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. 2020 Apr 3;177(14):3210–3224. doi: 10.1111/bph.15043

Hispidulin attenuates the social withdrawal in isolated disrupted‐in‐schizophrenia‐1 mutant and chronic phencyclidine‐treated mice

Akihiro Mouri 1,2,8, Hsin‐Jung Lee 3, Takayoshi Mamiya 4, Yuki Aoyama 4, Yurie Matsumoto 4, Hisayoshi Kubota 2, Wei‐Jan Huang 5, Lih‐Chu Chiou 3,6,7,✉, Toshitaka Nabeshima 1,4,8,✉
PMCID: PMC7312434  PMID: 32133633

Abstract

Background and Purpose

Hispidulin is a flavonoid isolated from Clerodendrum inerme that was found to inhibit intractable motor tics. Previously, we found that hispidulin attenuates hyperlocomotion and the disrupted prepulse inhibition induced by methamphetamine and N‐methyl‐d‐aspartate (NMDA) receptor antagonists, two phenotypes of schizophrenia resembling positive symptoms. Hispidulin can inhibit COMT, a dopamine‐metabolizing enzyme in the prefrontal cortex (PFC) that is important for social interaction. Here, we investigated whether hispidulin would affect social withdrawal, one of the negative symptoms of schizophrenia.

Experimental Approach

We examined whether acute administration of hispidulin would attenuate social withdrawal in two mice models, juvenile isolated disrupted‐in‐schizophrenia‐1 mutant (mutDISC1) mice and chronic phencyclidine (PCP)‐treated naïve mice.

Key Results

In chronic PCP‐treated mice, hispidulin (10 mg·kg−1, i.p.) attenuated social withdrawal similar to that observed with dopamine D1 receptor antagonist (SCH‐23390, 0.02 mg·kg−1, i.p.) and was mimicked by the selective COMT inhibitor, OR‐486 (10 mg·kg−1, i.p.). Hispidulin increased extracellular dopamine levels in the PFC of chronic PCP‐treated mice. In isolated mutDISC1 mice, hispidulin also reversed social withdrawal. In both models, intra‐PFC microinjection of a D1 agonist (SKF‐81297: 10 nmol/mouse/bilateral) reversed the impairment of Ser897phosphorylation at the GluN1 subunit of NMDA receptors, suggesting the association between GluN1 Ser897‐phosphorylation and D1 activation in the PFC exits in both models.

Conclusions and Implications

Hispidulin attenuated social withdrawal by activating D1 receptors indirectly through elevated dopamine levels in the PFC by COMT inhibition. This nature of hispidulin suggests that it a potential novel therapeutic candidate for the treatment of negative symptoms in schizophrenia.

Abbreviations

DISC1

disrupted‐in‐schizophrenia‐1

METH

methamphetamine

MIH

methamphetamine‐induced hyperlocomotion

PCP

phencyclidine

PFC

prefrontal cortex

PPI

prepulse inhibition

What is already known

  • Hispidulin is a trihydroxyflavone isolated from Clerodendrum inerme that alleviates intractable motor tics.

  • Hispidulin attenuates hyperlocomotion and prepulse inhibition disruptions in animal models mimicking positive symptoms of schizophrenia.

What this study adds

  • Hispidulin attenuated social withdrawal in isolated disrupted‐in‐schizophrenia‐1 mutant and phencyclidine‐treated mice (models of negative symptoms).

  • Hispidulin attenuated social withdrawal by activating D1 receptors through elevating dopamine by PFC COMT inhibition.

What is the clinical significance

  • Hispidulin could be a novel drug candidate to treat positive and negative symptoms in schizophrenia.

1. INTRODUCTION

The juice from the leaves of Clerodendrum inerme (L.) Gaertn (C. inerne) was found to relive intractable motor tics in a patient (Fan, Huang, & Chiou, 2009). We have identified the trihydroxyflavone hispidulin (Figure 1a) as the active constituent of the C. inerme leaf extract (Chen et al., 2012). As tic disorders have been attributed to hyperdopaminergic reactivity of the cortico‐thalamic‐striatal circuit (Singer, 2005) we have studied hispidulin in an animal model of schizophrenia, as hyperdopaminergic along with impaired sensorimotor gating function are observed in schizophrenia. We employed methamphetamine (METH), a dopamine releaser, as a tool to induce hyperlocomotion and found that hispidulin attenuated this induced hyperlocomotion (Huang et al., 2015). Further, in a prepulse inhibition (PPI) disruption mouse models we found that hispidulin attenuated PPI disruptions induced by methamphetamine and by N‐methyl‐d‐aspartate (NMDA) receptor antagonists, including phencyclidine (PCP; Chiou et al., 2018). Thus, given its ability to attenuate hyperlocomotion and PPI disruptions, two characteristics of schizophrenia which resemble the positive symptoms, we investigated whether hispidulin would also be effective in the treatment of other symptoms of schizophrenia, such as social withdrawal, one of the negative symptoms. We employed two different social withdrawal animal models, namely, juvenile isolated disrupted‐in‐schizophrenia‐1 mutant (mutDISC1) mice (Niwa et al., 2013) and chronic PCP‐treated mice (Qiao et al., 2001).

FIGURE 1.

FIGURE 1

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Experimental designs for evaluating the effects of hispidulin in isolated disrupted‐in‐schizophrenia‐1 mutant (mutDISC1) mice as well as acute and chronic phencyclidine (PCP)‐treated mice. (a) The chemical structure of hispidulin. (b) Isolated mutDISC1 mice: For this model, 5‐week‐old mice were subjected to juvenile isolation by singly housing each mouse for 2 weeks. Littermate wild‐type (WT) mice were group‐housed as controls. The social interaction test, prepulse inhibition (PPI) test and methamphetamine (METH)‐induced hyperlocomotion evaluation were conducted in 7‐week‐old mice after they had undergone juvenile isolation. Hispidulin (10 mg·kg−1) was administered intraperitoneally (i.p.) 15, 15 and 10 min before the social interaction test, PPI test and methamphetamine treatment. (c) Acute PCP‐treated mice: For this model, PCP (1 and 5 mg·kg−1) was acutely injected subcutaneously (s.c.) to 6‐week‐old male ddY mice. The PPI test was conducted 10 min after PCP injection. Hispidulin (10 and 30 mg·kg−1, i.p.) was administered to ddY mice 20 min before the PPI test. (d) Chronic PCP‐treated mice: Six‐week‐old male ddY mice were treated with PCP (10 mg·kg−1, s.c.) every day for 14 consecutive days. After undergoing 4 days of withdrawal from the chronic PCP treatment, mice performed the social interaction test or underwent microdialysis surgery. Hispidulin (10 mg·kg−1, i.p.), OR‐486 (10 mg·kg−1, i.p.), and SCH‐23390 (0.05 mg·kg−1, s.c.) was administered 15, 15 and 45 min before the social interaction test, respectively

DISC1, a gene disrupted in a hereditary balanced chromosome (1;11) (q42.1; q14.3) translocation, which co‐segregates with schizophrenia and related psychiatric diseases, as shown in a Scottish family with high degree of major mental illnesses (Millar et al., 2000), and plays important roles in neurodevelopment and synaptic functions (Ishizuka, Paek, Kamiya, & Sawa, 2006; Tomoda, Sumitomo, Jaaro‐Peled, & Sawa, 2016). The mutDISC1 mice, which carry the dominant‐negative mutation of human DISC1 gene (Ishizuka et al., 2006; Tomoda et al., 2016), displayed methamphetamine‐induced hyperlocomotion (MIH; Jaaro‐Peled et al., 2013), PPI disruption (Hikida et al., 2007), depression‐like behaviour (Hikida et al., 2007) and impaired cognitive performance (Shen et al., 2008). Based on the hypothesis that genetic and environmental risk factors work together during critical periods of neuronal development to ultimately lead to schizophrenia (Marenco & Weinberger, 2000), we have successfully developed an animal model mimicking schizophrenia by giving mutDISC1 mice mild isolation stress during the juvenile period (Matsumoto et al., 2017; Niwa et al., 2013). These juvenile isolated mutDISC1 mice displayed significant social withdrawal, impaired PPI and enhanced methamphetamine‐induced hyperlocomotion (Matsumoto et al., 2017; Niwa et al., 2013). It is possibly that this is due to hypofunction of dopaminergic transmission in the prefrontal cortex (PFC) resulting from a reduction in tyrosine hydroxylase, a dopamine synthesizing enzyme, due to increased DNA methylation in the limbic dopaminergic neurons that project to the prefrontal cortex (Niwa et al., 2013).

PCP induces psychotomimetic states have been found to closely resemble schizophrenia in human (Javitt & Zukin, 1991). In rodents, both acute and chronic PCP treatments induce hyperlocomotion (Nagai et al., 2003), depression‐like behaviour (Noda, Yamada, Furukawa, & Nabeshima, 1995), PPI disruption (Tanaka, Toriumi, Kubo, Nabeshima, & Nakajima, 2011), social withdrawal (Qiao et al., 2001) and impaired cognitive performance (Mouri, Noda, Noda, et al., 2007). Thus, PCP‐treated rodents are a well‐recognized animal model for schizophrenia based on the hypo‐glutamatergic transmission hypothesis (Carlsson, Hansson, Waters, & Carlsson, 1997; Mouri, Noda, Noda, et al., 2007). Especially the social withdraw behaviour in chronic PCP‐treated mice, which can mimic the negative symptom in patients (Qiao et al., 2001).

The most influential pathophysiological hypothesis for the antipsychotic development is the dopamine hypothesis, which advocates the involvement of a subcortical/cortical dopaminergic imbalance in the symptoms of schizophrenia (Brisch et al., 2014). The subcortical mesolimbic dopaminergic system is hyperactive, resulting in hyperstimulation of D2 receptor‐mediated signalling and the positive symptoms. This is supported by the finding of increased tyrosine hydroxylase (TH) in the substantia nigra of patients with schizophrenia (Howes et al., 2013). On the contrary, the mesocortical dopaminergic neurons projecting to the prefrontal cortex are hypoactive, resulting in hypofunction of D1 receptor‐mediated signalling in the prefrontal cortex and leading to negative symptoms and cognitive dysfunctions. Social withdrawal, a core feature of these negative symptoms which have been implicate in the development, course and outcome of schizophrenia (Couture, Penn, & Roberts, 2006), has been attributed to a hypofunction of D1 receptor‐mediated dopaminergic activity in the prefrontal cortex (Boyd & Mailman, 2012). In rodents, social deficits induced by chronic PCP treatment are associated with a dysregulation of D1 receptor‐mediated signalling in the prefrontal cortex (Aoyama et al., 2014). Thus, a general enhancement of dopaminergic transmission could not only attenuate negative symptoms and cognitive dysfunctions via D1 receptor stimulation in the prefrontal cortex but also cause undesired side effects, such as an exacerbation of positive symptoms and cause substance abuse through D2 receptor stimulation in the subcortical mesolimbic dopaminergic system (Arnsten, Girgis, Gray, & Mailman, 2017).

Among 92 screened neurotransmitter receptors, enzymes and transporters, hispidulin only displayed micromolar affinity at the COMT and GABAA receptor α6 subunit (Liao, Lee, Huang, Fan, & Chiou, 2016). Hispidulin may inhibit methamphetamine‐induced hyperlocomotion via acting as a positive allosteric modulator (PAM) of the GABAA receptor α6 subunit (Liao et al., 2016) and attenuated PPI disruptions by inhibiting COMT and modulating GABAA receptor α6 subunit (Chiou et al., 2018). Since COMT is relatively more important in controlling the extracellular dopamine level in the prefrontal cortex, where the dopamine transporter is sparse as compared to the striatum (Tunbridge, Weinberger, & Harrison, 2006), we hypothesized that hispidulin would also attenuate social withdrawal in these two animal models of schizophrenia mentioned above by activating D1 receptors and indirectly increasing the dopamine level in the prefrontal cortex through COMT inhibition without undesired side effects.

2. METHODS

2.1. Animals

Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010) and with the recommendations made by the British Journal of Pharmacology. Juvenile isolation was performed as reported previously in male mutDISC1 mice, which are C57BL/6N mice (RRID:IMSR_JAX:005304) that carry the schizophrenia susceptible gene disc1, generated by engineering the prion protein promoter of disc1 (Niwa et al., 2013). Briefly, each mutDISC1 mouse was singly housed in a wire‐topped opaque polypropylene cage (13 × 20 × 11 cm) from the age of 5 weeks for 2 weeks before conducting the behavioural tests. In contrast, wild‐type (WT) mice were group housed (n = 5 per cage) in a wire‐topped clear plastic cage (21 × 30 × 13 cm; Figure 1b). We prepared only these groups of mice, since isolated WT mice and group‐housed mutDISC1 mice did not show any abnormal behaviour (Niwa et al., 2013). Although the behavioural changes in isolated mutDISC1 mice was preserved in both sexes (Niwa et al., 2013), we used male mice to exclude females' hormone cycles. Male ddY mice (6 weeks old; Japan SLC Inc., Shizuoka, Japan, RRID:MGI:5652658) were used to prepare the acute and chronic PCP‐treated model. The mice were group housed (n = 5 per cage), unless otherwise stated, in a controlled environment (23 ± 1°C, 50 ± 5% humidity, 12‐hr light/dark cycle with lights on at 08:00), with free access to food and water, except during the behavioural experiments. Animals were moved to the behavioural room and acclimated for at least 1 hr before the experiments. All animal care and use were in accordance with the guidelines for the care and use of laboratory animals issued by the Japanese Pharmacological Society and approved by the Institutional Animal Care and Use Committee of Meijo University (2012‐Yaku‐Jitsu‐12).

2.2. Drugs

Hispidulin was isolated from the ethanol extract of C. inerme leaves (Chen et al., 2012) as reported previously (Huang et al., 2015). The purity of isolated hispidulin has been confirmed by NMR spectroscopy and in vivo activity of isolated hispidulin was comparable to commercial hispidulin (Huang et al., 2015). PCP was synthesized previously within the law of Japan (Nabeshima, Hiramatsu, Amano, Furukawa, & Kameyama, 1982) according to the methods published by Maddox, Godefroi, and Parcell (1965) and its purity was checked by NMR and IR analysis (Nabeshima, Ishikawa, Yamaguchi, Furukawa, & Kameyama, 1988). N‐methyl‐1‐phenylpropan‐2‐amine hydrochloride (methamphetamine hydrochloride: METH) was purchased from Dainippon Sumitomo Pharma Co. Ltd. (Osaka, Japan) under the permission of Ministry of Health, Labor and Welfare. The R(+)‐7‐chloro‐8‐hydroxy‐3‐methyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐hydrochloride (SCH‐23390), 3,5‐dinitrocatechol (OR‐486) and (±)‐6‐chloro‐2,3,4,5‐tetrahydro‐1‐phenyl‐1H‐3‐benzazepine‐7,8‐diol and monohydrobromide (SKF‐81297) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). PCP, methamphetamine, SCH‐23390 and SKF‐81297 were dissolved in saline. Hispidulin and OR‐486 were dissolved in a vehicle containing 20% DMSO, 20% Cremophor® EL (polyoxyethylene castor; Sigma‐Aldrich), and 60% normal saline. The mice were randomly divided to treatment groups. All compounds were administered intraperitoneally (i.p.) or subcutaneously (s.c.) in a volume of 0.1 ml/10 g of body weight. SK‐F81297 (10 nmol/1 μl/mouse) were microinjected bilaterally into the prefrontal cortex (anteroposterior [AP]: +1.7 mm, mediolateral [ML]: ±0.5 mm from the bregma, dorsoventral [DV]: −2.0 mm from the skull) according to the mouse brain atlas of Franklin and Paxinos (2007).

2.3. Behavioural experiments

Isolated mutDISC1 mice were subjected to several behavioural tests on each day sequentially (Figure 1b) arranged in the order of the stress level, from low to high, as follows:‐ social interaction test, PPI test and methamphetamine‐induced hyperlocomotion. Hispidulin was injected to isolated mutDISC1 mice before each test. The metabolic half‐life (t 1/2) of hispidulin is 46 min and the intrinsic clearance (CLint) is 0.0298 ml·min−1·mg−1 protein (Chen, Hsu, Chiou, Tseng, & Huang, 2017). To exclude any effect of remaining hispidulin in the previous behavioural test on the next test, hispidulin was injected 24 hr after the previous test.

PCP‐induced PPI deficits were observed soon after an acute treatment but not 2 days after withdrawal from chronic treatments (Martinez, Ellison, Geyer, & Swerdlow, 1999). Therefore, in the acute PCP‐treated model, mice were subjected to the PPI test 10 min after receiving a PCP injection (Figure 1c). Chronic PCP treatments produce both behavioural and neurological symptoms of schizophrenia, further underscoring its construct validity as an animal model of schizophrenia (Mouri, Noda, Enomoto, & Nabeshima, 2007). In the chronic PCP‐treated model, the mice were randomly divided into two groups for the social interaction test and the microdialysis study (Figure 1d).

2.3.1. The social interaction test

The social interaction test was performed as described previously (Koseki et al., 2012; Qiao et al., 2001), with some modifications. The apparatus was a square, topless, open arena (25 × 25 × 30 cm) made with grey non‐reflecting acrylics and was indirectly illuminated with lamps that could not be seen by the mice. The light was diffused to minimize shadows in the arena. Before the day for social interaction test, each mouse was placed alone in the test box for 10 min on two consecutive days for habituation.

Isolated mutDISC1 mice

On the test day, the mutDISC1 mouse (7‐week‐old) was pretreated with hispidulin (10 mg·kg−1, i.p.) 15 min before the test. Then the mutDISC1 mouse was placed in the test box and allowed to interact with a 7‐week‐old WT (C57BL/6N) mouse from a different home cage as an unfamiliar partner (Figure 1b).

Chronic PCP‐treated mice

Mice (ddY) were administered PCP (10 mg·kg−1, s.c.) once a day for 14 days (Figure 1d). After 4 days of withdrawal, the mice underwent the social interaction test (Qiao et al., 2001; Mouri et al., 2012). Hispidulin (10 mg·kg−1, i.p.) and SCH‐23390 (0.02 mg·kg−1, s.c.) were injected 15 and 45 min, respectively, before the social interaction test. A chronic PCP‐treated mouse was placed in the test box and allowed to interact with an age‐ and sex‐matched unfamiliar ddY mouse.

For the social interaction test, one mouse in the experimental group and an unfamiliar partner were placed in the same box for 10 min. The behaviours of paired mice were videotaped. The duration of social interaction behaviours (following, sniffing, grooming, mounting, and crawling), except for aggressive behaviours, was recorded and manually measured using a stopwatch for 5 and 10 min. Passive contact (sitting or lying with bodies in contact) was not considered or counted as a social interaction behaviour. The examiners were blind to drug treatments and genotypes of mice.

2.3.2. The PPI test

The PPI test was performed as described previously (Niwa et al., 2010; Niwa et al., 2013), with minor modifications. Acoustic startle and PPI responses were measured in a startle chamber (SR‐Laboratory Systems, San Diego Instruments, San Diego, CA, USA) adapted for mice. The animal enclosure was located in a ventilated and sound‐attenuated chamber. The animal enclosure consisted of a perspex® cylinder (40 mm in diameter) that was placed on a platform connected to a piezoelectric accelerometer that detected movement within the cylinder. A speaker was attached above the cylinder. The paradigm was used to assess the startle amplitude and PPI response with acoustic stimuli of 120 dB, a single prepulse interval (100 ms) and three different prepulse intensities (4, 8 and 16 dB above background noise [white noise, 70 dB]). Each mouse was placed in the startle chamber and initially acclimatized for 10 min with the background noise alone. The mouse was then subjected to 50 startle trials, with each trial consisting of one of the following five conditions:‐ (a) a 40‐ms, 120‐dB noise burst presented alone; (b)–(d) a prepulse (20‐ms noise burst) that was 4, 8 or 16 dB above the background noise (i.e. 74, 78 or 86 dB) followed 100 ms later by a 40‐ms 120‐dB noise burst or (e) no stimulus (background noise alone), which was used to measure the baseline movement in the chamber. Each of these five trial types was repeated 10 times in a pseudo‐random order to obtain 50 total trials and the total duration of a test session was approximately 25 min. The percentage of PPI induced by each prepulse intensity was calculated as follows: %PPI = (1 – [startle amplitude on prepulse trial/startle amplitude on pulse alone]) × 100.

Isolated mutDISC1 mice

One day after the social interaction test, PPI test was performed as described above. To equalize the pretreatment time of hispudulin in the PPI test in acute PCP‐treated mice and isolated mutDISC1 mice, hispidulin (10 mg·kg−1, i.p.) was injected 20 min before the test (Figure 1b).

Acute PCP‐treated mice

To equalize the pretreatment times of hispidulin and PCP in the PPI test and that of hispidulin and methamphetamine in the methamphetamine‐induced hyperlocomotion study, we administered PCP (1 and 5 mg·kg−1, s.c.) and hispidulin (10 and 30 mg·kg−1, i.p.) to ddY mice 10 and 20 min, respectively, before the PPI test (Figure 1c). The PPI test was then performed as described above.

2.3.3. The open field test

One day after the PPI test, the open field test was employed to measure the locomotor activity after exposure to a novel environment and MIH in isolated mutDISC1 mice (Figure 1b). The mouse was placed in a transparent acrylic cage (45 × 26 × 40 cm), and its locomotion was measured every 5 min for 4 hr using digital counters with IR sensors (Scanet SV‐20, Melquest, Toyama, Japan). For the first 2 hr, the baseline locomotor activity of the mouse was measured when it was habituated in the cage (habituation session). Then methamphetamine (1 mg·kg−1, s.c.) was injected to the mice and methamphetamine‐induced hyperlocomotion was measured for the remaining 2 h (methamphetamine session). Hispidulin (10 mg·kg−1, i.p.) was injected 10 min before the methamphetamine injection.

2.4. Microdialysis in free‐moving mice

The microdialysis procedure was conducted in free‐moving chronic PCP‐treated ddY mice as reported previously (Mouri, Noda, Noda, et al., 2007; Niwa et al., 2010; Matsumoto et al., 2017) with modifications. Mice were administered PCP (10 mg·kg−1, s.c.) or saline once a day for 14 days (Figure 1d). Three days after the last PCP treatment, mice were anaesthetized with sodium pentobarbital (40 mg·kg−1, i.p.) before the stereotaxic implantation of a guide cannula (AG‐6; Eicom, Kyoto, Japan) into the left prefrontal cortex (15° angle away from anteroposterior [AP] +1.7, medial lateral [ML] +1.0 from bregma, and dorsal ventral [DV] −1.5 from the skull). After the implantation, mice were placed under a heat lamp to maintain their normal body temperature after anesthesia. One day after the stereotaxic implantation, a dialysis probe (AI‐6‐1; 1‐mm membrane length; Eicom) was inserted through the guide cannula and perfused with artificial CSF (147‐mM NaCl, 4‐mM KCl, and 2.3‐mM CaCl2) at a flow rate of 1.2 μl·min−1. The mouse was placed in the apparatus (40 × 40 × 30 cm) equipped with a two‐channel swivel (TCS2–23; Eicom), which permits the mouse to move freely even with a microdialysis probe. The dialysates were collected every 10 min and assayed by HPLC equipped with electrochemical detection (HTEC‐500; Eicom) via an Eicompak PP‐ODS column and a graphite electrode set at 400 mV against an Ag/AgCl reference electrode. The mobile phase contained 100 mM sodium phosphate buffer (pH 6.0), 500 mg·L−1 sodium‐1‐decanesulfonic acid, 50 mg·L−1 EDTA and 1.5% (vol/vol) methanol. After the collection of three baseline fractions, mice were treated with hispidulin (10 mg·kg−1, i.p.).

2.5. Western blotting

The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology. Western blotting was performed as previously described (Mouri et al., 2017). The mice were killed by decapitation 10 min after injection of SK‐F81297 and the prefrontal cortex was dissected out according to the atlas of Franklin and Paxinos (2007). The brain tissue was homogenized by sonication in an ice‐cold lysis buffer (20‐mM Tris–HCl [pH 7.4], 150‐mM NaCl, 50‐mM NaF, 2‐mM EDTA, 0.1% sodium dodecyl sulfate [SDS], 1% sodium deoxycholate, 1% NP‐40, 1‐mM sodium orthovanadate, 20 μg·ml−1 pepstatin, 20 μg·ml−1 aprotinin, and 20 μg·ml−1 leupeptin). Tissue samples (20 μg of protein) were boiled in sample buffer (125‐mM Tris–HCl pH 6.8, 10% 2‐mercaptoethanol, 4% sodium diphosphate decahydrate, 10% sucrose, and 0.0004% bromophenol blue), separated on a polyacrylamide gel, and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). Rabbit anti‐phospho‐NMDA receptor subunit 1 (p‐GluN1 (NR1); 1:500, Millipore, 06–641, RRID:AB_310199) and goat anti‐total‐ GluN1 (t‐ GluN1 (NR1); 1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc‐1467, RRID:AB_670215) antibodies were used as primary antibodies. HRP‐conjugated anti‐rabbit (1:2,000, Kierkegaard & Perry Laboratories, Gaithersburg, MD, USA, 074‐1516) and anti‐goat IgG (1:2,000, Kierkegaard & Perry Laboratories, 14‐13‐06) were used as secondary antibodies. The immune complexes were detected based on chemiluminescence (ECL kit, Amersham Biosciences, Piscataway, NJ), and the band intensities were analysed by densitometry using the ATTO Densitograph Software Library Lane Analyzer (ATTO, Tokyo, Japan). After the protein phosphorylation of NR1 at Ser897 was detected, membranes were stripped with stripping buffer (100‐mM 2‐melcaptoehanol, 2% SDS, and 62.5‐mM Tris–HCl [pH 6.7]), and total GluN1 protein expression was measured. Then the GluN1 phosphorylation ratio was determined by the GluN1 protein phosphorylation/total GluN1 protein expression.

2.6. Statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology. Sample sizes subjected to statistical analysis at least 5 animal per group. Data are expressed as the mean ± SEM. Differences among the groups were analysed with Kruskal–Wallis test, followed by the Steel–Dwass test, or repeated measures ANOVAs, followed by the Bonferroni's test for multiple group comparisons.The post hoc tests were conducted only if F in ANOVA achieved P < .05 and there was no significant variance inhomogeneity. Differences were considered significant if P < .05.

3. RESULTS

3.1. Hispidulin attenuated social withdrawal in isolated mutDISC1 mice

As shown in our previous report (Matsumoto et al., 2017), juvenile isolation induced social withdrawal in mutDISC1 mice (Figure 2). The time spent in social interaction in isolated mutDISC1 mice was shorter than in group‐housed WT mice (Figure 2). Importantly, hispidulin (10 mg·kg−1, i.p.) increased the interaction time in the mutDISC1 group to a duration as observed in the group‐housed WT mice (Figure 2). However, hispidulin did not affect the interaction time in group‐housed WT mice (Figure 2). This suggests that hispidulin alleviates social withdrawal in isolated mutDISC1 mice.

FIGURE 2.

FIGURE 2

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Hispidulin restored social interaction in isolated disrupted‐in‐schizophrenia‐1 mutant (mutDISC1) mice. The time each mouse spent performing social interaction behaviours with an unfamiliar mouse was measured for 10 min. Hispidulin (10 mg·kg−1) was administered intraperitoneally to mice 15 min before the test. Note that the vehicle‐treated isolated mutDISC1 mice spent less time performing social interaction behaviours than did the vehicle‐treated group‐housed wild‐type (WT) mice. Hispidulin restored the social interaction time in isolated mutDISC1 mice to a level that was similar to that observed in group‐housed WT mice. Data are presented as the mean ± SEM (N = 12). *P < .05 versus vehicle‐treated/group‐housed WT mice; # P < .05 versus vehicle‐treated/isolated mutDISC1 mice: Kruskal–Wallis test followed by a comparison with the Steel–Dwass test

3.2. Hispidulin failed to attenuate the PPI deficits in isolated mutDISC1 mice

We previously reported that in addition to social withdrawal (Matsumoto et al., 2017), isolated mutDISC1 mice also show impaired PPI and enhanced methamphetamine‐induced hyperlocomotion (Niwa et al., 2013). As shown in Figure 3a, PPI magnitudes induced by the three prepulse acoustic intensities (4, 8 and 16 dB above background noise) in isolated mutDISC1 mice were all smaller than those in the group‐housed WT mice, Figure 3a. Hispidulin (10 mg·kg−1, i.p.) did not affect the PPI in isolated mutDISC1 or group‐housed WT mice (Figure 3a). Startle responses tended to be, but not statistically, different among groups (Figure 3b: Kruskal–Wallis test).

FIGURE 3.

FIGURE 3

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Hispidulin failed to attenuate the impaired prepulse inhibition (PPI) and enhanced methamphetamine (METH)‐induced hyperlocomotion (MIH) in isolated disrupted‐in‐schizophrenia‐1 mutant (mutDISC1) mice. (a) We induced PPI using three prepulse intensities, which were 4, 8 and 16 dB higher than the background noise and the pulse at 120 dB. (b) The startle response to a 120‐dB acoustic stimulation. We measured PPI and the startle response as described in Section 2. Hispidulin (10 mg·kg−1) or vehicle was injected intraperitoneally to the mouse 15 min before the PPI test. Note that isolated mutDISC1 mice showed impaired PPI while hispidulin failed to affect their PPI deficits. Each column represents the mean ± standard error (N = 12). * P < .05 versus vehicle‐treated/group‐housed wild‐type (WT) mice. (c) Time courses of the locomotor activity, measured as the number of interruptions of IR beams. We subcutaneously injected METH (1 mg·kg−1) into the mouse 2 hr after it was placed in the recording chamber. Hispidulin or vehicle was administered 10 min before the METH injection. Values are the mean ± standard error. (d) The total locomotor activity during the 2‐hr habituation period and the METH‐treated period. Data are presented as the mean ± SEM (N = 12). * P < .05 versus vehicle‐treated/group‐housed WT group

3.3. Hispidulin failed to affect the enhanced methamphetamine‐induced hyperlocomotion in isolated mutDISC1 mice

When being placed in the locomotor activity chamber, the mouse started to explore the environment and then gradually habituated to the environment over the initial 2‐hr exposure period (habituation session). Then its baseline locomotor activity of the mouse was reduced to a steady level by 2 hr in all groups and there was no difference among groups, Figure 3c. Methamphetamine (1 mg·kg−1, s.c.) was administered to all groups of mice after the habituation session. Methamphetamine did not affect the spontaneous locomotor activity of group‐housed WT mice but increased the locomotion of isolated mutDISC1 mice over the remaining 2‐hr period (METH session) Figure 3c. Therefore, methamphetamine‐induced hyperlocomotion in isolated mutDISC1 mice at the dose that is ineffective in the WT mice, as previously reported (Niwa et al., 2013). However, methamphetamine‐induced similar levels of hyperlocomotion in the hispidulin‐treated (10 mg·kg−1, i.p.) and vehicle‐treated isolated mutDISC1 mice (Figure 3c,d), suggesting that hispidulin does not affect the increased vulnerability to methamphetamine‐induced hyperlocomotion in isolated mutDISC1 mice.

3.4. Hispidulin attenuated the PPI deficits in acute PCP‐treated mice

Given that hispidulin failed to reduce the disrupted PPI in isolated mutDISC1 mice, despite prior evidence supporting its ability to reduce the PPI deficits that are induced by methamphetamine or a NMDA antagonists, including that induced by 1 mg·kg−1 PCP (Chiou et al., 2018), we re‐examined effect of hispidulin on PCP‐disrupted PPI by increasing the dose of PCP to 5 mg·kg−1 and the dose of hispidulin to 30 mg·kg−1. Mice that received PCP (s.c.) at the doses of 1 and 5 mg·kg−1 displayed reduced PPI compared to vehicle‐treated/saline‐treated mice . In agreement with our previous findings (Chiou et al., 2018), we found that hispidulin (10 mg·kg−1, i.p.) prevented the PPI deficits that were induced by PCP at 1 mg·kg−1. However, hispidulin did not prevent the PPI deficits when the dose of PCP was increased to 5 mg·kg−1 even at a higher dose of 30 mg·kg−1 (i.p.; Figure 4a). Startle responses were not different among groups (Figure 4b).

FIGURE 4.

FIGURE 4

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Effects of hispidulin on acute phencyclidine (PCP)‐induced prepulse inhibition (PPI) deficits. (a) We induced PPI using three prepulse intensities, which were 4, 8 and 16 dB higher than the background noise and the pulse at 120 dB. (b) The startle response to a 120‐dB acoustic stimulation. We measured PPI and the startle response as described in Section 2. Mice were treated with hispidulin (10 or 30 mg·kg−1, i.p.) for 10 min, followed by PCP (1 or 5 mg·kg−1, s.c.) for 10 min, and then subjected to the PPI test. Note that hispidulin prevented the PPI disruption that was induced by PCP (1 mg·kg−1). Hispidulin (10 and 30 mg·kg−1) failed to prevent the PPI deficits that were induced by PCP at the high dose (5 mg·kg−1). Data are presented as the mean ± SEM (N = 12) *P < .05 versus vehicle‐treated/saline‐treated mice

3.5. Hispidulin improved the social withdrawal in chronic PCP‐treated mice

In light of our finding that hispidulin attenuated the social withdrawal in isolated mutDISC1 mice, we further examined whether hispidulin would attenuate social withdrawal in another animal model, chronic PCP‐treated mice. As reported previously (Mouri et al., 2012; Qiao et al., 2001), the mice treated with PCP (10 mg·kg−1, s.c.) daily for 14 days and then withdrawn for 4 days displayed significantly shorter social interaction times than did the chronic saline‐treated mice (Figure 5a:). Hispidulin (10 mg·kg−1, i.p.) lengthened the social interaction time in chronic PCP‐treated mice to a level that was similar to that observed in chronic saline‐treated mice (Figure 5a). Hispidulin did not affect the social interaction time in chronic saline‐treated mice (Figure 5a). Thus, hispidulin attenuated the social withdrawal in chronic PCP‐treated mice at a dose that had no effect on the social interaction of chronic saline‐treated mice.

FIGURE 5.

FIGURE 5

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Hispidulin and OR‐486 attenuated social withdrawal in chronic phencyclidine (PCP)‐treated mice. Social interaction times were recorded in mice after 4 days of withdrawal from the repeated treatments with PCP (10 mg·kg−1, s.c.) or saline for 14 consecutive days. (a) Hispidulin (10 mg·kg−1, i.p.) was administered 15 min before the social interaction test. Note that hispidulin effectively attenuated social withdrawal in chronic PCP‐treated mic (b) OR‐486 (10 mg·kg−1, i.p.), a COMT inhibitor, 10 min before the social interaction test, which significantly restored the social interaction behaviour in chronic PCP‐treated mice. Data are presented as the mean ± SEM (a: N = 11, b: N = 12). *P < .05 versus the vehicle‐treated/saline‐treated group; # P < .05 versus the vehicle‐treated/PCP‐treated mice: Kruskal–Wallis test followed by acomparison with the Steel–Dwass test

3.6. OR‐486 also improved social withdrawal in chronic PCP‐treated mice

To examine whether the COMT inhibitory activity of hispidulin (Liao et al., 2016) contributes to hispidulin‐induced attenuation of social withdrawal, we investigated the effect of OR‐486, a selective COMT inhibitor (Nissinen et al., 1988), on chronic PCP‐induced social withdrawal. OR‐486 (10 mg·kg−1, i.p.) significantly lengthened the social interaction time in chronic PCP‐treated mice to the level as observed in the chronic saline‐treated control group (Figure 5b). Notably, OR‐486 itself did not affect the social interaction time in the chronic saline‐treated control group (Figure 5b).

3.7. Hispidulin elevated the extracellular dopamine level in the prefrontal cortex of chronic PCP‐treated mice

Dysregulation of mesocortical dopaminergic neurotransmission is thought to be correlated with the social deficits that are present in chronic PCP‐treated mice (Wang et al., 2007). Given that hispidulin can inhibit COMT (Liao et al., 2016), a dopamine degradation enzyme that is important for limiting the extracellular dopamine level in the prefrontal cortex (Sesack, Hawrylak, Matus, Guido, & Levey, 1998), we examined whether hispidulin would increase the dopamine levels in the prefrontal cortex microdialysates of chronic PCP‐treated mice. Systemic hispidulin (10 mg·kg−1, i.p.) increased the mean extracellular dopamine level in the prefrontal cortex of chronic PCP‐treated mice, Figure 6a. This effect reached its peak at 20 min after hispidulin injection and lasted for about 2 hr (Figure 7a).

FIGURE 6.

FIGURE 6

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Hispidulin increased the extracellular dopamine level in the prefrontal cortex (PFC) of chronic phencyclidine (PCP)‐treated mice and attenuated social withdrawal by activating dopamine D1 receptors in chronic PCP‐treated mice. (a) The dopamine level in PFC microdialysates was measured using a HPLC electrochemical detection system, as described in Section 2. Note that hispidulin (10 mg·kg−1, i.p.), but not vehicle, significantly increased the extracellular dopamine level in the PFC of chronic PCP‐treated mice. The basal levels of dopamine in the PFC of the vehicle‐ and hispidulin‐treated mice were 0.67 ± 0.11 and 0.45 ± 0.06 fmol/10 ml per 10 min, respectively. *P < .05 versus the vehicle‐treated group. Data are presented as the mean ± SEM (N = 6). (b) Hispidulin (10 mg·kg−1) was administered intraperitoneally 15 min before the social interaction test, and SCH‐23390 (0.02 mg·kg−1) was injected subcutaneously 30 min before hispidulin administration. Note that hispidulin effectively improved the social withdrawal in chronic PCP‐treated mice, and this effect was prevented by a D1 antagonist, SCH‐23390. Data are presented as the mean ± SEM (N = 11). * P < .05 versus the vehicle‐treated/saline‐treated mice; # P < .05 versus the vehicle‐treated/PCP‐treated mice; † P < .05 versus the PCP‐treated/hispidulin‐treated mice

FIGURE 7.

FIGURE 7

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Intra‐ prefrontal cortex(PFC) microinjection of a D1 agonist reversed the impairment of NR1 phosphorylation in both isolated mutDISC1 mice and chronic PCP‐treated mice. (a, b) The GluN1 phosphorylation ratio at Ser897 in the PFC was measured using a western blotting, as described in Section 2. The D1 agonist (SKF‐81297: 10 nmol/mouse/bilateral) was infused into the PFC of (a) isolated mutDISC1 mice and (b) chronic PCP‐treated mice 10 min before the decapitation. Representative western blotting of phosphorylated/total GluN1 immunoreactivity was shown. Note that SKF‐81297 significantly reversed the decrease of the GluN1 phosphorylation ratio in both mouse models. Data are presented as the mean ± SEM (a: N = 7, b: N = 8). The Western blotting data of vehicle‐infused/group‐housed WT mice, SKF81297‐ and vehicle‐infused/isolated mutDISC1 mice were cited from (Matsumoto et al., 2017, Figure 8b). * P < .05 versus the vehicle‐infuse/group‐housed WT mice and the vehicle‐infused/saline‐treated mice; # P < .05 versus the vehicle‐infused/isolated mutDISC1 mice and vehicle‐infused/PCP‐treated mice

3.8. D1 receptors are involved in hispidulin‐induced attenuation of social withdrawal

Previously, we showed that D1 receptor inactivation is involved in chronic PCP‐induced behavioural impairments in mice (Mouri, Noda, Noda, et al., 2007) and that clozapine, an antipsychotic that reduces negative symptoms in patients with schizophrenia, attenuated these impairments by indirectly activating D1 receptors (Aoyama et al., 2014). To clarify whether D1 receptor activation contributes to the beneficial effect of hispidulin on social withdrawal in chronic PCP‐treated mice, we investigated whether SCH‐23390, a D1 antagonist, would inhibit the attenuating effect of hispidulin on social withdrawal in chronic PCP‐treated mice. Interestingly, SCH‐23390 (0.02 mg·kg−1, s.c.) prevented hispidulin‐induced attenuation of social withdrawal in chronic PCP‐treated mice (Figure 6b), suggesting that hispidulin alleviates social withdrawal by enhancing D1 receptor‐mediated dopaminergic transmission indirectly through elevating the extracellular dopamine level in the prefrontal cortex (Figure 6a). Importantly, SCH‐23390 and hispidulin per se had no effect on the social interaction time in saline‐treated mice (Figure 6b). SCH‐23390 per se also did not affect PCP‐induced impairment of social interaction (Figure 6b).

3.9. Intra‐prefrontal cortex microinjection of a D1 receptor agonist reversed the impairment of GluN1 phosphorylation in both isolated mutDISC1 mice and chronic PCP‐treated mice

The GluN1 is an essential subunit of NMDA receptors and decrease of phosphorylated GluN1 at Ser897 has been reported in the frontal cortex of patients with schizophrenia (Emamian, Karayiorgou, & Gogos, 2004). Moreover, D1 receptor activation can increase the level of phosphorylated GluN1 at Ser897 and facilitate the function of NMDA receptors (Dudman et al., 2003). We therefore investigated whether impairment of D1 receptor‐mediated signalling in the prefrontal cortex is a common mechanism of behavioural impairments observed in both isolated mutDISC1 mice and chronic PCP‐treated mice. Mice in both models were infused a D1 agonist (SKF‐81297: 10 nmol/mouse/bilateral) into the prefrontal cortex.

In previous study, we have demonstrated a D1 receptor mediated impairment in GluN1 phosphorylation ratio (phosphorylated at Ser897/total NR1) in isolated mutDISC1 mice (Matsumoto et al., 2017) and chronic PCP‐treated mice (Mouri, Noda, Noda, et al., 2007). We further investigate whether impairment of D1 receptor‐mediated signalling in the prefrontal cortex is a common mechanism of behavioural impairments observed in both isolated mutDISC1 mice and chronic PCP‐treated mice. We compared the prefrontal cortex GluN1 phosphorylation ratio at Ser897 in both groups of mice in Figure 7. Western blotting data of vehicle‐infused/group‐housed WT mice, SKF‐81297‐ and vehicle‐infused/isolated mutDISC1 mice were cited from Matsumoto et al. (2017, Figure 8b), and the data of SKF‐81297‐ and vehicle‐infused/group‐housed WT mice and SKF‐81297‐ and vehicle‐infused/chronic saline‐ and PCP‐treated mice were taken from our unpublished data.

Previous data (Matsumoto et al., 2017, Figure 8b) showed that the phosphorylation ratio of GluN1 at Ser897 in their prefrontal cortex tissues was decreased in isolated mutDISC1 mice, and intra‐ prefrontal cortex microinjection of SKF‐81297 reversed the decrease of GluN1 phosphorylation ratio (Figure 7a). Similar results were obtained in chronic PCP‐treated mice (Figure 7b). However, intra‐prefrontal cortex microinjection of SKF‐81297 did not affect GluN1 phosphorylation in the group‐housed WT mice (Figure 7a) or chronic saline‐treated mice (Figure 7b).

4. DISCUSSION

Schizophrenia remains an unmet medical need due to a high refractory rate (~30%) to current antipsychotics and their intolerable side effects that lead to poor long‐term adherence and high risk of relapse in patients (Nose et al., 2009; Suzuki et al., 2011). Thus, it is an urgent need to develop new drugs with novel action mechanisms that may have a broader therapeutic window or fewer unwanted side effects. Previously, we have demonstrated that hispidulin attenuated methamphetamine‐induced hyperlocomotion (Liao et al., 2016) and PPI disruptions (Chiou et al., 2018), two symptoms of schizophrenia representing positive symptoms. Here, we revealed that hispidulin attenuated social withdrawal in both chronic PCP‐treated mice and isolated mutDISC1 mice. Thus, hispidulin has the potential to be the lead compound for the development of novel antipsychotic drugs for the treatment of both positive and negative symptoms in schizophrenia.

Hispidulin is a flavonoid isolated from an herb (Huang et al., 2015) that inhbited motor tics in a patient with refractory tic disorder (Fan et al., 2009), a neuropsychiatric disorder also manifesting disrupted PPI. We have demonstrated that hispidulin attenuated methamphetamine‐induced hyperlocomotion through the GABAA receptor α6 subunit positive allosteric modulator effect (Liao et al., 2016) and PPI disruptions also through the GABAA receptor α6 subunit positive allosteric modulator effect and COMT inhibition (Chiou et al., 2018). Both mechanisms may lead to elevated dopamine levels in the prefrontal cortex.

Chronic PCP‐treated mice have been used for screening compounds with potential antipsychotic properties (Mouri, Noda, Enomoto, & Nabeshima, 2007; Neill et al., 2010). These mice display long‐lasting social withdrawal even after withdrawing PCP (Qiao et al., 2001). The behavioural changes after chronic PCP treatment might be related to hypodopaminergic functions in the prefrontal cortex (Aoyama et al., 2014; Jentsch & Roth, 1999). Indeed, hispidulin increased dopamine levels of prefrontal cortex in chronic PCP‐treated mice (Figure 6a). This effect may contribute its improvement of social interaction in these mice.

The finding that hispidulin can inhibit COMT at micromolar concentrations (Liao et al., 2016) is noteworthy. COMT is relatively more important in controlling the extracellular dopamine level in the prefrontal cortex than in the striatum (Tunbridge, Weinberger, & Harrison, 2006). In humans, a functional single nucleotide polymorphism, namely, the Val158Met polymorphism, in the COMT gene has been shown to affect cognitive tasks that are related to executive functions (Bruder et al., 2005). Schizophrenic patients have a higher frequency of carrying the Val allele (higher activity) than do normal controls (Caspi et al., 2005; Wonodi, Stine, Mitchell, Buchanan, & Thaker, 2003). Acute PCP administration increases DA metabolism by COMT in the prefrontal cortex of rats (Rao, Kim, Lehmann, Martin, & Wood, 1989). Chronic administration of the non‐competitive NMDA antagonist MK‐801 increases COMT mRNA expression in the mouse prefrontal cortex (Paterlini et al., 2005). Although there is no report about COMT activity in the isolated mutDISC1 mice, higher COMT activity is implicated not only in the pathophysiology of the schizophrenia patients but also in the phenotypes of the animal models of schizophrenia.

We also investigated whether OR‐486, a selective COMT inhibitor with a potency that is 2.75 times higher than that of hispidulin (Liao et al., 2016), would attenuate the social withdrawal exhibited by chronic PCP‐treated mice. Indeed, like hispidulin, OR‐486 restored the social withdrawal in these mice. Further, the present in vivo microdialysis experiment showed that hispidulin increased the extracellular dopamine level in the prefrontal cortex of chronic PCP‐treated mice. Therefore, hispidulin may attenuate the social withdrawal in chronic PCP‐treated mice through its COMT inhibitory effect, which in turn elevates the dopamine levels in the prefrontal cortex. As compared with the prefrontal cortex, the dopamine transporter is densely concentrated in the striatum, and thus, COMT is relatively less important in the control the extracellular dopamine level (Tunbridge, Harrison, & Weinberger, 2006). Furthermore, COMT inhibition and deficiency have no effect on extracellular dopamine levels in the striatum (Acquas, Carboni, de Ree, Da Prada, & Di Chiara, 1992; Gogos et al., 1998). Although we did not measure dopamine levels in the striatum, hispidulin acting as a COMT inhibitor may not be expected to increase the level of dopamine in the striatum. Therefore, hispidulin may not exacerbate, and instead may have a beneficial effect on, the positive symptoms in schizophrenia since it effectively alleviated methamphetamine‐induced hyperlocomotion (Liao et al., 2016) and PPI disruptions induced by methamphetamine, ketamine, MK‐801 and PCP (Chiou et al., 2018).

Isolated mutDISC1 mice were developed to study how the interaction between genes and the environment during neurodevelopment in the genesis of schizophrenic phenotypes (Niwa et al., 2013). Previously, we have demonstrated a lower dopamine level in the prefrontal cortex of isolated mutDISC1 mice due to epigenetic impairments in tyrosine hydroxylase synthesis (Niwa et al., 2013). Hispidulin attenuated social withdrawal in these mutant mice probably through increasing prefrontal cortex dopamine levels. However, hispidulin failed to affect the PPI disruption and methamphetamine‐induced hyperlocomotion enhancement in these mice. It is possible that the ability of hispidulin to modulate the dopaminergic system is not sufficient to attenuate the PPI deficits in isolated mutDISC1 mice, perhaps because basal levels of dopamine in these mice are very low owing to the reduced expression of TH in the prefrontal cortex. Hispidulin (10 mg·kg−1) failed to attenuate the PPI disruption induced by 5 mg·kg−1 PCP although it worked when PCP was 1 mg·kg−1 (Chiou et al., 2018). Since higher dose of hispidulin (30 mg·kg−1) also failed to attenuate the high‐dose PCP‐induced PPI disruption, these data suggest that isolated mutDISC1 mice may be an animal model of schizophrenia resistant to GABAA receptor α6 subunit positive allosteric modulators, like hispidulin, compared to the PCP‐treated mice. Thus, additional studies are needed to examine whether increased doses and/or repeated administrations of hispidulin can attenuate the PPI impairments of the acute high‐dose PCP‐treated and isolated mutDISC1 mice.

The finding that hispidulin failed to attenuate the increased vulnerability to methamphetamine‐induced hyperlocomotion in isolated mutDISC1 mice is different from the positive finding in its effects on methamphetamine‐induced hyperlocomotion in normal mice (Liao et al., 2016). The reasons for this discrepancy are unclear. However, the increased methamphetamine‐induced hyperlocomotion vulnerability in isolated mutDISC1 mice may be resulted from the impaired glutamatergic transmission in the prefrontal cortex that exaggerates methamphetamine‐induced dopamine release in the striatum (Matsumoto et al., 2017). This mechanism might not be modified by hispidulin. There are some limitations in this study. First, single‐housed WT and group‐housed mutDISC1 mice were not included, because these two groups of mice did not show the phenotypes of social withdrawal (Data not shown), PPI disruption and methamphetamine‐induced hyperlocomotion enhancement (Niwa et al., 2013) seen in isolated mutDISC1 mice. Thus, the therapeutic widow for hispidulin in these two groups may be too small to be revealed. Second, isolated mutDISC1 mice were sequentially subjected to several behavioural tests on each day, and hispidulin was repeated injected every day before the test. These may be confounding factors that influence the results. Additional studies are needed to perform sequential behavioural tests with enough recovery time between each test or randomized crossover design to reduce the influence of previous behavioural tests and hispidulin injections.

We have reported the dysregulation of glutamatergic nervous system (i.e. decreased extracellular levels of glutamate, increased expression of glutamate transporters and impaired activation of NMDA‐Ca2+/calmodulin kinase II signalling) in the prefrontal cortex of both chronic PCP‐treated mice and isolated mutDISC1 mice (Mouri, Noda, Noda, et al., 2007; Murai et al., 2007; Matsumoto et al., 2017). Furthermore, there is functional linkage between glutamatergic and dopaminergic signalling in the prefrontal cortex. Stimulation of D1 receptor induces the Ser897 phosphorylation of the GluN1 subunit of NMDA receptors via PKA (Hida et al., 2015). We have previously demonstrated that an infusion of a D1 receptor agonist into the prefrontal cortex can attenuate the behavioural impairments in both chronic PCP‐treated mice (Mouri, Noda, Noda, et al., 2007) and isolated mutDISC1 mice (Matsumoto et al., 2017). Here, we further demonstrated that intra‐ prefrontal cortex injection of a D1 receptor agonist reversed the decrease of GluN1 phosphorylation (Ser897) ratio in both model mice. These data suggest that impaired D1 receptor‐mediated signals in the prefrontal cortex are common contributors of the behavioural impairments observed in both animal models. The finding that SCH‐23390, a D1 antagonist, inhibited the attenuating effects of hispidulin on the social withdrawal in chronic PCP‐treated mice suggests that this effect of hispidulin is mediated by an indirect activation of D1 receptors due to an increased dopamine level in the prefrontal cortex through COMT inhibition. Besides, we cannot exclude the contribution, at least in part, of the GABAA receptor α6 subunit positive allosteric modulator effect of hispidulin on its attenuation of social withdrawal, as this may also indirectly increase the dopamine level in the prefrontal cortex (Chiou et al., 2018).

5. CONCLUSIONS

Hispidulin attenuated social withdrawal in both isolated mutDISC1 and chronic PCP‐treated mice, two animal models of schizophrenia, by indirectly activating D1 receptors through elevated dopamine in the prefrontal cortex by COMT inhibition. This suggests that in addition to reducing PPI disruption (Chiou et al., 2018), hispidulin can also target and attenuate an important clinical dimension of schizophrenia, social withdrawal that is one of the negative symptoms of schizophrenia and is poorly treated by currently available antipsychotics (Buckley & Stahl, 2007; Harvey, Koren, Reichenberg, & Bowie, 2006). Hence, this two‐hit nature of hispidulin renders it a potential novel candidate for the treatment of schizophrenia.

AUTHOR CONTRIBUTIONS

L.‐C.C., H.‐J.L., Y.M., T.M., H.K., A.M., and T.N. participated in research design. H.‐J.L., Y.A., Y.M., T.M., and A.M. conducted experiments and performed data analysis. W.‐J.H. contributed new reagents. T.N., L.‐C.C., A.M., and T.M. wrote the manuscript.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry and Animal Experimentation, and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

Supporting information

Data S1 Supporting information

Click here for additional data file. (399KB, pdf)

ACKNOWLEDGEMENTS

This study was supported by Grants‐in‐Aids for Scientific Research from the Japan Society for the Promotion of Science (26460240, 15K08218, 16K10195, and 17H04252); the Private University Research Branding Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT); the Research Project from the Meijo Asian Research Center; and research grants from the Takeda Science Foundation, Nakatomi Foundation, and Smoking Research Foundation to A.M., T.M., and T.N. This study was also supported by the National Research Program for Biopharmaceuticals (NSC100‐2325‐B002‐050, NSC101‐2325‐B002‐048, NSC102‐2325‐B002‐047, MOST103‐2325‐B002‐037, MOST104‐2325‐B002‐010, MOST104‐2923‐B002‐006‐MY3, MOST108‐2321‐B002‐005, and MOST108‐2325‐B002‐029‐MY3) from the National Science Council, which is now known as the Ministry of Science and Technology, Taiwan, as well as the Innovative Research Grant (NHRI‐EX108‐10733NI) from National Health Research Institutes, Taiwan, to L.‐C.C.

Mouri A, Lee H‐J, Mamiya T, et al. Hispidulin attenuates the social withdrawal in isolated disrupted‐in‐schizophrenia‐1 mutant and chronic phencyclidine‐treated mice. Br J Pharmacol. 2020;177:3210–3224. 10.1111/bph.15043

Contributor Information

Lih‐Chu Chiou, Email: lcchiou@ntu.edu.tw.

Toshitaka Nabeshima, Email: tnabeshi@ccalumni.meijo-u.ac.jp.

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