Introduction

Schizophrenia is a devastating, complex, and costly neuropsychiatric disorder that affects ∼1% of the world’s population (Olesen et al, 2012). Schizophrenia is typically characterized by three symptom clusters: positive (eg, hallucinations, delusions, disordered thoughts), negative (eg, flattened affects, social withdrawal), and cognitive (eg, attention and working memory deficits) (Simpson et al, 2010). This disease has traditionally been associated with a deregulated dopaminergic system, mainly because of the fact that for many years typical antipsychotics (ie, strong dopamine (DA)-D2 receptor antagonists such as haloperidol) have served as the mainstream pharmacotherapy for psychotic patients (Keshavan et al, 2008). However, it has long been known that the serotonergic system is also implicated in the pathogenesis of schizophrenia (Meltzer and Massey, 2011). Indeed, atypical antipsychotics, of which clozapine is the prototype, consist of agents that are more potent antagonists of serotonin 2A (5-HT2A) receptors over D2 receptors (Gonzalez-Maeso et al, 2008). Many atypical antipsychotics show similar affinity for 5-HT2B and 5-HT2A receptors (eg, clozapine, amisulpride, asenapine, aripiprazole, or cariprazine) (Abbas et al, 2009; Kiss et al, 2010; Shahid et al, 2009; Shapiro et al, 2003), although the contribution of the 5-HT2B receptor to the action of antipsychotic compounds has never been reported. Notably, the elucidation of the intricate mechanisms underlying the neurobiology of schizophrenia is imperative in order to develop novel drugs with improved therapeutic efficacy (Kvajo et al, 2012). In this direction, models of genetic deletion in mice have been indispensable in characterizing the contribution of specific genes to disease pathophysiology (Arguello and Gogos, 2006).

Impulsivity, very broadly defined as action without foresight, novelty seeking, and hyperlocomotion share common ground with numerous mental disorders, including chronic substance abuse, attention deficit hyperactivity disorder, and schizophrenia (Fineberg et al, 2010; Humby and Wilkinson, 2011). In a recent study conducted in a Finish cohort, we reported that a population-specific 5-HT2B receptor stop codon (HTR2B Q20*) segregates with severe impulsivity, psychosis, and early-onset schizophrenia (Bevilacqua et al, 2010). Moreover, mice in which the Htr2B gene has been genetically inactivated (ie, Htr2B−/−) exhibit a hyperlocomotor phenotype as well as impulsivity, as assessed in the delay discounting task (Bevilacqua et al, 2010; Doly et al, 2008). However, the extent to which 5-HT2B receptor is implicated in the neurobiology of schizophrenia has never been investigated.

In this study, we assessed the effects of the genetic ablation of Htr2B in mice subjected to a comprehensive series of behavioral tests screening for positive, negative, and cognitive schizophrenic-like symptoms. Following the demonstration that loss of function of the 5-HT2B receptor in Htr2B−/− mice confers a wide spectrum of schizophrenic-like behavioral phenotypes, we show that selected phenotypes are induced by acute pharmacological blockade of 5-HT2B receptors with RS127445 (selective 5-HT2B receptor antagonist) and rescued by chronic antipsychotic treatment with the typical antipsychotic drug haloperidol. The dorsal striatum (dSTR) and its connections with the prefrontal cortex (PFC) have been implicated in the pathogenesis of both the positive and the cognitive symptoms of schizophrenia (Howes et al, 2009; Simpson et al, 2010). Indeed, neurochemical alterations in the dopaminergic and glutamatergic corticostriatal circuits underlie a substantial portion of the dysfunction seen in schizophrenic patients (Gordon, 2010; Howes et al, 2009; Keshavan et al, 2008; Simpson et al, 2010). Thus, we further investigated relevant dopaminergic and glutamatergic neurochemical alterations in the PFC and the dSTR of Htr2B−/− mice. Altogether, our findings present the first evidence for a role of 5-HT2B receptors in the neurobiology of schizophrenia.

Materials and Methods

Animals

Male Htr2B−/− mice used in these experiments were in a 129S2/SvPas (129S2) background, and wild-type (Htr2B+/+) 129S2 mice (8–12 weeks old) used as controls were bred in our animal facility under standard 12-h light/dark schedule, and housed in groups of 3–5 of the same genetic background and sex after weaning (Diaz et al, 2012). Animal experiments were conducted in accordance with standard ethical guidelines (National Institutes of Health’s ‘Guide for the Care and Use of Laboratory animals’, and European Directive 2010/63/UE) and were approved by the Ethics Committee for Animal Experiments (No. 1170.01).

Drug Treatments

The selective 5-HT2B receptor antagonist RS127445 (RS; 0.5 mg/kg; Tocris, France) was administered acutely (ie, 1 h before testing) and intraperitoneally (i.p.), as in previous studies from our group (Diaz et al, 2012; Doly et al, 2008). The glutamatergic psychostimulant and N-methyl-D-aspartic acid (NMDA) receptor antagonist dizocilpine (MK-801; 0.5 mg/kg; i.p.; Sigma-Aldrich, France) and the dopaminergic psychostimulant amphetamine (10 mg/kg; i.p.; Sigma-Aldrich, France) were dissolved in saline and administered at a rate 10 ml/kg. Different cohorts of Htr2B+/+ and Htr2B−/− mice were chronically treated (4 weeks) with haloperidol (Sigma-Aldrich) in drinking water (2.0 mg/kg/day) or vehicle, as described before (Terry et al, 2007). Following chronic antipsychotic treatment, locomotor activity and prepulse inhibition (PPI) were tested, as described below. Separate mouse cohorts were used for the assessment of sleep architecture and for social interaction behavior and neurochemical estimations.

Locomotor Response to Novelty and to Psychostimulants

Hyperactivity in response to novelty and locomotor hypersensitivity to acute administration of psychostimulants are considered useful correlates of positive schizophrenic-like symptoms in mice (Arguello and Gogos, 2006). Locomotor activity was measured in a circular corridor with four infrared beams placed at every 90° (Imetronic, Passac, France), as previously described (Blundell et al, 2010; Doly et al, 2008). Mice were injected with saline and individually placed in the activity box for 2 h during four consecutive days in order to habituate to the apparatus. On day 5, mice were placed in the activity box for 1 h and then treated with dizocilpine or amphetamine. Locomotor activity was then screened for 1–6 h.

PPI of Acoustic Startle

PPI has been considered an operational measure of sensorimotor gating and preattentive processing, and its deficit has been consistently reported in schizophrenic patients and in relevant animal models (Meyer et al, 2005). Sensorimotor gating was assessed as previously described (Yadav et al, 2011). PPI was indexed by the percentage inhibition of the startle response at each level of prepulse intensity by using the following formula: % PPI=((mean reactivity on pulse-alone trials−mean reactivity on prepulse-pulse trials)/mean reactivity on pulse-alone trials) × 100%. % Average PPI values were calculated from all PPI values across all the four prepulse intensities.

Sociability and Preference for Social Novelty

Decreased interactions with conspecifics are often used to model social withdrawal, a negative symptom of schizophrenia (Arguello and Gogos, 2006). The three-chamber social test was performed as previously described (Peca et al, 2011). Target subjects (social I and social II) were adult Htr2B+/+ males. For the sociability test, the test animal was introduced into the middle chamber and was left to habituate for 5 min, after what an unfamiliar mouse (social I) was introduced into a wire cage in one of the side -chambers and an empty wire cage (EC) on the other side chamber. The dividers were then opened and the test animal was allowed to freely explore all three chambers over a 10-min session. Preference for social novelty was assayed during a second 10-min session in which a novel stranger mouse (social II) was inserted into the previously empty wire cage. The time spent in close interaction with the two cages was scored manually.

Novel Object Recognition (NOR)

Cognitive deficits, including working memory impairment, are typically observed in animal models of schizophrenia (Arguello and Gogos, 2006; Meyer et al, 2005). The NOR task represents the most useful method for studying cognitive impairment in schizophrenia (Lyon et al, 2012). The NOR test was performed as previously described (Carlini et al, 2008) with retention tests performed at 1 and 24 h intervals in order to assess short- and long-term memory, respectively. Each trial lasted for 5 min. The time that the animals spent exploring the novel and the familiar objects were measured; results are expressed as % time of exploration of the novel object.

Contextual and Cued Fear Conditioning

Cognitive deficits in associative fear memory have been reported in mice displaying schizophrenic-like phenotypes (Arguello and Gogos, 2006; Gleason et al, 2012). Fear conditioning was assessed in computer-controlled operant chambers (Imetronic), as previously described (Blundell et al, 2010; Cai et al, 2006; Powell et al, 2004). All results were expressed as % time freezing.

Latent Inhibition (LI)

Latent inhibition (LI) refers to the retardation in learning about the significance of a stimulus as a result of its prior repeated preexposures without consequence (Lubow and Moore, 1959). LI was assessed in a conditioned freezing paradigm, as previously described (Meyer et al, 2005; Willi et al, 2010). The test procedures consisted of four phases: preexposure, conditioning, context test, and tone test. Animals were randomly allocated to either the preexposed (PE) or non-preexposed (NPE) condition. The establishment of LI (ie, difference in freezing between NPE and PE groups) reflects the ability to ignore stimuli that historically predict nonsignificant consequences. Thus, LI deficiency is a translational model that assesses attentional deficits in animal models of schizophrenia (Arguello and Gogos, 2006; Meyer et al, 2005).

Sleep/Wakefulness Patterns

A battery of evidence shows that schizophrenia is associated with alterations in sleep architecture (Keshavan et al, 2008; Monti and Monti, 2004). Sleep architecture and assessment and spectral analysis of the electroencephalogram (EEG) recording were performed as previously described (Boutrel et al, 1999). Polygraphic recordings were scored visually every 15 s epoch as wakefulness, nonrapid eye movement (NREM) sleep, or rapid eye movement (REM) sleep, following classical criteria (Lena et al, 2004; Tobler et al, 1997), using Somnologica. For analysis of the spontaneous sleep–wakefulness patterns, the amounts of vigilance states for each animal were calculated for every hour throughout 48 h and summed over 24 h. REM sleep latency was calculated as the time elapsing from sleep onset after the animal had been awakened to the first episode of REM sleep (Popa et al, 2005).

Neurochemical Assessments

DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and glutamate tissue concentrations in the PFC and the dSTR of Htr2B+/+ and Htr2B−/− mice were assessed ex vivo by means of high-performance liquid chromatography (HPLC) as previously described (Banas et al, 2011; Pitychoutis et al, 2011). A real-time qPCR-based approach was implemented to examine the mRNA expression of relevant key receptor genes pertaining to the dopaminergic and the glutamatergic neurochemical systems. qPCR was performed as previously described (Diaz et al, 2012).

Statistical Analysis

Differences between experimental groups were analyzed by unpaired t-tests and one-way or two-way analysis of variance (ANOVA) with genotype and treatment as main factors, depending on the experimental design. Bonferroni’s post hoc test was applied in order to elucidate specific differences between groups. Repeated measures ANOVAs were implemented in order to analyze locomotor activity and sleep architecture data. Statistical analyses of the results are summarized in Supplementary Tables S1 and S2. In graphs/tables, the values represent means±SEM and p<0.05 was considered statistically significant.

Results

Deficits in the PPI of the Startle Reflex

Genetic ablation of the 5-HT2B receptor induced a global deficit in the PPI of the startle reflex (Figure 1a) and a reduction in startle amplitude (Supplementary Figure S1a). Moreover, acute treatment of Htr2B+/+ mice with the 5-HT2B receptor antagonist RS127445 phenocopied the effects of genetic ablation of 5-HT2B receptor in the PPI (Figure 1b and Supplementary Figure S1b).

Figure 1
figure 1

Prepulse inhibition (PPI) is impaired in Htr2B−/− mice across prepulse intensities over 66 db and in average of all intensities (N=27 per group) (a). Acute injection of RS127445 (0.5 mg/kg), a selective 5-HT2B receptor antagonist (RS), also impaired PPI in Htr2B+/+ mice (N=8 per group) (b). Genetic ablation of the 5-HT2B receptor enhanced novelty-induced locomotion during the first 10 min of testing (N=8 per group) (c). Genetic blockade of the 5-HT2B receptor enhanced locomotor responses to the glutamatergic psychostimulant and noncompetitive NMDA receptor antagonist dizocilpine (MK-801; 0.5 mg/kg; i.p.) (N=8 per group) (d) and to the dopaminergic psychostimulant amphetamine (10 mg/kg; i.p.) (N=8 per group) (e). Genetic and pharmacological ablation of the 5-HT2B receptor impaired social interactions, as evidenced by the lack of preference for the social partner (social I vs the empty wire cage (EC)) (N=7–9 per group) (f). Htr2B+/+ mice spent significantly more time interacting with the novel social stimulus (social II vs social I), but this was not the case for Htr2B−/− mice or Htr2B+/+-RS-treated mice (N=7–9 per group) (g). Genetic ablation of 5-HT2B receptor impaired social memory, as Htr2B−/− mice spent similar time interacting with social I partner during both trials, whereas both Htr2B+/+ and Htr2B+/+-RS-treated mice spent significantly less time interacting with social I partner in the second trial (N=7–9 per group) (h). *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001 significantly different from vehicle-treated Htr2B+/+ mice as determined by unpaired t-test, one-way ANOVA, or two‐way ANOVA; full statistical analysis is presented in Supplementary Table S1.

PowerPoint slide

Full size image

Enhanced Locomotor Response to Psychostimulants

Administration of NMDA receptor antagonists (eg, dizocilpine) and DA releasers (eg, amphetamine) exacerbate existing symptoms in schizophrenic patients and cause psychotic-like symptoms in humans and in rodents (Arguello and Gogos, 2006). As shown herein and before, Htr2B−/− mice exhibit enhanced locomotor response to novelty (Figure 1c) (Doly et al, 2008). The locomotor response of Htr2B−/− mice to both dizocilpine (Figure 1d) and amphetamine (Figure 1e) was significantly enhanced as compared with Htr2B+/+ mice. Several other behaviors appear to be completely unaltered in Htr2B−/− mice, including motor coordination on the Rotarod and nociception assessed in the hot-plate test (Supplementary Figure S1c and d).

Defect in Social Interaction

According to our data, both Htr2B−/− mice and RS-treated Htr2B+/+ mice displayed dysfunctional social interaction behavior (Figure 1f), as measured by observing the time mice spent in close interaction with the social partner vs the inanimate stimulus (ie, EC). During the second session, preference for social novelty was evaluated by inserting a novel social stimulus (social II) in the previously empty cage and measuring the time spent interacting with social I and social II stimuli (Figure 1g). All three groups showed preference for social novelty as revealed by the greater % time interacting with social II vs social I (Supplementary Figure S1e). Social memory was assessed by comparing the time mice spent in close interaction with social I stimulus during the two different trials. This analysis showed that genetic ablation of 5-HT2B receptor impaired social memory, as Htr2B−/− mice spent similar time interacting with social I partner during both trials (Figure 1h). Social memory in both Htr2B+/+ and Htr2B+/+-RS-treated mice appeared to be intact. In addition, social interaction with a juvenile conspecific was also impaired in Htr2B−/− mice (Supplementary Figure S1f). Of note, the differences observed in social interaction could not be attributed to altered olfactory function because of the genetic ablation of 5-HT2B receptor in mice (Supplementary Figure S1g and h).

Memory Deficits in the NOR and the Fear Conditioning

5-HT2B receptor deficiency induced a significant impairment of both short-tem (1 h; Figure 2a) and long-term NOR memory (24 h; Figure 2b). Similarly, Htr2B−/− mice exhibited impaired fear learning and memory, as evidenced by the reduced time they spent freezing in response to the context (Figure 2c) and the cue in a fear conditioning paradigm (Figure 2d). These cognitive deficits appear to be independent of object exploration behavior or sensitivity to foot shocks. Indeed, Htr2B−/− and Htr2B+/+ mice spent similar time exploring the objects during both NOR trials (Supplementary Figure S1i) and showed comparable freezing behavior in response to foot shock during training in the fear conditioning paradigm (Supplementary Figure S1j).

Figure 2
figure 2

Htr2B−/− mice present with impaired short-term (a) and long-term (b) memory for a novel object, as assessed in the NOR test (N=8 per group). Contextual (c) and cued (d) fear memory is impaired in Htr2B−/− mice (N=6–7 per group). In the latent inhibition (LI) paradigm, (e) LI was established in Htr2B+/+ but not in Htr2B−/− mice as indicated by the absence of difference in freezing between NPE and PE groups (N=7 per group). *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001 significantly different from Htr2B+/+ mice following unpaired t-test or two‐way ANOVA; #p<0.05 significantly different from NPE in two‐way ANOVA; full statistical analysis is presented in Supplementary Table S1.

PowerPoint slide

Full size image

Attention Deficits in the LI Paradigm

In the test of the conditioned response to the tone, the presence of LI in Htr2B+/+ mice was demonstrated by a clear reduction in the amount of freezing to the tone in PE mice relative to the NPE mice. However, LI was not established in Htr2B−/− mice (Figure 2e).

Alterations in Sleep Architecture

Htr2B−/− mice exhibited increased wakefulness duration at the expense of NREM sleep (Figure 3a), as well as decreased latency to REM sleep (Figure 3b). Under baseline conditions, Htr2B+/+ and Htr2B−/− mice exhibited the typical polyphasic structure of vigilance states found in rodents and a diurnal rhythm of sleep and wakefulness, with larger amounts of sleep during the light period classically observed in nocturnal species (Figure 3c–e). However, Htr2B−/− mice exhibited significantly increased amounts of wakefulness, at the expense of NREM sleep, throughout the dark period and at the onset of light period (Figure 3c and d), whereas REM sleep was not affected (Figure 3e).

Figure 3
figure 3

In Htr2B−/− mice across 24 h, the total amount of wakefulness is increased while that of NREM sleep is decreased (a), as is the latency to REM sleep, calculated as the time elapsing from sleep onset after the animal had been awakened to the first episode of REM sleep (b) (N=4–5 per group). Htr2B−/− mice exhibit significantly increased amounts of wakefulness at the expense of NREM sleep throughout the dark period and at the onset of the light period as assessed by the amount of vigilance states, expressed as min per 3 h (c, d) but REM sleep is not affected (e). Chronic oral haloperidol treatment (Hal, 2 mg/kg/day during 4 weeks) normalizes sleep–wakefulness states across the light/dark cycle (N=4–5 per group) (c–e). The effects of chronic oral haloperidol treatment were also tested in the prepulse inhibition (f) and locomotor activity (g) (N=8–10 per group); social interaction was assessed in the three-chamber sociability test by comparing the time Htr2B+/+ (h) and Htr2B−/− mice (i) spent interacting with a novel mouse (Soc I) vs an empty wire cage (EC; N=7–9 per group). *P<0.05; **p<0.01; ***p<0.001 significantly different from the Veh-treated Htr2B+/+ mice; ##p<0.01; ####p<0.0001 significantly different from the Veh-treated Htr2B−/− mice; full statistical analysis is presented in Supplementary Table S1.

PowerPoint slide

Full size image

Chronic Haloperidol Treatment Rescued Selected Schizophrenic-Like Phenotypes in Htr2B−/− Mice

Haloperidol was selected herein on the basis of its minor binding capacity to 5-HT2 receptors including murine 5-HT2B receptors unlike many other antipsychotics (Meltzer, 2013) (Supplementary Figure S1k). Chronic oral intake of haloperidol (2 mg/kg/day) for 4 weeks restored the balance between wakefulness and NREM sleep (Figure 3c–e) in Htr2B−/− mice. Moreover, chronic haloperidol treatment normalized the PPI deficits (Figure 3f) and the psychomotor agitation (Figure 3g) conferred by 5-HT2B receptor deletion. However, haloperidol treatment did not ameliorate the social deficit observed in Htr2B−/− mice in the three-chamber social interaction test (Figure 3h and i).

Neurochemical Alterations in Htr2B−/− Mice

In our experimental setup, DA and glutamate tissue levels were found decreased in the dSTR of Htr2B−/− mice, whereas no major neurochemical alterations were observed in the PFC (Figure 4a and b). Moreover, our results showed that the genetic ablation of the 5-HT2B receptor resulted in a differential regulation of mRNA expression of DA and glutamate receptors in the PFC and the dSTR. In particular in the dSTR, D2R mRNA expression was decreased (Figure 4c), whereas GluR2 was upregulated in the PFC (Supplementary Figure S2f). It is noteworthy that chronic haloperidol treatment normalized the observed neurochemical alterations in DA and glutamate concentrations (Figure 4a and b) and D2R receptor expression in the dSTR (Figure 4c).

Figure 4
figure 4

Region-distinctive neurochemical profile in the prefrontal cortex (PFC-left) and the dorsal striatum (dSTR-right) of Htr2B+/+ (black circle) and Htr2B−/− (open square) mice. Htr2B−/− mice present lower tissue concentrations of DA (a) and glutamate (b) in the dSTR (N=6–8 per group). Genetic ablation of the 5-HT2B receptor was associated with a downregulation of D2R mRNA expression in dSTR (c) (N=4–5 per group). Neurochemical alterations in the dSTR are effectively reversed upon chronic haloperidol treatment (2 mg/kg/day 4 weeks) (Hal) (a–c). *p<0.05; **p<0.01; significantly different from vehicle (Veh)-treated Htr2B+/+ mice in unpaired t-test or two‐way ANOVA; #p<0.05, ###p<0.001 significant effect from Veh-treated Htr2B−/− mice in two‐way ANOVA; full statistical analysis is presented in Supplementary Table S1.

PowerPoint slide

Full size image

Discussion

Herein we investigated the effects of the genetic ablation of 5-HT2B receptor across a battery of translational behavioral paradigms relevant for assessing face validity of animal models of schizophrenia (Arguello and Gogos, 2006; Meyer et al, 2005; Willi et al, 2010). We report herein that loss of function of 5-HT2B receptor confers a wide spectrum of schizophrenic-like behavioral and psychopharmacological phenotypes in mice. Importantly, domains related to the positive, negative, and cognitive symptom clusters of schizophrenia appear to be affected upon 5-HT2B receptor gene ablation (Table 1). Htr2B−/− mice display a global deficit in sensorimotor gating (ie, impaired PPI performance) as well as psychomotor agitation (ie, novelty-induced hyperlocomotion) and psychostimulant hypersensitivity (ie, enhanced locomotor response to psychostimulants), all phenotypes that have been related to the positive symptoms of schizophrenia (Gunduz-Bruce, 2009; Meyer et al, 2005; Pratt et al, 2012). Moreover, 5-HT2B receptor gene ablation impaired social interaction behavior with conspecifics, a trait used commonly to model negative symptoms of schizophrenia in rodents (Arguello and Gogos, 2006; Pratt et al, 2012). Most importantly, Htr2B−/− mice present with selective attention deficits (ie, lack of LI establishment) and learning and memory impairments (ie, poor NOR and fear conditioning performance and social memory deficit) that closely resemble the cognitive deficits observed in schizophrenic patients (Keshavan et al, 2008). Remarkably, the alterations in sleep architecture observed in Htr2B−/− mice (ie, reduced duration of NREM sleep and decreased latency to REM sleep) mimic the sleep abnormalities often seen in schizophrenic patients (Keshavan et al, 2008). The list of behavioral paradigms implemented herein, although not exhaustive, is consistent with schizophrenia-like behavioral abnormalities. Moreover, selected schizophrenic-like phenotypes (ie, PPI and social interaction deficits) were phenocopied in Htr2B+/+ mice by acute pharmacological ablation of 5-HT2B receptors with the selective receptor antagonist RS127445.

Table 1 Schizophrenia-Like Behavioral Phenotypes in Htr2B−/− Mice
Full size table

We next assessed whether selected schizophrenic-like phenotypes could be rescued by chronic antipsychotic treatment. For this reason, Htr2B−/− and Htr2B+/+ were chronically treated with the typical antipsychotic haloperidol. Of note, chronic antipsychotic treatment completely abolished the observed PPI deficits and alterations in sleep architecture. As expected, haloperidol treatment suppressed psychomotor agitation in Htr2B−/− mice, but was ineffective against social withdrawal, a negative symptom of schizophrenia. Moreover, chronic haloperidol treatment also had an effect in Htr2B+/+ mice, normalizing the differences observed between the two genotypes during locomotor activity and social interaction testing.

How 5-HT2B receptor gene deficiency may lead to the observed schizophrenic-like phenotype can only be speculated at present as the function of this receptor in the brain remains largely uninvestigated. It is noteworthy that 5-HT2B receptors have been shown to modulate aspects of both serotonergic and dopaminergic neurotransmission (Bevilacqua et al, 2010; Diaz and Maroteaux, 2011; Doly et al, 2008). In particular, ex vivo studies have indicated that 5-HT2B receptors may serve as positive autoregulator of the serotonergic tone through modulating the 5-HT transporter (SERT) in raphe neurons (Launay et al, 2006), and in vivo studies in mice further confirmed that 5-HT2B receptors contribute to the behavioral effects of the SERT-targeting 5-HT releasers, MDMA (ie, ecstasy) and dexfenfluramine (Banas et al, 2011; Doly et al, 2009; Doly et al, 2008). Recently, Auclair et al (2010) reported that 5-HT2B receptors control the mesoaccumbal DA pathway activity, as an acute systemic administration of the 5-HT2B subtype-selective antagonist RS127445 in rats induced a region-selective reduction of extracellular DA levels in the NAC in response to amphetamine challenge.

In light of the schizophrenic-like phenotype observed herein, the relevant neurochemical alterations found are not surprising. Indeed, the enhanced psychomotor response of Htr2B−/− mice to dizocilpine and to amphetamine indicates that the glutamatergic and/or the dopaminergic systems must, at least to some extent, be altered in Htr2B−/− mice. Schizophrenia has been largely associated with dopaminergic hypofunction in the PFC and hyperfunction in the basal ganglia; indeed numerous post-mortem studies report increased D2R densities in the dSTR (Hirvonen et al, 2005; Seeman and Kapur, 2000) and decreased D2R levels in the PFC of schizophrenic patients (Takahashi et al, 2006). Moreover, aberrations in glutamate-mediated neurotransmission through NMDA receptors in schizophrenia is supported by the reduced glutamate levels in the cerebrospinal fluid (CSF) and NMDA receptor expression in the PFC of schizophrenic patients (Gordon, 2010; Keshavan et al, 2008). In our experimental setup, DA and glutamate tissue concentrations and D2R mRNA expression were found decreased in the dSTR of Htr2B−/− mice. In accordance with our findings, DA tissue concentrations were also found decreased in the dSTR of Nogo-A−/− mice that also present with a schizophrenic-like phenotype (Willi et al, 2010). Intriguingly, the observed dopaminergic and glutamatergic alterations in the dSTR were corrected upon chronic antipsychotic treatment. The latter finding indicates that both D2R-related and unrelated defects in Htr2B−/− mice are reversed by haloperidol treatment and provide evidence that the effects of 5-HT2B receptors extend beyond the serotonergic and the dopaminergic systems.

Acute pharmacological ablation of 5-HT2B receptors (ie, RS127445 treatment) phenocopied both positive (ie, PPI deficit) and negative (ie, impaired social interaction) symptoms observed in Htr2B−/− mice. Moreover, in an earlier study, administration of another 5-HT2B receptor-selective antagonist (ie, SB-215505) was shown to increase wakefulness at the expense of NREM and REM sleep in rats (Kantor et al, 2004), and in another study it was concluded that 5-HT exerts a 5-HT2B receptor-mediated facilitation of NREM sleep (Popa et al, 2005), in accordance with our findings in Htr2B−/− mice. Thus, based on these observations, we propose that the schizophrenic-like phenotype of Htr2B−/− mice results from a combination of both the direct absence of 5-HT2B receptor signaling and the neural adaptations triggered by the permanent lack of this receptor.

Antipsychotic drugs ameliorate hallucinations and delusions in patients with neuropsychiatric disorders, particularly schizophrenia and bipolar disorders. The two main classes are known as typical and atypical antipsychotics. Many atypical antipsychotics show similar affinity for 5-HT2B and 5-HT2A receptors (eg, clozapine, amisulpride, asenapine) (Abbas et al, 2009; Shahid et al, 2009) or even higher affinity for 5-HT2B receptors (eg, aripiprazole, cariprazine) (Kiss et al, 2010; Shapiro et al, 2003). Notably, it was recently shown that the efficacy of clozapine, but not haloperidol, is diminished in Pet1−/− mice that lack 5-HT neurons, and thus depends on an intact presynaptic serotonergic system (Yadav et al, 2011). Given that many marketed atypical antipsychotic drugs present high affinity for 5-HT2B receptors (Shapiro et al, 2003) and that these receptors are expressed in 5-HT neurons (Diaz et al, 2012), our findings bear broader significance for the elucidation of the effects of these drugs in the treatment of psychotic disorders.

It should be borne in mind that the behavioral phenotype of Htr2B−/− mice portrayed herein is not exclusive of a putative role of the 5-HT2B receptor in the neurobiology of a broader range of neuropsychiatric diseases that share common domains of dysfunction with psychotic disorders. Interestingly, it was recently reported that two polymorphisms of the HTR2B gene were associated with intelligence quotation, intellectual disability, and language onset delay in a cohort of children and young adults suffering from autistic spectrum disorders (Hervas et al, 2014). In agreement, the present data strongly support a novel role for 5-HT2B in the regulation of cognitive function and social behavior. Despite the fact that Htr2B−/− mice display deficits in social interaction and in learning and memory processes that closely resemble autistic-like behavior, they also present with deficits in sensorimotor gating and locomotor hypersensitivity to psychostimulants that are core endophenotypes of schizophrenia (Arguello and Gogos, 2006; Powell et al, 2009).

Overall, this study revealed that 5-HT2B receptor deficiency induces a wide spectrum of antipsychotic-sensitive schizophrenic-like phenotypes in mice. Further pharmacological validations with selective 5-HT2B receptor antagonists (given acutely or chronically) that extend present findings to other behavioral tests, mouse strains and species, or other brain regions and neurochemical systems are clearly warranted to further dissect the role of the 5-HT2B receptor in the neurobiology of psychotic spectrum disorders. This genetic mouse model holds additional value in further elucidating the schizophrenia-relevant neurodevelopmental, epigenetic, and physiological mechanisms that may be sensitive to 5-HT2B receptor polymorphisms.

Funding and Disclosure

The authors declare no conflict of interest.