*Review* **5-HT Receptors and the Development of New Antidepressants**

**Grzegorz Slifirski ´ , Marek Król \* and Jadwiga Turło**

Department of Drug Technology and Pharmaceutical Biotechnology, Faculty of Pharmacy, Medical University of Warsaw, 1 Banacha Street, 02-097 Warsaw, Poland; gslifirski@wum.edu.pl (G.S.); jturlo@wum.edu.pl (J.T.) ´ **\*** Correspondence: mkrol@wum.edu.pl

**Abstract:** Serotonin modulates several physiological and cognitive pathways throughout the human body that affect emotions, memory, sleep, and thermal regulation. The complex nature of the serotonergic system and interactions with other neurochemical systems indicate that the development of depression may be mediated by various pathomechanisms, the common denominator of which is undoubtedly the disturbed transmission in central 5-HT synapses. Therefore, the deliberate pharmacological modulation of serotonergic transmission in the brain seems to be one of the most appropriate strategies for the search for new antidepressants. As discussed in 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 activity towards the 5-HT system. The aim of this article is to summarize the search for new antidepressants in recent years, focusing primarily on the possibility of benefiting from interactions with various 5-HT receptors in the pharmacotherapy of depression.

**Keywords:** antidepressants; drug design; dual 5-HT1A/SERT activity; multimodal activity

**Citation:** Slifirski, G.; Król, M.; Turło, ´ J. 5-HT Receptors and the Development of New Antidepressants. *Int. J. Mol. Sci.* **2021**, *22*, 9015. https://doi.org/10.3390/ ijms22169015

Academic Editor: Philippe De Deurwaerdère

Received: 26 July 2021 Accepted: 19 August 2021 Published: 20 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Depression is a mental illness that affects over 250 million people worldwide [1]. Emotional (depressed mood, irritability, anhedonia), somatic (sleep, appetite, libido), and functional disorders (suicidal thoughts, slowed speech and movement, learning, memory and attention deficits) [2] make this disease the main cause of disabilities in the general population [3,4].

An important step in the treatment of depressive disorders is the introduction of SSRIs (serotonin reuptake inhibitors), which are currently first-line antidepressants (e.g., fluoxetine, sertraline, escitalopram). Their mechanism of action is based on the serotonergic system, and the molecular target is the serotonin transporter protein (SERT). The effectiveness of these therapeutics, unfortunately, leaves much to be desired; 60–70% of patients do not experience a remission of symptoms, and 30–40% do not respond to the treatment at all [5]. A serious drawback of selective serotonin reuptake inhibitors is their latency period, i.e., a delay in the therapeutic response by 2–6 weeks. Common side effects for SSRIs are sexual dysfunction, anxiety, and food intolerances.

Apart from SSRIs, other selective monoamine reuptake inhibitors are also used in pharmacotherapy. Reboxetine, a selective norepinephrine reuptake inhibitor, appears to be less effective than the SSRIs. These observations may, however, result from its relatively low tolerance [6]. Bupropion, on the other hand, is a norepinephrine and dopamine reuptake inhibitor and, therefore, has a more activating profile than SSRI drugs. Two drugs, venlafaxine and duloxetine, are classified as dual serotonin-norepinephrine reuptake inhibitors (SNRIs). However, the efficacy of the norepinephrine reuptake blocking at clinical doses of duloxetine is unclear [7]. Clinical guidelines often recommend the use of SNRIs in patients who do not respond to SSRIs [8–10].

There is a need for the further exploration of the neurochemical causes of depression. Recent studies report the influence of many various types of neurosignaling on

the mechanism of depression [11–14]. The search for new generations of antidepressants using the triple reuptake inhibition mechanism (SSRI/SNRI/SDARI), or the combination of serotonin reuptake inhibition with affinities for various 5-hydroxytryptamine (5-HT) receptor subtypes, broadens the knowledge in this field [15–17]. anism of depression [11–14]. The search for new generations of antidepressants using the triple reuptake inhibition mechanism (SSRI/SNRI/SDARI), or the combination of serotonin reuptake inhibition with affinities for various 5-hydroxytryptamine (5-HT) receptor subtypes, broadens the knowledge in this field [15–17].

There is a need for the further exploration of the neurochemical causes of depression. Recent studies report the influence of many various types of neurosignaling on the mech-

*Int. J. Mol. Sci.* **2021**, *22*, 9015 2 of 32

A significant part of recent studies proves that serotonergic dysfunction, especially related to the postsynaptic 5-HT1A receptor, plays an important role in the pathomechanism of Major Depressive Disorder (MDD) [18–24]. Clinical trials show that the combination of SSRIs with both partial agonism and antagonism of the 5-HT1A receptor may result in an improvement in the speed and efficacy of the antidepressant effect [23,25,26]. This can be confirmed by the drugs recently introduced into the pharmacotherapy of depression– vilazodone and vortioxetine (Figure 1). Vilazodone exhibits partial agonist activity at the 5-HT1A receptor, while vortioxetine binds to several 5-HT receptor subtypes (5-HT1A, 5-HT1B, 5-HT1D, 5-HT3, and 5-HT7). For example, the degree of sexual dysfunction associated with the use of vilazodone has been found to be relatively low [27]. Vortioxetine, on the other hand, positively influences cognitive impairment related to depression [10,28]. A significant part of recent studies proves that serotonergic dysfunction, especially related to the postsynaptic 5-HT1A receptor, plays an important role in the pathomechanism of Major Depressive Disorder (MDD) [18–24]. Clinical trials show that the combination of SSRIs with both partial agonism and antagonism of the 5-HT1A receptor may result in an improvement in the speed and efficacy of the antidepressant effect [23,25,26]. This can be confirmed by the drugs recently introduced into the pharmacotherapy of depression–vilazodone and vortioxetine (Figure 1). Vilazodone exhibits partial agonist activity at the 5-HT1A receptor, while vortioxetine binds to several 5-HT receptor subtypes (5-HT1A, 5-HT1B, 5-HT1D, 5-HT3, and 5-HT7). For example, the degree of sexual dysfunction associated with the use of vilazodone has been found to be relatively low [27]. Vortioxetine, on the other hand, positively influences cognitive impairment related to depression [10,28].

**Figure 1.** Novel antidepressants: vilazodone and vortioxetine. **Figure 1.** Novel antidepressants: vilazodone and vortioxetine.

The targeted pharmacological modulation of serotonergic transmission in the brain continues to be a leading strategy in the search for new antidepressants. The careful selection of molecular targets for the proper use of the mechanisms of serotonergic modulation, which 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 the receptors and receptor signaling responsible for the effects of serotonin on neurogenesis can also help in the development of new and more effective drugs. The aim of this article is to summarize the search for new antidepressants in recent years, focusing primarily on the possibility of benefiting from interactions with various 5-HT receptors in the pharmacotherapy of depression. The targeted pharmacological modulation of serotonergic transmission in the brain continues to be a leading strategy in the search for new antidepressants. The careful selection of molecular targets for the proper use of the mechanisms of serotonergic modulation, which 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 the receptors and receptor signaling responsible for the effects of serotonin on neurogenesis can also help in the development of new and more effective drugs. The aim of this article is to summarize the search for new antidepressants in recent years, focusing primarily on the possibility of benefiting from interactions with various 5-HT receptors in the pharmacotherapy of depression.

#### **2. The Serotonergic System and Depression 2. The Serotonergic System and Depression**

Serotonin, or 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter found throughout the human body [19,29]. Serotonin is synthesized in the midbrain in a small population of raphe nucleus neurons where tryptophan hydroxylase is expressed [30]. However, serotonin synthesis is not limited to the central nervous system (CNS), as tryptophan hydroxylase is also found in enterochromaffin cells in the gastrointestinal tract [31]. In fact, it should be noted that most of the serotonin in the human body is produced by this cell type [32]. Serotonin binds to more than 14 receptor proteins, most of which are G-protein coupled receptors [30,33]. This molecule mediates the transmission of several physiological and cognitive systems throughout the body that affect emotions, memory, Serotonin, or 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter found throughout the human body [19,29]. Serotonin is synthesized in the midbrain in a small population of raphe nucleus neurons where tryptophan hydroxylase is expressed [30]. However, serotonin synthesis is not limited to the central nervous system (CNS), as tryptophan hydroxylase is also found in enterochromaffin cells in the gastrointestinal tract [31]. In fact, it should be noted that most of the serotonin in the human body is produced by this cell type [32]. Serotonin binds to more than 14 receptor proteins, most of which are G-protein coupled receptors [30,33]. This molecule mediates the transmission of several physiological and cognitive systems throughout the body that affect emotions, memory, sleep, and thermal regulation [34].

sleep, and thermal regulation [34]. Serotonin is synthesized in the body from an essential amino acid—L-tryptophan. Ingested with food, L-tryptophan is converted into serotonin through a series of reactions. Serotonin is synthesized in the body from an essential amino acid—L-tryptophan. Ingested with food, L-tryptophan is converted into serotonin through a series of reactions. The first step, which simultaneously limits the rate of serotonin synthesis, is the hydroxylation of L-tryptophan to 5-hydroxy-L-tryptophan (5-HTP) by tryptophan hydroxylase

(TPH) using oxygen and tetrahydropteridine as co-factors. There are two isoforms of TPH that can participate in this reaction: TPH1, expressed predominantly peripherally; and TPH2, expressed only in the brain. L-aromatic amino acid decarboxylase (AADC) then converts 5-HTP to serotonin [19,31].

The crossing of the blood–brain barrier (BBB) by serotonin is impossible due to its acid dissociation [35]; therefore, the amount of serotonin present in the CNS depends on the amount of centrally present L-tryptophan. The L-tryptophan present in the systemic circulation is actively transported by the BBB to the CNS using a carrier protein, where it is then converted into serotonin. Serotonin synthesized in the central nervous system is stored in secretory vesicles, where it remains until neuronal depolarization triggers its release into the synaptic cleft, allowing postsynaptic binding. Once released into the synapse, the serotonin molecules are eventually taken up by the serotonin transporter (5-HTT), which is located on the presynaptic axonal membrane. After the above-mentioned reuptake occurs, serotonin molecules are metabolized by monoamine oxidase (MAO) to 5-hydroxyindole acetic acid (5-HIAA) [29]. There are two isoforms of MAO (MAO-A and MAO-B), and both break down serotonin into neurons through oxidative deamination. The serotonin metabolite (5-HIAA) is actively transported from the CNS to the periphery and then excreted in the urine [19].

Already by the 1950s, it was noted that several mental illnesses showed abnormalities in the serotonergic system. The relationship between the serotonergic system and depression has been confirmed in clinical trials. They showed that an acute, transient relapse of depressive symptoms can be produced in subjects in remission using p-chlorophenylalanine (an irreversible inhibitor of serotonin synthesis). L-tryptophan depletion, causing a temporary reduction in central serotonin levels, had similar consequences. These findings have shown that the clinical efficacy of antidepressants depends on the presynaptic serotonergic function. Other studies have demonstrated a reduced concentration of the major metabolite of serotonin (5-HIAA) in the cerebrospinal fluid of untreated depressed patients and a reduced concentration of 5-HT and its major metabolite (5-HIAA) in the postmortem brain tissue of depressed and/or suicidal patients [20].

The serotonergic neurons of the mammalian brain constitute the most extensive and complex neurochemical network in the CNS after the glutamatergic system, which is the brain's primary transmission network. It has been estimated that the human brain contains approximately 250,000 5-HT neurons. For comparison, the total number of all neurons is around 10<sup>11</sup> [36]. While serotonergic neurons originate mainly in the brainstem dorsal and median raphe nuclei, they arborise over large areas such that they innervate almost every area of the brain with high densities of axonal varicosities. Some serotonergic projections create classical chemical synapses, but many release 5-HT in a paracrine manner (sometimes referred to as "volumetric transmission"). In addition, serotonin neurons exhibit slow (~1 Hz) and regular tonic activity that ceases during the rapid eye movement sleep phase (REM-off neurons). This activity is parallel to the noradrenergic neurons of the locus coeruleus [34]. Under normal conditions, the activity of serotonergic neurons is tightly controlled by a number of mechanisms, including: (i.) glutamatergic inputs from the forebrain (mainly the prefrontal cortex) [37], (ii.) the tonic noradrenergic input from the pontine nuclei [38], (iii.) inhibitory GABAergic signals from local interneurons [39], and (iv.) dopamine signals from the dopaminergic nuclei of the midbrain [40]. Moreover, the serotonin system is, in a way, self-regulating. The key control mechanism of 5-HT neurons is negative feedback through the 5-HT1A autoreceptors [20]. This mechanism is currently being studied in great detail in the context of the treatment of CNS diseases.

The aforementioned anatomical and electrophysiological picture shows that changes in the activity of serotonergic neurons affect a large population of target neurons in the forebrain. The complex nature of the serotonergic system and interactions with other neurochemical systems indicate that the development of MDD may be mediated by various pathomechanisms. Currently suggested mechanisms include: (i.) low neuronal production of serotonin or of postsynaptic receptors, (ii.) decreased excitatory inputs or excessive

system self-control, and (iii.) decreased 5-HT synthesis and/or tryptophan deficiency. The common denominator of these phenomena in depression is undoubtedly the disturbed transmission in the central 5-HT synapses. Therefore, the deliberate pharmacological modulation of serotonergic transmission in the brain seems to be one of the appropriate strategies for the search for new antidepressants.

#### **3. The 5-HT Receptors**

The serotonergic system affects various physiological functions, including psychoemotional expression, sensorimotor integration, and the regulation of the autonomic, cardiovascular, respiratory, and digestive systems. Within the CNS, 5-HT is involved in the regulation of higher mental functions and emotions, extrapyramidal motor functions, and cognitive functions (e.g., learning and memory).

At least 14 different serotonin receptors have been identified. These receptors can be divided into distinct families, which are labelled 1, 2, 3, 4, 5, 6, and 7, and the subtypes in each family are labelled with letters (e.g., a, b, c). Many of these receptors are thought to be involved in the pathogenesis of various CNS disorders [41].

### *3.1. The 5-HT1A Receptors*

The 5-HT1A receptors are located primarily in the following populations: (i.) presynaptic neurons of the raphe nuclei of the midbrain and (ii.) postsynaptic neurons, mainly in the hippocampus, septum, amygdala, and corticolimbic regions [42]. Autoreceptors are located within the bodies and dendrites of serotonin neurons. Their activation inhibits neuronal discharges and reduces the release of serotonin [43]. Thus, 5-HT1A autoreceptors play an important role in the self-regulation of the serotonergic system; they partially inhibit the activity of adenylate cyclase [44] and activate G protein-dependent rectifying potassium channels (GIRK) with the use of the βγ subunit of G protein [45]. This causes membrane hyperpolarization, a reduction in neuronal excitability, and the inhibition of potentialdependent calcium channels, reducing the influx of calcium ions. The consequence is a reduction in the neural discharge rate. Given the significant influence of these neuronal discharges on the overall activity of the entire serotonergic system, it can be concluded that the reduction in the firing rate evoked by serotonin and other 5-HT1A agonists immediately translates into an overall reduction in 5-HT release in most areas of the brain, particularly in regions innervated by the dorsal raphe [20].

The activation of 5-HT1A autoreceptors by endogenous serotonin, therefore, plays an essential role in the physiological control of the activity of the 5-HT ascending neurons. The 5-HT neurons during waking periods show a slow and regular rate of discharge [36]. Under conditions of excessive excitatory input (e.g., stress), there is an increased release of serotonin in the vicinity of neuronal bodies. It activates 5-HT1A autoreceptors, which allow low and regular neuronal activity to be maintained [40]. Thus, 5-HT1A autoreceptors act as negative feedback physiological "safety valves" to maintain homeostasis.

The expression of 5-HT1A heteroreceptors, in turn, takes place in populations of non-serotonin receptors, mainly in the limbic system within: (i.) bodies and dendrites of glutamatergic neurons [43] or (ii.) axons of GABA-ergic [46], and (iii.) cholinergic neurons [47]. These receptors are involved in regulating the release of various neurotransmitters: acetylcholine in the medial septum [48], glutamate in the prefrontal cortex [49], and dopamine in the ventral tegmental area [50]. In most regions of the brain, the inhibition of adenylate cyclase occurs due to the activation of the Gαi protein. The GIRK channels in the hippocampus are activated by the βγ subunits of the Gαo isoform [51]. The 5-HT1A receptors in the cortex and hypothalamus bind to both the Gαi and Gαo subunits, while their preferential binding to the Gαi3 protein occurs within the raphe nucleus.

The differences in the properties of 5-HT1A auto- and hetero-receptors are manifested in their different functional selectivity [52]: 5-HT1A heteroreceptors stimulate [53], while 5-HT1A autoreceptors inhibit ERK1/2 transmission [54]. The 5-HT1A-biased agonism appears to result in the preferential activation of a specific signaling pathway without

affecting or even blocking other pathways associated with this receptor subtype [55]. It has also been shown that there is an agonist-dependent modulation of G-protein coupling and a transduction of 5-HT1A receptors in rat dorsal raphe nucleus. Moreover, 8-hydroxy-2- (di-n-propylamino)tetralin (8-OH-DPAT, a full 5-HT1A receptor agonist) compared with buspirone (a partial 5-HT1A receptor agonist) fails to modify forskolin-stimulated cAMP accumulation [56]. ing or even blocking other pathways associated with this receptor subtype [55]. It has also been shown that there is an agonist-dependent modulation of G-protein coupling and a transduction of 5-HT1A receptors in rat dorsal raphe nucleus. Moreover, 8-hydroxy-2-(din-propylamino)tetralin (8-OH-DPAT, a full 5-HT1A receptor agonist) compared with buspirone (a partial 5-HT1A receptor agonist) fails to modify forskolin-stimulated cAMP accumulation [56]. In general, 5-HT1A receptor-deficient mice show a shorter immobility time in the

5-HT1A autoreceptors inhibit ERK1/2 transmission [54]. The 5-HT1A-biased agonism appears to result in the preferential activation of a specific signaling pathway without affect-

*Int. J. Mol. Sci.* **2021**, *22*, 9015 5 of 32

In general, 5-HT1A receptor-deficient mice show a shorter immobility time in the forced swim test than wild-type control animals [57]. The lack of functional 5-HT1A autoreceptors may, therefore, favor a less-depressed phenotype. The whole-life suppression of 5-HT1A heteroreceptor expression in adolescence results in a broad depression-like phenotype. In addition, the group showed physiological and cellular changes within medial prefrontal cortex–dorsal raphe proper circuitry: (i.) increased basal serotonin levels in the medial prefrontal cortex, which is hyporeactive to stress and (ii.) decreased basal serotonin levels and firing rates in a dorsal raphe hyperactivated by the same stressor [57]. forced swim test than wild-type control animals [57]. The lack of functional 5-HT1A autoreceptors may, therefore, favor a less-depressed phenotype. The whole-life suppression of 5-HT1A heteroreceptor expression in adolescence results in a broad depression-like phenotype. In addition, the group showed physiological and cellular changes within medial prefrontal cortex–dorsal raphe proper circuitry: (i.) increased basal serotonin levels in the medial prefrontal cortex, which is hyporeactive to stress and (ii.) decreased basal serotonin levels and firing rates in a dorsal raphe hyperactivated by the same stressor [57].

Animal studies show that both the stimulation and blockade of 5-HT1A receptors can cause or accelerate the antidepressant effect [17]. It is difficult not to associate this with the above-described functional differences of 5-HT1A auto- and hetero-receptors and the phenomenon of the biased 5-HT1A agonism. Many studies have demonstrated the antidepressant effect of 8-OH-DPAT reversed by 5-HT1A receptor antagonists [58]. Moreover, 5-HT1A receptor-deficient mice showed no increase in adult neurogenesis in the hippocampus after chronic treatment with fluoxetine (SSRI) and not with imipramine (TCA) [59]. The preferential activation of postsynaptic 5-HT1A receptors by F15599 (Figure 2), a biased 5-HT1A agonist, resulted in an antidepressant-like effect [60]. Similar activity was shown by F13714, a non-selective agonist of 5-HT1A receptors, but it induced a deeper "serotonin syndrome", hypothermia, and corticosterone release in rats. Elevated corticosterone levels accompany chronic stress in animals, leading to depression [61]. Moreover, the activation of 5-HT1A receptors in the prefrontal cortex (PFC) by F15599 produces strong antidepressant-like effects in the forced swim test (FST) in rats, with a distinctive bimodal dose–response pattern. These data suggest that F15599 may target specific 5-HT1A receptor subpopulations in the PFC, possibly located on the GABAergic and/or glutaminergic neurons [62]. Animal studies show that both the stimulation and blockade of 5-HT1A receptors can cause or accelerate the antidepressant effect [17]. It is difficult not to associate this with the above-described functional differences of 5-HT1A auto- and hetero-receptors and the phenomenon of the biased 5-HT1A agonism. Many studies have demonstrated the antidepressant effect of 8-OH-DPAT reversed by 5-HT1A receptor antagonists [58]. Moreover, 5- HT1A receptor-deficient mice showed no increase in adult neurogenesis in the hippocampus after chronic treatment with fluoxetine (SSRI) and not with imipramine (TCA) [59]. The preferential activation of postsynaptic 5-HT1A receptors by F15599 (Figure 2), a biased 5-HT1A agonist, resulted in an antidepressant-like effect [60]. Similar activity was shown by F13714, a non-selective agonist of 5-HT1A receptors, but it induced a deeper "serotonin syndrome", hypothermia, and corticosterone release in rats. Elevated corticosterone levels accompany chronic stress in animals, leading to depression [61]. Moreover, the activation of 5-HT1A receptors in the prefrontal cortex (PFC) by F15599 produces strong antidepressant-like effects in the forced swim test (FST) in rats, with a distinctive bimodal dose– response pattern. These data suggest that F15599 may target specific 5-HT1A receptor subpopulations in the PFC, possibly located on the GABAergic and/or glutaminergic neurons [62].

**Figure 2.** 5-HT1A receptor agonists: 8-OH-DPAT, F15599, and F13714. **Figure 2.** 5-HT1A receptor agonists: 8-OH-DPAT, F15599, and F13714.

The previously described physiological function of 5-HT1A autoreceptors and their regulation of depressive behavior seem to be unfavorable in the context of the mechanism of action of antidepressants [20,63]. The negative feedback pathway through 5-HT1A autoreceptors may decrease the efficacy of the SSRI as the dose increases, thus creating a second, anomalous part of the dose–response curve. This effect may also be responsible for the so-called therapeutic window for such antidepressants [64]. The prolonged use of SSRIs translates into significantly higher levels of extracellular 5-HT than after a single administration [65]. The negative feedback loop is believed to be the cause of the slow and delayed clinical efficacy of antidepressant drugs [66]. Administration of antidepressants The previously described physiological function of 5-HT1A autoreceptors and their regulation of depressive behavior seem to be unfavorable in the context of the mechanism of action of antidepressants [20,63]. The negative feedback pathway through 5-HT1A autoreceptors may decrease the efficacy of the SSRI as the dose increases, thus creating a second, anomalous part of the dose–response curve. This effect may also be responsible for the so-called therapeutic window for such antidepressants [64]. The prolonged use of SSRIs translates into significantly higher levels of extracellular 5-HT than after a single administration [65]. The negative feedback loop is believed to be the cause of the slow and delayed clinical efficacy of antidepressant drugs [66]. Administration of antidepressants (tricyclic drugs, monoamine oxidase inhibitors, and SSRIs) significantly increases the level of extracellular 5-HT in the midbrain raphe [67]. This leads to: (i.) the activation of 5-HT1A receptors, (ii.) the reduction in 5-HT cell firing [68], and (iii.) the terminal release of

5-HT [69]. The inhibition of SSRIs in the negative feedback pathway clearly decreases with the duration of treatment. This is most likely due to the serotonin-induced desensitization of raphe 5-HT1A autoreceptors discussed earlier [70]. Thus, the desensitization of 5-HT1A autoreceptors may accelerate the onset and/or enhance the antidepressant effect [71]. Mice with higher levels of 5-HT1A autoreceptors showed a blunted physiological response to acute stress, increased behavioral despair, and no behavioral response to fluoxetine [72]. Moreover, mice with lower autoreceptor levels showed a strong behavioral response to fluoxetine after both chronic and subchronic administration [72]. Thus, lowering the level of 5-HT1A autoreceptors prior to antidepressant treatment may accelerate and increase the effectiveness of antidepressant therapy. Combining SSRI treatment with the 5-HT1A receptor antagonist pindolol significantly reduces the latency of the antidepressant response and improves the clinical response in previously untreated MDD patients (Table 1) [20,21,73]. The above data indicate that the stimulation of postsynaptic 5-HT1A receptors or the blockade of presynaptic 5-HT1A receptors results in antidepressant-like activity. (-)-pindolol may also stimulate somatodendritic 5-HT1A receptors. Then, its accelerating antidepressant effect might stem from the accelerated adaptive changes like autoreceptor desensitization in response to both serotonin and pindolol. This mechanism can also be achieved by initiating the treatment with high-dose SSRI when a patient is suicidal. The antidepressant action of pindolol may also be related to its agonistic activity at the β1-adrenoreceptor as this drug possesses the strongest intrinsic sympathicomimetic activity among other β-blockers [74].


**Table 1.** Clinical effects of augmentation of SERT inhibition with different activities towards 5-HT receptors.

According to the neurotrophic hypothesis of depression, decreased neurotrophic support causes neuronal atrophy, which in turn reduces hippocampal neurogenesis and leads to depression. Clinical data support this theory: postmortem analysis has shown reduced volumes of the hippocampus and prefrontal cortex in depressed patients [78,79]. Persons diagnosed with MDD showed decreased levels of BDNF (brain-derived neurotrophic factor) and NGF (nerve growth factor) in the hippocampus. A deficit of these neurotrophins may promote neuronal loss [80,81]. This phenomenon was confirmed by in vivo studies [82–85], which showed that antidepressants reversed these changes [86]. Chronic treatment with 8-OH-DPAT, in turn reduced the feeding delay in the noveltysuppressed feeding test and increased adult hippocampal neurogenesis in wild-type mice, but showed no effect in the 5-HT1A receptor knockout group [59]. Thus, 5-HT1A receptors mediate the action of 8-OH-DPAT, from which it can be concluded that the postsynaptic 5-HT1A receptors mediate the antidepressant-like action of 8-OH-DPAT [87]. The specific deletion of the 5-HT1A heteroreceptors from mature granular cells in the dentate gyrus of

the hippocampus has also been found to abolish the effects of SSRIs in various behavioral tests [88]. It also attenuated the effects of SSRIs on adult neurogenesis and the expression of hippocampal neurotrophic factors: BDNF and VEGF (vascular endothelial growth factor). Whole-life 5-HT1A heteroreceptor-knockout (but not autoreceptor-knockout) mice showed decreased mobility in the forced swim test [89]. Such a depression-like phenotype was not observed when the suppression of heteroreceptors was initiated in adulthood. Therefore, serotonergic signaling in the forebrain during development may stably influence the circuits underlying the behavioral response to the FST [89].

The STAR\*D clinical trial shows that in patients unsuccessfully treated with SSRIs, the augmentation with buspirone resulted in symptom remission [75]. Buspirone (a partial agonist of the 5-HT1A receptor) enhances the desensitization of 5-HT1A autoreceptors, increasing the effectiveness of the SSRI treatment. Recently, a single transcription factor, Freud-1, has been found to be crucial for the expression of the 5-HT1A autoreceptor [90]. Mice with a conditional knockout of Freud-1 in serotonin neurons were shown to have elevated levels of 5-HT1A autoreceptors and exhibited the enhanced anxiety and depressive behavior in adulthood that was refractory to chronic SSRI treatment [90]. Interestingly, the double knockout of the Freud-1/5-HT1A gene did not produce such effects. In this case, the depressive-like behavior was even reduced [90]. The study suggests that targeting specific transcription factors may increase the response to antidepressant treatment. These reports indicated the need to search for compounds targeting only the population of 5-HT1A autoor heteroreceptors.

The results of postmortem and neuroimaging studies suggest an increased density of 5-HT1A autoreceptors in patients with MDD compared to the control group [91–93]. Genetic studies have shown that individuals with an increased density or activity of 5-HT1A autoreceptors are more prone to mood disorders and respond poorly to antidepressant treatment [94,95]. However, the number and density of postsynaptic 5-HT1A receptors have been shown to be unaltered or reduced in depressed patients, and this alteration is not sensitive to antidepressant treatment [96]. Long-term antidepressant therapy causes the tonic activation of 5-HT1A receptors in the dorsal hippocampus [97], and activation of 5- HT1A receptors in the dentate gyrus increases hippocampal neurogenesis [98]. In light of the cited reports, the use of 5-HT1A agonists as antidepressants seems natural [99]. Some agents possessing such activity (e.g., buspirone and gepirone) show antidepressant efficacy in placebo-controlled trials, but their potency is lower than that of SSRIs. Most 5-HT1A agonists (especially azapirones, Figure 3) show the preferential activation of presynaptic 5-HT1A receptors. Moreover, these agents tend to have a reduced efficacy at postsynaptic 5-HT1A receptors. Thus, endogenous serotonin competes in the postsynaptic sites with an exogenous substance (with lower agonism), which causes a paradoxical reduction in the tone at the postsynaptic 5-HT1A receptors. Higher doses of 5-HT1A agonists (such as those used in experimental animals) are likely to result in the greater activation of postsynaptic 5-HT1A receptors, which may explain the positive results of efficacy studies in animal models. Conversely, the administration of the selective 5-HT1A receptor antagonist DU125530 with fluoxetine did not accelerate or increase the efficacy of fluoxetine in a double-blind, randomized, placebo-controlled clinical trial. DU125530 had similar binding to pre- and post-synaptic 5-HT1A receptors [100], and the blockade of postsynaptic 5-HT1A receptors likely offset the benefits of enhancing presynaptic serotonergic function [101]. This may show the importance of the activation of postsynaptic 5-HT1A receptors in the mechanism of antidepressant action.

**Figure 3.** Azapirones: buspirone and gepirone. **Figure 3.** Azapirones: buspirone and gepirone.

Observations on the 5-HT1A receptor population contributed to a fruitful search for potential multimodal antidepressants that incorporate 5-HT1A receptor activity into their mechanism of action [102]. Recently developed compounds seem to overcome the aforementioned therapeutic problems of azapirones and other first-generation 5-HT1A agonists. Two new antidepressants, vilazodone [27,103] and vortioxetine [104,105], inhibit 5-HT reuptake and show the partial agonism at 5-HT1A receptors. Observations on the 5-HT1A receptor population contributed to a fruitful search for potential multimodal antidepressants that incorporate 5-HT1A receptor activity into their mechanism of action [102]. Recently developed compounds seem to overcome the aforementioned therapeutic problems of azapirones and other first-generation 5-HT1A agonists. Two new antidepressants, vilazodone [27,103] and vortioxetine [104,105], inhibit 5-HT reuptake and show the partial agonism at 5-HT1A receptors.

The 5-HT1A receptor ligands also possess their own potentially therapeutic activity. The 5-HT1A partial agonists show antianxiety [106,107], antidepressant [108], antiaggressive [109], anticraving [110], and anticataleptic properties [111]: The 5-HT1A receptor ligands also possess their own potentially therapeutic activity. The 5-HT1A partial agonists show antianxiety [106,107], antidepressant [108], antiaggressive [109], anticraving [110], and anticataleptic properties [111]:


#### 5-HT1A receptor activity into their mechanism of action. *3.2. The 5-HT1B Receptors*

cortex [112].

gression [120].

*3.2. The 5-HT1B Receptors*  The 5-HT1B receptors, like 5-HT1A receptors, are located pre- and post-synaptically and are also negatively coupled to adenylate cyclase. Their highest densities are in the striatum, pallidum, nucleus accumbens, substantia nigra, and ventral tegmental area. The 5-HT1B receptors, like 5-HT1A receptors, are located pre- and post-synaptically and are also negatively coupled to adenylate cyclase. Their highest densities are in the striatum, pallidum, nucleus accumbens, substantia nigra, and ventral tegmental area. Lower levels of 5-HT1B receptors are found in the hippocampus, amygdala, and cingulate cortex [112].

Lower levels of 5-HT1B receptors are found in the hippocampus, amygdala, and cingulate Unlike somatodendritic 5-HT1A autoreceptors, 5-HT1B autoreceptors are located on serotonergic axons, where they regulate the synthesis and release of 5-HT locally. The 5- HT1B postsynaptic receptors are located mainly in the centers of motor control (such as the basal ganglia), where they control the synaptic transmission of other neurotransmitters [112]. Studies have shown that 5-HT1B receptors play a role in depression, anxiety, mi-Unlike somatodendritic 5-HT1A autoreceptors, 5-HT1B autoreceptors are located on serotonergic axons, where they regulate the synthesis and release of 5-HT locally. The 5-HT1B postsynaptic receptors are located mainly in the centers of motor control (such as the basal ganglia), where they control the synaptic transmission of other neurotransmitters [112]. Studies have shown that 5-HT1B receptors play a role in depression, anxiety, migraines, locomotor activity, aggressive behavior, and the potentiation of the action of other drugs [112–114].

graines, locomotor activity, aggressive behavior, and the potentiation of the action of other drugs [112–114]. Animal studies show that the involvement of 5-HT1B receptors in the pathophysiology of depression is partly related to their responsiveness to environmental stress as well as their exposure to antidepressants [115]. The 5-HT1B heteroreceptors are involved in hippocampal neurogenesis, which may explain their importance for the antidepressant-like effect [116]. Mice lacking 5-HT1B autoreceptors showed an increased mobility in the FST as well as an increased preference for lower-sucrose concentrations in the sucrose preference test compared to the control group. After SSRI administration, elevated levels of serotonin in the hippocampus were observed [117]. Moreover, two common genetic poly-Animal studies show that the involvement of 5-HT1B receptors in the pathophysiology of depression is partly related to their responsiveness to environmental stress as well as their exposure to antidepressants [115]. The 5-HT1B heteroreceptors are involved in hippocampal neurogenesis, which may explain their importance for the antidepressant-like effect [116]. Mice lacking 5-HT1B autoreceptors showed an increased mobility in the FST as well as an increased preference for lower-sucrose concentrations in the sucrose preference test compared to the control group. After SSRI administration, elevated levels of serotonin in the hippocampus were observed [117]. Moreover, two common genetic polymorphisms of 5-HT1B receptors, G861C [118] and C129T [119], were associated with MDD and affective disorders. The 5-HT1B receptor gene knockout mice showed increased aggression [120].

morphisms of 5-HT1B receptors, G861C [118] and C129T [119], were associated with MDD and affective disorders. The 5-HT1B receptor gene knockout mice showed increased ag-The p11 protein, which colocalizes with 5-HT1B and 5-HT<sup>4</sup> receptors [121], plays a key role in modulating the function of the 5-HT1B receptor. Its dysregulation has been reported

in preclinical models of depression and in postmortem samples from MDD patients [122]. The p11 protein improves 5-HT1B receptor function in various regions of the brain and contributes to an antidepressant-like effect in animal behavioral tests [123]. P11 knockout mice showed depression-like behavior and demonstrated a reduced responsiveness to 5-HT1B receptor agonists and tricyclic antidepressants [123]. in preclinical models of depression and in postmortem samples from MDD patients [122]. The p11 protein improves 5-HT1B receptor function in various regions of the brain and contributes to an antidepressant-like effect in animal behavioral tests [123]. P11 knockout mice showed depression-like behavior and demonstrated a reduced responsiveness to 5- HT1B receptor agonists and tricyclic antidepressants [123].

The p11 protein, which colocalizes with 5-HT1B and 5-HT4 receptors [121], plays a key role in modulating the function of the 5-HT1B receptor. Its dysregulation has been reported

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Studies in the learned helplessness model showed that 5-HT1B receptors were upregulated in various regions of the brain following stress exposure. A reduced 5-HT1B autoreceptor function and, thus, increased serotonin release, has also been demonstrated after chronic antidepressant treatment [124]. Moreover, chronic treatment with SSRIs induced a negative regulation and/or desensitization of 5-HT1B autoreceptors [125] and facilitated the effect of SSRIs in serotonin neurotransmission [126]. Compounds exhibiting 5-HT1B antagonism, administered alone or with antidepressants, have been shown to be effective in preclinical models of depression [127]. The pretreatment with 5-HT1B receptor antagonists [128] or the genetic inactivation of the 5-HT1B receptor [129] increased the SSRI-induced effect in mice. Therefore, the blockade of 5-HT1B autoreceptors may promote the antidepressant effect. It has been suggested that the 5-HT1B receptor antagonists themselves may be attributed to an antidepressant-like effect. SB-616234-A, a 5-HT1B receptor antagonist, decreased immobility in a forced swim test in mice (Figure 4) [130]. The selective 5-HT1B receptor inverse agonist, SB236057A, increased, in turn, the extracellular concentration of serotonin in the dentate gyrus of a guinea pig. This effect was comparable to that of 14 days of paroxetine therapy [131]. The acute blockade of the 5-HT1B receptor might cause a rapid antidepressant effect [131]. It appears that the agonist activation of 5-HT1B heteroreceptors may also induce antidepressant-like effects [132]. CP94253, a selective 5-HT1B receptor agonist, showed an antidepressant-like activity in a forced swimming test in mice [133]. Anpirtoline, as a selective 5-HT1B receptor agonist, also reduced immobility in control mice but had no effect in 5-HT1B knockout mice [132]. The effect of this compound in the FST was, therefore, due to the activation of the 5-HT1B receptor. The above studies suggest that 5-HT1B receptors play a role in antidepressant-like activity. Ther stimulation of postsynaptic receptors and the inhibition of presynaptic 5-HT1B receptors may be beneficial in the treatment of depression [134]. Studies in the learned helplessness model showed that 5-HT1B receptors were upregulated in various regions of the brain following stress exposure. A reduced 5-HT1B autoreceptor function and, thus, increased serotonin release, has also been demonstrated after chronic antidepressant treatment [124]. Moreover, chronic treatment with SSRIs induced a negative regulation and/or desensitization of 5-HT1B autoreceptors [125] and facilitated the effect of SSRIs in serotonin neurotransmission [126]. Compounds exhibiting 5-HT1B antagonism, administered alone or with antidepressants, have been shown to be effective in preclinical models of depression [127]. The pretreatment with 5-HT1B receptor antagonists [128] or the genetic inactivation of the 5-HT1B receptor [129] increased the SSRI-induced effect in mice. Therefore, the blockade of 5-HT1B autoreceptors may promote the antidepressant effect. It has been suggested that the 5-HT1B receptor antagonists themselves may be attributed to an antidepressant-like effect. SB-616234-A, a 5-HT1B receptor antagonist, decreased immobility in a forced swim test in mice (Figure 4) [130]. The selective 5-HT1B receptor inverse agonist, SB236057A, increased, in turn, the extracellular concentration of serotonin in the dentate gyrus of a guinea pig. This effect was comparable to that of 14 days of paroxetine therapy [131]. The acute blockade of the 5-HT1B receptor might cause a rapid antidepressant effect [131]. It appears that the agonist activation of 5- HT1B heteroreceptors may also induce antidepressant-like effects [132]. CP94253, a selective 5-HT1B receptor agonist, showed an antidepressant-like activity in a forced swimming test in mice [133]. Anpirtoline, as a selective 5-HT1B receptor agonist, also reduced immobility in control mice but had no effect in 5-HT1B knockout mice [132]. The effect of this compound in the FST was, therefore, due to the activation of the 5-HT1B receptor. The above studies suggest that 5-HT1B receptors play a role in antidepressant-like activity. Ther stimulation of postsynaptic receptors and the inhibition of presynaptic 5-HT1B receptors may be beneficial in the treatment of depression [134].

**Figure 4.** 5-HT1B receptor ligands: anpirtoline, SB-616234-A, and SB236057A. **Figure 4.** 5-HT1B receptor ligands: anpirtoline, SB-616234-A, and SB236057A.

As with 5-HT1A receptors, acute SSRI therapy activates terminally localized 5-HT1B receptors, thus reducing 5-HT synthesis and release. The long-term administration of SSRIs desensitizes terminal 5-HT1B autoreceptors [135], suggesting that the plasticity of the autoregulatory function of both 5-HT1A and 5-HT1B receptors may be important with respect to the therapeutic profile of SSRIs. Again, as with 5-HT1A receptor antagonists, the administration of 5-HT1B receptor antagonists increases the neurochemical and behavioral effects of SSRIs [128,136]. Interestingly, the co-administration of the selective 5-HT1A antagonist WAY-100635 and the 5-HT1B receptor antagonist SB-224289 has an additive effect, enhancing the neurochemical effects of fluoxetine. This has led to the suggestion that the As with 5-HT1A receptors, acute SSRI therapy activates terminally localized 5-HT1B receptors, thus reducing 5-HT synthesis and release. The long-term administration of SSRIs desensitizes terminal 5-HT1B autoreceptors [135], suggesting that the plasticity of the autoregulatory function of both 5-HT1A and 5-HT1B receptors may be important with respect to the therapeutic profile of SSRIs. Again, as with 5-HT1A receptor antagonists, the administration of 5-HT1B receptor antagonists increases the neurochemical and behavioral effects of SSRIs [128,136]. Interestingly, the co-administration of the selective 5-HT1A antagonist WAY-100635 and the 5-HT1B receptor antagonist SB-224289 has an additive effect, enhancing the neurochemical effects of fluoxetine. This has led to the suggestion that the combination of the 5-HT1A and 5-HT1B receptor antagonism may increase CNS serotonin levels and, therefore, potentially be an effective treatment strategy for depression [20]:


### *3.3. The 5-HT1D, 5-HT1E, and 5-HT1F Receptors*

The clinical significance of the remaining 5-HT<sup>1</sup> receptors (5-HT1D, 5-HT1E, 5-HT1F) is less clear. There is limited preclinical evidence linking some of the receptors with depressive states. The sensitivity of postsynaptic 5-HT1D receptors in patients after treatment with SSRIs has been found to be impaired [137]. On the other hand, a postmortem study of untreated suicidal victims with a confirmed history of depression showed a much higher density of 5-HT1D receptors in the globus pallidus [138]. The observed high expression of the 5-HT1E receptor in the frontal cortex and hippocampus may indicate the relationship between 5-HT1E receptors and cognitive functions and memory [20,139].
