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Glucocorticoid receptor dysregulation underlies 5-HT2AR-dependent synaptic and behavioral deficits in a mouse neurodevelopmental disorder model

Open AccessPublished:September 10, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102481
      Prenatal environmental insults increase the risk of neurodevelopmental psychiatric conditions in the offspring. Structural modifications of dendritic spines are central to brain development and plasticity. Using maternal immune activation (MIA) as a rodent model of prenatal environmental insult, previous results have reported dendritic structural deficits in the frontal cortex. However, very little is known about the molecular mechanism underlying MIA-induced synaptic structural alterations in the offspring. Using prenatal (E12.5) injection with polyinosinic–polycytidylic acid potassium salt as a mouse MIA model, we show here that upregulation of the serotonin 5-HT2A receptor (5-HT2AR) is at least in part responsible for some of the effects of prenatal insults on frontal cortex dendritic spine structure and sensorimotor gating processes. Mechanistically, we report that this upregulation of frontal cortex 5-HT2AR expression is associated with MIA-induced reduction of nuclear translocation of the glucocorticoid receptor (GR) and, consequently, a decrease in the enrichment of GR at the 5-HT2AR promoter. The translational significance of these preclinical findings is supported by data in postmortem human brain samples suggesting dysregulation of GR translocation in frontal cortex of schizophrenia subjects. We also found that repeated corticosterone administration augmented frontal cortex 5-HT2AR expression and reduced GR binding to the 5-HT2AR promoter. However, virally (adeno-associated virus) mediated augmentation of GR function reduced frontal cortex 5-HT2AR expression and improved sensorimotor gating processes via 5-HT2AR. Together, these data support a negative regulatory relationship between GR signaling and 5-HT2AR expression in the mouse frontal cortex that may carry implications for the pathophysiology underlying 5-HT2AR dysregulation in neurodevelopmental psychiatric disorders.

      Keywords

      Abbreviations:

      AAV (adeno-associated virus), AP (antipsychotic), BSA (bovine serum albumin), cDNA (complementary DNA), ChIP (chromatin immunoprecipitation), CRL (Charles River Laboratories), DEPC (diethyl pyrocarbonate), DMSO (dimethyl sulfoxide), eYFP (enhanced YFP), GR (glucocorticoid receptor), HRP (horseradish peroxidase), 5-HT2AR (serotonin 5-HT2A receptor), IL-6 (interleukin 6), JAX (Jackson Laboratories), MIA (maternal immune activation), NIH (National Institutes of Health), PFA (paraformaldehyde), poly-(I:C) (polyinosinic–polycytidylic acid potassium salt), PPI (prepulse inhibition), qPCR (quantitative PCR), s.c. (subcutaneously), SFB (segmented filamentous bacteria), SSC (saline sodium citrate), Tac (Taconic Biosciences), TBST (Tris-buffered saline with Tween-20)
      Neurodevelopmental psychiatric disorders, such as schizophrenia and autism, are severe and usually cause lifelong disability (
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      Fetomaternal immune cross-talk and its consequences for maternal and offspring's health.
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      ). Accordingly, converging lines of evidence from humans (
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      Activation of the maternal immune system during pregnancy alters behavioral development of rhesus monkey offspring.
      ) animal models suggest a causal relationship between maternal immune activation (MIA) during pregnancy and neuropathological/behavioral abnormalities consistent with a range of neurodevelopmental psychiatric disorders. Previous results based on microarray and RNA-Seq assays have shown changes in gene expression levels that accompany behavioral alterations in rodent MIA models (
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      Gene expression changes in brains of mice exposed to a maternal virus infection.
      ). However, there is not presently a clear study assessing the translatability of any of these MIA-affected genes in offspring deficits relevant to neurodevelopmental psychiatric conditions.
      The serotonin 5-HT2A receptor (5-HT2AR) is a class A G protein–coupled receptor involved in processes related to cognition, perception, and mood (
      • Sharp T.
      • Barnes N.M.
      Central 5-HT receptors and their function; present and future.
      ,
      • McCorvy J.D.
      • Roth B.L.
      Structure and function of serotonin G protein-coupled receptors.
      ). Our previous data suggested upregulation of 5-HT2AR in postmortem frontal cortex samples of untreated schizophrenia subjects as compared with individually matched controls (
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      Serotonin 5-HT2A receptor expression and functionality in postmortem frontal cortex of subjects with schizophrenia: selective biased agonism via Galphai1-proteins.
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      • Callado L.F.
      • et al.
      Evaluation of 5-HT2A and mGlu2/3 receptors in postmortem prefrontal cortex of subjects with major depressive disorder: effect of antidepressant treatment.
      ,
      • Muguruza C.
      • Moreno J.L.
      • Umali A.
      • Callado L.F.
      • Meana J.J.
      • Gonzalez-Maeso J.
      Dysregulated 5-HT(2A) receptor binding in postmortem frontal cortex of schizophrenic subjects.
      ,
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      • Ang R.L.
      • Yuen T.
      • Chan P.
      • Weisstaub N.V.
      • Lopez-Gimenez J.F.
      • et al.
      Identification of a serotonin/glutamate receptor complex implicated in psychosis.
      ). Interestingly, we also reported a similar phenotype—that is, upregulation of frontal cortex 5-HT2AR density—using three independent mouse MIA models that included maternal infection with a mouse-adapted influenza virus (
      • Saunders J.M.
      • Moreno J.L.
      • Ibi D.
      • Sikaroodi M.
      • Kang D.J.
      • Munoz-Moreno R.
      • et al.
      Gut microbiota manipulation during the prepubertal period shapes behavioral abnormalities in a mouse neurodevelopmental disorder model.
      ,
      • Moreno J.L.
      • Kurita M.
      • Holloway T.
      • Lopez J.
      • Cadagan R.
      • Martinez-Sobrido L.
      • et al.
      Maternal influenza viral infection causes schizophrenia-like alterations of 5-HT2A and mGlu2 receptors in the adult offspring.
      ), maternal variable stress, and MIA with polyinosinic–polycytidylic acid potassium salt (poly-(I:C)) (
      • Holloway T.
      • Moreno J.L.
      • Umali A.
      • Rayannavar V.
      • Hodes G.E.
      • Russo S.J.
      • et al.
      Prenatal stress induces schizophrenia-like alterations of serotonin 2A and metabotropic glutamate 2 receptors in the adult offspring: role of maternal immune system.
      ). This has been followed by numerous reports revealing upregulation of cortical 5-HT2AR in rodent models of environmental insults, such as MIA with poly-(I:C) (
      • Malkova N.V.
      • Gallagher J.J.
      • Yu C.Z.
      • Jacobs R.E.
      • Patterson P.H.
      Manganese-enhanced magnetic resonance imaging reveals increased DOI-induced brain activity in a mouse model of schizophrenia.
      ) or lipopolysaccharide (
      • Wischhof L.
      • Irrsack E.
      • Dietz F.
      • Koch M.
      Maternal lipopolysaccharide treatment differentially affects 5-HT and mGlu2/3 receptor function in the adult male and female rat offspring.
      ), maternal stress (
      • Wang Y.
      • Ma Y.
      • Hu J.
      • Cheng W.
      • Jiang H.
      • Zhang X.
      • et al.
      Prenatal chronic mild stress induces depression-like behavior and sex-specific changes in regional glutamate receptor expression patterns in adult rats.
      ,
      • Akatsu S.
      • Ishikawa C.
      • Takemura K.
      • Ohtani A.
      • Shiga T.
      Effects of prenatal stress and neonatal handling on anxiety, spatial learning and serotonergic system of male offspring mice.
      ), and lipopolysaccharide administration in adult mice (
      • Savignac H.M.
      • Couch Y.
      • Stratford M.
      • Bannerman D.M.
      • Tzortzis G.
      • Anthony D.C.
      • et al.
      Prebiotic administration normalizes lipopolysaccharide (LPS)-induced anxiety and cortical 5-HT2A receptor and IL1-beta levels in male mice.
      ), which further strengthens the validity of MIA models to replicate biochemical alterations observed in postmortem schizophrenia frontal cortex samples. It remains unknown, however, whether this 5-HT2AR upregulation is involved in the negative effects on disorder-related phenotypes such as dendritic structural plasticity and sensory processes. Furthermore, the upstream signaling mechanisms leading to 5-HT2AR upregulation have not been resolved.
      Dendritic spines are a fundamental component of synaptic structural plasticity with important roles in cognitive and sensory processes (
      • Spruston N.
      Pyramidal neurons: dendritic structure and synaptic integration.
      ). Neuroanatomical studies from postmortem brains of subjects with neurodevelopmental disorders demonstrate altered density and morphology of dendritic spines (
      • Black J.E.
      • Kodish I.M.
      • Grossman A.W.
      • Klintsova A.Y.
      • Orlovskaya D.
      • Vostrikov V.
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      Pathology of layer V pyramidal neurons in the prefrontal cortex of patients with schizophrenia.
      ,
      • Glantz L.A.
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      Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia.
      )—particularly in the frontal cortex; a brain region involved in processes related to perception and cognition (
      • Kellendonk C.
      • Simpson E.H.
      • Kandel E.R.
      Modeling cognitive endophenotypes of schizophrenia in mice.
      ). Our data suggest that MIA-induced upregulation of frontal cortex 5-HT2AR occurs through a mechanism that requires dysregulation in nuclear localization and consequently binding of the glucocorticoid receptor (GR) to the promoter region of the 5-HT2AR gene. In addition, we propose that MIA-induced upregulation of frontal cortex 5-HT2AR is at least in part responsible for some of the effects of prenatal insults on frontal cortex dendritic spine structure and sensorimotor gating deficits in the offspring.

      Results

      Poly-(I:C)-induced activation of the immune system in mice

      Poly-(I:C) is a synthetic analog of dsRNA, a molecular pattern associated with viral infections, that is widely used to provoke an immune response in rodent models, including administration of the viral mimetic to induce immune activation in pregnant mice (
      • Kentner A.C.
      • Bilbo S.D.
      • Brown A.S.
      • Hsiao E.Y.
      • McAllister A.K.
      • Meyer U.
      • et al.
      Maternal immune activation: reporting guidelines to improve the rigor, reproducibility, and transparency of the model.
      ). Recent studies suggest that the immunogenicity and alterations observed in the offspring following administration of poly-(I:C) to pregnant mice may vary in intensity depending on both source and lot of the dsRNA synthetic analogs (
      • Mueller F.S.
      • Richetto J.
      • Hayes L.N.
      • Zambon A.
      • Pollak D.D.
      • Sawa A.
      • et al.
      Influence of poly(I:C) variability on thermoregulation, immune responses and pregnancy outcomes in mouse models of maternal immune activation.
      ,
      • Careaga M.
      • Taylor S.L.
      • Chang C.
      • Chiang A.
      • Ku K.M.
      • Berman R.F.
      • et al.
      Variability in PolyIC induced immune response: implications for preclinical maternal immune activation models.
      ). To assess the effect of poly-(I:C) on the immune response within our experimental system, we tested immunoreactivity and expression of interleukin 6 (IL-6), a key proinflammatory mediator induced by poly-(I:C) administration (
      • Smith S.E.
      • Li J.
      • Garbett K.
      • Mirnics K.
      • Patterson P.H.
      Maternal immune activation alters fetal brain development through interleukin-6.
      ), in serum and frontal cortex samples of nonpregnant adult female 129S6/SvEv (in-house) mice and C57BL6/N mice from Charles River Laboratories (CRL) (see also Table 1 for information regarding inclusion of male and/or female mice throughout). Mice were administered (i.p.) poly-(I:C) (20 mg/kg) or vehicle and sacrificed 2.5 h afterward. Our data show that this model of immune activation leads to an increase in IL-6 mRNA expression in frontal cortex samples as well as an increase in IL-6 immunoreactivity in serum samples in both 129S6/SvEv (in-house) and C57BL6/N (CRL) animals (Fig. 1, A–D). In addition, poly-(I:C)-treated C57BL6/N (CRL) mice showed reduced body weight 24 h after immune activation (Fig. 1E).
      Table 1Number of litters per group and inclusion of male and/or female mice in the experimental groups
      FigureNumber of litters per groupMouse strainSex
      Figure 1, ADN/AC57BL6/N and 129S6/SvEvFemale
      Figure 1EN/AC57BL6/NFemale
      Figure 2, ACN/AC57BL6/N, C57BL6/J, 129S6/SvEvFemale
      Figure 3, A and B2 (Mock) – 3 (MIA)C57BL6/NMale
      Figure 3, C and D3 (Mock) – 4 (MIA)C57BL6/NMale and female
      Figure 5AN/A129S6/SvEvMale
      Figure 5, BF7 (Mock) – 10 (MIA)129S6/SvEvMale and female
      Figure 6CN/AC57BL6/NMale
      Figure 6, D and E3 (Mock) – 4 (MIA)C57BL6/NMale
      Figure 7, C and D3 (Mock) – 4 (MIA)C57BL6/NMale
      Figure 8, ACN/A129S6/SvEvMale
      Figure 9, AGN/A129S6/SvEvMale
      Figure 10DN/A129S6/SvEvMale
      Figure 11, ADN/A129S6/SvEvMale and female
      Figure 11, EHN/A129S6/SvEvMale
      Figure 12, ADN/A129S6/SvEvMale
      Fig. S1, B and CN/AC57BL6/NFemale
      Abbreviation: N/A, not applicable.
      Figure thumbnail gr1
      Figure 1Poly-(I:C) induces an immune response in nonpregnant female mice. AD, adult female 129S6/SvEv (in-house) (A and C) and C57BL6/N (CRL) (B and D) mice were injected (i.p.) with poly-(I:C) (20 mg/kg) or vehicle, and sacrificed 2.5 h after administration. IL-6 mRNA was assessed by quantitative RT–PCR (qRT–PCR) in mouse frontal cortex samples (A and B), and IL-6 immunoreactivity was assessed by ELISAs in serum samples (C and D) (n = 3–4 female mice/group). E, adult female C57BL6/N (CRL) mice were injected (i.p.) with poly-(I:C) (20 mg/kg) or vehicle, and percentage change in weight was evaluated 24 h after (n = 8 female mice/group). Data show mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Unpaired two-tailed Student’s t test (A: t4 = 5.21, p < 0.01; B: t4 = 3.25, p < 0.05; C: t4 = 10.65, p < 0.001; D: t6 = 3.07, p < 0.05; and E: t14= 5.17, p < 0.001). CRL, Charles River Laboratories; poly-(I:C), polyinosinic–polycytidylic acid potassium salt.
      Previous findings have also suggested that maternal gut bacteria composition represents an additional source of variation in neurodevelopmental abnormalities in poly-(I:C)-induced MIA models (
      • Kim S.
      • Kim H.
      • Yim Y.S.
      • Ha S.
      • Atarashi K.
      • Tan T.G.
      • et al.
      Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring.
      ). Particularly, it has been reported that MIA-induced behavioral alterations were more evident in C57BL6/N mice from Taconic Biosciences (Tac) as compared with C57BL6/J from Jackson Laboratories (JAX), and these MIA-induced deficits correlated with the presence of segmented filamentous bacteria (SFB) in the pregnant mother. To evaluate the presence of SFB in female mice, we extracted DNA from cecum contents of 129S6/SvEv (in-house), C57BL6/N (CRL), C57BL6/J (JAX), and C57BL6/N (Tac) animals and performed quantitative PCR (qPCR) assays targeting the 16S rRNA gene of SFB. Consistent with previous findings (
      • Kim S.
      • Kim H.
      • Yim Y.S.
      • Ha S.
      • Atarashi K.
      • Tan T.G.
      • et al.
      Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring.
      ), our data show that C57BL6/J (JAX) animals were SFB deficient, whereas the presence of SFB was more evident in C57BL6/N (CRL) mice as compared with 129S6/SvEv (in-house) and C57BL6/N (Tac) females (Fig. 2, A and B). Evaluating the total abundance of the bacterial 16S rRNA gene using universal qPCR primers that detect this gene across the vast majority of bacterial taxa, we found that C57BL6/N (JAX) mice exhibited a significantly greater abundance of 16S rRNA gene copies in the cecum as compared with mice from any of the other three sources (Fig. 2C).
      Figure thumbnail gr2
      Figure 2Gut microbiota are enriched for segmented filamentous bacteria (SFB) in 129S6/SvEv (in-house), C57BL6/N (CRL), and C57BL6/N (Tac), but not C57BL6/J (JAX) adult female mice. AC, DNA was extracted from mouse cecum contents, and the 16S rRNA gene was assessed by qPCR using primer pairs targeting either SFB (A) or universal primer pairs that detect this gene across the vast majority of bacterial taxa (C) (n = 3 female mice/group). B, the same data as in “A” are depicted, but the C57BL6/N (CRL) mice were excluded from the data analysis. Data show mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. One-way ANOVA followed by Bonferroni’s post hoc test (A: F[3,8] = 8.40, p < 0.01; B: F[2,6] = 30.95, p < 0.001; C: F[3,8] = 11.9, p < 0.01). CRL, Charles River Laboratories; JAX, Jackson Laboratories; qPCR, quantitative PCR; Tac, Taconic Biosciences.

      MIA-induced upregulation of 5-HT2AR and deficits in sensorimotor gating

      Our previous work suggested upregulation of both 5-HT2AR density by radioligand-binding assays and 5-HT2AR mRNA expression by qRT–PCR assays in the frontal cortex of adult mice born to mothers infected with a mouse-adapted influenza virus during pregnancy (
      • Saunders J.M.
      • Moreno J.L.
      • Ibi D.
      • Sikaroodi M.
      • Kang D.J.
      • Munoz-Moreno R.
      • et al.
      Gut microbiota manipulation during the prepubertal period shapes behavioral abnormalities in a mouse neurodevelopmental disorder model.
      ,
      • Moreno J.L.
      • Kurita M.
      • Holloway T.
      • Lopez J.
      • Cadagan R.
      • Martinez-Sobrido L.
      • et al.
      Maternal influenza viral infection causes schizophrenia-like alterations of 5-HT2A and mGlu2 receptors in the adult offspring.
      ). Using a FISH approach, our data here show that frontal cortex tissue sections from adult MIA offspring exhibit an increase in 5-HT2AR mRNA relative to mock C57BL6/N offspring mice (Fig. 3, A and B). Controls to validate the specificity and selectivity of FISH assay included decreased fluorescent signal upon dilution of the mRNA probe (Fig. S1, A and B) as well as the absence of fluorescent signal in frontal cortex tissue sections exposed to a sense strand 5-HT2AR mRNA probe (Fig. S1C).
      Figure thumbnail gr3
      Figure 3MIA increases frontal cortex 5-HT2AR mRNA expression and disrupts sensorimotor gating in the adult offspring. A and B, increased expression of 5-HT2AR mRNA in the frontal cortex of adult MIA mice. Representative FISH images with the antisense 5-HT2AR probe (A), and quantification of fluorescence intensity (n = 61 cells from four male C57BL6/N CRL mice/group; 2–3 litters/group) (B). C and D, effect of MIA on %PPI (C) and startle amplitude (D) in the adult offspring (n = 21–32 male and female C57BL6/N CRL mice/group; 3–4 litters/group). Data show mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001. Unpaired two-tailed Student’s t test (B: t120 = 2.12, p < 0.05; D: t51 = 2.25, p < 0.05). Two-way ANOVA followed by Bonferroni’s post hoc test (C: MIA versus mock F[1,153] = 12.52, p < 0.001; prepulse intensity F[2,153] = 46.90, p < 0.001; interaction F[2,153] = 0.04, p > 0.05). Nuclei were stained in blue with Hoechst (A). The scale bar represents 200 μm (A). CRL, Charles River Laboratories; 5-HT2AR, serotonin 5-HT2A receptor; MIA, maternal immune activation; PPI, prepulse inhibition.
      Prepulse inhibition (PPI) of startle is a conserved sensorimotor gating behavioral model diminished in individuals with certain psychiatric conditions (
      • Siegel S.J.
      • Talpos J.C.
      • Geyer M.A.
      Animal models and measures of perceptual processing in schizophrenia.
      ). Similar to previous findings (
      • Shi L.
      • Fatemi S.H.
      • Sidwell R.W.
      • Patterson P.H.
      Maternal influenza infection causes marked behavioral and pharmacological changes in the offspring.
      ), we observed PPI deficits in MIA adult offspring relative to C57BL6/N wildtype controls (Fig. 3C). In addition, adult MIA mice exhibited a decrease in startle amplitude during the initial five trials of the PPI paradigm (Fig. 3D). A three-way ANOVA demonstrated no sex-related effect on %PPI (Table S1). Similarly, a two-way ANOVA demonstrated no sex-related effect on startle amplitude (MIA: F[1,49] = 5.13, p < 0.05; sex: F[1,49] = 0.04, p > 0.05).
      Together with our results in Figures 1 and 2, these data corroborate that 129S6/SvEv (in-house) and C57BL6/N (CRL) mice can serve as a preclinical model to evaluate the effects of MIA on neurodevelopmental processes in the offspring.

      MIA-induced reduction of mature dendritic spines via 5-HT2AR

      Previous findings suggest that MIA affects offspring’s frontal cortex dendritic spine structure (
      • Cotter D.
      • Takei N.
      • Farrell M.
      • Sham P.
      • Quinn P.
      • Larkin C.
      • et al.
      Does prenatal exposure to influenza in mice induce pyramidal cell disarray in the dorsal hippocampus?.
      ,
      • Coiro P.
      • Padmashri R.
      • Suresh A.
      • Spartz E.
      • Pendyala G.
      • Chou S.
      • et al.
      Impaired synaptic development in a maternal immune activation mouse model of neurodevelopmental disorders.
      ,
      • Baharnoori M.
      • Brake W.G.
      • Srivastava L.K.
      Prenatal immune challenge induces developmental changes in the morphology of pyramidal neurons of the prefrontal cortex and hippocampus in rats.
      ). To determine whether alterations in frontal cortex 5-HT2AR-dependent signaling are involved in the effects of MIA on offspring’s frontal cortex dendritic structural plasticity, heterozygous 5-HT2AR+/− 129S6/SvEv pregnant mice were injected with poly-(I:C) or vehicle, and cortical dendritic structure assays were tested in adult 5-HT2AR+/+ or 5-HT2AR−/− offspring (Fig. 4A). To target cortical pyramidal neurons, adult MIA and mock mice were stereotaxically injected into the frontal cortex with adeno-associated virus serotype 8 (AAV8) preparations expressing enhanced YFP (eYFP) under the control of the CaMKIIα promoter (Fig. 5A). Using a similar viral vector, our previous findings reported a robust overexpression of eYFP in excitatory pyramidal CaMKIIα-positive but not inhibitory parvalbumin-positive neurons (
      • Ibi D.
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      • Muguruza C.
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      • et al.
      Antipsychotic-induced Hdac2 transcription via NF-kappaB leads to synaptic and cognitive side effects.
      ). In slices prepared approximately 3 weeks after viral injection, when transgene expression is maximal, this approach enabled us to explicitly discriminate single pyramidal cells and their dendritic spines. Notably, MIA offspring mice showed a selective reduction of mature mushroom spines in CaMKIIα-positive neurons (Fig. 5, B and E). There was also a trend toward MIA effect on stubby spines (Fig. 5, B and C) and total spine density (Fig. 5, B and F), whereas immature thin spines remained unaffected (Fig. 5, B and D). This effect of MIA required expression of 5-HT2AR since the frontal cortex synaptic remodeling alteration was not observed in 5-HT2AR−/− littermates (Fig. 5, B–F). Similar to previous reports (
      • de la Fuente Revenga M.
      • Zhu B.
      • Guevara G.A.
      • Naler L.B.
      • Saunders J.M.
      • Zhou Z.
      • et al.
      Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.
      ), adult mice with selective deletion of the 5-HT2AR gene presented a trend toward reduction in the density of frontal cortex dendritic spines (Fig. 5, B–F). A three-way ANOVA demonstrated no sex-related effect on MIA-induced alterations in the density of mature mushroom spines. However, a three-way ANOVA suggested sex-related differences in immature thin spines and total spine density (Tables S2–S5).
      Figure thumbnail gr4
      Figure 4Breeding 5-HT2AR−/− strategy in MIA mice. A, Heterozygous 5-HT2AR+/− mice were bred to obtain 5-HT2AR+/+ and 5-HT2AR−/− offspring. Pregnant females (E12.5) received a single injection of poly-(I:C) (20 mg/kg) or vehicle. Adult 5-HT2AR+/+ and 5-HT2AR−/− mice born to either mock or MIA mothers were stereotaxically injected with AAV-eYFP and sacrificed for analysis 3 weeks after surgery. AAV, adeno-associated virus; eYFP, enhanced YFP; 5-HT2AR, serotonin 5-HT2A receptor; MIA, maternal immune activation; poly-(I:C), polyinosinic–polycytidylic acid potassium salt.
      Figure thumbnail gr5
      Figure 5MIA decreases mature synaptic structural elements in mouse frontal cortex via 5-HT2AR. A, eYFP expression by immunohistochemistry. B, representative three-dimensional reconstructions of AAV-injected cortical dendritic segments. CF, stubby (C), thin (D), mushroom (E), and total (F) frontal cortex spine density in adult 5-HT2AR+/+ and 5-HT2AR−/− mice born to either mock or MIA mothers (n = 73–109 neurons from four to six male and female 129S6/SvEv in-house mice/group; 467-10 litters/group). Data show mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001, ns, not significant. Two-way ANOVA followed by Bonferroni’s post hoc test (C: stubby MIA versus mock F[1,363] = 3.64, p = 0.057; stubby genotype F[1,363] = 1.96, p > 0.05; stubby interaction F[1,363] = 0.01, p > 0.05; D: thin MIA versus mock F[1,363] = 0.48, p > 0.05; thin genotype F[1,363] = 1.97, p > 0.05; thin interaction F[1,363] = 0.11, p > 0.05; E: mushroom MIA versus mock F[1,363] = 6.58, p < 0.05; mushroom genotype F[1,363] = 3.01, p = 0.08; mushroom interaction F[1,363] = 8.45, p < 0.05; F: total MIA versus mock F[1,363] = 3.72, p = 0.054; total genotype F[1,363] = 3.58, p = 0.059; total interaction F[1,363] = 0.54, p > 0.05). The scale bars represent 100 μm (A) and 5 μm (B). AAV, adeno-associated virus; eYFP, enhanced YFP; 5-HT2AR, serotonin 5-HT2A receptor; MIA, maternal immune activation.

      Reduced GR binding to the 5-HT2AR promoter in MIA mice

      GRs, which belong to an evolutionary conserved nuclear receptor superfamily, are involved in the mechanism by which steroid hormones, such as cortisol in most mammals including humans and corticosterone in laboratory rodents, regulate stress responsiveness as ligand-dependent transcription factors across different species (
      • Stolte E.H.
      • van Kemenade B.M.
      • Savelkoul H.F.
      • Flik G.
      Evolution of glucocorticoid receptors with different glucocorticoid sensitivity.
      ). Previous reports raise the possibility that the GR regulates 5-HT2AR transcription in vitro in different experimental systems in a manner expected to be conserved from mice to humans (
      • Falkenberg V.R.
      • Rajeevan M.S.
      Identification of a potential molecular link between the glucocorticoid and serotonergic signaling systems.
      ), but the basic molecular mechanism underlying crosstalk between the GR and serotonin receptor systems in vivo remains unsolved. Using the consensus sequence for the GR obtained from transcription factor–binding datasets (
      • Sandelin A.
      • Alkema W.
      • Engstrom P.
      • Wasserman W.W.
      • Lenhard B.
      JASPAR: an open-access database for eukaryotic transcription factor binding profiles.
      ), we detected a predicted GR-binding site at the promoter region of the mouse 5-HT2AR gene (Fig. 6A). Based on these findings, we performed a series of chromatin immunoprecipitation followed by qPCR (ChIP–qPCR) assays with an anti-GR antibody in mouse frontal cortex samples throughout different regions of the 5-HT2AR gene (Fig. 6B). Our ChIP–qPCR data revealed enrichment of GR at the predicted binding site but not at other locations along the 5-HT2AR gene (Fig. 6C).
      Figure thumbnail gr6
      Figure 6MIA dysregulates binding of GR to the 5-HT2AR promoter in mouse frontal cortex. A, alignment of the vertebrate GR consensus binding site with the promoter of the mouse 5-HT2AR gene. B, map of the 5-HT2AR gene showing position of primers used for qPCR assays. Yellow box indicates location of the predicted GR-binding site. C, GR binds to the promoter region of the 5-HT2AR gene in mouse frontal cortex (n = 8 male C57BL6/N CRL mice/group). D and E, binding of GR to the region of the 5-HT2AR gene containing a putative GR-binding site (D), but not within exon 1 (E), is decreased in frontal cortex samples of adult MIA mice (n = 4–8 male C57BL6/N CRL mice/group; 3–4 litters/group). Data show mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant. Two-way ANOVA followed by Bonferroni’s post hoc test (C: antibody F[1,73] = 3.83, p = 0.054; gene site F[5,73] = 2.80, p < 0.05). Unpaired two-tailed Student’s t test (D: t10 = 3.57, p < 0.01; E: t10 = 0.23, p > 0.05). CRL, Charles River Laboratories; GR, glucocorticoid receptor; 5-HT2AR, serotonin 5-HT2A receptor; MIA, maternal immune activation; qPCR, quantitative PCR; TSS, transcriptional start site.
      Following this finding in naïve mice, we next sought to determine the effect of MIA on GR enrichment at the 5-HT2AR promoter in mouse frontal cortex. Notably, ChIP–qPCR data reveal that prenatal MIA leads to a significant decrease in the enrichment of GR at the 5-HT2AR promoter (Fig. 6D) but not at a different location within exon 1 (Fig. 6E).

      Dysregulated GR translocation in MIA mice and schizophrenia brain samples

      To gain insights into a potential mechanism underlying these findings, and given that activation of the GR pathway can induce GR nuclear translocation, we tested whether prenatal MIA affects subcellular localization of GR in mouse frontal cortex. Consistent with previous studies, GR immunoreactivity was observed in both nuclear and cytoplasmic preparations of frontal cortex samples from control mice (Fig. 7, A and B). Importantly, MIA offspring animals showed decreased immunoreactivity against nuclear (Fig. 7, A and C), but not cytoplasmic (Fig. 7, B and D), GR in the frontal cortex.
      Figure thumbnail gr7
      Figure 7Dysregulation of GR localization in MIA mice and postmortem frontal cortex samples of schizophrenia subjects. AD, immunoblots showed downregulation of GR protein in nuclear (C) but not cytoplasmic (D) preparations from the frontal cortex of adult MIA mice as compared with mock animals. Representative immunoblots are shown (A and B) (n = 6–7 male C57BL6/N CRL mice/group; 3–4 litters/group). EH, immunoblots showed upregulation of GR protein in cytoplasmic (H) but not nuclear (G) preparations from postmortem human brain samples of schizophrenia subjects as compared with controls (for demographic information, see and ). Representative immunoblots are shown (E and F). Data show mean ± SEM. ∗p < 0.05, ns, not significant. Unpaired two-tailed Student’s t test (C: t11 = 2.34, p < 0.05; D: t11 = 0.57, p > 0.05; G: t62 = 0.47, p > 0.05; H: t59 = 2.35, p < 0.05). GR, glucocorticoid receptor; MIA, maternal immune activation.
      To test for potential dysregulation of GR trafficking in individuals with neurodevelopmental psychiatric conditions, we performed cellular fractionation followed by immunoblot assays with anti-GR antibodies in postmortem frontal cortex samples of schizophrenia subjects and individually matched controls (Tables 2 and 3). Interestingly, we observed a statistically significant difference related to GR subcellular localization in frontal cortex samples from schizophrenia subjects as compared with controls (Fig. 7, E–H). This alteration in GR nuclear translocation was also apparent as a trend in both antipsychotic-free and antipsychotic-treated schizophrenia subjects versus their respective control groups (nuclear: antipsychotic-free versus controls, Student’s t test, t30 = 1.09, p > 0.05; antipsychotic-treated versus controls, Student’s t test, t30 = 0.45, p > 0.05) (cytoplasmic: antipsychotic-free versus controls, Student’s t test, t28 = 1.49, p = 0.14; antipsychotic-treated versus controls, Student’s t test, t29 = 1.85, p = 0.07).
      Table 2Demographic characteristics of antipsychotic-free schizophrenia subjects and their respective control subjects
      Sex (F/M)Age at death (years)Postmortem delay (h)Storage time (months)Antipsychotic treatmentAdditional drugs
      Schizophrenia 1M3051203(−)(−)
      Control 1M291868(−)
      Schizophrenia 2M4820122(−)(−)
      Control 2M4718142BDZ
      Schizophrenia 3M3111113(−)(−)
      Control 3M311399ETH (0.96 g/l)
      Schizophrenia 4M453106(−)BDZ
      Control 4M442192(−)
      Schizophrenia 5M2724104(−)(−)
      Control 5M2830178(−)
      Schizophrenia 6M462269(−)(−)
      Control 6M462467(−)
      Schizophrenia 7M481150(−)(−)
      Control 7M49843(−)
      Schizophrenia 8M451859(−)(−)
      Control 8M471544(−)
      Schizophrenia 9M341562(−)(−)
      Control 9M3648117(−)
      Schizophrenia 10M52766(−)BDZ
      Control 10M511346(−)
      Schizophrenia 11M491233(−)BDZ
      Control 11M46628ETH (1.91 g/l)
      Schizophrenia 12M601243(−)BDZ
      Control 12M62966(−)
      Schizophrenia 13F672252(−)(−)
      Control 13F661624(-)
      Schizophrenia 14M3423116(−)(−)
      Control 14M332828ETH (0.97 g/l)
      Schizophrenia 15M3252121(−)BDZ
      Control 15M32444ETH (2.98 g/l)
      Schizophrenia 16M3221122(−)(−)
      Control 16M321633(−)
      Schizophrenia1F/15M42.5 ± 2.920.3 ± 3.490.1 ± 10.9
      Control1F/15M42.4 ± 2.917.9 ± 2.769.9 ± 11.2
      Abbreviations: BDZ, benzodiazepine; ETH, ethanol; F, female; M, male.
      Antipsychotics were not detected in blood samples of schizophrenia subjects at the time of death.
      Table 3Demographic characteristics of antipsychotic-treated schizophrenia subjects and their respective control subjects
      Sex (F/M)Age at death (years)Postmortem delay (h)Storage time (months)Antipsychotic treatmentAdditional drugs
      Schizophrenia 17M3018130OLZ(−)
      Control 17M2936185ETH (1.71 g/l)
      Schizophrenia 18M328125QTPBDZ
      Control 18M32424(−)
      Schizophrenia 19M2316120SUL(−)
      Control 19M231793(−)
      Schizophrenia 20M353120QTPBDZ
      Control 20M362363(−)
      Schizophrenia 21M3323107CLZ(−)
      Control 21M33499(−)
      Schizophrenia 22M351170CLZBDZ
      Control 22M3618140ETH (1.69 g/l)
      Schizophrenia 23F4238129CLZ, QTP, SULBDZ
      Control 23F382246(−)
      Schizophrenia 24M37875OLZBDZ, ETH (0.90 g/l)
      Control 24M4124180(−)
      Schizophrenia 25F602354CLZ, SULBDZ
      Control 25F604860BDZ
      Schizophrenia 26F5613119CLZ(−)
      Control 26F5820176(−)
      Schizophrenia 27M43521OLZBDZ
      Control 27M442924(−)
      Schizophrenia 28M58621CLP(−)
      Control 28M57339(−)
      Schizophrenia 29M511822PALBDZ
      Control 29M50233(−)
      Schizophrenia 30M512825OLZ,CLPBDZ
      Control 30M502420(−)
      Schizophrenia 31M342128HALBDZ
      Control 31M331733(−)
      Schizophrenia 32M581633APZ(−)
      Control 32M561518(−)
      Schizophrenia3F/13M42.4 ± 2.915.9 ± 2.374.9 ± 11.4
      Control3F/13M42.3 ± 2.919.1 ± 3.177.1 ± 15.3
      Abbreviations: BDZ, benzodiazepine; ETH, ethanol.
      Therapeutic levels of aripriprazole (APZ), clotiapine (CLP), clozapine (CLZ), haloperidol (HAL), olanzapine (OLZ), paliperidone (PAL), quetiapine (QTP), and sulpiride (SUL) were detected in blood samples of schizophrenia subjects at the time of death.

      Repeated corticosterone administration upregulates 5-HT2AR expression in mice

      Following our data suggesting that MIA produces both upregulation of 5-HT2AR mRNA expression and decreased GR binding to the 5-HT2AR promoter in mouse frontal cortex, we focused our investigation on the effects of direct manipulation of the GR system on 5-HT2AR-related behavioral phenotypes. Previous investigation has reported that a regimen of corticosterone twice a day for 4 days produces an increase in head-twitch behavior—a rodent behavioral proxy of human hallucinogenic potential (
      • Hanks J.B.
      • Gonzalez-Maeso J.
      Animal models of serotonergic psychedelics.
      )—in response to the psychedelic 5-HT2AR agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane in rats (
      • Berendsen H.H.
      • Kester R.C.
      • Peeters B.W.
      • Broekkamp C.L.
      Modulation of 5-HT receptor subtype-mediated behaviours by corticosterone.
      ). To evaluate and optimize this experimental system in 129S6/SvEv mice, we first administered (subcutaneously [s.c.]) corticosterone (50 mg/kg) twice a day for up to 8 days and sacrificed 8.5 to 13 h after the last injection. Control groups included mice injected with vehicle as well as injection-naïve mice. Previous findings have established FKBP5 as an interactor that opposes nuclear translocation of the GR and hence decreases GR-dependent transcriptional activity. Upon glucocorticoid binding, however, FKBP5 is exchanged for FKBP4, a cochaperone that promotes GR nuclear translocation and transcriptional regulation (
      • Zannas A.S.
      • Wiechmann T.
      • Gassen N.C.
      • Binder E.B.
      Gene-stress-epigenetic regulation of FKBP5: clinical and translational implications.
      ). Our data suggest that neither GR (Fig. 8A) nor FKBP4 (Fig. 8B) mRNA expression was affected upon repeated corticosterone administration. However, FKBP5 mRNA (Fig. 8C) showed upregulation in the frontal cortex of mice after either 1, 2, 4, 6, or 8 days of repeated corticosterone administration, as compared with vehicle; the injection-naïve group was not significantly different from vehicle. These findings suggest that repeated corticosterone administration activates and elicits negative feedback within the GR pathway in the mouse frontal cortex. We therefore elected to use 4 days as the treatment duration for our studies.
      Figure thumbnail gr8
      Figure 8A time course of repeated corticosterone administration reveals FKBP5 mRNA induction in mice. AC, adult male mice were administered (s.c.) corticosterone (50 mg/kg) twice a day for either 1, 2, 4, 6, or 8 days and sacrificed to collect samples 8.5 to 13 h after the last injection. Control groups included mice injected with vehicle as well as injection-naïve mice. Expression of GR (A), FKBP4 (B), and FKBP5 (C) mRNAs in frontal cortex was assessed by qRT–PCR (n = 4 male 129S6/SvEv in-house mice/group). Data show mean ± SEM. ∗p < 0.05, ∗∗p < 0.01. One-way ANOVA followed by Dunnett’s post hoc test (A: F[6,21] = 2.17, p > 0.05; B: F[6,21] = 1.38, p > 0.05; C: F[6.21] = 6.26, p < 0.001). s.c., subcutaneously; qRT, quantitative RT.
      Upon repeated (twice a day for 4 days) administration (s.c.) of corticosterone (50 mg/kg), we found upregulation of 5-HT2AR mRNA expression in the mouse frontal cortex, an effect that was not observed with the closely related 5-HT2CR or the dopamine D2 receptor (Fig. 9A). In addition, this paradigm of repeated corticosterone administration led to a decrease of GR enrichment at its predicted binding site within the 5-HT2AR promoter (Fig. 9B), an effect that was not observed at a region within exon 1 (Fig. 9C).
      Figure thumbnail gr9
      Figure 9Repeated corticosterone administration upregulates frontal cortex 5-HT2AR mRNA expression and disrupts sensorimotor gating in mice. A, increased expression of 5-HT2AR mRNA in frontal cortex after repeated corticosterone administration. Mice were treated (twice a day for 4 days) with corticosterone (50 mg/kg; s.c.) or vehicle and sacrificed for analysis 8.5 to 13.5 h after the last injection. Expression of 5-HT2AR, 5-HT2CR, and D2R mRNAs in frontal cortex was assessed by qRT–PCR (n = 9 male 129S6/SvEv mice/group). B and C, enrichment of the GR at the region of the 5-HT2AR gene containing a putative GR-binding site (B), but not within exon 1 (C), is decreased upon repeated corticosterone administration in mouse frontal cortex (n = 4 male 129S6/SvEv mice/group). DG, effect of repeated corticosterone administration on %PPI (D and E) and startle amplitude (F and G) in 5-HT2AR+/+ (n = 15 male 129S6/SvEv mice/group) and 5-HT2AR−/− (n = 11–13 male 129S6/SvEv mice/group) animals. Data show mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Unpaired two-tailed Student’s t test (A: 5-HT2AR t16 = 2.98, p < 0.01; 5-HT2CR t16 = 0.06, p > 0.05; D2R t16 = 0.23, p > 0.05; B: t6 = 6.95, p < 0.001; C: t6 = 0.38, p > 0.05; F: t28 = 1.28, p > 0.05; G: t22 = 1.30, p > 0.05). Two-way ANOVA followed by Bonferroni’s post hoc test (B: corticosterone versus vehicle F[1,84] = 4.87, p < 0.05; prepulse intensity F[2,84] = 67.28, p < 0.001; interaction F[2,84] = 0.47, p > 0.05; E: corticosterone versus vehicle F[1,66] = 4.20, p < 0.05; prepulse intensity F[2,66] = 66.62, p < 0.001; interaction F[2,66] = 0.24, p > 0.05; for three-way ANOVA, see ). D2R, dopamine D2 receptor; GR, glucocorticoid receptor; 5-HT2AR, serotonin 5-HT2A receptor; PPI, prepulse inhibition; qRT, quantitative RT; s.c., subcutaneously.
      Using the same model of repeated corticosterone administration, we next evaluated the effects on sensorimotor gating and the role of 5-HT2AR-dependent signaling in mediating this behavior. Corticosterone treatment reduced %PPI in 5-HT2AR+/+ mice (Fig. 9D), an effect that was also observed in 5-HT2AR−/− mice (Fig. 9E). Startle magnitude was unaffected upon repeated corticosterone treatment (Fig. 9, F and G), whereas 5-HT2AR−/− mice showed a statistically significant increase in %PPI as a genotype effect compared with 5-HT2AR+/+ controls (Fig. 9, D and E, and Table 4). These data suggest that although repeated corticosterone treatment augments 5-HT2AR mRNA expression in mouse frontal cortex—an effect that occurs in parallel with decreased binding of the GR the 5-HT2AR promoter—the negative effect of this repeated pharmacological activation of the GR on sensorimotor gating processes does not seem to be dependent on 5-HT2AR-dependent signaling mechanisms.
      Table 4Three-way ANOVA of the effect of repeated corticosterone versus vehicle treatment on %PPI in male 129S6/SvEv (in-house) mice
      Source of variationANOVAp
      Prepulse intensityF[2,150] = 125.7p < 0.001
      TreatmentF[1,150] = 8.55p < 0.01
      GenotypeF[1,150] = 6.84p < 0.01
      Prepulse intensity × treatmentF[2,150] = 0.17p > 0.05
      Prepulse intensity × genotypeF[2,150] = 0.70p > 0.05
      Genotype × treatmentF[1,150] = 0.095p > 0.05
      Prepulse intensity × genotype × treatmentF[2,150] = 0.54p > 0.05

      AAV-mediated augmentation of GR nuclear translocation affects PPI via 5-HT2AR

      Systemically administered glucocorticoids exert a wide variety of physiological effects and pharmacological actions. To further test the functional significance of changes in frontal cortex GR-dependent transcriptional activity on 5-HT2AR expression, we next evaluated the effects of AAV-mediated gene transfer of a previously described GR construct (ΔGR) (
      • Godowski P.J.
      • Rusconi S.
      • Miesfeld R.
      • Yamamoto K.R.
      Glucocorticoid receptor mutants that are constitutive activators of transcriptional enhancement.
      ,
      • Sher L.
      • Oquendo M.A.
      • Galfalvy H.C.
      • Grunebaum M.F.
      • Burke A.K.
      • Zalsman G.
      • et al.
      The relationship of aggression to suicidal behavior in depressed patients with a history of alcoholism.
      ), which lacks the hormone-binding domain and is therefore able to constitutively translocate to the nucleus where it has the potential to affect transcription. The ΔGR construct generated from mouse complementary DNA (cDNA) was subcloned into the AAV8 viral vector under the control of the CaMKIIα promoter, which, as mentioned previously, allows selective expression of the transgene in frontal cortex glutamatergic pyramidal, but not GABAergic inhibitory, neurons. In addition, the inclusion of the p2A peptide allows for expression of ΔGR and eYFP as independent proteins from the same transcript (
      • Szymczak-Workman A.L.
      • Vignali K.M.
      • Vignali D.A.
      Design and construction of 2A peptide-linked multicistronic vectors.
      ) (Fig. 10A). This was validated by immunoblot (Fig. 10B) and immunocytochemical (Fig. 10C) assays in Neuro-2a cells, which endogenously express the CaMKIIα gene, as well as immunohistochemical assays in mouse frontal cortex tissue sections (Fig. 10D). First, we confirmed that mice injected with the AAV-CaMKIIα:ΔGR-p2A-eYFP construct (AAV-ΔGR-eYFP) showed elevated expression of GR in the frontal cortex (Fig. 11A), as well as upregulation of FKBP5 (Fig. 11C), but not FKBP4 (Fig. 11B), mRNA expression. Notably, such overexpression of ΔGR decreased 5-HT2AR mRNA but not 5-HT2CR or dopamine D2 receptor mRNAs in this brain region (Fig. 11D).
      Figure thumbnail gr10
      Figure 10Virally mediated augmentation of GR function. A, schematic representation of the recombinant AAV vector used to overexpress both ΔGR and eYFP under the CaMKIIα promoter in mouse frontal cortex pyramidal neurons. The arrowhead indicates p2A cleave site. B, representative immunoblot of Neuro-2a cells transfected with mock or AAV-ΔGR-p2A-eYFP plasmids. C, representative immunocytochemical images of Neuro-2a cells transfected with mock, pCMV-hGR, or AAV-ΔGR-p2A-eYFP plasmids. D, representative immunohistochemical images of the mouse frontal cortex injected with the AAV-CaMKIIα::ΔGR-p2A-eYFP (AAV-ΔGR-eYFP) construct. Note that signals of ΔGR (red) and eYFP (green) do not overlap, indicating high cleavage efficacy of the p2A site (C and D). The scale bars represent 10 μm (C and D). AAV, adeno-associated virus; eYFP, enhanced YFP; GR, glucocorticoid receptor.
      Figure thumbnail gr11
      Figure 11Virally mediated augmentation of GR function downregulates frontal cortex 5-HT2AR mRNA expression and disrupts sensorimotor gating in mice. AD, AAV-ΔGR-eYFP or AAV-eYFP empty vector was injected into the frontal cortex. Mice were sacrificed for analysis 3 weeks after surgery, and expression of GR (A), FKBP4 (B), FKBP5 (C), and 5-HT2AR, 5-HT2CR, and D2R mRNAs (D) in frontal cortex was assessed by qRT-PCR (n = 4 male and female 129S6/SvEv mice/group). EH, AAV-ΔGR-eYFP or AAV-eYFP empty vector was injected into the frontal cortex of 5-HT2AR+/+ (n = 14–16 male 129S6/SvEv mice/group) and 5-HT2AR−/− (n = 15–16 male 129S6/SvEv mice/group) animals. %PPI (E and F) and startle amplitude (G and H) was tested 3 weeks after surgery. Data show mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ns, not significant. Unpaired two-tailed Student’s t test (A: t6 = 3.93, p < 0.01; B: t6 = 0.098, p > 0.05; C: t6 = 3.51, p < 0.05; D: 5-HT2AR t6 = 2.93, p < 0.05; 5-HT2CR t6 = 0.66, p > 0.05; D2R t6 = 0.32, p > 0.05; G: t28 = 0.27, p > 0.05; H: t29 = 1.22, p > 0.05). Two-way ANOVA followed by Bonferroni’s post hoc test (E: AAV-ΔGR-eYFP versus AAV-eYFP F[1,84] = 8.30, p < 0.01; prepulse intensity F[2,84] = 159.6, p < 0.001; interaction F[2,84] = 0.43, p > 0.05; F: AAV-ΔGR-eYFP versus AAV-eYFP F[1,87] = 1.32, p > 0.05; prepulse intensity F[2,87] = 92.77, p < 0.001; interaction F[2,87] = 0.36, p > 0.05; for three-way ANOVA, see ). AAV, adeno-associated virus; D2R, dopamine D2 receptor; eYFP, enhanced YFP; GR, glucocorticoid receptor; 5-HT2AR, serotonin 5-HT2AR receptor; PPI, prepulse inhibition; qRT, quantitative RT.
      To determine whether overexpression of ΔGR influences behavioral responses that involve 5-HT2AR-mediated signaling pathways, we tested the effects of AAV-ΔGR-eYFP compared with the empty AAV-eYFP vector on sensorimotor gating behavior in 5-HT2AR+/+ and 5-HT2AR−/− mice. As before (Fig. 9, D and E, and Table 4), %PPI was increased in 5-HT2AR−/− animals as compared with 5-HT2AR+/+ controls (Fig. 11, E and F and Table 5), whereas startle amplitude was similar in both groups of mice (Fig. 11, G and H). Notably, AAV-mediated overexpression of ΔGR in the frontal cortex increased %PPI behavior in 5-HT2AR+/+ animals (Fig. 11E), whereas a similar manipulation of GR-dependent function failed to affect sensorimotor gating processes when this behavior model was tested in 5-HT2AR−/− mice (Fig. 11F).
      Table 5Three-way ANOVA of the effect of AAV-ΔGR-eYFP versus AAV-eYFP on %PPI in male 129S6/SvEv (in-house) mice
      Source of variationANOVAp
      Prepulse intensityF[2,171] = 241.5p < 0.001
      Viral vectorF[1,150] = 8.55p < 0.01
      GenotypeF[1,171] = 7.64p < 0.001
      Prepulse intensity × viral vectorF[2,171] = 0.78p > 0.05
      Prepulse intensity × genotypeF[2,171] = 1.71p > 0.05
      Genotype × viral vectorF[1,171] = 1.10p > 0.05
      Prepulse intensity × genotype × viral vectorF[2,171] = 0.0028p > 0.05
      We also tested whether AAV-dependent manipulation of GR transcriptional activity affected the correlation between the magnitude of the startle amplitudes during the five pulse-only trials at the beginning of the PPI session and the average %PPI in both genotypes. Importantly, frontal cortex injection of the AAV-ΔGR-eYFP construct led to a negative correlation between startle amplitudes prior to the PPI test and average %PPI in 5-HT2AR+/+ mice (Fig. 12, A and B), an effect that was not observed in 5-HT2AR−/− littermates (Fig. 12, C and D). This suggests that virally mediated augmentation of frontal cortex GR function promotes behavioral sensorimotor gating processes via 5-HT2AR.
      Figure thumbnail gr12
      Figure 12AAV-dependent manipulation of GR function affects correlation between startle amplitude and sensorimotor gating via 5-HT2AR. AD, effect of AAV-ΔGR-eYFP (B and D) versus AAV-eYFP (A and C) on Pearson’s correlation between startle magnitudes within the five stimulus-only trials at the beginning of the PPI session and %PPI in 5-HT2AR+/+ (A and B) and 5-HT2AR−/− (C and D) mice (n = 14–16 male 129S6/SvEv mice/group) and 5-HT2AR−/− (n = 15–16 male 129S6/SvEv mice/group) animals. Correlation analysis was conducted using Pearson’s r (A–D). AAV, adeno-associated virus; eYFP, enhanced YFP; GR, glucocorticoid receptor; 5-HT2AR, serotonin 5-HT2AR receptor; PPI, prepulse inhibition.

      Discussion

      Prior to these studies, although 5-HT2AR dysregulation in frontal cortex of rodent MIA models had been demonstrated (
      • Saunders J.M.
      • Moreno J.L.
      • Ibi D.
      • Sikaroodi M.
      • Kang D.J.
      • Munoz-Moreno R.
      • et al.
      Gut microbiota manipulation during the prepubertal period shapes behavioral abnormalities in a mouse neurodevelopmental disorder model.
      ,
      • Moreno J.L.
      • Kurita M.
      • Holloway T.
      • Lopez J.
      • Cadagan R.
      • Martinez-Sobrido L.
      • et al.
      Maternal influenza viral infection causes schizophrenia-like alterations of 5-HT2A and mGlu2 receptors in the adult offspring.
      ,
      • Holloway T.
      • Moreno J.L.
      • Umali A.
      • Rayannavar V.
      • Hodes G.E.
      • Russo S.J.
      • et al.
      Prenatal stress induces schizophrenia-like alterations of serotonin 2A and metabotropic glutamate 2 receptors in the adult offspring: role of maternal immune system.
      ), the underlying cause of this alteration remained unsolved. Our data using three independent models suggest that alterations in GR signaling, including both decreased nuclear immunoreactivity and reduced GR binding to the 5-HT2AR promoter, underlie increased 5-HT2AR expression in MIA offspring mouse frontal cortex. Dysregulation of 5-HT2AR, in turn, was necessary for MIA-induced reduction in mature mushroom frontal cortex dendritic spine density—implicating this process of synaptic structure alterations in the MIA model. Together, these data provide key insights into mechanisms leading to 5-HT2AR dysregulation in the context of prenatal insults, clarifying the GR-dependent control of 5-HT2AR abnormal expression and elucidating potential mechanisms involved in synaptic and sensorimotor gating deficits within the preclinical model.
      One of the most intriguing findings of our study was the potential repressive role of the transcription factor GR in the promoter activity of the 5-HT2AR gene. Using ChIP assays in frontal cortex samples from naïve mice, we showed enrichment of GR binding at a region of the 5-HT2AR gene containing a putative GR-binding site. However, in frontal cortex samples of adult mice born to mothers injected with poly-(I:C) during pregnancy, we found a significantly decreased enrichment of GR at the 5-HT2AR gene promoter. This MIA-dependent epigenetic modification was associated with augmentation of 5-HT2AR mRNA transcription in the mouse frontal cortex, which suggests that GR plays a repressive role in the transcriptional activity of the 5-HT2AR gene. This concept is further supported by our findings suggesting downregulation of 5-HT2AR expression upon continued activation of the GR pathway via AAV-ΔGR, which provides a crucial advancement to work on the relationship between the two receptors, and yields information about a potential mechanism for the exquisite sensitivity of 5-HT2ARs to stress.
      The relationship between stress and frontal cortex 5-HT2AR expression and related phenotypes is a robustly demonstrated phenomenon, observed following a variety of stressors, such as methamphetamine binge (
      • Chiu H.Y.
      • Chan M.H.
      • Lee M.Y.
      • Chen S.T.
      • Zhan Z.Y.
      • Chen H.H.
      Long-lasting alterations in 5-HT2A receptor after a binge regimen of methamphetamine in mice.
      ), maternal separation (
      • Sood A.
      • Pati S.
      • Bhattacharya A.
      • Chaudhari K.
      • Vaidya V.A.
      Early emergence of altered 5-HT2A receptor-evoked behavior, neural activation and gene expression following maternal separation.
      ), and sleep deprivation (
      • Zhao X.
      • Ozols A.B.
      • Meyers K.T.
      • Campbell J.
      • McBride A.
      • Marballi K.K.
      • et al.
      Acute sleep deprivation upregulates serotonin 2A receptors in the frontal cortex of mice via the immediate early gene Egr3.
      ). It has been speculated that this relationship between stress and the 5-HT2AR serves as a mechanism by which organisms under stress can upregulate a receptor associated with synaptic plasticity, potentially altering behavior in an advantageous manner (
      • Carhart-Harris R.L.
      • Nutt D.J.
      Serotonin and brain function: a tale of two receptors.
      ,
      • Murnane K.S.
      Serotonin 2A receptors are a stress response system: implications for post-traumatic stress disorder.
      ). In the context of gestational insults in mouse (
      • Saunders J.M.
      • Moreno J.L.
      • Ibi D.
      • Sikaroodi M.
      • Kang D.J.
      • Munoz-Moreno R.
      • et al.
      Gut microbiota manipulation during the prepubertal period shapes behavioral abnormalities in a mouse neurodevelopmental disorder model.
      ,
      • Moreno J.L.
      • Kurita M.
      • Holloway T.
      • Lopez J.
      • Cadagan R.
      • Martinez-Sobrido L.
      • et al.
      Maternal influenza viral infection causes schizophrenia-like alterations of 5-HT2A and mGlu2 receptors in the adult offspring.
      ,
      • Holloway T.
      • Moreno J.L.
      • Umali A.
      • Rayannavar V.
      • Hodes G.E.
      • Russo S.J.
      • et al.
      Prenatal stress induces schizophrenia-like alterations of serotonin 2A and metabotropic glutamate 2 receptors in the adult offspring: role of maternal immune system.
      ,
      • Malkova N.V.
      • Gallagher J.J.
      • Yu C.Z.
      • Jacobs R.E.
      • Patterson P.H.
      Manganese-enhanced magnetic resonance imaging reveals increased DOI-induced brain activity in a mouse model of schizophrenia.
      ,
      • Akatsu S.
      • Ishikawa C.
      • Takemura K.
      • Ohtani A.
      • Shiga T.
      Effects of prenatal stress and neonatal handling on anxiety, spatial learning and serotonergic system of male offspring mice.
      ,
      • Savignac H.M.
      • Couch Y.
      • Stratford M.
      • Bannerman D.M.
      • Tzortzis G.
      • Anthony D.C.
      • et al.
      Prebiotic administration normalizes lipopolysaccharide (LPS)-induced anxiety and cortical 5-HT2A receptor and IL1-beta levels in male mice.
      ) and rat (
      • Wischhof L.
      • Irrsack E.
      • Dietz F.
      • Koch M.
      Maternal lipopolysaccharide treatment differentially affects 5-HT and mGlu2/3 receptor function in the adult male and female rat offspring.
      ,
      • Wang Y.
      • Ma Y.
      • Hu J.
      • Cheng W.
      • Jiang H.
      • Zhang X.
      • et al.
      Prenatal chronic mild stress induces depression-like behavior and sex-specific changes in regional glutamate receptor expression patterns in adult rats.
      ) MIA models, upregulation of frontal cortex 5-HT2AR expression may be maladaptive and instead promote phenotypes related to perception and sensory processing. Interestingly, increased FKBP5 mRNA has been reported in postmortem prefrontal cortex samples from an Australian cohort of schizophrenia subjects relative to controls (
      • Sinclair D.
      • Fillman S.G.
      • Webster M.J.
      • Weickert C.S.
      Dysregulation of glucocorticoid receptor co-factors FKBP5, BAG1 and PTGES3 in prefrontal cortex in psychotic illness.
      ), whereas 5-HT2AR density is upregulated in frontal cortex of samples from subjects with schizophrenia (
      • Garcia-Bea A.
      • Miranda-Azpiazu P.
      • Muguruza C.
      • Marmolejo-Martinez-Artesero S.
      • Diez-Alarcia R.
      • Gabilondo A.M.
      • et al.
      Serotonin 5-HT2A receptor expression and functionality in postmortem frontal cortex of subjects with schizophrenia: selective biased agonism via Galphai1-proteins.
      ,
      • Muguruza C.
      • Miranda-Azpiazu P.
      • Diez-Alarcia R.
      • Morentin B.
      • Gonzalez-Maeso J.
      • Callado L.F.
      • et al.
      Evaluation of 5-HT2A and mGlu2/3 receptors in postmortem prefrontal cortex of subjects with major depressive disorder: effect of antidepressant treatment.
      ,
      • Muguruza C.
      • Moreno J.L.
      • Umali A.
      • Callado L.F.
      • Meana J.J.
      • Gonzalez-Maeso J.
      Dysregulated 5-HT(2A) receptor binding in postmortem frontal cortex of schizophrenic subjects.
      ,
      • Gonzalez-Maeso J.
      • Ang R.L.
      • Yuen T.
      • Chan P.
      • Weisstaub N.V.
      • Lopez-Gimenez J.F.
      • et al.
      Identification of a serotonin/glutamate receptor complex implicated in psychosis.
      )—suggesting that certain components of the GR-5-HT2AR pathway are dysregulated within the human disorder. We previously reported upregulation of frontal cortex 5-HT2AR and augmentation of psychedelic-induced head-twitch behavior in adult, but not prepubertal, MIA offspring as compared with controls (
      • Moreno J.L.
      • Kurita M.
      • Holloway T.
      • Lopez J.
      • Cadagan R.
      • Martinez-Sobrido L.
      • et al.
      Maternal influenza viral infection causes schizophrenia-like alterations of 5-HT2A and mGlu2 receptors in the adult offspring.
      ,
      • Holloway T.
      • Moreno J.L.
      • Umali A.
      • Rayannavar V.
      • Hodes G.E.
      • Russo S.J.
      • et al.
      Prenatal stress induces schizophrenia-like alterations of serotonin 2A and metabotropic glutamate 2 receptors in the adult offspring: role of maternal immune system.
      ), which supports construct validity of our model of neurodevelopmental psychiatric conditions since, as an example, the onset of schizophrenia usually occurs during adolescence or early adulthood (
      • Freedman R.
      Schizophrenia.
      ). While we have demonstrated an association between decreased GR enrichment at the 5-HT2AR promoter (a more proximal evaluation of GR interaction with the 5-HT2AR promoter than GR protein in the nuclear compartment) with increased 5-HT2AR expression in two completely independent experimental systems, as well as decreased 5-HT2AR expression with direct brain region–specific GR overexpression, further work needs to be completed to clarify the mechanisms by which GR dysregulation itself occurs within MIA models. Similarly, follow-up work could explore both the timing of these dysregulations throughout prenatal and postnatal development and adulthood as well as the level at which GR dysregulation occurs. As mentioned previously, there is a well-established relationship between GR signaling and 5-HT2AR expression and related phenotypes. To our knowledge, our work is the first to explore this associated relationship within the context of an MIA model.
      Although sex-related differences are not the main objective of this study and consequently not all the experimental techniques were confirmed in both sexes, our data suggest that MIA has a primarily sex-independent effect on two key alterations in the adult offspring (PPI deficits and density of mature mushroom spines). However, our data also suggest that dendritic structural elements such as density of immature thin spines and total spine density may vary among sexes. Further investigation will be focused on a full exploration of sex-specific differences related to MIA models.
      Regarding validation of our MIA model, our data suggested induction of IL-6 and weight loss in nonpregnant female mice following poly-(I:C) administration. It has also been proposed that maternal SFB are necessary for induction of maternal IL-17A (
      • Kim S.
      • Kim H.
      • Yim Y.S.
      • Ha S.
      • Atarashi K.
      • Tan T.G.
      • et al.
      Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring.
      ), which is necessary for at least part of the MIA-induced phenotypes (
      • Choi G.B.
      • Yim Y.S.
      • Wong H.
      • Kim S.
      • Kim H.
      • Kim S.V.
      • et al.
      The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring.
      ). Using qPCR on 16S ribosomal RNA from murine cecal contents, we demonstrate the presence of SFB in the gut of mice from sources used for our studies, but not in mice from JAX, which have previously been shown to lack gut SFB and whose offspring did not exhibit MIA-induced phenotypes following maternal poly-(I:C) administration (
      • Kim S.
      • Kim H.
      • Yim Y.S.
      • Ha S.
      • Atarashi K.
      • Tan T.G.
      • et al.
      Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring.
      ). While this supports the validity of our experimental design, we also recognize that the stress of shipping could contribute to maternal stress and potentially impact offspring phenotypes.
      Our current data raise the question of why repeated administration of systemic corticosterone and local frontal cortex AAV-ΔGR injection exert opposite effects on both 5-HT2AR mRNA expression and %PPI. While these studies were all conducted in male 129S6/SvEv mice (Table 1), one possible explanation is the ability of negative feedback to constrain GR-dependent signaling. Although both repeated corticosterone and ΔGR result in increased FKBP5 expression, suggesting the existence of negative feedback within both experimental systems, AAV-ΔGR injection results in a roughly fourfold, at least at the mRNA level, induction of GR expression. In addition, the ΔGR truncation is capable of constitutive nuclear translocation and is not expressed under the control of the endogenous GR locus, both of which could render its resistance to endogenous negative feedback mechanisms. Our observation that repeated corticosterone administration results in decreased enrichment of the GR at the 5-HT2AR promoter further supports the hypothesis that negative feedback may explain this discrepancy. Thus, sustained AAV-mediated ΔGR occupancy at the 5-HT2AR promoter would suppress 5-HT2AR transcription, whereas negative feedback upon repeated corticosterone administration would reduce endogenous GR occupancy of the 5-HT2AR promoter and allow stimulation of 5-HT2AR expression. In addition, while ΔGR-induced PPI improvements were absent in 5-HT2AR−/− mice, corticosterone-induced PPI deficits were observable in both genotypes. While this might be explained by the systemic nature of corticosterone administration, in contrast to the frontal cortex–specific AAV-mediated ΔGR expression, further studies are needed to clarify what cellular target(s) and molecular changes underlie sensorimotor gating alterations induced by this model of repeated pharmacological activation of the GR.
      We report that MIA-induced alterations in mushroom dendritic spine density are 5-HT2AR dependent. Of note, 5-HT2AR−/− mice exhibited lower mature mushroom spine density compared with 5-HT2AR+/+ controls regardless of the maternal treatment. Within a different experimental paradigm, we have recently observed similar decreases in frontal cortex spine density in 5-HT2AR−/− mice as compared with 5-HT2AR+/+ controls (
      • de la Fuente Revenga M.
      • Zhu B.
      • Guevara G.A.
      • Naler L.B.
      • Saunders J.M.
      • Zhou Z.
      • et al.
      Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.
      ). Given the implication of ligands that activate (
      • de la Fuente Revenga M.
      • Zhu B.
      • Guevara G.A.
      • Naler L.B.
      • Saunders J.M.
      • Zhou Z.
      • et al.
      Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.
      ,
      • Ly C.
      • Greb A.C.
      • Cameron L.P.
      • Wong J.M.
      • Barragan E.V.
      • Wilson P.C.
      • et al.
      Psychedelics promote structural and functional neural plasticity.
      ) or block (
      • Ibi D.
      • de la Fuente Revenga M.
      • Kezunovic N.
      • Muguruza C.
      • Saunders J.M.
      • Gaitonde S.A.
      • et al.
      Antipsychotic-induced Hdac2 transcription via NF-kappaB leads to synaptic and cognitive side effects.
      ) 5-HT2AR-dependent signaling in processes related to synaptic structural and functional plasticity, our findings corroborate the fundamental role of this serotonin receptor in these processes. Follow-up studies building on this work, such as overexpressing ΔGR and/or 5-HT2AR in the frontal cortex of MIA and control offspring to determine if these manipulations are sufficient to affect dendritic spine density, could further explore this relationship and would be useful for confirming the mechanisms suggested by the data presented here.
      Our data support a negative regulatory relationship between GR-dependent signaling and 5-HT2AR expression with implications for dendritic structural plasticity, functional GR nuclear translocation, and behavior phenotypes observed in mouse MIA models. These findings advance our understanding of the molecular mechanisms by which prenatal insults upregulate 5-HT2AR expression in the frontal cortex of the adult offspring.

      Experimental procedures

      Materials, drug administration, and mouse brain and blood samples

      Poly-(I:C) (MilliporeSigma; catalog no.: P9582) was dissolved in normal saline (0.9% NaCl) and, after a brief centrifugation (800g), the resulting solution (4 mg/ml) was aliquoted and stored at −20 °C until the day of the experiment.
      Corticosterone (MilliporeSigma; catalog no.: 46148) was diluted in dimethyl sulfoxide (DMSO) (100 mg/ml), and aliquots were stored at −20 °C until the day of the experiment. Adult male mice were injected (s.c.) with corticosterone (50 mg/kg) dissolved in a final solution of DMSO:ethanol (1:3). For time-course assays, all mice (excluding the no-injection group) were injected (s.c.) twice a day over 8 days with corticosterone (50 mg/kg), or vehicle, and sacrificed 8.5 to 13 h after the last injection. For the short-term experiments, adult male mice received (s.c.) corticosterone (50 mg/kg), or vehicle, twice a day for 4 days. Mice were either sacrificed or tested for behavior 8.5 to 13 h after the last injection.
      For brain samples, mice were sacrificed for analysis by cervical dislocation, and bilateral frontal cortex (bregma 1.40–1.90 mm) was dissected and either frozen at −80 °C or immediately processed for RNA extraction, ChIP, and/or biochemical assays. The coordinates were taken according to a published atlas of the C57BL/6 and 129Sv mouse brains (
      • Hof P.R.
      • Young W.G.
      • Bloom F.E.
      • Belichenko P.V.
      • Celio M.R.
      Comparative Cytoarchitectonic Atlas of the C57BL/6 and 129/Sv Mouse Brains.
      ).
      For serum samples, following decapitation, trunk blood was collected and allowed to clot at room temperature for at least 30 min. The blood was then centrifuged (1000g) at 4 °C for 10 min after which the serum supernatant was collected and stored at −80 °C until the day of the assays.
      All other chemicals were obtained from standard sources.

      Animals

      Experiments were performed on adult (10–22 week old) male C57BL6/N, C57BL6/J, and/or 129S6/SvEv mice (Table 1). Animals were housed on a 12 h light/dark cycle (lights on 6 AM to 6 PM) at 23 °C with food and water ad libitum, except during behavioral testing, which took place during the light cycle. All procedures were conducted in accordance with the National Institutes of Health (NIH) guidelines and were approved by the Virginia Commonwealth University Animal Care and Use Committee. Behavioral testing took place between 9:00 AM and 6:00 P.M.
      5-HT2AR (Htr2a) knockout (5-HT2AR−/−) mice on a 129S6/SvEv background have been previously described (
      • Gonzalez-Maeso J.
      • Weisstaub N.V.
      • Zhou M.
      • Chan P.
      • Ivic L.
      • Ang R.
      • et al.
      Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior.
      ). For experiments involving 5-HT2AR−/− mice, wildtype (5-HT2AR+/+) littermates on a 129S6/SvEv background were used as controls. All subjects were offspring of heterozygote breeding.

      Immune activation with poly-(I:C)

      To test the effect of poly-(I:C) administration on female immune response (data presented in Fig. 1), naïve adult animals (C57BL6/N from CRL and 129S6/SvEv generated via in-house breeding) were injected (i.p.) with poly-(I:C) (20 mg/kg), or vehicle, and observed for sickness behavior, such as lethargy, ptosis, and hunched posture. After 2.5 h, mice were sacrificed by either decapitation and exsanguination (for experiments involving serum samples) or by cervical dislocation for collection of frontal cortex samples.
      To test the effect of MIA during pregnancy (data presented in Figs. 3, 5, 6, D, E, and 7, A–D), pregnant (E12.5) female mice were injected (i.p.) with poly-(I:C) (20 mg/kg), or vehicle, and then monitored for sickness behavior. This timing was chosen because the first and second trimesters have been shown to be a critical period for increased risk of neurodevelopmental conditions including schizophrenia and autism following influenza virus infection in humans (
      • Brown A.S.
      • Derkits E.J.
      Prenatal infection and schizophrenia: a review of epidemiologic and translational studies.
      ), and MIA at E12.5 has been demonstrated to produce MIA-induced phenotypes in offspring (
      • Kentner A.C.
      • Bilbo S.D.
      • Brown A.S.
      • Hsiao E.Y.
      • McAllister A.K.
      • Meyer U.
      • et al.
      Maternal immune activation: reporting guidelines to improve the rigor, reproducibility, and transparency of the model.
      ). To reduce litter loss, MIA or mock mice were pair-housed with another nonpregnant female of the same strain and provided with enrichment Bio-Huts (Bio-Serv) as shelter in the cage. It has been shown that litter effects are substantial (
      • Mueller F.S.
      • Scarborough J.
      • Schalbetter S.M.
      • Richetto J.
      • Kim E.
      • Couch A.
      • et al.
      Behavioral, neuroanatomical, and molecular correlates of resilience and susceptibility to maternal immune activation.
      ). To avoid litter effects, animals from at least two separate litters were subjected to the different protocols (Table 1). For MIA experiments in “wild-type” animals, assays were carried out with either timed pregnant C57BL6/N mice ordered from CRL (Figs. 3, A, B, 6, D, E, and 7, A–D) or C57BL6/N females in-house bred with C57BL6/N males from CRL (Fig. 3, C and D). For MIA experiments in 5-HT2AR−/− mice and 5-HT2AR+/+ controls (Fig. 5), 129S6/SvEv female 5-HT2AR+/− (heterozygote) mice (Taconic background) were bred in-house; females were monitored for mating plug, and pregnancy was evaluated using both weight gain and visual appearance of the flanks. All offspring (C57BL6/N and 129S6/SvEv) were weaned between 3 and 4 weeks of age, and littermates were evaluated in biochemical, dendritic spine structure, and/or behavioral assays as adult animals (10–22 weeks old).

      PPI of startle

      PPI of startle experiments were conducted following a previously described paradigm (
      • Vohra H.Z.
      • Saunders J.M.
      • Jaster A.M.
      • de la Fuente Revenga M.
      • Jimenez J.
      • Fernandez-Teruel A.
      • et al.
      Sex-specific effects of psychedelics on prepulse inhibition of startle in 129S6/SvEv mice.
      ). Briefly, mice were placed in SR-Lab Startle Response System (San Diego Instruments) chambers and allowed to habituate to background noise for 5 min. Following habituation, mice were exposed to five startle-only trials. They were then subjected to 65 pseudorandomized trials consisting of interspersed no-stimulus trials, startle-only trials, or 73 dB, 77 dB, or 85 dB prepulse trials. The session concluded with five additional startle-only trials. %PPI was calculated based on averages for each trial type during those 65 trials by the equation %PPI = (1 – [Vmax of prepulse trials/Vmax of stimulus-only trials]) × 100. Startle magnitude was calculated as the average of the Vmax value for the five initial startle-only trials. Background noise was maintained at 69 dB for the duration of the experiment. The startle-inducing stimulus was a 120 dB pulse lasting for 20 ms. For prepulse trials, a 20 ms prepulse of 73 dB, 77 dB, or 85 dB was administered prior to the startle stimulus. The interstimulus interval was 100 ms from the start of the prepulse to the start of the startle stimulus. The intertrial interval was an average of 15 s, with a range of 12 to 18 s. Each PPI session lasted a total of about 30 min.

      Plasmid construction

      The AAV-CaMKIIα::eYFP construct has been described previously (
      • Ibi D.
      • de la Fuente Revenga M.
      • Kezunovic N.
      • Muguruza C.
      • Saunders J.M.
      • Gaitonde S.A.
      • et al.
      Antipsychotic-induced Hdac2 transcription via NF-kappaB leads to synaptic and cognitive side effects.
      ). The pCMV-GR11 construct was obtained from Addgene (catalog no.: 89105). To generate the AAV-CaMKIIα::ΔGR-p2A-eYFP construct, a PCR fragment was obtained from mouse frontal cortex cDNA as a template using the primers mΔGR-BamHI/S and mΔGR-Ascl/A (mΔGR cloning forward and reverse, respectively; Table S6). The resulting PCR product was digested with BamHI and AscI and subcloned into the same restriction sites of AAV-CaMKIIα-c-Myc-5-HT2AR-p2A-eYFP. All PCR construct amplifications were performed with PfuUltra high-fidelity DNA polymerase (Agilent) in a Mastercycler EP Gradient Auto thermal cycler (Eppendorf). All the constructs were confirmed by DNA sequencing.

      Virally mediated gene transfer

      The construct vectors CaMKIIα::eYFP and CaMKIIα::ΔGR-p2A-eYFP were packaged into AAV serotype 8 vector particles, all produced by the University of North Carolina at Chapel Hill Vector Core. The AAV-eYFP and AAV-ΔGR-eYFP construct vectors were injected into the frontal cortex (+1.6 mm rostrocaudal, −2.4 mm dorsoventral, and +2.6 mm mediolateral relative to bregma) of adult mice by stereotaxic surgery according to standard methods (
      • Ibi D.
      • de la Fuente Revenga M.
      • Kezunovic N.
      • Muguruza C.
      • Saunders J.M.
      • Gaitonde S.A.
      • et al.
      Antipsychotic-induced Hdac2 transcription via NF-kappaB leads to synaptic and cognitive side effects.
      ,
      • de la Fuente Revenga M.
      • Zhu B.
      • Guevara G.A.
      • Naler L.B.
      • Saunders J.M.
      • Zhou Z.
      • et al.
      Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.
      ). Mice were anesthetized with isoflurane (2% initial dose, 1–2% maintenance) during the surgery.

      Dendritic spine analysis

      Frontal cortex dendritic spine structural assays were performed as previously reported (
      • Ibi D.
      • de la Fuente Revenga M.
      • Kezunovic N.
      • Muguruza C.
      • Saunders J.M.
      • Gaitonde S.A.
      • et al.
      Antipsychotic-induced Hdac2 transcription via NF-kappaB leads to synaptic and cognitive side effects.
      ,
      • de la Fuente Revenga M.
      • Zhu B.
      • Guevara G.A.
      • Naler L.B.
      • Saunders J.M.
      • Zhou Z.
      • et al.
      Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.
      ). Briefly, male and female mice were deeply anesthetized (ketamine/xylazine) after which transcardial perfusion was performed with PBS (about 10 ml) followed by 30 ml of 4% paraformaldehyde (PFA) in PBS. Following perfusion, the brain was dissected and stored in 30% sucrose in PBS at 4 °C until sectioning. The frontal cortex samples were cut into 50 μm sections (VT1000S vibratome; Leica) after which sections were stored at 4 °C until immunofluorescence. For immunofluorescence assays, frontal cortex tissue sections were incubated in 4% PFA in PBS for 20 min. Sections were then washed with PBS for 10 min and permeabilized in 0.1% Triton X-100 in PBS for 15 min. After an additional two washes in PBS, sections were blocked in blocking buffer (5% bovine serum albumin [BSA] in 0.1% Triton X-100 in PBS). Sections were then incubated with primary antibody (rabbit anti-GFP; Invitrogen; catalog no.: A-11122, 1:1000 dilution) in blocking buffer overnight at 4 °C. The sections were then washed in PBS and incubated with secondary antibody (anti-rabbit; Alexa-488, Invitrogen A-11008; 1:2000 dilution) in blocking buffer at room temperature for 1 h. After this, the sections were washed three times with PBS and mounted on #1.5 coverslips (Fisher) in ProLong Diamond antifade mountant (Invitrogen). After placement of slides, the sections were allowed to cure overnight before sealing with nail polish top coat (Seche Vite). Slides were stored at 4 °C in the dark until imaging.
      Slides were imaged by confocal laser scanning microscopy using the 488 nm laser line of an LSM 710 microscope (Zeiss). Z-stacks were acquired for layer 5 dendrites in mouse frontal cortex using a 63× objective with 2.5× zoom, yielding stacks of 632 × 632 pixel images with a voxel size of 0.09 μm × 0.09 μm × 0.2 μm. Files were converted to TIFF format for analysis. Dendrite image stacks were then analyzed using NeuronStudio (a non-commercial software created at Mount Sinai School of Medicine [
      • Ibi D.
      • de la Fuente Revenga M.
      • Kezunovic N.
      • Muguruza C.
      • Saunders J.M.
      • Gaitonde S.A.
      • et al.
      Antipsychotic-induced Hdac2 transcription via NF-kappaB leads to synaptic and cognitive side effects.
      ,
      • de la Fuente Revenga M.
      • Zhu B.
      • Guevara G.A.
      • Naler L.B.
      • Saunders J.M.
      • Zhou Z.
      • et al.
      Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice.
      ]) by a blinded experimenter. Dendritic lengths were determined by tracing the dendrite images within the software. With its Rayburst sampling algorithm, NeuronStudio was used to classify dendritic spines as stubby, thin, or mushroom in morphology.

      Transient transfection of Neuro-2a cells

      Neuro-2a cells (American Type Culture Collection; CCL-131) were maintained in a growth medium of Dulbecco's modified Eagle's medium with glucose, l-glutamine, and sodium pyruvate (Corning) supplemented with 5% qualified fetal bovine serum (Gibco) and 1% 10,000 U/ml penicillin/10,000 μg/ml streptomycin solution (Gibco) at 37 °C in a 5% CO2 humidified atmosphere. To prepare a surface for adherence of Neuro-2a cells, coverslips (#1) (Fisherbrand) were incubated in 10 M NaOH at room temperature overnight with gentle shaking, washed, and autoclaved. The coverslips were coated in poly-d-lysine (Sigma–Aldrich) in borate buffer (50.1 mM BH3O3 and 12.5 mM Na2B4O7·10H2O) overnight. The next day, coverslips were washed three times in sterile water and then allowed to dry. For transfection, Neuro-2a cells were plated on poly-d-lysine-coated coverslips and allowed to grow. Neuro-2a cells on coverslips in 6-well plates were transfected with 4 μg of plasmid DNA with 5 μl of polyethyleneimine made up to a final volume of 300 μl with Opti-MEM reduced serum medium (Gibco) that were added to growth medium. Prior to transfection, DNA, polyethyleneimine, and Opti-MEM were combined, shaken, and incubated for 10 min at room temperature. The medium was replaced with regular growth medium the day after transfection. Cells were processed for immunofluorescence 48 h after transfection as described later. For immunoblot assays, Neuro-2a cells in 10 cm plates were transfected with 6 μg of plasmid DNA (AAV-ΔGR-eYFP plasmid construct, see above, or a construct expressing the full-length human GR under control of the cytomegalovirus promoter, pCMV-GR11; Addgene; catalog no.: 89105) with 10 μl of polyethyleneimine made up to a final volume of 300 μl with Opti-MEM as described above. To harvest, transfected Neuro-2a cells were washed with Dulbecco’s PBS three times before being dislodged with a cell scraper in cold Dulbecco’s PBS. Cells were then pooled and spun down for 5 min at 188g at room temperature. The supernatant was removed, and pelleted cells were frozen at −80 °C until they were subjected to subcellular fractionation followed by immunoblot assays as described later.

      Immunohistochemistry

      In Neuro-2a cells, experiments were performed as previously reported with minor modifications (
      • Ibi D.
      • de la Fuente Revenga M.
      • Kezunovic N.
      • Muguruza C.
      • Saunders J.M.
      • Gaitonde S.A.
      • et al.
      Antipsychotic-induced Hdac2 transcription via NF-kappaB leads to synaptic and cognitive side effects.
      ). Briefly, GR immunoreactivity was assayed by using an anti-GR antibody (Santa Cruz; catalog no.: sc-393232; 1:50 dilution) and Alexa 568 dye-conjugated anti-mouse secondary antibody (Invitrogen; catalog no.: A-11004; 1:1000 dilution).
      In mouse frontal cortex, brains of 39-week-old female mice that had been stereotaxically injected with AAV-eYFP versus AAV-ΔGR-eYFP were cut into 50 μm sections, and the resulting slices were then subjected to the immunofluorescence staining protocol (see above): primary antibody (mouse anti-GR; Santa Cruz; catalog no.: sc-393232; 1:50 dilution), secondary antibody (anti-mouse Alexa-488; Invitrogen; catalog no.: A-11004; 1:2000 dilution). Images were acquired using the 405, 488, and 561 nm laser lines of an LSM 710 confocal laser scanning microscope. A 63× objective was used, resulting in 1912 × 1912 pixel images with a 0.07 μm × 0.07 μm pixel size.

      FISH

      Using a subcloned and sequenced PCR fragment (162 bp; Table S6) of the mouse 5-HT2AR cDNA (Zero Blunt TOPO PCR Cloning kit; Invitrogen), high-specific activity RNA probes were produced from 3 μg of linearized plasmid (HindIII) and labeled using the Fluorescein RNA Labeling Mix (Roche), following the manufacturer's instructions. All work surfaces and dissection tools were thoroughly cleaned with RNAse Zap (Life Technologies), and great care was taken to maintain an RNase-free environment during tissue sample collection. Adult mice were perfused, and brain samples were sectioned as described previously with minor modifications that included transcardial perfusion with diethyl pyrocarbonate (DEPC)-PBS (about 10 ml) followed by 30 ml of 4% microscopy grade PFA (Electron Microscopy Sciences) in DEPC-PBS at 4 °C. Following perfusions, the brain was dissected and stored in cryoprotectant (2% DMSO and 20% glycerol in DEPC-PBS) until sectioning. The frontal cortex samples were cut into 20 μm sections (VT1000S vibratome) after which sections were stored at 4 °C until hybridization. Coronal brain sections were washed with DEPC-PBS and incubated with saline sodium citrate (SSC) 5× buffer (748 mM NaCl, 74.8 mM Na3C6H5O7·H2O in DEPC-H2O, pH 7) with shaking. A border was drawn around the sections with a Liquid Blocker hydrophobic pen (Daido Sangyo Co), and slides were placed in a preheated humidified StainTray Slide Staining System (Simport). The slides were covered with blocking solution (50% formamide, 25% SSC 20× buffer [2.99 M NaCl, 299 mM Na3C6H5O7·H2O in DEPC-H2O, pH 7.0], 25% DEPC-H2O, 0.8% salmon sperm DNA; Invitrogen) and incubated at 37 °C for 2 h. The fluorescein-labeled RNA probe was diluted at the appropriate concentration (1:20–1:100) in hybridization buffer (50% formamide, 40% dextran sulfate solution; Millipore, 10% SSC 20×), and the resulting solution was heated at 77 °C for 5 min. The blocking solution was removed from the sections, and the probe in hybridization buffer was added; the slides were then covered with HybriSlip hybridization covers (Grace Bio-Labs), sealed with Fixogum rubber cement (Marabu), and incubated overnight at 37 °C. The next day, the slide covers were removed and slides were washed at room temperature in SSC 2× buffer (299 mM NaCl, 29.9 mM Na3C6H5O7·H2O in ddH2O, pH 7.0) for 5 min three times in Coplin jars with shaking. The slides were then washed for 30 min with shaking in SSC 2× buffer that had been heated to 43 °C, after which the slides were washed again for 30 min with shaking in SSC 0.1× buffer (15 mM NaCl, 1.5 mM Na3C6H5O7·H2O in ddH2O, pH 7.0) that had been heated to 43 °C. Autofluorescence was then reduced by incubating slides with shaking in Coplin jars containing 0.1% Sudan Black B that had been dissolved in 70% ethanol in the dark overnight and syringe filtered using a 0.45 μm filter (Corning). Slides were then washed three times in PBS with shaking. Sections were dried, mounted in Duolink In Situ Mounting Medium with 4′,6-diamidino-2-phenylindole (Sigma–Aldrich), covered with #1.5 coverslips, and sealed. The 488 nm laser line of an LSM 710 confocal laser scanning microscope was used to acquire images from mouse frontal cortex (delineated by +1.4 to +1.9 mm relative to bregma). Images were acquired with a 63× objective, yielding 1912 × 1912 pixel images with a pixel size of 0.07 μm × 0.07 μm. Files were converted to TIFF format for analysis. Fluorescence intensity was evaluated by a blinded experimenter using ImageJ (NIH): a region of interest was drawn around the border for each cell, and mean fluorescence intensity in the region of interest was used for data analysis—cells were selected from among two fields from each animal.

      ELISAs

      ELISAs were performed using the mouse IL-6 ELISA-MAX kit (BioLegend) using the manufacturer’s instructions. Briefly, 100 μl of a 1:200 dilution of capture antibody in coating buffer (100 mM NaHCO3, 33.6 mM Na2CO3 in ddH2O, pH 9.5) was added to wells of a 96-well Nunc MaxiSorp plate (Invitrogen) and incubated at 4 °C overnight. The next day, the wells were washed in 300 μl wash buffer (137 mM NaCl, 2.68 mM KCl, 10.1 mM Na2HPO4, 1.76 mM KH2PO4, 0.05% Tween-20, pH 7.4) three times. The wells were then blocked in 200 μl assay diluent (137 mM NaCl, 2.68 mM KCl, 10.1 mM Na2HPO4, 1.76 mM KH2PO4, 1% BSA in ddH2O, pH 7.4) at room temperature for 1 h with shaking. The wells were then washed four times in wash buffer. After this, 100 μl of 1:100 dilutions of serum samples in assay diluent were added to wells in triplicate, as were mouse IL-6 standards (7.8–500 pg/ml) diluted in assay diluent, before sealing and incubation with shaking at room temperature for 2 h. The wells were then washed four times in wash buffer after which 100 μl of a 1:200 dilution of detection antibody in assay diluent was added to each well and the plate was sealed and incubated at room temperature for 1 h with shaking. The wells were then washed four times in wash buffer before addition of 100 μl of a 1:1000 dilution of Avidin-horseradish peroxidase (HRP) in assay diluent, sealing, and incubation at room temperature for 30 min with shaking. The plate was then washed five times in wash buffer, with soaking of wells for 30 s to 1 min during each wash. After this, 100 μl of TMB substrate solution (BioLegend, a 1:1 mixture of TMB substrate A and TMB substrate B) was added to each well and incubated in the dark for 5 to 10 min. The reaction was stopped by addition of 100 μl stop solution (BioLegend), and absorbance was read at 450 nm with subtraction of the absorbance at 570 nm using a VICTOR Nivo multimode plate reader (PerkinElmer).

      Quantitative real-time PCR

      Real-time qPCR (qRT-PCR) assays were carried out as previously described. Analysis was done using the ΔΔCT method (
      • de la Fuente Revenga M.
      • Ibi D.
      • Cuddy T.
      • Toneatti R.
      • Kurita M.
      • Ijaz M.K.
      • et al.
      Chronic clozapine treatment restrains via HDAC2 the performance of mGlu2 receptor agonism in a rodent model of antipsychotic activity.
      ). Primer pair sequences are listed in Table S6—see also (
      • Kim S.
      • Kim H.
      • Yim Y.S.
      • Ha S.
      • Atarashi K.
      • Tan T.G.
      • et al.
      Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring.
      ,
      • Ibi D.
      • de la Fuente Revenga M.
      • Kezunovic N.
      • Muguruza C.
      • Saunders J.M.
      • Gaitonde S.A.
      • et al.
      Antipsychotic-induced Hdac2 transcription via NF-kappaB leads to synaptic and cognitive side effects.
      ,
      • Chow K.H.
      • Yan Z.
      • Wu W.L.
      Induction of maternal immune activation in mice at mid-gestation stage with viral mimic poly(I:C).
      ,
      • Kurita M.
      • Holloway T.
      • Garcia-Bea A.
      • Kozlenkov A.
      • Friedman A.K.
      • Moreno J.L.
      • et al.
      HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity.
      ,
      • Nguyen V.T.
      • Farman N.
      • Maubec E.
      • Nassar D.
      • Desposito D.
      • Waeckel L.
      • et al.
      Re-epithelialization of pathological cutaneous wounds is improved by local mineralocorticoid receptor antagonism.
      ,
      • Lebsack T.W.
      • Fa V.
      • Woods C.C.
      • Gruener R.
      • Manziello A.M.
      • Pecaut M.J.
      • et al.
      Microarray analysis of spaceflown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors.
      ,
      • Zheng D.
      • Sabbagh J.J.
      • Blair L.J.
      • Darling A.L.
      • Wen X.
      • Dickey C.A.
      MicroRNA-511 binds to FKBP5 mRNA, which encodes a chaperone protein, and regulates neuronal differentiation.
      ).

      Microbiome evaluation

      Adult female mice (C57BL6/N from CRL, C57BL6/N from Taconic, C57BL6/J from JAX, and 129S6/SvEv generated via in-house breeding) were sacrificed, and cecum samples were collected based on previously described protocols (
      • Saunders J.M.
      • Moreno J.L.
      • Ibi D.
      • Sikaroodi M.
      • Kang D.J.
      • Munoz-Moreno R.
      • et al.
      Gut microbiota manipulation during the prepubertal period shapes behavioral abnormalities in a mouse neurodevelopmental disorder model.
      ). Briefly, Eppendorf tubes filled with cecum contents were then weighed to obtain wet weights, and samples were frozen at –80 °C until use. DNA was isolated using a PowerFecal DNA Isolation kit (MO BIO Laboratories), and DNA samples were then stored at −80 °C until use. After this, DNA samples (20 ng) were loaded into each reaction for qPCR (for primer pair sequences, see Table S6). Values are reported based on amplification of the 16S rRNA gene normalized to wet weight of cecum contents.

      Cellular fractionation

      Neuro-2a cells, mouse frontal cortex, or postmortem human frontal cortex samples were homogenized in 1 ml of Tris–HCl (50 mM, pH 7.4) supplemented with 0.25 M sucrose (Neuro-2a and mouse) or 6 ml of Tris–HCl (50 mM, pH 7.4) supplemented with 0.32 M sucrose (postmortem human frontal cortex) using a Teflon-glass homogenizer. The samples were then centrifuged at 1000g for 5 min (mouse frontal cortex) or 15 min (postmortem human frontal cortex) at 4 °C. The supernatant (S1 fraction) was transferred to a new tube. The pellet (P1 fraction) was resuspended in Tris–HCl and centrifuged under the same conditions. The final pellet was resuspended in Tris–HCl and transferred to a new tube for use as the nuclear fraction. The S1 fraction was centrifuged at 40,000g for 20 min at 4 °C. The resulting supernatant (S2 fraction) was retained as the cytoplasmic fraction. The cytoplasmic and nuclear fractions were stored at −80 °C.

      Immunoblot assays

      Western blot experiments were performed as previously reported, with minor modifications (
      • Ibi D.
      • de la Fuente Revenga M.
      • Kezunovic N.
      • Muguruza C.
      • Saunders J.M.
      • Gaitonde S.A.
      • et al.
      Antipsychotic-induced Hdac2 transcription via NF-kappaB leads to synaptic and cognitive side effects.
      ). Briefly, samples were loaded onto polyacrylamide gel (12%) and submitted to sodium dodecyl sulfate-PAGE. After transfer to nitrocellulose membranes, samples were blocked with 2.5% nonfat dry milk and 0.5% BSA in TBST buffer (65 mM Tris–HCl, 150 mM NaCl, 0.05 Tween-20, pH 7.6), followed by overnight incubation with primary antibody at 4 °C or 1 h at room temperature. In Neuro-2a cells and mouse frontal cortex samples, the following primary antibodies were used: mouse anti-GR (Santa Cruz; catalog no.: sc-393232, 1:100 dilution), mouse anti-α-tubulin (Abcam; catalog no.: ab7291, 1:3000 dilution), rabbit anti-β-actin (Abcam; catalog no.: ab8227, 1:3000–1:20,000 dilution). In Neuro-2a cells and mouse frontal cortex samples, incubation with secondary antibodies (HRP-linked mouse IgG; Amersham, catalog no.: NA931, or HRP-linked rabbit IgG; Amersham, catalog no.: NA934, 1:5000 dilution) was performed at room temperature for 60 to 90 min, followed by repeated washing with TBST. Immunoreactive proteins were visualized using a ChemiDoc MP Imaging System (Bio-Rad). In each case, the blots were stripped and reprobed for a control protein to control loading amounts. All immunoblots were quantified by densitometry using ImageJ (NIH). In postmortem frontal cortex samples, incubation with secondary antibodies (Alexa 680-conjugated anti-mouse IgG, Invitrogen; catalog no.: A21057, 1:6000 dilution, or DyLight 800-conjugated anti-rabbit IgG, Rockland, catalog no.: 611-745-127; 1:10,000 dilution) was performed at room temperature for 60 to 90 min, followed by repeated washing with TBST. Immunoreactive proteins were visualized using an Odyssey infrared imaging system (LI-COR Biosciences). Signal was quantified as integrated intensity using Image Studio Lite version 5.2 (LI-COR Biosciences).

      ChIP assay

      ChIP experiments were performed as previously reported with minor modifications (
      • Ibi D.
      • de la Fuente Revenga M.
      • Kezunovic N.
      • Muguruza C.
      • Saunders J.M.
      • Gaitonde S.A.
      • et al.
      Antipsychotic-induced Hdac2 transcription via NF-kappaB leads to synaptic and cognitive side effects.
      ,
      • de la Fuente Revenga M.
      • Ibi D.
      • Cuddy T.
      • Toneatti R.
      • Kurita M.
      • Ijaz M.K.
      • et al.
      Chronic clozapine treatment restrains via HDAC2 the performance of mGlu2 receptor agonism in a rodent model of antipsychotic activity.
      ), using a MAGnify Chromatin Immunoprecipitation System (Invitrogen). Primer pair sequences are listed in Table S6.

      Postmortem human brain samples

      Human brains were obtained at autopsies performed in the Basque Institute of Legal Medicine, Bilbao, Spain. The study was developed in compliance with policies of research and ethical review boards for postmortem brain studies (Basque Institute of Legal Medicine, Spain). Retrospective searches were conducted for previous medical diagnosis and treatment using examiners’ information and records of hospitals and mental health centers. After searching antemortem information, 32 subjects who had met criteria of schizophrenia according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV and/or DSM-IV TR) were selected (
      American Psychiatric Association
      Diagnostic and Statistical Manual of Mental Disorders.
      ). Toxicological screening for antipsychotics, other drugs, and ethanol was performed in blood, urine, liver, and gastric content samples. The toxicological assays were performed at the National Institute of Toxicology, Madrid, Spain, using a variety of standard procedures including radioimmunoassay, enzymatic immunoassay, high-performance liquid chromatography, and gas chromatograph mass spectrometry. The schizophrenia subjects were divided into 16 antipsychotic-free (AP-free) and 16 antipsychotic-treated (AP-treated) subjects according to the presence or the absence of antipsychotics in blood at the time of death. Controls for the present study were chosen among the collected brains on the basis, whenever possible, of the following cumulative criteria: (i) negative medical information on the presence of neuropsychiatric disorders or drug abuse and (ii) appropriate sex, age, postmortem delay (time between death and autopsy), and freezing storage time to match each subject in the schizophrenia group. Causes of death among the schizophrenia patients included suicide (n = 19), accidents (n = 2), and natural deaths (n = 11). Controls’ causes of death included accidents (n = 20) and natural deaths (n = 12). Specimens of prefrontal cortex (Brodmann’s area 9) were dissected at autopsy (0.5–1.0 g tissue) on an ice-cooled surface and immediately stored at −80 °C until use. Tissue pH values were within a relatively narrow range (control subjects: 6.48 ± 0.08; schizophrenia subjects: 6.28 ± 0.05). The definitive pairs of AP-free schizophrenia subjects and respective matched controls are shown in Table 2, and the definitive pairs of AP-treated schizophrenia subjects and respective matched controls are shown in Table 3. Pairs of schizophrenia patients and matched controls were processed simultaneously and under the same experimental conditions.

      Statistical analysis

      Animals were randomly allocated into the different experimental groups. Statistical significance was assessed by Student’s t test, and one-way or two-way (or multiway) ANOVA, depending upon the number of experimental conditions and independent variables. Following a significant ANOVA, specific comparisons were made using the Bonferroni’s post hoc test. For the corticosterone time course, Dunnett’s post hoc test was used because the goal was to determine which groups were different from vehicle. Male and female mice were tested in Figures 3, C, D, 5, B–F, and 11, A–D (Table 1). For sex-related effects in Figures 3, C, D and 5, see Tables S1–S5. Sex as an independent variable was not tested in Figure 11, A–D because two mice per sex per group were utilized. Datapoints were excluded based on previously established criterion and were set to ±2 SD from the group mean. Correlation analysis was conducted using the Pearson’s r. All values in the figures represent mean ± SEM. All statistical analyses were performed with GraphPad Prism software, version 9 (GraphPad Software, Inc), and comparisons were considered statistically significant if p < 0.05.

      Data availability

      All data presented in this article are available upon request.

      Supporting information

      This article contains supporting information (
      • Hof P.R.
      • Young W.G.
      • Bloom F.E.
      • Belichenko P.V.
      • Celio M.R.
      Comparative Cytoarchitectonic Atlas of the C57BL/6 and 129/Sv Mouse Brains.
      ).

      Conflict of interest

      J. G.-M. had a sponsored research contract with NeuRistic. All the other authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank Mario de la Fuente for the critical review of the article, Terrell Holloway for the early help in PPI experiments, Dexter Jandres Rivera for the technical assistance, and the staff members of the Basque Institute of Legal Medicine for the cooperation in the study.

      Author contributions

      J. M. S. and J. G.-M. conceptualization; J. M. S, J. J. M., P. M. B., and J. G.-M. methodology; J. M. S. and J. G.-M. formal analysis; J. M. S., C. M., S. S., and J. L. M. investigation; L. F. C., J. J. M., and P. M. B. resources; J. M. S. and J. G.-M. writing–original draft; J. J. M., P. M. B, and J. G.-M. supervision; J. G.-M. funding acquisition.

      Funding and additional information

      National Institutes of Health R01MH084894 (to J. G.-M.), R01MH111940 (to J. G.-M.), NIH-N01DA-17-8932 (to P. M. B.), NIH-N01DA-19-8949 (to P. M. B.), and F30MH116550 (to J. M. S.), and Basque Government IT1211-19 (to J. J. M.) participated in the funding of this study. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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