in the FST and in the TST in rodents (rats and mice) [260]. In addition, the combination of an ineffective dose of SB-399885 with ineffective doses of imipramine, desipramine, bupropion, or moclobemide has been shown to exert antidepressant effects in the rat FST [261]. This suggests that the inhibition of the 5-HT<sup>6</sup> receptor potentiates the effects of clinically used antidepressants. This synergistic effect is interesting in the search for a multimodal antidepressant therapy with minimized side effects or a faster onset of action. Other preclinical studies show that 5-HT<sup>6</sup> agonism can be used in the treatment of depression. The reduction in the immobility

of mice in the FST after the administration of WAY208466, a selective potent agonist of the 5-HT<sup>6</sup> receptor, has been demonstrated [262]. The 5-HT<sup>6</sup> receptor partial agonist, EMD386088, caused antidepressant- and anxiolytic-like effects after intrahippocampal administration [263]. This also occurred after acute and chronic treatment in rats [264], possibly because it directly stimulated the receptor. The stimulation of the 5-HT<sup>6</sup> receptor may initiate the biochemical and behavioral effects induced by SSRIs (fluoxetine) [122]. On the other hand, the 5-HT<sup>6</sup> receptor agonist LY-586713 increases the expression of BDNF (a marker of cellular antidepressant activity) in the hippocampus after just a single administration [265]. In comparison, SSRIs require multiple applications to produce the same effect [240]. Therefore, it is unclear what functional 5-HT<sup>6</sup> receptor ligand profile (antagonism or agonism) will be more beneficial in the treatment of depression. Moreover, the exact mechanism by which 5-HT<sup>6</sup> ligands induce antidepressant effects is unknown and may include effects on other neurotransmission systems [260,266]: FST after the administration of WAY208466, a selective potent agonist of the 5-HT6 receptor, has been demonstrated [262]. The 5-HT6 receptor partial agonist, EMD386088, caused antidepressant- and anxiolytic-like effects after intrahippocampal administration [263]. This also occurred after acute and chronic treatment in rats [264], possibly because it directly stimulated the receptor. The stimulation of the 5-HT6 receptor may initiate the biochemical and behavioral effects induced by SSRIs (fluoxetine) [122]. On the other hand, the 5-HT6 receptor agonist LY-586713 increases the expression of BDNF (a marker of cellular antidepressant activity) in the hippocampus after just a single administration [265]. In comparison, SSRIs require multiple applications to produce the same effect [240]. Therefore, it is unclear what functional 5-HT6 receptor ligand profile (antagonism or agonism) will be more beneficial in the treatment of depression. Moreover, the exact mechanism by which 5-HT6 ligands induce antidepressant effects is unknown and may include effects on other neurotransmission systems [260,266]:

5-HT6 receptor potentiates the effects of clinically used antidepressants. This synergistic effect is interesting in the search for a multimodal antidepressant therapy with minimized side effects or a faster onset of action. Other preclinical studies show that 5-HT6 agonism can be used in the treatment of depression. The reduction in the immobility of mice in the

**Figure 9.** 5-HT6 receptor ligands: SB-399885, WAY-208466, and EMD386088. **Figure 9.** 5-HT<sup>6</sup> receptor ligands: SB-399885, WAY-208466, and EMD386088.

*Int. J. Mol. Sci.* **2021**, *22*, 9015 18 of 32


#### *3.10. The 5-HT7 Receptors 3.10. The 5-HT<sup>7</sup> Receptors*

The 5-HT7 receptor is highly expressed in the thalamus, hypothalamus, hippocampus, and cortex [267]. The results of immunolocation and autoradiography studies are generally consistent with the pattern of mRNA distribution [268,269], suggesting a dominant somatodendritic localization. The physiological role of 5-HT7 receptors is to regulate The 5-HT<sup>7</sup> receptor is highly expressed in the thalamus, hypothalamus, hippocampus, and cortex [267]. The results of immunolocation and autoradiography studies are generally consistent with the pattern of mRNA distribution [268,269], suggesting a dominant somatodendritic localization. The physiological role of 5-HT<sup>7</sup> receptors is to regulate circadian rhythm, sleep, and mood [270].

circadian rhythm, sleep, and mood [270]. As with the 5-HT6 receptors, several antidepressants [271] and antipsychotics [272] have been found to have a high affinity for the 5-HT7 receptor, leading to much further research into its antidepressant activity. One preclinical study in rats showed that several antidepressants, both tricyclic and SSRIs, induce *c-fos* expression in a manner consistent As with the 5-HT<sup>6</sup> receptors, several antidepressants [271] and antipsychotics [272] have been found to have a high affinity for the 5-HT<sup>7</sup> receptor, leading to much further research into its antidepressant activity. One preclinical study in rats showed that several antidepressants, both tricyclic and SSRIs, induce *c-fos* expression in a manner consistent with 5-HT<sup>7</sup> receptor activation within the suprachiasmatic nucleus, and that chronic treatment with antidepressant drugs downregulates 5-HT<sup>7</sup> receptor binding [271].

Preclinical studies also indicate the antidepressant and anxiolytic effects of the selective 5-HT<sup>7</sup> receptor antagonist, SB-269970, in rodents [273], as well as a synergistic interaction between subeffective doses of this agent and antidepressants, leading to a reduction in immobility in both the FST and the TST [274,275]. The intrahippocampal administra-

tion of SB-269970 (Figure 10) induced an antidepressant effect in the FST in rats [276]. The co-administration of citalopram and SB-269970 increased the activity of serotonin neurons in rats and improved the antidepressant effect in the TST [274]. SB-269970 enhanced the antidepressant effect of antidepressants (citalopram, imipramine, desipramine, and moclobemide) in the FST in mice [275]. It was also shown that the administration of SB-269970 for only one week caused the behavioral, electrophysiological, and neuroanatomical changes that usually occur after a long-term treatment with SSRIs. Therefore, 5-HT<sup>7</sup> receptor antagonists might represent a new class of antidepressants with a faster therapeutic effect. JNJ-18038683, another 5-HT<sup>7</sup> receptor antagonist, was also effective in mice TST [277]. Moreover, the compound potentiated serotonin transmission, REM suppression, and antidepressant-like behaviour induced by citalopram in rodents [277]. The above studies indicate that the participation of the 5-HT<sup>7</sup> receptor in the antidepressant-like action, and blockade of the 5-HT<sup>7</sup> receptor may not only induce but accelerate this action. for only one week caused the behavioral, electrophysiological, and neuroanatomical changes that usually occur after a long-term treatment with SSRIs. Therefore, 5-HT7 receptor antagonists might represent a new class of antidepressants with a faster therapeutic effect. JNJ-18038683, another 5-HT7 receptor antagonist, was also effective in mice TST [277]. Moreover, the compound potentiated serotonin transmission, REM suppression, and antidepressant-like behaviour induced by citalopram in rodents [277]. The above studies indicate that the participation of the 5-HT7 receptor in the antidepressant-like action, and blockade of the 5-HT7 receptor may not only induce but accelerate this action. In addition, the genetic and pharmacological inactivation of 5-HT7 receptors partially reversed phencyclidine-induced deficits of pre-pulse inhibition, an animal model for antipsychotic activity [278]. Similarly, it should also be noted that the atypical antipsychotic aripiprazole, which has a high affinity for the 5-HT7 receptor, is sometimes used to enhance the effects of traditional antidepressants [279]. Similarly, there are reports that the antidepressant effect of amisulpride is mediated by its action on 5-HT7 receptors [280].

with 5-HT7 receptor activation within the suprachiasmatic nucleus, and that chronic treat-

Preclinical studies also indicate the antidepressant and anxiolytic effects of the selective 5-HT7 receptor antagonist, SB-269970, in rodents [273], as well as a synergistic interaction between subeffective doses of this agent and antidepressants, leading to a reduction in immobility in both the FST and the TST [274,275]. The intrahippocampal administration of SB-269970 (Figure 10) induced an antidepressant effect in the FST in rats [276]. The coadministration of citalopram and SB-269970 increased the activity of serotonin neurons in rats and improved the antidepressant effect in the TST [274]. SB-269970 enhanced the antidepressant effect of antidepressants (citalopram, imipramine, desipramine, and moclobemide) in the FST in mice [275]. It was also shown that the administration of SB-269970

ment with antidepressant drugs downregulates 5-HT7 receptor binding [271].

*Int. J. Mol. Sci.* **2021**, *22*, 9015 19 of 32

**Figure 10.** 5-HT7 receptor antagonists: SB-269970 and JNJ-18038683. **Figure 10.** 5-HT<sup>7</sup> receptor antagonists: SB-269970 and JNJ-18038683.

Vortioxetine is a high affinity inhibitor of the human 5-HT transporter, 5-HT3 and 5- HT7 receptors, and a 5-HT1A agonist [281]. Although the affinity of vortioxetine for the rat 5-HT7 receptor is lower compared to the human receptor [282], subacute administration (within 3 days) of an effective dose of vortioxetine rapidly lowers rat 5-HT7 receptor levels [283]. This preclinical evidence suggests that vortioxetine has a relatively low affinity for the 5-HT7 receptor compared to other 5-HT receptor subtypes but inhibits its action with In addition, the genetic and pharmacological inactivation of 5-HT<sup>7</sup> receptors partially reversed phencyclidine-induced deficits of pre-pulse inhibition, an animal model for antipsychotic activity [278]. Similarly, it should also be noted that the atypical antipsychotic aripiprazole, which has a high affinity for the 5-HT<sup>7</sup> receptor, is sometimes used to enhance the effects of traditional antidepressants [279]. Similarly, there are reports that the antidepressant effect of amisulpride is mediated by its action on 5-HT<sup>7</sup> receptors [280].

a rapid 5-HT7 receptor downregulation as an inverse agonist, similar to other 5-HT7 receptor-inhibiting mood-stabilizing atypical antipsychotics: clozapine, lurasidone, and olanzapine [283,284]. In other words, the rapid-acting antidepressant and anxiolytic actions of 5-HT7 receptor antagonism are worth reassessing in the context of drug development after future clinical data have been accumulated. Overall, the 5-HT7 receptor is currently considered a promising target for the development of antidepressants [285]. Recent clinical studies have shown that both the intravenous and oral administration of vortioxetine resulted in a significant improvement in depression (Montgomery Åsberg Depression Rating Scale and Hospital Depression Scale) and anxiety (Hospital Anxiety Scale) after 3 days [77]: Vortioxetine is a high affinity inhibitor of the human 5-HT transporter, 5-HT<sup>3</sup> and 5-HT<sup>7</sup> receptors, and a 5-HT1A agonist [281]. Although the affinity of vortioxetine for the rat 5-HT<sup>7</sup> receptor is lower compared to the human receptor [282], subacute administration (within 3 days) of an effective dose of vortioxetine rapidly lowers rat 5-HT<sup>7</sup> receptor levels [283]. This preclinical evidence suggests that vortioxetine has a relatively low affinity for the 5-HT<sup>7</sup> receptor compared to other 5-HT receptor subtypes but inhibits its action with a rapid 5-HT<sup>7</sup> receptor downregulation as an inverse agonist, similar to other 5-HT<sup>7</sup> receptor-inhibiting mood-stabilizing atypical antipsychotics: clozapine, lurasidone, and olanzapine [283,284]. In other words, the rapid-acting antidepressant and anxiolytic actions of 5-HT<sup>7</sup> receptor antagonism are worth reassessing in the context of drug development after future clinical data have been accumulated. Overall, the 5-HT<sup>7</sup> receptor is currently considered a promising target for the development of antidepressants [285]. Recent clinical studies have shown that both the intravenous and oral administration of vortioxetine resulted in a significant improvement in depression (Montgomery Åsberg Depression Rating Scale and Hospital Depression Scale) and anxiety (Hospital Anxiety Scale) after 3 days [77]:


oral administration of vortioxetine resulted in a significant improvement in depression and anxiety after 3 days.

#### **4. Conclusions**

The development of new antidepressants is based on monoamine systems. The targeted pharmacological modulation of serotonergic transmission in the brain continues to be a leading strategy in the search for new antidepressants. As can be seen from this review, the serotonergic system offers great potential for the development of new antidepressant therapies based on the combination of SERT inhibition with different pharmacological activities towards the 5-HT system. The careful selection of molecular targets for the proper use of the mechanisms of serotonergic autoregulation and selective/biased activation or the blockade of relevant receptors (e.g., stimulation of postsynaptic 5-HT1A, postsynaptic 5-HT1B, 5-HT2B and 5-HT<sup>4</sup> receptors; or the blockade of presynaptic 5-HT1A, presynaptic 5-HT1B, 5-HT2A, 5-HT3, and 5-HT7), which also influences other neurotransmission systems, seems to be the most effective strategy for supplementing the activity of "serotonin-enhancing" drugs in the near future. A better understanding of receptors and the receptor signaling responsible for the effects of serotonin on neurogenesis could also help in the development of new and more effective drugs.

**Author Contributions:** Conceptualization, G.S.; writing—original draft preparation, G. ´ S.; writing— ´ review and editing, M.K. and J.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Medical University of Warsaw. This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

