Keywords

Serotonin-2A Receptor: An Introduction

The 5-HT2A receptors belong to the 5-HT2 family consists of two more subtypes: 5-HT2B and 5-HT2C receptors. These subtypes have similar molecular structure, amino acid sequence, and signaling properties. The 5-HT2B receptors have a restricted expression in CNS; they play an important role during the embryonic development [1]. The 5-HT2A and 5-HT2C receptors are widely distributed across the CNS and have multiple functions. All members of the 5-HT2 receptor family primarily couple to PLC on activation. Like other G-protein coupled receptors (GPCRs) , 5-HT2 functional regulation also involves sensitization and desensitization-regulatory processes that help prevent overstimulation and allow recuperation of signaling competence, respectively [2].

Serotonin-2 receptor subtypes have been cloned from various species and tissues. The 5-HT2A receptor from hamster, human, monkey, mouse, pig, rat, and sheep all have the same length of 471 amino acid. The 5-HT2B receptor from human, mouse and rat have a length of 481, 504, and 479 amino acids and the 5-HT2C receptor from human, mouse and rat have a length of 458, 459, and 460 amino acids, respectively [3]. The 5-HT2A and 5-HT2C receptors are glycosylated on multiple sites. The genes for the 5-HT2A and 5-HT2B receptor have 3 introns; the 5-HT2C receptor gene has two introns. In humans, the genes are located on chromosome 13q14-q21 for the 5-HT2A receptor, chromosome position 2q36.3–2q37.1 for the 5-HT2B receptor, and chromosome X q24 for the 5-HT2C receptor [1].

It has been shown that some GPCRs , including the 5-HT2A receptor, exhibit critical differences in some aspects of functional regulation from those seen in conventionally studied model GPCRs such as the β2-adrenergic receptor . This receptor couples to a number of intracellular signaling cascades, making it an important receptor to study. Therefore, the 5-HT2A receptor could well serve as an important alternate paradigm in the study of GPCR function [2].

Though the receptor has been studied largely in relation to its multiple functions in the CNS, high levels of receptor expression in other areas such as the intestine, platelets, and endothelial cells suggest that it could play crucial roles in other aspects of physiology, as well. They mediate contractile responses in many vascular smooth muscle preparations (e.g. bronchial, uterine and urinary smooth muscle), and part of the contractile effects of 5-HT in the guinea pig ileum. In addition, platelet aggregation and increased capillary permeability following exposure to 5-HT have been attributed to 5-HT2A receptor-mediated process. Moreover, 5-HT2 receptor agonists, in addition to precursors of 5-HT and 5-HT releasing agents, mediate certain behavioral syndromes in vivo (e.g. head twitching in mice, and wet-dog shakes and back muscle contractions in rats) [4]. Centrally, these receptors are principally located in the cortex, claustrum and basal ganglia. 5-HT2A receptor activation stimulates hormone secretion (e.g. ACTH, corticosterone, oxytocin, renin and prolactin) [5]. Considering the broad expression of 5-HT2A receptors across the brain and their involvement in multiple CNS functions, it is expected that these receptors will play a role pathophysiology of brain disorders . Indeed, the CNS disorders in which the 5-HT2A receptor seems to be involved range from schizophrenia, depression, obsessive compulsive disorder (OCD), and attention deficit–hyperactivity disorder (ADHD), to eating disorders such as anorexia nervosa, to autism spectrum disorders [2]. Implication of 5-HT2A receptors in mental disorders with complex etiologies is still not clearly understood. There are a large number of drugs targeted to this receptor.

Molecular Genetics and Epigenetics of Serotonin-2A Receptor

Serotonin-2A Gene Polymorphism

The 5-HT2A receptor, encoded by HTR2AR gene, is a widely-distributed post-synaptic target for 5-HT in the human brain. Serotonin-2A receptor heterogenity is affected by alternative polymorphisms and alternative splicing. The 5-HT2A receptor is a target for atypical antipsychotics and antidepressants. The role of genetic variants of HTR2AR in signaling modulation remains unclear, despite positive clinical associations [6]. Methods for detecting genetic polymorphisms are advancing rapidly and now allow simultaneous genotyping of several nucleotide polymorphisms. The Genetic Association Database [7] reports 346 unique association studies between single nucleotide polymorphisms (SNPs) in HTR2AR gene and human phenotypes and more than half of these studies find positive genotype-phenotype associations. Most are related to cognition or risk for neuropsychiatric disorders , supporting the presence of functional genetic variants in HTR2AR gene. Some of SNPs (e.g., T102C, C516T, A1438G) are silent mutations and do not cause a change in the protein. Other SNPs (e.g., W25S, I197V, S421F, A447V, H452Y) result in a change in an amino acid. Although the A1438G mutation is silent and does not result in alteration of the amino acid sequence of 5-HT2A receptor, it is located within promoter region of the gene. Thus was proposed that this mutation alters promoter activity and even so expression of 5-HT2A receptors [8]. Lower 5-HT2A receptor densities in some brain areas may cause another silent mutation, T102C [9]. On the other hand, mutation H452Y which caused change in protein has no effect on receptor expression, but reduces intracellular signaling capacity [10].

Numbers of studies have been conducted on the association between HTR2AR gene T102C polymorphism and major depressive disorder (MDD) [11,12,13]. To clarify the effects of HTR2AR gene T102C polymorphism on the risk of depression, Lin et al. [11] performed a meta-analysis in the Chinese population. Results have shown that HTR2AR gene T102C polymorphism is not associated with susceptibility to MDD in these population. Another study [14] demonstrated an association between T102C polymorphism of HTR2AR gene, lifespan, and the risk of age-related CNS disorders. Their results suggest that T102C is associated with mean life span, and thus this gene becomes a possible candidate for the group of adaptive genes to meat consumption proposed in the literature.

The 5HT2A receptor gene polymorphisms rs7997012 and rs6311 has been suggested to be involved in major depressive disorder. Htr2a knock-out mice (Htr2a−/−) displayed an increase in depressive-like behavior, compared to wild type, thus suggesting , that lowered 5-HT2A receptor transmission may favor the susceptibility and severity of major depressive episodes [15].

It is seems that genetic variants in the HTR2A gene affect the therapeutic effects of andtidepressant drugs but mechanism underlying the regulation of such response remains poorly described. According to study of Qesseveur et al. [16] the HTR2A gene may represent a relevant marker to predict the efficacy of antidepressant drugs . The effect of three HTR2A single nucleotide polymorphisms (SNPs- rs6313, rs6314 and rs7333412) was investigated. These three SNPs have potential functional consequences on 5-HT2A receptor, on response and remission rates after 3 months of antidepressant treatments. Their clinical data indicated that GG patients for the rs7333412 SNP were less prone to respond to antidepressant drugs than AA/AG patients.

T102C and A1438G polymorphisms were associated with risk for schizophrenia [17,18,19]. The T102C polymorphism is also related to tobacco use [20] and the A1438G polymorphism of HTR2AR gene is involved in the development of alcohol dependence [21]. Polymorphisms of the HTR2AR gene are associated with hallucinatory symptoms and delusions in demented and non-demented cohorts. The study of Craig et al. [22] examined the role of the HTR2AR gene T102C polymorphism in influencing psychotic symptoms in a large Northern Ireland Alzheimer’s disease (AD) population. No significant association was found either in frequency of genotype or allelic variation for either set of symptoms. On the other hand, Lam et al. [23] demonstrated significant association between neuropsychiatric symptoms in AD and HTR2AR gene polymorphisms.

Methylation

Differential DNA methylation has been suggested to contribute to differential activity of alleles C and T and thereby to genetic associations between the C/T(102) polymorphism in the HTR2AR gene and psychiatric disorders [24]. This study demonstrated methylation in two CpG sites, which are specific to allele C. The majority of allele C-specific CpG sites were methylated in human temporal cortex and peripheral leukocytes. Findings that methylation of allele C-specific CpG sites in the first exon correlated significantly with the expression of DNA methylase 1 but not S-adenosylhomocysteine hydrolase, support the hypothesis that allele-specific DNA methylation is involved in regulation of HTR2AR gene expression , influencing expression differences between alleles C and T.

De Luca et al. [25] developed an improved quantitative assay for the measurement of allele-specific methylation of the HTR2AR gene and genetic association between the HTR2AR gene T102C silent polymorphism and suicidality in patients with mood disorders and schizophrenia.

Falkenberg et al. [26] used functional and structural equation modeling (SEM) approaches to assess the contributions of the polymorphism (R6311S) to DNA methylation and HTR2AR gene expression in chronic fatigue syndrome (CFS) subjects from a population-based study. Their study suggests that the promoter polymorphism (rs6311) can affect both transcription factor binding and promoter methylation, and this along with an individual’s stress response can impact the rate of HTR2A transcription in a genotype and methylation-dependent manner.

Alternative Splicing

The first alternatively spliced isoform of 5-HT2A receptor was identified by Huang et al. [27] in the parasitic nematode species, Ascaris Suum. The 5-HT2A-s1 and 5-HT2A-s2 exhibited identical pharmacological profiles when stably expressed in human embryonic kidney (HEK) 293 cells . Both 5-HT2As isoforms had higher affinity for 5-HT than their closely related Caenorhabditis Elegans homolog (5-HT2C-e).

Table 1 5-HT2A ligands and their selectivity towards the 5-HT receptor family
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Guest et al. [28] identified an alternatively spliced HTR2AR gene transcript by PCR of human brain cDNA using degenerate oligonucleotide primers to transmembrane domains. PCR analysis showed that truncated (5HT2ARtr) and native HTR2AR genes were co-expressed in most brain tissues, with the highest levels being found in hippocampus, corpus callosum, amygdala, and caudate nucleus. Western blot analysis of HEK-293 cells transfected transiently with a 5HT2ARtr construct showed that a 30-kDa protein was expressed in cell membranes. Co-transfection studies showed no effect of the 5HT2ARtr variant on 3H-ketanserin binding to the native HTR2AR or on functional coupling of the HTR2AR to 5-HT-stimulated calcium influx.

Molecular Pharmacology of and Serotonin-2A Receptors

Signal Transduction Pathways of Serotonin-2A Receptor

The activation of 5-HT2A receptor leads to the dissociation of GαQ/Z protein into α and βγ subunits. The α subunit of GαQ/Z protein activates the phospholipase C (PLC) , which in turn catalyzes the dissociation of inositol 1,4,5-trisphosphate (IP3)-di-acylglycerol (DAG) complex into the IP3 and DAG. The DAG activates protein kinase C (PKC), and IP3 stimulates calcium (Ca2+) release from endoplasmic reticulum (ER) into the cytoplasm, a characteristic activation signature of many GPCRs [29, 30]. This cascade has been the most extensively studied and is perhaps the most important signal transduction pathway regulated by this receptor (Fig. 1).

Fig. 1
figure 1

Detailed signal transduction pathways of serotonin-2A receptors. Serotonin-2A (5-HT2A) receptor activates protein kinase Cβ (PLCβ). Protein kinase Cβ hydrolysis phosphatidylinositol 4,5 bisphosphate (PIP2) to diacylglycerol (DAG) which activates protein kinesis A (PKA) and inositol trisphosphate (IP3) which acts through inositol trisphosphate receptors (IP3R) localize on endoplasmic reticulum. Activation of this signaling pathway leads to increase in intracellular calcium concentration which affects ion channels, enzyme activity, and neurotransmission or gene expression. Intracellular calcium can also lead to activation of calmodulin which activates extracellular signal-regulated kinases (ERK) and activation of calcineurin leading to inhibition of voltage-dependent calcium channels. Activation of ERK signaling pathway suppresses 5-HT2A receptor signaling through RSK2 kinase. Extracellular signal-regulated kinases can be activated by TGFβ receptor signaling pathway involving Ras GTP-ases interacting with Raf kinases and mitogen-activated protein kinase kinases (MEK) which phosphorylates mitogen-activated protein kinase (MAPK)

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Stimulation of the 5-HT2A receptor leads to the activation of at least three distinct signal transduction pathways: IP3/DAG-, arachidonic acid (AA)-, and 2-arachidonylglycerol (2-AG)-mediated. In addition to PLC, 5-HT2A receptors were also reported to activate phospholipase A2 (PLA2), so-called phospholipase B (PLB) [31].

Besides phospholipases-mediated calcium signaling , 5-HT2A receptor activation also induces extracellular signal-regulated kinase (ERK) phosphorylation via diverse intracellular signaling mechanisms [32]. Src and calmodulin (CaM) promote 5-HT2A receptor-mediated phosphorylation of ERK. In the PC12 cells, ERK phosphorylation by 5-HT2A receptor may not depend on PLC/PKC signaling, and instead requires an increase in intracellular calcium , and the activation of CaM and Src [33]. The ERK target p90 ribosomal S6 kinase 2 (RSK2) directly acts on the third intracellular (i3) loop of 5-HT2A receptor protein [34], leading to direct phosphorylation of the i3 loop at the conserved residue Ser-314 and to suppression of 5-HT2A receptor signaling.

In addition, RSK2 is required for tyrosine kinases, such as the epidermal growth factor receptor and the platelet-derived growth factor receptor, both of which have been demonstrated to attenuate 5-HT2A receptor functioning in primary cortical neurons [35, 36].

The 5-HT2A receptors, like other members of 5-HT2 family , couple preferentially via GαQ/Z-11 to the IP3/PKC/Ca2+ pathway, although inhibition of cyclic adenosine monophosphate (cAMP) production has been reported [37].

The 5-HT2A receptor also regulates the tyrosine kinase pathway activity [33]. Activation of neuronal 5-HT2A receptor activates transglutaminase which leads to transamidation of Rac1, a small G protein, resulting in constitutive activation of Rac1 [38]. Chronic treatment with olanzapine, an atypical antipsychotic drug, causes the desensitization of 5-HT2A receptor signaling. In rat frontal cortex, stimulation of the JAK-STAT pathway desensitizes the 5-HT2A receptor-mediated PLC activation induced by olanzapine [39]. Furthermore, constitutive activation of 5-HT2A receptor induces GαQ/Z-11 phosphorylation and desensitization (uncoupling) [40].

Functional Selectivity and Internalization of Serotonin-2A Receptors

Interestingly, different agonists of 5-HT2A receptors vary in the efficacy with which they stimulate individual signal transduction pathways [2, 41]. This phenomena is called functional selectivity and the 5-HT2A receptor was one of the first receptors for which this was described [29, 42]. This discovery was based of the observation that hallucinogenic effects of drugs such as LSD do not correlate with their activation of the IP3/DAG pathway [2].

It has been suggested that hallucinogen, but not nonhallucinogen, 5-HT2A receptor agonist induce phosphorylation of the 5-HT2A receptor at S280 located in the third intracellular loop. Importantly, these authors also demonstrated that pretreating cells with pertussis toxin (PTX) decreased PLC activation induced by the hallucinogens 2,5-Dimethoxy-4-iodoamphetamine (DOI) and LSD, whereas PTX treatment did not affect lisuride and ergotamine responses [43]. Jones et al. [44] discovered, that application of the 5-HT2A receptor agonist DOI to cultured cortical neurons induced phosphorylation of p21-activated kinase (PAK) via Rac guanine nucleotide exchange factor (RacGEF) kalirin-7 [44]. Taken together, these observations suggest that hallucinogens selectively activate GαI/O-dependent signaling , whereas non-hallucinogen 5-HT2A receptor agonists do not [45].

Both in vitro and studies in vivo have shown receptor redistribution in response to exposure to antagonists. The 5-HT2A receptor is internalized in response to both agonists and antagonists, adding a very interesting twist to its signaling properties [46, 47]. This feature of the 5-HT2A receptor may play important roles in its signaling and in the actions of antipsychotic medications. The antagonist-mediated internalization of the rat 5-HT2A receptor, unlike 5-HT-mediated internalization , is independent of protein kinase C (PKC) activation [47]. Bhatnagar and colleagues [46] examined the internalization process of this receptor in detail, demonstrating that both agonist- and antagonist-induced internalization of the 5-HT2A receptor were dynamin-dependent and via clathrin-mediated endocytosis. Activation of the 5-HT2A receptor by agonists, but not antagonists, induced greater translocation of arrestin-3 than arrestin-2 to the plasma membrane, and resulted in differential sorting of arrestin-2, arrestin-3, and 5-HT2A receptors into distinct plasma membrane and intracellular compartments. It is likely that these differences in distribution of the various signaling components induced by agonists and antagonists may be important in the “ligand-directed” of second messenger signals by the 5-HT2A receptor, depending upon which ligand is used to stimulate the receptor. Authors discovered, that in vitro knockdown of Caveolin-1 (Cav-1, a scaffolding protein) nearly abolished 5-HT2A receptor-mediated signal transduction as measured by calcium flux assays. Cav-1 appeared to modulate 5-HT2A receptor signaling by facilitating the interaction of 5-HT2A receptors with Gαq.

Serotonin-2A-Acting Drugs

Several drugs that have been developed for treatment of psychiatric disorders selectively bind to the 5-HT2A receptor and modulate its signaling pathways (Table 1). The antipsychotic drugs spiperone and methiothepin with antipsychotic properties are nonselective antagonists of 5-HT1 and 5-HT2 receptors. Both prevent the 5-HT-dependent PLC activation at 10 μM concentration. However, cyproheptadine (10 μM), another antagonist of 5-HT1 and 5-HT2 receptors, had no effect on PLC activity [48].

Brexpriprazole is an antagonist of 5-HT2A, 5-HT1A and D2 receptors, is approved for the clinical use as a main pharmacotherapy in schizophrenia and as an adjunct in antidepressant-resistant depression. This drug demonstrated robust antipsychotic, antidepressant-like and anxiolytic activities, and limited extrapyramidal symptom liability with pro-cognitive efficacy in animal models [49]. Accumulating evidence suggests that antipsychotic drugs act by promoting neurite outgrowth. In the study of Ishima and colleagues [50] authors examined whether brexpiprazole can affect neurite outgrowth in cell culture. They found that brexpiprazole significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells, in a concentration dependent manner. Moreover, inhibitors of inositol IP3 receptors , xestospongin C and 2-aminoethoxydiphenyl borate (2-APB), significantly blocked the effects of brexpiprazole. These findings suggest that brexpiprazole-induced neurite outgrowth is mediated through 5-HT1A and 5-HT2A receptors , and subsequent Ca2+ signaling via IP3 receptors [50].

Role Serotonin-2A Receptors in the Regulation of CNS Circuits

Role of Serotonin-2A Receptors in the Interactions Between Serotonin and Glutamate and GABA Systems

DOI (1-[2,5-dimethoxy-4-iodophenyl-2-aminopropane]) is a hallucinogen acting as agonist of 5-HT2A receptors, similarly to lysergic acid diethylamide (LSD) . It was reported that DOI causes a dose-related inhibition of 5-HT neuronal activity, with the highest dose reducing firing rates by >80%. Pretreatment with the 5-HT2 receptor antagonist ritanserin completely blocked the action of DOI [51]. Study of Quesseveur et al. [52] confirms this inhibitory effect of DOI on dorsal raphe (DR) nucleus 5-HT neuronal activity. DOI’s response is dependent on 5-HT2A receptors because it diminished in 5-HT2A receptors lacking mice. Possible way of DOI inhibitory effect on DR 5-HT neuronal activity is via increasing of GABA release in DR. Other study shows that activation of 5-HT2A receptors in the PFC by DOI increased the firing activity of DR 5-HT neurons. DOI administration also affected the firing rate of pyramidal neurons while most of them were excited, 11% were inhibited and rest was unaffected [53] In this case, excitatory and inhibitory actions of DOI on pyramidal cell firing are likely mediated by receptors located on pyramidal neurons and GABA interneurons, respectively. DOI also stimulates 5-HT release in the PFC, probably via a mechanism involving interaction between 5-HT2A and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors [54] (Fig. 2).

Fig. 2
figure 2

Interactions between 5-HT2A receptors and the other system. (a) Excitatory pyramidal neurons in the medial prefrontal cortex (mPFC) control activity of 5-HT neurons in dorsal raphe (DR) Fig. 2 (continued) through 3 different mechanisms: N-methyl-d-aspartate (NMDA) and 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) receptors- mediated excitation; GABAA receptors- mediated inhibition; and 5-HT1A autoreceptors- mediated inhibition. (b) Regulation of the dopaminergic system through 5-HT2A receptors. In the ventral tegmental area (VTA) or in medial prefrontal cortex (mPFC), 5-HT2A receptors have also been identified in GABAergic interneurons. Their activation leads to the inhibition of dopaminergic activity. 5-HT2A receptors might also be expressed in dopaminergic neurons in VTA region and their activation would stimulate dopaminergic activity. (c) Locus coeruleus (LC) receives dense 5-HT projections coming from dorsal raphe (DR), which have an inhibitory effect on noradrenergic neurons. Increased 5-HT levels act also on excitatory 5-HT2A receptors on GABAergic neurons which lead to an inhibition of norepinephrine release

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The PFC seems to play crucial role in depression. PFC is involved in higher brain functions and carries a control of brain functions through the processing and integration of signals from other brain areas, such as neocortex, several thalamic nuclei, and the brain stem. The apical and basal dendrites of pyramidal neurons of the PFC are highly enriched with 5-HT2A receptors. These receptors are present also on large and medium-sized GABAergic interneurons that control the activity of local microcircuits [55]. The mPFC in rodents innervates via long glutamatergic axons various brain areas involved in depression, such as nucleus accumbens (NAcc), amygdala, and PFC [56]. As well, activity of dopaminergic neurons in ventral tegmental area (VTA) is under the excitatory control of 5-HT2A receptors in mPFC. Neurons in mPFC excited through 5-HT2A receptors increase the firing rate and burst firing of dopaminergic neuron and dopamine release in VTA [57].

The 5-HT2A receptor activation located on thalamocortical afferents could increase glutamate release and increase spontaneous excitatory postsynaptic currents (EPSCs) through the activation of pyramidal AMPA receptors, however, this suggestion is based by the recent anatomical data indicating that the terminal 5-HT2A receptors are not located on glutamate axons [58].

Role of Serotonin-2A Receptors in the Interactions Between Serotonin and Dopamine Systems

The 5-HT2A receptor stimulation results in enhanced dopamine (DA) release in rat PFC, presumably via facilitation of 5-HT1A receptor stimulation. Ability of clozapine to increase DA release may be boosted by antagonism of 5-HT2A receptors [59].

The local infusion of DOI into the PFC dampened potassium (K+)-mediated DA release in a dose-dependent manner. Regular intracortical administration of MDL 100907 caused an increase in cortical DA efflux, suggesting that cortical 5-HT2A receptors potentiate the phasic release of DA [60]. The stimulatory effect of 5-HT on efflux of dopamine in the striatum is effective only when nigro-striatal DA transmission is elevated above basal levels [61]. Antagonism of 5-HT2A receptors may modulate the activity of dopamine neurons in different areas. For the nigro-striatal dopaminergic pathway was suggested a model in which blockade of these receptors led to increased output of dopaminergic neurons into the striatum [62].

Brexpiprazole has higher affinity to D2 than to the 5-HT2A receptors . While other antipsychotic drugs act as D2 antagonists, brexpiprazole is a partial agonist of the D2 receptors [63, 64]. The D2 receptor agonistic features could alter DA neurotransmission by stimulating D2 receptors when the levels of DA are lowered, while decreasing their activation when DA levels are increased [65].

Increase in 5-HT levels inhibits dopaminergic neurons as the lesion of 5-HT neurons results in an increase of dopaminergic neuronal activity in the VTA [66]. Thus, an increase in the availability of 5-HT cause by SSRIs might result in attenuation of the firing of dopaminergic neurons. Neuronal activity of dopaminergic neurons has a critical role in the VTA in motivation, hedonia and reward, so the inhibition of this firing might contribute to SSRI resistance in some patients [67].

Role of Serotonin 2A Receptors in the Interactions Between Serotonin and Norepinephrine Systems

The 5-HT2A receptor is likely to play an important role in the interaction between norepinephrine (NE) and serotonin (5-HT) systems [68]. Increased 5-HT levels act on excitatory 5-HT2A receptors on GABA neurons, thus leading to an inhibition of NE release [69].

Acute brexpiprazole administration reduced inhibition of two important interaction nodes between the 5-HT and NE systems. The blockade of 5-HT2A receptors revokes the tonic inhibition of NE neuronal firing activity, and the blocking of α2-adrenergic receptors on the nerve terminals of NE neurons stimulates NE release [70].

YM992 [(S)-2-[[(7-fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride] is a selective serotonin reuptake inhibitor (SSRI) and a potent 5-HT2A receptor antagonist. Acute injection of YM992 significantly decreased NE neuron firing activity and blocked the inhibitory effect of a subsequent injection of the 5-HT2 receptor agonist DOI. After 2-day treatment the firing activity was elevated even more significantly, however after 7-day and 21-day treatment a partial recovery was observed. This NE activity may be a result of 5-HT reuptake inhibition plus 5-HT2A receptor antagonism [69].

The activation of 5-HT2A and 5-HT1A receptors suppresses the firing of 5-HT and noradrenergic neurons of the locus coeruleus (LC). Serotoninergic neurons recover their firing rate with prolonged treatment, because of the desensitization of 5-HT1A autoreceptors , but the firing rate of noradrenergic neurons does not recover over time [68].

Role of Serotonin-HT2A in the Response to Antidepressant and Mood Stabilizing Drugs

Selective serotonin reuptake inhibitors (SSRIs) induce inhibition of NE neuron firing [71]. It was reported in several open-label and blind studies that antagonists of 5-HT2A receptors, such as atypical antipsychotic drugs, potentiate the therapeutic effect of SSRIs in patients with depression [72]. It is also reported that antidepressants induce down-regulation of 5-HT2A receptors after repeated treatment [55].

Risperidone is 5-HT2A and dopamine D2 receptor antagonist which is the only antagonist known to saturate the 5-HT2A receptors even at low doses (0.5–1 mg/day) [73]. It was reported that risperidone reverses SSRI-induced inhibition of NE neurons due to its 5-HT2A receptor antagonistic property [71]. Co-administration of risperidone with venlafaxine or fluoxetine may enhance their antidepressant effects. Addition of yohimibine to the combination of risperidone with venlafaxine or fluoxetine augmented the antidepressant-like action proposing an interaction of α2-adrenergic and 5-HT2A receptor in mediating their action [74]. Palperidone is the main metabolite of risperidone. Although they share the same receptor binding profile, it seems that they have different effects on 5-HT and NE firing in vivo. Co-administration of paliperidone did not interfere with the effect of SSRIs, but still managed to inhibit the NE firing inhibition induced by the SSRIs which leads to assumption that it may be an effective enhancement of the treatment [75].

Amibegron (SR58611A)—selective β3 adrenergic agonist [76] interacts with serotonergic system in the brain resulting in an antidepressant effect [77]. It increases the synthesis of 5-HT and tryptophan levels in several brain areas, such as hippocampus, cortex, hypothalamus and striatum. Amibegron did not modify noradrenaline synthesis and metabolism, but it did increase its release [78]. A 5-HT2A receptor antagonist ketanserin significantly reversed the effect of amibegron which leads to conclusion that these antidepressant-like effects are partially caused by the 5-HT2A receptor activation, more precisely by interaction with 5-HT1A, 5-HT2A/2C and 5-HT3 serotonin receptors [79, 80].

Function of cortical 5-HT2A receptors has a specific role in the modulation of conflict anxiety. Weisstaub et al. [81] demonstrated that global disruption of 5-HT2A receptor signaling in mice reduced inhibition in conflict anxiety paradigms without affecting fear-conditioned and depression-related behaviors. Selective restoration of 5HT2A receptor signaling to the cortex normalized conflict anxiety behaviors.

The serotonergic system appears to play a role in episodic memory which is affected in pathologies such as schizophrenia, Alzheimer and depression. The 5-HT2A receptors as one of the principal post-synaptic receptors for 5-HT in the brain are involved in neuropsychiatric and neurological disorders associated with memory deficits. Results of Morici et al. [82] showed that the 5-HT2A and also 5-HT1A receptors can be a novel target for drug development to improve episodic memory retrieval in psychiatric and neurological disorders.

Serotonin-2A Receptors in Pathophysiology and Treatment of Depression

Expression and Function of Serotonin-2A Receptors in the Hippocampus

Hippocampus is a brain structure which plays role in a spatial learning and declarative memory. It receives robust serotonergic innervation from medial and dorsal raphe nuclei. There is some evidence indicating role of 5-HT and its receptors in various aspects of cognitive functions including learning and memory. Nowadays, exact role of 5-HT in hippocampus is not fully understood. Results of functional studies are contradictory. One of possible explanation for these contradictory results is that 5-HT acts through different types of 5-HT receptors. The 5-HT2A receptor subtype is related to memory disorders [83] and several neurological diseases like Alzheimer disease [84, 85] and schizophrenia [86,87,88].

The presence of 5-HT2A receptors in hippocampus was demonstrated in different studies by multiple methods including immunohistochemistry, in situ hybridization, autoradiography and quantitative reverse transcription-polymerase chain reaction (RT-PCR) . Results from these studies are quite different and depending on methodology which was used. Minimal levels of 5-HT2A receptors were detected in human hippocampus by RT-PCR and autoradiography. They were barely detected in pyramidal cells in Cornu ammonis (CA) regions, and were not detected in dentate gyrus (DG) [89]. In rat hippocampus mRNA for 5-HT2A receptors was detected in both CA regions and in DG [90]. In CA area of rat hippocampus low levels of 5-HT2A receptors were detected by in situ hybridization and autoradiography methods. In ventral DG moderate levels of specific 5-HT2A receptors binding were detected [91]. Immunohistochemistry studies showed that 5-HT2A receptors expressed both excitatory glutamatergic and inhibitory GABAergic neurons [92,93,94,95]. Virtually all main hippocampal excitatory neurons (granular and pyramidal cells) expressed 5-HT2A receptors . Strong expression is localized in apical dendrites of pyramidal cells, where 5-HT receptors can increase excitatory postsynaptic currents (EPSP) [92, 94]. Electrophysiological studies demonstrated that outward current induced by 5-HT and α-methyl-serotonin (5-HT2A receptors agonist) in pyramidal cells of rat CA1 hippocampal area is blocked by ketanserin and spiperon (5-HT2A receptors antagonist) in dose dependent manner [96]. The 5-HT2A receptors are also expressed in mossy fiber in rat hippocampus [92]. Receptors localized on presynaptic side of mossy fibers could regulate excitatory neurotransmission and as result affect release of glutamate in hippocampus [97, 98]. On the other hand, colocalization analyses show that 5-HT2A receptors are expressed in GABAergic neurons located in different rat hippocampal regions . This colocalization is similar in different hippocampal areas: in DG, CA1, CA2 and CA3 field. In hippocampal CA areas are 5-HT2A receptors widespread in number of GABAergic interneurons distributed in pyramidal cell layer, in strata oriens, radiatum and lacunosum-moleculare.

The 5-HT2A receptors are expressed on 90% of GABAergic neurons in hippocampus [92]. Electrophysiology studies showed that activation of 5-HT2A receptors activate GABAergic neurons in rat DG [99] and in CA1 field [100]. High density of 5-HT2A receptor in deeper layers of granular cell layer corresponds with study demonstrating that 5-HT receptors can regulate neurogenesis in subgranular zone of DG [101]. Because GABA regulates progenitor turnover and integration of newly synthetized neurons in DG [102], it can be assumed that GABA neurons distributed in subgranular zone can be involved in hippocampal progenitor proliferation mediated by 5-HT2A receptors [103].

Function of 5-HT2A Receptors in Hippocampus in Health

Recent studies suggested that 5-HT2A receptors are included in several hippocampal functions although underlying mechanisms are still unclear. Activity of hippocampal pyramidal neurons can be modulated by 5-HT2A receptors in different ways: directly, by activation of 5-HT2A receptors in pyramidal cells, or indirectly, by activation of 5-HT2A receptors in GABA interneurons [96]. Serotonin 5-HT2A receptors can participate in information processing in hippocampus by participating in neurotransmission in different neuronal populations. Strong and widespread expression of 5-HT2A receptors in hippocampus is prerequisite for critical involvement of 5-HT receptors in number of brain functions including learning and memory [92]. It was shown that an application of M100907 (highly selective 5-HT2A receptors antagonist) to brain slices facilitates induction of long term potentiation (LTP) in CA1 field of rat hippocampus [104].

As a critical factor modulating brain plasticity is considered brain-derived neurotrophic factor (BDNF) . Hippocampal BDNF mRNA expression was induced by physical activity which positively regulated neurogenesis and induced LTP [105]. This factor can acutely influence synaptic efficiency of neurons. Some electrophysiological studies demonstrate that application of BDNF on hippocampal slices results in increase of synaptic strength [106,107,108,109,110]. In hippocampus 5-HT2A receptors participate in regulation of BDNF levels as their agonist DOI decreased the expression of BDNF mRNA in granular cell layer in DG, but not in CA regions. Effect of agonist was blocked by pretreatment with selective antagonist of 5-HT2A receptors. Same decrease of BDNF expression in hippocampus is observed during stress and it is possible that this effect is mediated by 5-HT2A receptors. This hypothesis is supported by an observation that pretreatment with ketaserin significantly blocked stress induced decrease in BDNF expression [111].

Involvement of 5-HT2A receptors in process of learning and memory is supported by study where systematic activation of 5-HT2A receptors with agonist (TCB-2) enhanced the consolidation of both fear memory and object memory [112]. The memory strengthening effect of TCB-2 was blocked by pretreatment with 5-HT2A receptors antagonist (MDL11,939). Local microinfusion of TCB-2 into CA1 field of dorsal hippocampus had similar effect on memory consolidation observed after systemic treatment [113]. Postsynaptic 5-HT2A receptors can modulate memory storage associated with object also by influencing on N-Methyl-d-Aspartate (NMDA) receptors . It is supported by fact that hippocampal 5-HT2A receptors are predominantly expressed in dendritic part of pyramidal neurons [93, 114] and dendrites which expressed 5-HT2A receptors expressed also NMDAR subunit NR1 and GluR2 [114]. Activation of 5-HT2A receptors causes an increase of intracellular Ca2+ concentration which in combination with NMDA receptor-mediated calcium influx can strengthen the synaptic plasticity. These observations suggest that an activation of 5-HT2A receptors induces facilitation of object memory storage and can result from potentiating of glutamate release in hippocampus, temporal dynamics of pyramidal neurons and critical post-training period. These receptors may serve as a drug target for pharmacological intervention in the treatment of memory disorders [115]. It is known that new neurons are generated in mammal DG. These new neurons are later during life integrated into hippocampal circuit. Serotonin belongs to important factors influencing neurogenesis. Among others 5-HT receptor subtypes (5-HT1A, 5-HT1B and 5-HT2C), activation of 5-HT2A receptors is involved in the positive regulation of adult neurogenesis in DG caused by regulation of cell proliferation in this region [103]. It was reported that some animal models of depression produce decrease in hippocampal cell proliferation and neurogenesis. Unlike the depression , chronic treatment with antidepressants, such as SSRIs, seem to have the positive effect on neurogenesis which is sufficient to reduce anxiety and depression-related behavior [116].

Role of Hippocampal Serotonin-2A Receptors in Pathophysiology and Treatment of Depression

The main effect of antidepressants is increasing of synaptic 5-HT levels. There is some evidence suggesting that hippocampus can be influenced by depression. It is known that hypercorticosolemia, an animal model of depression, results in the death of hippocampal neurons [117]. Change of serotonergic function in hippocampus is likely to be involved in defects of mood regulation associated with the major depressive disorder (MDD) . Serotonin 5-HT2A receptors play role in these changes. Postmortem studies in depressed suicide completers documented changes in 5-HT2A receptors binding in hippocampus [118, 119]. Magnetic resonance imaging (MRI) studies showed changes in 5-HT2A receptors binding potential in hippocampus in patients with MDD [120, 121]. Magnetic resonance imaging studies also demonstrated decrease of hippocampal volume in patients with MDD which correlated with duration of depression [120, 121]. However, decrease in 5-HT2A receptors binding potential is higher than volume loss and indicates that both conditions can coexist. Not only depression itself, but also the total number of days with depression inversely correlates with hippocampal volume [121, 122]. Serotonin 5-HT2A receptor binding is not influenced by depression phase. However, patients not previously treated for depression have lower 5-HT2A receptor binding than patients with previous medication treatment . It is possible that medication treatment provides compensatory upregulation of 5-HT2A receptors [123]. It is well established that decreased 5-HT2A receptor transmission is associated with depression [124]. It is also possible that decreased 5-HT2A receptor-mediated neurotransmission has special importance. Indeed, decreased 5-HT2A receptors binding was reported in patients with depression [123]. In addition, antidepressants treatment may cause changes in expression and binding of 5-HT2A receptors and these changes can persist for a long time after treatment [1, 125,126,127,128,129].

Nowadays, the role of astrocytes in depression has been intensively studied [130]. 5-HT2A receptors are expressed not only in hippocampal neurons, but also in astrocytes. This suggests the possibility that also 5-HT2A receptors express in astrocyte have functional implications in psychiatric disorders [95]. Beside their housekeeping functions, astrocytes are dynamic regulators of synaptogenesis, synaptic strength and control neurogenesis in the adult DG [131]. Astrocytes synthesize and release many neurotrophic factors vital for neuronal health such as BDNF, glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and neurotrophins 3 and 4/5 [132, 133]. Brain-derivated neurotrophic factor blocks neurogenesis in depression which is opposite to healthy condition. Its function has been implicated in the neurogenesis hypothesis of depression in which the antidepressants enhance neurogenesis, and BDNF is a key regulator of this mechanism. Antidepressants (including SSRIs) induce the CREB phosphorylation, CREB binds to the BDNF 13 promoter and induces BDNF transcription. Moreover, stress can reduce the expression of BDNF in the hippocampus and this reduction can be prevented by long-term chronic antidepressant treatment [134, 135]. In vitro studies reported that SSRIs stimulate the expression of BDNF , GDNF and vascular endothelial growth factor (VEGF) in primary culture of astrocytes [136,137,138]. In vivo data showed that the specific over-expression of BDNF in hippocampal astrocytes produced antidepressant-like effect accompanied by an increase in cell proliferation, maturation and survival of new neurons by generated cells in the DG of the hippocampus [139]. It is possible that astrocytes contribute to the enhancement in neurotrophic support and associated augmentation in synaptic plasticity that may form the basis for antidepressant efficacy. Several reports suggested that fluoxetine and other drugs can modulate the structural plasticity of astrocytes. Following chronic administration of lithium and some antipsychotic drugs , increased numbers of glia have been reported in the hippocampi of rats and nonhuman primates [140, 141]. In another study fluoxetine prevented the stress-induced decrease on a number of hippocampal astrocytes , but had no effect in nonstressed animals [142]. It demonstrates that fluoxetine, a prominent member of the SSRI family, can significantly modify the structural plasticity of astrocytes, and it is very likely that these morphological alterations either reflect or induce functional changes within the glial–neuronal interaction [142]. In particular, it is well accepted that SSRIs activate 5-HT2A receptors and stimulate signaling intracellular cascades leading to the phosphorylation/activation of extracellular signal regulated kinases (ERK1/2). Hence, antidepressants may exert their therapeutic activity by stimulating this pathway. In the hippocampus ERK1/2 have been implicated in mood regulation [143] as suggested by their blunted activation and/or expression in both depressed patient [144] and animal models of depression [145].

Conclusion

The 5-HT2A receptors belong to the 5-HT2 receptor family, the only known group of 5-HT receptors which are coupled to GαQ/Z proteins . The primary signal transduction mechanism of 5-HT2A receptors involves activation of PLC and calcium signaling . However, 5-HT2A receptor-mediated alteration of cAMP levels has also been reported. The 5-HT2A receptor is a product of 5HT2AR gene. Genetic polymorphism of 5HT2AR gene, its epigenetic regulation, and post-translational modifications of 5HT2AR mRNA have been reported. Furthermore, pre- and post-translational 5HT2AR alterations correlate with certain CNS disorders, such as depression, schizophrenia, dementia, and alcohol and nicotine dependence. On the functional level, 5-HT2A receptors play a central role in the interaction between 5-HT and norepinephrine systems and they are also involved in 5-HT-glutamate, 5-HT-GABA, and 5-HT-dopamine interactions. In addition, 5-HT2A receptors are fundamental in the modulation of hippocampal neuronal circuits. These lines of evidence, taken together, indicate that 5-HT2A receptors are one of the primary targets for antidepressant and mood stabilizing drugs and other CNS medications. And indeed, atypical antidepressant drugs act as antagonist of 5-HT2A receptors.