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Intake of 7,8-Dihydroxyflavone During Juvenile and Adolescent Stages Prevents Onset of Psychosis in Adult Offspring After Maternal Immune Activation

Prenatal infection and subsequent abnormal neurodevelopment of offspring is involved in the etiology of schizophrenia. Brain-derived neurotrophic factor (BDNF) and its high affinity receptor, tropomyosin receptor kinase B (TrkB) signaling plays a key role in the neurodevelopment. Pregnant mice exposed to polyriboinosinic-polyribocytidylic acid [poly(I:C)] causes schizophrenia-like behavioral abnormalities in their offspring at adulthood. Here we found that the juvenile offspring of poly(I:C)-treated mice showed cognitive deficits, as well as reduced BDNF-TrkB signaling in the prefrontal cortex (PFC). Furthermore, the adult offspring of poly(I:C)-treated mice showed cognitive deficits, prepulse inhibition (PPI) deficits, reduced BDNF-TrkB signaling, immunoreactivity of parvalbumin (PV) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in the prelimbic (PrL) of medial PFC and CA1 of hippocampus. Supplementation of a TrkB agonist 7,8-dihydroxyflavone (1 mg/mL in drinking water) during juvenile and adolescent stages could prevent these behavioral abnormalities, reduced BDNF-TrkB signaling in PFC and CA1, and immunoreactivity of PV and PGC-1α in the PrL of medial PFC and CA1 in the adult offspring from poly(I:C)-treated mice. These findings suggest that early intervention by a TrkB agonist in subjects with ultra-high risk for psychosis may reduce the risk of subsequent transition to schizophrenia.

Two-way analysis of p-TrkB/TrkB ratio data revealed significant effects (PFC, poly(I:C): F 1,20 = 5.824, P = 0.026; 7,8-DHF: F 1,20 = 5.305, P = 0.032, interaction: F 1,20 = 9.683, P = 0.005) (CA1, poly(I:C): F 1,20 = 5.578, P = 0.028, 7,8-DHF: F 1,20 = 5.24, P = 0.033, interaction: F 1,20 = 4.89, P = 0.039). Post-hoc test showed that the p-TrkB/TrkB ratio in the PFC and CA1 of adult offspring from poly(I:C) + VEH group was significantly lower than that of PBS + VEH or poly(I:C) + 7,8-DHF groups (Fig. 3b). In contrast, there were no differences among the four groups in the other regions including NAc, CA3, and DG. These data suggest that adult offspring from prenatal mice exposed to poly(I:C) showed decreased BNDF-TrkB signaling in the PFC and CA1 of hippocampus, but not NAc, CA3, DG of hippocampus, and that supplementation with 7,8-DHF could normalize decreased BDNF-TrkB signaling in these regions of adult offspring from poly(I:C)-treated group. . (c) PPI: there were no differences between poly I:C offspring and controls in the PPI tasks. The value is expressed as the mean ± SEM (n = 16). (d) NORT: the exploratory preferences were significantly lower in the poly(I:C) offspring than controls in the retention session, but there was no difference between the two groups in the training session. ***P < 0.001 compared with PBS treated group. The value is expressed as the mean ± SEM (n = 14). (e) BDNF: **P < 0.01 compared with PBS treated group. The value is expressed as the mean ± SEM (n = 6). (f) p-TrkB/TrkB ratio: **P < 0.01 compared with PBS treated group. The value is expressed as the mean ± SEM (n = 6). Post-hoc test showed that PV-immunoreactivity in the PrL of medial PFC (mPFC) and CA1 of hippocampus (not IL, NAc, CA3, and DG) of poly(I:C) + VEH group was significantly lower than that of PBS + VEH or poly(I:C) + 7,8-DHF groups ( Fig. 4b-d). These findings suggest that adult offspring from prenatal mice exposed to poly(I:C) showed the loss of PV-immunoreactivity in the PrL of mPFC and CA1, but not NAc, CA3, and DG, and that supplementation with 7,8-DHF could prevent the loss of PV-immunoreactivity in these brain regions after MIA.

Discussion
The major findings of the present study are as follows. Supplementation with 7,8-DHF during the juvenile and adolescent stages of offspring of prenatal mice exposed to poly(I:C) led to the prevention of behavioral changes (e.g., cognitive deficits and PPI deficits), decreased BDNF-TrkB signaling in PFC and CA1 of hippocampus, and loss of PV and PGC-1α immunoreactivity in the PrL of mPFC and CA1 of hippocampus at adulthood after MIA. Therefore, it is likely that supplementation with a TrkB agonist such as 7,8-DHF during the prodromal stage has prophylactic effects on the behavioral abnormalities relevant to schizophrenia and related disorders at adulthood.
In this study, we identified the cognitive deficits of offspring from poly(I:C)-treated mice at 4 weeks and 10 weeks of age. Since cognitive deficits are seen in young subjects with ultra-high risk for psychosis 8,9 , the juvenile offspring from poly(I:C)-treated mice may be at the prodromal stage for psychosis. Furthermore, we noticed PPI deficits in offspring from poly(I:C)-treated mice at adulthood, but not in those at the juvenile stage. Because PPI deficits in neural circuits might cause some symptoms of schizophrenia 38,39 , PPI deficits of adult offspring from poly(I:C)-treated mice mimic PPI deficits in patients with schizophrenia 40 . Our findings are similar to the behavioral abnormalities relevant to human psychiatric disorders including schizophrenia 6,10,25 . We found that decreased BDNF-TrkB signaling in the PFC and CA1 of the hippocampus may play a role in the cognitive deficits observed in offspring from poly(I:C)-treated mice and that supplementation of 7,8-DHF at 4-8 weeks of age (similar to juvenile and adolescent stages in human) in poly(I:C) offspring could treat or prevent cognitive deficits and PPI deficits at adulthood after MIA. In contrast, we found an increase expression of Bdnf gene in the PFC of offspring after poly(I:C) injection (Supplemental Fig. 1). It is likely that increase in Bdnf gene expression in the PFC may be compensatory response to decreased expression of BDNF protein in the PFC of offspring from poly(I:C)-treated mice. Prenatal infection may cause neurodevelopmental disorders in their offspring 41 , and downregulation of BDNF-TrkB signaling may be involved in this abnormal brain neurodevelopment 21,42,43 . We have reported that 7,8-DHF could attenuate behavioral abnormalities (e.g., hyperlocomotion, PPI deficits, and behavioral sensitization) and dopaminergic neurotoxicity after methamphetamine administration 44,45 . These results suggest that early treatment with a TrkB agonist during juvenile and adolescent stages may have prophylactic and therapeutic effects on behavioral abnormalities in several psychiatric disorders, including schizophrenia and substance abuse.
Adolescence is a critical period of neurodevelopment, and is also more vulnerable to psychiatric disorders 5,7 . The study provides support for the deleterious effects of early brain insult on adult behaviors and brain neurodevelopment abnormalities 46 . Therefore, although adolescence is the peak time for the onset of a number of psychiatric disorders, cognitive deficits at an early age such as the juvenile stage may present prodromal symptoms for later onset of psychiatric disorders. Supplementation with 7,8-DHF in young subjects at ultra-high risk for psychosis may play an important role in preventing the onset of psychosis.
We observed loss of PV immunoreactivity in the PrL, but not IL (infralimbic), of mPFC and CA1 of hippocampus at adulthood after MIA, which is consistent with the findings of other rodent studies 47,48 . Loss of PV-positive cells in the PFC might contribute to the pathogenesis of schizophrenia 33,34,49 . Since TrkB is predominantly expressed by PV-containing neurons in the PFC and hippocampus, decreased BDNF-TrkB signaling in PV-positive interneurons of PrL of mPFC and CA1 of hippocampus may play an important role in modulating behavioral abnormalities in the offspring after MIA.
Further, we found loss of PGC-1α immunoreactivity in the PrL, but not IL, of mPFC and CA1 of hippocampus in the adult offspring after MIA. The mPFC is a heterogeneous cortical structure composed of several nuclei, including PrL and IL cortices 50 . PrL is mainly involved in cognitive function, whereas IL appears to represent a visceromotor center homologous in primates 50 . PrL and IL play an important role in cognitive processes and visceromotor functions, respectively. Therefore, loss of PV immunoreactivity in the PrL, but not IL, of mPFC of adult offspring might be associated with cognitive deficits in MIA offspring. A recent study demonstrated a reduction in PGC-1α -dependent transcripts (e.g., PV, synaptotagmin, and complexin 1) in the anterior cingulate cortex from schizophrenia 32 . Furthermore, a recent study reported an abnormal alteration in the PGC-1α mRNA expression in the PFC from patients with schizophrenia 51 . Taken together, it is likely that reductions in the PV and PGC-1α immunoreactivity in the PrL of mPFC and CA1 of offspring from poly(I:C)-treated mice may be associated with reduced BDNF-TrkB signaling in these regions, supporting the results of a previous study showing BDNF expression through PGC-1α 52 . However, precise studies reporting the underlying BDNF-TrkB signaling through PGC-1α in neurodevelopment are needed.
In conclusion, supplementation of 7,8-DHF during juvenile and adolescent stages could prevent the onset of behavioral abnormalities and loss of PV and PGC-1α immunoreactivity in the PrL of mPFC and CA1 in the offspring after MIA. Therefore, supplementation of a TrkB agonist in young subjects at ultra-high risk for psychosis may prevent conversion to psychosis.
The first day after copulation was defined as embryonal day 0 (E0) of the pregnancy. Every 6 consecutive days from E12 to E17, the pregnant mice were injected intraperitoneally (IP) with poly(I:C) (5.0 mg/kg) dissolved in 0.2 ml 1% PBS per 20 g body weight or an equivalent volume of PBS, as previously reported 14 . The offspring were separated from their mothers after 3 weeks, and male mice were used for the experiment and caged separately in groups of three to five. The mice were treated with VEH or 7,8-DHF (1 mg/mL) in drinking water for consecutive from 4 to 8-weeks old. Subsequently, normal water was given into all mice from 8 to 10-weeks old. The mice were housed in clear polycarbonate cages (22.5 × 33.8 × 14.0 cm), under a controlled 12/12 hour light-dark cycle (lights on from 07:00 am to 07:00 pm), with room temperature at 23 ± 1 °C and humidity at 55 ± 5%. The mice were given free access to water and food pellets. All experiments were carried out in accordance with the Guide for Animal Experimentation of Chiba University. The protocol was approved by the Chiba University Institutional Animal Care and Use Committee. Chemical Industry Co., Ltd. (Tokyo, Japan), and were dissolved in phosphate buffered saline containing 17% dimethyl sulfoxide (DMSO) to generate a stock solution at 100 mg/mL concentration. The stock solution (1 ml) was added to 100 ml of Hydropac water that contained 1% sucrose (pH = 7.4), and subsequently was given to mice as drinking water (1 mg/mL of 7,8-DHF). The dose of 7,8-DHF was selected as previously reported 53 . The VEH contained the same concentration of sucrose and DMSO. The drinking water that contained either 7,8-DHF or VEH was replaced every 2 days. Other drugs were purchased from commercial sources.

Behavioral tests. Locomotion (LMT).
The mice were measured locomotor activity using an animal movement analysis system (SCANET MV-40; Melquest, Toyama, Japan) as reported previously 54 . The locomotion activity was measured 60 min.
Prepulse inhibition (PPI). The mice were tested for their acoustic startle responses in a startle chamber (SR-LAB, San Diego Instruments, San Diego, California, USA) using standard methods described previously 54 . The mice were subjected to one of six trial types: (1) pulse alone, 40-millisecond broadband burst; pulse preceded 100 milliseconds by a 20-millisecond prepulse that was (2) 4 dB (PP69), (3) 8 dB (PP73), (4) 12 dB (PP77) and (5) 16 dB (PP81) over background (65 dB); and (6) background only (no stimulus). The amount of PPI was expressed as the percentage decrease in the amplitude of the startle response caused by presentation of the prepulse (%PPI).

Novel object recognition test (NORT).
To assess the cognitive function, the mice were examined by NORT as previously reported 55 . Each mouse habituated in the open field 10 minutes every time for 3 days before the training session. During the training session, two novel objects (differing in shape and colour but of similar size) were placed into the box 35.5 cm apart (symmetrically), and each mouse was allowed to explore freely in the open field for 10 minutes. The mice were considered to be exploring the object when the head of the mouse was touching or standing on the object. The time that mice spent exploring each object was recorded. After training, mice were immediately returned to their home cages, and the box and objects were cleaned with 75% ethanol, to avoid any possible instinctive odorant cues. Retention tests were carried out at the same box 24 hour after the training session, and one of the familiar objects during training was replaced by a novel object. The mice were then allowed to explore freely for 5 minutes, and the time spent exploring each object was recorded. Throughout the experiments, the objects were counter-balanced, in terms of their physical complexity and emotional neutrality. A preference index, a ratio of the amount of time spent exploring either of the two objects (training session) or the novel one (retention session) over the total time spent exploring both objects, was used to measure recognition memory. Western blot analysis. The brain samples of prefrontal cortex (PFC), CA1, CA3 and dentate gyrus (DG) of the hippocampus, and nucleus accumbens (NAc) were prepared and Western blot analysis was performed as described previously [56][57][58] . Basically, tissue samples were homogenized in Laemmli lysis buffer. Aliquots (10 μ g) of protein were measured using the DC protein assay kit (Bio-Rad, Hercules, CA, USA), and incubated for 5 min at 95 °C, with an equal volume of 125 mM Tris/HCl, pH 6.8, 20% glycerol, 0.1% bromophenol blue, 10% β -mercaptoethanol, 4% sodium dodecyl sulfate, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, using 10% mini-gels (Mini-PROTEAN ® TGX ™ Precast Gel; Bio-Rad, CA, USA). Proteins were transferred onto polyvinylidenedifluoride (PVDF) membranes using a Trans Blot Mini Cell (Bio-Rad). For immunodetection, the blots were blocked with 2% bovine serum albumin (BSA) in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature (RT), and kept with primary antibodies overnight at 4 °C. The following primary antibodies were used: BDNF (1:1000, H-117, Santa Cruz Biotechnology, Inc., CA, USA), phosphorylated-TrkB (Tyr 706) (1:200, Santa Cruz Biotechnology), TrkB (80E3) (1:1000, Cell Signalling Technology, Danvers, MA, USA) and β -actin (1:10000, Sigma-Aldrich). The next day, blots were washed three times in TBST and incubated with horseradish peroxidase conjugated anti-rabbit antibody or anti-mouse antibody for 1 hour at room temperature. After final three washes with TBST, bands were detected using enhanced chemiluminescence (ECL) plus the Western Blotting Detection system (GE Healthcare Bioscience, Tokyo, Japan). Images were captured with a Fuji LAS3000-mini imaging system (Fujifilm, Tokyo, Japan) and immunoreactive bands were quantified.
Immunohistochemistry. Immunohistochemistry on the mouse brain sections was performed as the reported previously 57,59,60 . Mice were deeply anesthetized with sodium pentobarbital and perfused transcardially with 10 ml of isotonic saline, followed by 40 ml of ice-cold, 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4). Brains were removed from the skulls and postfixed overnight at 4 °C in the same fixative. For the immunohistochemical analysis, 50 μ m-thick serial, coronal sections of brain tissue were cut in ice-cold, 0.01 M phosphate buffered saline (pH 7.5) using a vibrating blade microtome (VT1000s, Leica Microsystems AG, Wetzlar, Germany). Free-floating sections were treated with 0.3% H 2 O 2 in 50 mM Tris-HCL saline (TBS) for 30 min and were blocked in TBS containing 0.2% Triton X-100 (TBST) and 1.5% normal goat serum for 1 h at room temperature. The samples were then incubated for 24 h at 4 °C with rabbit polyclonal anti-PV antibody (1:2,500, Swant, Bellinzona, Switzerland) or rabbit polyclonal anti-PGC-1α antibody (1:500, Abcam, Cambridge, MA, USA). The sections were washed three times in TBS and then processed using the avidin-biotin-peroxidase method (Vectastain Elite ABC, Vector Laboratories, Inc., Burlingame, CA, USA). Sections were incubated for 3 min in a solution of 0.25 mg/mL diaminobenzidine containing 0.01% H 2 O 2 . Then, sections were mounted on gelatinized slides, dehydrated, cleared, and coverslipped under Permount ® (Fisher Scientific, Fair Lawn, NJ, USA). The sections were imaged, and the staining intensity of PV or PGC-1α immunoreactivity in the infralimbic (IL) and prelimbic (PrL) of the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and hippocampus (CA1, CA3, DG) regions were analyzed using a light micro-scope equipped with a CCD camera (Olymups IX70, Tokyo, Japan) and the SCION IMAGE software package. Images of sections within mPFC (IL, PrL), NAc and hippocampal (CA1, CA3, DG) regions were captured using a 100× objective with a Keyence BZ-9000 Generation II microscope (Osaka, Japan). Statistical Analysis. The data are expressed as the mean ± standard error of the mean (SEM). Analysis was performed using PASW Statistics 20 (formerly SPSS Statistics; SPSS, Tokyo, Japan). For the offspring at 4 week study, the locomotion, NORT and western blot were analysed between the control and poly(I:C) groups by using a Student t-test. The PPI data were analysed by multivariate analysis of variance (MANOVA), followed by Student t-test. At 10 weeks of study all data collected from the offspring including the locomotion, NORT, western blot and immunohistochemistry results, were analysed by two-way ANOVA, followed by post hoc Fisher's least significant difference (LSD) test. The PPI data was analysed by MANOVA, followed by a post hoc Fisher's LSD test. Statistical significance was set at P < 0.05.